Process for synthesizing cyclic peptide

Abstract

Provided is a process for synthesizing a cyclic peptide. Specifically provided is a method for synthesizing a cyclic peptide as represented by the following formula (I). The method significantly improves the purity and yield of the cyclic peptide by means of optimizing the route and condition of solid-phase peptide synthesis, and effectively reduce the number of impurities and the maximum single impurity content, thereby providing the target cyclic peptide with a purity of 99.5% or more.

Description

Cyclic peptide synthesis process

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 202510354192.4, filed on March 24, 2025, the contents of which are incorporated herein by reference in their entirety and for all purposes.

[0003] sequence list

[0004] This application contains a sequence list in computer-readable form, which is submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence list XML file submitted with this application is named "IEC260133PCT", created on March 17, 2026, and is 7KB in size. Technical Field

[0005] This invention belongs to the field of peptide synthesis, and more specifically, this invention relates to the synthesis process of cyclic peptides. Background Technology

[0006] Since Merrifield successfully developed the solid-phase peptide synthesis (SPPS) method in 1963, it has been continuously improved and refined, and today it has become a commonly used technique in peptide and protein synthesis. The basic principle is to first covalently link the carboxyl group of the first amino acid at the C-terminus (carboxyl terminus) of the desired peptide chain to a solid support. Then, subsequent amino acids are progressively linked to extend the peptide chain until the desired peptide sequence is reached. Each step involves repeated deprotection and condensation operations. Finally, the peptide chain is cleaved from the resin and purified to obtain a high-purity linear peptide.

[0007] Cyclic peptides have become a hot topic in peptide research in recent years due to their superior conformational stability, bioavailability, and metabolic stability compared to linear peptides. The synthesis of cyclic peptides, in addition to linear peptide synthesis, also includes an intramolecular cyclization step. Steric hindrance of side chain groups, rigid constraints of the cyclic structure, and intermolecular side reactions are all important factors affecting the efficiency of the cyclization reaction and the purity of the target cyclic peptide. Summary of the Invention

[0008] The cyclic peptide shown in Formula I is a novel cyclic peptide with significant biological activity developed by the applicant. It significantly increases the levels of Collagen I, hyaluronic acid (HA), and elastin in fibroblasts, and exhibits good safety, demonstrating great potential in the pharmaceutical and cosmetic fields. Based on the above research results, this paper further optimizes the synthesis process of the cyclic peptide, laying the foundation for its industrial application. Specifically, this invention provides a method for synthesizing the cyclic peptide shown in Formula I or its salts, the method comprising coupling amino acids and / or polypeptide substrates through 1-6 peptide bond condensation reactions to obtain the cyclic peptide or its fragments; optionally, the amino acids or polypeptide substrates independently have protecting groups at the side chain, amino terminus, and / or carboxyl terminus;

[0009] The cyclic peptides described in this article can be synthesized using a strategy of first synthesizing a linear precursor peptide and then cyclizing it end-to-end. Based on this, the purpose of peptide bond condensation reactions is to generate and elongate peptides to obtain linear precursor peptides, or to cyclize the C-terminus (carboxyl terminus) and N-terminus (amino terminus) of a linear precursor peptide end-to-end to obtain a cyclic peptide.

[0010] To obtain a linear precursor peptide, an amino acid substrate and an amino acid substrate, an amino acid substrate and a polypeptide substrate, or a polypeptide substrate and a polypeptide substrate can be coupled via peptide bond condensation reactions. In some embodiments, the peptide bond condensation reaction can be performed 1, 2, 3, 4, or 5 times, with each peptide bond condensation reaction yielding the cyclic peptide fragment described herein. In some embodiments, the linear precursor peptide is obtained by sequentially coupling an amino acid substrate via peptide bond condensation reactions. In other embodiments, the linear precursor peptide is obtained by peptide bond condensation reactions of polypeptide substrates. In other embodiments, polypeptide substrates are subjected to peptide bond condensation reactions, followed by further coupling of an amino acid substrate via peptide bond condensation reactions to obtain the linear precursor peptide. In still other embodiments, an amino acid substrate and a polypeptide substrate are first subjected to peptide bond condensation reactions, followed by further coupling of an amino acid substrate via peptide bond condensation reactions to obtain the linear precursor peptide. In some embodiments, the amino acid substrate is commercially available or pretreated. In some embodiments, the pretreatment involves immobilizing the amino acid substrate on a support. In some embodiments, the polypeptide substrate is commercially available or pre-synthesized, or is the product of a preceding one or more peptide bond condensation reactions. In some embodiments, the amino acid substrate independently has protecting groups on its side chains and / or amino terminals. In some embodiments, the polypeptide substrate independently has protecting groups on its side chains and / or amino terminals. In some embodiments, the polypeptide substrate independently has protecting groups on its side chains.

[0011] The linear precursor peptide can be cyclized by linking its C-terminus and N-terminus through a peptide bond condensation reaction to obtain the cyclic peptide. Generally, to reduce the influence of side-chain functional groups on the reaction, the linear precursor peptide has protecting groups on its side chains. In this case, the cyclization yields an intermediate of the cyclic peptide. The cyclic peptide or its salt can be obtained through one or more steps of deprotection reactions.

[0012] In some implementations, the method includes the following steps:

[0013] (1) Using any amino acid residue in the cyclic peptide as the first amino acid in the synthesis, the amino acid substrate corresponding to the first amino acid is coupled to the carrier, and the remaining amino acid substrates are coupled sequentially according to the structure of the cyclic peptide to obtain a linear peptide-carrier.

[0014] (2) The linear peptide-carrier is cleaved to obtain the linear peptide;

[0015] (3) The linear peptide is subjected to a head-to-tail cyclization reaction to obtain a cyclic peptide intermediate;

[0016] (4) Remove the side chain protecting group of the cyclic peptide intermediate to obtain the cyclic peptide or its salt.

[0017] In some embodiments, the salt of the cyclic peptide is the trifluoroacetate salt of the cyclic peptide.

[0018] In some implementations, the method further includes the following steps:

[0019] (5) Purify the cyclic peptide or its salt obtained in step (4), preferably, obtain the trifluoroacetate salt of the cyclic peptide;

[0020] (6) Salt conversion yields salt conversion products;

[0021] (7) Dry the salt-transfer product.

[0022] In some embodiments, the salt transfer product is not a trifluoroacetate.

[0023] Synthesis of linear peptide-carrier

[0024] The present invention uses a solid-phase synthesis method to synthesize the cyclic peptide, coupled the amino acid substrate corresponding to the first amino acid to the carrier, and then coupled the remaining amino acid substrates sequentially according to the structure of the cyclic peptide to obtain a linear peptide-carrier.

[0025] In some embodiments, the amino acid substrate is a protected form of the amino acid, for example, having a protecting group on the side chain and / or amino terminus of the amino acid.

[0026] In some embodiments, the carrier is a resin, such as Wang-Resin or CTC-Resin.

[0027] In some embodiments, the carrier is CTC-Resin, such as 2-CTC. In some embodiments, prior to coupling, a swelling treatment of the CTC-Resin is included, for example, in DCM, DMF, NMP, or THF. In a particular embodiment, the swelling treatment is performed in DCM. The degree of substitution of the resin refers to the amount of active reactive sites per gram of resin, expressed in mmol / g. In some embodiments, the degree of substitution of CTC-Resin used herein is 0.5-1.0 mmol / g, for example, 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g, 0.9 mmol / g, or 1.0 mmol / g. After coupling with the first amino acid, the degree of substitution of CTC-Resin used herein is preferably 0.7 mmol / g.

[0028] Solid-phase synthesis methods are generally divided into Boc synthesis and Fmoc synthesis. Both methods use N-terminal (amino-terminal) Boc or Fmoc-protected amino acids as starting materials. First, the C-terminus (carboxyl-terminus) of the first amino acid substrate is immobilized on a support. Then, the amino protecting group of this amino acid is removed. Next, the deprotected amino group of this amino acid is linked to the carboxyl group of the second amino acid through a condensation reaction. This process of deprotection, washing, deprotection, washing, and condensation of the next amino acid is repeated until all amino acids have been condensed. In a specific embodiment, the Fmoc synthesis method is used for the synthesis.

[0029] To avoid side reactions and improve peptide synthesis efficiency and product purity, it is necessary to protect the side chain functional groups of certain amino acids. Based on the amino acid residue structure constituting the target cyclic peptide of this invention, it is essential to protect the side chain functional groups of the histidine and lysine starting materials during synthesis. In some embodiments, the amino acid substrate is an Fmoc-amino acid optionally containing a side chain protecting group. In some embodiments, both the histidine and lysine substrates contain side chain protecting groups. In some embodiments, the side chain protecting group of the histidine substrate is selected from Boc (tert-butoxycarbonyl) and Trt (triphenylmethyl). In some embodiments, the side chain protecting group of the lysine substrate is selected from Boc and Alloc (allyloxycarbonyl). In some embodiments, the side chain protecting group of the histidine substrate is Trt. In some embodiments, the side chain protecting group of the lysine substrate is Boc.

