Preparation method of retaglutide and intermediate thereof

Abstract

The invention relates to the technical field of polypeptide drug preparation methods, and provides a preparation method of retaglutide and a preparation method of a retaglutide intermediate.The preparation method of the retaglutide comprises the steps that a fragment 1-resin is prepared through a solid-phase synthesis method, then a fragment 2 and the fragment 1-resin are coupled through the solid-phase synthesis method, and the retaglutide is obtained. And cracking the retaglutide-resin, so as to obtain the retaglutide. Wherein the fragment 1 is a 15th-39th amino acid polypeptide fragment, and the fragment 2 is a 1st-14th amino acid polypeptide fragment. According to the preparation method disclosed by the invention, the production cost of the retaglutide can be greatly reduced, the synthesis steps are few, the subsequent treatment is simple, and the purity and the yield of the retaglutide are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drug preparation methods, and particularly relates to a preparation method of retaglutide and its intermediates. Background Art

[0002] Retatrutide, an active compound released by Eli Lilly in June 2023, is based on the GLP / GIP dual receptor agonist Tirzepatide. It utilizes a hybrid glucagon (Gcg)-related structure to achieve triple (GLP-1R / GCGR / GIPR) agonist activity. It is a drug in development for the treatment of obesity and type 2 diabetes. This active compound activates receptor agonists in the human body, contributing to blood sugar control and weight loss management.

[0003] The structure of retaglutide is as follows: H-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-eicosanedioic acid)-Ala 18 -Gln 19 -Aib 20 -Ala 21 -Phe 22 -Ile 23 -Glu 24 -Tyr 25 -Leu 26 -Leu 27 -Glu 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro37 -Pro 38 -Ser 39 -NH2.

[0004] Current peptide synthesis methods generally involve sequential coupling of single amino acids or coupling using multiple peptide fragments. Continuous coupling involves coupling amino acids one by one onto an amino resin via solid-phase synthesis. Starting from the amino terminus of the peptide, the first amino acid, deprotected from its carboxyl group, is attached to the resin. The amino protecting group is then removed. The next amino acid is coupled according to the peptide sequence, the amino protecting group is removed, and the coupling cycle is repeated until the amino acid assembly is complete. The peptide is cleaved from the resin and all protecting groups are removed to produce a crude peptide. This is then purified and lyophilized to obtain the peptide active pharmaceutical ingredient (API). Given the spatial structure of retaglutide and its large number of amino acids, the sequential coupling process can result in low or no coupling efficiency for individual amino acids, leading to the production of a large number of missing peptides and low retaglutide synthesis efficiency and purity. To overcome the issues of low synthesis efficiency and purity associated with single amino acid coupling, technologies for preparing retaglutide using multiple peptide fragments have been developed. In the prior art, 3, 4 or more peptide fragments are generally used for coupling. Each peptide fragment is prepared by solid-phase synthesis. After synthesis, the peptide fragments are completely cleaved from the resin and purified. The purified peptide fragments are coupled with each other by liquid-phase synthesis to prepare retaglutide. In the synthesis process of retaglutide, the resin accounts for a high proportion of the total material cost. The more peptide fragments are used, the more resin is used, and the higher the cost. In addition, coupling between peptide fragments by liquid-phase synthesis will produce more impurities, and the post-processing process is more complicated than the solid-phase synthesis method, which will also lead to low purity of retaglutide and high synthesis cost.

[0005] Therefore, designing and developing a low-cost, simple synthetic process and high-quality preparation method of retaglutide has important practical significance and industrial value. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing retaglutide and its intermediates with high yield and suitable for industrial production. The preparation method of the present invention can reduce the use of resin in the preparation process, simplify subsequent processing steps, and significantly reduce production costs. In addition, the yield and purity of retaglutide are high, making it suitable for large-scale production.

[0007] The present invention provides a method for preparing retaglutide, wherein the amino acid sequence of the retaglutide is H-Tyr 1 -Aib 2 -Gln 3 -Gly 4-Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-eicosanedioic acid)-Ala 18 -Gln 19 -Aib 20 -Ala 21 -Phe 22 -Ile 23 -Glu 24 -Tyr 25 -Leu 26 -Leu 27 -Glu 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH2, characterized in that the preparation method comprises: Step 1: Prepare the fragment 1-resin of formula I by solid phase synthesis method, Fmoc-Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-eicosanedioic acid)-Ala 18 -Gln 19 -Aib 20 -Ala 21 -Phe 22 -Ile 23 -Glu 24 -Tyr 25 -Leu 26 -Leu 27 -Glu 28 -Gly 29 -Gly 30-Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -resin; Formula I Step 2: Using a solid phase synthesis method, the fragment 2 of formula II is coupled with the fragment 1-resin to obtain retaglutide-resin. Fmoc-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -NH2; Formula II as well as Step 3: cleaving the retaglutide-resin to obtain retaglutide.

[0008] Preferably, the step 1 comprises: using an amino resin as a starting resin, sequentially coupling amino acids and small fragment peptides from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide to obtain the fragment 1-resin, wherein the amino acids and small fragment peptides and the coupling order are as follows: ; The coupling agent in step 1 is selected from Oxyma and DIC, TPTU and DIEA, COMU and DIEA, COMU and TMP, and PyOxim and DIEA.

[0009] Preferably, in the step 1, the coupling agents used for the amino acids or small fragment peptides with a coupling order of 1-1, 1-3, and 1-5 to 1-7 are Oxyma and DIC, the coupling agents used for the small fragment peptides with a coupling order of 1-2 and 1-4 are TPTU and DIEA, the coupling agents used for the amino acids or small fragment peptides with a coupling order of 1-8 to 1-11 and 1-13 to 1-15 are COMU and DIEA, the coupling agents used for the amino acids with a coupling order of 1-12 and 1-18 are COMU and TMP, and the coupling agents used for the amino acids with a coupling order of 1-16 and 1-17 are PyOxim and DIEA.

[0010] Preferably, the step 2 further includes: Step 2-A: Using 2-CTC resin as the starting resin, amino acids and small peptide fragments are sequentially coupled from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide by a solid-phase synthesis method to obtain Fragment 2-resin, and the Fragment 2-resin is cleaved to obtain Fragment 2, wherein the amino acids and small peptide fragments and the coupling order are as follows: ; The coupling agent in step 2-A is selected from Oxyma and DIC, and TPTU and DIEA.

