A preparation method of tilpotide

Abstract

The present invention provides a preparation method of tilpoitide, belongs to the technical field of polypeptide synthesis, the preparation method adopts a small amount of lysate for cracking, and uses a small amount of poor solution composed of methyl tert-butyl ether and n-heptane in a volume ratio of 1~2:1 to precipitate crude peptide, the precipitated particles are uniform and large, not easy to agglomerate, the crude peptide impurity level is lower, easy to subsequent purification steps, and the amount of waste liquid generated is small, the present invention also uses activator 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazole-1-ol to activate amino acids, the coupling reaction degree is high, and the target peptide yield is high. A preparation method of tilpoitide provided by the present invention has the advantages of being simple, economical, capable of mass production, low impurity content, high purification yield, and high yield.

Description

Technical Field

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

[0002] Tirzepatide is a dual agonist of the gastric inhibitory polypeptide and glucagon-like peptide-1 receptors. It is the world's first and currently only dual agonist of the GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) receptors. By combining the actions of two incretin hormones, GIP and GLP-1, this drug, administered once a week, is designed to improve glycemic control and assist with weight management. Tirzepatide has demonstrated excellent glucose-lowering and weight-reducing effects in multiple clinical trials, particularly in head-to-head comparisons with similar drugs. In the first half of 2024 alone, global sales of the drug reached $6.658 billion, and sales continue to grow. Due to its significant market demand, the drug remains designated as a critically needed drug by the FDA as of August 2024.

[0003] The current demand for tilpotide API (active pharmaceutical ingredient) is enormous, and the traditional cup centrifuge cannot meet the needs of large-scale commercial production. Therefore, it is urgent to change the current fragmentation and precipitation process to increase the particle size of the tilpotide precipitated solid. The use of a centrifuge with a filter medium such as a filter bag can greatly improve the filtration efficiency of the crude peptide precipitate, increase batch production, and thus meet commercial needs.

[0004] Prior art, such as Chinese invention patent publication number CN113330024A, discloses novel intermediates and methods useful in the manufacture of tirzepatide or a pharmaceutically acceptable salt thereof. It also discloses a method for precipitating a crude peptide from a lysis solution, comprising adding methyl tert-butyl ether (precooled to below -20°C) to a precooled lysis solution. The precipitation process is controlled at a temperature between -18°C and 5°C, and the resulting crude tirzepatide suspension is separated using a bag centrifuge to obtain a tirzepatide solid. This method requires continuous cooling of a large amount of solvent, placing high demands on the cooling equipment. Furthermore, the precipitation process is prone to the generation of large lumps, which deposit at the bottom of the reactor, making discharge difficult and requiring manual crushing or removal. Residual acids and ethers in the reactor and the material pose significant hazards to personnel. Summary of the Invention

[0005] The present invention provides a method for preparing tilpotide, which is simple, economical, and has the advantages of large production batches, low cost, low impurity content, and high purification yield.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0007] The present invention discloses a method for preparing tilpotide, comprising the following steps:

[0008] S1. Using the resin as a solid phase support, the main chain is synthesized sequentially from the C-terminus to the N-terminus, and then the side chain protecting group of lysine is removed and the side chain of telpotide is coupled; or the side chain of telpotide is combined with the side chain of lysine and directly introduced into the sequence during the solid phase resin synthesis process to obtain a fully protected peptide resin of telpotide;

[0009] S2. adding a cleavage solution to cleave the fully protected tilpotide peptide resin, filtering, and slowly adding the filtrate to a poor solution at -20 to 10° C., controlling the temperature of the sedimentation solution to not exceed 15° C. during the dropwise addition, to precipitate a tilpotide solid precipitate, filtering, beating, washing, and drying the tilpotide solid precipitate to obtain a crude tilpotide product;

[0010] S3, separating and purifying the crude product, converting the salt, and freeze-drying to obtain the finished product of tilpotide;

[0011] The poor solution in step S2 comprises methyl tert-butyl ether and n-heptane in a volume ratio of 1-2:1.