[0030] This invention has found that the choice of the first amino acid has a significant impact on the yield and subsequent cyclization of the linear peptide. In some embodiments, the first amino acid is leucine (Leu), alanine (Ala), proline (Pro), glycine (Gly), histidine (His), or lysine (Lys). In some embodiments, the first amino acid is selected from one of leucine (Leu), alanine (Ala), proline (Pro), glycine (Gly), or lysine (Lys). In some embodiments, the first amino acid is selected from one of leucine (Leu), alanine (Ala), glycine (Gly), or lysine (Lys). In some embodiments, the first amino acid is selected from one of alanine (Ala), glycine (Gly), or lysine (Lys). In some embodiments, the first amino acid is selected from both alanine (Ala) and lysine (Lys). In some embodiments, the first amino acid is alanine (Ala).

[0031] In some implementations, in step (1), the Ala substrate, Leu substrate, Lys substrate, His substrate, Gly substrate and Pro substrate are coupled sequentially from C end to N end.

[0032] In some implementations, in step (1), Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH and Fmoc-Pro-OH are coupled sequentially from C-terminus to N-terminus.

[0033] In some embodiments, the first amino acid substrate is coupled to the support via a substitution reaction. In some embodiments, the substitution reaction is carried out under the following conditions:

[0034] (1-i) The substitution reaction is carried out in the presence of a base; and / or

[0035] (1-ii) The molar ratio of the carrier (in terms of degree of substitution), the first amino acid substrate and the base is 1:(1-5):(1-10), for example 1:(1.5-3.5):(3-7), or even 1:3:6.

[0036] In some embodiments, the base is selected from organic bases. In some embodiments, the base is selected from N-methylmorpholine, DIEA, and triethylamine. In some embodiments, the base is DIEA.

[0037] In some embodiments, the substitution reaction is carried out at 15-30°C, for example, 15°C, 20°C, 25°C, or 30°C.

[0038] In some embodiments, the substitution reaction is carried out for 1-12 hours, for example 2-10 hours, or even 4-6 hours.

[0039] In some embodiments, the substitution reaction is carried out in an organic solvent. In some embodiments, the organic solvent is selected from DCM, DCE (1,2-dichloroethane), THF, DMF, and DMSO. In some embodiments, the organic solvent is DCM.

[0040] In some embodiments, the remaining amino acid substrate is coupled via a condensation reaction. In some embodiments, the condensation reaction is carried out under the following conditions:

[0041] (2-i) The condensation reaction is carried out in the presence of a condensing agent, preferably selected from HATU, PyBop, HBTU, TBTU, HOBT, DIC, and any combination thereof; preferably selected from HATU, PyBop, HBTU, TBTU, HOBT, and combinations of HOBT and DIC; preferably, the condensing agent is a combination of HOBT and DIC; and / or

[0042] (2-ii) The molar ratio of the amino acid substrate and each condensing agent is 1:(1-5), for example 1:(1-3), or even 1:1.

[0043] In some embodiments, the condensing agent is a combination of HOBT and DIC, and the molar ratio of the amino acid substrate to HOBT and DIC is 1:(1-5):(1-5), for example 1:(1-3):(1-3), or even 1:1:1.

[0044] In some embodiments, the molar ratio of the support (in terms of degree of substitution) to the remaining amino acid substrates (excluding the first amino acid substrate) is 1:(1-5), for example 1:(1.5-3.5), or even 1:(2-3). In some embodiments, DIC is added to a mixed solution of the amino acid substrate and HOBT at a low temperature (e.g., -5 to 5°C), reacted for 5-10 min, and then the resulting mixed solution is added to the support system coupled with the amino acid substrate for a condensation reaction.

[0045] In some embodiments, the condensation reaction is carried out at 15-30°C, for example, 15°C, 20°C, 25°C or 30°C.

[0046] In some embodiments, the condensation reaction is carried out for 1-6 hours, for example 1-4 hours, or even 2-3 hours.

[0047] In some embodiments, the condensation reaction is carried out in an organic solvent or a mixture of solvents with water. In some embodiments, the condensation reaction is carried out in an organic solvent. In some embodiments, the organic solvent is selected from DMF, DMSO, NMP (N-methylpyrrolidone), THF, acetonitrile, toluene, and any combination thereof. In some embodiments, the organic solvent is DMF.

[0048] In some embodiments, after coupling each amino acid substrate and before coupling the next amino acid substrate or before performing the cleavage described in step (2), the step of removing the N-terminal or C-terminal protecting group is further included.

[0049] In some embodiments, after coupling each amino acid substrate and before coupling the next amino acid substrate or before performing the cleavage described in step (2), the step of removing the N-terminal Fmoc protecting group is further included.

[0050] In some embodiments, the N-terminal Fmoc protecting group is removed under alkaline conditions. In some embodiments, the base is selected from piperidine and DBU. In some embodiments, the base is selected from piperidine.

[0051] In some embodiments, the N-terminal Fmoc protecting group is removed under the following conditions: a 20% (v / v) DMF solution of piperidine or a 5% (v / v) DBU DMF solution, preferably a 20% (v / v) DMF solution of piperidine.

[0052] Cleavage of linear peptide-carrier

[0053] After obtaining the linear peptide-resin, the linear peptide needs to be cleaved off the resin for subsequent synthesis of cyclic peptides.

[0054] In some embodiments, in step (2), the lysis is performed in the presence of a cleavage agent.

[0055] In some embodiments, the cleavage agent is selected from acids and alcohols, and combinations thereof. In some embodiments, the cleavage agent is selected from acids and trifluoroethanol, and combinations thereof.

[0056] In some embodiments, the cleavage agent is an acid. In some embodiments, the acid is selected from TFA and AcOH. In some embodiments, the acid is TFA. In some embodiments, the cleavage is carried out in a DCM solution of 1% (v / v) TFA.

[0057] In some embodiments, the cleavage agent is an alcohol. In some embodiments, the alcohol is selected from hexafluoroisopropanol and trifluoroethanol. In some embodiments, the alcohol is hexafluoroisopropanol. In some embodiments, the cleavage is carried out in a 30% (v / v) hexafluoroisopropanol DCM solution. Using 30% hexafluoroisopropanol / DCM as the cleavage agent, the target product can be obtained simply by filtration after cleavage and vacuum distillation, which is simple and convenient and conducive to industrial production.

[0058] In some embodiments, the pyrolysis is carried out at 15-30°C, for example, 15°C, 20°C, 25°C, or 30°C.

[0059] In some embodiments, the pyrolysis is carried out for 0.5-6 hours, for example 0.5-3 hours, or even 0.5-1.5 hours. In some embodiments, the pyrolysis is carried out for 1-6 hours, for example 1-4 hours, or even 2-3 hours.

[0060] Cyclization reaction

[0061] The linear peptide obtained by cleavage undergoes head-to-tail cyclization via a peptide bond formed by the N-terminal amino group and the C-terminal carboxyl group to obtain a cyclic peptide intermediate.

[0062] In some embodiments, the cyclization reaction in step (3) is characterized by one or more of the following:

[0063] (3-i) The concentration of the linear peptide is 1 mg / ml to 30 mg / ml, for example, 1 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml or 30 mg / ml; preferably, the concentration of the linear peptide is 10 mg / ml to 20 mg / ml; more preferably, the concentration of the linear peptide is 10 mg / ml;

[0064] (3-ii) The solvent for the cyclization reaction is selected from DMF and Dioxane; preferably, the solvent for the cyclization reaction is DMF;

[0065] (3-iii) The cyclization reaction is carried out in the presence of a condensing agent and a base; preferably, the condensing agent is selected from HOBT, HBTU, TBTU, HATU, DCC, DIC, PyBOP and any combination thereof; preferably, the condensing agent is PyBOP; preferably, the base is selected from triethylamine, pyridine, 2,4,6-trimethylpyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, morpholine and N-methylmorpholine; preferably, the base is N,N-diisopropylethylamine.

[0066] In some embodiments, the linear peptide is cyclized in DMF under the action of a condensing agent (e.g., PyBOP) and a base (e.g., DIEA) to obtain the cyclic peptide intermediate. In some embodiments, the molar ratio of the linear peptide to the condensing agent and base is 1:(1-5):(1-5), for example 1:(1-3):(1-3), or even 1:(1-2):(1-2). In some embodiments, the concentration of the linear peptide is 10 mg / ml. In some embodiments, the cyclization temperature is 15-30°C, for example 20-30°C. In some embodiments, the cyclization is carried out for 1-6 hours, for example 2-3 hours.

[0067] Synthesis of cyclic peptides

[0068] After obtaining the cyclic peptide intermediate, the protecting groups of the amino acid (e.g., His, Lys) side chain functional groups are removed to obtain the cyclic peptide described in this invention.

[0069] In some embodiments, in step (4), the side chain protecting group is removed under the following conditions:

[0070] (4-i) Remove the side chain protecting group under acidic conditions;

[0071] (4-ii) The removal reaction is carried out for 1-6 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours; preferably, the removal reaction is carried out for 2-4 hours; more preferably, the removal reaction is carried out for 3 hours.

