A method for preparing retaliglutide and its intermediates

Abstract

This invention relates to the field of polypeptide drug preparation techniques, and provides a method for preparing retaglutide and a method for preparing retaglutide intermediates. The method for preparing retaglutide includes preparing fragment 1-resin using a solid-phase synthesis method, followed by coupling fragment 2 to fragment 1-resin using a solid-phase synthesis method to obtain retaglutide-resin, which is then cleaved to obtain retaglutide. Fragment 1 is a polypeptide fragment of amino acids 15-39, and fragment 2 is a polypeptide fragment of amino acids 1-14. The preparation method of this invention can significantly reduce the production cost of retaglutide, with fewer synthesis steps, simpler subsequent processing, and improved purity and yield.

Description

Technical Field

[0001] This invention belongs to the technical field of polypeptide drug preparation methods, and specifically relates to the preparation method of retaglutide and its intermediates. Background Technology

[0002] Retatrutide, an active compound announced by Eli Lilly in June 2023, is a triple (GLP-1R / GCGR / GIPR) agonist based on the GLP / GIP dual receptor agonist Tirzepatide, utilizing a hybrid glucagonGcg-related structure. It is being developed as a drug for the treatment of obesity and type 2 diabetes. This active compound activates receptor agonists in the body, helping to control blood sugar and manage weight loss.

[0003] The structure of retaglutide is as follows:

[0004] 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 -Pro36 -Pro 37 -Pro 38 -Ser 39 -NH2.

[0005] Currently, peptide synthesis methods generally include sequential coupling of single amino acids or coupling of multiple peptide fragments. Sequential coupling of single amino acids involves solid-phase synthesis, where amino acids are coupled one by one onto an amino resin. Starting from the amino terminus of the peptide, the first amino acid to be decarboxylated is attached to the resin, followed by decarboxylation of the amino group. This process is repeated sequentially until amino acid assembly is complete. The peptide is then cleaved from the resin, and all protecting groups are removed to obtain the crude peptide. After purification and lyophilization, the peptide active pharmaceutical ingredient (API) is obtained. Considering the spatial structure of retaglutide and its large number of amino acids, the coupling efficiency of some amino acids is low or they may not couple at all during sequential coupling, easily resulting in a large number of missing peptides, leading to low synthesis efficiency and purity. To avoid the low synthesis efficiency and purity issues caused by single-amino acid coupling, technologies for preparing retaglutide using multiple peptide fragment coupling have been developed. Current technologies commonly employ the coupling of three, four, or more peptide fragments. Each fragment is prepared using a solid-phase synthesis method. After synthesis, all peptide fragments are cleaved from the resin and purified. The purified peptide fragments are then coupled together using a liquid-phase synthesis method to prepare ratalutide. In the synthesis of ratalutide, resin accounts for a significant portion of the overall material cost; the more peptide fragments used, the more resin is required, resulting in higher costs. Furthermore, coupling peptide fragments via liquid-phase synthesis introduces more impurities, and the post-processing is more complex than with solid-phase synthesis, leading to lower purity and higher synthesis costs for ratalutide.

[0006] Therefore, designing and developing a low-cost, simple synthetic process and high-quality preparation method for retaglutide has significant practical and industrial value. Summary of the Invention

[0007] To address the shortcomings of the prior art, this invention provides a high-yield method for preparing retaglutide and its intermediates suitable for industrial production. The preparation method of this invention reduces the use of resin in the preparation process and simplifies subsequent processing steps, significantly reducing production costs. Furthermore, the retaglutide has high yield and purity, making it suitable for large-scale production.

[0008] This invention provides 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 includes:

[0009] Step 1: Prepare fragment 1-resin of formula I using a solid-state synthesis method.

[0010] 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 -Glu28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 - Resin type I;

[0011] Step 2: Using a solid-phase synthesis method, fragment 2 of Formula II is coupled with fragment 1-resin to obtain retaliglutide-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; and

[0012] Step 3: Cleavage the ratalutide-resin to obtain ratalutide.

[0013] Preferably, step one includes: using an amino resin as the starting resin, sequentially coupling amino acids and small peptide fragments according to the amino acid sequence of the retaglutide from the N-terminus to the C-terminus to obtain fragment 1-resin, wherein the amino acids and small peptide fragments and the coupling sequence are as follows:

[0014] ;

[0015] The coupling agent in step one is selected from Oxyma and DIC, TPTU and DIEA, COMU and DIEA, COMU and TMP, and PyOxim and DIEA.

[0016] Preferably, in step one, the coupling agents used for amino acids or small peptide fragments with coupling sequences of 1-1, 1-3, and 1-5~1-7 are Oxyma and DIC; the coupling agents used for small peptide fragments with coupling sequences of 1-2 and 1-4 are TPTU and DIEA; the coupling agents used for amino acids or small peptide fragments with coupling sequences of 1-8~1-11 and 1-13~1-15 are COMU and DIEA; the coupling agents used for amino acids with coupling sequences of 1-12 and 1-18 are COMU and TMP; and the coupling agents used for amino acids with coupling sequences of 1-16 and 1-17 are PyOxim and DIEA.

[0017] Preferably, step two further includes:

[0018] 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 the said retaliglutide using a solid-phase synthesis method to obtain fragment 2-resin. The fragment 2-resin is then cleaved to obtain fragment 2. The amino acids, small peptide fragments, and coupling sequence are as follows:

[0019] ;

[0020] The coupling agent in step two-A is selected from Oxyma and DIC, as well as TPTU and DIEA.

