Preparation method of tilpotide

Abstract

The invention provides a preparation method of tiall peptide, and belongs to the technical field of polypeptide synthesis, according to the preparation method, a small amount of lysis solution is adopted for lysis, a small amount of poor solution composed of methyl tert-butyl ether and n-heptane with the volume ratio of 1-2: 1 is used for crude peptide precipitation, precipitated particles are uniform, large and not prone to caking, the crude peptide impurity level is lower, and the purity of the crude peptide is higher. The method has the advantages that the method is simple in process and easy in subsequent purification step, the amount of generated waste liquid is small, the activating agent 7-(1H-pyrrolo [2, 3-b] pyridine-1-yl)-1H-benzo [d] [1, 2, 3] triazole-1-alcohol is used for activating amino acid, the coupling reaction degree is high, and the target peptide yield is high. The preparation method of the tilpotide has the advantages of being simple, economical, capable of achieving mass production, low in impurity content, high in purification yield, high in yield and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and particularly relates to a preparation method of tirzepatide. Background Art

[0002] Tirzepatide is a dual agonist of gastric inhibitory polypeptide and glucagon-like peptide-1 receptor. It is the world's first and currently the only dual agonist of GIP (glucose-dependent insulinotropic polypeptide) / GLP-1 (glucagon-like peptide-1) receptor. By integrating the actions of two incretins, GIP and GLP-1, and being injected once a week, this drug aims to improve blood glucose control and assist in weight management. Tirzepatide has demonstrated excellent hypoglycemic and weight loss effects in multiple clinical trials, especially showing outstanding performance in head-to-head comparisons with similar drugs. In the first half of 2024 alone, the global sales of this drug reached as high as $6.658 billion, and the sales volume continues to increase. The market demand for this drug is huge. As of August 2024, it is still listed as a scarce drug by the FDA.

[0003] Currently, the demand for the API (active pharmaceutical ingredient) of tirzepatide is huge. The use of traditional cup centrifuges cannot meet large-scale commercial production. Therefore, it is urgent to change the current cleavage and precipitation process to increase the particle size of the precipitated solid of tirzepatide. Only by using a centrifuge with a filter medium such as a filter bag can the filtration efficiency of the crude peptide precipitate be greatly improved, the batch production be increased, and thus the commercial demand be met.

[0004] Existing technologies such as the Chinese invention patent with the publication number CN113330024A disclose novel intermediates and methods useful in the manufacture of tirzepatide or its pharmaceutically acceptable salts, and disclose a method for precipitating a crude peptide precipitate from a cleavage solution, that is, adding pre-cooled methyl tert-butyl ether at less than -20°C to the pre-cooled cleavage solution, controlling the temperature at -18 to 5°C during the precipitation process, and separating the obtained crude peptide suspension of tirzepatide with a filter bag centrifuge to obtain tirzepatide solid. The method of this invention requires continuous cooling of a large amount of solvent, has high requirements for cooling equipment, and is prone to generating large lumps during the precipitation process, which deposit at the bottom of the reaction kettle, making it difficult to discharge the material. Even manual tools are needed to crush or fish out and grind them. Moreover, the residual acids, ethers, etc. in the reaction kettle and the materials are highly harmful to personnel. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of tirzepatide, which has the advantages of simplicity, economy, large production batch, low cost, low impurity content, high purification yield, etc.

[0006] The technical solution adopted by the present invention to achieve the above purpose is as follows: The present invention discloses a preparation method of tirzepatide, comprising the following steps: S1. Using resin as a solid-phase carrier, the main chain is synthesized sequentially from the C-terminus to the N-terminus, then the side-chain protecting group of lysine is removed, and the side chain of telotristat ethyl is coupled; or the side chain of telotristat ethyl is combined with the side chain of lysine and directly introduced into the sequence during the solid-phase synthesis resin process to obtain a fully protected telotristat ethyl peptide resin; S2. Add a cleavage solution to cleave the fully protected telotristat ethyl peptide resin, filter, and slowly add the filtrate into a poor solution at -20 to 10 °C, controlling the temperature of the sedimentation solution not to exceed 15 °C during dropping to precipitate a telotristat ethyl solid precipitate. Filter, slurry wash, and dry the telotristat ethyl solid precipitate to obtain a crude telotristat ethyl product; S3. Separate, purify, transfer the salt, and lyophilize the crude product to obtain a finished telotristat ethyl product; In the above step S2, the poor solution includes methyl tert-butyl ether and n-heptane with a volume ratio of 1 to 2:1.

