Semi-synthesis method of semeglutide

Abstract

The invention relates to a semi-synthesis method of semeglutide, and belongs to the field of chemical synthesis. The semi-synthesis method mainly comprises the following steps: (1) activating a side chain; (2) connecting an activated side chain with an Arg34GLP-1 (9-37) 29 peptide; (3) activating the dipeptide; (4) connection of the activated dipeptide and the side chain 29 peptide conjugate; (5) removing a protecting group to obtain a final product, namely a crude product of the semeglutide; meanwhile, pretreatment of materials and post-treatment of products are optimized in the synthesis process. On one hand, a side chain and dipeptide activation method which is short in time consumption and high in conversion rate is provided, on the other hand, an intermediate pretreatment and post-treatment method which is convenient, rapid, beneficial to reaction and beneficial to cost reduction and efficiency improvement is provided, the characters of substrates / products in all the steps are improved, cost reduction, production specification expansion and efficiency improvement in industrial production are facilitated, and the method is suitable for industrial production. And the method has very important significance on industrial production of a semeglutide bulk drug.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis and relates to a semi-synthetic method of smegglutinin. Background Technology

[0002] Smeglucopyrithione, chemically named N-ε26-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)peptide, has the molecular formula C 187 H 291 N 45 O 59 Smegglutinin, with a molecular weight of 4113.64, is a long-acting GLP-1 (glucagon-like peptide-1) receptor agonist developed by Novo Nordisk. It is a once-weekly hypoglycemic drug with weight-loss effects. The main peptide chain structure of smegglutinin is a 29-peptide of Arg34GLP-1(9-37), which can be obtained through solid-phase peptide synthesis or gene recombination-based fermentation. Compared to human GLP-1, smegglutinin replaces lysine at position 34 with arginine to prevent the octadecanoic acid fatty chain from binding at the wrong position; lysine at position 26 (Lys) connects to a side chain composed of two aminoethoxyethoxyacetic acid, glutamic acid, and octadecanoic acid fatty chains; and the original alanine (Ala) at position 8 is replaced with the non-natural amino acid 2-aminoisobutyric acid. Smeglucopyrithione modified with the AEEA side chain can bind tightly to albumin, mask the hydrolysis site of DPP-4 enzyme, reduce renal excretion, prolong biological half-life, and achieve a long-lasting hypoglycemic effect.

[0003] The structure of smegglutinin is as follows:

[0004] Currently, there are patents related to the semi-synthetic preparation method of semaglutide raw material, namely CN115322250A, CN114790474A, CN118702800A and CN120365409A. The specific preparation method includes coupling the side chain with the main peptide chain, coupling the dipeptide with the main peptide chain, and removing the protecting group to obtain crude semaglutide. In the preparation of activated dipeptides, patent CN115322250A uses one or a combination of N-hydroxysuccinimide, dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, and p-nitrophenol as activators, with a reaction time of up to 12 hours. The solvent used in the coupling reaction is N,N-dimethylformamide, which is highly toxic to humans and incompatible with many acids, bases, and redox reagents. It poses a safety hazard of explosion in industrial production and has the disadvantages of being difficult to purify and prone to residues in scale-up production. The proportion of trifluoroacetic acid in the deprotection lysis solution is as high as 95%. Trifluoroacetic acid is highly volatile and corrosive, posing a significant safety hazard in industrial production.

[0005] In patent CN114790474A, to improve product purity, high-performance liquid chromatography (HPLC) is used to purify the intermediate dipeptide-main peptide chain conjugate, followed by concentration, dilution, and acid precipitation to obtain the corresponding solid for subsequent reactions. However, the cost consumed is disproportionate to the achieved effect. Patent CN118702800A also uses HPLC to purify the intermediate side chain-main peptide chain conjugate and freeze-dry it, which greatly reduces efficiency, and each step takes 10-24 hours, resulting in high time costs. Patent CN120365409A uses a side chain with both carboxyl groups protected by tert-butyl groups for activation, followed by a series of operations under nitrogen protection, including deprotection, crystallization, washing, and drying. The starting materials are expensive, the process is cumbersome, and takes about 10 hours. In the conjugation of the dipeptide and the main peptide chain, the molar ratio of the dipeptide to the main peptide chain is 3:1, resulting in low dipeptide utilization and significant material loss.

[0006] In addition, the post-processing methods of the above patents are not conducive to industrial production. For example, in the post-processing of dipeptide-main peptide chain conjugates, solids are obtained by direct acid precipitation. However, because the reaction solvent system contains a large amount of organic solvent, the precipitated solids will agglomerate and become sticky during the sedimentation process due to interference from the organic solvent. This can easily cause blockages and even increase cleaning costs during pipeline transportation in industrial production. In the post-processing of crude smegglutinin, methyl tert-butyl ether is used as a detergent for impurity removal. Methyl tert-butyl ether has the characteristics of low flash point, easy anesthetic and flammable and explosive properties. Its use should be minimized in industrial production to control safety risks.

[0007] In summary, a semi-synthetic method for semaglutide API is proposed to improve upon existing patented technologies, which suffer from cumbersome steps, long processing times, and low material utilization. Furthermore, methods for pretreatment and post-treatment of intermediates during the preparation process are developed. This method is of great significance for reducing the industrial production cost of semaglutide API, improving industrial efficiency, and expanding production specifications. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a semi-synthetic method for smegglutinin.