[0021] Preferably, in step two-A, the coupling agents used for coupling amino acids in sequences 2-1 and 2-3 to 2-9 are Oxyma and DIC, and the coupling agents used for coupling small peptide fragments in sequences 2-2 and 2-10 are TPTU and DIEA.

[0022] Preferably, the coupling agents for coupling fragment 1-resin and fragment 2 are TPTU and DIEA.

[0023] Preferably, the preparation method includes:

[0024] Step 1:

[0025] (a1) Deprotecting the amino resin with a deprotecting agent;

[0026] (b1) Add the coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acids;

[0027] (c1) The activated amino acid obtained in step (b1) is added to the resin obtained in step (a1) and coupled to obtain Fmoc-Ser(tBu)-amide resin;

[0028] (d1) Repeat the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain fragment 1-resin;

[0029] The amino acids and small peptide fragments, coupling sequence, and coupling agent in step one are as follows:

[0030] ;

[0031] Step Two:

[0032] Step Two - A:

[0033] (a2) Deprotecting 2-CTC resin with a deprotecting agent;

[0034] (b2) Add the coupling agent to Fmoc-Leu-OH to activate the amino acids;

[0035] (c2) The activated amino acid obtained in step (b2) is added to the resin obtained in step (a2) and coupled to obtain Fmoc-Leu-ester resin;

[0036] (d2) Repeat the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain fragment 2-resin;

[0037] (e2) The fragment 2 resin obtained in step (d2) is added to the pyrolysis solution for pyrolysis to obtain fragment 2;

[0038] The amino acids and small peptide fragments, coupling sequence, and coupling agent in step two-A are as follows:

[0039] ;

[0040] Step Two-B:

[0041] (a3) Deprotect the fragment 1-resin with a deprotecting agent;

[0042] (b3) Add TPTU and DIEA to fragment 2 to activate fragment 2;

[0043] (c3) The activated fragment 2 obtained in step (b3) is coupled with the fragment 1-resin obtained in step (a3) ​​to obtain retaliglutide-resin;

[0044] Step 3:

[0045] The ratalutide-resin was added to a lysis buffer and lysed to obtain ratalutide.

[0046] Preferably, the deprotectant is a 20% PIP / DMF mixture, wherein the volume ratio of PIP to DMF is 20:80.

[0047] Preferably, the pyrolysis solution in steps two-A and three is a TFA / EDT / Tis / H2O / phenol pyrolysis solution, which 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 phenol is 2% of the mass of the first solution.

[0048] Preferably, steps two-A and three further include a crystallization step after pyrolysis, wherein the precipitate used in the crystallization step is selected from MTBE and EA, MTBE and IPAc, and diethyl ether and EA, wherein the volume ratio of the two components in MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1:1.

[0049] Preferably, step three further includes liquid chromatography purification, wherein the chromatographic system uses a reversed-phase C18 column, and the purification includes:

[0050] (f-1): The rataglutide-resin obtained in step (c3) is pyrolyzed, crystallized, filtered, and dried to obtain crude rataglutide. The crude rataglutide is dissolved in a 30% acetonitrile aqueous solution, filtered, and the filtrate is collected.

[0051] (f-2): The filtrate is purified for the first time. Mobile phase A is a phosphoric acid aqueous solution with pH 3, and mobile phase B is acetonitrile. The qualified fraction with a purity greater than 96% is collected.

[0052] (f-3): The qualified fraction collected in step (f-2) is purified a second time. Mobile phase A is sodium acetate solution with pH 8.5 and mobile phase B is acetonitrile. The fraction with a purity greater than 99.5% is collected.

[0053] Preferably, the resin in step one is selected from Rink Amind-AM resin, Rink Amide resin and RinkAmide-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 two is 0.3-1.2 mmol / g.

[0054] 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).

[0055] This invention also provides a method for preparing a retaglutide intermediate, characterized in that the retaglutide intermediate is fragment 2 of formula II.

[0056] Fmoc-Tyr1 -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

[0057] The preparation method includes:

[0058] 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 the said retaliglutide using a solid-phase synthesis method to obtain fragment 2-resin. The fragment 2-resin is then cleaved to obtain fragment 2, wherein the amino acids, small peptide fragments, and coupling sequence are as follows:

[0059] ;

[0060] The coupling agents for the coupling reaction are selected from Oxyma and DIC, as well as TPTU and DIEA.

[0061] Preferably, the coupling agents used for amino acids with coupling sequences of 2-1 and 2-3 to 2-9 are Oxyma and DIC, and the coupling agents used for small peptide fragments with coupling sequences of 2-2 and 2-10 are TPTU and DIEA.

[0062] This invention prepares retaglutide via a solid-phase reaction between two peptide fragments, fragment 1 and fragment 2. Fragment 1 is prepared using a solid-phase synthesis method and coupled to fragment 2 in the form of fragment 1-resin. Compared to a liquid-phase synthesis method for coupling fragments 1 and 2, this method is simpler, produces fewer impurities, and simplifies post-processing, thus improving synthesis efficiency, retaglutide purity, and reducing costs. Furthermore, the method for preparing retaglutide intermediates using this invention yields retaglutide intermediates with high purity and yield. Subsequent reactions with fragment 1-resin can further improve the yield and purity of retaglutide. This method for preparing retaglutide and its intermediates is particularly suitable for large-scale production. Attached Figure Description

[0063] Figure 1 The HPLC chromatogram of fragment 2 from Example 1;

[0064] Figure 2The HPLC chromatogram of crude retaloglutide from Example 1 is shown below.