[0011] Preferably, in step 2-A, the coupling agents used for the amino acids in the coupling order of 2-1 and 2-3 to 2-9 are Oxyma and DIC, and the coupling agents used for the small fragment peptides in the coupling order of 2-2 and 2-10 are TPTU and DIEA.

[0012] Preferably, the coupling agent for coupling the fragment 1-resin and the fragment 2 is TPTU and DIEA.

[0013] Preferably, the preparation method comprises: Step 1: (a1) deprotecting the amino resin using a deprotecting agent; (b1) Adding coupling agent to Fmoc-Ser(tBu)-OH to activate amino acid; (c1) adding the activated amino acid obtained in step (b1) to the resin obtained in step (a1) to couple to obtain Fmoc-Ser(tBu)-amide resin; (d1) Repeating the deprotection, amino acid activation, and coupling reaction steps, sequentially coupling the remaining amino acids and the small peptide fragment to obtain fragment 1-resin; The amino acids and small peptide fragments, coupling sequence and coupling agent in step 1 are as follows: ; Step 2: Step 2-A: (a2) deprotecting the 2-CTC resin using a deprotecting agent; (b2) Adding a coupling agent to Fmoc-Leu-OH to activate the amino acid; (c2) adding the activated amino acid obtained in step (b2) to the resin obtained in step (a2) to couple to obtain Fmoc-Leu-ester resin; (d2) Repeating the deprotection, amino acid activation, and coupling reaction steps to sequentially couple the remaining amino acids and the small peptide fragment to obtain fragment 2-resin; (e2) adding the fragment 2-resin obtained in step (d2) to a cleavage solution for cleavage to obtain fragment 2; The amino acids and small peptide fragments, coupling sequence, and coupling agents of step 2-A are as follows: ; Step 2-B: (a3) deprotecting the fragment 1-resin using a deprotecting agent; (b3) adding TPTU and DIEA to the fragment 2 to activate the fragment 2; (c3) coupling the activated fragment 2 obtained in step (b3) with the fragment 1-resin obtained in step (a3) ​​to obtain retaglutide-resin; Step 3: The retaglutide-resin is added to a cleavage solution for cleavage to obtain retaglutide.

[0014] Preferably, the deprotecting agent is PIP / DMF with a concentration of 20%, and the volume ratio of PIP to DMF is 20:80.

[0015] Preferably, the lysis solution in step 2-A and step 3 is a TFA / EDT / Tis / H2O / phenol lysis solution, and the TFA / EDT / Tis / H2O / phenol lysis solution is prepared by the following method: preparing a first solution with a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5, and then adding phenol to the first solution, the mass of the phenol being 2% of the mass of the first solution.

[0016] Preferably, step 2-A and step 3 further include a crystallization step after cracking, and the precipitate used in the crystallization step is selected from MTBE and EA, MTBE and IPAc, and diethyl ether and EA, and the volume ratio of the two components in MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1:1.

[0017] Preferably, the step three further comprises liquid chromatography purification, the chromatography system adopts a reverse phase C18 chromatography column, and the purification comprises: (f-1): cracking, crystallizing, filtering, and drying the retaglutide-resin obtained in step (c3) to obtain a crude retaglutide product, dissolving the crude retaglutide product in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; (f-2): The filtrate was subjected to a first purification, with mobile phase A being a phosphoric acid aqueous solution with a pH of 3 and mobile phase B being acetonitrile, and the qualified fractions exceeding 96% were collected; (f-3): The qualified fractions collected in step (f-2) are purified for the second time, with mobile phase A being sodium acetate solution with a pH of 8.5 and mobile phase B being acetonitrile, and fractions greater than 99.5% are collected.

[0018] Preferably, the resin in step 1 is selected from Rink Amind-AM resin, Rink Amide resin and Rink Amide-MBHA resin, and the degree of substitution of the resin is 0.4-1.0 mmol / g; the degree of substitution of the resin in step 2 is 0.3-1.2 mmol / g.

[0019] Preferably, the solvent in the preparation method is at least one selected from DMF, DMF / DCM=2 / 1 (V / V), DMAC / DMF / DCM=1 / 1 / 1 (V / V / V), and DMF / NPM=1 / 1 (V / V).

[0020] The present invention also provides a method for preparing a retaglutide intermediate, characterized in that the retaglutide intermediate is fragment 2 of formula II, Fmoc-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -NH2 Formula II The preparation method comprises: Using 2-CTC resin as the starting resin, amino acids and small peptide fragments were sequentially coupled from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide by a solid-phase synthesis method to obtain Fragment 2-resin, and the Fragment 2-resin was cleaved to obtain Fragment 2, wherein the amino acids and small peptide fragments and the coupling order are as follows: ; The coupling agent for the coupling reaction is selected from Oxyma and DIC, and TPTU and DIEA.

[0021] Preferably, the coupling agents used for the amino acids with a coupling sequence of 2-1 and 2-3 to 2-9 are Oxyma and DIC, and the coupling agents used for the small fragment peptides with a coupling sequence of 2-2 and 2-10 are TPTU and DIEA.

[0022] The present invention prepares retaglutide by a solid phase reaction between two fragment peptides, namely fragment 1 and fragment 2, wherein fragment 1 is prepared by a solid phase synthesis method and coupled with fragment 2 in the form of fragment 1-resin. Compared with the coupling of fragment 1 and fragment 2 by liquid phase synthesis, the operation is simple, the impurities generated are small, and the post-processing operation is simple, which not only improves the synthesis efficiency and the purity of retaglutide, but also reduces the cost. In addition, fragment 2 is prepared by the synthesis method of the retaglutide intermediate of the present invention, and the purity and yield of the prepared retaglutide intermediate are high. Subsequent reaction with fragment 1-resin can improve the yield and purity of retaglutide. The method for preparing retaglutide and its intermediate of the present invention is particularly suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the HPLC spectrum of fragment 2 of Example 1; Figure 2 This is the HPLC spectrum of the crude retaglutide of Example 1; Figure 3 This is the HPLC spectrum of the pure retaglutide of Example 1; Figure 4 This is the mass spectrum of retaglutide prepared in Example 1; Figure 5 This is the HPLC spectrum of the crude retaglutide of Comparative Example 1; Figure 6 HPLC spectrum of pure retaglutide of Comparative Example 1; Figure 7 This is the HPLC spectrum of the crude product of Retaglutide in Comparative Example 2; Figure 8 This is the HPLC spectrum of pure retaglutide in Comparative Example 2; Figure 9 This is the mass spectrum of the impurities involved in Comparative Example 2. DETAILED DESCRIPTION

[0024] The technical solutions of the present invention are further described below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. The methods used in the present invention are all conventional production methods unless otherwise specified; the raw materials used are all conventional commercially available products unless otherwise specified. In the following examples, the small peptide fragments of the present invention were purchased from Sichuan Tongsheng Biopharmaceutical Co., Ltd.

