The pure gonadotropin-releasing hormone antagonist cetrorelix

Abstract

This invention relates to purified cetrorelix acetate, a gonadotropin-releasing hormone antagonist. One aspect relates to a method for preparing cetrorelix acetate using a solid-phase synthesis method, comprising the following steps: using Fmoc-linker-MBHA-resin resin as a starting material for a peptide initiation reaction, sequentially linking Fmoc-protected amino acids such as Fmoc-D-Ala-OH; before each peptide initiation reaction, sequentially removing fluorene methoxycarbonyl groups using a decapping reagent to obtain a protected decapeptide resin; after removing the fluorene methoxycarbonyl groups using a decapping reagent, performing an acetylation reaction, followed by simultaneous removal of side-chain protecting groups and peptide cleavage to obtain crude cetrorelix acetate; purifying and desalting, and drying the collected eluent to obtain purified cetrorelix acetate. The invention also relates to cetrorelix acetate prepared by this method and its pharmaceutical uses. The method of this invention exhibits excellent technical effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a gonadotropin-releasing hormone (LHRH) antagonist for assisted reproductive technology. Specifically, it relates to a gonadotropin-releasing hormone (GnRH) antagonist for use in patients undergoing controlled ovarian stimulation to prevent premature ovulation, thereby facilitating oocyte retrieval and assisted reproductive technology treatment. The gonadotropin-releasing hormone antagonist is typically cetrorelix or its pharmaceutical salts, such as acetate. This invention particularly relates to pure cetrorelix acetate. Background Technology

[0002] Cetrorex acetate is a synthetic decapeptide with gonadotropin-releasing hormone (GnRH) antagonist activity. Cetrorex acetate is a natural GnRH analog with non-natural D-amino acid substituents at positions 1, 2, 3, 6, and 10. The Chinese chemical name of cetrorex acetate is: N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine)-D-alanyl-L-seryl-L-tyrosyl-N 5 -Carbamoyl-D-guanine-L-leucine-L-arginine-L-proline-D-alanine-amide acetate, a typical English chemical name for cetrorex is: Acetyl-D-3-(2′-naphtyl)-alanine-D-4-chlorophenylalanine-D-3-(3′-pyridyl)alanine-L-serine-L-tyrosin eD-citruline-L-leucine-L-arginine-L-proline-D-alanine-amide. The chemical structural formula, molecular formula, and molecular weight of cetrorex acetate are as follows:

[0003]

[0004] C 70 H 92 ClN 17 O 14 ·xC₂H₄O₂, 1431.06·x₆0.05

[0005] Gonadotropin-releasing hormone (GnRH) binds to receptors on pituitary cell membranes, stimulating the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Cetrorelix acetate competes with endogenous GnRH for receptors on pituitary cells, thereby inhibiting the release of endogenous LH and FSH. The inhibitory effect of cetrorelix acetate occurs immediately after injection and has no stimulatory effect. In women, the effect of cetrorelix acetate is to delay the LH surge, thereby controlling ovulation.

[0006] As a peptide composed of 10 amino acids, cetrorex acetate is usually prepared by chemical synthesis, and many synthetic methods have been documented in the literature. For example, CN101284863B (200810043454.1) discloses a solid-phase synthesis method for cetrorex, which includes the following steps: using Fmoc-Linker-MBHA-Resin as the starting material, amino acids with Fmoc-protecting groups are sequentially linked according to the solid-phase synthesis method to obtain a protected decapeptide resin. During this process, the Fmoc-protecting groups are sequentially removed, and a peptide linking reaction is carried out using HBTU / HOBT or DIC / HOBT as a condensing agent. After obtaining the protected decapeptide resin, an acetylation reaction is performed, followed by simultaneous removal of side-chain protecting groups and peptide cleavage to obtain cetrorex acetate. The cetrorex acetate is then separated and purified by C18 or C8 chromatographic column and freeze-dried to obtain cetrorex acetate or trifluoroacetate.

[0007] CN101863960B (200910134943.2) discloses a method for preparing cetrorex, using Boc amino acids and Wang resin as raw materials. C-terminal amino acids are added sequentially, followed by ammonolysis with 10wt% ammonia water. Then, H2 and Pd are used as deprotecting agents to obtain high-quality cetrorex. The BOC method yields a crude cetrorex yield as high as 60-70%. Using Wang resin as a carrier avoids the use of highly toxic HF acid during final cleavage, and adheres to the principle of minimum protection, allowing for greater flexibility in raw material selection. It also results in fewer synthetic byproducts and easier separation and purification.

[0008] CN104086632A (201410265578.X) discloses a method for preparing cetrorex, the specific steps of which are as follows: A) Using AM resin as the starting resin, amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled according to the cetrorex backbone peptide sequence, wherein the amino acid at position 6 of the peptide sequence is coupled using Fmoc-D-Orn(Dde)-OH; B) After coupling, the Fmoc protecting group is removed, and then the N-terminus is acetylated; C) The D-ornithine side chain Dde protecting group is removed using a mixed solution of hydrazine hydrate and DMF in a volume ratio of 3:97, and then tert-butyl isocyanate is added to modify the D-ornithine side chain in the decapeptide resin to obtain a fully protected cetrorex peptide resin; D) Cetrorex can be obtained after cleavage, purification, desalting, and lyophilization of the peptide resin.

[0009] CN104610433A (201510083267.6) discloses a method for preparing cetrarolix using Fmoc-Linker-amino resin as the starting material. Nine amino acids with Fmoc-protecting groups are sequentially linked from the C-terminus to the N-terminus. After removing the Fmoc protecting groups, Ac-D-2-Nal-OH is added to obtain an N-terminal acetylated peptide resin, i.e., an Ac-fully protected decapeptide resin. The peptide resin is cleaved using a cleavage reagent, and the cleavage solution is precipitated with ice-cold ether to obtain crude cetrarolix peptide. This is then purified by HPLC preparative column chromatography, and freeze-dried to obtain cetrarolix trifluoroacetate or cetrarolix acetate. It is believed that this invention adopts the principle of minimum protection and uses pre-acetylated N-terminal amino acids Ac-D-2-Nal-OH instead of Fmoc-D-2-Nal-OH, eliminating the side reaction of acetic anhydride in the acetylation reagent on the D-Cit side-chain urea group, improving product purity, increasing crude peptide yield, and facilitating large-scale industrial production.

[0010] CN104861042A (201510275629.1) relates to a method for synthesizing cetrorex acetate. Using RinkAmide-AMResin resin as a carrier and low-cost HBTU / DIEA as a condensing agent, a specific microwave synthesis technique was employed, which improved the condensation efficiency, achieving a crude product yield of 92%. The obtained crude cetrorex was purified by reversed-phase high-performance liquid chromatography (rHPLC) and then treated with a strong anion exchange resin to convert to acetate, resulting in a pure cetrorex acetate yield of over 40%, with acetic acid content meeting international standards. This invention is believed to significantly shorten reaction time, improve the final product yield, and possess considerable economic value and broad application prospects.

[0011] The method for preparing cetrorex disclosed in CN107778355B (201610728868.2) uses a novel amino resin as a carrier and a protected D-Orn as a precursor for D-Cit. The side-chain protecting group is then removed, and the cetrorex peptide resin is reacted with tert-butyl isocyanate to generate D-Cit(tBu). The cetrorex peptide resin is acidified using a unique trifluoroacetic acid solution containing hydrogen bromide, which maximizes the removal of the side-chain protecting group tBu from the generated D-Cit(tBu). The resulting cetrorex has high purity and overall yield, avoiding the generation of the toxic impurity [D-Cit(Ac)]-cetrorex. The entire method is simple to operate, operates under mild conditions, and produces a high-quality product.

[0012] CN108264540A (201611265510.7) discloses a method for preparing cetrarolix, which includes the following steps: (1) using resin as a carrier, esterifying the C-terminal amino acid of cetrarolix to prepare an amino acid-resin complex; (2) using the Fmoc strategy solid-phase peptide synthesis method to prepare a protected cetrarolix-resin complex; (3) removing the side chain protecting group with TFA to prepare a deprotected cetrarolix-resin complex; (4) obtaining crude cetrarolix by ammonolysis; (5) purifying the crude product by high performance liquid chromatography to obtain a refined cetrarolix. It is believed that this invention is suitable for the simple and efficient preparation of cetrarolix, solving the problems of numerous side reactions, low yield, and high cost associated with existing methods.

[0013] CN110903352A (201911376082.9) discloses a method for preparing cetrorex, which involves sequentially linking fragments 1-9 of the cetrorex sequence from the C-terminus to the N-terminus using a solid-phase synthesis method under the action of a coupling reagent. After fully protecting and cleaving fragments 1-9, fragment condensation is performed in the liquid phase with DAla-NH2.HCl to obtain fully protected cetrorex. The cetrorex is then cleaved with a lysis buffer, and the cleavage buffer is precipitated with ice-cold diethyl ether to obtain crude cetrorex peptide. Finally, the cetrorex product is purified by reversed-phase high-performance liquid chromatography. This invention is believed to have the advantages of simple method, suitability for industrial production, and high production efficiency.

[0014] CN112159461A (202011049746.3) discloses a method for preparing cetrorex, using Knorr-2-Cl-Resin resin as the starting material. Following a solid-phase synthesis method, ten different amino acid resins are sequentially linked to obtain a protected decapeptide resin. In the synthesis, fluorene methoxycarbonyl groups are sequentially removed, followed by a condensation reaction using a solution of benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate and N,N-diisopropylethylamine in N,N-dimethylformamide as a condensing agent to obtain the protected decapeptide resin. After the condensation reaction, an acetylation reaction is performed to remove the side-chain protecting groups. After cleavage, crude cetrorex is obtained, which is then purified to obtain the final product. It is believed that this invention uses Knorr-2-Cl-Resin resin as the starting material and PyBoP / DIPEA as the condensing agent, making peptide sequence synthesis easier and resulting in a crude cetrorex peptide with high purity.

[0015] CN114920804A(202210672194.4) relates to a cetrilac synthesis process that can be directly used for pilot-scale amplification, comprising: a) obtaining a nonapeptide resin with free terminal amino groups by sequential coupling on a resin support via solid-phase synthesis; b) adding dissolved Ac-D-2-Nal-OAt / OSu / OBt to the nonapeptide resin obtained in step a), adding a catalyst dropwise, and coupling to obtain a fully protected decapeptide resin; c) obtaining crude cetrilac by pyrolysis, precipitation, washing and drying.

[0016] The inventors have discovered that, when preparing cetrilac using existing methods, unavoidable impurities [D-Arg] are routinely detected. 8 D-Arg-Citrolax has been found to be introduced not only from the raw materials used, but also possibly from the process.