[0012] In some embodiments, the poor solution in step S2 is composed of methyl tert-butyl ether and n-heptane in a volume ratio of 1:1 or 2:1. Whether solid-phase stepwise coupling or long fragment condensation, when cleaving peptide resin or fully protected peptide, the crude peptide precipitation process is basically to pour the peptide-containing cleavage solution into 8-15 times the volume ratio of diethyl ether or methyl tert-butyl ether, and obtain the crude peptide solid through precipitation and sedimentation centrifugation. This type of operation generally requires the use of a cleavage solution with a volume of 8 to 15 times the weight of the peptide resin or fully protected peptide for the cleavage step, and then using ether with a volume of 8 to 15 times the volume of the cleavage solution to precipitate the crude peptide, and washing with ether 30 to 90 times the weight of the co-peptide resin three times, resulting in a final waste liquid volume of up to 102 to 330 times the weight of the peptide resin or fully protected peptide, which is extremely wasteful; in addition, the precipitate obtained by this precipitation process is viscous and has fine particles, which are easy to clog or pass through the filter medium. Therefore, a cup centrifuge is generally selected to collect the crude peptide wet product. However, this collection method not only causes the crude product to be wrapped with more impurities, affecting the purification and product quality of the subsequent stage, but also often leads to poor separation effect due to the small difference in the relative gravity between the solid and liquid, and it is easy to cause product loss due to pouring out the turbid upper liquid. The present invention adopts a poor solution of methyl tert-butyl ether and n-heptane in a specific ratio to precipitate the crude peptide, and can use a smaller amount of the poor solution to precipitate the crude tilpotide. The obtained crude tilpotide solid particles are uniform and larger and are not prone to agglomeration. The crude product can be collected by using a centrifuge with a filter medium, thereby improving the filtration efficiency of the crude peptide precipitation. The obtained crude peptide has fewer impurities and is convenient for subsequent purification steps.

[0013] In some embodiments, the lysis solution in step S2 is selected from two or more of TFA, Tis, EDT, DODT, DTT, PhOH, and H2O.

[0014] More preferably, the lysis solution in step S2 is composed of TFA, Tis, and DODT in a volume ratio of 78-82:7-8:12-13.

[0015] In some embodiments, the lysis time in step S2 is 2.0 to 5.0 hours.

[0016] Preferably, the lysis time in step S2 is 3.0 to 4.0 hours.

[0017] In some embodiments, the pyrolysis temperature in step S2 is 20-30°C.

[0018] In some embodiments, the resin in step S1 is a Sieber amide resin.

[0019] In some embodiments, the degree of substitution of the resin in step S1 is 0.4-0.6 mmol / g.

[0020] In some embodiments, the ratio of the lysate to the fully protected telportide peptide resin in step S2 is 4-6 mL / g.

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

[0022] In some embodiments, the filtration method used for filtering the solid precipitate of tilpotide in step S2 is filtering through a G3-G5 sand core funnel or a 500-1500 mesh filter medium.

[0023] Preferably, the filtration method used for filtering the solid precipitate of telpotide in the above step S2 is 800 mesh filter cloth.

[0024] In some embodiments, the temperature of the poor solution in step S2 is -5 to 0°C.

[0025] In some embodiments, in the above step S2, the temperature of the sedimentation liquid is controlled not to exceed 10°C during the dropwise addition.

[0026] In some embodiments, the volume ratio of the poor solution to the lysis solution in step S2 is 4-6:1.

[0027] In some embodiments, the solvent used for pulping and washing in step S2 is methyl tert-butyl ether.

[0028] In some embodiments, the ratio of methyl tert-butyl ether to telpotide fully protected peptide resin used in the beating and washing in step S2 is 2-4 mL / g, preferably 3 mL / g.

[0029] In some embodiments, the method for preparing the above-mentioned fully protected telpotide peptide resin comprises:

[0030] a. Remove the Fmoc protection on the resin with a deprotecting agent to obtain a deprotected resin;

[0031] b. Prepare an amino acid reagent activation solution of Fmoc-Ser(tBu)-OH, add it to the deprotected resin obtained in step a to carry out a coupling reaction to obtain Fmoc-Ser(tBu)-resin;

[0032] c. 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)-O H, 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, the resin is shrunk with methanol and dried in vacuo to obtain the fully protected peptide resin of tebulate.

[0033] In some embodiments, the preparation method of the amino acid activation solution in step c is: adding the amino acid reagent and the activator to a solvent, adding a condensing agent at 0-10° C. and standing for activation for 5-15 minutes to obtain the amino acid activation solution.

[0034] In some embodiments, the preparation method of the amino acid activation solution of Fmoc-Ser(tBu)-OH in the above step b is: adding the amino acid reagent Fmoc-Ser(tBu)-OH and the activator to a solvent, adding a condensing agent at 0~10°C and standing for activation for 5~15 minutes to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH.

[0035] In some embodiments, in the preparation of the amino acid activation solution in step c above, the amino acid reagent is selected according to the coupling order from 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)-O One of H, 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.

[0036] In some embodiments, the deprotecting agent is a Pip / DMF solution with a volume ratio of 20% to 30%.

[0037] In some embodiments, the 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 activating agent comprises 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 on the structure of the activator 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 component. The structure of pyrrolopyridine and the specific site where it is located greatly increase the activity of the activated ester, have a high ester aminolysis rate, and a high degree of amino acid coupling reaction, which can increase the yield of the target peptide. When used together with HOBT as an activator, the effect is better.

[0038] In some embodiments, the condensing agent is DCC or DIC.

[0039] In some embodiments, the solvent includes DMF.

[0040] In some embodiments, the solvent comprises DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a volume ratio of 7-9:2-3:1-2. A certain ratio of DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene as a solvent can facilitate the activation of the amino acid reagent, increase the degree of amino acid coupling reaction, and thereby increase the yield of the target peptide.