[0025] The English abbreviations and Chinese meanings of the reagents used in the present invention are shown in Table 1: Table 1 .

[0026] The preparation method of retaglutide of the present invention comprises the steps of: preparing a fragment 1-resin of formula I by a solid phase synthesis method; step 2: coupling a fragment 2 of formula II with the fragment 1-resin by a solid phase synthesis method to obtain retaglutide-resin; and step 3: cleaving the retaglutide-resin to obtain retaglutide. In the prior art, a solid phase synthesis method is generally used to prepare multiple fragments, and then the multiple fragments are cleaved from the resin, and then the multiple fragments are coupled by a liquid phase synthesis method to produce retaglutide. The above-mentioned prior art method requires that each fragment be cleaved from the resin, and after cleavage, it must be purified before it can be put into the liquid phase reaction. In addition, the liquid phase synthesis reaction is more likely to produce impurities. In addition, it is more difficult to separate the target product dissolved in the reaction system from the liquid phase reaction system. After the reaction is completed, it is necessary to go through the steps of extraction, concentration, column purification, pH adjustment, etc. The operation is complicated, many reagents and equipment are used, and the production cost is high. The present invention uses a solid-phase synthesis method to couple fragment 2 with fragment 1-resin. On the one hand, it reduces the steps of cleaving all fragments from the resin and purifying them and the corresponding reagents. On the other hand, since the synthesized retaglutide is connected to the resin, it is relatively simple to separate it from the reaction system, reducing a series of steps such as extraction, concentration, column purification, and the corresponding reagents in the liquid phase reaction. In addition, compared with the use of liquid phase reaction, the solid phase reaction has fewer impurities, which improves the purity and yield of the retaglutide product. In addition, fragment 1 of the present invention is a polypeptide fragment of amino acids 15 to 39, and fragment 2 is a polypeptide fragment of amino acids 1 to 14. When synthesizing retaglutide, only one coupling is performed, avoiding the accumulation of impurities in multiple coupling reactions. In addition, the two fragment peptides of the present invention are coupled between Leu 14 and Asp 15 of retaglutide. The coupling reactivity between fragment 1 and fragment 2 is good, and it is difficult to produce racemic impurities. The product has high purity and high coupling efficiency.

[0027] In a preferred embodiment of the present invention, step 1 comprises: using an amino resin as a starting resin, sequentially coupling amino acids and small fragment peptides from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide to obtain the fragment 1-resin, wherein the amino acids and small fragment peptides and the coupling order are as follows: The coupling agent in step 1 is selected from Oxyma and DIC, TPTU and DIEA, COMU and DIEA, COMU and TMP, and PyOxim and DIEA. Preferably, in step 1, the coupling agents used for the amino acids or small peptide fragments with a coupling order of 1-1, 1-3, and 1-5 to 1-7 are Oxyma and DIC, the coupling agents used for the small peptide fragments with a coupling order of 1-2 and 1-4 are TPTU and DIEA, the coupling agents used for the amino acids or small peptide fragments with a coupling order of 1-8 to 1-11 and 1-13 to 1-15 are COMU and DIEA, the coupling agents used for the amino acids with a coupling order of 1-12 and 1-18 are COMU and TMP, and the coupling agents used for the amino acids with a coupling order of 1-16 and 1-17 are PyOxim and DIEA.

[0028] In a preferred embodiment of the present invention, step 2 further comprises: step 2-A: using 2-CTC resin as the starting resin, sequentially coupling amino acids and small peptide fragments from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide by a solid-phase synthesis method to obtain fragment 2-resin, and cleaving the fragment 2-resin to obtain the fragment 2, wherein the amino acids and small peptide fragments and the coupling order are as follows: ; The coupling agent in step 2-A is selected from Oxyma and DIC, and TPTU and DIEA. Preferably, in step 2-A, the coupling agents used for the amino acids in the coupling order of 2-1 and 2-3 to 2-9 are Oxyma and DIC, and the coupling agents used for the small peptide fragments in the coupling order of 2-2 and 2-10 are TPTU and DIEA.

[0029] In a preferred embodiment of the present invention, the preparation method comprises: Step 1: (a1) deprotecting the amino resin using a deprotecting agent; (b1) Adding coupling agent to Fmoc-Ser(tBu)-OH to activate amino acid; (c1) adding the activated amino acid obtained in step (b1) to the resin obtained in step (a1) to couple to obtain Fmoc-Ser(tBu)-amide resin; (d1) Repeating the deprotection, amino acid activation, and coupling reaction steps, sequentially coupling the remaining amino acids and the small peptide fragment to obtain fragment 1-resin; The amino acids and small peptide fragments, coupling sequence and coupling agent in step 1 are as follows: ; Step 2: Step 2-A: (a2) deprotecting the 2-CTC resin using a deprotecting agent; (b2) Adding a coupling agent to Fmoc-Leu-OH to activate the amino acid; (c2) adding the activated amino acid obtained in step (b2) to the resin obtained in step (a2) to couple to obtain Fmoc-Leu-ester resin; (d2) Repeating the deprotection, amino acid activation, and coupling reaction steps to sequentially couple the remaining amino acids and the small peptide fragment to obtain fragment 2-resin; (e2) adding the fragment 2-resin obtained in step (d2) to a cleavage solution for cleavage to obtain fragment 2; The amino acids and small peptide fragments, coupling sequence and coupling agents of step 2-A are as follows: ; Step 2-B: (a3) deprotecting the fragment 1-resin using a deprotecting agent; (b3) adding TPTU and DIEA to the fragment 2 to activate the fragment 2; (c3) coupling the activated fragment 2 obtained in step (b3) with the fragment 1-resin obtained in step (a3) ​​to obtain retaglutide-resin; Step 3: The retaglutide-resin is added to a cleavage solution for cleavage to obtain retaglutide.