[0007] In some embodiments, the poor solution in the above step S2 is composed of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 or 2:1. Whether it is solid-phase stepwise coupling or long-fragment condensation, when cleaving the peptide resin or fully protected peptide, the precipitation process of the crude peptide basically involves pouring the peptide-containing cleavage solution into 8 to 15 times the volume of ether or methyl tert-butyl ether, followed by precipitation, sedimentation, and centrifugation to obtain the crude peptide solid. Such operations generally require using 8 to 15 times the weight of the peptide resin or fully protected peptide in cleavage solution for the cleavage step, then using 8 to 15 times the volume of the cleavage solution in ether for crude peptide precipitation, and 3 times a total of 30 to 90 times the weight of the peptide resin in ether for washing, resulting in a final waste liquid volume as high as 102 to 330 times the volume of the peptide resin or fully protected peptide, which is extremely wasteful; in addition, the precipitate obtained by this precipitation process has a viscous property and fine particles, which are prone to clogging or passing through the filter medium. Therefore, a cup centrifuge is generally selected to collect the wet crude peptide product. However, this collection method not only causes the crude product to be wrapped with more impurities, affecting the purification and product quality in the subsequent stage, but also often results in poor separation due to a small specific gravity difference between the solid and liquid phases, and is prone to product loss due to pouring the turbid upper layer liquid. The present invention uses a mixed poor solution of methyl tert-butyl ether and n-heptane in a specific ratio for crude peptide precipitation, which can precipitate the crude telotristat ethyl product with a smaller amount of poor solution. The obtained telotristat ethyl crude product has uniform and larger solid particles, is not easy to agglomerate, can be collected by using a centrifuge with a filter medium to improve the filtration efficiency of the crude peptide precipitate, and the obtained crude peptide has less impurities, facilitating the subsequent purification steps.

[0008] In some embodiments, the cleavage solution in the above step S2 is selected from two or more of TFA, Tis, EDT, DODT, DTT, PhOH, and H2O.

[0009] More preferably, in the above step S2, the lysis solution is composed of TFA, Tis, and DODT in a volume ratio of 78-82:7-8:12-13.

[0010] In some embodiments, the lysis time in the above step S2 is 2.0-5.0 h.

[0011] Preferably, the lysis time in the above step S2 is 3.0-4.0 h.

[0012] In some embodiments, the lysis temperature in the above step S2 is 20-30 °C.

[0013] In some embodiments, the resin in the above step S1 is Sieber amide resin.

[0014] In some embodiments, the substitution degree of the resin in the above step S1 is 0.4-0.6 mmol / g.

[0015] In some embodiments, the ratio of the lysis solution to the telotristat fully protected peptide resin in the above step S2 is 4-6 mL / g.

[0016] In some embodiments, the volume ratio of the waste solution to the lysis solution in the above step S2 is 4-6:1.

[0017] In some embodiments, the filtration method used for filtering the telotristat solid precipitate in the above step S2 is filtration with a G3-G5 sintered glass funnel or a filtration medium with 500-1500 mesh.

[0018] Preferably, the filtration method used for filtering the telotristat solid precipitate in the above step S2 is an 800-mesh filter cloth.

[0019] In some embodiments, the temperature of the waste solution in the above step S2 is -5-0 °C.

[0020] In some embodiments, in the above step S2, the temperature of the sedimentation solution is controlled not to exceed 10 °C during dropping.

[0021] In some embodiments, the volume ratio of the waste solution to the lysis solution in the above step S2 is 4-6:1.

[0022] In some embodiments, the solvent used for slurry washing in the above step S2 is methyl tert-butyl ether.

[0023] In some embodiments, the ratio of methyl tert-butyl ether to the telotristat fully protected peptide resin used for slurry washing in the above step S2 is 2-4 mL / g, preferably 3 mL / g.

[0024] In some embodiments, the preparation method of the above telotristat fully protected peptide resin includes: a. Deprotect the Fmoc protection on the resin with a deprotecting agent to obtain the deprotected resin; b. Prepare an activated solution of the amino acid reagent Fmoc-Ser(tBu)-OH and add it to the deprotected resin obtained in step a for a coupling reaction to obtain Fmoc-Ser(tBu)-resin; c. Repeat the deprotection, preparation of the amino acid activated solution and coupling reaction steps to sequentially couple Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH. After the coupling is completed, contract the resin with methanol and dry it under vacuum to obtain the fully protected peptide resin of telotristat.

[0025] In some embodiments, the method for preparing the amino acid activated solution in step c above is: add the amino acid reagent and the activator to a solvent, and add the condensing agent at 0 - 10 °C and let it stand for activation for 5 - 15 min to obtain the amino acid activated solution.

[0026] In some embodiments, the method for preparing the amino acid activated solution of Fmoc-Ser(tBu)-OH in step b above is: add the amino acid reagent Fmoc-Ser(tBu)-OH and the activator to a solvent, and add the condensing agent at 0 - 10 °C and let it stand for activation for 5 - 15 min to obtain the amino acid activated solution of Fmoc-Ser(tBu)-OH.

[0027] In some embodiments, in the preparation of the amino acid activation solution in step c above, the amino acid reagent selects one of Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH according to the coupling sequence.

[0028] In some embodiments, the above deprotecting agent is a Pip / DMF solution with a volume ratio of 20% - 30%.

[0029] In some embodiments, the above activating agent is HOBT and / or 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol. Preferably, the above activating agent includes HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol, and the molar ratio of HOBT to 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol is 2 - 3:1 - 2. The hydroxyl group in the structure of the activating agent 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol can form an ester with the carboxyl group component. The structure of pyrrolopyridine and its specific position make the activity of the activated ester greatly increased, with a high rate of ester aminolysis, a relatively high degree of amino acid coupling reaction, which can improve the yield of the target peptide. Using it together with HOBT as the activating agent has a better effect.