[0072] In some embodiments, the acid described in (4-i) is selected from HCl, TFA, and AcOH. In some embodiments, the side-chain protecting group is removed in a mixed solution of the following: TFA / H2O / EDT (1,2-ethylenedithiol) / TA (aniline sulfide) / TIPS (triisopropylsilane), TFA / phenol / H2O / TIPS, TFA / phenol / H2O / TA / EDT, TFA / phenol / H2O / TA / dodecyl mercaptan, or TFA / DTT (dithiothreitol) / H2O / TIPS.

[0073] In some embodiments, the side-chain protecting groups are removed in a mixed solution of TFA / phenol / H2O / TIPS. In some embodiments, the volume ratio of TFA:phenol:H2O:TIPS is (50-150):5:5:2, for example (70-100):5:5:2, or even more specifically 88:5:5:2.

[0074] In some embodiments, the side-chain protecting groups are removed in a mixed solution of TFA / phenol / H2O / TA / EDT. In some embodiments, the volume ratio of TFA:phenol:H2O:TA:EDT is (50-150):5:5:5:2.5, for example (70-100):5:5:5:2.5, and even more specifically 82.5:5:5:5:2.5.

[0075] In some embodiments, the side-chain protecting groups are removed in a mixed solution of TFA / phenol / H2O / TA / dodecyl mercaptan. In some embodiments, the volume ratio of TFA:phenol:H2O:TA:dodecyl mercaptan is (50-150):5:5:5:2.5, for example (70-100):5:5:5:2.5, and even more specifically 82.5:5:5:5:2.5.

[0076] In some embodiments, the side-chain protecting groups are removed in a mixed solution of TFA / DTT / H2O / TIPS. In some embodiments, the volume ratio of TFA:DTT:H2O:TIPS is (50-150):5:5:2, for example (70-100):5:5:2, or even more specifically 88:5:5:2.

[0077] In some embodiments, the side-chain protecting groups are removed in a mixed solution of TFA / H2O / EDT / TA / TIPS. In some embodiments, the volume ratio of TFA:H2O:EDT:TA:TIPS is (50-150):2:2:1:1. In some embodiments, the volume ratio of TFA:H2O:EDT:TA:TIPS is (90-100):2:2:1:1.

[0078] In some embodiments, step (4) yields a salt of the cyclic peptide. In some embodiments, step (4) yields a trifluoroacetic acid salt of the cyclic peptide.

[0079] In some embodiments, the trifluoroacetate of the cyclic peptide is represented as:

[0080] mCF3COOH, where m represents the amount of trifluoroacetic acid bound to each molecule of the cyclic peptide. When discussing the amount of trifluoroacetic acid bound to a single cyclic peptide molecule, m is an integer, which can be 1, 2, or 3, for example, 1; when discussing the amount of trifluoroacetic acid bound to a group of two or more cyclic peptides, m is an average, which can be an integer or decimal selected from 0.5 to 3.5, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5.

[0081] Salts of cyclic peptides

[0082] After obtaining the trifluoroacetate of the cyclic peptide described in step (4), the method of the present invention further includes steps (5)-(7):

[0083] (5) Purify the trifluoroacetate of the cyclic peptide obtained in step (4) to obtain the purified trifluoroacetate of the cyclic peptide;

[0084] (6) Salt conversion yields salt conversion products;

[0085] (7) Dry the salt-transfer product.

[0086] In some embodiments, the trifluoroacetate of the cyclic peptide is converted to the acetate, hydrochloride, or citrate of the cyclic peptide.

[0087] In some embodiments, the product obtained in step (7) is not the trifluoroacetate of the cyclic peptide.

[0088] In some embodiments, column chromatography is used for the purification described in step (5) and / or the salt conversion described in step (6). In some embodiments, the column chromatography is reversed-phase high-performance liquid chromatography (RP-HPLC). In some embodiments, the stationary phase of the R-HPLC is C18.

[0089] In some embodiments, in step (5), the amount of the cyclic peptide loaded is 0.1%-1% (wt.) of the stationary phase, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1.0%. In some embodiments, the amount of the cyclic peptide loaded is 0.25%-0.75% (wt.) of the stationary phase. In some embodiments, the amount of the cyclic peptide loaded is 0.75% (wt.) of the stationary phase.

[0090] In some embodiments, in step (5), a trifluoroacetic acid / water-acetonitrile system is used as the mobile phase, for example, 0.1% trifluoroacetic acid / water as mobile phase A; and acetonitrile as mobile phase B.

[0091] In some embodiments, in step (6), an acid / water-acetonitrile system and / or an ammonium acetate / water-acetonitrile system are used as the mobile phase. The choice of acid will be determined by the target salting product, and it will be the same acid used to form the target salting product. For example, when the salting product is an acetate, the acid in the mobile phase is acetic acid; when the salting product is a hydrochloride, the acid in the mobile phase is hydrochloric acid; and when the salting product is a citrate, the acid in the mobile phase is citric acid. In some embodiments, in step (6), 0.3% acetic acid / water or 100 mmol / L ammonium acetate aqueous solution is used as mobile phase A; and acetonitrile is used as mobile phase B.

[0092] In some implementations, the salt conversion includes the following three stages:

[0093] In the first stage, 100 mmol / L ammonium acetate aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B for isocratic elution.

[0094] In the second stage, isocratic elution was performed using 0.3% acetic acid / water as mobile phase A and acetonitrile as mobile phase B.

[0095] In the third stage, gradient elution was performed using 0.3% acetic acid / water as mobile phase A and acetonitrile as mobile phase B.

[0096] In some embodiments, the drying in step (7) is freeze drying.

[0097] In some embodiments, the acetate of the cyclic peptide is represented as:

[0098] nCH3COOH, where n represents the number of acetic acid molecules bound to each cyclic peptide. When discussing the number of acetic acid molecules bound to a single cyclic peptide molecule, n is an integer, which can be 1, 2, or 3, for example, 1; when discussing the number of acetic acid molecules bound to a group of two or more cyclic peptides, n is an average, which can be an integer or decimal selected from 0.5 to 3.5, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5.

[0099] The total yield of the method described in this invention, from coupling the second amino acid substrate to drying in step (7), is 40% or more, preferably 50% or more, and more preferably 60% or more. The purity of the resulting SEQ1 acetate final product is 98% or more, preferably 98.5% or more, more preferably 99% or more, and more preferably 99.5% or more, for example 99.6%, 99.7%, 99.8%, or 99.9%; the maximum single impurity content is less than 0.2%, for example less than 0.15%; and the number of impurities is less than 10, for example less than 8, less than 6, and less than 5.

[0100] Terminology Definition

[0101] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art, and laboratory procedures refer to terms and routine procedures widely used in the relevant fields. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0102] The 20 common amino acids and their abbreviations used in this article follow their usual usage. See Immunology-A Synthesis (2nd edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this article, unless otherwise specified, all amino acids are L-forms.

[0103] In this article, when amino acids are mentioned, their meanings should be appropriately adjusted according to the specific context in which they appear; this is a consensus among those skilled in the art. Specifically,

[0104] Ala (Alanine, A), when it appears in the sequence, represents an alanine residue, while when it is a starter / substrate or reaction product, it represents alanine or its protected form, such as N-terminal protected alanine (e.g., Fmoc-alanine).

[0105] Leu (Leucine, L) represents a leucine residue when it appears in the sequence, and when it is a starter / substrate or reaction product, it represents leucine or its protected form, such as N-terminal protected leucine (e.g., Fmoc-leucine).

[0106] Lys(Lysine, K), when it appears in the sequence, represents a lysine residue, while when it is a starter / substrate or reaction product, it represents lysine or its protected form, such as N-terminal and / or side-chain protected lysine (e.g., Fmoc-lysine, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH).

[0107] His(Histidine, H), when it appears in the sequence, represents a histidine residue, while when it is a starting material / substrate or reaction product, it represents histidine or its protected form, such as histidine protected at the N-terminus and / or on the side chain (e.g., Fmoc-histidine, Fmoc-His(Trt)-OH, Fmoc-His(Boc)-OH).

[0108] Gly (Glycine, G), when it appears in the sequence, represents a glycine residue, while when it is a starter / substrate or reaction product, it represents glycine or its protected form, such as N-terminal protected glycine (e.g., Fmoc-glycine).

[0109] Pro (Proline, P), when it appears in the sequence, represents a proline residue, while when it is a starter / substrate or reaction product, it represents proline or its protected form, such as N-terminal protected proline (e.g., Fmoc-proline).

[0110] As is known to those skilled in the art, the term "amino acid residue" refers to the amino acid unit in a protein or polypeptide. An amino acid residue is the structure formed when a hydrogen atom is removed from the amino group and a hydroxyl group is removed from the carboxyl group during the formation of a protein or polypeptide. Attached Figure Description

[0111] Figure 1 shows the results of the synthetic route screening experiment for the cyclic peptide SEQ1 in Example 1.