[0030] The present invention uses a specific small fragment peptide to prepare fragment 1-resin and fragment 2, which reduces the total number of coupling reactions when preparing the two fragments, avoids the problem of missing amino acids in the product due to the inability to couple or the high difficulty of coupling using a single amino acid at certain sites, improves the synthesis efficiency, and reduces the accumulation of impurities generated by multiple couplings. In addition, depending on the difference in carboxyl components and amino components, the condensation reaction has different levels of difficulty and speed. For example, Pro with a secondary amine group is an amino acid that is more difficult to condense. In addition to the influence of the structure of the amino acids participating in the reaction, factors such as the length of the assembled peptide chain and the hydrophobicity of the peptide chain also have an important influence on the condensation reaction. The present invention improves the synthesis efficiency by selecting a specific coupling agent in each step of the coupling reaction. In addition, in the above-mentioned preferred embodiment, the method of the present invention only needs to prepare two fragments, namely fragment 1 and fragment 2, which can reduce the use of resin in the solid phase synthesis method and reduce production costs. In addition, the preparation method of the retaglutide intermediate fragment 2 of the present invention has high purity and yield of the obtained fragment 2 and is suitable for mass production.

[0031] In a preferred embodiment of the present invention, the deprotecting agent is PIP / DMF with a concentration of 20%, and the volume ratio of PIP to DMF is 20:80.

[0032] In a preferred embodiment of the present invention, the lysis solution used is a TFA / EDT / Tis / H2O / phenol lysis solution. The lysis solution is prepared by preparing a first solution having a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5, and then adding phenol to the first solution, wherein the mass of the phenol is 2% of the mass of the first solution. For example, if 90 mL of TFA / EDT / Tis / H2O is prepared at a volume ratio of 90 / 5 / 2.5 / 2.5 and the weight is 100 grams, 2 grams of phenol are added. Phenol acts as an antioxidant, preventing oxidation of Tyr and Trp.

[0033] In a preferred embodiment of the present invention, step 2-A and step 3 further include a crystallization step after cleavage, and the precipitate used in the crystallization step is selected from MTBE and EA, MTBE and IPAc, and diethyl ether and EA, and the volume ratio of the two components in MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1:1.

[0034] In a preferred embodiment of the present invention, the method of the present invention further comprises liquid chromatography purification, the chromatography system adopts a reverse phase C18 chromatography column, and the purification comprises: Dissolving: cracking, crystallizing, filtering, and drying the retaglutide-resin prepared in step (c3) to obtain a crude retaglutide product, dissolving the crude retaglutide product in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; First purification: the filtrate was subjected to first purification, mobile phase A was a phosphoric acid aqueous solution with a pH of 3, mobile phase B was acetonitrile, and qualified fractions with a rate of more than 96% were collected; Second purification: The collected qualified fractions were subjected to a second purification, with mobile phase A being sodium acetate solution with a pH of 8.5 and mobile phase B being acetonitrile, and fractions with a concentration greater than 99.5% were collected.

[0035] Through the above purification method, a high-purity pure product of retaglutide can be obtained.

[0036] In a preferred embodiment of the present invention, the starting resin in step 1 includes Rink Amind-AM resin, Rink Amide resin, or Rink Amide-MBHA resin, the degree of substitution of the starting resin is 0.4-1.0 mmol / g, and the degree of substitution of the resin in step 2 is 0.3-1.2 mmol / g.

[0037] In a preferred embodiment of the present invention, the solvent in the method of the present invention is at least one selected from DMF, DMF / DCM=2 / 1 (V / V), DMAC / DMF / DCM=1 / 1 / 1 (V / V / V), and DMF / NPM=1 / 1 (V / V).

[0038] Example 1: Preparation of Retaglutide Step 1: Preparation of fragment 1-resin (a1) 30.0 g of Rink Amide-AM resin (degree of substitution 0.58 mmol / g) was placed in a solid-phase reactor and swelled with DMF solution for 30 minutes. The mixture was filtered and deprotected with 20% PIP / DMF solution (PIP:DMF volume ratio 20:80) for 10 minutes. The mixture was filtered and deprotected with 20% PIP / DMF solution for another 10 minutes. The mixture was filtered and washed with DMF six times, stirring for 3 minutes each time.

[0039] (b1) Add 300 ml of DMF to another reaction bottle (hereinafter referred to as the "amino acid activation reaction bottle"), add 20.01 g of Fmoc-Ser(tBu)-OH and 7.42 g of Oxyma to the amino acid activation reaction bottle and stir to dissolve, then slowly add 6.59 ml of DIC (Suzhou Haofan Biotechnology Co., Ltd.) and stir for at least 5 minutes to activate the amino acid.

[0040] (c1) The activated amino acid solution was added to a solid phase reactor and reacted for 2 hours. A small amount of resin was taken for detection with ninhydrin. The resin was colorless and filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0041] (d1) Add 20% PIP / DMF to the resin obtained in step (c1) for deprotection for 10 minutes, filter, add 20% PIP / DMF again for deprotection for 10 minutes, filter, and wash with DMF six times, stirring for 3 minutes each time.

[0042] (e1) In an amino acid activation reaction flask, add 300 mL of a DMF / DCM (2 / 1 by volume) mixed solvent, add 18.50 g of Fmoc-Pro-Pro-Pro-OH, and dissolve and clarify in 10.34 g of TPTU (Suzhou Haofan Biotechnology Co., Ltd.). Cool to 15°C, then dropwise add 6.75 mL of DIEA (Annage (Shanghai) Pharmaceutical Chemical Co., Ltd.). After stirring and activating at 15°C for at least 10 minutes, add the mixture to a solid-phase reactor and stir for 2 hours. A small amount of the resin was tested with ninhydrin, indicating that the resin was colorless. Filter and wash twice with DMF to obtain Fmoc-Pro-Pro-Pro-Ser(tBu)-amide resin.