[0009] To achieve the above objectives, the present invention provides the following technical solution: 1. A semi-synthetic method for smegglutinin, characterized by comprising the following steps: (1) Under alkaline catalyst conditions, the activated side chain solution (intermediate I) was mixed with Arg34GLP-1(9-37)29 peptide solution to obtain intermediate II. The specific synthetic route is as follows:

[0010] After the reaction is complete, the organic solvent is evaporated, acid precipitation is performed, and the precipitate is collected to obtain intermediate II wet solid; or the precipitate is dried, slurried with dichloromethane to remove impurities, and dried again to obtain intermediate II powder. (2) Under alkaline catalyst conditions, the wet solid or powder of intermediate II obtained in step (1) is dissolved and mixed with the activated dipeptide solution (intermediate III) to react and obtain intermediate IV. The specific synthetic route is as follows:

[0011] After the reaction was completed, the reaction solution was poured into methyl tert-butyl ether, centrifuged and the solid was collected. The solid was then slurried with dichloromethane to remove impurities and dried to obtain intermediate IV powder. (3) Deprotecting intermediate IV with a deprotecting agent to obtain smegglutinin. The specific synthetic route is as follows:

[0012] After the reaction was completed, the reaction solution was poured into methyl tert-butyl ether, centrifuged and the solid was collected. The solid was then slurried with dichloromethane to remove impurities and dried to obtain smegglutinin powder. The alkaline catalyst in steps (1) and (2) is N,N-diisopropylethylamine; the solvent used to dissolve intermediate II in step (2) is N-methylpyrrolidone; and the deprotecting agent in step (3) is a mixed solvent of trifluoroacetic acid, water and triisopropylsilane.

[0013] Preferably, the 29 peptide in step (1) is initially in one of the following states: dry powder, wet solid or solution. It is prepared into a 29 peptide solution by adding water / N,N-diisopropylethylamine mixed solvent for reaction. Preferably, in step (1), the molar ratio of intermediate I to peptide 29 is 1~2:1, the reaction time is 0.5~2h, and the reaction temperature is 2~25℃; Preferably, the acid precipitation reagent in step (1) is glacial acetic acid, and the acid precipitation conditions are pH 4.7~4.9.

[0014] Preferably, in step (2), the molar ratio of intermediate II to intermediate III is 1:1~3, the reaction time is 4~24h, and the reaction temperature is 20~25℃; Preferably, in step (2), the volume ratio of the reaction solution to methyl tert-butyl ether is 1:6~12; and the treatment temperature is 0~20℃.

[0015] Preferably, in step (3), the volume ratio of trifluoroacetic acid, water and triisopropylsilane is 13.5:0.75:0.75; the reaction time is 2~4 h; the reaction temperature is 20~25℃; the volume ratio of the reaction solution to methyl tert-butyl ether is 1:6~12; and the treatment temperature is 0~20℃.

[0016] 2. Based on the semi-synthetic method of smegglutinin, this invention further proposes a method for activating the required side chain, as follows: under the condition of a coupling condensing agent, the side chain undergoes an esterification reaction to obtain intermediate I; The synthesis route is as follows:

[0017] After the reaction was completed, the reaction solution was filtered, evaporated to dryness, acetonitrile was added, filtered again, and acetonitrile was added to obtain intermediate I solution; The coupling condensing agents are N-hydroxysuccinimide and dicyclohexylcarbodiimide, and the reaction solvent used is dichloromethane.

[0018] Preferably, in the method, the molar ratio of the side chain, N-hydroxysuccinimide, and dicyclohexylcarbodiimide is 1:1.0~1.8:1.0~1.6, the reaction time is 2~5 h, and the reaction temperature is 2~25℃; Preferably, the volume ratio of acetonitrile added to the reaction liquid in the method is 2~10:1, and the concentration of intermediate I solution is 8~12 mg / mL.

[0019] 3. Based on the semi-synthetic method of smegglutinin, this invention also proposes an activation method for the required dipeptide, as detailed below: Under the conditions of a carboxylic acid activator, the dipeptide is activated to give intermediate III; The specific synthesis route is as follows:

[0020] After the reaction was completed, the organic solvent was evaporated, and N-methylpyrrolidone was added to prepare intermediate III solution; The carboxylic acid activator is N,N'-carbonyldiimidazole.

[0021] Preferably, in the method, the molar ratio of the dipeptide to N,N'-carbonyldiimidazole is 1:1~2, the reaction time is 0.3~2h, the reaction temperature is 20~25℃, and the concentration of intermediate III solution is 0.2 mol / mL.