[0030] In some embodiments, the above condensing agent is DCC or DIC.

[0031] In some embodiments, the above solvent includes DMF.

[0032] In some embodiments, the above solvent is composed of DMF / 1,1 - diisopropoxytrimethylamine / 4 - phenyl - 1 - butene in a volume ratio of 7 - 9:2 - 3:1 - 2. A certain proportion of DMF / 1,1 - diisopropoxytrimethylamine / 4 - phenyl - 1 - butene as the solvent can facilitate the activation of amino acid reagents, improve the degree of amino acid coupling reaction, and thus increase the yield of the target peptide.

[0033] In some embodiments, the molar ratio of amino acid reagent: activator: condensing agent in the amino acid activation solution is 1:1:1.

[0034] In some embodiments, in each coupling reaction, the molar ratio of resin: amino acid reagent is 1:3.

[0035] Since the present invention optimizes the cleavage solution, it can complete cleavage with a smaller amount of cleavage solution, overcoming the problem of a large amount of waste liquid in the traditional cleavage step in the industry. The present invention also optimizes the precipitation solution, and can precipitate the crude telotristat ethyl with a smaller amount of precipitation solution. The obtained crude telotristat ethyl solid particles are uniform and large, not easily caked, and the filtration efficiency of the crude peptide precipitate can be greatly improved by using a centrifuge with a filter medium, increasing the batch production, and thus meeting the commercial demand.

[0036] Since the present invention uses 7 - (1H - pyrrolo[2,3 - b]pyridin - 1 - yl)-1H - benzotriazol - 1 - ol and HOBT together as activators in the coupling reaction system for solid - phase synthesis of telotristat ethyl, it can improve the degree of coupling reaction and the yield of the target peptide. Since the present invention uses DMF / 1,1 - diisopropoxytrimethylamine / 4 - phenyl - 1 - butene with a volume ratio of 7 - 9:2 - 3:1 - 2 as the solvent for preparing the amino acid activation solution, it is more conducive to the activation of amino acid reagents, can improve the degree of amino acid coupling reaction, and thus increase the yield of the target peptide.

[0037] The present invention provides a method for preparing telotristat ethyl, which is simple and economical, and has the advantages of large production batch, low cost, low impurity content, high purification yield, high yield, etc.

[0038] As used herein, the following abbreviations have the meanings given herein: "FMOC" means 9-fluorenylmethyloxycarbonyl, "Trt" means triphenylmethyl, "Boc" means tert-butoxycarbonyl, "tBu" means tert-butyl, "OtBu" means tert-butyl ester group, "HOBt" means 1-hydroxybenzotriazole, "EDT" means 1,2-ethanedithiol, "DODT" means 3,6-dioxa-1,8-octanedithiol, "DTT" means dithiothreitol, "Pip" means piperidine, "DCC" means N,N'-dicyclohexylcarbodiimide, "DIC" means N,N'-diisopropylcarbodiimide, "TFA" means trifluoroacetic acid, "Tis" means triisopropylsilane, "PhOH" means phenol, "DMF" means N,N'-dimethylformamide, "AEEA-AEEA" means 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azapentadecanoic acid, "PEG-600" means polyethylene glycol with an average molecular weight of 600. Brief Description of the Drawings

[0039] Figure 1 This is the measurement result of the crude peptide weight yield in Test Example 1 of the present invention.

[0040] Figure 2 This is the measurement result of the crude peptide purity in Test Example 1 of the present invention.

[0041] Figure 3 This is the measurement result of the total yield of tirzepatide in Test Example 1 of the present invention. Detailed Description of the Invention

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Next, the concepts involved in the present application will be described first in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments: In the embodiments of the present invention, unless otherwise stated, the equipment and materials used in the present invention are obtained by purchasing on the market.

[0044] Example 1: A method for preparing crude teplizumab peptide: Deprotection: Weigh 80.0 g of Sieber amide resin with a substitution degree of 0.5 mmol / g (synthesis scale: 40.0 mmol) into a solid-phase reaction column. Swell the resin with DMF for 30 minutes and then evacuate the solution under vacuum. Add 20% (v / v) Pip / DMF solution to deprotect Fmoc for 30 minutes, and then add DMF to wash 6 times. Preparation of the deprotection solution: Add Pip to DMF and mix to obtain the deprotection solution. The volume ratio of Pip to DMF in the deprotection solution is 1:5.

[0045] Preparation of the amino acid reagent activation solution: Add the amino acid reagent Fmoc-Ser(tBu)-OH and the activator HOBT to DMF, add the condensing agent DIC at 5°C and let it stand for activation for 10 minutes to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of the amino acid reagent: activator: condensing agent is 1:1:1.