[0043] The remaining amino acids and small fragment peptides were coupled in sequence to obtain fragment 1-resin. The amino acids and small fragment peptides, coupling agents, and solvents used in the coupling process are shown in Table 2 below: Table 2 .

[0044] The temperature for activating amino acids or small molecule peptides using coupling agents Oxyma and DIC, COMU and DIEA, TPTU and DIEA, COMU and DIEA, PyOxim and DIEA, and COMU and TMP is 10-20°C.

[0045] Step 2-A: Preparation of Fragment 2 (a2) 25.0 g of 2-CTC resin (degree of substitution 0.98 mmol / g) was placed in a solid-phase reactor and swelled with DMF solution for 30 min. The mixture was filtered and deprotected by adding 20% ​​PIP / DMF (PIP:DMF volume ratio 20:80) for 10 min. The mixture was filtered and deprotected by adding 20% ​​PIP / DMF again for 10 min. The mixture was filtered and washed with DMF six times, stirring for 3 min each time.

[0046] (b2) Add 300 ml of DMF to another reaction bottle (hereinafter referred to as the "amino acid activation reaction bottle"), add 25.97 g of Fmoc-Leu-OH and 10.44 g of Oxyma to the amino acid activation reaction bottle and stir to dissolve, then slowly add 9.28 ml of DIC (Suzhou Haofan Biotechnology Co., Ltd.) and stir for at least 5 minutes to activate the amino acid.

[0047] (c2) The activated amino acid solution was added to a solid phase reactor and reacted for 2 hours. A small amount of resin was taken for detection with ninhydrin. The resin was colorless and filtered and washed twice with DMF to obtain Fmoc-Leu-ester resin.

[0048] (d2) Add 20% PIP / DMF to the resin obtained in step (c2) for deprotection for 10 minutes, filter, add 20% PIP / DMF again for deprotection for 10 minutes, filter, and wash with DMF six times, stirring for 3 minutes each time.

[0049] In an amino acid activation reaction flask, add 300 ml of a 2 / 1 DMF / DCM mixed solvent, add 37.92 g of Fmoc-Ile-αMeLeu-OH, and dissolve and clarify in 14.56 g of TPTU (Suzhou Haofan Biotechnology Co., Ltd.). Cool to 15°C, then dropwise add 9.50 ml of DIEA (Annaiji (Shanghai) Pharmaceutical Chemical Co., Ltd.). After stirring and activating at 15°C for at least 10 minutes, add the mixture to a solid-phase reactor and stir for 2 hours. A small amount of the resin was tested with ninhydrin, indicating it was colorless. Filter and wash twice with DMF to obtain Fmoc-Ile-αMeLeu-Leu-ester resin.

[0050] The remaining amino acids and small peptide fragments were coupled in sequence. The amino acids and small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 3 below: Table 3 .

[0051] The temperature for activating amino acids or small molecule peptides using coupling agents Oxyma and DIC, COMU and DIEA, and TPTU and DIEA is 10-20°C.

[0052] After all amino acids and small peptide fragments were coupled, methanol was added for shrinkage three times, each time for 10 minutes, filtered, and vacuum-dried for 4 hours. Fragment 2-resin was collected and weighed 85.5 g, of which fragment 2 was 85.5-25=60.5 g. The theoretical yield of fragment 2 was: 25×(0.98 / 1000)×2324.40 (molecular weight of fragment 2)=56.95 g, and the yield of fragment 2 was 60.5 / 56.95=106.2%.

[0053] (e2) Prepare the cleavage solution in the following manner: prepare a first solution with a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5, then add phenol to the first solution, the mass of the phenol being 2% of the mass of the first solution. Add the fragment 2-resin prepared in step (d2) to the cleavage solution, the amount of the cleavage solution being 10 times the weight of the fragment 2-resin, stir for 3 hours, filter, collect the filtrate, cool to below 0°C, and slowly add the filtrate to a mixed system with a volume ratio of MTBE / EA=1 / 1 for precipitation, the amount of the mixed system being 60 times the weight of the fragment 2-resin, filter, wash the filter cake with MTBE, and dry the solid in an oven for 4 hours to obtain 52g of fragment 2 solid, with a yield of 52 / 56.95×100%=91.315%. The HPLC spectrum of fragment 2 is as follows: Figure 1 As shown, the HPLC purity was 93.80%.

[0054] Step 2-B: (a3) Add 20% PIP / DMF (PIP:DMF volume ratio is 20:80) to the above fragment 1-resin for deprotection for 10 minutes, filter with suction, add 20% PIP / DMF again for deprotection for 10 minutes, filter with suction, and wash with DMF six times, stirring for 3 minutes each time.

[0055] (b3) The solid fragment 2 obtained in step 2 above was dissolved in 300 mL of a mixed solution of DMF / NPM (volume ratio = 1 / 1), 6.7 g of TPTU was added to dissolve and clarify, the mixture was cooled to 15°C, 4.5 mL of DIEA was added, and the mixture was stirred at 15°C for at least 10 minutes to activate fragment 2.

[0056] (c3) adding the activated fragment 2 solid obtained in step (b3) to the resin obtained in step (a3) ​​and reacting for 6 h. The resulting product was washed three times with DMF and three times with methanol to collect 135 g of retaglutide resin; Step 3: The lysis solution was prepared as in step (e2), and the retaglutide resin in the above step (c3) was added to the lysis solution, the amount of the lysis solution was 10 times the weight of the retaglutide resin, and the mixture was stirred for 3 hours. The mixture was filtered and the filtrate was collected. The temperature was lowered to below 0°C, and the filtrate was slowly added to a mixed system with a volume ratio of MTBE / EA = 1 / 1 for precipitation. The amount of the mixed system was 60 times the weight of the retaglutide resin. The mixture was filtered, the filter cake was washed with MTBE, and the solid was dried in an oven for 4 hours to obtain 75 g of crude retaglutide. The theoretical yield of crude retaglutide was: 30 × (0.58 / 1000) × 4731.42 (molecular weight of retaglutide) = 82.33 g, and the yield was: 75 / 82.33 × 100% = 91.1%. The HPLC spectrum of crude retaglutide was as shown below. Figure 2 As shown, the HPLC purity is 89.54%, wherein the purity is the area ratio in the HPLC spectrum. The chromatographic detection is performed using the following method: Liquid chromatograph: Waters 2695 Separations Module; Specifications: UV (ALLLANCE E2695 / ACQUITY Arc); Column: Kinetex 2.6 μm C18 100 Å 150 x 4.6 mm; Column temperature: 30°C; Detection wavelength: 210nm; Injection volume: 10 μL; Flow rate: 0.7 mL / min; Mobile phase A: Weigh 18.4 g of ammonium dihydrogen phosphate, dissolve in 1600 g of ultrapure water, adjust the pH to 3.6 with phosphoric acid, then add 150 g of acetonitrile and 200 g of ultrapure water and mix well. Mobile phase B: Weigh 930 g of acetonitrile, 310 g of isopropanol, and 400 g of ultrapure water and mix well; The elution gradient is shown in Table 4 below: Table 4 .