[0017] Therefore, those skilled in the art still anticipate new methods for preparing cetrirexate acetate, and still expect such methods to achieve one or more technical effects, such as enabling the removal of specific miscellaneous substances, such as [D-Arg], from the product. 8 The amount of strychnine should be kept as low as possible. Summary of the Invention

[0018] The purpose of this invention is to provide a novel method for preparing cetrilac acetate, with the aim of achieving one or more technical advantages, such as reducing the presence of specific impurities in the product, such as [D-Arg]. 8 The amount of cetrorex is controlled to be as low as possible. It has been unexpectedly discovered that the cetrorex acetate prepared by the method of this invention exhibits one or more of the excellent technical effects described in this invention, upon which this invention is based.

[0019] Therefore, the first aspect of the present invention provides a method for preparing cetrorex acetate using a solid-phase synthesis method, wherein amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled according to the cetrorex backbone peptide sequence, comprising the following steps:

[0020] (i) Using Fmoc-linker-MBHA-resin resin as the starting material, a mixture of HBTU / HOBT / DIEA was used as a condensing agent to carry out peptide linking reactions, sequentially linking Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Cit-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-3(3-Pyridyl)-D-Ala-OH, Fmoc-4-Chloro-D-Phe-OH, and Fmoc-3-(2-naphthyl)-D-Ala-OH. Before each peptide linking reaction, a decapping agent was used to remove the fluorene methoxycarbonyl group to obtain a protected decapeptide resin.

[0021] (ii) After removing the fluorene methoxycarbonyl group from the protected decapping resin with a decapping agent, the resin was acetylated with acetic anhydride, then the side chain protecting group was removed and the peptide was cleaved to obtain crude cetrilac acetate.

[0022] (iii) The crude cetrilac was purified and desalted sequentially using preparative liquid chromatography, and the collected eluent fraction was dried to obtain purified cetrilac acetate.

[0023] According to the method of the first aspect of the invention, the decapping agent used is 20% piperidine / DMF.

[0024] According to the method of the first aspect of the present invention, the amount of the decapping agent used is such that its feeding ratio with the resin is 8 to 15 ml / g resin.

[0025] According to the method of the first aspect of the present invention, the conditions for the decapping reaction are a reaction at 40-50°C for 20-30 minutes, for example, a decapping reaction at 45°C for 25 minutes.

[0026] According to the method of the first aspect of the present invention, the peptide-addition reagent used in the peptide-addition reaction is DMF.

[0027] According to the method of the first aspect of the present invention, the amount of Fmoc-protected amino acids used in each step of the peptide-linking reaction is 1 to 3 times the amount of resin, for example, 2 times the amount of resin.

[0028] According to the method of the first aspect of the present invention, in the peptide reaction, the molar amounts of HBTU and HOBT used are both twice that of the resin, and the molar amounts of DIEA are four times that of the resin.

[0029] According to the method of the first aspect of the present invention, the conditions for the peptide inoculation reaction are 35-50°C for 1-3 hours, for example, 40°C for 2 hours.

[0030] According to the method of the first aspect of the invention, the acetylation reaction is carried out using a mixed solution of acetic anhydride, pyridine, and dichloromethane, for example, in a volume ratio of 1:2:2.

[0031] According to the method of the first aspect of the present invention, the acetylation reaction is carried out at 35 to 50°C for 1 to 2 hours, for example at 40°C for 1.5 hours.

[0032] According to the method of the first aspect of the present invention, a mixture of trifluoroacetic acid / thiol / triisopropylsilane / water is used as a peptide cleaving reagent to carry out the peptide cleavage reaction.

[0033] According to the method of the first aspect of the present invention, the peptide cleavage reagent is pre-cooled to -15°C before use.

[0034] According to the method of the first aspect of the invention, the peptide cleavage reagent is a mixture of trifluoroacetic acid / thiol / triisopropylsilane / water in a volume ratio of 87.5 / 5 / 5 / 2.5.

[0035] According to the method of the first aspect of the present invention, the peptide cleavage reaction conditions are as follows: peptide cleavage reaction at 40-50°C for 2-3 hours, for example, peptide cleavage reaction at 42°C for 2.5 hours.

[0036] According to the method of the first aspect of the present invention, the precipitate obtained by the peptide cleavage reaction is precipitated and washed with diethyl ether, and then dried to obtain crude cetrirexate.

[0037] According to the method of the first aspect of the invention, a preparative C18 column is used for purification and desalting.

[0038] According to the method of the first aspect of the present invention, the conditions for purification using a preparative C18 column are as follows: mobile phase A is 0.1% TFA, mobile phase B is acetonitrile, starting from 20% mobile phase B and increasing to 55% mobile phase B within 60 minutes, detection wavelength is 260 nm, and the target peak fraction is collected.

[0039] According to the method of the first aspect of the present invention, the conditions for desalting the preparative C18 column are as follows: mobile phase A is 30 mmol / L ammonium acetate aqueous solution / acetonitrile = 95 / 5, mobile phase B is water / acetonitrile = 95 / 5, mobile phase C is 2% acetic acid aqueous solution / acetonitrile = 90 / 10, mobile phase D is 2% acetic acid aqueous solution / acetonitrile = 50 / 50, elution is first performed with mobile phase A for 15 minutes, then with mobile phase B for 15 minutes, then with mobile phase C for 15 minutes, and finally with mobile phase D for 25 minutes, the detection wavelength is 260 nm, and the target peak fraction is collected.

[0040] According to the method of the first aspect of the present invention, the desalted target peak fraction is freeze-dried to a moisture content of less than 5% to obtain refined cetrilac acetate.

[0041] According to a method of a first aspect of the present invention, in the process of incorporating Fmoc-protected arginine, Fmoc-Arg(Tos)-OH is used as a raw material and a mixture of HBTU / 6-Cl-HOBT / DIEA is used as a condensing agent to perform a peptide incorporation reaction, thereby obtaining Fmoc-Arg(Tos)-Pro-D-Aal- resin for subsequent reactions. In one embodiment, the molar amounts of HBTU and 6-Cl-HOBT in the condensing agent are both twice that of the resin, and the molar amount of DIEA is four times that of the resin.

[0042] According to the method of the first aspect of the present invention, it is prepared by a method comprising the following steps:

[0043] (1) Soak Fmoc-linker-MBHA-resin in DCM 3 to 5 times to fully swell the resin. Add decapping reagent (the ratio of reagent to resin is 10 to 15 ml / g resin) and decapping reaction at 42 to 48°C for 25 minutes. Dry the resin and wash it 3 times with DMF. Add Fmoc-D-Ala-OH (the molar amount of which is twice the amount of resin) dissolved in peptide-linking reagent and HBTU / HOBT / DIEA mixture in sequence. React peptide-linking reaction at 40°C for 2 hours. Dry the resin and wash it with DMF. Vacuum dry the resin to obtain Fmoc-D-Aal-resin.

[0044] (2) Add a decapping agent (the ratio of the agent to the resin is 8-12 ml / g resin) to the Fmoc-D-Aal- resin obtained in step (1), and decapping reaction at 42-48℃ for 25 minutes. Dry the resin and wash it three times each with DMF, MeOH and DCM. Dry the resin and add Fmoc-Pro-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA. React the resin at 40℃ for 2 hours. Dry the resin and wash it with DMF. Dry the resin to obtain Fmoc-Pro-D-Aal- resin.

[0045] (3) Add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin) to the Fmoc-Pro-D-Aal- resin obtained in step (2), and decapping reaction is carried out at 42-48℃ for 25 minutes. After drying, wash with DMF, MeOH and DCM three times each, and then dry. Add Fmoc-Arg(Tos)-OH (the molar amount of which is 3 times the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / 6-Cl-HOBT / DIEA in sequence. React with peptide inoculation at 40℃ for 2 hours, and then dry. Wash the resin with DMF and then dry to obtain Fmoc-Arg(Tos)-Pro-D-Aal- resin. The molar amounts of HBTU and 6-Cl-HOBT are both twice that of the resin, and the molar amount of DIEA is four times that of the resin.

[0046] (4) Add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin) to the Fmoc-Arg(Tos)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 42-48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin;

[0047] (5) Add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin) to the Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 42-48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin;

[0048] (6) Add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (5), and decapping reaction at 42-48℃ for 25 minutes. Dry the resin and wash it three times each with DMF, MeOH, and DCM. Dry the resin and add a mixture of Fmoc-Tyr(tBu)-OH (the molar amount of which is twice the amount of resin) and HBTU / HOBT / DIEA dissolved with peptide inoculation reagent. React the resin at 40℃ for 2 hours and dry the resin. Wash the resin with DMF and dry the resin to obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0049] (7) Add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin) to the Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (6), and decapping reaction at 42-48℃ for 25 minutes. Dry the resin and wash it three times each with DMF, MeOH, and DCM. Dry the resin and add a mixture of Fmoc-Ser(tBu)-OH (the molar amount of which is twice the amount of resin) and HBTU / HOBT / DI EA dissolved with peptide inoculation reagent. React the resin at 40℃ for 2 hours and dry the resin. Wash the resin with DMF and dry the resin to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0050] (8) Add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin) to the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (7), and decapping reaction is carried out at 42-48℃ for 25 minutes. After drying, wash with DMF, MeOH and DCM three times each, and then dry. Add Fmoc-3(3-Pyridyl)-D-Ala-OH (the molar amount of which is twice the amount of resin) and HBTU / HOBT / DIEA mixture dissolved with peptide inoculation reagent in sequence, and peptide inoculation reaction is carried out at 40℃ for 2 hours. After drying, wash the resin with DMF and then dry to obtain Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0051] (9) To the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (8), add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin), and react at 42-48℃ for 25 minutes. Dry the mixture under vacuum, wash it three times each with DMF, MeOH, and DCM, and then dry it under vacuum. Add Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. A mixture of moc-4-Chloro-D-Phe-OH (molar amount of which is twice the amount of resin) and HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin;

[0052] (10) To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(To s)-Pro-D-Aal- resin obtained in step (9), add a decapping reagent (the ratio of the reagent to the resin is 8-12 ml / g resin), and react at 42-48℃ for 25 minutes. Dry the mixture under vacuum, wash it three times each with DMF, MeOH, and DCM, and then dry it under vacuum. Add Fmoc-3-(2-naphthyl) dissolved in a peptide-integrating reagent. A mixture of 3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was reacted with peptides at 40°C for 2 hours, dried, and the resin was washed with DMF and dried to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin;

[0053] (11) To the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (10), add a decapping agent (the ratio of the agent to the resin is 13-15 ml / g resin), and react at 42-48℃ for 25 minutes to remove the cap. Dry the mixture and then react it with DMF, MeOH, DCM was washed three times in sequence, dried, and a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents was 1:2:2) was added. The mixture was reacted at 40°C for 1.5 hours, dried, and the resin was washed with DMF and dried to obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0054] (12) To the AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin prepared in step (11), add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃. The peptide-cleaving reaction is carried out at 42℃ for 2.5 hours. The solvent is filtered out, and diethyl ether is added to precipitate the product. The precipitate is collected, washed twice with diethyl ether, and dried under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin. e-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2 is the crude form of cetrirexate;

[0055] (13) Preparation of purified cetrorex acetate: The crude cetrorex obtained in the previous step was purified and desalted using a preparative C18 column.