[0112] Figure 2 shows an exemplary synthetic route for the cyclic peptide of the present invention. Detailed Implementation

[0113] The present invention will now be clearly and completely described in conjunction with embodiments. Obviously, the described embodiments are merely illustrative and are not intended to limit the scope of protection of the present invention in any way. Based on the embodiments, all other alternatives obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0114] Table 1 Sequence Information

[0115] Table 2 Abbreviation Notes:

[0116] Example 1. Screening of synthetic routes for cyclic peptide SEQ1

[0117] The synthetic routes of the cyclic peptide SEQ1 were screened by using Gly, Leu, Pro, Ala, Lys, or His as the first amino acid in solid-phase synthesis, respectively, and following the assembly order from C-terminus to N-terminus.

[0118] Table 3 Synthetic route of cyclic peptide SEQ1

[0119] General synthesis operations for routes 1-6:

[0120] 1. Linear peptide intermediate-resin synthesis

[0121] 1.1 Resin pretreatment and first amino acid coupling

[0122] Weigh 2-CTC resin (1 eq, i.e., degree of substitution (0.6 mmol / g) * resin weight (g)) into a solid-phase reaction flask, and add DCM (10 Vol, i.e., 10 mL / g). 2-CTC resin was swollen at room temperature with stirring for 30 min, and the liquid was removed under reduced pressure. DCM (10 vol) was then added, and the resin was washed with stirring for 3 min. The washing of the resin was repeated twice. The first amino acid raw materials (3 eq) of routes 1-6 were weighed into the reaction flask, and DCM (10 vol) and DIPEA (6 eq) were added and stirred until the raw materials were completely dissolved. Then, the mixture was added to the solid-phase reaction flask and stirred at room temperature for 4-6 h. After the reaction was completed, MeOH (1 vol) was added and stirred at room temperature for 0.5-1 h. The reaction liquid was removed under reduced pressure, DMF (10 vol) was added, and the resin was washed with stirring for 3 min. The solution was removed under reduced pressure and repeated twice. A 20% PIPE / DMF (10 vol) solution was added and stirred at room temperature for 30 min to remove the Fmoc protecting group. After the reaction was completed, DMF (10 vol × 6) was added and the resin was washed 6 times. The obtained product was used for the next reaction.

[0123] 1.2 Coupling of other amino acids

[0124] Following the amino acid assembly sequence listed in routes 1 to 6, the remaining amino acids were coupled sequentially from the C-terminus to the N-terminus. The amino acid raw material (3 eq), HOBT (3 eq), and DMF were weighed and added to a reaction flask. After stirring and dissolving, the mixture was cooled to -5 to 5°C, and DIC (3 eq) was added. The reaction was activated for 5 min. After activation, the mixture was added to a reaction flask containing the product obtained in step 1.1 and reacted at room temperature for 2 h. After the reaction was complete, the resin was washed 6 times with DMF (10 Vol × 6). A 20% PIPE / DMF (10 Vol, i.e., 10 mL / g 2-CTC resin) solution was added, and the mixture was stirred at room temperature for 30 min to remove the Fmoc protecting group. After the reaction was complete, the resin was washed 6 times with DMF (10 Vol × 6). This process was repeated until all amino acids were coupled. Finally, the resin was washed 3 times with DCM (10 Vol × 3) and 3 times with MeOH (10 Vol × 3). The resin was then air-dried at room temperature to obtain the linear peptide intermediate resin.

[0125] 2. Synthesis of linear peptide intermediates

[0126] The linear peptide intermediate-resin was added to a reaction flask, followed by 0.5% TFA / DCM solution (10 Vol, i.e., 10 mL / g linear peptide intermediate-resin). The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the resin was removed by filtration. DIEA was added to the filtrate to adjust the pH to 7-8, and the solution was concentrated under reduced pressure at 30°C until no fraction flowed out. DMF (5 Vol) was added to dissolve and concentrate the product. The product was then added dropwise to a mixed solution of n-Hex (35 Vol) and MTBE (15 Vol) (total 50 Vol). The solution was stirred at room temperature for 1-2 hours to crystallize, and then filtered. The filter cake was washed three times with n-Hex and dried at room temperature to obtain the linear peptide intermediate as a solid powder.

[0127] 3. Synthesis of the cyclic peptide intermediate INT2

[0128] The linear peptide intermediate (1 eq) was added to a reaction flask, and DMF (500 Vol, i.e., 500 mL / g linear peptide intermediate) was added and stirred to dissolve it, so that the concentration of the linear peptide intermediate was 2 mg / mL. PyBop (1.0 eq) and DIEA (2 eq) were added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was added dropwise to purified water (5000 Vol), and a white solid precipitated. After stirring and crystallizing for 3 hours, the mixture was filtered. The filter cake was washed three times with purified water and then placed in a forced-air drying oven and dried at 40°C overnight to obtain a solid powder (INT2).

[0129] 4. Synthesis of cyclic peptide SEQ1

[0130] In a reaction flask, prepare a lysis buffer (10 Vol, i.e., 10 mL / g INT2) according to the formula (TFA / EDT / TA / H2O / TIPS = 94 / 2 / 1 / 2 / 1, v / v), i.e., TFA (9.4 Vol), EDT (0.2 Vol), TA (0.1 Vol), H2O (0.2 Vol), TIPS (0.1 Vol). Add INT2 (1 eq) to the lysis buffer and react at room temperature for 3 h. After the reaction is complete, add MTBE (100 Vol, i.e., 100 mL / g INT2) cooled to -5 to 5 °C. A white solid precipitates. Stir and crystallize for 30 min, then transfer to a centrifuge tube and centrifuge (4500 rpm, 3 min). After centrifugation, discard the supernatant. Add MTBE (5 Vol) to the filter cake, shake and wash, centrifuge again, discard the supernatant, and repeat the washing twice. Let the precipitate air dry at room temperature overnight. The crude trifluoroacetate of SEQ1 is obtained as a block or powder solid.

[0131] Experimental results

[0132] The yield and purity of SEQ1 trifluoroacetate obtained from each route were calculated, and the results are shown in Figure 1. The yield calculation starts from the coupling of the first amino acid in the linear peptide intermediate-resin synthesis step to the total yield of crude SEQ1 trifluoroacetate.

[0133] Theoretical weight gain (g) = Resin substitution degree (0.6 mmol / g) × Resin feed amount (g) × [Linear peptide intermediate molecular weight (964.18 g / mol) - 36.5 g / mol] ÷ 1000

[0134] As shown in Figure 1, the purities of the trifluoroacetate of SEQ1 obtained from routes 1, 2, 3, and 6 are 45.19%, 33.96%, 30.32%, and 15.13%, respectively, all of which are relatively low. Among them, the weight gain rate of the peptide resin obtained from route 3 is 68.8%, and the weight gain rate of the peptide resin obtained from route 5 is 45.8%, indicating that using Pro and Lys as the first amino acids will reduce the yield. The purity of the trifluoroacetate of SEQ1 obtained from route 4 is 79.06%, and the weight gain rate of the peptide resin is 106.1%, both of which are good. Therefore, route 4 was selected as the synthetic route for SEQ1, and subsequent process optimization was carried out.

[0135] Example 2. Process Optimization

[0136] 1. Investigation of the concentration of linear peptide intermediate INT1 (SEQ ID NO:2) in the cyclization reaction

[0137] Referring to step 3 of Example 1, "Synthesis of cyclic peptide intermediate INT2", the effect of INT1 concentration on the cyclization reaction was investigated.

[0138] Filtering criteria:

[0139] Condition 1: The concentration of INT1 in DMF is 30 mg / ml;

[0140] Condition 2: The concentration of INT1 in DMF is 20 mg / ml;

[0141] Condition 3: The concentration of INT1 in DMF is 10 mg / ml.

[0142] Table 4. Effect of INT1 concentration on cyclization reaction results

[0143] Conclusion: During the experiment, it was found that INT1 was difficult to dissolve under condition 1 (30 mg / ml), while it was soluble under conditions 2 and 3. Comparing the INT2 data obtained under conditions 2 and 3, it can be seen that the purity of the product obtained under condition 3 (91.21%) is better than that of the product obtained under condition 2 (90.75%), and the dimer impurities of the product obtained under condition 3 (1.83%) are significantly less than those of the product obtained under condition 2 (5.11%).

[0144] 2. Investigation of INT2 cleavage time

[0145] Referring to step 4 of Example 1, "Synthesis of Cyclic Peptide SEQ1", the effect of INT2 cleavage time on the yield and purity of SEQ1 trifluoroacetate was investigated.

[0146] Filtering criteria:

[0147] Condition 1: The pyrolysis reaction time is 2 hours;

[0148] Condition 2: The pyrolysis reaction time is 3 hours;

[0149] Condition 3: The pyrolysis reaction time is 4 hours.

