Preparation method of semeglutide

Abstract

The invention relates to the technical field of medicines, in particular to a preparation method of semeglutide, which comprises the following steps: by optimizing a coupling sequence, firstly connecting a main peptide chain compound 1 with a side chain compound 5 without carboxyl protection, and then performing subsequent coupling with a dipeptide structure compound 6 with high steric hindrance, thereby obtaining the semeglutide. The problem of excessive modification caused by coexistence of a plurality of active sites in a traditional route is effectively avoided, especially for coupling difficulty brought by the structure of a compound 6, the types of a condensation reagent and a side-chain compound 5 are optimized, dipeptide carboxyl of the compound 5 is remarkably activated, amido bonds can be efficiently formed by the compound 5 and a compound 2 under the mild condition, and the compound 6 can be efficiently synthesized. In the path of synthesizing the compound 3 from the compound 1, one-pot operation is realized, and the reaction liquid in each step can be used for the next step without refining, so that the reaction time and the consumption of raw materials are reduced, and the connection efficiency and selectivity of key steps are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing smegglutinin. Background Technology

[0002] Semaglutide, a long-acting GLP-1 receptor agonist, is widely used in the treatment of type 2 diabetes and obesity. Its complex chemical structure, containing multiple amino acid residues and specific side chain modifications, makes its synthesis challenging. While several patent documents have reported synthetic routes for semaglutide, problems such as low reaction efficiency, numerous byproducts, and imprecise process control still hinder its industrial production.

[0003] In the synthesis method disclosed in patent CN111378028A, the two carboxyl groups on the side chain were not protected, which made it easy for over-modification to occur in the coupling reaction, generating a variety of impurities. The purity of the final compound was only 67.5%, of which the over-modified impurities, which were modified at both the 26-position and the terminal position of the main peptide chain, accounted for as high as 5.42%, which seriously affected the purity of the product and the efficiency of subsequent purification. Furthermore, there was a lack of effective impurity control methods.

[0004] Another patent, CN105154498A, has the problem of excessively long reaction time. Key coupling steps, such as the reaction of [Arg34]GLP-1(9-37)) with the side-chain activated ester, and the subsequent connection with the dipeptide structure, all require 16 to 24 hours. This not only results in low production efficiency and high energy consumption, but also makes it difficult to meet the timeliness requirements of industrial continuous production.

[0005] Furthermore, existing synthetic processes generally lack systematic research on the stability of key intermediates and corresponding process monitoring strategies. The instability of intermediates in the reaction system may lead to deviations between actual feed and theoretical values, resulting in increased side reactions, more complex impurity profiles, and batch-to-batch quality inconsistencies. These are also key potential factors restricting further improvements in the purity and yield of smegglutinin synthesis.

[0006] Therefore, the existing methods for synthesizing smegglutinin still have the following prominent problems: (1) There is a lack of effective monitoring and control methods for key over-modified impurities, resulting in unsatisfactory product purity; (2) The reaction time of the key coupling step is too long, which is not conducive to industrial scale-up; (3) The process as a whole lacks precise control over the reaction process, resulting in insufficient repeatability and robustness. Summary of the Invention

[0007] To address the shortcomings of existing methods for synthesizing semaglutide, such as limited methods for monitoring and controlling over-modification impurities, long production time, high production costs, and difficulty in product purification, this invention proposes a method for preparing semaglutide, comprising the following steps: ; S1: After mixing compound 1 and water, adjust the pH of the mixture, add a mixture of compound 5 and the first organic solvent to carry out the first coupling reaction, and obtain a reaction solution containing compound 2, which is directly used in step S2. S2: Mix compound 6, base, condensing agent and second organic solvent, and perform a pre-activation reaction to form a pre-reaction solution containing activated intermediate compound 6; After adjusting the pH of the reaction solution containing compound 2, it was mixed with the pre-reaction solution containing compound 6 to undergo a second coupling reaction, resulting in a reaction solution containing compound 3. S3: The reaction solution containing compound 3 undergoes one or more steps of reaction to obtain compound 4; The structural formula of compound 5 is as follows: .