[0056] The purification conditions were as follows: mobile phase A was 0.1% TFA, mobile phase B was acetonitrile, starting from 20% mobile phase B and increasing to 55% mobile phase B within 60 minutes, with a detection wavelength of 260 nm, and the target peak fraction was collected.

[0057] The desalting conditions were as follows: mobile phase A was 30 mmol / L ammonium acetate aqueous solution / acetonitrile = 95 / 5, mobile phase B was water / acetonitrile = 95 / 5, mobile phase C was 2% acetic acid aqueous solution / acetonitrile = 90 / 10, and mobile phase D was 2% acetic acid aqueous solution / acetonitrile = 50 / 50. The solution was eluted with mobile phase A for 15 minutes, then with mobile phase B for 15 minutes, then with mobile phase C for 15 minutes, and finally with mobile phase D for 25 minutes. The detection wavelength was 260 nm, and the target peak fraction was collected.

[0058] The target peak fraction collected after desalting was freeze-dried until the moisture content was less than 5% to obtain refined cetrilac acetate.

[0059] Furthermore, a second aspect of the present invention provides cetrorex acetate prepared by the method described in any one of the first aspects of the present invention.

[0060] Furthermore, a third aspect of the present invention provides the use of cetrorex acetate prepared by the method described in any one of the first aspects of the present invention, or cetrorex acetate described in the second aspect, in the preparation of a gonadotropin-releasing hormone antagonist for assisted reproductive technology.

[0061] For example, a third aspect of the present invention provides the use of cetrorex acetate prepared by the method of any one of the first aspects of the present invention or cetrorex acetate of the second aspect in the preparation of a medicament for preventing premature ovulation in patients undergoing controlled ovarian stimulation.

[0062] The cetrorex acetate prepared by the method of the present invention exhibits one or more excellent technical effects as described in the context of this paper. Attached Figure Description

[0063] Figure 1 This is a chromatogram of a typical system suitability test solution for related substance testing. Detailed Implementation

[0064] The present invention provides the following examples to further illustrate various aspects of the invention.

[0065] Some abbreviations used in this article have their general meanings in this field, such as: Fmoc: fluorenemethoxycarbonyl, tBu: tert-butyl, HOBT: 1-hydroxybenzotriazole, HBTU: benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate, DIC: N,N-diisopropylcarbodiimide, DIEA: N,N-diisopropylethylamine, Leu: leucine, Tyr: tyrosine, Arg: arginine, D-Cit: D-citrulline, Pro: proline, Ser: serine, 3-Pyridyl-D-Ala: 3-D-pyridinealanine, 4-Chloro-D-Phe: 4-chloro-D-phenylalanine, Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Boc: tert-butyloxycarbonyl, DMF: N,N-dimethylformamide, Fmoc-Rink-MBH A-resin: fluorenemethyloxycarbonyl-4-toluenehydroamine resin, TFA: trifluoroacetic acid, MeOH: methanol, DCM: dichloromethane, Kniser tes The reagents for the t assay are: ninhydrin, D-Ala: D-alanine, D-Orn: D-ornithine, D-3Pal: D-3-pyridinealanine, D-Phe(4Cl): D-4-chloro-phenylalanine, D-2Nal: D-2-naphthylalanine, Dde: N-1-(4,4-dimethyl-2,6-dioxane-1)ethyl, PyBop: benzotriazol-1-yl-oxytripyrrolidine hexafluorophosphate, NMP: N-methylpyrrolidone, Fmoc-Arg(Pbf)-OH, N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, Fmoc-Arg(Mts)-OH, N-fluorenylmethoxycarbonyl-N'-(2,4 Fmoc-Arg(Mtr)-OH, N-fluorenylmethoxycarbonyl-N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine, Fmoc-Arg(Tos)-OH, N-fluorenylmethoxycarbonyl-N'-toluenesulfonyl-L-arginine, and other unlisted abbreviations or designations have meanings well-known in the art. These materials are readily available from commercial sources; for example, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Mts)-OH, Fmoc-Arg(Mtr)-OH, and Fmoc-Arg(Tos)-OH were purchased from GLPBIO and their stated HPLC purities are all greater than 98%. All materials used herein, unless otherwise stated, are from the same batch.

[0066] This invention employs a solid-phase synthesis method, using Fmoc-linker-MBHA-resin as the starting resin, and sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the cetrorex backbone peptide sequence. Unless otherwise specified herein, the decapping agent used is 20% piperidine / DMF, the peptide linking agent is DMF, the molar amounts of HBTU and HOBT are twice that of the resin, and the molar amount of DIEA is four times that of the resin.

[0067] Example 1: Preparation of cetrorex acetate

[0068] (1) Preparation of Fmoc-D-Aal-resin: Fmoc-linker-MBHA-resin (50g of material was added, and the amount of each material was expressed as a ratio below) was soaked in DCM 4 times to allow the resin to swell fully. Decapping reagent (the ratio of the reagent to the resin was 12ml / g of resin) was added, and the decapping reaction was carried out at 45℃ for 25 minutes. The resin was dried under vacuum, washed 3 times with DMF, and dried under vacuum. Fmoc-D-Ala-OH (the molar amount of which was twice the amount of resin) dissolved with peptide-linking reagent and a mixture of HBTU / HOBT / DIEA were added in sequence. The peptide-linking reaction was carried out at 40℃ for 2 hours, and dried under vacuum. The resin was washed with DMF and dried under vacuum to obtain Fmoc-D-Aal-resin.

[0069] (2) Preparation of Fmoc-Pro-D-Aal-resin: Add a decapping agent (the ratio of the agent to the resin is 10 ml / g resin) to the Fmoc-D-Aal-resin obtained in step (1), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Pro-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Pro-D-Aal-resin.

[0070] (3) Preparation of Fmoc-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin) to the Fmoc-Pro-D-Aal- resin obtained in step (2), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Arg(pbf)-OH (the molar number of which is 3 times the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Arg(pbf)-Pro-D-Aal- resin.

[0071] (4) Preparation of Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin) to the Fmoc-Arg(pbf)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and HBT U / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0072] (5) Preparation of Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin) to the Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0073] (6) Preparation of Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (5), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Tyr(tBu)-OH (the molar number is twice the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0074] (7) Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: The Fmoc-Tyr(tBu) resin obtained in step (6) Add a decapping reagent (at a ratio of 10 ml / g resin) to Fmoc-Ser(tBu)-OH dissolved in a peptide-injecting reagent at 45°C for 25 minutes. Dry the resin under vacuum. Wash it three times each with DMF, MeOH, and DCM, and dry it under vacuum. Then add a mixture of Fmoc-Ser(tBu)-OH dissolved in a peptide-injecting reagent (molar amount twice the resin volume) and HBTU / HOBT / DIEA. Inject the resin under vacuum at 40°C for 2 hours. Dry the resin under vacuum. Wash the resin with DMF and dry it under vacuum to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0075] (8) Preparation of Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: Add a decapping agent (the ratio of the agent to the resin is 10 ml / g resin) to the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin obtained in step (7), and decapping reaction at 45°C for 25 minutes. Dry the resin, wash it three times each with DMF, MeOH, and DCM, and dry it. Then add Fmoc-3(3-Pyridyl)-D-Ala-OH (the molar amount of which is twice the amount of resin) dissolved in the peptide-linking reagent, HBTU / HOBT / DIE, etc. The mixture of A was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3(3-Pyridyl)-D-Ala-Se r(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0076] (9) Preparation of Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping agent (with a feeding ratio of 10 ml / g resin) to the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (8), and perform a decapping reaction at 45°C for 25 minutes. After 2 minutes, the resin was dried and washed three times each with DMF, MeOH, and DCM, then dried again. A mixture of Fmoc-4-Chloro-D-Phe-OH (dissolved with peptide induction reagent in a molar ratio twice the resin amount) and HBTU / HOBT / DIEA was added sequentially. The reaction was carried out at 40°C for 2 hours, then dried. The resin was washed with DMF and dried again to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0077] (10) Preparation of Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin obtained in step (9), add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin), and decapping reaction at 45°C for 25 minutes. Dry the resin under vacuum, wash it three times each with DMF, MeOH, and DCM, dry it under vacuum, and then add Fm dissolved in peptide-injecting reagent. A mixture of oc-3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0078] (11) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add to the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (10) Add the decapping reagent (the ratio of reagent to resin is 15 ml / g resin), react at 45°C for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, add a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents is 1:2:2), react at 40°C for 1.5 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, to obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0079] (12) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2: The AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-tree obtained in step (11) Add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃ to the lipid, and perform peptide cleavage reaction at 42℃ for 2.5 hours. Filter out the solvent, add diethyl ether to precipitate, collect the precipitate, wash twice with diethyl ether, and dry under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac.

[0080] (13) Preparation of refined cetrorex acetate

[0081] Weigh 15g of the crude cetrirexate obtained in the previous step, dissolve it in 1.5L of 30% HAc aqueous solution, and then purify it by passing it through a preparative C18 column (UltraAqueous C18 preparative chromatographic column, 9178579, id 30mm×250mm, 5μm).

[0082] Purification conditions: Mobile phase A was 0.1% TFA, mobile phase B was acetonitrile, starting from 20% mobile phase B and increasing to 55% mobile phase B within 60 minutes, detection wavelength was 260 nm, and the target peak fraction was collected.

[0083] Desalting conditions: Mobile phase A was 30 mmol / L ammonium acetate aqueous solution / acetonitrile = 95 / 5, mobile phase B was water / acetonitrile = 95 / 5, mobile phase C was 2% acetic acid aqueous solution / acetonitrile = 90 / 10, and mobile phase D was 2% acetic acid aqueous solution / acetonitrile = 50 / 50. Elution was performed first with mobile phase A for 15 minutes, then with mobile phase B for 15 minutes, then with mobile phase C for 15 minutes, and finally with mobile phase D for 25 minutes. The detection wavelength was 260 nm, and the target peak fraction was collected.