[0065] Figure 3 The HPLC chromatogram of pure retaglutide from Example 1 is shown below.

[0066] Figure 4 The mass spectrum of the retaglutide prepared in Example 1;

[0067] Figure 5 The HPLC chromatogram of crude retaloglutide from Comparative Example 1;

[0068] Figure 6 The HPLC chromatogram of pure Retaglutide from Comparative Example 1;

[0069] Figure 7 The HPLC chromatogram of crude retaloupeptide from Comparative Example 2 is shown below.

[0070] Figure 8 The HPLC chromatogram of pure Retaglutide from Comparative Example 2;

[0071] Figure 9 The mass spectrum of the impurities involved in Comparative Example 2. Detailed Implementation

[0072] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the invention. Unless otherwise specified, the methods used in the present invention are conventional production methods; the raw materials used, unless otherwise specified, are conventional commercially available products. In the following embodiments, the small fragment peptides of the present invention were purchased from Sichuan Tongsheng Biomedical Co., Ltd.

[0073] The English abbreviations and Chinese meanings of the reagents used in this invention are shown in Table 1:

[0074] Table 1

[0075] .

[0076] The method for preparing retaglutide of the present invention includes step one: preparing fragment 1-resin of formula I by solid-phase synthesis; step two: coupling fragment 2 of formula II with the fragment 1-resin by solid-phase synthesis to obtain retaglutide-resin; and step three: cleaving the retaglutide-resin to obtain retaglutide. In the prior art, multiple fragments are typically prepared by solid-phase synthesis, then cleaved from the resin, and then coupled together by liquid-phase synthesis to produce retaglutide. The above-mentioned prior art methods require cleaving each fragment from the resin, followed by purification before being added to the liquid-phase reaction. Furthermore, liquid-phase synthesis reactions are prone to generating impurities. Separating the target substance dissolved in the reaction system from the liquid-phase reaction system is also difficult. After the reaction is complete, steps such as extraction, concentration, column purification, and pH adjustment are required, making the operation complex, requiring numerous reagents and equipment, and resulting in high production costs. This invention employs a solid-phase synthesis method, coupling fragment 2 with fragment 1 via resin. This reduces the steps and reagents required for cleaving and purifying all fragments from the resin. Furthermore, since the synthesized retaglutide is attached to the resin, its separation from the reaction system is simpler, reducing the steps and reagents required in liquid-phase reactions such as extraction, concentration, and column purification. Additionally, solid-phase reactions produce fewer impurities compared to liquid-phase reactions, improving the purity and yield of the retaglutide product. Moreover, fragment 1 is a polypeptide fragment containing amino acids 15-39, and fragment 2 is a polypeptide fragment containing amino acids 1-14. Only one coupling is required during the synthesis of retaglutide, avoiding impurity accumulation from multiple coupling reactions. The two fragment peptides are coupled between Leu at position 14 and Asp at position 15 of the retaglutide, resulting in good coupling reactivity between fragment 1 and fragment 2, minimal racemic impurities, high product purity, and high coupling efficiency.

[0077] In a preferred embodiment of the present invention, step one includes: using an amino resin as the starting resin, sequentially coupling amino acids and small peptide fragments from the N-terminus to the C-terminus of the amino acid sequence of the retaglutide to obtain fragment 1-resin, wherein the amino acids and small peptide fragments and the coupling sequence are as follows: ;

[0078] The coupling agent in step one is selected from Oxyma and DIC, TPTU and DIEA, COMU and DIEA, COMU and TMP, and PyOxim and DIEA. Preferably, in step one, the coupling agent used for amino acids or small peptide fragments with coupling sequences of 1-1, 1-3, and 1-5~1-7 is Oxyma and DIC; the coupling agent used for small peptide fragments with coupling sequences of 1-2 and 1-4 is TPTU and DIEA; the coupling agent used for amino acids or small peptide fragments with coupling sequences of 1-8~1-11 and 1-13~1-15 is COMU and DIEA; the coupling agent used for amino acids with coupling sequences of 1-12 and 1-18 is COMU and TMP; and the coupling agent used for amino acids with coupling sequences of 1-16 and 1-17 is PyOxim and DIEA.

[0079] In a preferred embodiment of the present invention, step two further includes: Step two-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 the retaglutide using a solid-phase synthesis method to obtain fragment 2-resin, and fragment 2-resin is cleaved to obtain fragment 2, wherein the amino acids and small peptide fragments and the coupling sequence are as follows: ;

[0080] The coupling agent in step two-A is selected from Oxyma and DIC, and TPTU and DIEA. Preferably, the coupling agents used for coupling amino acids with a coupling sequence of 2-1 and 2-3 to 2-9 in step two-A are Oxyma and DIC, and the coupling agents used for coupling small peptide fragments with a coupling sequence of 2-2 and 2-10 are TPTU and DIEA.