[0046] Coupling reaction: Add the prepared amino acid activation solution of Fmoc-Ser(tBu)-OH to the deprotected resin obtained. The molar ratio of the resin to the amino acid reagent is 1:3. The coupling temperature is 30°C and the reaction time is 3 hours. After the coupling reaction, wash with DMF 3 times to obtain Fmoc-Ser(tBu)-resin; Repeat the deprotection, preparation of amino acid activation solution and coupling reaction steps, and sequentially couple Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH. After coupling is completed, contract the resin with methanol and dry it under vacuum to obtain 333.1 g of the fully protected peptide resin of tirzepatide.

[0047] Preparation of crude tirzepatide: Weigh 10.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleave at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, wash the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrates into 250 mL of methyl tert-butyl ether at -5 to 0 °C, controlling the temperature during the dripping process to be less than 5 °C to precipitate a white solid. Stir at 0 to 5 °C in a water bath for 10 min, filter under reduced pressure with a 1000-mesh filter cloth. The filtrate is slightly turbid and the filtration speed is extremely slow. It takes about 3 h to filter 300 mL of the precipitated solution; take out the filter cake and wash it by slurrying three times with 30 mL / portion of methyl tert-butyl ether, filter under reduced pressure and dry to constant weight to obtain 5.6 g of crude tirzepatide solid, with a weight yield of 96.88% and a purity of 75.12%.

[0048] Example 2: The difference between this example and Example 1 lies in the preparation of crude tirzepatide.

[0049] Preparation of crude teplizumab peptide: Weigh 10.0 g of the dried fully protected teplizumab peptide resin and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into a 250 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C, controlling the temperature during the dripping process to be less than 5 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with a 1000-mesh filter cloth, and the filtrate is clear. The filtration speed is fast, and it takes about 5 min to filter 300 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to constant weight to obtain 5.7 g of crude teplizumab peptide solid, with a weight yield of 98.62% and a purity of 76.42%; compared with Example 1, the filtration speed is greatly improved, the filtrate is clear, and the loss is reduced.

[0050] Example 3: The difference between this example and Example 1 lies in the preparation of the crude teplizumab peptide.

[0051] Preparation of crude teplizumab peptide: Weigh 10.0 g of the dried fully protected teplizumab peptide resin and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into a 250 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 2:1 at -5 to 0 °C, controlling the temperature during the dripping process to be less than 5 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with a 1000-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 8 min to filter 300 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to constant weight to obtain 5.7 g of crude teplizumab peptide solid, with a weight yield of 98.62% and a purity of 76.11%; compared with Example 2, the solid particles in the precipitated solution are slightly smaller, which affects the filtration speed, but the effect of the mixed solution of methyl tert-butyl ether / n-heptane = 2 / 1 used in this example method is within an acceptable range.

[0052] Example 4: The difference between this example and Example 1 lies in the preparation of the crude teplizumab peptide.

[0053] Preparation of Tirzepatide crude peptide: Weigh 10.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into a 250 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at 0-5 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate white solid. Stir in a water bath at 0-5 °C for 10 min, filter under reduced pressure with a 1000-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast. It takes about 8 min to filter 300 mL of the precipitated solution. Take out the filter cake and wash it three times by slurrying with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to constant weight to obtain 5.7 g of tirzepatide crude peptide solid, with a weight yield of 98.62% and a purity of 75.84%. Compared with Example 3, the solid particles in the precipitated solution are slightly smaller, which affects the filtration speed. However, the initial temperature of the mixed solution designed in the method of this example and the maximum temperature during the dripping process of the cleavage filtrate are both within an acceptable range. Considering the large heat release when dripping the cleavage solution, a certain temperature fluctuation space is reserved. It is preferred that the temperature does not exceed 10 °C when dripping at -5 to 0 °C for the poorly mixed solution.

[0054] Example 5: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0055] Preparation of Tirzepatide crude peptide: Weigh 10.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into a 200 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate white solid. Stir in a water bath at 0-5 °C for 10 min, filter under reduced pressure with a 1000-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast. It takes about 4 min to filter 250 mL of the precipitated solution. Take out the filter cake and wash it three times by slurrying with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to constant weight to obtain 5.6 g of tirzepatide crude peptide solid, with a weight yield of 96.88% and a purity of 76.12%. The difference is relatively small compared with Examples 2 and 4.

[0056] Example 6: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0057] Preparation of crude teplizumab peptide: Weigh 10.0 g of the dried fully protected teplizumab peptide resin and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into 300 mL of a mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with a 1000-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 7 min to filter 350 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to a constant weight to obtain 5.8 g of crude teplizumab peptide solid, with a weight yield of 100.35% and a purity of 75.87%; compared with Examples 2, 4, and 5, the weight of the crude peptide is higher, and the other differences are smaller.

[0058] Example 7: The difference between this example and Example 1 lies in the preparation of crude teplizumab peptide.

[0059] Preparation of crude teplizumab peptide: Weigh 10.0 g of the dried fully protected teplizumab peptide resin and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into 250 mL of a mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 5 min to filter 300 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to a constant weight to obtain 5.7 g of crude teplizumab peptide solid, with a weight yield of 98.62% and a purity of 76.03%, which is basically the same as that of Examples 2 and 4.