[0041] In some embodiments, the molar ratio of amino acid reagent:activating agent:condensing agent in the amino acid activation solution is 1:1:1.

[0042] In some embodiments, the molar ratio of resin to amino acid reagent in each coupling reaction is 1:3.

[0043] By optimizing the lysis solution, the present invention can use a smaller amount of lysis solution to complete the lysis, overcoming the problem of large amounts of waste liquid in the lysis step in the traditional industry. Furthermore, by optimizing the precipitate solution, the present invention can use a smaller amount of precipitate solution to precipitate crude tilpoitide. The resulting crude tilpoitide solid particles are uniform, large, and less prone to agglomeration. The use of a centrifuge equipped with a filter medium can greatly improve the filtration efficiency of the crude peptide precipitate, thereby increasing batch production and meeting commercial needs.

[0044] The present invention utilizes 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazole-1-ol and HOBT as activators in a coupling reaction system for solid-phase synthesis of telportide, thereby enhancing the degree of coupling reaction and the yield of the target peptide. Furthermore, the present invention utilizes DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a volume ratio of 7-9:2-3:1-2 as a solvent for preparing an amino acid activation solution, thereby facilitating the activation of the amino acid reagent, enhancing the degree of amino acid coupling reaction, and thereby increasing the yield of the target peptide.

[0045] The invention provides a preparation method of tilpotide, which is simple and economical and has the advantages of large production batch, low cost, low impurity content, high purification yield, high productivity, and the like.

[0046] As used herein, the following abbreviations have the meanings as given herein: “FMOC” means 9-fluorenylmethyloxycarbonyl, “Trt” means trityl, “Boc” means tert-butyloxycarbonyl, “tBu” means tert-butyl, “OtBu” means tert-butyl carbothiol, “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-azaheptadecanoic acid, and “PEG-600” means polyethylene glycol with an average molecular weight of 600. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The results are for determining the weight yield of the crude peptide in Test Example 1 of the present invention.

[0048] Figure 2 This is the result of measuring the purity of the crude peptide in Test Example 1 of the present invention.

[0049] Figure 3 This is the measurement result of the total yield of tilpotide in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is only for the purpose of making the content of this application easier to understand, and does not limit the scope of protection of this application; at the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following application will be described in detail with reference to the accompanying drawings and in combination with the embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments:

[0052] In the embodiments of the present invention, unless otherwise stated, the equipment and materials used in the present invention are purchased from the market.

[0053] Example 1:

[0054] A method for preparing crude tilpoxetine peptide:

[0055] Deprotection: Weigh 80.0 g of Sieber amide resin (40.0 mmol on a synthetic scale) with a substitution degree of 0.5 mmol / g into a solid-phase reaction column. Swell the resin with DMF for 30 minutes, then remove the solvent under vacuum. Add a 20% (v / v) Pip / DMF solution to remove the Fmoc residue for 30 minutes, then wash six times with DMF. 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.

[0056] Preparation of amino acid reagent activation solution: Add the amino acid reagent Fmoc-Ser(tBu)-OH and the activating agent 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, wherein the molar ratio of amino acid reagent: activating agent: condensing agent is 1:1:1.

[0057] Coupling reaction: Add the prepared Fmoc-Ser(tBu)-OH amino acid reagent activation solution to the obtained deprotected resin at a resin to amino acid reagent molar ratio of 1:3, coupling temperature 30°C, reaction for 3 hours, and wash with DMF three times after the coupling reaction to obtain Fmoc-Ser(tBu)-resin;

[0058] Repeat the deprotection, preparation of amino acid activation 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- After the coupling was completed, the resin was shrunk with methanol and dried in vacuo to obtain 333.1 g of telpotide fully protected peptide resin.

[0059] Preparation of crude telport peptide: Weigh 10.0 g of dried telport peptide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25 ° C for 3.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 250 mL of -5~0 ° C methyl tert-butyl ether. In butyl ether, the temperature of the dropwise addition process was controlled to be less than 5°C, a white solid precipitated, stirred in a water bath at 0~5°C for 10 minutes, and filtered under reduced pressure using a 1000-mesh filter cloth. The filtrate was slightly turbid and the filtration speed was extremely slow. It took 3 hours to filter about 300 mL of precipitate. The filter cake was taken out and then slurried and washed three times with 30 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 5.6 g of crude telportipeptide solid with a weight yield of 96.88% and a purity of 75.12%.