[0081] In a preferred embodiment of the present invention, the preparation method includes:

[0082] Step 1:

[0083] (a1) Deprotecting the amino resin with a deprotecting agent;

[0084] (b1) Add the coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acids;

[0085] (c1) The activated amino acid obtained in step (b1) is added to the resin obtained in step (a1) and coupled to obtain Fmoc-Ser(tBu)-amide resin;

[0086] (d1) Repeat the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain fragment 1-resin;

[0087] The amino acids and small peptide fragments, coupling sequence, and coupling agent in step one are as follows:

[0088] ;

[0089] Step Two:

[0090] Step Two - A:

[0091] (a2) Deprotecting 2-CTC resin with a deprotecting agent;

[0092] (b2) Add the coupling agent to Fmoc-Leu-OH to activate the amino acids;

[0093] (c2) The activated amino acid obtained in step (b2) is added to the resin obtained in step (a2) and coupled to obtain Fmoc-Leu-ester resin;

[0094] (d2) Repeat the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain fragment 2-resin;

[0095] (e2) The fragment 2 resin obtained in step (d2) is added to the pyrolysis solution for pyrolysis to obtain fragment 2;

[0096] The amino acids and small peptide fragments, coupling sequence, and coupling agent in step two-A are as follows: ;

[0097] Step Two-B:

[0098] (a3) Deprotect the fragment 1-resin with a deprotecting agent;

[0099] (b3) Add TPTU and DIEA to fragment 2 to activate fragment 2;

[0100] (c3) The activated fragment 2 obtained in step (b3) is coupled with the fragment 1-resin obtained in step (a3) ​​to obtain retaliglutide-resin;

[0101] Step 3:

[0102] The ratalutide-resin was added to a lysis buffer and lysed to obtain ratalutide.

[0103] This invention uses specific small peptide fragments to prepare fragment 1 (resin) and fragment 2, reducing the total number of coupling reactions required to prepare the two fragments. This avoids the problem of missing amino acids in the product due to the inability to couple individual amino acids at certain sites or the high difficulty of coupling, thus improving synthesis efficiency and reducing the accumulation of impurities from multiple couplings. Furthermore, depending on the carboxyl and amino groups, the condensation reaction varies in ease and speed; for example, Pro, with a secondary amino group, is a more difficult amino acid to condense. Besides the influence of the structure of the amino acids involved in the reaction, factors such as the length of the assembled peptide chain and the degree of hydrophobicity of the peptide chain also significantly affect the condensation reaction. This invention improves synthesis efficiency by using specific coupling agents in each coupling step. In addition, in the above preferred embodiment, the method of this invention only requires the preparation of two fragments, fragment 1 and fragment 2, which reduces the use of resin in solid-phase synthesis methods and lowers production costs. Moreover, the method for preparing the retaliglutide intermediate fragment 2 of this invention yields fragment 2 with high purity and yield, making it suitable for mass production.

[0104] In a preferred embodiment of the present invention, the deprotecting agent is a 20% concentration of PIP / DMF, wherein the volume ratio of PIP to DMF is 20:80.

[0105] In a preferred embodiment of the present invention, the lysis buffer used is a TFA / EDT / Tis / H2O / phenol lysis buffer, which is prepared by the following method: A first solution with a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5 is prepared, and then phenol is added to the first solution, wherein the mass of phenol is 2% of the mass of the first solution. For example, if 90 mL of solution is prepared according to the TFA / EDT / Tis / H2O = 90 / 5 / 2.5 / 2.5 volume ratio, and the weight is 100 grams, then 2 grams of phenol are added. Phenol acts as an antioxidant to prevent Tyr and Trp from being oxidized.

[0106] In a preferred embodiment of the present invention, steps two-A and three further include a crystallization step after pyrolysis. The precipitate used in the crystallization step is selected from MTBE and EA, MTBE and IPAc, and diethyl ether and EA, wherein the volume ratio of the two components in MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1:1.

[0107] In a preferred embodiment of the present invention, the method further includes liquid chromatography purification, wherein the chromatographic system employs a reversed-phase C18 column, and the purification includes:

[0108] Dissolution: The rataglutide-resin obtained in step (c3) is pyrolyzed, crystallized, filtered, and dried to obtain crude rataglutide. The crude rataglutide is dissolved in a 30% acetonitrile aqueous solution, filtered, and the filtrate is collected.

[0109] First purification: The filtrate is purified for the first time. Mobile phase A is a phosphoric acid aqueous solution with pH 3, and mobile phase B is acetonitrile. The qualified fraction with a purity greater than 96% is collected.

[0110] Second purification: The collected qualified fractions were purified a second time. Mobile phase A was sodium acetate solution with pH 8.5, and mobile phase B was acetonitrile. Fractions with a purity greater than 99.5% were collected.

[0111] The above purification method can yield high-purity refined retaliutide.

[0112] In a preferred embodiment of the present invention, the starting resin in step one includes Rink Amind-AM resin, Rink Amide resin, and Rink Amide-MBHA resin, and 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 two is 0.3-1.2 mmol / g.

[0113] 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).

[0114] Example 1: Preparation of Retaglutide

[0115] Step 1: Preparation of Fragment 1 - Resin

[0116] (a1) Take 30.0g of Rink Amide-AM resin (degree of substitution 0.58mmol / g) and place it in a solid-phase reactor. Add DMF solution to swell for 30 minutes, filter, add 20% PIP / DMF solution (PIP:DMF volume ratio of 20:80) to protect for 10 minutes, filter, add 20% PIP / DMF solution again to protect for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0117] (b1) Add 300 mL of DMF to another reaction flask (hereinafter referred to as the "amino acid activation reaction flask"), add 20.01 g of Fmoc-Ser(tBu)-OH and 7.42 g of Oxyma to the amino acid activation reaction flask and stir to dissolve. Slowly add 6.59 mL of DIC (Suzhou Haofan Biotechnology Co., Ltd.) and stir for at least 5 min to activate the amino acids.