[0022] The beneficial effects of this invention are as follows: 1. The synthesis process of this invention requires less time and has higher production efficiency: for example, the side chain activation time is only 2-5 hours, which is much shorter than the 12-hour reaction time in patent CN115322250A; in the dipeptide activation method, N,N'-carbonyldiimidazole is used as a carboxylic acid activator. Compared with the activator combination in Chinese patent CN115322250A, the reagents of this invention are simple, reducing the types of reagents, saving material consumption, and reducing the activation reaction time. The shortest reaction time is only 20 minutes, which is much shorter than the reaction time of up to 10 hours in existing patents. 2. This invention also discloses pretreatment / posttreatment methods suitable for industrial production and capable of improving production efficiency and yield: (1) Pretreatment: The 29 peptide and the side chain-main peptide chain conjugate (intermediate II) are treated into a solution of a predetermined concentration to make the subsequent feeding amount more accurate and facilitate industrial pipeline transportation. (2) Post-treatment of side chain-main peptide chain conjugate (intermediate II): Existing patents do not remove organic solvents, which may cause the precipitate to be sticky and difficult to remove during acid precipitation; In this technology, the organic solvent is removed first in the post-treatment and then acid precipitation is carried out. The removal of organic solvents can ensure that the precipitate is uniform and loose, which is conducive to the smooth transportation of materials in pipelines in industrial production and reduces the difficulty of cleaning. (3) Post-treatment of the deprotected precursor (intermediate IV) of smegglutide: The post-treatment of existing patents is mostly acid precipitation, but because the solvent is a highly polar organic solvent such as N,N-dimethylformamide / N-methylpyrrolidone, it is easy for the precipitate to interact with the organic solvent during acid precipitation to form an adhesive body, which is difficult to handle due to wall adhesion, resulting in large physical losses and is not suitable for industrial production; This technology uses 6 to 12 times the volume of methyl tert-butyl ether as the precipitant, the steps are simple, and the precipitate is uniform and loose, which is conducive to the smooth transportation of materials in pipelines in industrial production; (4) Post-processing of the final product smegglutide: Existing patents often use methyl tert-butyl ether in the pulping and impurity removal step, which is flammable and explosive. Its use frequency should be minimized in industrial production. This technology selects dichloromethane as the pulping solvent, which can avoid the unnecessary use of hundreds of liters of methyl tert-butyl ether in industrial-scale production, saving costs and reducing safety risks.

[0023] In summary, this invention proposes a semi-synthetic method for semaglutide. On the one hand, it employs a side-chain and dipeptide activation method that is time-efficient and has a high conversion rate. On the other hand, it provides convenient, rapid, reaction-friendly, cost-effective, and efficiency-enhancing intermediate pretreatment and post-treatment methods, improving the properties of substrates / products at each step. This method is of great significance for reducing costs, expanding production scale, and improving efficiency in industrial production, and is of great importance to the industrial production of semaglutide active pharmaceutical ingredient.

[0024] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 The HPLC spectrum of intermediate I prepared in Example 1a; Figure 2 The HPLC spectrum of intermediate I prepared in Example 1b is shown. Figure 3 The HPLC spectrum of intermediate I prepared in Example 1c; Figure 4 The HPLC spectrum of intermediate I prepared in Example 1d is shown below. Figure 5 The HPLC spectrum of intermediate II prepared in Example 2a; Figure 6 The HPLC spectrum of intermediate II prepared in Example 2b is shown. Figure 7 The HPLC spectrum of intermediate II prepared in Example 2c; Figure 8 The HPLC spectrum of intermediate II prepared in Example 3; Figure 9 The HPLC spectrum of intermediate II prepared in Example 4; Figure 10The HPLC spectrum of intermediate II prepared in Example 5; Figure 11 The HPLC spectrum of intermediate III prepared in Example 6a; Figure 12 The HPLC spectrum of intermediate III prepared in Example 6b is shown. Figure 13 The HPLC spectrum of intermediate III prepared in Example 6c; Figure 14 The HPLC spectrum of intermediate IV prepared in Example 7a; Figure 15 The HPLC spectrum of intermediate IV prepared in Example 7b is shown. Figure 16 The HPLC spectrum of intermediate IV prepared in Example 7c is shown. Figure 17 The HPLC spectrum of intermediate IV prepared in Example 8; Figure 18 The HPLC chromatogram of the final product smegglutinin prepared in Example 9a is shown. Figure 19 The image shows the HPLC chromatogram of the final product smegglutinin prepared in Example 9b. Detailed Implementation

[0026] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0027] Note: The following examples are based on the amount of peptide 29 used in step (2), and the experimental scale is 50 g; the side chain in the examples is tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu purchased through commercial channels, peptide 29 is Arg34GLP-1 (9-37) fermented by our company, and dipeptide is Boc-His(Trt)-Aib-OH purchased through commercial channels.

[0028] Example 1a Activation of the side chain (synthesis of intermediate I) The specific preparation process is as follows: (1) Activation of side chain: In a reaction flask, the side chain (20.0 g, 23.6 mmol) was dissolved in 400.0 mL of dichloromethane. After stirring evenly, N-hydroxysuccinimide powder (2.72 g, 23.6 mmol) was added. Dicyclohexylcarbodiimide solution (4.8 g, 23.6 mmol, dissolved in 20 mL of dichloromethane) was added dropwise to the above reaction flask at a uniform rate. The reaction was stirred for 2 h, and the temperature was controlled at 2~25℃ throughout the process. (2) Post-treatment of reaction solution: After the reaction is completed, the reaction solution is filtered, the filtrate is collected, dichloromethane is evaporated to dryness, 100 mL of acetonitrile is added, the mixture is stirred evenly and filtered again, 3.0 L of acetonitrile is added to the filtrate, the mixture is shaken evenly and the concentration is standardized to 8.9 mg / mL to prepare intermediate I solution.

[0029] Experimental results: The HPLC chromatogram of intermediate I solution is shown below. Figure 1 As shown, the intermediate solution had a purity of 93.9% and a yield of 89%.