[0084] The desalted fraction was freeze-dried until the moisture content was less than 5%, yielding 6.13 g of refined cetrilac acetate [M]. + =1429.654, which matches the theoretical value.

[0085] Example 2: Preparation of cetrorex acetate

[0086] (1) Preparation of Fmoc-D-Aal-resin: Fmoc-linker-MBHA-resin (50g of material was added, and the amount of each material was expressed as a ratio below) was soaked in DCM 5 times to allow the resin to swell fully. Decapping reagent (the ratio of the reagent to the resin was 15ml / g of resin) was added, and the decapping reaction was carried out at 45℃ for 25 minutes. The resin was dried under vacuum, washed 3 times with DMF, and dried under vacuum. Fmoc-D-Ala-OH (the molar amount of which was twice the amount of resin) dissolved with peptide-linking reagent and a mixture of HBTU / HOBT / DIEA were added in sequence. The peptide-linking reaction was carried out at 40℃ for 2 hours, and dried under vacuum. The resin was washed with DMF and dried under vacuum to obtain Fmoc-D-Aal-resin.

[0087] (2) Preparation of Fmoc-Pro-D-Aal-resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-D-Aal-resin obtained in step (1), decapping reaction at 42°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Pro-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / HOBT / DIEA, react with peptide inoculation at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Pro-D-Aal-resin.

[0088] (3) Preparation of Fmoc-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-Pro-D-Aal- resin obtained in step (2), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Arg(pbf)-OH (the molar number of which is 3 times the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Arg(pbf)-Pro-D-Aal- resin.

[0089] (4) Preparation of Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-Arg(pbf)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and HBT U / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0090] (5) Preparation of Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0091] (6) Preparation of Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (5), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Tyr(tBu)-OH (the molar number is twice the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0092] (7) Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: The Fmoc-Tyr(tBu) resin obtained in step (6) Add a decapping reagent (at a ratio of 12 ml / g resin) to Fmoc-Ser(tBu)-OH dissolved in a peptide-injecting reagent at 42°C for 25 minutes. Dry the resin and wash it three times each with DMF, MeOH, and DCM. Dry the resin again and add a mixture of Fmoc-Ser(tBu)-OH dissolved in a peptide-injecting reagent (molar amount twice that of the resin) and HBTU / HOBT / DIEA. Inject the resin at 40°C for 2 hours. Dry the resin and wash it with DMF. Dry the resin again to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0093] (8) Preparation of Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: To the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin obtained in step (7), add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin), and decapping reaction at 42℃ for 25 minutes. Dry the solution, wash it three times each with DMF, MeOH, and DCM, and dry it again. Then add Fmoc-3(3-Pyridyl)-D-Ala-OH (the molar amount of which is twice the amount of resin) dissolved in the peptide-linking reagent, HBTU / HOBT / DIE, etc. The mixture of A was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3(3-Pyridyl)-D-Ala-Se r(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0094] (9) Preparation of Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: To the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (8), a decapping agent (with a feeding ratio of 12 ml / g resin) was added, and the decapping reaction was carried out at 42°C for 25 minutes. After 2 minutes, the resin was dried and washed three times each with DMF, MeOH, and DCM, then dried again. A mixture of Fmoc-4-Chloro-D-Phe-OH (dissolved with peptide induction reagent in a molar ratio twice the resin amount) and HBTU / HOBT / DIEA was added sequentially. The reaction was carried out at 40°C for 2 hours, then dried. The resin was washed with DMF and dried again to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0095] (10) Preparation of Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin obtained in step (9), add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin), and decapping reaction at 42℃ for 25 minutes. Dry the resin under vacuum, wash it three times each with DMF, MeOH, and DCM, dry it under vacuum, and then add Fm dissolved in peptide-injecting reagent. A mixture of oc-3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0096] (11) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add to the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (10) Add the decapping reagent (the ratio of reagent to resin is 13 ml / g resin), react at 42℃ for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, add a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents is 1:2:2), react at 40℃ for 1.5 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0097] (12) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2: The AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-tree obtained in step (11) Add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃ to the lipid, and perform peptide cleavage reaction at 42℃ for 2.5 hours. Filter out the solvent, add diethyl ether to precipitate, collect the precipitate, wash twice with diethyl ether, and dry under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac.

[0098] (13) Preparation of refined cetrorex acetate: Weigh 15g of crude cetrorex obtained in the previous step and refine it according to step (13) of Example 1 to obtain 6.21g of refined cetrorex acetate.

[0099] Example 3: Preparation of cetrorex acetate

[0100] (1) Preparation of Fmoc-D-Aal-resin: Fmoc-linker-MBHA-resin (50g of material was added, and the amount of each material is expressed by proportion below) was soaked in DCM 3 times to make the resin fully swollen. Decapping reagent (the ratio of the reagent to the resin was 10ml / g of resin) was added, and the decapping reaction was carried out at 48℃ for 25 minutes. The resin was dried under vacuum, washed 3 times with DMF, and dried under vacuum. Fmoc-D-Ala-OH (the molar amount of which was twice the amount of resin) dissolved with peptide-linking reagent and a mixture of HBTU / HOBT / DIEA were added in sequence. The peptide-linking reaction was carried out at 40℃ for 2 hours, and dried under vacuum. The resin was washed with DMF and dried under vacuum to obtain Fmoc-D-Aal-resin.

[0101] (2) Preparation of Fmoc-Pro-D-Aal-resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-D-Aal-resin obtained in step (1), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Pro-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Pro-D-Aal-resin.

[0102] (3) Preparation of Fmoc-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-Pro-D-Aal- resin obtained in step (2), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Arg(pbf)-OH (the molar number of which is 3 times the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DI EA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Arg(pbf)-Pro-D-Aal- resin.

[0103] (4) Preparation of Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-Arg(pbf)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DC M three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0104] (5) Preparation of Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0105] (6) Preparation of Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (5), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Tyr(tBu)-OH (the molar number is twice the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0106] (7) Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin: To the Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-resin obtained in step (6), add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin), and decapping reaction at 48℃ for 25 minutes. Dry the resin under vacuum, wash it three times each with DMF, MeOH, and DCM, dry it under vacuum, and then add Fm dissolved in peptide-injecting reagent. A mixture of oc-Ser(tBu)-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation reaction at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0107] (8) Preparation of Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: To the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (7), add a decapping agent (the ratio of the agent to the resin is 8 ml / g resin), and decapping reaction at 48℃ for 25 minutes. Dry the mixture and then use DMF and M... Wash three times each with eOH and DCM, then dry the resin. Add a mixture of Fmoc-3(3-Pyridyl)-D-Ala-OH (twice the amount of resin) dissolved in a peptide inoculation reagent and HBTU / HOBT / DIEA. Inoculate the resin at 40°C for 2 hours, then dry the resin. Wash the resin with DMF and dry the resin to obtain Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0108] (9) Preparation of Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add a decapping agent (at a ratio of 8 ml / g resin) to the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (8), and decapping reaction at 48℃ for 25 minutes. Dry the mixture and then react it with DMF and MeO2. H and DCM were washed three times each, and the mixture was dried. Then, a mixture of Fmoc-4-Chloro-D-Phe-OH (twice the amount of resin) dissolved in peptide inoculation reagent and HBTU / HOBT / DIEA was added sequentially. The peptide inoculation reaction was carried out at 40°C for 2 hours, and the mixture was dried. The resin was washed with DMF and dried to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0109] (10) Preparation of Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (9), add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g of resin), react at 48°C for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, and then add the peptide-integrating reagent to dissolve the resin. A mixture of Fmoc-3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0110] (11) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin: Add to the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin obtained in step (10) Add the decapping reagent (the ratio of reagent to resin is 15 ml / g resin), react at 48℃ for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, add a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents is 1:2:2), react at 40℃ for 1.5 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal- resin.

[0111] (12) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2: The AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(pbf)-Pro-D-Aal-tree obtained in step (11) Add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃ to the lipid, and perform peptide cleavage reaction at 42℃ for 2.5 hours. Filter out the solvent, add diethyl ether to precipitate, collect the precipitate, wash twice with diethyl ether, and dry under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac.

[0112] (13) Preparation of refined cetrorex acetate: Weigh 15g of crude cetrorex obtained in the previous step and refine it according to step (13) of Example 1 to obtain 6.07g of refined cetrorex acetate.

[0113] Example 4: Preparation of cetrorex acetate

[0114] (1) Preparation of Fmoc-D-Aal-resin: Fmoc-linker-MBHA-resin (50g of material was added, and the amount of each material was expressed as a ratio below) was soaked in DCM 4 times to allow the resin to swell fully. Decapping reagent (the ratio of the reagent to the resin was 12ml / g of resin) was added, and the decapping reaction was carried out at 45℃ for 25 minutes. The resin was dried under vacuum, washed 3 times with DMF, and dried under vacuum. Fmoc-D-Ala-OH (the molar amount of which was twice the amount of resin) dissolved with peptide-linking reagent and a mixture of HBTU / HOBT / DIEA were added in sequence. The peptide-linking reaction was carried out at 40℃ for 2 hours, and dried under vacuum. The resin was washed with DMF and dried under vacuum to obtain Fmoc-D-Aal-resin.

[0115] (2) Preparation of Fmoc-Pro-D-Aal-resin: Add a decapping agent (the ratio of the agent to the resin is 10 ml / g resin) to the Fmoc-D-Aal-resin obtained in step (1), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Pro-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Pro-D-Aal-resin.

[0116] (3) Preparation of Fmoc-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Pro-D-Aal-resin obtained in step (2), a decapping reagent (with a feeding ratio of 10 ml / g resin) was added. The decapping reaction was carried out at 45°C for 25 minutes, then dried. The resin was washed three times each with DMF, MeOH, and DCM, and dried again. A mixture of Fmoc-Arg(Tos)-OH dissolved with a peptide-inoculating reagent (its molar amount was three times that of the resin), HBTU / 6-Cl-HOBT / DIEA was added sequentially. The peptide-inoculating reaction was carried out at 40°C for 2 hours, then dried. The resin was washed with DMF and dried again to obtain Fmoc-Arg(Tos)-Pro-D-Aal-resin. The 6-Cl-HOBT and HOBT used in this study were both from the Abbexa brand. The molar amounts of HBTU and 6-Cl-HOBT were twice that of the resin, and the molar amount of DIEA was four times that of the resin.