[0060] Example 2:

[0061] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0062] Preparation of crude tilpoitide peptide: Weigh 10.0 g of dried tilpoitide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25°C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 250 mL of a mixed solution of -5~0°C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The temperature during the addition process was controlled to be less than 5°C, a white solid precipitated, and the mixture was stirred in a water bath at 0-5°C for 10 minutes. The mixture was filtered under reduced pressure using a 1000-mesh filter cloth, and the filtrate was clarified. The filtration speed was fast, and it took 5 minutes to filter about 300 mL of the precipitate. The filter cake was removed and then slurried and washed three times with 30 mL of methyl tert-butyl ether, filtered under reduced pressure, and dried to a constant weight to obtain 5.7 g of a crude telportipeptide solid with a weight yield of 98.62% and a purity of 76.42%. Compared with Example 1, the filtration speed was greatly improved, the filtrate was clarified, and the loss was reduced.

[0063] Example 3:

[0064] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0065] Preparation of crude tilpoitide peptide: Weigh 10.0 g of dried tilpoitide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25°C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 250 mL of a mixed solution of -5~0°C methyl tert-butyl ether and n-heptane in a volume ratio of 2:1. The temperature of the addition process is controlled to be less than 5°C, and a white solid is precipitated. , stirred in a water bath at 0-5°C for 10 minutes, filtered under reduced pressure with a 1000-mesh filter cloth, and the filtrate was clarified. The filtration speed was relatively fast, and it took 8 minutes to filter about 300 mL of precipitate. The filter cake was taken out and then slurried and washed three times with 30 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 5.7 g of crude telportipeptide solid, with a weight yield of 98.62% and a purity of 76.11%. Compared with Example 2, the solid particles in the precipitate were slightly smaller, which affected the filtration speed, but the effect of the mixed solution of methyl tert-butyl ether / n-heptane = 2 / 1 used in the method of this embodiment was within an acceptable range.

[0066] Example 4:

[0067] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0068] Preparation of crude telport peptide: Weigh 10.0 g of dried telport peptide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is obtained by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Crack at 25°C for 3.0 h. After the cracking time is reached, filter the cracking reaction solution, rinse the filter cake once with a small amount of TFA, combine the filtrates, and then slowly drop the filtrate into 250 mL of a mixed solution of 0~5°C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The temperature of the addition process is controlled to be less than 10°C. A white solid is precipitated, stirred in a water bath at 0~5°C for 10 min, and filtered under reduced pressure with a 1000 mesh filter cloth. , the filtrate was clear and the filtration speed was fast. It took 8 minutes to filter about 300 mL of precipitate. The filter cake was taken out and then washed three times with 30 mL / time of methyl tert-butyl ether, filtered under reduced pressure and dried to constant weight to obtain 5.7 g of crude telportide peptide solid with a weight yield of 98.62% and a purity of 75.84%. Compared with Example 3, the solid particles in the precipitate were slightly smaller, which affected the filtration speed. However, the initial temperature of the mixed solution and the maximum temperature of the cleavage filtrate addition process designed by the method of this embodiment were within an acceptable range. Considering the large amount of heat released when the cleavage solution was added, a certain temperature fluctuation space was reserved. It is preferred that the temperature of the poorly mixed solution be -5~0°C and the temperature not exceed 10°C when added dropwise.

[0069] Example 5:

[0070] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0071] Preparation of crude telport peptide: Weigh 10.0 g of dried telport peptide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25 ° C for 3.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 200 mL of -5~0 ° C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. In the mixed solution, the temperature of the dropwise addition process was controlled to be less than 10°C, a white solid precipitated, and the mixture was stirred in a water bath at 0-5°C for 10 min. The mixture was filtered under reduced pressure using a 1000 mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 4 min to filter about 250 mL of the precipitate. The filter cake was removed and then slurried and washed three times with 30 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 5.6 g of crude telportin peptide solid with a weight yield of 96.88% and a purity of 76.12%. The difference was small compared with Examples 2 and 4.

[0072] Example 6:

[0073] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0074] Preparation of crude tilpoitide peptide: Weigh 10.0 g of dried tilpoitide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25°C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 300 mL of a mixed solution of -5~0°C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The temperature during the addition process was controlled to be less than 10°C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5°C for 10 minutes. The mixture was filtered under reduced pressure using a 1000-mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 7 minutes to filter about 350 mL of the precipitate. The filter cake was removed and then slurried and washed three times with 30 mL of methyl tert-butyl ether, filtered under reduced pressure, and dried to a constant weight to obtain 5.8 g of a crude telportipeptide solid with a weight yield of 100.35% and a purity of 75.87%. The weight of the crude peptide was higher than that of Examples 2, 4, and 5, and the other differences were minor.

[0075] Example 7:

[0076] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0077] Preparation of crude tilpoitide peptide: Weigh 10.0 g of dried tilpoitide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25 ° C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 250 mL of -5~0 ° C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The mixed solution was added, the temperature of the dropwise addition process was controlled to be less than 10 ° C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5 ° C for 10 min. The mixture was filtered under reduced pressure with an 800 mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 5 min to filter about 300 mL of the precipitate. The filter cake was taken out and then slurried and washed three times with 30 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 5.7 g of crude telportipeptide solid with a weight yield of 98.62% and a purity of 76.03%, which was basically the same as that of Examples 2 and 4.