[0030] Example 1b Activation of the side chain (synthesis of intermediate I) The preparation process is as follows: (1) Activation of side chain: In a reaction flask, the side chain (20.0 g, 23.6 mmol) was dissolved in 400.0 mL of dichloromethane. After stirring evenly, N-hydroxysuccinimide powder (4.0 g, 34.7 mmol) was added. Dicyclohexylcarbodiimide solution (5.8 g, 28.3 mmol, dissolved in 20 mL of dichloromethane) was added dropwise to the above reaction flask at a uniform rate. The reaction was stirred for 4 h, and the temperature was controlled at 2~25℃ throughout the process. (2) Post-treatment of reaction solution: After the reaction is completed, the reaction solution is filtered, the filtrate is collected, dichloromethane is evaporated to dryness, 100 mL of acetonitrile is added, the mixture is stirred evenly and filtered again, 3.0 L of acetonitrile is added to the filtrate, the mixture is shaken evenly and the concentration is standardized to 9.6 mg / mL to prepare intermediate I solution.

[0031] Experimental results: The HPLC chromatogram of intermediate I solution is shown below. Figure 2 As shown, the purity of intermediate I solution was 95.9%, and the yield was 96%.

[0032] Example 1c Activation of the side chain (synthesis of intermediate I) The specific preparation process is as follows: (1) Activation of side chain: In a reaction flask, the side chain (20.0 g, 23.6 mmol) was dissolved in 400.0 mL of dichloromethane. After stirring evenly, N-hydroxysuccinimide powder (4.9 g, 42.5 mmol) was added. Dicyclohexylcarbodiimide solution (7.6 g, 37.8 mmol, dissolved in 20 mL of dichloromethane) was added dropwise to the above reaction flask. The reaction was stirred for 5 h, and the temperature was controlled at 2~25℃ throughout the process. (2) Post-treatment of reaction solution: After the reaction is completed, the reaction solution is filtered, the filtrate is collected, dichloromethane is evaporated to dryness, 100 mL of acetonitrile is added, the mixture is stirred evenly and filtered again, 3.0 L of acetonitrile is added to the filtrate, the mixture is shaken evenly and the concentration is standardized to 9.2 mg / mL to prepare intermediate I solution.

[0033] Experimental results: The HPLC chromatogram of intermediate I solution is shown below. Figure 3 As shown, the purity of intermediate I solution was 94.6%, and the yield was 92%.

[0034] Example 1d Activation of the side chain (synthesis of intermediate I) The specific preparation process is as follows: (1) Activation of side chain: In a reaction flask, the side chain (60.0 g, 70.8 mmol) was dissolved in 1200.0 mL of dichloromethane. After stirring evenly, N-hydroxysuccinimide powder (12 g, 104.1 mmol) was added. Dicyclohexylcarbodiimide solution (17.4 g, 84.9 mmol, dissolved in 60 mL of dichloromethane) was added dropwise to the above reaction flask. The reaction was stirred for 5 h, and the temperature was controlled at 2~25℃ throughout the process. (2) Post-treatment of reaction solution: After the reaction is completed, the reaction solution is filtered, the filtrate is collected, dichloromethane is evaporated to dryness, 300 mL of acetonitrile is added, the mixture is stirred evenly and filtered again, 9.0 L of acetonitrile is added to the filtrate, the mixture is shaken evenly and the concentration is standardized to 9.5 mg / mL to prepare intermediate I solution.

[0035] Experimental results: The HPLC chromatogram of intermediate I solution is shown below. Figure 4 As shown, the purity of intermediate I solution was 95.8%, and the yield was 95%.

[0036] Example 2a The activation of the side chain and the connection of peptide 29 (synthesis of intermediate II) involves the production of peptide 29 in-house, derived from upstream purification processes, and the material is in the form of lyophilized powder.

[0037] The specific preparation process is as follows: (1) Pretreatment of 29 peptide: Prepare 5 L of water-N,N-diisopropylethylamine solution with a volume ratio of 100:1, add 50.0 g of 29 peptide (15.7 mmol) lyophilized powder, dissolve it completely, and the pH of the 29 peptide solution is 11.5; (2) Activation of the side chain and connection of peptide 29: Pour the peptide 29 solution into the reaction flask, slowly add 1.53 L (15.7 mmol) of intermediate I solution prepared in Example 1a, stir the reaction for 1 h, and control the temperature at 2~25℃ throughout the process; (3) Post-treatment of reaction solution: After the reaction is completed, the acetonitrile in the reaction solution is evaporated. After rotary evaporation, the pH of the reaction solution is measured to be 9.6. Glacial acetic acid is added dropwise to the reaction solution. After the pH is adjusted to 4.8, a precipitate appears. The precipitate is collected by centrifugation, dried, and then slurried twice with dichloromethane to remove impurities. After drying again, intermediate II powder is prepared.

[0038] Experimental results: 48.3 g of intermediate II powder was prepared, with a yield of 48.4%; the HPLC chromatogram of intermediate II is shown below. Figure 5 As shown, its purity is 56.5%.

[0039] Example 2b The activation of the side chain and the connection of peptide 29 (synthesis of intermediate II) involves the production of peptide 29 in-house, derived from upstream purification processes, and the material is in the form of lyophilized powder.