[0117] (4) Preparation of Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin) to the Fmoc-Arg(Tos)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HB TU / HOBT / DIEA, peptide inoculation reaction at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0118] (5) Preparation of Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping agent (the ratio of the agent to the resin is 10 ml / g resin) to the Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0119] (6) Preparation of Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (5), decapping reaction at 45°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Tyr(tBu)-OH (the molar number is twice the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0120] (7) Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (6), add a decapping agent (the ratio of the agent to the resin is 10 ml / g resin), and react at 45°C for 25 minutes to remove the cap. Then, dry the mixture and use DMF... MeOH and DCM were washed three times each, and the mixture was dried. Then, a mixture of Fmoc-Ser(tBu)-OH (twice the amount of resin) dissolved in peptide inoculation reagent and HBTU / HOBT / DIEA was added sequentially. The peptide inoculation reaction was carried out at 40°C for 2 hours, and the mixture was dried. The resin was washed with DMF and dried to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0121] (8) Preparation of Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: Add a decapping agent (the ratio of the agent to the resin is 10 ml / g resin) to the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (7), and decapping reaction at 45℃ for 25 minutes. Dry the resin, wash it three times each with DMF, MeOH, and DCM, and dry it. Then add Fmoc-3(3-Pyridyl)-D-Ala-OH (the molar amount of which is twice the amount of resin) dissolved in the peptide-linking reagent, HBTU / HOBT / DIE, etc. The mixture of A was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3(3-Pyridyl)-D-Ala-Se r(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0122] (9) Preparation of Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: To the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (8), a decapping agent (with a feeding ratio of 10 ml / g resin) was added, and the decapping reaction was carried out at 45°C for 25 minutes. After 2 minutes, the resin was dried and washed three times each with DMF, MeOH, and DCM, then dried again. A mixture of Fmoc-4-Chloro-D-Phe-OH (dissolved with peptide induction reagent in a molar ratio twice the resin amount) and HBTU / HOBT / DIEA was added sequentially. The reaction was carried out at 40°C for 2 hours, then dried. The resin was washed with DMF and dried again to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0123] (10) Preparation of Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (9), add a decapping reagent (the ratio of the reagent to the resin is 10 ml / g resin), react at 45°C for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, and then add Fm dissolved in peptide-injecting reagent. A mixture of oc-3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0124] (11) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add to the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (10) Add the decapping reagent (the ratio of reagent to resin is 15 ml / g resin), react at 45°C for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, add a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents is 1:2:2), react at 40°C for 1.5 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, to obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0125] (12) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2: The AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-tree obtained in step (11) Add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃ to the lipid, and perform peptide cleavage reaction at 42℃ for 2.5 hours. Filter out the solvent, add diethyl ether to precipitate, collect the precipitate, wash twice with diethyl ether, and dry under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac.

[0126] (13) Preparation of refined cetrorex acetate: Weigh 15g of crude cetrorex obtained in the previous step, and refine it according to step (13) of Example 1 to obtain 6.31g of refined cetrorex acetate. Cetrorex [M] + =1429.654, which matches the theoretical value.

[0127] Example 5: Preparation of cetrorex acetate

[0128] (1) Preparation of Fmoc-D-Aal-resin: Fmoc-linker-MBHA-resin (50g of material was added, and the amount of each material was expressed as a ratio below) was soaked in DCM 5 times to allow the resin to swell fully. Decapping reagent (the ratio of the reagent to the resin was 15ml / g of resin) was added, and the decapping reaction was carried out at 45℃ for 25 minutes. The resin was dried under vacuum, washed 3 times with DMF, and dried under vacuum. Fmoc-D-Ala-OH (the molar amount of which was twice the amount of resin) dissolved with peptide-linking reagent and a mixture of HBTU / HOBT / DIEA were added in sequence. The peptide-linking reaction was carried out at 40℃ for 2 hours, and dried under vacuum. The resin was washed with DMF and dried under vacuum to obtain Fmoc-D-Aal-resin.

[0129] (2) Preparation of Fmoc-Pro-D-Aal-resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-D-Aal-resin obtained in step (1), decapping reaction at 42°C for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Pro-OH (the molar amount of which is twice the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / HOBT / DIEA, react with peptide inoculation at 40°C for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Pro-D-Aal-resin.

[0130] (3) Preparation of Fmoc-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Pro-D-Aal-resin obtained in step (2), a decapping reagent (with a feeding ratio of 12 ml / g resin) was added. The decapping reaction was carried out at 42°C for 25 minutes, then dried. The resin was washed three times each with DMF, MeOH, and DCM, and dried again. A mixture of Fmoc-Arg(Tos)-OH dissolved with a peptide-inoculating reagent (its molar amount was three times that of the resin) and HBTU / 6-Cl-HOBT / DIEA was added sequentially. The peptide-inoculating reaction was carried out at 40°C for 2 hours, then dried. The resin was washed with DMF and dried again to obtain Fmoc-Arg(Tos)-Pro-D-Aal-resin. The molar amounts of HBTU and 6-Cl-HOBT were both twice that of the resin, and the molar amount of DIEA was four times that of the resin.

[0131] (4) Preparation of Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-Arg(Tos)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HB TU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0132] (5) Preparation of Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0133] (6) Preparation of Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (5), decapping reaction at 42℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Tyr(tBu)-OH (the molar number is twice the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0134] (7) Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (6), add a decapping agent (the ratio of the agent to the resin is 12 ml / g resin), and react at 42°C for 25 minutes to remove the cap. Then, dry the mixture and use DMF... MeOH and DCM were washed three times each, and the mixture was dried. Then, a mixture of Fmoc-Ser(tBu)-OH (twice the amount of resin) dissolved in peptide inoculation reagent and HBTU / HOBT / DIEA was added sequentially. The peptide inoculation reaction was carried out at 40°C for 2 hours, and the mixture was dried. The resin was washed with DMF and dried to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0135] (8) Preparation of Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (7), add a decapping reagent (the ratio of the reagent to the resin is 12 ml / g resin), and decapping reaction at 42℃ for 25 minutes. Dry the solution, wash it three times each with DMF, MeOH, and DCM, and dry it again. Then add Fmoc-3(3-Pyridyl)-D-Ala-OH (the molar amount of which is twice the amount of resin) dissolved in the peptide-linking reagent, HBTU / HOBT / DIE, etc. The mixture of A was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3(3-Pyridyl)-D-Ala-Se r(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0136] (9) Preparation of Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: To the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (8), a decapping agent (the ratio of the agent to the resin is 12 ml / g resin) is added, and the decapping reaction is carried out at 42℃ for 25 minutes. After 2 minutes, the resin was dried and washed three times each with DMF, MeOH, and DCM, then dried again. A mixture of Fmoc-4-Chloro-D-Phe-OH (dissolved with peptide induction reagent in a molar ratio twice the resin amount) and HBTU / HOBT / DIEA was added sequentially. The reaction was carried out at 40°C for 2 hours, then dried. The resin was washed with DMF and dried again to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0137] (10) Preparation of Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping agent (at a ratio of 12 ml / g resin) to the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (9), and react at 42°C for 25 minutes. Dry the resin under vacuum, wash three times each with DMF, MeOH, and DCM, and then dry it under vacuum. Add Fm-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. A mixture of oc-3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0138] (11) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add to the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (10) Add the decapping reagent (the ratio of reagent to resin is 13 ml / g resin), react at 42℃ for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, add a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents is 1:2:2), react at 40℃ for 1.5 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0139] (12) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2: The AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-tree obtained in step (11) Add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃ to the lipid, and perform peptide cleavage reaction at 42℃ for 2.5 hours. Filter out the solvent, add diethyl ether to precipitate, collect the precipitate, wash twice with diethyl ether, and dry under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac.

[0140] (13) Preparation of refined cetrorex acetate: Weigh 15g of crude cetrorex obtained in the previous step and refine it according to step (13) of Example 1 to obtain 6.09g of refined cetrorex acetate.

[0141] Example 6: Preparation of cetrorex acetate

[0142] (1) Preparation of Fmoc-D-Aal-resin: Fmoc-linker-MBHA-resin (50g of material was added, and the amount of each material is expressed by proportion below) was soaked in DCM 3 times to make the resin fully swollen. Decapping reagent (the ratio of the reagent to the resin was 10ml / g of resin) was added, and the decapping reaction was carried out at 48℃ for 25 minutes. The resin was dried under vacuum, washed 3 times with DMF, and dried under vacuum. Fmoc-D-Ala-OH (the molar amount of which was twice the amount of resin) dissolved with peptide-linking reagent and a mixture of HBTU / HOBT / DIEA were added in sequence. The peptide-linking reaction was carried out at 40℃ for 2 hours, and dried under vacuum. The resin was washed with DMF and dried under vacuum to obtain Fmoc-D-Aal-resin.

[0143] (2) Preparation of Fmoc-Pro-D-Aal-resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-D-Aal-resin obtained in step (1), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Pro-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and a mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Pro-D-Aal-resin.

[0144] (3) Preparation of Fmoc-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Pro-D-Aal-resin obtained in step (2), add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin), and react at 48°C for 25 minutes. Dry the resin under vacuum, wash it three times each with DMF, MeOH, and DCM, and dry it under vacuum. Then, add a mixture of Fmoc-Arg(Tos)-OH dissolved with a peptide-inoculating reagent (its molar amount is 3 times the amount of resin), HBTU / 6-Cl-HOBT / DIEA, and react at 40°C for 2 hours. Dry the resin under vacuum, wash it with DMF, and dry it under vacuum to obtain Fmoc-Arg(Tos)-Pro-D-Aal-resin. The molar amounts of HBTU and 6-Cl-HOBT are both twice that of the resin, and the molar amount of DIEA is four times that of the resin.