[0118] (c1) The activated amino acid solution was added to a solid-phase reactor and reacted for 2 hours. A small amount of resin ninhydrin was tested. The resin was colorless. The resin was filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0119] (d1) Add 20% PIP / DMF to the resin obtained in step (c1) for protection for 10 minutes, filter, add 20% PIP / DMF again for protection for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0120] (e1) In an amino acid activation reaction flask, 300 mL of a mixed solvent with a volume ratio of DMF / DCM = 2 / 1 was added, along with 18.50 g of Fmoc-Pro-Pro-Pro-OH and 10.34 g of TPTU (Suzhou Haofan Biotechnology Co., Ltd.). The mixture was dissolved and clarified, cooled to 15°C, and 6.75 mL of DIEA (Annegi (Shanghai) Pharmaceutical Chemical Co., Ltd.) was added dropwise. After activation at 15°C with stirring for at least 10 minutes, the mixture was added to a solid-phase reactor and stirred for 2 hours. A small amount of resin was tested for ninhydrin; the resin was colorless. After filtration and washing twice with DMF, Fmoc-Pro-Pro-Pro-Ser(tBu)-amide resin was obtained.

[0121] The remaining amino acids and small peptide fragments were sequentially coupled to obtain fragment 1-resin. The amino acids, small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 2 below:

[0122] Table 2

[0123] .

[0124] The temperature for activating amino acids or small 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.

[0125] Step 2-A: Preparation of Fragment 2

[0126] (a2) Take 25.0g of 2-CTC resin (degree of substitution 0.98mmol / g) and place it in a solid-phase reactor. Add DMF solution to swell for 30 minutes, filter, add 20% PIP / DMF (PIP:DMF volume ratio of 20:80) to protect for 10 minutes, filter, add 20% PIP / DMF again to protect for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0127] (b2) Add 300 mL of DMF to another reaction flask (hereinafter referred to as the "amino acid activation reaction flask"), add 25.97 g of Fmoc-Leu-OH and 10.44 g of Oxyma to the amino acid activation reaction flask and stir to dissolve. Slowly add 9.28 mL of DIC (Suzhou Haofan Biotechnology Co., Ltd.) and stir for at least 5 min to activate the amino acids.

[0128] (c2) The activated amino acid solution was added to a solid-phase reactor and reacted for 2 hours. A small amount of resin ninhydrin was tested. The resin was colorless. The resin was filtered and washed twice with DMF to obtain Fmoc-Leu-ester resin.

[0129] (d2) Add 20% PIP / DMF to the resin obtained in step (c2) for protection for 10 minutes, filter, add 20% PIP / DMF again for protection for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0130] In an amino acid activation reaction flask, 300 mL of a DMF / DCM mixed solvent (2 / 1 volume ratio) was added, along with 37.92 g of Fmoc-Ile-αMeLeu-OH and 14.56 g of TPTU (Suzhou Haofan Biotechnology Co., Ltd.). The mixture was dissolved and clarified, then cooled to 15°C. 9.50 mL of DIEA (Annegi (Shanghai) Pharmaceutical Chemical Co., Ltd.) was added dropwise. After activation at 15°C with stirring for at least 10 minutes, the mixture was added to a solid-phase reactor and stirred for 2 hours. A small amount of resin was tested for ninhydrin; the resin was colorless. After filtration and washing twice with DMF, Fmoc-Ile-αMeLeu-Leu-ester resin was obtained.

[0131] The remaining amino acids and small peptide fragments were coupled sequentially. The amino acids, small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 3 below:

[0132] Table 3

[0133] .

[0134] The temperature for activating amino acids or small peptides using coupling agents Oxyma and DIC, COMU and DIEA, and TPTU and DIEA is 10–20 °C.

[0135] After all amino acids and small peptide fragments were coupled, methanol was added and condensed three times, 10 minutes each time. The mixture was then filtered, vacuum dried for 4 hours, and fragment 2-resin was collected. The weight was 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. Therefore, the yield of fragment 2 was 60.5 / 56.95 = 106.2%.

[0136] (e2) The lysis buffer was prepared as follows: a first solution with a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5 was prepared, and phenol was added to the first solution, the mass of which was 2% of the mass of the first solution. Fragment 2-resin prepared in step (d2) was added to the lysis buffer, the volume of which was 10 times the weight of fragment 2-resin. The mixture was stirred for 3 hours, filtered, and the filtrate was collected. The filtrate was cooled to below 0°C, and the filtrate was slowly added to a mixture with a volume ratio of MTBE / EA = 1 / 1 to precipitate the fragment. The volume of this mixture was 60 times the weight of fragment 2-resin. The mixture was filtered, the filter cake was washed with MTBE, and the mixture was dried in a solid oven for 4 hours to obtain 52 g of fragment 2 solid. The yield of fragment 2 was 52 / 56.95 × 100% = 91.315%. The HPLC chromatogram of fragment 2 is shown below. Figure 1 As shown, the HPLC purity is 93.80%.

[0137] Step Two-B:

[0138] (a3) Add 20% PIP / DMF (PIP:DMF volume ratio of 20:80) to the above fragment 1-resin for protection for 10 minutes, filter, add 20% PIP / DMF again for protection for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0139] (b3) Dissolve the solid fragment 2 obtained in step two above in 300 mL of a mixed solution with a volume ratio of DMF / NPM = 1 / 1, add 6.7 g of TPTU to dissolve and clarify, cool to 15 °C, add 4.5 mL of DIEA, stir at 15 °C for at least 10 minutes to activate fragment 2.