[0150] Table 5 Results of INT2 pyrolysis time investigation

[0151] Conclusion: As shown in the table above, there is no significant difference in the purity and impurities of the trifluoroacetate obtained from the pyrolysis reaction at 2h, 3h, or 4h. To ensure process stability, the preferred reaction time is 3h.

[0152] Example 3. Synthesis of SEQ1 acetate

[0153] As shown in Figure 2, SEQ1 trifluoroacetate was synthesized under the optimized conditions of Examples 1 and 2, and then further purified and converted to obtain SEQ1 acetate.

[0154] 1. INT1 synthesis

[0155] 1.1 Resin pretreatment and coupling of Fmoc-Ala-OH

[0156] Table 6 Resin pretreatment and Fmoc-Ala-OH coupling experiments

[0157] 1) Weigh 5.0728 g of 2-CTC resin and add it to a 100 mL solid-phase reaction flask. Add 50 mL of DCM and swell the resin under stirring for 30 min. Then remove the liquid under reduced pressure.

[0158] 2) Add DCM (50 mL), stir and wash the resin for 3 minutes, then remove the liquid under reduced pressure. Repeat the washing process once more.

[0159] 3) Add DCM (50 mL) to another reaction flask and start stirring;

[0160] 4) Add Fmoc-Ala-OH (2.8027 g) and DIPEA (3.2 mL) and stir until the raw materials are completely dissolved;

[0161] 5) Add the solution obtained in 4) to the solid-phase reaction flask, turn on the stirrer, control the temperature at 15-30℃, and stir for 4-6 hours;

[0162] 6) Add MeOH (5 mL) and stir at 15–30 °C for 0.5–1 h;

[0163] 7) Remove the reaction solution under reduced pressure;

[0164] 8) Add DMF (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process twice.

[0165] 9) Add MeOH (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process twice more. After the resin is dried, calculate the degree of substitution based on the resin weight using the following formula:

[0166] Calculation formula:

[0167] The calculated degree of resin substitution was 0.70 mmol / g, and all subsequent feed additions were calculated based on this.

[0168] 1.2 Coupling of Fmoc-Leu-OH

[0169] Table 7 Coupling experiments of Fmoc-Leu-OH

[0170] 1) Add 20% PIPE / DMF (50mL) solution to the reaction flask, turn on the stirrer, control the temperature at 15-30℃ and react for 30min, then remove the reaction solution under reduced pressure.

[0171] 2) Add DMF (50 mL) to the reaction flask, stir and wash the resin for 3 min, then remove the liquid under reduced pressure. Repeat the washing process 5 times.

[0172] 3) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser test reagents A, B, and C to the test tube and heat at 100°C for 5 minutes. Remove the test tube and observe the resin. If it turns blue, proceed to the next step.

[0173] 4) Add DMF (50 mL) to another reaction flask and start stirring;

[0174] 5) Add Fmoc-Leu-OH (3.7147g) and HOBT (1.4259g) and stir until the raw materials are completely dissolved, then cool to -5 to 5℃;

[0175] 6) Add DIC (1.6 mL) and stir for 5–10 minutes;

[0176] 7) Add the solution obtained in 6) to the reaction flask in 2), and under stirring conditions, control the temperature at 15-30℃ and stir for 2-3 hours;

[0177] 8) Remove the reaction solution under reduced pressure;

[0178] 9) Add DMF (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process 5 times.

[0179] 10) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser reagent A, B, and C to the test tube and heat at 100°C for 5 minutes. The resin is colorless and transparent, and the liquid is pale yellow and transparent. Proceed to the next step.

[0180] 1.3 Coupling of Fmoc-Lys(Boc)-OH

[0181] Table 8 Coupling experiments of Fmoc-Lys(Boc)-OH

[0182] 1) Add 20% PIPE / DMF (50mL) solution to the reaction flask, turn on the stirrer, control the temperature at 15-30℃ and react for 30min, then remove the reaction solution under reduced pressure.

[0183] 2) Add DMF (50 mL) to the reaction flask, stir and wash the resin for 3 min, then remove the liquid under reduced pressure. Repeat the washing process 5 times.

[0184] 3) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser test reagents A, B, and C to the test tube and heat at 100°C for 5 minutes. Remove the test tube and observe the resin. If it turns blue, proceed to the next step.

[0185] 4) Add DMF (50 mL) to another reaction flask and start stirring;

[0186] 5) Add Fmoc-Lys(Boc)-OH (4.9264g) and HOBT (1.4728g) and stir until the raw materials are completely dissolved, then cool to -5 to 5℃;

[0187] 6) Add DIC (1.6 mL) and stir for 5–10 minutes;

[0188] 7) Add the solution obtained in 6) to the reaction flask in 2), and under stirring conditions, control the temperature at 15-30℃ and stir for 2-3 hours;

[0189] 8) Remove the reaction solution under reduced pressure;

[0190] 9) Add DMF (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process 5 times.

[0191] 10) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser reagent A, B, and C to the test tube and heat at 100°C for 5 minutes. The resin is colorless and transparent, and the liquid is pale yellow and transparent. Proceed to the next step.

[0192] 1.4 Coupling of Fmoc-His(Trt)-OH

[0193] Table 9 Coupling of Fmoc-His(Trt)-OH

[0194] 1) Add 20% PIPE / DMF (50mL) solution to the reaction flask, turn on the stirrer, control the temperature at 15-30℃ and react for 30min, then remove the reaction solution under reduced pressure.

[0195] 2) Add DMF (50 mL) to the reaction flask, stir and wash the resin for 3 min, then remove the liquid under reduced pressure. Repeat the washing process 5 times.

[0196] 3) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser test reagents A, B, and C to the test tube and heat at 100°C for 5 minutes. Remove the test tube and observe the resin. If it turns blue, proceed to the next step.

[0197] 4) Add DMF (50 mL) to another reaction flask and start stirring;

[0198] 5) Add Fmoc-His(Trt)-OH (6.5137g) and HOBT (1.4206g) and stir until the raw materials are completely dissolved, then cool to -5 to 5℃;

[0199] 6) Add DIC (1.6 mL) and stir for 5–10 minutes;

[0200] 7) Add the solution obtained in 6) to the reaction flask in 2), and under stirring conditions, control the temperature at 15-30℃ and stir for 2-3 hours;

[0201] 8) Remove the reaction solution under reduced pressure;

[0202] 9) Add DMF (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process 5 times.

[0203] 10) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser reagent A, B, and C to the test tube and heat at 100°C for 5 minutes. The resin is colorless and transparent, and the liquid is pale yellow and transparent. Proceed to the next step.

[0204] Coupling of 1,5Fmoc-Gly-OH

[0205] Table 10 Coupling of Fmoc-Gly-OH

[0206] 1) Add 20% PIPE / DMF (50mL) solution to the reaction flask, turn on the stirrer, control the temperature at 15-30℃ and react for 30min, then remove the reaction solution under reduced pressure.

[0207] 2) Add DMF (50 mL) to the reaction flask, stir and wash the resin for 3 min, then remove the liquid under reduced pressure. Repeat the washing process 5 times.

[0208] 3) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser test reagents A, B, and C to the test tube and heat at 100°C for 5 minutes. Remove the test tube and observe the resin. If it turns blue, proceed to the next step.

[0209] 4) Add DMF (50 mL) to another reaction flask and start stirring;

[0210] 5) Add Fmoc-Gly-OH (3.1258g) and HOBT (1.4016g) and stir until the raw materials are completely dissolved, then cool to -5 to 5℃;

[0211] 6) Add DIC (1.6 mL) and stir for 5–10 minutes;

[0212] 7) Add the solution obtained in 6) to the reaction flask in 2), and under stirring conditions, control the temperature at 15-30℃ and stir for 2-3 hours;

[0213] 8) Remove the reaction solution under reduced pressure;

[0214] 9) Add DMF (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process 5 times.

[0215] 10) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser reagent A, B, and C to the test tube and heat at 100°C for 5 minutes. The resin is colorless and transparent, and the liquid is pale yellow and transparent. Proceed to the next step.

[0216] Coupling of 1,6Fmoc-Pro-OH

[0217] Table 11 Coupling of Fmoc-Pro-OH

[0218] 1) Add 20% PIPE / DMF (50mL) solution to the reaction flask, turn on the stirrer, control the temperature at 15-30℃ and react for 30min, then remove the reaction solution under reduced pressure.

[0219] 2) Add DMF (50 mL) to the reaction flask, stir and wash the resin for 3 min, then remove the liquid under reduced pressure. Repeat the washing process 5 times.

[0220] 3) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser test reagents A, B, and C to the test tube and heat at 100°C for 5 minutes. Remove the test tube and observe the resin. If it turns blue, proceed to the next step.

[0221] 4) Add DMF (50 mL) to another reaction flask and start stirring;

[0222] 5) Add Fmoc-Pro-OH (3.5490g) and HOBT (1.4116g) and stir until the raw materials are completely dissolved, then cool to -5 to 5℃;

[0223] 6) Add DIC (1.6 mL) and stir for 5–10 minutes;

[0224] 7) Add the solution obtained in 6) to the reaction flask in 2), and under stirring conditions, control the temperature at 15-30℃ and stir for 2-3 hours;

[0225] 8) Remove the reaction solution under reduced pressure;

[0226] 9) Add DMF (50 mL), stir and wash the resin for 3 min, then remove the solution under reduced pressure; repeat the washing process 5 times.