[0040] The specific preparation process is as follows: (1) Pretreatment of 29 peptide: Prepare 5 L of water-N,N-diisopropylethylamine solution with a volume ratio of 100:1, add 50.0 g of 29 peptide (15.7 mmol) lyophilized powder, dissolve it completely, and the pH of the 29 peptide solution is 11.8; (2) Activation of the side chain and connection of peptide 29: Pour the peptide 29 solution into the reaction flask, slowly add 2.5 L (25.2 mmol) of intermediate I solution prepared in Example 1b, stir the reaction for 0.5 h, and control the temperature at 2~25℃ throughout the process; (3) Post-treatment of reaction solution: After the reaction is completed, the acetonitrile in the reaction solution is evaporated. After rotary evaporation, the pH of the reaction solution is measured to be 9.8. Glacial acetic acid is added dropwise to the reaction solution. After the pH is adjusted to 4.8, a precipitate appears. The precipitate is collected by centrifugation, dried, and then slurried twice with dichloromethane to remove impurities. After drying again, intermediate II powder is prepared.

[0041] Experimental results: 50.7 g of intermediate II powder was prepared, with a yield of 80.1%; the HPLC chromatogram of intermediate II is shown below. Figure 6 As shown, its purity is 89.1%.

[0042] Example 2c The activation of the side chain and the connection of peptide 29 (synthesis of intermediate II) involves the production of peptide 29 in-house, derived from upstream purification processes, and the material is in the form of lyophilized powder.

[0043] The specific preparation process is as follows: (1) Pretreatment of 29 peptide: Prepare 5 L of water-N,N-diisopropylethylamine solution with a volume ratio of 100:1, add 50.0 g of 29 peptide (15.7 mmol) lyophilized powder, dissolve it completely, and the pH of the 29 peptide solution is 11.6; (2) Activation of the side chain and connection of peptide 29: Pour the peptide 29 solution into the reaction flask, slowly add 3.1 L (31.4 mmol) of intermediate I solution prepared in Example 1c, stir the reaction for 2 h, and control the temperature at 2~25℃ throughout the process; (3) Post-treatment of reaction solution: After the reaction is completed, the acetonitrile in the reaction solution is evaporated. After rotary evaporation, the pH of the reaction solution is measured to be 9.5. Glacial acetic acid is added dropwise to the reaction solution. After the pH is adjusted to 4.8, a precipitate appears. The precipitate is collected by centrifugation, dried, and then slurried twice with dichloromethane to remove impurities. After drying again, intermediate II powder is obtained.

[0044] Experimental results: 45.6 g of intermediate II powder was prepared, with a yield of 68.9%; the HPLC chromatogram of intermediate II is shown below. Figure 7 As shown, its purity is 85.0%.

[0045] Example 3 The activation of the side chain and the connection of peptide 29 (synthesis of intermediate II) involves peptide 29, which is self-made by our company, derived from upstream purification process, and the material is in the state of wet solid.

[0046] The specific preparation process is as follows: (1) Pretreatment of 29 peptide: A water-N,N-diisopropylethylamine solution with a volume ratio of 100:1 was prepared to fully dissolve the wet solid of 29 peptide obtained upstream, and the concentration was determined to be 9.8 mg / mL. N,N-diisopropylethylamine was added to bring the pH of the solution to 11.6. The total volume of the 29-peptide solution was 5.1 L, and the total amount of 29-peptide was 50.0 g (15.7 mmol). (2) Activation of the side chain and connection with peptide 29: 2.5 L (25.2 mmol) of intermediate I solution prepared in Example 1d was slowly added dropwise to peptide 29 solution, and the reaction was stirred for 0.5 h with the temperature controlled at 2~25℃ throughout the process; (3) Post-treatment of reaction solution: After the reaction is completed, acetonitrile in the reaction solution is evaporated; glacial acetic acid is added dropwise to the reaction solution, and after the pH is adjusted to 4.8, a precipitate appears. The precipitate is collected by centrifugation, dried, and then slurried twice with dichloromethane to remove impurities. After drying again, intermediate II powder is obtained.

[0047] Experimental results: 51.2 g of intermediate II powder was prepared, with a yield of 72.3%; the HPLC chromatogram of intermediate II is shown below. Figure 8 As shown, its purity is 88.8%.

[0048] Example 4 The activation of the side chain and the connection of peptide 29 (synthesis of intermediate II) involves the production of peptide 29 in-house, derived from upstream purification processes, and the material is in liquid form.

[0049] The specific preparation process is as follows: (1) Pretreatment of 29 peptide: 9.3 L of 29 peptide purified solution was obtained from the upstream process. The concentration of 29 peptide in the purified solution was 5.4 mg / mL. The amount of 29 peptide added was 50.0 g (15.7 mmol). 93 mL of N,N-diisopropylethylamine was added at a ratio of 100:1 (v / v) and stirred evenly. (2) Activation of the side chain and connection with peptide 29: 2.5 L (25.2 mmol) of intermediate I solution prepared in Example 1d was slowly added dropwise to peptide 29 solution, and the reaction was stirred for 0.5 h with the temperature controlled at 2~25℃ throughout the process; (3) Post-treatment of reaction solution: After the reaction is completed, acetonitrile in the reaction solution is evaporated; glacial acetic acid is added dropwise to the reaction solution, and after the pH is adjusted to 4.8, a precipitate appears. The precipitate is collected by centrifugation, dried, and then slurried twice with dichloromethane to remove impurities. After drying again, intermediate II powder is obtained.