[0060] Example 8: The difference between this example and Example 1 lies in the preparation of crude teplizumab peptide.

[0061] Preparation of Tirzepatide crude peptide: Weigh 10.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, wash the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into a 250 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C, controlling the temperature during the dripping process to be less than 10 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with a 500-mesh filter cloth, and the filtrate is slightly turbid with a relatively fast filtration speed. It takes about 3 min to filter 300 mL of the precipitated solution; take out the filter cake and wash it three times by pulping with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to a constant weight to obtain 5.6 g of tirzepatide crude peptide solid, with a weight yield of 96.88% and a purity of 75.89%. There may be a small amount of loss during the filtration of the precipitated solution compared with Examples 2, 4, and 7.

[0062] Example 9: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0063] Preparation of Tirzepatide crude peptide: Weigh 10.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 50.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, wash the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drip the filtrate into a 250 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C, controlling the temperature during the dripping process to be less than 10 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with a 1500-mesh filter cloth, and the filtrate is clear with a relatively slow filtration speed. It takes about 14 min to filter 300 mL of the precipitated solution; take out the filter cake and wash it three times by pulping with 30 mL of methyl tert-butyl ether each time, filter under reduced pressure, and dry to a constant weight to obtain 5.7 g of tirzepatide crude peptide solid, with a weight yield of 98.62% and a purity of 75.96%. The filtration speed of the precipitated solution is slower compared with Examples 2, 4, 7, and 8.

[0064] Example 10: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0065] Preparation of Tirzepatide crude peptide: Weigh 20.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 100.0 mL of the cleavage solution. The cleavage solution is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 20 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrates into 500 mL of a mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 10 min to filter 600 mL of the precipitated solution. Take out the filter cake and wash it by pulping with 60 mL of methyl tert-butyl ether each time for three times, filter under reduced pressure, and dry to constant weight to obtain 11.3 g of tirzepatide crude peptide solid, with a weight yield of 97.75% and a purity of 70.16%. Compared with Example 2, the purity of the crude peptide is lower.

[0066] Example 11: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0067] Preparation of Tirzepatide crude peptide: Weigh 20.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 100.0 mL of the cleavage solution. The cleavage solution is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 30 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrates into 500 mL of a mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate a white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 10 min to filter 600 mL of the precipitated solution. Take out the filter cake and wash it by pulping with 60 mL of methyl tert-butyl ether each time for three times, filter under reduced pressure, and dry to constant weight to obtain 11.4 g of tirzepatide crude peptide solid, with a weight yield of 98.62% and a purity of 76.02%. Compared with Example 2 and 10, the purity of the crude peptide is lower.

[0068] Example 12: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0069] Preparation of Tirzepatide crude peptide: Weigh 20.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 100.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 2.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrates into a 500 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dropping process to be less than 10 °C to precipitate white solids. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 10 min to filter 600 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 60 mL / time of methyl tert-butyl ether, filter under reduced pressure, and dry to constant weight to obtain 11.4 g of tirzepatide crude peptide solid, with a weight yield of 98.62% and a purity of 72.25%. Compared with Example 2, the purity of the crude peptide is lower.

[0070] Example 13: The difference between this example and Example 1 lies in the preparation of the tirzepatide crude peptide.

[0071] Preparation of Tirzepatide crude peptide: Weigh 20.0 g of the dried fully protected peptide resin of tirzepatide and pour it into 100.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 4.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrates into a 500 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dropping process to be less than 10 °C to precipitate white solids. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 10 min to filter 600 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 60 mL / time of methyl tert-butyl ether, filter under reduced pressure, and dry to constant weight to obtain 11.4 g of tirzepatide crude peptide solid, with a weight yield of 98.62% and a purity of 76.13%.

[0072] Example 14: The difference between this example and Example 1 lies in the preparation of the tirzepatide crude peptide.

[0073] Preparation of Tirzepatide crude peptide: Weigh 20.0 g of the dried fully protected peptide resin of tirzepatide, pour it into 100.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5, and carry out cleavage at 25 °C for 5.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrate into a 500 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 10 min to filter 600 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 60 mL / time of methyl tert-butyl ether, filter under reduced pressure, and dry to constant weight to obtain 11.5 g of tirzepatide crude peptide solid, with a weight yield of 99.48% and a purity of 75.87%.

[0074] Example 15: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0075] Preparation of Tirzepatide crude peptide: Weigh 30.0 g of the dried fully protected peptide resin of tirzepatide, pour it into 120.0 mL of the cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5, and carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrate into a 600 mL mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C. Control the temperature during the dripping process to be less than 10 °C to precipitate white solid. Stir in a water bath at 0 to 5 °C for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear. The filtration speed is relatively fast, and it takes about 10 min to filter 720 mL of the precipitated solution; take out the filter cake and wash it three times by slurrying with 90 mL / time of methyl tert-butyl ether, filter under reduced pressure, and dry to constant weight to obtain 17.3 g of tirzepatide crude peptide solid, with a weight yield of 99.77% and a purity of 74.35%. Compared with Example 2, the differences in the weight yield and purity of the crude peptide are not significant.