[0078] Example 8:

[0079] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0080] Preparation of crude tilpoitide peptide: Weigh 10.0 g of dried tilpoitide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25°C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 250 mL of a mixed solution of -5~0°C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The temperature during the addition process was controlled to be less than 10°C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5°C for 10 min. The mixture was filtered under reduced pressure using a 500-mesh filter cloth. The filtrate was slightly turbid and the filtration speed was fast. It took 3 min to filter about 300 mL of the precipitate. The filter cake was removed and then slurried and washed three times with 30 mL of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 5.6 g of crude telportipeptide solid with a weight yield of 96.88% and a purity of 75.89%. Compared with Examples 2, 4, and 7, there may be a small amount of loss during filtration of the precipitate.

[0081] Example 9:

[0082] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0083] Preparation of crude telport peptide: Weigh 10.0 g of dried telport peptide fully protected peptide resin and pour it into 50.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25 ° C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 250 mL of a mixed solution of -5~0 ° C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. , the temperature of the dropwise addition process was controlled to be less than 10°C, a white solid was precipitated, stirred in a water bath at 0-5°C for 10 min, filtered under reduced pressure with a 1500 mesh filter cloth, the filtrate was clarified, the filtration speed was slow, and it took 14 min to filter about 300 mL of precipitate; the filter cake was taken out and then slurried and washed three times with 30 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 5.7 g of crude telportipeptide peptide solid, with a weight yield of 98.62% and a purity of 75.96%. Compared with Examples 2, 4, 7, and 8, the filtration speed of the precipitate was slower.

[0084] Example 10:

[0085] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0086] Preparation of crude telport peptide: Weigh 20.0 g of dried telport peptide fully protected peptide resin and pour it into 100.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 20 ° C for 3.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 500 mL of -5~0 ° C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. In the mixed solution, the temperature of the dropwise addition process was controlled to be less than 10 ° C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5 ° C for 10 min. The mixture was filtered under reduced pressure with an 800 mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 10 min to filter about 600 mL of precipitate. The filter cake was taken out and then slurried and washed three times with 60 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 11.3 g of crude telportin peptide solid with a weight yield of 97.75% and a purity of 70.16%. The purity of the crude peptide was lower than that in Example 2.

[0087] Example 11:

[0088] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0089] Preparation of crude telport peptide: Weigh 20.0 g of dried telport peptide fully protected peptide resin and pour it into 100.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 30°C for 3.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 500 mL of a mixture of -5~0°C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The mixture was added to the solution under a temperature of less than 10 ° C. The mixture was stirred in a water bath at 0-5 ° C for 10 min, filtered under reduced pressure with an 800-mesh filter cloth, and the filtrate was clarified. The filtration speed was fast, and it took 10 min to filter about 600 mL of precipitate. The filter cake was taken out and washed three times with 60 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 11.4 g of crude telportipeptide solid with a weight yield of 98.62% and a purity of 76.02%. The purity of the crude peptide was lower than that of Examples 2 and 10.

[0090] Example 12:

[0091] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0092] Preparation of crude telport peptide: Weigh 20.0 g of dried telport peptide fully protected peptide resin and pour it into 100.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25 ° C for 2.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 500 mL of -5~0 ° C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. In the mixed solution, the temperature of the dropwise addition process was controlled to be less than 10 ° C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5 ° C for 10 min. The mixture was filtered under reduced pressure with an 800 mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 10 min to filter about 600 mL of precipitate. The filter cake was taken out and then slurried and washed three times with 60 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 11.4 g of crude telportin peptide solid with a weight yield of 98.62% and a purity of 72.25%. The purity of the crude peptide was lower than that in Example 2.

[0093] Example 13:

[0094] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0095] Preparation of crude telport peptide: Weigh 20.0 g of dried telport peptide fully protected peptide resin and pour it into 100.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25°C for 4.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 500 mL of a mixture of -5~0°C methyl tert-butyl ether and n-heptane. In a mixed solution with a volume ratio of 1:1, the temperature of the dropwise addition process was controlled to be less than 10°C, a white solid was precipitated, and the mixture was stirred in a water bath at 0~5°C for 10 minutes. The mixture was filtered under reduced pressure with an 800-mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 10 minutes to filter about 600 mL of precipitate. The filter cake was taken out and then slurried and washed three times with 60 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 11.4 g of crude telportin peptide solid with a weight yield of 98.62% and a purity of 76.13%.