[0227] 10) Dip a glass rod into the reaction flask and place a small amount of resin into a test tube. Add 3 drops each of Kaiser reagent A, B, and C to the test tube and heat at 100°C for 5 minutes. The resin is colorless and transparent, and the liquid is pale yellow and transparent. Proceed to the next step.

[0228] 1.7 INT1 Peptide Resin Post-treatment

[0229] Table 12 Post-treatment of INT1 peptide resin

[0230] 1) Add 20% PIPE / DMF solution (50mL) to the reactor, stir and react at 15-30℃ for 30min, then remove the reaction solution under reduced pressure.

[0231] 2) Add DMF (50 mL) to the reactor, stir and wash the resin for 3 min, remove the liquid under reduced pressure; repeat the washing process twice.

[0232] 3) Dip a glass rod into the reaction vessel and place a small amount of resin into a test tube. Add 3 drops each of Kaiser test reagents A, B, and C to the test tube and heat at 100°C for 5 minutes. Remove the test tube and observe the resin. If it turns blue, proceed to the next step.

[0233] 4) Add MeOH (50 mL) to the reactor, stir and wash the resin for 3 min, remove the liquid under reduced pressure; repeat the washing process 3 times.

[0234] 5) Continue to dry the peptide resin for 1-2 hours.

[0235] 6) Remove the semi-dry peptide resin and air-dry it at room temperature until constant weight (weigh it every 10-20 hours, with the change between two weighings being less than or equal to 1.0%). 8.8450g of INT1 peptide resin was obtained.

[0236] 1.8INT1 peptide resin lysis

[0237] Table 13. Cleavage of INT1 peptide resin

[0238] 1) Add DCM (44 mL) and TFA (0.44 mL) to a 250 mL reaction flask with stirring. After stirring and mixing, add INT1 peptide resin (4.4023 g) and react at 15-30 °C for 3 h.

[0239] 2) After the reaction is complete, filter the resin using a G3 sintered glass funnel. Wash the resin with DCM (44 mL) and combine the filtrates.

[0240] 3) Add DIPEA (0.10 mL) dropwise to adjust the pH of the filtrate to 7-8, and concentrate under reduced pressure at 35-40℃ until no distillate flows out;

[0241] 4) Add DMF (22 mL) to the rotary evaporation flask containing the residue (INT1) and swirl to dissolve;

[0242] 5) Add n-Hex (44 mL) and MTBE (176 mL) to another reaction flask, and stir to mix well;

[0243] 6) Add the solution from 4) dropwise to the reaction flask from 5), a white solid will precipitate, and continue stirring for 1-2 hours;

[0244] 7) Filter the filter cake, rinse it three times with n-Hex (44 mL), and continue to dry it for 2-3 hours. Let it dry at room temperature for 10-20 hours. The material yields 2.0299 g (INT1) of solid powder, with a yield of 118.6% (containing residual solvent and salt or other impurities that cannot be removed by the above post-treatment methods).

[0245] Calculation formula:

[0246] Reaction process monitoring methods

[0247] Mobile phase A: 0.1% trifluoroacetic acid / aqueous solution

[0248] Mobile phase B: 0.1% trifluoroacetic acid / acetonitrile

[0249] Gradient elution, detection wavelength: 214nm.

[0250]

[0251] 2. INT2 synthesis

[0252] Table 14 Experimental conditions for the synthesis of INT2

[0253] 1) Add DMF (100 mL) and INT1 (1.0027 g) to a 250 mL reaction flask and stir to dissolve;

[0254] 2) Add PyBop (0.5402g) and DIEA (0.2723g), and react at 15-30℃ for 2-3 hours;

[0255] 3) Add purified water (1000 mL) to a 3 L reaction flask;

[0256] 4) After the reaction is complete, add the reaction solution obtained in 2) dropwise to the reaction flask in 3), and stir to allow crystals to precipitate for 2-3 hours;

[0257] 5) Filtration: The filter cake was rinsed three times with purified water; the filter cake was dried in a forced-air drying oven at 40°C for 10-30 hours, and 0.8050 g (INT2) of solid powder was collected, with a yield of 81.8%.

[0258] Reaction process monitoring methods

[0259] Mobile phase A: 0.1% trifluoroacetic acid / aqueous solution

[0260] Mobile phase B: 0.1% trifluoroacetic acid / acetonitrile

[0261] Gradient elution, detection wavelength: 214nm.

[0262] 3. Synthesis of SEQ1

[0263] Table 15 Experimental conditions for the synthesis of SEQ1

[0264] 1) Add trifluoroacetic acid (7.52 mL), 1,2-ethylenedithiol (0.16 mL), pure water (0.16 mL), anisole sulfide (0.08 mL) and triisopropylsilane (0.08 mL) to the reaction flask and stir to mix well;

[0265] 2) Add INT2 (0.8002 g), stir and react at 10–30 °C for 2–3 h;

[0266] 3) Add 80 mL of MTBE to a 250 mL reaction flask, stir and cool to -5 to 5 °C for later use;

[0267] 4) After the reaction is complete, add the reaction solution dropwise to the 250mL reaction flask of step 3), and stir at 15-30℃ for 10-30 minutes to allow crystals to precipitate.

[0268] 5) Filtration: The filter cake was washed three times with MTBE (10 mL), dried under vacuum, and air-dried at room temperature for 15–24 h. 0.5740 g of solid powder (trifluoroacetate of SEQ 1) was collected, with a yield of 112.4% (containing residual solvent and salt or other impurities that could not be removed by the above post-treatment methods).

[0269] Reaction process monitoring methods

[0270] Mobile phase A: 0.1% trifluoroacetic acid / aqueous solution

[0271] Mobile phase B: 0.1% trifluoroacetic acid / acetonitrile

[0272] Gradient elution, detection wavelength: 214nm.

[0273] 4. Purification of SEQ 1 trifluoroacetate

[0274] 1) Preparation of mobile phase: 0.1% trifluoroacetic acid / water as mobile phase A; acetonitrile (chromatographic grade) as mobile phase B.

[0275] 2) Preparation of crude trifluoroacetate solution (SEQ1): Dissolve the crude trifluoroacetate (SEQ1) obtained in step 3 in water to prepare a solution with a concentration of approximately 50 mg / mL. Filter the solution through a 0.45 μm microporous membrane (organic system). Seal the filtrate and store it at 2–8 °C for later use.

[0276] 3) Separation and purification conditions: Preparative high-performance liquid chromatography (Agilent 1260, 50 mL / min), column (C18, 9.6 × 250 mm, 5 μm). ); Flow rate 3 mL / min; Detection wavelength λ 214 nm.

[0277] Equilibrate the column for 5 min using phase A / phase B (v / v, 92:8).

[0278] After equilibration, begin injection at 0.25%, 0.50%, or 0.75% of the column packing material (SEQ 1 trifluoroacetate: 25 mg, 50 mg, or 75 mg, respectively). Elute using the gradients listed in the table below.

[0279] The target peak was collected in segments, and after collection, it was sent to HPLC for analysis. The sample was sealed and stored at 2–8°C.

[0280] Based on the HPLC results, samples with a normalized purity of ≥98.0% were combined, pre-frozen with liquid nitrogen, and then freeze-dried in a suspended freeze dryer for 48–72 hours to obtain solid powder.

[0281] Reaction process monitoring methods

[0282] Mobile phase A: 0.1% trifluoroacetic acid / aqueous solution

[0283] Mobile phase B: 0.1% trifluoroacetic acid / acetonitrile

[0284] Gradient elution, detection wavelength: 214nm.

[0285] Table 16 Effect of sample loading amount of SEQ1 trifluoroacetate crude product on purification results

[0286] Conclusion: As shown in the table above, the purity of SEQ1 trifluoroacetate obtained under the three conditions was 99.87%, 99.71%, and 99.51%, respectively, all of which met the standard requirement of ≥98.0% and showed no significant difference. The yield of condition 3 was 67.9%, which was higher than that of condition 1 (60.1%) and condition 2 (62.8%). Moreover, the higher sample loading volume helped to reduce the number of sample loadings, shorten the purification cycle, and reduce production costs.

[0287] Based on the purification experimental conditions described above, the crude trifluoroacetate product SEQ1 obtained earlier was purified by loading 75 mg (0.75% of the column packing material) to finally obtain 0.0509 g of solid powder, with a yield of 67.9%.

[0288] 5. Preparation of SEQ1 acetate (salt conversion)

[0289] 1) Preparation of mobile phase: 0.3% glacial acetic acid / water as mobile phase A1; 100 mmol / L ammonium acetate aqueous solution as mobile phase A2; acetonitrile (chromatographic grade) as mobile phase B.