[0050] Experimental results: 49.8 g of intermediate II powder was prepared, with a yield of 70.0%; the HPLC chromatogram of intermediate II is shown below. Figure 9 As shown, its purity is 88.4%.

[0051] Example 5 The activation of the side chain and the connection of peptide 29 (synthesis of intermediate II) involves the production of peptide 29 in-house, derived from upstream purification processes, and the material is in liquid form.

[0052] The specific preparation process is as follows: (1) Pretreatment of 29 peptide: 9.3 L of 29 peptide purified solution was obtained from the upstream process. The concentration of 29 peptide in the purified solution was 5.4 mg / mL. The amount of 29 peptide added was 50.0 g (15.7 mmol). 93 mL of N,N-diisopropylethylamine was added at a ratio of 100:1 (v / v) and stirred evenly. (2) Activation of the side chain and connection with peptide 29: 2.5 L (25.2 mmol) of intermediate I solution prepared in Example 1d was slowly added dropwise to peptide 29 solution, and the reaction was stirred for 0.5 h with the temperature controlled at 2~25℃ throughout the process; (3) Post-treatment of reaction solution: After the reaction is completed, acetonitrile in the reaction solution is evaporated; glacial acetic acid is added dropwise to the reaction solution, and after the pH is adjusted to 4.8, a precipitate appears. The precipitate is collected by centrifugation and directly carried out subsequent experiments.

[0053] Experimental results: The content of intermediate II obtained was 53.6 g (13.4 mmol), and the yield was 85.3%; the HPLC chromatogram of intermediate II is shown below. Figure 10 As shown, its purity is 88.4%.

[0054] Example 6a Activation of dipeptide (synthesis of intermediate III) The specific preparation process is as follows: (1) Activation of dipeptide: In a reaction flask, the dipeptide (17.5 g, 30.0 mmol) was dissolved in 350 mL of dichloromethane, stirred evenly, and then 4.9 g of N,N'-carbonyldiimidazole powder (30.0 mmol) was added. The mixture was stirred at 20~25℃ for 20 min. (2) Post-treatment of reaction solution: Dichloromethane in the reaction solution was evaporated, and 135 mL of N-methylpyrrolidone was added to dilute and stir the rotary evaporation residue to prepare intermediate III solution with a concentration of 0.2 mmol / mL.

[0055] Experimental results: The HPLC spectrum of intermediate III solution is shown below. Figure 11 As shown, the purity is 61.0%.

[0056] Example 6b Activation of dipeptide (synthesis of intermediate III) The specific preparation process is as follows: (1) Activation of dipeptide: In a reaction flask, the dipeptide (17.5 g, 30.0 mmol) was dissolved in 350 mL of dichloromethane, stirred evenly, and then 7.4 g of N,N'-carbonyldiimidazole powder (45.0 mmol) was added. The mixture was stirred at 20~25℃ for 1 h. (2) Post-treatment of reaction solution: Dichloromethane in the reaction solution was evaporated, and 135 mL of N-methylpyrrolidone was added to dilute and stir the rotary evaporation residue to prepare intermediate III solution with a concentration of 0.2 mmol / mL.

[0057] Experimental results: The HPLC spectrum of intermediate III solution is shown below. Figure 12 As shown, the purity is 88.8%.

[0058] Example 6c Activation of dipeptide (synthesis of intermediate III) The specific preparation process is as follows: (1) Activation of dipeptide: In a reaction flask, the dipeptide (17.5 g, 30.0 mmol) was dissolved in 350 mL of dichloromethane, stirred evenly, and then 9.8 g of N,N'-carbonyldiimidazole powder (60.0 mmol) was added. The mixture was stirred at 20~25℃ for 2 h. (2) Post-treatment of reaction solution: Dichloromethane in the reaction solution was evaporated, and 135 mL of N-methylpyrrolidone was added to dilute and stir the rotary evaporation residue to prepare intermediate III solution with a concentration of 0.2 mmol / mL.

[0059] Experimental results: The HPLC spectrum of intermediate III solution is shown below. Figure 13 As shown, the purity is 79.6%.

[0060] Example 7a Linkage of activated dipeptide to side-chain-29 peptide conjugate (synthesis of intermediate IV) The specific preparation process is as follows: (1) Connection of activated dipeptide with side chain-29 peptide conjugate: In a reaction flask, add 1.3 L N-methylpyrrolidone, 125.0 mL water and 50.0 mL N,N-diisopropylethylamine, stir well and then add 49.6 g (12.4 mmol) of dry powder of intermediate II obtained in Example 4 in small amounts several times, and stir well; add 62.0 mL (12.4 mmol) of intermediate III solution prepared in Example 6a into the reaction flask all at once, and continue to stir the reaction at 20~25℃ for 4 h; (2) After the reaction is completed, the reaction solution is poured into 13 L of methyl tert-butyl ether, the temperature is controlled at 0~20℃, the precipitated solid is collected by centrifugation, the obtained solid is slurried with dichloromethane twice (1 L / time) to remove impurities, and then dried to obtain intermediate IV powder.

[0061] Experimental results: 38.6 g of intermediate IV powder was prepared, with a yield of 49.2%; the HPLC chromatogram of intermediate IV is shown below. Figure 14 As shown, the purity is 56.7%.