[0096] Example 14:

[0097] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0098] Preparation of crude telport peptide: Weigh 20.0 g of dried telport peptide fully protected peptide resin and pour it into 100.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. The cleavage reaction is carried out at 25 ° C for 5.0 h. After the cleavage time is reached, the cleavage reaction solution is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 500 mL of a mixture of -5~0 ° C methyl tert-butyl ether and n-heptane. In a mixed solution with a volume ratio of 1:1, the temperature of the dropwise addition process was controlled to be less than 10°C, a white solid was precipitated, and the mixture was stirred in a water bath at 0~5°C for 10 minutes. The mixture was filtered under reduced pressure with an 800-mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 10 minutes to filter about 600 mL of precipitate. The filter cake was taken out and then slurried and washed three times with 60 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 11.5 g of crude telportin peptide solid with a weight yield of 99.48% and a purity of 75.87%.

[0099] Example 15:

[0100] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0101] Preparation of crude telport peptide: Weigh 30.0 g of dried telport peptide fully protected peptide resin and pour it into 120.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25 ° C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 600 mL of a mixed solution of -5~0 ° C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1 The temperature of the dropwise addition process was controlled to be less than 10°C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5°C for 10 min. The mixture was filtered under reduced pressure with an 800-mesh filter cloth and the filtrate was clarified. The filtration speed was relatively fast, and it took 10 min to filter about 720 mL of the precipitate. The filter cake was taken out and then slurried and washed three times with 90 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 17.3 g of a crude telportipeptide solid with a weight yield of 99.77% and a purity of 74.35%. Compared with Example 2, the weight yield and purity of the crude peptide were not much different.

[0102] Example 16:

[0103] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0104] Preparation of crude tilpoitide peptide: Weigh 30.0 g of dried tilpoitide fully protected peptide resin and pour it into 180.0 mL of cleavage solution, which is prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. Cleavage is carried out at 25°C for 3.0 h. After the cleavage time is reached, the cleavage reaction liquid is filtered, the filter cake is rinsed once with a small amount of TFA, the filtrate is combined, and the filtrate is slowly dripped into 900 mL of a mixed solution of -5~0°C methyl tert-butyl ether and n-heptane in a volume ratio of 1:1. The temperature of the dropwise addition process was controlled to be less than 10°C, a white solid was precipitated, and the mixture was stirred in a water bath at 0-5°C for 10 min. The mixture was filtered under reduced pressure using an 800-mesh filter cloth and the filtrate was clarified. The filtration speed was moderate, and it took 22 min to filter about 1080 mL of the precipitate. The filter cake was taken out and then slurried and washed three times with 90 mL / time of methyl tert-butyl ether, filtered under reduced pressure, and dried to constant weight to obtain 17.3 g of crude telportipeptide solid with a weight yield of 99.77% and a purity of 76.78%. Compared with Examples 2 and 14, the weight yield and purity of the crude peptide were not much different.

[0105] Example 17:

[0106] This example differs from Example 1 in the preparation of crude tilpoxetine peptide.

[0107] Preparation of crude telpotide peptide: 30.0 g of dried telpotide fully protected peptide resin was weighed and poured into 240.0 mL of cleavage solution, which was prepared by mixing TFA, Tis, and DODT in a volume ratio of 80:7.5:12.5. The mixture was cleaved at 25°C for 2.0 h. After the cleavage time was reached, the cleavage reaction liquid was filtered, the filter cake was rinsed once with a small amount of TFA, the filtrate was combined, and the filtrate was poured into 2000 mL of pre-cooled <-10°C methyl tert-butyl ether to precipitate a white solid. The mixture was centrifuged and the supernatant was discarded. The obtained solid was slurried and washed with 700 mL / methylene tert-butyl ether and centrifuged three times. The solid was dried to constant weight to obtain 17.8 g of crude telpotide peptide solid with a weight yield of 102.65% and a purity of 66.42%.

[0108] Example 18:

[0109] Purification of crude tilpotide: Dissolve crude tilpotide in 30% acetonitrile / water and filter the solution through a 0.45µm filter. Use a 150mm ID C18 preparative column with a mobile phase consisting of 0.1% TFA / water-0.1% TFA / acetonitrile. Load 30g / sample at a flow rate of 500mL / min for gradient elution. Repeated injections were performed before and after the peak to obtain a refined peptide solution. After desalting, the purified tilpotide was lyophilized.

[0110] Example 19:

[0111] The difference between this embodiment and embodiment 2 lies in the preparation of the amino acid reagent activation solution.

[0112] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of the activating agent 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol were added to 2 L of DMF. The condensing agent DIC was added and the mixture was incubated at 5°C for 10 minutes to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1. 348.8 g of the fully protected telportide peptide resin was obtained.

[0113] Preparation of 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol:

[0114] A 250 mL flask was added with 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. The mixture was refluxed with magnetic stirring for 10 h. The solid was cooled to room temperature. The solid was precipitated and recrystallized from ethanol, filtered, and dried under an infrared lamp to obtain 1-(3-chloro-2-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine. 1 H NMR (CDCl3, 400 MHz): δ8.51 (1H, d), δ8.43 (1H, d), δ7.75-7.78 (3H, m), δ7.36 (1H, m), δ7.26 (1H, d), δ6.79 (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%.