[0140] (c3) The activated fragment 2 solid obtained in step (b3) is added to the resin obtained in step (a3) ​​and reacted for 6 hours. The resulting product is washed three times with DMF and three times with methanol, and 135g of retaliptide resin is collected.

[0141] Step 3:

[0142] Prepare the lysis buffer as in step (e2), and add the retaliatory resin from step (c3) to the lysis buffer. The amount of lysis buffer is 10 times the weight of the retaliatory resin. Stir for 3 hours, filter, collect the filtrate, cool to below 0°C, and slowly add the filtrate to a mixture with a volume ratio of MTBE / EA = 1 / 1 to precipitate the resin. The amount of the mixture is 60 times the weight of the retaliatory resin. Filter, wash the filter cake with MTBE, and dry in a solid oven for 4 hours to obtain 75g of crude retaliatory resin. The theoretical yield of crude retaliatory resin is: 30 × (0.58 / 1000) × 4731.42 (retaglutide molecular weight) = 82.33g, and the yield is: 75 / 82.33 × 100% = 91.1%. The HPLC chromatogram of crude retaliatory resin is shown below. Figure 2 As shown, the HPLC purity is 89.54%, where purity is the area ratio in the HPLC chromatogram. Chromatographic detection was performed using the following method:

[0143] Liquid Chromatography System: Waters 2695 Separations Module;

[0144] Specifications: UV (ALLLANCE E2695 / ACQUITY Arc);

[0145] Column: Kinetex 2.6μm C18 100Å 150x4.6mm;

[0146] Column temperature: 30℃;

[0147] Detection wavelength: 210nm;

[0148] Injection volume: 10 μL;

[0149] Flow rate: 0.7 mL / min;

[0150] Mobile phase A: Weigh 18.4g of ammonium dihydrogen phosphate, add 1600g of ultrapure water to dissolve, adjust the pH to 3.6 with phosphoric acid, then add 150g of acetonitrile and 200g of ultrapure water, and mix well;

[0151] Mobile phase B: Weigh 930g of acetonitrile, 310g of isopropanol, and 400g of ultrapure water, and mix well;

[0152] The elution gradient is shown in Table 4 below:

[0153] Table 4

[0154] .

[0155] (e3) The crude retaliculide was purified by two injections using a high-performance liquid chromatograph (HPLC) system. The HPLC system used in the purification process and its parameters are shown in Table 5 below:

[0156] Table 5

[0157] .

[0158] The purification steps include:

[0159] (f-1): Dissolve the above crude retaloupeptide in acetonitrile water with a volume concentration of 30%, filter, and collect the filtrate;

[0160] (f-2): The filtrate was subjected to a first purification. Mobile phase A was an aqueous solution of phosphoric acid at pH 3, and mobile phase B was acetonitrile. The elution gradient is shown in Table 6 below:

[0161] Table 6

[0162] .

[0163] Collect qualified fractions with a purity greater than 96%;

[0164] (f-3): The qualified fraction collected in step (f-2) is subjected to a second purification. Mobile phase A is a sodium acetate solution with a pH of 8.5, and mobile phase B is acetonitrile. The elution gradient is shown in Table 7 below:

[0165] Table 7

[0166] .

[0167] Collect qualified fractions with a purity greater than 99.5%.

[0168] Subsequently, the qualified fraction was subjected to salt exchange using a multifunctional membrane experimental device (Hangzhou Ruina Membrane Engineering Co., Ltd., RNM-18G). The filter membrane pore size was selected as 1-2 nm, and the pressure was set to 1.5 MPa. The concentrate was collected. After lyophilization, pure retaglutide was obtained, totaling 63 g, with a yield of 63 / 75 × 100% = 84%. The HPLC chromatogram of pure retaglutide is shown below. Figure 3 As shown, the HPLC purity is 99.89%.

[0169] The obtained pure retaliculide was analyzed by mass spectrometry using liquid chromatography-mass spectrometry (LC-MS). The instruments and their parameters were as follows:

[0170] Instrument Name: Ultra-High Performance Liquid Chromatography (UHHPLC) System;

[0171] Brand and Model: ACQUIIY UPLC H-CLASS PIUS;

[0172] Supplier: Waters Technology (Shanghai) Co., Ltd.;

[0173] Column: 1.7μm PEPTIDE XB-C18 10C LC Column 150x2.1nm;

[0174] Column temperature: 30℃;

[0175] Injection volume: 10 μL;

[0176] Flow rate: 0.4 mL / min;

[0177] Phase A: 0.1% formic acid aqueous solution;

[0178] Phase B: Acetonitrile;

[0179] The elution gradient is shown in Table 8 below:

[0180] Table 8

[0181] .

[0182] The mass spectrum of the pure retaliculide is as follows: Figure 4 As shown, 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 of 1183.77 corresponds to the product prepared in Example 1, which is retaloupeptide.

[0183] Comparative Example 1

[0184] Take 30.0g of Rink Amide-AM resin (degree of substitution 0.58mmol / g) and place it in a solid-phase reactor. Add DMF solution to swell for 30 minutes, filter, add 20% PIP / DMF (PIP:DMF volume ratio of 20%:80%) to protect for 10 minutes, filter, add 20% PIP / DMF again to protect for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0185] Add 300 mL of DMF to another reaction flask (hereinafter referred to as "amino acid activation reaction flask"), add 20.01 g of Fmoc-Ser(tBu)-OH and 7.42 g of Oxyma to the amino acid activation reaction flask and stir to dissolve. Slowly add 6.59 mL of DIC and stir for at least 5 min to activate the amino acids.