[0076] Example 16: The difference between this example and Example 1 lies in the preparation of tirzepatide crude peptide.

[0077] Preparation of Tirzepatide crude peptide: Weigh 30.0 g of dried fully protected Tirzepatide peptide resin and pour it into 180.0 mL of cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 3.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then slowly drip the filtrates into 900 mL of a mixed solution of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 at -5 to 0 °C, controlling the temperature during the dropping process to be less than 10 °C to precipitate a white solid. Stir at 0 to 5 °C in a water bath for 10 min, filter under reduced pressure with an 800-mesh filter cloth, and the filtrate is clear with a moderate filtration rate. It takes about 22 min to filter 1080 mL of the precipitated solution; take out the filter cake and wash it by slurrying three times with 90 mL / portion of methyl tert-butyl ether, filter under reduced pressure, and dry to constant weight to obtain 17.3 g of Tirzepatide crude peptide solid, with a weight yield of 99.77% and a purity of 76.78%. Compared with Example 2 and Example 14, there is little difference in the weight yield and purity of the crude peptide.

[0078] Example 17: The difference between this example and Example 1 lies in the preparation of Tirzepatide crude peptide.

[0079] Preparation of Tirzepatide crude peptide: Weigh 30.0 g of dried fully protected Tirzepatide peptide resin and pour it into 240.0 mL of cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Carry out cleavage at 25 °C for 2.0 h. After reaching the cleavage time, filter the cleavage reaction solution, rinse the filter cake with a small amount of TFA once, combine the filtrates, and then pour the filtrates into 2000 mL of pre-cooled methyl tert-butyl ether at <-10 °C to precipitate a white solid, centrifuge, discard the upper clear liquid, wash the obtained solid by slurrying three times with 700 mL / portion of methyl tert-butyl ether and centrifuge, and dry the solid to constant weight to obtain 17.8 g of Tirzepatide crude peptide solid, with a weight yield of 102.65% and a purity of 66.42%.

[0080] Example 18: Purification of Tirzepatide crude peptide: Dissolve the Tirzepatide crude peptide in acetonitrile water with a volume fraction of 30%, and filter the solution with a 0.45 µm filter membrane for standby. Use a C18 preparative column with an inner diameter of 150 mm, the mobile phase is a system of 0.1% TFA / water - 0.1% TFA / acetonitrile with a volume fraction, the sample loading amount is 30 g / portion, the flow rate is 500 mL / min, and gradient elution is carried out; inject samples cyclically before and after the peak to obtain a purified peptide solution, and after desalting, lyophilize to obtain Tirzepatide purified peptide.

[0081] Example 19: The difference between this example and Example 2 lies in the preparation of the amino acid reagent activation solution.

[0082] Preparation of the activation solution of the amino acid reagent Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol to 2 L of DMF. Add the condensing agent DIC at 5 °C and let it stand for activation for 10 min to obtain the activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of the amino acid reagent: activator: condensing agent is 1:1:1. 348.8 g of the fully protected peptide resin of telotristat was prepared.

[0083] Preparation of 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol: Add 17.6 g of 2-chloro-6-fluoronitrobenzene, 12 g of 7-azaindole, 14 g of anhydrous K2CO3, 35 mL of acetonitrile, and 5 mmol of PEG-600 to a 250 mL flask. Stir magnetically and reflux for 10 h. Cool to room temperature, and a solid precipitates. Recrystallize with ethanol, filter by suction, and dry with an infrared lamp to obtain 1-(3-chloro-2-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine. 1 1H NMR (CDCl3, 400 MHz): δ 8.51 (1H, d), δ 8.43 (1H, d), δ 7.75 - 7.78 (3H, m), δ 7.36 (1H, m), δ 7.26 (1H, d), δ 6.79 (1H, d). EI-MS m / z: 273.03 M peak, relative intensity 100%, 275.03 M+2 peak, relative intensity 32.5%, 274.03 M+1 peak, relative intensity 15.2%.

[0084] Take 251 mg of 1-(3-chloro-2-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine, 150 mg of hydrazine hydrate, and 250 mg of isopropanol. Heat to 110 °C and react for 5 h. After the reaction, neutralize with 40 wt% NaOH solution, distill off the excess hydrazine hydrate and the solvent, adjust the pH value to 3.2 with hydrochloric acid, filter by suction and wash with 5 wt% ice saline. After drying, separate and purify by silica gel column chromatography, using chloroform / methanol with a volume ratio of 3:1 - 95:5 as the eluent to obtain 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol. 11H NMR (CDCl3, 400 MHz): δ 8.51 (1H, d), δ 8.43 (1H, d), δ 7.96 (1H, m), δ 7.40 - 7.43 (2H, m), δ 7.36 (1H, m), δ 7.26 (1H, d), δ 6.79 (1H, d), δ 2.67 (1H, s). EI-MS m / z: 251.08 M peak, relative intensity 100%, 252.08 M+1 peak, relative intensity 15.9%.