[0057] (e3) The crude retaglutide was purified twice using a liquid chromatography system. The HPLC instrument and its parameters used in the purification process are shown in Table 5 below: Table 5 .

[0058] The purification step comprises: (f-1): dissolving the crude retaglutide in 30% acetonitrile water by volume, filtering, and collecting the filtrate; (f-2): The filtrate was purified for the first time, mobile phase A was a phosphoric acid aqueous solution with a pH of 3, mobile phase B was acetonitrile, and the elution gradient was as shown in Table 6 below: Table 6 .

[0059] Collect more than 96% qualified fractions; (f-3): The qualified fractions collected in step (f-2) were purified for the second time. Mobile phase A was sodium acetate solution with a pH of 8.5, and mobile phase B was acetonitrile. The elution gradient was as shown in Table 7 below: Table 7 .

[0060] Collect more than 99.5% qualified fractions.

[0061] Afterwards, the qualified fractions were salt-exchanged using a multifunctional membrane experimental device (RNM-18G, Hangzhou Reina Membrane Engineering Co., Ltd.) with a pore size of 1-2 nm and a pressure of 1.5 MPa. The concentrate was collected and lyophilized to obtain 63 g of pure retaglutide, with a yield of 63 / 75 × 100% = 84%. The HPLC chromatogram of pure retaglutide is shown below. Figure 3 As shown, the HPLC purity was 99.89%.

[0062] The obtained pure retaglutide was subjected to mass spectrometry analysis using a liquid chromatography-mass spectrometry instrument. The instrument and its parameters were as follows: Instrument name: Ultra-high performance liquid chromatography; Brand model: ACQUIIY UPLC H-CLASS PIUS; Supplier: Waters Technology (Shanghai) Co., Ltd. Column: 1.7 μm PEPTIDE XB-C18 10C LC Column 150 x 2.1 nm; Column temperature: 30°C; Injection volume: 10 μL; Flow rate: 0.4 mL / min; Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; The elution gradient is shown in Table 8 below: Table 8 .

[0063] The mass spectrum of the pure retaglutide is as follows Figure 4 As shown in Figure 2, the precise molecular weight of retaglutide is 4728.47, and its mass-to-charge ratio should be 4728.47 / 4+1=1183.12, which is consistent with Figure 4 The detection value in SEQ ID NO: 1183.77 corresponds to 1183.77, indicating that the product prepared in Example 1 is retaglutide.

[0064] Comparative Example 1 30.0 g of Rink Amide-AM resin (degree of substitution 0.58 mmol / g) was placed in a solid phase reactor, and DMF solution was added to swell for 30 minutes. The mixture was filtered, and 20% PIP / DMF (the volume ratio of PIP:DMF was 20%:80%) was added for deprotection for 10 minutes. The mixture was filtered, and 20% PIP / DMF was added again for deprotection for 10 minutes. The mixture was filtered, and washed with DMF 6 times, stirring for 3 minutes each time.

[0065] Add 300 ml of DMF to another reaction bottle (hereinafter referred to as the "amino acid activation reaction bottle"), add 20.01 g of Fmoc-Ser(tBu)-OH and 7.42 g of Oxyma to the amino acid activation reaction bottle and stir to dissolve, then slowly add 6.59 ml of DIC and stir for at least 5 minutes to activate the amino acid.

[0066] The activated amino acid solution was added to a solid phase reactor and reacted for 2 hours. A small amount of resin was taken for ninhydrin detection. The resin was colorless and filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0067] Deprotect the Fmoc-Ser(tBu)-amide resin by adding 20% ​​PIP / DMF for 10 minutes, filter, and then add 20% PIP / DMF for another 10 minutes. Filter and wash with DMF six times, stirring for 3 minutes each time. To activate the amino acid, add 300 mL of a 2 / 1 DMF / DCM mixture, add 18.50 g of Fmoc-Pro-Pro-Pro-OH, and dissolve and clarify with 10.34 g of TPTU. Cool to 15°C, then dropwise add 6.75 mL of DIEA. After activation with stirring at 15°C for at least 10 minutes, add the resin to the solid-phase reactor and stir for 2 hours. A small amount of the resin was tested with ninhydrin, indicating it was colorless. Filter and wash twice with DMF to obtain Fmoc-Pro-Pro-Pro-Ser(tBu)-amide resin.

[0068] The remaining amino acids and small peptide fragments were coupled in sequence. The amino acids and small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 9 below: Table 9 .

[0069] The temperature for activating amino acids or small molecule peptides using coupling agents Oxyma and DIC, COMU and DIEA, TPTU and DIEA, COMU and DIEA, PyOxim and DIEA, and COMU and TMP is 10-20°C.

[0070] After the coupling, 132 g of retaglutide resin was obtained by washing with methanol. The retaglutide resin was cleaved in the same manner as in Example 1 to obtain 71 g of crude retaglutide. The theoretical yield of retaglutide was 30 × (0.58 / 1000) × 4731.42 = 82.33 g, and the yield was 71 / 82.33 × 100% = 86.23%. The HPLC spectrum of the crude retaglutide is shown in FIG. Figure 5 As shown, the HPLC purity of the crude product was 77.97%; the crude retaglutide was purified twice in the same manner as in Example 1 to obtain 58 g of pure product with a yield of 58 / 71×100%=81.7%. The HPLC spectrum of the pure product is shown in FIG. Figure 6 As shown, the HPLC purity of the pure product is 99.55%.