[0145] (4) Preparation of Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-Arg(Tos)-Pro-D-Aal- resin obtained in step (3), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Leu-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and HB TU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0146] (5) Preparation of Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (4), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-D-Cit-OH (the molar number of which is twice the amount of resin) dissolved with peptide inoculation reagent and the mixture of HBTU / HOBT / DIEA, peptide inoculation reaction at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0147] (6) Preparation of Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g resin) to the Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (5), decapping reaction at 48℃ for 25 minutes, dry under vacuum, wash with DMF, MeOH and DCM three times each, dry under vacuum, add Fmoc-Tyr(tBu)-OH (the molar number is twice the amount of resin) dissolved with peptide inoculation reagent and HBTU / HOBT / DIEA mixture in sequence, react with peptide inoculation at 40℃ for 2 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0148] (7) Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (6), add a decapping agent (the ratio of the agent to the resin is 8 ml / g resin), and react at 48°C for 25 minutes to remove the cap. Then, dry the mixture and use DMF... MeOH and DCM were washed three times each, and the mixture was dried. Then, a mixture of Fmoc-Ser(tBu)-OH (twice the amount of resin) dissolved in peptide inoculation reagent and HBTU / HOBT / DIEA was added sequentially. The peptide inoculation reaction was carried out at 40°C for 2 hours, and the mixture was dried. The resin was washed with DMF and dried to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0149] (8) Preparation of Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin: To the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (7), add a decapping agent (the ratio of the agent to the resin is 8 ml / g resin), and decapping reaction at 48℃ for 25 minutes. Dry the mixture and then use DMF and M... The resin was washed three times each with eOH and DCM, dried, and then a mixture of Fmoc-3(3-Pyridyl)-D-Ala-OH (twice the amount of resin) dissolved in a peptide inoculation reagent and HBTU / HOBT / DIEA was added sequentially. The reaction was carried out at 40°C for 2 hours, dried, and the resin was washed with DMF and dried to obtain Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0150] (9) Preparation of Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add a decapping agent (at a ratio of 8 ml / g resin) to the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (8), and react at 48°C for 25 minutes. Dry the mixture and then react it with DMF and MeO2. H and DCM were washed three times each, and the mixture was dried. Then, a mixture of Fmoc-4-Chloro-D-Phe-OH (twice the amount of resin) dissolved in peptide inoculation reagent and HBTU / HOBT / DIEA was added sequentially. The peptide inoculation reaction was carried out at 40°C for 2 hours, and the mixture was dried. The resin was washed with DMF and dried to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0151] (10) Preparation of Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (9), add a decapping reagent (the ratio of the reagent to the resin is 8 ml / g of resin), and decapping reaction at 48℃ for 25 minutes. Dry the resin under vacuum, wash it three times each with DMF, MeOH, and DCM, dry it under vacuum, and then add the peptide-integrating reagent to dissolve the resin. A mixture of Fmoc-3-(2-naphthyl)-D-Ala-OH (molar amount of which is twice the amount of resin), HBTU / HOBT / DIEA was subjected to peptide inoculation at 40°C for 2 hours, dried under vacuum, and the resin was washed with DMF and dried under vacuum to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0152] (11) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin: Add to the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (10) Add the decapping reagent (the ratio of reagent to resin is 15 ml / g resin), react at 48℃ for 25 minutes, dry under vacuum, wash three times each with DMF, MeOH, and DCM, dry under vacuum, add a mixed solution of acetic anhydride, pyridine, and dichloromethane (volume ratio of the three reagents is 1:2:2), react at 40℃ for 1.5 hours, dry under vacuum, wash the resin with DMF, dry under vacuum, and obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin.

[0153] (12) Preparation of AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2: The AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-tree obtained in step (11) Add a peptide-cleaving reagent (trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, volume ratio) pre-cooled to -15℃ to the lipid, and perform peptide cleavage reaction at 42℃ for 2.5 hours. Filter out the solvent, add diethyl ether to precipitate, collect the precipitate, wash twice with diethyl ether, and dry under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac.

[0154] (13) Preparation of refined cetrorex acetate: Weigh 15g of crude cetrorex obtained in the previous step and refine it according to step (13) of Example 1 to obtain 6.14g of refined cetrorex acetate.

[0155] Experimental Example 1: Quality Inspection of Cetrorex Acetate

[0156] The quality inspection of the cetrorex acetate refined products obtained in step (13) of Examples 1-6 was carried out, and the detection methods / results of some key quality indicators are as follows:

[0157] Appearance: All 6 samples were white powders.

[0158] Solubility: All 6 samples showed that they were readily soluble in dimethyl sulfoxide, soluble in 30% acetic acid solution, and slightly soluble in water.

[0159] Specific rotation: Weigh the sample accurately, dissolve it in 10% acetic acid solution and dilute quantitatively to prepare a solution containing approximately 1 mg per ml. Determine the specific rotation according to the method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0621). Calculated based on anhydrous and acetic acid-free conditions, the specific rotation is generally required to be between -34.0° and -44.0°. The specific rotation of all 6 samples was measured to be between -38.3° and -41.1°. For example, the specific rotations of the two samples in Examples 1 and 4 were -39.6° and -40.3°, respectively.

[0160] Identification: Take about 1 mg of this product, add 1 ml of water to dissolve it, add 1 ml of biuret test solution (take 0.15 g of copper sulfate, add 0.6 g of potassium sodium tartrate, add 50 ml of water to dissolve it, add 30 ml of 10% sodium hydroxide solution while stirring, and add water to 100 ml). The test result should show a blue-purple color. All 6 samples tested showed a blue-purple color.

[0161] Acidity: Take an appropriate amount of this product, dissolve and dilute it with water to prepare a solution containing about 1 mg per 1 ml, and determine it according to the method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0631). The pH value is generally required to be 4.0 to 6.0. The pH values ​​of the 6 samples were measured and were all in the range of 4.6 to 5.1. For example, the pH values ​​of the two samples in Examples 1 and 4 were 4.92 and 4.83, respectively.

[0162] Clarity and color of the solution: Take an appropriate amount of this product, dissolve and dilute it with water to prepare a solution containing about 0.25 mg per ml, and test it according to the law (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0901, Method I and General Chapter 0902, Method I). Generally, the solution should be clear and colorless. All 6 samples tested showed that the solution was clear and colorless.

[0163] Acetic acid: The acetic acid content was determined according to the method for determination of acetic acid in synthetic polypeptides (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0872). The content was calculated by peak area using the external standard method. Generally, the acetic acid content should be 5.0% to 12.0%. The acetic acid content of the 6 samples was found to be in the range of 7.3% to 7.7%. For example, the acetic acid content of samples 1 and 4 was 7.62% and 7.56%, respectively.

[0164] Moisture content: Take this product and determine the moisture content according to the method of determination of moisture (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0832, Method 1, 2). Generally, the moisture content should not exceed 6.0%. The moisture content of the 6 samples were all within the range of 3.1% to 3.6%. For example, the moisture content of the two samples in Examples 1 and 4 was 3.42% and 3.36%, respectively.

[0165] Bacterial endotoxins: Take this product and test it according to the law (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 1143). The amount of endotoxin in each 1 mg of cetrorex should generally be less than 50 EU. The results of the test of 6 samples were all <50 EU / mg.

[0166] Microbial limits: Take 3g of this product and test it according to the method described in the Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapters 1105, 1106 and 1107. The total number of aerobic bacteria should generally not exceed 10. 3 cfu / g (all 6 samples were tested and the results were <10 cfu / g), and the total number of molds and yeasts should generally not exceed 10. 2 cfu / g (all 6 samples were measured to be <10 cfu / g).

[0167] Acetic acid: Determined according to the method for the determination of acetic acid in synthetic polypeptides (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0872);

[0168] Test solution: Accurately weigh an appropriate amount of prepared cetrarolix acetate, dissolve and quantitatively dilute it with a mixed solution of mobile phase A and mobile phase B (95:5) to prepare a solution containing approximately 1.25 mg of cetrarolix per ml; Reference solution: Accurately weigh an appropriate amount of glacial acetic acid, quantitatively dilute it with a mixed solution of mobile phase A and mobile phase B (95:5) to prepare a solution containing approximately 0.1 mg per ml; Chromatographic conditions: Use octadecylsilane-bonded silica gel as the stationary phase (4.6 mm × 250 mm, 5 μm); use phosphoric acid solution (0.7 ml of phosphoric acid added to 1000 ml of water, adjusted to pH 3.0 with 0.42% sodium hydroxide solution) as mobile phase A, and methanol as mobile phase B; perform linear gradient elution according to the table below; flow rate is 1.2 ml per minute; detection wavelength is 210 nm; injection volume is 10 μl;

[0169] Gradient elution program: Mobile phase A is linearly reduced from 95% to 50% during the 0-5 minute period, mobile phase A is maintained at 50% during the 5-20 minute period, and mobile phase A is maintained at 95% during the 20-30 minute period;

[0170] System suitability requirements: In the chromatogram of the reference solution, the retention time of the acetic acid peak should be approximately 3–4 minutes, and the theoretical plate number calculated based on the acetic acid peak should be no less than 2000; Assay method: Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms; Limits: Calculate the acetic acid content based on the peak area using the external standard method. The acetic acid content of the six samples from Examples 1–6 was determined to be within the range of 6.8%–8.3%, for example, the acetic acid content of samples 1 and 4 was 7.16% and 7.34%, respectively.

[0171] Content determination: determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512);

[0172] Test solution: Take an appropriate amount of the prepared cetrorex acetate, accurately weigh it, dissolve it in 30% acetic acid solution and quantitatively dilute it to prepare a solution containing about 50 μg of cetrorex per 1 ml.

[0173] Reference solution: Take an appropriate amount of cetrorex acetate reference standard, accurately weigh it, dissolve it in 30% acetic acid solution and dilute it quantitatively to prepare a solution containing about 50 μg of cetrorex per 1 ml;

[0174] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; 0.25% sulfuric acid solution (adjusted to pH 2.7 with 2M sodium hydroxide solution) was used as mobile phase A, and acetonitrile was used as mobile phase B; linear gradient elution was performed according to the table below; column temperature was 30℃; flow rate was 1.0 ml per minute; detection wavelength was 226 nm; injection volume was 20 μl.

[0175] Gradient elution program: Mobile phase A is linearly reduced from 75% to 70% during 0-10 minutes, mobile phase A is linearly reduced from 70% to 60% during 10-30 minutes, mobile phase A is increased from 60% to 75% during 30-31 minutes, and mobile phase A is maintained at 75% during 31-35 minutes;

[0176] System applicability requirements: The theoretical plate number, calculated based on the Sittrielle peak, shall not be less than 5000;

[0177] Determination method: Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms; calculate the results by peak area according to the external standard method;

[0178] According to the analysis, the six samples from Examples 1 to 6, calculated based on anhydrous and acetic acid-free conditions, contained cetrarolix acetate at a concentration of cetrarolix (C... 70 H 92 ClN 17 O 14 The content was all in the range of 98.0% to 102.0%, for example, the two samples in Examples 1 and 4 were 100.13% and 99.87%, respectively. It should be noted that since the content was calculated using the external standard method with cetrirexate acetate reference standard, the result is not directly related to the results of related substances.

[0179] Related substances: determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512);

[0180] Test solution: Take an appropriate amount of the prepared cetrorex acetate, accurately weigh it, dissolve it in 30% acetic acid solution and quantitatively dilute it to prepare a solution containing about 0.25 mg of cetrorex per 1 ml;

[0181] Reference stock solution: Take appropriate amounts of cetrorex acetate reference standard and reference standards of impurities 32, 28, 6, and 8, accurately weigh them, dissolve them in 30% acetic acid solution, and quantitatively dilute them to prepare a solution containing 0.125 mg of each in 1 ml.