[0085] Example 20: The difference between this example and Example 19 lies in the preparation of the amino acid reagent activation solution.

[0086] Preparation of the amino acid reagent activation solution of Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator to 2 L of DMF, add the condensing agent DIC at 5°C and let it stand for activation for 10 min to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of amino acid reagent: activator: condensing agent is 1:1:1, and the activator is composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol with a molar ratio of 2:1. 345.6 g of the fully protected peptide resin of tezepelumab was prepared.

[0087] Compared with the 333.1 g of the fully protected peptide resin of tezepelumab prepared in Example 2, those in Example 19 and Example 20 are higher, and Example 19 is slightly higher than Example 20.

[0088] Example 21: The difference between this example and Example 20 lies in the preparation of the amino acid reagent activation solution.

[0089] Preparation of the amino acid reagent activation solution of Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator to 2 L of a solvent composed of DMF / 1,1-diisopropoxytriethylamine / 4-phenyl-1-butene with a volume ratio of 8:2:1, add the condensing agent DIC at 5°C and let it stand for activation for 10 min to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of amino acid reagent: activator: condensing agent is 1:1:1, and the activator is composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol with a molar ratio of 2:1. 341.5 g of the fully protected peptide resin of tezepelumab was prepared.

[0090] Example 22: The difference between this example and Example 20 lies in the preparation of the amino acid reagent activation solution.

[0091] Preparation of the amino acid reagent activation solution of Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator to 2 L of a solvent composed of DMF / 1,1-diisopropoxytriethylamine with a volume ratio of 4:1. Add the condensing agent DIC and let it stand for activation at 5 °C for 10 min to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of the amino acid reagent: activator: condensing agent is 1:1:1. The activator is composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol with a molar ratio of 2:1. 344.4 g of the fully protected peptide resin of tezepelumab is obtained.

[0092] Example 23: The difference between this example and Example 20 lies in the preparation of the amino acid reagent activation solution.

[0093] Preparation of the amino acid reagent activation solution of Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator to 2 L of a solvent composed of DMF / 4-phenyl-1-butene with a volume ratio of 8:1. Add the condensing agent DIC and let it stand for activation at 5 °C for 10 min to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of the amino acid reagent: activator: condensing agent is 1:1:1. The activator is composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol with a molar ratio of 2:1. 347.4 g of the fully protected peptide resin of tezepelumab is obtained.

[0094] Example 24: The difference between this example and Example 2 lies in the preparation of the amino acid reagent activation solution.

[0095] Preparation of the amino acid reagent activation solution of Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator to 2 L of a solvent composed of DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene with a volume ratio of 8:2:1. Add the condensing agent DIC at 5°C and let it stand for activation for 10 min to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of the amino acid reagent:activator:condensing agent is 1:1:1. The activator is composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol with a molar ratio of 2:1. 330.7 g of the fully protected peptide resin of telotristat ethyl is prepared.

[0096] Example 25: The difference between this example and Example 19 lies in the preparation of the amino acid reagent activation solution.

[0097] Preparation of the amino acid reagent activation solution of Fmoc-Ser(tBu)-OH: Add 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activator to 2 L of a solvent composed of DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene with a volume ratio of 8:2:1. Add the condensing agent DIC at 5°C and let it stand for activation for 10 min to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH, where the molar ratio of the amino acid reagent:activator:condensing agent is 1:1:1. The activator is composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol with a molar ratio of 2:1. 350.6 g of the fully protected peptide resin of telotristat ethyl is prepared.

[0098] Test Example 1: 1. Measure the crude peptide weight yields of the preparation steps of the telotristat ethyl crude peptides in Example 2 and Examples 19 - 25. The measurement results are shown in Figure 1 .

[0099] It can be seen from Figure 1 that there are no significant differences in the crude peptide yields of Example 2, Examples 19, 20, 21, 22, 23, 24, and 25.

[0100] 2. Measure the purities of the telotristat ethyl crude peptides obtained from the preparation steps of the telotristat ethyl crude peptides in Example 2 and Examples 19 - 25. The measurement results are shown in Figure 2 .

[0101] It can be seen from Figure 2It can be seen that the purity of the crude peptide prepared in Example 19 has no obvious difference from that in Example 2, and the purity of the crude peptide in Example 20 is higher than that in Example 19, indicating that during the coupling reaction of amino acids with 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol as the activator, the by-products do not increase significantly. When HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol are used together as activators in a certain proportion, the generation of by-products can be reduced; compared with Examples 20, 22, and 23, the purity of the crude peptide prepared in Example 21 is higher, while there is no obvious difference among Examples 20, 22, and 23. Compared with Example 2, there is no obvious difference in the purity of the crude peptide between Example 24 and Example 19. Compared with Example 19, the purity of the crude peptide prepared in Example 25 is higher, indicating that when the activator in the preparation of the amino acid reagent activation solution includes 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol, using DMF / 1,1-diisopropoxytriethylamine / 4-phenyl-1-butene in a ratio of 7-9:2-3:1-2 as the solvent can reduce the generation of by-products.