[0071] Comparative Example 2 30.0 g of Rink Amide-AM resin (degree of substitution 0.58 mmol / g) was placed in a solid phase reactor, and DMF solution was added to swell for 30 minutes. The mixture was filtered, and 20% PIP / DMF (the volume ratio of PIP:DMF was 20%:80%) was added for deprotection for 10 minutes. The mixture was filtered, and 20% PIP / DMF was added again for deprotection for 10 minutes. The mixture was filtered, and washed with DMF 6 times, stirring for 3 minutes each time.

[0072] Add 300 ml of DMF to another reaction bottle (hereinafter referred to as the "amino acid activation reaction bottle"), add 20.01 g of Fmoc-Ser(tBu)-OH and 7.42 g of Oxyma to the amino acid activation reaction bottle and stir to dissolve, then slowly add 8.08 ml of DIC and stir for at least 5 minutes to activate the amino acid.

[0073] The activated amino acid solution was added to a solid phase reactor and reacted for 2 hours. A small amount of resin was taken for ninhydrin detection. The resin was colorless and filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0074] Add 20% PIP / DMF to the Fmoc-Ser(tBu)-amide resin for deprotection for 10 minutes, filter, and then add 20% PIP / DMF for deprotection again for 10 minutes. Filter and wash with DMF six times, stirring for 3 minutes each time. To the amino acid activation reaction flask, add 300 ml of DMF. Add 17.61 g of Fmoc-Pro-OH and 7.42 g of Oxyma and stir to dissolve. Slowly add 8.08 ml of DIC and stir for at least 5 minutes to activate the amino acid. Add the activated amino acid to the solid phase reactor and stir for 2 hours. A small amount of the resin was tested with ninhydrin, indicating that the resin was colorless. Filter and wash twice with DMF to obtain the Fmoc-Pro-Ser(tBu)-amide resin.

[0075] The remaining amino acids and small peptide fragments were coupled in sequence. The amino acids and small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 10 below: Table 10

[0076] After coupling, 133 g of retaglutide resin was obtained by washing with methanol.

[0077] The same method as in Example 1 was used to cleave the retaglutide resin to obtain 70 g of crude retaglutide. The theoretical yield of retaglutide was 30 × (0.58 / 1000) × 4731.42 = 82.33 g, and the yield was 70 / 82.33 × 100% = 85.0%. The HPLC spectrum of the crude retaglutide was as follows: Figure 7 As shown in FIG, the crude product HPLC purity was 70.00%. The crude product of retaglutide was purified twice in the same manner as in Example 1 to obtain 52 g of pure product with a yield of 52 / 70×100%=74.3%. The HPLC spectrum of the pure product is shown in FIG. Figure 8 As shown in the figure, the HPLC purity of the pure product is 98.96%. The sequential coupling of single amino acids is prone to produce obvious missing impurities. Figure 9 As shown, the product of Comparative Example 2 contains an impurity with a molecular weight of 1155.31. As mentioned above, the detected molecular weight of retaglutide by mass spectrometry is 1183.77. Compared with retaglutide, the molecular weight of the impurity is reduced by (1183.77-1155.31)×4=113.84. It can be seen that the impurity is a deleted peptide with a missing amino acid with a molecular weight of 113.84.

[0078] It can be seen from the above examples and comparative examples that in Comparative Example 1, a single amino acid and a small fragment peptide are sequentially coupled to form retaglutide, while in the present invention, the same single amino acid and small fragment are first coupled to form fragment 1-resin and fragment 2, and then fragment 2 is coupled to fragment 1 on fragment 1-resin. Although the single amino acid and small fragment peptide used in Comparative Example 1 are the same as those in Example 1, the coupling steps in Comparative Example 1 are many, time-consuming, and prone to impurities. The yield and purity of the crude retaglutide and pure retaglutide in Comparative Example 1 are both lower than those in Example 1. The purity of the crude product in Comparative Example 1 is low, which is not conducive to purification and easily leads to excessively high single impurities after purification. In Comparative Example 2, except for Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-OH, the other reaction steps are all single amino acids for coupling. There are many coupling steps, poor reaction effect, long reaction time, high raw material consumption, and easy generation of missing peptide impurities, resulting in yield loss and increased purification difficulty. The yield and purity of the crude retaglutide and pure retaglutide in Comparative Example 2 were lower than those in Example 1. The preparation method of the present invention consumes less resin, has fewer reaction steps, simplifies subsequent treatment, greatly reduces costs, and improves the purity and yield of the product, making it more suitable for industrial production.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing retaglutide, wherein the amino acid sequence of retaglutide is H-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-eicosanedioic acid)-Ala 18 -Gln 19 -Aib 20 -Ala 21 -Phe 22 -Ile 23 -Glu 24 -Tyr 25 -Leu 26 -Leu 27 -Glu 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH2, characterized in that The preparation method comprises: Step 1: Prepare the fragment 1-resin of formula I by solid phase synthesis method, Fmoc-Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-eicosanedioicacid)-Ala 18 -Gln 19 -Aib 20 -Ala 21 -Phe 22 -Ile 23 -Glu 24 -Tyr 25 -Leu 26 -Leu 27 -Glu 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -Resin; Formula I Step 2: Using a solid phase synthesis method, the fragment 2 of formula II is coupled with the fragment 1-resin to obtain retaglutide-resin. Fmoc-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -NH2; Formula II as well as Step 3: cleaving the retaglutide-resin to obtain retaglutide.

2. The preparation method according to claim 1, characterized in that The step 1 comprises: using amino resin as the starting resin, sequentially coupling amino acids and small fragment peptides from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide to obtain the fragment 1-resin, wherein the amino acids and small fragment peptides and the coupling order are as follows: ； The coupling agent in step 1 is selected from Oxyma and DIC, TPTU and DIEA, COMU and DIEA, COMU and TMP, and PyOxim and DIEA.