[0186] The activated amino acid solution was added to a solid-phase reactor and reacted for 2 hours. A small amount of resin ninhydrin was tested, and the resin was colorless. The resin was filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0187] Add 20% PIP / DMF to Fmoc-Ser(tBu)-amide resin for protection for 10 minutes, filter, add another 20% PIP / DMF for protection for 10 minutes, filter again, wash with DMF 6 times, stirring for 3 minutes each time. In an amino acid activation reaction flask, add 300 mL of a DMF / DCM (2 / 1 volume ratio) mixed solvent, add 18.50 g of Fmoc-Pro-Pro-Pro-OH, add 10.34 g of TPTU to dissolve and clarify, cool to 15°C, add 6.75 mL of DIEA dropwise, stir and activate at 15°C for at least 10 minutes, then add to a solid-phase reactor and stir to react for 2 hours. Take a small amount of resin ninhydrin for testing; the resin is colorless. Filter, wash twice with DMF to obtain Fmoc-Pro-Pro-Pro-Ser(tBu)-amide resin.

[0188] The remaining amino acids and small peptide fragments were coupled sequentially. The amino acids, small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 9 below:

[0189] Table 9

[0190] .

[0191] The temperature for activating amino acids or small 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.

[0192] After coupling, 132g of retaglutide resin was obtained by washing with methanol. The resin was then cleaved in the same manner as in Example 1 to obtain 71g of crude retaglutide. The theoretical yield of retaglutide was 30 × (0.58 / 1000) × 4731.42 = 82.33g, and the yield was 71 / 82.33 × 100% = 86.23%. The HPLC chromatogram of the crude retaglutide is shown below. Figure 5 As shown, the crude product had an HPLC purity of 77.97%. The crude retaglutide was purified twice in the same manner as in Example 1, yielding 58 g of pure product, with a yield of 58 / 71 × 100% = 81.7%. The HPLC chromatogram of the pure product is shown below. Figure 6 As shown, the HPLC purity of the pure product is 99.55%.

[0193] Comparative Example 2

[0194] Take 30.0g of Rink Amide-AM resin (degree of substitution 0.58mmol / g) and place it in a solid-phase reactor. Add DMF solution to swell for 30 minutes, filter, add 20% PIP / DMF (PIP:DMF volume ratio of 20%:80%) to protect for 10 minutes, filter, add 20% PIP / DMF again to protect for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.

[0195] Add 300 mL of DMF to another reaction flask (hereinafter referred to as "amino acid activation reaction flask"), add 20.01 g of Fmoc-Ser(tBu)-OH and 7.42 g of Oxyma to the amino acid activation reaction flask and stir to dissolve. Slowly add 8.08 mL of DIC and stir for at least 5 min to activate the amino acids.

[0196] The activated amino acid solution was added to a solid-phase reactor and reacted for 2 hours. A small amount of resin ninhydrin was tested, and the resin was colorless. The resin was filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0197] To protect Fmoc-Ser(tBu)-amide resin, add 20% PIP / DMF for 10 minutes, filter, and then add another 20% PIP / DMF for 10 minutes. Filter again, wash with DMF 6 times, stirring for 3 minutes each time. In an amino acid activation reaction flask, add 300 mL of DMF, then 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 acids. Add the activated amino acids to a solid-phase reactor and stir for 2 hours. Test a small amount of resin for ninhydrin; the resin is colorless. Filter, wash twice with DMF, and obtain Fmoc-Pro-Ser(tBu)-amide resin.

[0198] The remaining amino acids and small peptide fragments were coupled sequentially. The amino acids, small peptide fragments, coupling agents, and solvents used in the coupling process are shown in Table 10 below:

[0199] Table 10

[0200]

[0201] After coupling, the resin was washed with methanol to obtain 133g of retaliptide resin.

[0202] The resin for retaglutide was cleaved using the same method as in Example 1, yielding 70 g of crude retaglutide. The theoretical yield of retaglutide is 30 × (0.58 / 1000) × 4731.42 = 82.33 g, and the yield is 70 / 82.33 × 100% = 85.0%. The HPLC chromatogram of the crude retaglutide is shown below. Figure 7 As shown, the crude product had an HPLC purity of 70.00%. The crude retaglutide was purified twice in the same manner as in Example 1, yielding 52 g of pure product, with a yield of 52 / 70 × 100% = 74.3%. The HPLC chromatogram of the pure product is shown below. Figure 8 As shown, the HPLC purity of the pure product is 98.96%. Sequential coupling of single amino acids easily produces significant deletion impurities. (See attached image.) Figure 9 As shown, the product of Comparative Example 2 contained an impurity with a molecular weight of 1155.31. As mentioned above, the molecular weight of retaliglutide detected by mass spectrometry was 1183.77. Compared with retaliglutide, the molecular weight of the above impurity was reduced by (1183.77-1155.31)×4=113.84. It can be seen that the above impurity is a peptide lacking an amino acid with a molecular weight of 113.84.