[0008] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the reaction solution containing compound 3 is subjected to one or more steps of reaction to obtain compound 4 by adjusting the pH of the reaction solution containing compound 3, isoelectric point precipitation, filtration, pulping, pyrolysis reaction and purification to obtain compound 4.

[0009] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the mass of compound 1 added per 1L of water is 80~120g.

[0010] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the pH of the mixture is adjusted to 10-12.

[0011] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, after the pH of the mixture is adjusted, the temperature is stabilized at 8~12℃.

[0012] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the first organic solvent is selected from any one or a mixture of N,N-dimethylformamide, toluene, xylene, methanol, ethanol, and acetonitrile.

[0013] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the mixture of compound 5 and the first organic solvent is added to the mixture dropwise.

[0014] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the first coupling reaction is carried out at 8-15°C.

[0015] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S2, the condensing reagent of the pre-activation reaction is selected from any or a combination of p-toluenesulfonic anhydride, methanesulfonic anhydride, and benzenesulfonic anhydride, or a combination of HONB and DIC, or a combination of Sulfo-NHS and EDC·HCl.

[0016] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S2, the base is selected from one or more of N,N-diisopropylethylamine, sodium hydroxide, and ammonia.

[0017] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S2, the second organic solvent is selected from one or more of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, and acetone.

[0018] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the pre-activation reaction time in step S2 is 10-30 minutes.

[0019] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the temperature of the second coupling reaction is 15~30.0 °C.

[0020] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step 2, the pH of the reaction solution containing compound 2 is adjusted to 8-10.

[0021] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the pH is 4.0 to 6.0.

[0022] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the solvent for pulping is selected from one or more of acetonitrile, methyl tert-butyl ether, tetrahydrofuran, acetone, dichloromethane, and N,N-dimethylformamide.

[0023] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the lysis buffer comprises trifluoroacetic acid, an organic solvent, and at least one cation scavenger; the organic solvent is selected from dichloromethane, methyl tert-butyl ether, or mixtures thereof; the cation scavenger is selected from triisopropylsilane, water, or combinations thereof.

[0024] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the lysis buffer comprises trifluoroacetic acid, dichloromethane, and triisopropylsilane.

[0025] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the volume ratio of trifluoroacetic acid, dichloromethane and triisopropylsilane in the lysis solution is 1:(0.8~1.2):(0.05~0.1).

[0026] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the mass-to-volume ratio of compound 3 to the lysis buffer is 1 g: 8-10 ml.

[0027] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the temperature of the cleavage reaction is 15.0~25.0 °C.

[0028] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the temperature of the cleavage reaction is 15~30.0 °C.

[0029] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, the cleavage reaction time is 1.0~2.0 h.

[0030] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the reaction process is monitored by high performance liquid chromatography, and the corresponding reaction is terminated when the following conditions are met simultaneously: (i) The peak area of ​​the feed compound decreased to ≤ 2.0%; and (ii) The peak area of ​​the corresponding key overmodified impurities is ≤ 5.0%.

[0031] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, the key overmodified impurity is overmodified impurity 1, and the structure of overmodified impurity 1 is as follows: ; The reaction process in step S1 is monitored by liquid chromatography. The termination condition of step S1 is that the peak area of ​​compound 1 decreases to ≤2.0% and the peak area of ​​overmodified impurity 1 is ≤5.0% at the same time.

[0032] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S2, the key overmodified impurity is overmodified impurity 2, and the structure of overmodified impurity 2 is as follows: ; The reaction process in step S2 is monitored by HPLC. The termination condition of step S2 is that the peak area of ​​compound 2 decreases to ≤2.0% and (ii) the peak area of ​​overmodified impurity 2 is ≤2.0%.

[0033] In some specific embodiments of the preparation method of smegglutinin described in the first aspect, in step S1, compound 5 is prepared by a method comprising the following steps: mixing compound 7, N-hydroxy-5-norbornene-2,3-dicarboximide, an organic solvent and a dehydrating condensing agent, and reacting to obtain the compound 7, the structure of which is as follows: .