[0182] Reference solution: Accurately measure appropriate amounts of each reference stock solution and dilute with 30% acetic acid solution to prepare a mixed solution containing 0.5 μg each of cetrirexate, impurity 32 and impurity 28 and 1.25 μg each of impurity 6 and impurity 8 per 1 ml;

[0183] System suitability solution: Take about 25 mg of cetrorex acetate reference standard and place it in a 100 ml volumetric flask. Add 1 ml each of the stock solutions of impurity 32, impurity 28, impurity 6, and impurity 8 reference standards. Dilute with 30% acetic acid solution to prepare a mixed solution containing about 0.25 mg of cetrorex and 1.25 μg each of impurity 32, impurity 28, impurity 6, and impurity 8 per ml.

[0184] Sensitivity solution: Accurately measure an appropriate amount of cetrorex acetate reference standard stock solution and quantitatively dilute it with 30% acetic acid solution to prepare a solution containing approximately 0.125 μg of cetrorex per ml;

[0185] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (YMC Triart C18 column, 4.6 mm × 250 mm, 5 μm); mobile phase A was 0.4% sulfuric acid solution (4 ml sulfuric acid and 2 ml tetrabutylammonium bromide solution were added to 1000 ml water, and the pH was adjusted to 2.7 with sodium hydroxide solution) - acetonitrile (90:10), and mobile phase B was acetonitrile; linear gradient elution was performed according to the table below; column temperature was 35℃; flow rate was 1.0 ml per minute; detection wavelength was 226 nm; injection volume was 20 μl.

[0186] Gradient elution program: Mobile phase A is linearly reduced from 80% to 75% from 0 to 25 minutes; from 75% to 70% from 25 to 34 minutes; from 70% to 65% from 34 to 40 minutes; from 65% to 80% from 40 to 41 minutes; and from 80% from 41 to 49 minutes.

[0187] System suitability requirements: In the system suitability solution chromatogram, generally, impurities 32, 28, cetrorex, 6, and 8 should elute sequentially. The resolution between cetrorex and impurity 28 should be greater than 1.5, and the theoretical plate number calculated based on the cetrorex peak should be no less than 5000 (actually greater than 8000). (See a typical system suitability solution chromatogram for reference.) Figure 1 In the sensitivity solution chromatogram, the signal-to-noise ratio of the cetrirek peak should be greater than 10 (measured to be greater than 15); the above system suitability requirements are met.

[0188] Assay: Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms;

[0189] Limits: Generally, if there are impurity peaks (excluding solvent peaks and acetic acid peaks) in the chromatogram of the test solution, the peak area of ​​impurity 32, impurity 28, impurity 6 and impurity 8 shall not exceed 0.20% each, calculated by external standard method for impurity reference; the peak area of ​​other individual impurities shall not exceed 0.10%, and the total impurities shall not exceed 1.5%, calculated by external standard method for principal components.

[0190] Results: In the purified cetrorex acetate obtained in step (13) of Example 4, the impurities 32, 28, 6, and 8 were 0.018%, 0.035%, 0.041%, and 0.029%, respectively, and no other individual impurities were detected; In the purified cetrorex acetate obtained in step (13) of Examples 1-3, the impurities 28 were 0.381%, 0.397%, and 0.374%, respectively, and In the purified cetrorex acetate obtained in step (13) of Examples 5-6, the impurities 28 were 0.041% and 0.033%, respectively; In the purified cetrorex acetate obtained in step (13) of Examples 1-6, the impurities 32 were all below 0.025%, the impurities 6 were all in the range of 0.035-0.060%, and the impurities 8 were all in the range of 0.025-0.050%, and no other individual impurities were detected.

[0191] The chemical names of the substances are as follows: Cetrilac: N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine)-D-alanyl-L-seryl-L-tyrosyl-N 5 -Carbamoyl-D-ornithine-L-leucyl-L-arginyl-L-prolyl-D-propanamide; Impurity 32([D-Orn) 6 ]-Sydryk): C 69 H 91 ClN 16 O 13 ,1388.01,Ac-3-(2-Nal)-D-Ala-D-Phe(4-Cl)-3-(3-pyridyl)-D-Ala-Ser-Tyr-D-Orn-Leu-Arg-Pro-D-Ala-NH2,N-acetyl-3-(2-naphthalene)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine)-D-alanyl-L-seryl-L-tyrosyl-D-guanyl-L-leucyl-L-arginyl-L-prolyl-D-alanylamide; Impurity 28([D-Arg 8 ]-Sydryk): C 70 H 92 ClN 17 O 14 1431.06, Ac-3-(2-Nal)-D-Ala-D-Phe(4-Cl)-3-(3-pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-D-Arg-Pro-D-Ala-NH2,N-acetyl-3-(2-naphthalene)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine)-D-alanyl-L-seryl-L-tyrosyl-N 5-Carbamoyl-D-guanyl-L-leucyl-D-arginyl-L-prolyl-D-alanylamide; Impurity 6([D-Ala 10 -OH]-Cyclorek):C 70 H 91 ClN 16 O 15 1432.02, Ac-3-(2-Nal)-D-Ala-D-Phe(4-Cl)-3-(3-pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-OH, N-acetyl-3-(2-naphthalene)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine)-D-alanyl-L-seryl-L-tyrosyl-N 5 -Carbamoyl-D-ornithine-L-leucyl-L-arginyl-L-prolyl-D-alanine; Impurity 8 ([Ser(Ac) 4 ]-Sydryk): C 72 H 94 ClN 17 O 15 1473.08, Ac-3-(2-Nal)-D-Ala-D-Phe(4-Cl)-3-(3-pyridyl)-D-Ala-Ser(Ac)-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2,N-acetyl-3-(2-naphthalene)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine)-D-alanyl-(O-acetyl)-L-seryl-L-tyrosyl-N 5 -Carbamoyl-D-ornithine-L-leucyl-L-arginyl-L-prolyl-D-propanamide.

[0192] The above results appear to indicate that using the combination of Fmoc-Arg(Tos)-OH and HBTU / 6-Cl-HOBT / DIEA in step (3) significantly reduces the amount of impurity 28 in the final product compared to using the combination of Fmoc-Arg(pbf)-OH and HBTU / HOBT / DIEA. To verify this hypothesis, the present invention further prepares the following purified cetrilac acetate. Example 7: Referring to Example 4, but using Fmoc-Arg(pbf)-OH instead of Fmoc-Arg(Tos)-OH in step (3) to obtain refined cetrorex acetate; Example 8: Referring to Example 4, but using Fmoc-Arg(Mts)-OH instead of Fmoc-Arg(Tos)-OH in step (3) to obtain refined cetrorex acetate; Example 9: Referring to Example 4, but using Fmoc-Arg(Mtr)-OH instead of Fmoc-Arg(Tos)-OH in step (3) to obtain refined cetrorex acetate; Examples 10: Referring to Example 4, but using HOBT instead of 6-Cl-HOBT in step (3), refined cetrorex acetate was prepared; the four refined cetrorex acetate samples from Examples 7 to 10 were determined using the above-mentioned related substances detection method. The results showed that impurity 28 was 0.373%, 0.406%, 0.348%, and 0.393%, respectively; impurity 32 was in the range of 0.012-0.020%; impurity 6 was in the range of 0.037-0.058%; and impurity 8 was in the range of 0.022-0.056%. No other individual impurities were detected. The results were not significantly different from those of the products in Examples 1 to 4. Since all the raw materials used in each example were from the same batch, the difference in the content of impurity 28 in the products of different examples should not be related to the batch of materials, but rather to the type of raw materials used in step (3). This finding was completely unexpected.

[0193] Experimental Example 2: Stability of Cetirizine Acetate

[0194] Accelerated stability tests were conducted by placing the cetrorex acetate obtained in Examples 4-6 at 25°C for 6 months under light-protected conditions. Samples were taken at 0 and 6 months, and their contents and related substances were determined according to the method described in Test Example 1. The results for 0 months are shown in Test Example 1; the results for cetrorex acetate obtained in Examples 4-6 at 6 months showed no significant difference from those at 0 months, specifically as follows: the contents were 99.79%, 99.93%, and 99.67%, respectively, with no significant difference from the results at 0 months; impurity 32 was all below 0.034%, impurity 6 was all in the range of 0.041-0.056%, and impurity 8 was all in the range of 0.047-0.064%, with no other individual impurities detected; impurity 28 was 0.038%, 0.033%, and 0.047%, respectively. The above test results indicate that the cetrorex acetate prepared by this invention has excellent stability.

[0195] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing cetrorex acetate using a solid-phase synthesis method, comprising the following steps: Ceturorex acetate is prepared by sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the main peptide sequence of the cetrorex backbone. (i) Using Fmoc-linker-MBHA-resin resin as the starting material, a mixture of HBTU / HOBT / DIEA was used as the condensing agent for peptide linking reactions. Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Arg(Tos)-OH, Fmoc-Leu-OH, Fmoc-D-Cit-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-3(3-Pyridyl)-D-Ala-OH, Fmoc-4-Chloro-D-Phe-OH, and Fmoc-3-(2-naphthyl)-D-Ala-OH were linked sequentially. Before each peptide linking reaction, a decapping agent was used to remove the fluorene methoxycarbonyl group to obtain a protected decapeptide resin. The peptide linking agent used in the peptide linking reaction was DMF, and the decapping agent used was 20% piperidine / DMF. (ii) After removing the fluorene methoxycarbonyl group from the protected decapping resin with a decapping agent, an acetylation reaction is carried out with a mixed solution of acetic anhydride, pyridine, and dichloromethane in a volume ratio of 1:2:

2. Then, the side chain protecting groups are removed simultaneously and the peptide is cleaved to obtain crude cetrilac acetate. A mixture of trifluoroacetic acid / thiol / triisopropylsilane / water is used as the peptide cleaving agent. Before use, the solution is pre-cooled to -15°C and the peptide cleaving reaction is carried out at 40~50°C for 2~3 hours. (iii) The crude cetrorex was purified and desalted sequentially using preparative C18 column liquid chromatography. The collected eluent fraction was dried to obtain purified cetrorex acetate. in, In step (i), when incorporating Fmoc-protected arginine, Fmoc-Arg(Tos)-OH is used as the raw material and a mixture of HBTU / 6-Cl-HOBT / DIEA is used as the condensing agent to carry out the peptide incorporation reaction, thereby obtaining Fmoc-Arg(Tos)-Pro-D-Aal- resin for subsequent reactions. The molar amounts of HBTU, HOBT, and 6-Cl-HOBT in the condensing agent are all twice that of the resin, and the molar amount of DIEA is four times that of the resin. The ratio of the decapping agent to the resin is 8~15ml / g resin; The amount of Fmoc-protected amino acids used in each step of the peptide initiation reaction is 1 to 3 times the amount of resin. The peptide cleavage reagent is a mixture of trifluoroacetic acid / thiol / triisopropylsilane / water in a volume ratio of 87.5 / 5 / 5 / 2.