[0062] Example 7b Linkage of activated dipeptide to side-chain-29 peptide conjugate (synthesis of intermediate IV) The specific preparation process is as follows: (1) Activation of dipeptide and side chain-29 peptide conjugate: In a reaction flask, add 1.3 L N-methylpyrrolidone, 125.0 mL water and 50.0 mL N,N-diisopropylethylamine, stir well and then add 49.8 g (12.4 mmol) of dry powder of intermediate II obtained in Example 4 in small amounts several times, and stir well; add 124.0 mL (24.8 mmol) of intermediate III solution prepared in Example 6b into the reaction flask all at once, and continue to stir the reaction at 20~25℃ for 8 h; (2) After the reaction is completed, the reaction solution is poured into 13 L of methyl tert-butyl ether, the temperature is controlled at 0~20℃, the precipitated solid is collected by centrifugation, the obtained solid is slurried twice with dichloromethane (1 L / time), and dried to obtain intermediate IV powder.

[0063] Experimental results: 42.2 g of intermediate IV powder was prepared, with a yield of 74.5%; the HPLC chromatogram of intermediate IV is shown below. Figure 15 As shown, the purity is 78.6%.

[0064] Example 7c Linkage of activated dipeptide to side-chain-29 peptide conjugate (synthesis of intermediate IV) The specific preparation process is as follows: (1) Activation of dipeptide and side chain-29 peptide conjugate: In the reaction flask, add 1.3 L N-methylpyrrolidone, 125.0 mL water and 50.0 mL N,N-diisopropylethylamine, stir well and then add 49.9 g (12.4 mmol) of dry powder of intermediate II obtained in Example 4 in small amounts several times, and stir well; add 186.0 mL (37.2 mmol) of intermediate III solution prepared in Example 6c into the reaction flask at one time, and continue to stir the reaction at 20~25℃ for 24 h; (2) Post-treatment of reaction solution: After the reaction is completed, the reaction solution is poured into 13 L of methyl tert-butyl ether, the temperature is controlled at 0~20℃, the precipitated solid is collected by centrifugation, the obtained solid is slurried twice with dichloromethane (1 L / time), and dried to obtain intermediate IV powder.

[0065] Experimental results: 39.7 g of intermediate IV powder was prepared, with a yield of 65.1%; the HPLC spectrum of intermediate IV is shown below. Figure 16 As shown, the purity is 72.5%.

[0066] Example 8 Linkage of activated dipeptide to side-chain-29 peptide conjugate (synthesis of intermediate IV) The specific preparation process is as follows: (1) Connection of activated dipeptide with side chain-29 peptide conjugate: Based on the wet solid of intermediate II obtained in Example 5 (net weight of intermediate II is 53.6 g, 13.4 mmol), 1.3 L of N-methylpyrrolidone, 125.0 mL of water and 50.0 mL of N,N-diisopropylethylamine were added and stirred until homogeneous; 124.0 mL (24.8 mmol) of intermediate III solution prepared in Example 6b was added to the reaction flask at once, and the reaction was continued to be stirred for 4 h. (2) Post-treatment of reaction solution: After the reaction is completed, the reaction solution is poured into 13 L of methyl tert-butyl ether, the temperature is controlled at 0~20℃, the precipitated solid is collected by centrifugation, the obtained solid is slurried twice with dichloromethane (1 L / time), and dried to obtain intermediate IV powder.

[0067] Experimental results: 45.0 g of intermediate IV powder was prepared, with a yield of 78.8%; the HPLC chromatogram of intermediate IV is shown below. Figure 17 As shown, the purity is 77.8%.

[0068] Example 9a The removal of protecting groups yielded smegglutinin. The specific preparation process is as follows: (1) Removal of protecting groups: In a reaction flask, add 630 mL of trifluoroacetic acid, 35 mL of triisopropylsilane and 35 mL of water, stir well and then add 47.0 g of intermediate IV (10.3 mmol) prepared in Example 7b, and react at 20°C for 2 h; (2) Post-treatment of reaction solution: Pour the reaction solution into 5.6 L of methyl tert-butyl ether, control the temperature at 0~20℃, centrifuge to collect the solid, and beat the obtained solid twice with dichloromethane. Use 4.9 L of water and 7 mL of ammonia to prepare a crude peptide solution with pH 7.7 for subsequent purification.

[0069] Experimental results: The HPLC spectrum of the purified crude peptide solution is shown below. Figure 18 As shown, the purity of the purified crude peptide solution was 99.6%, the net weight of smegglutinin was 28.3 g (10.1 mmol), and the yield was 85.1%.

[0070] Example 9b The removal of protecting groups yielded smegglutinin. The specific preparation process is as follows: (1) Removal of protecting groups: In a reaction flask, add 630 mL of trifluoroacetic acid, 35 mL of triisopropylsilane and 35 mL of water, stir well and then add 47.0 g of intermediate IV (10.3 mmol) prepared in Example 7b, and react at 20°C for 4 h; (2) Post-treatment of reaction solution: Pour the reaction solution into 5.6 L of methyl tert-butyl ether, control the temperature at 0~20℃, centrifuge to collect the solid, and beat the obtained solid twice with dichloromethane. Use 4.9 L of water and 7 mL of ammonia to prepare a crude peptide solution with pH 7.6 for subsequent purification.

[0071] Experimental results: The HPLC spectrum of the purified crude peptide solution is shown below. Figure 19 As shown, the purity of the purified crude peptide solution was 99.3%, the net weight of smegglutinin was 25.8 g (8.2 mmol), and the yield was 78.3%.