[0203] As can be seen from the above examples and comparative examples, Comparative Example 1 uses a single amino acid and a small peptide fragment to be sequentially coupled to form retaloglutide, while the present invention uses the same single amino acid and small fragment to first couple to form fragment 1-resin and fragment 2, and then couples fragment 2 to fragment 1 on fragment 1-resin. Although the single amino acid and small peptide fragment used in Comparative Example 1 are the same as those in Example 1, the coupling steps in Comparative Example 1 are more numerous, time-consuming, and prone to generating impurities. The yield and purity of crude and pure retaloglutide in Comparative Example 1 are lower than those in Example 1. The low purity of the crude product in Comparative Example 1 is not conducive to purification and easily leads to excessive single impurities after purification. In Comparative Example 2, except for Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-OH, all other reaction steps involve coupling single amino acids. This results in numerous coupling steps, poor reaction effect, long reaction time, high raw material consumption, and easy generation of missing peptide impurities, causing yield loss and increasing purification difficulty. Comparative Example 2 showed lower yields and purities of crude and pure retaglutide compared to Example 1. The preparation method of this invention consumes less resin, involves fewer reaction steps, simplifies post-processing, significantly reduces costs, and improves product purity and yield, making it more suitable for industrial production.

[0204] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A process for the preparation of raloxifene having the amino acid sequence 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-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 -NH2, characterized in that, The preparation method comprises: Step one: preparing 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 two: coupling fragment 2 of formula II with the fragment 1-resin by solid phase synthesis method to obtain a resin of remogliflozin, 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; and Step three: cleaving the resin of remogliflozin to obtain remogliflozin, The step one comprises: taking an amino resin as a starting resin, and sequentially coupling amino acids and small fragment peptides from N-terminal to C-terminal according to the amino acid sequence of the remogliflozin to obtain the fragment 1-resin, wherein the amino acids and small fragment peptides and the coupling sequence are as follows: The coupling agent used for the amino acids or small fragment peptides of the coupling sequence of 1-1, 1-3 and 1-5~1-7 in the step one is Oxyma and DIC, the coupling agent used for the small fragment peptides of the coupling sequence of 1-2 and 1-4 is TPTU and DIEA, the coupling agent used for the amino acids or small fragment peptides of the coupling sequence of 1-8~1-11 and 1-13~1-15 is COMU and DIEA, the coupling agent used for the amino acids of the coupling sequence of 1-12 and 1-18 is COMU and TMP, and the coupling agent used for the amino acids of the coupling sequence of 1-16 and 1-17 is PyOxim and DIEA; The step two further comprises: Step two-A: taking 2-CTC resin as a starting resin, and sequentially coupling amino acids and small fragment peptides from N-terminal to C-terminal according to the amino acid sequence of the remogliflozin by solid phase synthesis method to obtain a fragment 2-resin, and cleaving the fragment 2-resin to obtain the fragment 2, wherein the amino acids and small fragment peptides and the coupling sequence are as follows: The coupling agent used for the amino acids of the coupling sequence of 2-1 and 2-3~2-9 in the step two-A is Oxyma and DIC, and the coupling agent used for the small fragment peptides of the coupling sequence of 2-2 and 2-10 is TPTU and DIEA; The coupling agent for coupling the fragment 1-resin and the fragment 2 is TPTU and DIEA.

2. The production method according to claim 1, characterized by, The preparation method comprises: Step one: (a1) deprotecting the amino resin by using a deprotecting agent; (b1) adding a coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acid; (c1) adding the activated amino acid obtained in the step (b1) to the resin obtained in the step (a1) to couple to obtain Fmoc-Ser(tBu)-amide resin; (d1) repeating the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small fragment peptides to obtain the fragment 1-resin; The amino acids and small fragment peptides, the coupling sequence and the coupling agent of the step one are as follows: ； Step two: Step two-A: (a2) deprotecting the 2-CTC resin by 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 the step (b2) to the resin obtained in the 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 small fragment peptides to obtain the fragment 2-resin; (e2) cleaving the fragment 2-resin obtained in step (d2) to obtain fragment 2; In step two-A, the amino acid and small fragment peptide, the coupling sequence and the coupling agent are as follows: ； Step two-B: (a3) deprotecting the fragment 1-resin with 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 a resin of rilatlimab; Step three: cleaving the resin of rilatlimab to obtain rilatlimab.

3. The preparation method according to claim 2, 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.

4. The preparation method according to claim 2, characterized in that, The cleavage solution in step two-A and step three is TFA / EDT / Tis / H2O / phenol cleavage solution, which 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.

5. The preparation method according to claim 2, characterized in that, The step two-A and step three further comprise a crystallization step after cleavage, and the crystallization solution used in the crystallization step is selected from MTBE and EA, MTBE and IPAc, and ether and EA, and the volume ratio of the two components in the MTBE and EA, MTBE and IPAc, ether and EA is 1:

1.

6. The preparation method according to claim 2, characterized in that, In step three, liquid chromatography purification is further included, and the chromatography system adopts a reversed-phase C18 chromatography column, and the purification includes: (f-1): cleaving, crystallizing, filtering and drying the resin of rilatlimab prepared in step (c3) to obtain crude rilatlimab, dissolving the crude rilatlimab in 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; (f-2): purifying the filtrate for the first time, mobile phase A is pH 3 phosphoric acid aqueous solution, mobile phase B is acetonitrile, and collect the qualified fraction of more than 96%; (f-3): purifying the qualified fraction collected in step (f-2) for the second time, mobile phase A is pH 8.5 sodium acetate solution, mobile phase B is acetonitrile, and collect the fraction of more than 99.5%.

7. The production method according to any one of claims 1 to 6, characterized by, The resin in step one is selected from Rink Amind-AM resin, Rink Amide resin and Rink Amide-MBHA resin, and the substitution degree of the resin is 0.4-1.0 mmol / g; the substitution degree of the resin in step two is 0.3-1.2 mmol / g.

8. The production method according to any one of claims 1 to 6, characterized by, 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).

Images