[0290] 2) Sample dissolution: Dissolve the SEQ1 trifluoroacetate obtained from the previous purification step in water to prepare a solution with a concentration of approximately 50 mg / mL for later use.

[0291] 3) Separation and purification conditions: Preparative high-performance liquid chromatography (Agilent Preparative Liquid Chromatography 1260, 50 mL / min), chromatographic column (C18, 9.6 × 250 mm, 5 μm). ); Flow rate 3 ml / min; Detection wavelength set λ 214 nm.

[0292] Equilibrate the column for 5 min using phase A1 / phase B (v / v, 100:0).

[0293] After equilibration, begin injection. The injection volume per injection should not exceed 0.75% of the column packing mass (i.e., not more than 0.075g, actual injection volume 0.0509g). After injection, perform the following procedures:

[0294] ① Pump A feeds into phase A2, and pump B feeds into phase B, operating at the following gradient.

[0295] ② Pump A enters phase A1, and pump B enters phase B, operating at the following gradient.

[0296] ③ Pump A enters phase A1, and pump B enters phase B, operating at the following gradient.

[0297] Collect all target peaks, and based on the HPLC detection results, combine samples with an area normalized purity greater than or equal to 98.0%, seal them, and store them at 2–8℃.

[0298] Reaction process monitoring methods

[0299] Mobile phase A: 0.1% trifluoroacetic acid / aqueous solution

[0300] Mobile phase B: 0.1% trifluoroacetic acid / acetonitrile

[0301] Gradient elution, detection wavelength: 214nm.

[0302] 6. Drying of SEQ1 acetate

[0303] The salt concentrate was placed in a freeze-drying bottle, pre-frozen with liquid nitrogen, and then freeze-dried on a hanging freeze dryer for 48–72 hours to obtain 0.0421 g of solid powder.

[0304] Combined yield of salt-free freeze-drying: 82.7%

[0305] Total yield: 118.6% × 81.8% × 112.4% × 67.9% × 82.7% = 61.2%

[0306] Yield calculations begin with the total yield from the coupling of the second amino acid in the INT1 peptide resin synthesis step to the final freeze-dried product (the coupling of the first amino acid is used to determine the final degree of substitution, which is used for subsequent feed amount calculations).

[0307] The final product quality was tested and found to be as follows:

[0308] In addition, the trifluoroacetic acid content in the acetate of the parallel batch final product SEQ1 was tested, and it was found that the trifluoroacetic acid content (%) was not detected, and the product quality met the standard requirements.

[0309] Methods for detecting trifluoroacetic acid residues:

[0310] Instruments and reagents:

[0311] Metrohm Compat792 ion chromatograph (Metrosep A Supp 5-250 analytical column).

[0312] Trifluoroacetic acid: Sigama chromatographic grade; Na2CO3: standard, Tianjin Chemical Reagent Research Institute; NaHCO3: standard, Tianjin Guangfu Fine Chemical Research Institute; all solutions were prepared with ultrapure water with a resistivity greater than 18Ω.

[0313] Chromatographic conditions:

[0314] Chromatographic column: Metrosep A SUPP 5-250 anion analysis column.

[0315] Mobile phase: 3.2 mmol / L Na₂CO₃ / 1.0 mmol / L NaHCO₃ 3； Flow rate: 0.7 mL / min; injection volume: 20 μL.

[0316] Weigh a certain amount of sample, dilute it with pure water, make up to volume, filter it with a 0.45um filter head, and enter the chromatograph.

[0317] Example 4: Bioactivity test of cyclic peptide SEQ1

[0318] 1. Testing Method

[0319] A. Fibroblast toxicity test

[0320] Test materials: Human fibroblasts (Guangdong Boxi Biotechnology Co., Ltd., batch number Fb20081902), DMEM culture medium (Gibco, 23042301, expiry date 230522), PBS (Solepro, P1010), MTT (Sigma, M5655), DMSO (Sigma, D4540-1L), CO2 incubator (Thermo, 150I), laminar flow hood (Sujing Antai, SW-CJ-1F), inverted microscope (Olympus, CKX53);

[0321] Sample to be tested: SEQ1 trifluoroacetate, from PCT / CN2024 / 120959 (which is incorporated herein by reference), purity >95%.

[0322] Test method:

[0323] 1) Cell seeding: After reviving the cells, when the plating rate reaches about 60%, seed the cells into 96-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0324] 2) Experimental Groups: The experiment included a zeroing group, a solvent control group, a positive control group, and a sample group. In the sample group, each sample was configured with 7 concentration gradients, and each concentration gradient had 3 replicate wells.

[0325] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentrations of 0.0781, 0.1563, 0.3125, 0.625, 1.25, 2.5 and 5 μM.

[0326] 4) Drug administration: Drug administration was performed when the cell seeding rate in the 96-well plate reached 50%–60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of the sample was added to each well; for the zeroing group, no cells were seeded, only 200 μL of cell culture medium was added. After drug administration, the 96-well plate was placed in an incubator (37℃, 5% CO2) and incubated for 24 h.

[0327] 5) Detection: After culturing cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 150 μL of DMSO to each well, and read the OD value at 490 nm.

[0328] 6) Calculation of relative cell viability: Calculated according to the following formula

[0329] The test results are shown in Table 17.

[0330] Table 17: Detection results of cyclic peptide SEQ1

[0331] As shown in Table 17, in the fibroblast toxicity test, the cyclic peptide SEQ1 did not show obvious cytotoxicity in the concentration range of 5 μM, and has good prospects for pharmaceutical and cosmetic applications.

[0332] B. Fibroblast Collagen I (Type I Collagen) Enhancement Effect, Fibroblast HA (Hyaluronic Acid) Enhancement Effect, Fibroblast Elastin (ELN) Enhancement Effect

[0333] Test materials: Human fibroblasts (Guangdong Boxi Biotechnology Co., Ltd., batch number Fb20081902), DMEM culture medium (Gibco, 23042301, expiry date 230522), PBS (Solepro, P1010), TGF-β1 (Peprotech, 230504), Collagen I ELISA kit (Cusabio, B21025824), Elastin ELISA kit (Abcam, GR3446070-2), HA ELISA kit (RD, P325066). CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch).

[0334] Test method:

[0335] 1) Inoculation: After cell resuscitation, when the cell plating rate reaches about 60%, inoculate the cells into 6-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0336] 2) Solution preparation: Sample group cyclic peptide SEQ1, Yangshen group PC2 (Biotinoyl-GHK, ) and PC3 All concentrations were 1 μM, and working solutions of the test substances were prepared. The blank control group (BC) was prepared with only cell culture medium.

[0337] 3) Drug administration: When the cell deposition rate in the 6-well plate reaches 40% to 60%, administer the drug, with 2 mL per well. Each group has 3 replicates. Incubate in an incubator (37℃, 5% CO2) for 24 h.

[0338] 4) ELISA detection: Perform ELISA detection according to the ELISA kit instructions. The detection indicators are the enhancement of Collagen I in fibroblasts, the enhancement of HA in fibroblasts, and the enhancement of elastin in fibroblasts.

[0339] The activity test data of cyclic peptide SEQ1 are shown in Tables 18A, 18B, and 18C below.

[0340] Table 18A: Data on the effect of cyclic peptide SEQ1 on enhancing Collagen I in fibroblasts.

[0341] As shown in Table 18A, compared with BC, the content of type I collagen in cyclic peptide SEQ1 was significantly increased, indicating that cyclic peptide SEQ1 can achieve anti-wrinkle effect by increasing the content of collagen I.

[0342] Table 18B: Data on the fibroblast HA-enhancing effect of cyclic peptide SEQ1

[0343] As shown in Table 18B, compared with BC, the hyaluronic acid (HA) content of cyclic peptide SEQ1 is significantly increased, indicating that cyclic peptide SEQ1 can achieve a firming effect by increasing HA content.

[0344] Table 18C: Data on the effect of cyclic peptide SEQ1 on enhancing fibroblast elastin.

[0345] As shown in Table 18C, compared with BC, the elastin content of cyclic peptide SEQ1 was significantly increased, indicating that cyclic peptide SEQ1 can achieve a firming effect by increasing the elastin content.

[0346] In addition, as shown in Tables 18A, 18B, and 18C, compared with BC, the content of type I collagen (Collagen I), hyaluronic acid (HA), and elastin in cyclic peptide SEQ1 is significantly increased, indicating that cyclic peptide SEQ1 can achieve firming and anti-wrinkle effects by increasing the content of collagen I, HA, and elastin, and has excellent pharmaceutical and cosmetic effects.