[0072] In summary, this technology proposes a semi-synthetic method for semaglutide active pharmaceutical ingredient (API). On the one hand, it employs a side-chain and dipeptide activation method that is time-efficient and has a high conversion rate. On the other hand, it provides convenient, rapid, reaction-friendly, cost-effective, and efficiency-enhancing intermediate pretreatment and post-treatment methods, improving the properties of substrates / products at each step. This method is of great significance for reducing costs, expanding production scale, and improving efficiency in industrial production, and is of great importance to the industrial production of semaglutide API.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method of semisynthesis of semaglutide characterized in that, Comprise the following steps: (1) under the condition of base catalyst, the activated side chain solution (intermediate I) is mixed with Arg34GLP-1 (9-37) 29 peptide solution, reaction obtains intermediate II, the specific synthesis route is as follows: After the reaction is completed, the organic solvent is distilled off, acid is precipitated, the precipitate is collected, and intermediate II wet solid is obtained; or the precipitate is dried, impurities are removed by methylene chloride slurry, and intermediate II powder is obtained by drying again; (2) under the condition of base catalyst, the intermediate II wet solid or powder obtained in step (1) is dissolved, and then mixed with the activated dipeptide solution (intermediate III), to obtain intermediate IV, and the specific synthesis route is as follows: After the reaction is completed, the reaction liquid is poured into methyl tert-butyl ether, the solid is collected after centrifugation, impurities are removed by methylene chloride slurry, and intermediate IV powder is obtained by drying; (3) the deprotection agent is used for deprotection of intermediate IV to obtain semeglutide, and the specific synthesis route is as follows: After the reaction is completed, the reaction liquid is poured into methyl tert-butyl ether, the solid is collected after centrifugation, impurities are removed by methylene chloride slurry, and semeglutide powder is obtained by drying; The base catalyst in steps (1) and (2) is N,N-diisopropyl ethylamine; the solvent used for dissolving intermediate II in step (2) is N-methyl pyrrolidone; the deprotection agent in step (3) is a mixed solvent of trifluoroacetic acid, water and triisopropylsilane.

2. The semi-synthetic process according to claim 1, characterized in that, The 29 peptide in step (1) is in one of dry powder, wet solid or solution, and is prepared into 29 peptide solution by adding water / N,N-diisopropyl ethylamine mixed solvent for reaction.

3. The semi-synthetic process according to claim 1, wherein, The molar ratio of intermediate I and 29 peptide in step (1) is 1-2:1, the reaction time is 0.5-2 h, and the reaction temperature is 2-25 DEG C.

4. The semi-synthetic process according to claim 1, wherein, The acid precipitation reagent in step (1) is glacial acetic acid, and the acid precipitation condition is pH 4.7-4.

9.

5. The semi-synthetic process according to claim 1, wherein, The molar ratio of intermediate II and intermediate III in step (2) is 1:1-3, the reaction time is 4-24 h, and the reaction temperature is 20-25 DEG C.

6. The semi-synthetic process according to claim 1, wherein, The volume ratio of reaction liquid and methyl tert-butyl ether in step (2) is 1:6-12, and the treatment temperature is 0-20 DEG C.

7. The semi-synthetic process according to claim 1, wherein, The volume ratio of trifluoroacetic acid, water and triisopropylsilane in step (3) is 13.5:0.75:0.75; the reaction time is 2-4 h, the reaction temperature is 20-25 DEG C, the volume ratio of reaction liquid and methyl tert-butyl ether is 1:6-12, and the treatment temperature is 0-20 DEG C.

8. The semi-synthetic process of claim 1, wherein, The activation method of side chain in step (1) is that the side chain is esterified under the condition of coupling condensing agent to obtain intermediate I; The specific synthesis route is as follows: After the reaction is completed, the reaction liquid is filtered, distilled, acetonitrile is added again, filtered, and acetonitrile is added to obtain intermediate I solution; The coupling condensing agent is N-hydroxy succinimide and dicyclohexyl carbodiimide, and the reaction solvent used is dichloromethane.

9. The semi-synthetic process according to claim 8, wherein, The molar ratio of side chain, N-hydroxy succinimide and dicyclohexyl carbodiimide in the activation method of side chain is 1:1.0-1.8:1.0-1.6, the reaction time is 2-5 h, and the reaction temperature is 2-25 DEG C.

10. The semi-synthetic process of claim 8, wherein, The volume ratio of acetonitrile to reaction liquid in the activation method of the side chain is 2-10:1, and the concentration of the intermediate I solution is 8-12 mg / mL.

11. The semi-synthetic process according to claim 1, wherein, The activation method of the dipeptide in the step (2) is that the dipeptide is activated under the condition of a carboxylic acid activator to obtain an intermediate III; The specific synthesis route is as follows: After the reaction is completed, the organic solvent is evaporated, and N-methyl pyrrolidone is added to prepare an intermediate III solution. The carboxylic acid activator is N,N'-carbonyldiimidazole.

12. The semi-synthetic process according to claim 11, wherein, In the activation method of the dipeptide, the molar ratio of the dipeptide to N,N'-carbonyldiimidazole is 1:1-2, the reaction time is 0.3-2 h, the reaction temperature is 20-25 DEG C, and the concentration of the intermediate III solution is 0.2 mol / mL.

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