[0102] 3. Purify the crude telotristat peptides prepared in Examples 2 and 19-25, and determine the total yield of telotristat. The determination results are shown in Figure 3 .

[0103] From Figure 3It can be seen that the overall yield of tirzepatide in Example 19 is higher than that in Example 2, and the overall yield of tirzepatide in Example 20 is significantly higher than that in Example 19, indicating that compared with HOBT, 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol as an activator can promote the amino acid coupling reaction and increase the yield of the target peptide. When HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol are used together as activators in a certain proportion, they can play a synergistic role, not only promoting the amino acid coupling reaction but also reducing the generation of by-products, thereby increasing the yield of the target peptide. Compared with Example 20, Example 22, and Example 23, the overall yield of tirzepatide prepared in Example 21 is higher, while the differences among Example 20, Example 22, and Example 23 are not obvious. Compared with Example 2, the overall yield of tirzepatide in Example 24 has no obvious difference. Compared with Example 19, the overall yield of tirzepatide prepared in Example 25 is relatively high, indicating that when 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol is used as the activator in the preparation of the amino acid reagent activation solution, using DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a ratio of 7-9:2-3:1-2 as the solvent can effectively promote the activation of amino acids, increase the degree of the coupling reaction, and improve the yield of the target peptide.

[0104] The above-described embodiments and / or implementation manners are merely used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0105] Specific examples are used in this article to elaborate on the principles and implementation manners of the present application. The descriptions of the above embodiments are only used to help understand the method and its core idea of the present application. The above are only the preferred implementation manners of the present application. It should be noted that due to the limited nature of written expression and the objectively infinite specific structures, for those of ordinary skill in the art in this technical field, without departing from the principles of the present application, several improvements, refinements, or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes, or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. A method for preparing tirzepatide, characterized in that, It includes the following steps: S1. Using resin as a solid-phase carrier, the main chain is synthesized sequentially from the C-terminus to the N-terminus, then the side-chain protecting group of lysine is removed, and the side chain of telotristat ethyl is coupled; or the side chain of telotristat ethyl is combined with the side chain of lysine and directly introduced into the sequence during the solid-phase synthesis resin process to obtain the fully protected telotristat ethyl peptide resin; S2. Adding a cleavage solution to cleave the fully protected telotristat ethyl peptide resin, filtering, slowly adding the filtrate into a poor solution at -20~10°C, controlling the temperature of the sedimentation solution not to exceed 15°C during dropping, precipitating the solid precipitate of telotristat ethyl, filtering, slurrying and washing, and drying the solid precipitate of telotristat ethyl to obtain the crude telotristat ethyl; S3. Separating, purifying, transsalting, and freeze-drying the crude product to obtain the finished telotristat ethyl; In step S2, the poor solution includes methyl tert-butyl ether and n-heptane with a volume ratio of 1~2:

1.

2. The preparation method according to claim 1, characterized in that: In step S2, the poor solution is composed of methyl tert-butyl ether and n-heptane with a volume ratio of 1:1 or 2:

1.

3. The preparation method according to claim 1, characterized in that: The preparation method of the fully protected telotristat ethyl peptide resin includes: a. Removing the Fmoc protection on the resin with a deprotecting agent to obtain the deprotected resin; b. Preparing an amino acid activation solution of Fmoc-Ser(tBu)-OH, adding it to the deprotected resin obtained in step a for coupling reaction to obtain Fmoc-Ser(tBu)-resin; c. Repeating the steps of deprotection, preparing the amino acid activation solution and coupling reaction, coupling Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH in turn. After the coupling is completed, the resin is shrunk with methanol and vacuum dried to obtain the fully protected telotristat ethyl peptide resin; The preparation method of the amino acid activation solution of Fmoc-Ser(tBu)-OH in step b is as follows: Add the amino acid reagent Fmoc-Ser(tBu)-OH and the activator into a solvent, add the condensing agent at 0-10°C, and let it stand for activation for 5-15 minutes to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH.

4. The preparation method according to claim 3, characterized in that, The activator is HOBT and / or 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol.

5. The preparation method according to claim 1, characterized in that, The substitution degree of the resin in step S1 is 0.4-0.6 mmol / g.

6. The preparation method according to claim 1, characterized in that: The cleavage solution in step S2 is selected from two or more of TFA, Tis, EDT, DODT, DTT, PhOH, and H2O.

7. The preparation method according to claim 1, characterized in that: The cleavage solution in step S2 is composed of TFA, Tis, and DODT in a volume ratio of 78-82:7-8:12-13.

8. The preparation method according to claim 1, characterized in that: The proportional relationship between the cleavage solution and the telotristat fully protected peptide resin in step S2 is 4-6 mL / g.

9. The preparation method according to claim 1, characterized in that: The volume ratio of the waste solution to the cleavage solution in step S2 is 4-6:

1.

10. The preparation method according to claim 1, characterized in that: The filtration method used for filtering the telotristat solid precipitate in step S2 is filtration with a G3-G5 sintered glass funnel or a filtration medium with 500-1500 mesh.

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