[0115] 251 mg of 1-(3-chloro-2-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine, 150 mg of hydrazine hydrate, and 250 mg of isopropanol were heated to 110°C for 5 h. After the reaction, the mixture was neutralized with 40 wt% NaOH solution, and the excess hydrazine hydrate and solvent were evaporated. The pH was adjusted to 3.2 with hydrochloric acid, and the mixture was filtered and washed with 5 wt% ice brine. After drying, the mixture was separated and purified by silica gel column chromatography using chloroform / methanol in a volume ratio of 3:1 to 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. 1H NMR (CDCl3, 400 MHz): δ8.51 (1H, d), δ8.43 (1H, d), δ7.96 (1H, m), δ7.40-7.43 (2H, m), δ7.36 (1H, m), δ7.26 (1H, d), δ6.79 (1H, d), δ2.67 (1H, s). EI-MS m / z: 251.08 M peak relative intensity 100%, 252.08 M+1 peak relative intensity 15.9%.

[0116] Example 20:

[0117] The difference between this example and Example 19 lies in the preparation of the amino acid reagent activation solution.

[0118] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of an activating agent were added to 2 L of DMF. The condensing agent DIC was added and the mixture was activated at 5°C for 10 min to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1, and the activating agent was composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol at a molar ratio of 2:1. 345.6 g of fully protected telpotide peptide resin was obtained.

[0119] Compared with 333.1 g of the fully protected telpoitide peptide resin prepared in Example 2, the yields of Examples 19 and 20 were higher, and Example 19 was slightly higher than Example 20.

[0120] Example 21:

[0121] The difference between this example and Example 20 lies in the preparation of the amino acid reagent activation solution.

[0122] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of an activating agent were added to 2 L of a solvent consisting of DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a volume ratio of 8:2:1. The condensing agent DIC was added and the mixture was activated at 5°C for 10 minutes to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1, and the activating agent was composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol in a molar ratio of 2:1. 341.5 g of the fully protected telpotide peptide resin was obtained.

[0123] Example 22:

[0124] The difference between this example and Example 20 lies in the preparation of the amino acid reagent activation solution.

[0125] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of an activating agent were added to 2 L of a solvent consisting of DMF / 1,1-diisopropoxytrimethylamine in a volume ratio of 4:1. The condensing agent DIC was added and the mixture was activated at 5°C for 10 minutes to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1, and the activating agent was composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol in a molar ratio of 2:1. 344.4 g of fully protected telpotide peptide resin was obtained.

[0126] Example 23:

[0127] The difference between this example and Example 20 lies in the preparation of the amino acid reagent activation solution.

[0128] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of an activating agent were added to 2 L of a solvent consisting of DMF / 4-phenyl-1-butene in a volume ratio of 8:1. DIC, a condensing agent, was added and activated at 5°C for 10 minutes to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1, and the activating agent was composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol in a molar ratio of 2:1. 347.4 g of the fully protected telpotide peptide resin was obtained.

[0129] Example 24:

[0130] The difference between this embodiment and embodiment 2 lies in the preparation of the amino acid reagent activation solution.

[0131] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of an activating agent were added to 2 L of a solvent consisting of DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a volume ratio of 8:2:1. The condensing agent DIC was added and the mixture was activated at 5°C for 10 minutes to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1, and the activating agent was composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol in a molar ratio of 2:1. 330.7 g of fully protected telportide peptide resin was obtained.

[0132] Example 25:

[0133] The difference between this example and Example 19 lies in the preparation of the amino acid reagent activation solution.

[0134] Preparation of an amino acid reagent activation solution for Fmoc-Ser(tBu)-OH: 120 mmol of the amino acid reagent Fmoc-Ser(tBu)-OH and 120 mmol of an activating agent were added to 2 L of a solvent consisting of DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a volume ratio of 8:2:1. The condensing agent DIC was added and the mixture was activated at 5°C for 10 minutes to obtain an amino acid activation solution for Fmoc-Ser(tBu)-OH. The molar ratio of amino acid reagent: activating agent: condensing agent was 1:1:1, and the activating agent was composed of HOBT and 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol in a molar ratio of 2:1. 350.6 g of fully protected telpotide peptide resin was obtained.

[0135] Test Example 1:

[0136] 1. The crude peptide weight yield of the crude peptide preparation steps of Example 2 and Examples 19-25 was measured. The results are shown in Figure 1 .

[0137] Depend on Figure 1 It can be seen that there is no significant difference in the crude peptide yields of Example 2, Example 19, Example 20, Example 21, Example 22, Example 23, Example 24, and Example 25.

[0138] 2. The purity of crude telpotide peptide obtained by the preparation steps of crude telpotide peptide in Example 2 and Examples 19-25 was determined. The results are shown in FIG. Figure 2 .