[0347] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for synthesizing a cyclic peptide of Formula I or a salt thereof, the method comprising coupling an amino acid and / or a polypeptide substrate via 1-6 peptide bond condensation reactions to obtain the cyclic peptide or a fragment thereof; optionally, the amino acid or polypeptide substrate independently has a protecting group at its side chain, amino terminus, and / or carboxyl terminus; 2. The method of claim 1, wherein the method comprises the following steps: (1) Using any amino acid residue in the cyclic peptide as the first amino acid in the synthesis, the amino acid substrate corresponding to the first amino acid is coupled to the carrier, and the remaining amino acid substrates are coupled sequentially according to the structure of the cyclic peptide to obtain a linear peptide-carrier. (2) The linear peptide-carrier is cleaved to obtain the linear peptide; (3) The linear peptide is subjected to a head-to-tail cyclization reaction to obtain a cyclic peptide intermediate; (4) Remove the side chain protecting group of the cyclic peptide intermediate to obtain the cyclic peptide or its salt.

3. The method according to claim 1 or 2, wherein it is a solid-phase synthesis method; Preferably, the carrier is a resin, such as Wang-Resin or CTC-Resin; Preferably, the carrier is CTC-Resin; preferably, before coupling, the method further includes a swelling treatment of CTC-Resin, for example, in DCM, DMF, NMP or THF (preferably DCM); preferably, the degree of substitution of CTC-Resin is 0.5-1.0 mmol / g, for example 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g, 0.9 mmol / g or 1.0 mmol / g.

4. The method of claim 3, wherein the solid-phase synthesis is carried out using the Boc synthesis method or the Fmoc synthesis method; Preferably, the solid-phase synthesis is performed using the Fmoc synthesis method; preferably, the amino acid substrate is an Fmoc-amino acid optionally containing a side-chain protecting group; preferably, the histidine substrate and the lysine substrate contain side-chain protecting groups; preferably, the side-chain protecting group of the histidine substrate is selected from Boc (tert-butoxycarbonyl) and Trt (triphenylmethyl); preferably, the side-chain protecting group of the lysine substrate is selected from Boc and Alloc (allyloxycarbonyl); preferably, the side-chain protecting group of the histidine substrate is Trt; preferably, the side-chain protecting group of the lysine substrate is Boc.

5. The method according to any one of claims 2-4, wherein the first amino acid is leucine (Leu), alanine (Ala), proline (Pro), glycine (Gly), histidine (His), or lysine (Lys); Preferably, the first amino acid is alanine; preferably, in step (1), Ala substrate, Leu substrate, Lys substrate, His substrate, Gly substrate and Pro substrate are coupled sequentially from C-terminus to N-terminus.

6. The method according to any one of claims 2-5, wherein the first amino acid substrate is coupled to the support by a substitution reaction; Preferably, the substitution reaction is carried out under the following conditions: (1-i) The substitution reaction is carried out in the presence of a base, preferably an organic base; preferably, the base is selected from N-methylmorpholine, DIEA, and triethylamine; more preferably, the base is DIEA; and / or (1-ii) The molar ratio of the carrier (in terms of degree of substitution), the first amino acid substrate and the base is 1:(1-5):(1-10), for example 1:(1.5-3.5):(3-7), or even 1:3:

6.

7. The method according to any one of claims 2-6, wherein the remaining amino acid substrate is coupled via a condensation reaction; Preferably, the condensation reaction is carried out under the following conditions: (2-i) The condensation reaction is carried out in the presence of a condensing agent, preferably selected from HATU, PyBop, HBTU, TBTU, HOBT, DIC, and any combination thereof; preferably, the condensing agent is selected from HATU, PyBop, HBTU, TBTU, HOBT, and a combination of HOBT and DIC; preferably, the condensing agent is a combination of HOBT and DIC; and / or (2-ii) The molar ratio of the amino acid substrate and each condensing agent is 1:(1-5), for example 1:(1-3), or even 1:

1.

8. The method according to any one of claims 2-7, after coupling each amino acid substrate and before coupling the next amino acid substrate or before performing the cleavage described in step (2), further includes the step of removing the N-terminal or C-terminal protecting group.

9. The method of claim 8, after coupling each amino acid substrate and before coupling the next amino acid substrate or before performing the cleavage described in step (2), further includes the step of removing the N-terminal Fmoc protecting group; Preferably, the N-terminal Fmoc protecting group is removed in the presence of a base; preferably, the base is selected from piperidine and DBU; preferably, the base is piperidine. Preferably, the N-terminal Fmoc protecting group is removed under the following conditions: a 20% (v / v) DMF solution of piperidine or a 5% (v / v) DBU DMF solution; preferably a 20% (v / v) DMF solution of piperidine.

10. The method according to any one of claims 2-9, wherein in step (2), the lysis is carried out in the presence of a cleavage agent; Preferably, the cleavage reagent is selected from acids and trifluoroethanol and combinations thereof, with acids being preferred; Preferably, the acid is selected from TFA and AcOH; preferably, the acid is TFA. Preferably, the lysis is carried out in a DCM solution of 1% (v / v) TFA.

11. The method according to any one of claims 2-10, wherein the cyclization reaction in step (3) is characterized by one or more of the following: (3-i) The concentration of the linear peptide is 1 mg / ml to 30 mg / ml, for example, 1 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml or 30 mg / ml; preferably, the concentration of the linear peptide is 10 mg / ml to 20 mg / ml; more preferably, the concentration of the linear peptide is 10 mg / ml; (3-ii) The solvent for the cyclization reaction is selected from DMF and Dioxane; preferably, the solvent for the cyclization reaction is DMF; (3-iii) The cyclization reaction is carried out in the presence of a condensing agent and a base; preferably, the condensing agent is selected from HOBT, HBTU, TBTU, HATU, DCC, DIC, PyBOP and any combination thereof; preferably, the condensing agent is PyBOP; preferably, the base is selected from triethylamine, pyridine, 2,4,6-trimethylpyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, morpholine and N-methylmorpholine; preferably, the base is N,N-diisopropylethylamine.

12. The method according to any one of claims 2-11, wherein in step (4), the side chain protecting group is removed under the following conditions: (4-i) Remove the side chain protecting group under acidic conditions; preferably, the acid is selected from HCl, TFA, and AcOH; Preferably, the side-chain protecting group is removed in the following mixed solutions: TFA / H2O / EDT (1,2-ethylenedithiol) / TA (anisole) / TIPS (triisopropylsilane), TFA / phenol / H2O / TIPS, TFA / phenol / H2O / TA / EDT, TFA / phenol / H2O / TA / dodecyl mercaptan, or TFA / DTT (dithiothreitol) / H2O / TIPS; Preferably, the side chain protecting groups are removed in a mixed solution of TFA / H2O / EDT / TA / TIPS; preferably, the volume ratio of TFA:H2O:EDT:TA:TIPS is (50-150):2:2:1:1; preferably, the volume ratio of TFA:H2O:EDT:TA:TIPS is (90-100):2:2:1:1; (4-ii) The removal reaction is carried out for 1-6 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours; preferably, the removal reaction is carried out for 2-4 hours; more preferably, the removal reaction is carried out for 3 hours.

13. The method according to any one of claims 2-12, wherein step (4) yields a salt of the cyclic peptide; preferably, step (4) yields a trifluoroacetate salt of the cyclic peptide; Preferably, the method further includes steps (5)-(7): (5) Purify the trifluoroacetate of the cyclic peptide obtained in step (4) to obtain the purified trifluoroacetate of the cyclic peptide; (6) Salting to obtain salt-transfer product, preferably, the trifluoroacetate of the cyclic peptide is converted into the acetate, hydrochloride or citrate of the cyclic peptide, preferably into the acetate of the cyclic peptide; (7) Dry the salt-transfer product.

14. The method of claim 13, wherein column chromatography is used for the purification described in step (5) and / or the salt conversion described in step (6); Preferably, the column chromatography is reversed-phase high-performance liquid chromatography; Preferably, the stationary phase of the reversed-phase high-performance liquid chromatography is C18.

15. The method of claim 14, wherein in step (5), the amount of the cyclic peptide loaded is 0.1%-1% (wt.) of the stationary phase, for example 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1.0%; preferably, the amount of the cyclic peptide loaded is 0.25%-0.75% (wt.) of the stationary phase; more preferably, the amount of the cyclic peptide loaded is 0.75% (wt.) of the stationary phase; Preferably, in step (5), a trifluoroacetic acid / water-acetonitrile system is used as the mobile phase, for example, 0.1% trifluoroacetic acid / water as mobile phase A; and acetonitrile as mobile phase B.

16. The method of claim 14 or 15, wherein in step (6), an acid / water-acetonitrile system and / or an ammonium acetate / water-acetonitrile system is used as the mobile phase, preferably, the acid is the same as the acid used to form the salt conversion product in step (6), for example, 0.3% acetic acid / water or 100 mmol / L ammonium acetate aqueous solution as mobile phase A; and acetonitrile as mobile phase B; Preferably, the salt conversion includes the following three stages: In the first stage, 100 mmol / L ammonium acetate aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B for isocratic elution. In the second stage, isocratic elution was performed using 0.3% acetic acid / water as mobile phase A and acetonitrile as mobile phase B. In the third stage, gradient elution was performed using 0.3% acetic acid / water as mobile phase A and acetonitrile as mobile phase B.

17. The method according to any one of claims 13-16, wherein in step (7), the drying is freeze drying.

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