5.

2. According to the method of claim 1, the decapping reaction is carried out at 40-50°C for 20-30 minutes.

3. According to the method of claim 1, the peptide inoculation reaction is carried out at 35-50°C for 1-3 hours.

4. According to the method of claim 1, the acetylation reaction is carried out at 35~50°C for 1~2 hours.

5. According to the method of claim 1, the precipitate obtained from the peptide cleavage reaction is precipitated and washed with diethyl ether, and then dried to obtain crude cetrilac.

6. According to the method of claim 1, the conditions for purification using a preparative C18 column are as follows: mobile phase A is 0.1% TFA, mobile phase B is acetonitrile, starting from 20% mobile phase B and increasing to 55% mobile phase B within 60 minutes, detection wavelength is 260 nm, and the target peak fraction is collected.

7. According to the method of claim 1, the conditions for desalting the preparative C18 column are as follows: mobile phase A is 30 mmol / L ammonium acetate aqueous solution / acetonitrile = 95 / 5, mobile phase B is water / acetonitrile = 95 / 5, mobile phase C is 2% acetic acid aqueous solution / acetonitrile = 90 / 10, mobile phase D is 2% acetic acid aqueous solution / acetonitrile = 50 / 50, first elute with mobile phase A for 15 minutes, then with mobile phase B for 15 minutes, then with mobile phase C for 15 minutes, and finally with mobile phase D for 25 minutes, with a detection wavelength of 260 nm, and the target peak fraction is collected.

8. The method according to claim 1, wherein the product is prepared by a method comprising the following steps: (1) Soak Fmoc-linker-MBHA-resin in DCM 3-5 times to fully swell the resin. Add decapping reagent at a ratio of 10-15 ml / g resin. Decapping reaction at 42-48℃ for 25 minutes. Dry the resin and wash it 3 times with DMF. Add Fmoc-D-Ala-OH dissolved in peptide-linking reagent (molar number twice the amount of resin) and HBTU / HOBT / DIEA mixture. React peptide-linking reaction at 40℃ for 2 hours. Dry the resin and wash it with DMF. Vacuum dry the resin to obtain Fmoc-D-Aal-resin. (2) Add a decapping agent to the Fmoc-D-Aal-resin obtained in step (1), with a feeding ratio of 8~12 ml / g resin. Decapping reaction is carried out at 42~48℃ for 25 minutes. The solution is dried and washed three times each with DMF, MeOH and DCM. The solution is dried and then Fmoc-Pro-OH dissolved with peptide inoculation reagent is added sequentially, with a molar number twice that of the resin, and a mixture of HBTU / HOBT / DIEA. Peptide inoculation reaction is carried out at 40℃ for 2 hours. The solution is dried and the resin is washed with DMF and dried to obtain Fmoc-Pro-D-Aal-resin. (3) Add a decapping agent to the Fmoc-Pro-D-Aal- resin obtained in step (2), with a feeding ratio of 8~12 ml / g resin. Decapping reaction is carried out at 42~48℃ for 25 minutes. After drying, wash with DMF, MeOH and DCM three times each. After drying, add Fmoc-Arg(Tos)-OH dissolved with peptide inoculation reagent, with a molar amount of 3 times that of the resin, and a mixture of HBTU / 6-Cl-HOBT / DIEA. Peptide inoculation reaction is carried out at 40℃ for 2 hours. After drying, the resin is washed with DMF and dried to obtain Fmoc-Arg(Tos)-Pro-D-Aal- resin. The molar amounts of HBTU and 6-Cl-HOBT are both twice that of the resin, and the molar amount of DIEA is four times that of the resin. (4) Add a decapping agent to the Fmoc-Arg(Tos)-Pro-D-Aal- resin obtained in step (3). The ratio of the decapping agent to the resin is 8~12 ml / g resin. The decapping reaction is carried out at 42~48℃ for 25 minutes. The resin is then dried and washed three times each with DMF, MeOH, and DCM. The resin is then dried and added sequentially with Fmoc-Leu-OH dissolved with peptide inoculation reagent (the molar amount of which is twice the amount of resin) and a mixture of HBTU / HOBT / DIEA. The peptide inoculation reaction is carried out at 40℃ for 2 hours. The resin is then dried and washed with DMF. The resin is then dried to obtain Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin. (5) Add a decapping agent to the Fmoc-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (4), with a feeding ratio of 8~12 ml / g resin. Decapping reaction is carried out at 42~48℃ for 25 minutes. The resin is then dried and washed three times each with DMF, MeOH, and DCM. The resin is then dried and added sequentially with Fmoc-D-Cit-OH dissolved with peptide inoculation reagent, with a molar number twice that of the resin, and a mixture of HBTU / HOBT / DIEA. The peptide inoculation reaction is carried out at 40℃ for 2 hours. The resin is then dried and washed with DMF. The resin is then dried to obtain Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (6) Add a decapping agent to the Fmoc-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (5). The ratio of the decapping agent to the resin is 8~12 ml / g. The resin is decapped at 42~48℃ for 25 minutes, dried, and washed three times each with DMF, MeOH, and DCM. The resin is dried, and Fmoc-Tyr(tBu)-OH dissolved with peptide inoculation reagent is added sequentially. The molar amount of OH is twice the amount of resin, and a mixture of HBTU / HOBT / DIEA is added. The peptide inoculation reaction is carried out at 40℃ for 2 hours, dried, and the resin is washed with DMF and dried to obtain Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (7) Add a decapping agent to the Fmoc-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (6). The ratio of the decapping agent to the resin is 8~12 ml / g resin. The decapping reaction is carried out at 42~48℃ for 25 minutes. The resin is then dried and washed three times each with DMF, MeOH, and DCM. The resin is then dried and Fmoc-Ser(tBu)-OH dissolved with peptide inoculation reagent is added sequentially. The molar amount of OH is twice the amount of resin. A mixture of HBTU / HOBT / DIEA is also added. The peptide inoculation reaction is carried out at 40℃ for 2 hours. The resin is then dried and washed with DMF. The resin is then dried to obtain Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (8) Add a decapping agent to the Fmoc-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (7). The ratio of the decapping agent to the resin is 8~12 ml / g resin. The decapping reaction is carried out at 42~48℃ for 25 minutes. The resin is then dried and washed three times each with DMF, MeOH, and DCM. The resin is then dried and Fmoc-3(3-Pyridyl)-D-Ala-OH dissolved with peptide-inoculating reagent is added sequentially. The molar amount of Fmoc-3(3-Pyridyl)-D-Ala-OH is twice the amount of resin. A mixture of HBTU / HOBT / DIEA is also added. The resin is then inoculated at 40℃ for 2 hours. The resin is then dried and washed with DMF. The resin is then dried to obtain Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (9) To the Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (8), add a decapping reagent at a ratio of 8~12 ml / g resin. React at 42~48℃ for 25 minutes, then dry under vacuum. Wash three times each with DMF, MeOH, and DCM, then dry under vacuum again. Add Fmoc-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. A mixture of moc-4-Chloro-D-Phe-OH (twice the amount of resin) and HBTU / HOBT / DIEA was inoculated with peptides at 40°C for 2 hours, dried, and the resin was washed with DMF and dried to obtain Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (10) To the Fmoc-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (9), add a decapping agent at a ratio of 8~12 ml / g resin. React at 42~48℃ for 25 minutes, then dry under vacuum. Wash three times each with DMF, MeOH, and DCM, then dry under vacuum again. Add Fmoc-3 dissolved in a peptide-linking reagent sequentially. -(2-naphthyl)-D-Ala-OH, with a molar amount twice that of the resin, and a mixture of HBTU / HOBT / DIEA, were subjected to peptide inoculation at 40°C for 2 hours. The mixture was then dried, and the resin was washed with DMF and dried again to obtain Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (11) To the Fmoc-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin obtained in step (10), add a decapping agent at a ratio of 13~15 ml / g resin. The decapping reaction is carried out at 42~48℃ for 25 minutes, then dried under vacuum and treated with DMF, MeOH, DCM was washed three times in sequence, dried, and a mixed solution of acetic anhydride, pyridine, and dichloromethane was added in a volume ratio of 1:2:

2. The mixture was reacted at 40°C for 1.5 hours, dried, and the resin was washed with DMF and dried to obtain AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal- resin. (12) To the AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser(tBu)-Tyr(tBu)-D-Cit-Leu-Arg(Tos)-Pro-D-Aal-resin obtained in step (11), add a peptide-cleaving reagent, trifluoroacetic acid / thiol / triisopropylsilane / water = 87.5 / 5 / 5 / 2.5, pre-cooled to -15℃. The peptide was cleaved at 42°C for 2.5 hours by volume ratio. The solvent was filtered out, and ether was added to precipitate the product. The precipitate was collected, washed twice with ether, and dried under reduced pressure in a desiccator at room temperature for 4 hours to obtain the final product AC-3-(2-naphthyl)-D-Ala-4-Chloro-D-Phe-3(3-Pyridyl)-D-Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2, i.e., crude cetrilac. (13) Preparation of purified cetrorex acetate: The crude cetrorex obtained in the previous step was purified and desalted using a preparative C18 column. The purification conditions were as follows: mobile phase A was 0.1% TFA, mobile phase B was acetonitrile, starting from 20% mobile phase B and increasing to 55% mobile phase B within 60 minutes, with a detection wavelength of 260 nm, and the target peak fraction was collected. The desalting conditions were as follows: mobile phase A was 30 mmol / L ammonium acetate aqueous solution / acetonitrile = 95 / 5, mobile phase B was water / acetonitrile = 95 / 5, mobile phase C was 2% acetic acid aqueous solution / acetonitrile = 90 / 10, and mobile phase D was 2% acetic acid aqueous solution / acetonitrile = 50 / 50. The mobile phase was first eluted with mobile phase A for 15 minutes, then with mobile phase B for 15 minutes, then with mobile phase C for 15 minutes, and finally with mobile phase D for 25 minutes. The detection wavelength was 260 nm, and the target peak fraction was collected. The desalted and collected target peak fraction was freeze-dried until the moisture content was less than 5% to obtain refined cetrilac acetate.

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