[0139] Depend on Figure 2 It can be seen that the purity of the crude peptide obtained in Example 19 is not significantly different from that in Example 2, and the purity of the crude peptide in Example 20 is higher than that in Example 19, indicating that compared with HOBT, when 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol is used as an activator for the coupling reaction of amino acids, there is no significant increase in by-products, and 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 ratio to reduce the generation of by-products; compared with Examples 20 and 20, the purity of the crude peptide in Example 20 is higher than that in Example 19, indicating that when 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol is used as an activator, there is no significant increase in by-products. Compared with Example 22 and Example 23, the crude peptide obtained in Example 21 has higher purity, while there is no obvious difference between Example 20, Example 22 and Example 23. The purity of the crude peptide in Example 2 is not significantly different from that in Example 24. Compared with Example 19, the purity of the crude peptide obtained in Example 25 is higher, indicating that in the preparation of the amino acid reagent activation solution, when the activator includes 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazole-1-ol, using DMF / 1,1-diisopropoxytrimethylamine / 4-phenyl-1-butene in a ratio of 7~9:2~3:1~2 as the solvent can reduce the formation of by-products.

[0140] 3. The crude telpotide peptide prepared in Example 2 and Examples 19-25 was purified and the total yield of telpotide was determined. The results are shown in Table 3. Figure 3 .

[0141] Depend on Figure 3It can be seen that the total yield of tilpotide in Example 19 is higher than that in Example 2, and the total yield of tilpotide 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 improve the coupling reaction of amino acids and improve the yield of the target peptide. 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, which can play a synergistic role, not only improving the coupling reaction of amino acids, but also reducing the formation of by-products, thereby improving the yield of the target peptide. Compared with Example 20 and Example 20, Compared with Example 22 and Example 23, the total yield of tilpoitide obtained in Example 21 is higher, while the difference between Example 20, Example 22 and Example 23 is not obvious. The total yield of tilpoitide in Example 2 is not obvious compared with Example 24. Compared with Example 19, the total yield of tilpoitide obtained in Example 25 is higher, indicating that in the preparation of the amino acid reagent activation solution, when the activator is 7-(1H-pyrrolo[2,3-b]pyridin-1-yl)-1H-benzo[d][1,2,3]triazol-1-ol, 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 the amino acid, improve the degree of coupling reaction, and improve the yield of the target peptide.

[0142] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0143] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing tilpoitide, characterized in that: The steps include: S1, combining the tulposide side chain with the lysine side chain and directly introducing it into the sequence during the solid phase resin synthesis process to obtain the tulposide fully protected peptide resin; S2. adding a cleavage solution to cleave the fully protected tilpotide peptide resin, filtering, and slowly adding the filtrate to a poor solution at -20 to 10° C., controlling the temperature of the sedimentation solution to not exceed 15° C. during the dropwise addition, to precipitate a tilpotide solid precipitate, filtering, beating, washing, and drying the tilpotide solid precipitate to obtain a crude tilpotide product; S3, separating and purifying the crude product, converting the salt, and freeze-drying to obtain the finished product of tilpotide; In step S2, the poor solution is composed of methyl tert-butyl ether and n-heptane in a volume ratio of 1 to 2:1; The preparation method of the telpotide fully protected peptide resin comprises: a. Remove the Fmoc protection on the resin with a deprotecting agent to obtain a deprotected resin; b. Prepare an amino acid activation solution of Fmoc-Ser(tBu)-OH, add it to the deprotected resin obtained in step a to carry out a coupling reaction to obtain Fmoc-Ser(tBu)-resin; c. 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 the coupling is completed, the resin is shrunk with methanol and dried in vacuo to obtain the fully protected tebulate peptide resin; The preparation method of the amino acid activation solution of Fmoc-Ser(tBu)-OH in the step b is as follows: adding the amino acid reagent Fmoc-Ser(tBu)-OH and the activator to a solvent, adding a condensing agent and standing for activation at 0-10° C. for 5-15 minutes to obtain the amino acid activation solution of Fmoc-Ser(tBu)-OH; The activating agents are 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.

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

1.

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

4. The preparation method according to claim 1, wherein: In step S2, the lysis solution is selected from two or more of TFA, Tis, EDT, DODT, DTT, PhOH, and H2O.

5. The preparation method according to claim 1, wherein: In step S2, the lysis solution is composed of TFA, Tis, and DODT in a volume ratio of 78-82:7-8:12-13.

6. The preparation method according to claim 1, wherein: In step S2, the ratio of the lysate to the fully protected telportipeptide resin is 4-6 mL / g.

7. The preparation method according to claim 1, wherein: In step S2, the volume ratio of the poor solution to the lysis solution is 4-6:

1.

8. The preparation method according to claim 1, wherein: The filtration method used for filtering the tilpotide solid precipitate in step S2 is G3-G5 sand core funnel or 500-1500 mesh filter medium filtration.

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