3. The preparation method according to claim 2, characterized in that In the step 1, the coupling agents used for the amino acids or small fragment peptides with a coupling order of 1-1, 1-3, and 1-5 to 1-7 are Oxyma and DIC, the coupling agents used for the small fragment peptides with a coupling order of 1-2 and 1-4 are TPTU and DIEA, the coupling agents used for the amino acids or small fragment peptides with a coupling order of 1-8 to 1-11 and 1-13 to 1-15 are COMU and DIEA, the coupling agents used for the amino acids with a coupling order of 1-12 and 1-18 are COMU and TMP, and the coupling agents used for the amino acids with a coupling order of 1-16 and 1-17 are PyOxim and DIEA.

4. The preparation method according to claim 1, characterized in that The second step also includes: Step 2-A: Using 2-CTC resin as the starting resin, amino acids and small peptide fragments are sequentially coupled from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide by a solid-phase synthesis method to obtain Fragment 2-resin, and the Fragment 2-resin is cleaved to obtain Fragment 2, wherein the amino acids and small peptide fragments and the coupling order are as follows: ； The coupling agent in step 2-A is selected from Oxyma and DIC, and TPTU and DIEA.

5. The preparation method according to claim 4, characterized in that In step 2-A, the coupling agents used for coupling the amino acids in the order of 2-1 and 2-3 to 2-9 are Oxyma and DIC, and the coupling agents used for coupling the small fragment peptides in the order of 2-2 and 2-10 are TPTU and DIEA.

6. The preparation method according to claim 1, characterized in that The coupling agents for coupling the fragment 1-resin and the fragment 2 are TPTU and DIEA.

7. The preparation method according to claim 1, characterized in that The preparation method comprises: Step 1: (a1) deprotecting the amino resin using a deprotecting agent; (b1) Adding coupling agent to Fmoc-Ser(tBu)-OH to activate amino acid; (c1) adding the activated amino acid obtained in step (b1) to the resin obtained in step (a1) to couple to obtain Fmoc-Ser(tBu)-amide resin; (d1) Repeating the deprotection, amino acid activation, and coupling reaction steps, sequentially coupling the remaining amino acids and the small peptide fragment to obtain fragment 1-resin; The amino acids and small peptide fragments, coupling sequence and coupling agent in step 1 are as follows: ； Step 2: Step 2-A: (a2) deprotecting the 2-CTC resin using a deprotecting agent; (b2) Adding a coupling agent to Fmoc-Leu-OH to activate the amino acid; (c2) adding the activated amino acid obtained in step (b2) to the resin obtained in step (a2) to couple to obtain Fmoc-Leu-ester resin; (d2) Repeating the deprotection, amino acid activation, and coupling reaction steps, sequentially coupling the remaining amino acids and the small peptide fragment to obtain fragment 2-resin; (e2) adding the fragment 2-resin obtained in step (d2) to a cleavage solution for cleavage to obtain fragment 2; The amino acids and small peptide fragments, coupling sequence, and coupling agents of step 2-A are as follows: ； Step 2-B: (a3) deprotecting the fragment 1-resin using a deprotecting agent; (b3) adding TPTU and DIEA to the fragment 2 to activate the fragment 2; (c3) coupling the activated fragment 2 obtained in step (b3) with the fragment 1-resin obtained in step (a3) ​​to obtain retaglutide-resin; Step 3: The retaglutide-resin is added to a cleavage solution for cleavage to obtain retaglutide.

8. The preparation method according to claim 7, characterized in that The deprotecting agent is PIP / DMF with a concentration of 20%, and the volume ratio of PIP to DMF is 20:

80.

9. The preparation method according to claim 7, characterized in that The lysis solution in step 2-A and step 3 is a TFA / EDT / Tis / H2O / phenol lysis solution, and the TFA / EDT / Tis / H2O / phenol lysis solution is prepared by the following method: preparing a first solution with a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5, and then adding phenol to the first solution, wherein the mass of the phenol is 2% of the mass of the first solution.

10. The preparation method according to claim 7, characterized in that The step 2-A and step 3 also include a crystallization step after cracking, and the precipitate used in the crystallization step is selected from MTBE and EA, MTBE and IPAc, and diethyl ether and EA, and the volume ratio of the two components in the MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1:

1.

11. The preparation method according to claim 7, characterized in that The step three further includes liquid chromatography purification, wherein the chromatography system uses a reverse phase C18 chromatography column, and the purification includes: (f-1): cracking, crystallizing, filtering, and drying the retaglutide-resin obtained in step (c3) to obtain a crude retaglutide product, dissolving the crude retaglutide product in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; (f-2): The filtrate was subjected to a first purification, with mobile phase A being a phosphoric acid aqueous solution with a pH of 3 and mobile phase B being acetonitrile, and the qualified fractions exceeding 96% were collected; (f-3): The qualified fractions collected in step (f-2) are purified for the second time, with mobile phase A being sodium acetate solution with a pH of 8.5 and mobile phase B being acetonitrile, and fractions greater than 99.5% are collected.

12. The preparation method according to any one of claims 1 to 11, characterized in that The resin in step 1 is selected from Rink Amind-AM resin, Rink Amide resin and Rink Amide-MBHA resin, and the degree of substitution of the resin is 0.4-1.0 mmol / g; the degree of substitution of the resin in step 2 is 0.3-1.2 mmol / g.

13. The preparation method according to any one of claims 1 to 11, characterized in that The solvent in the preparation method is at least one selected from DMF, DMF / DCM=2 / 1 (V / V), DMAC / DMF / DCM=1 / 1 / 1 (V / V / V), and DMF / NPM=1 / 1 (V / V).

14. A method for preparing a retaglutide intermediate, characterized in that: The retaglutide intermediate is fragment 2 of formula II, Fmoc-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -NH2 of formula II The preparation method comprises: Using 2-CTC resin as the starting resin, amino acids and small peptide fragments were sequentially coupled from the N-terminus to the C-terminus according to the amino acid sequence of retaglutide by a solid-phase synthesis method to obtain Fragment 2-resin, and the Fragment 2-resin was cleaved to obtain Fragment 2, wherein the amino acids and small peptide fragments and the coupling order are as follows: ； The coupling agent for the coupling reaction is selected from Oxyma and DIC, and TPTU and DIEA.

15. The preparation method according to claim 14, characterized in that The coupling agents used for the amino acids with coupling sequences of 2-1 and 2-3 to 2-9 were Oxyma and DIC, and the coupling agents used for the small fragment peptides with coupling sequences of 2-2 and 2-10 were TPTU and DIEA.

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