[0034] definition To make the claims and description of this invention clearer and more explicit, specific terms used herein are defined. Unless otherwise stated, all terms used in this application shall be subject to the following definitions.

[0035] Compound 1: refers to the main peptide chain, namely Arg34-GLP-1(9-37), CAS: 1169630-82-3.

[0036] Compound 2: refers to the product obtained after the first coupling reaction of compound 1 and compound 5.

[0037] Compound 3: refers to the smegglutinin precursor obtained after the second coupling reaction of compound 2 and compound 6, still bearing the protecting group.

[0038] Compound 4: refers to the crude smegglutinin obtained by cleavage and purification of compound 3.

[0039] Compound 6: refers to the carboxylic acid form of the dipeptide, namely Boc-His(Trt)-Aib-OH.

[0040] Compound 7: refers to the parent structure of the side chain.

[0041] "First coupling reaction": refers to the process in which compound 1 reacts with compound 5 to form compound 2.

[0042] "Second coupling reaction": refers to the process by which compound 2 reacts with pre-activated compound 6 to generate compound 3.

[0043] "Pre-activation reaction": refers to the step of mixing compound 6 with a condensing agent and an organic base in a solvent before the second coupling reaction to generate an intermediate compound 6 with higher reactivity.

[0044] "Standard mild range": refers to the temperature range in which the reaction described in this invention takes place, typically from 10 °C to 25 °C. This temperature range is distinct from the harsh reaction conditions that require deep cooling (e.g., -78 °C) or heating (e.g., >40 °C), reflecting the ease of operation and low energy consumption of this invention.

[0045] "Pulping": refers to a process for purifying solid compounds, in which the obtained solid is stirred in a specific solvent (such as acetonitrile, methyl tert-butyl ether, etc.) to dissolve and remove residual soluble impurities, thereby improving the purity of the product.

[0046] "Isoelectric point precipitation" refers to a purification method that reduces the solubility of the target product (such as compound 3) by adjusting the pH of the reaction solution to near its isoelectric point (such as pH 3.5-5.0), thereby causing it to precipitate from the solution.

[0047] "High-performance liquid chromatography (HPLC) monitoring method": refers to the analytical method adopted in this invention, which uses specific chromatographic conditions (such as a C4 column and an ammonium formate-acetonitrile mobile phase system) to monitor the reaction process and impurity content in real time.

[0048] "Rotary drying" refers to the process of removing volatile solvents from a solution under reduced or normal pressure using equipment such as a rotary evaporator, thereby obtaining a concentrate or solid residue. This term is synonymous with "concentration to dryness under reduced pressure" and "rotary evaporation to dryness," and is a standard technique for separating and purifying products in the field of chemical synthesis.

[0049] "Over-modified impurity 1": refers to the byproduct generated in the first coupling reaction due to the modification of multiple possible reaction sites (such as the N-terminal amino group and the Lys26 side chain amino group) on the main peptide chain (compound 1) by compound 5.

[0050] "Over-modified impurity 2": refers to the byproduct generated when other sites on compound 2, other than the target site (N-terminus), (such as the amino group on the Lys26 side chain) are linked to compound 6 in the second coupling reaction.

[0051] "Peak area": ​​In this invention, the peak area of ​​a target component at a specific detection wavelength (e.g., 210-220 nm) is used to quantify the approximate content of the component in the mixture by high performance liquid chromatography analysis.

[0052] "Equivalent" or abbreviation "eq": refers to molar equivalent.

[0053] The structural formula of compound 6 is as follows: ; The structure of the overmodified impurity 1 is as follows: The structure of overmodified impurity 2 is as follows:

[0054] The overmodified impurity 3 is a byproduct formed by the cleavage of overmodified impurity 2 to remove tert-butyloxycarbonyl and triphenylmethyl groups.

[0055] The structure of compound 5 is as follows:

[0056] Using the same method as in Example 1, N-hydroxy-5-norbornene-2,3-dicarboximide was replaced with N-hydroxysulfonate succinimide, and an esterification reaction was carried out to obtain compound 5-1.

[0057] The structure of compound 5-1 is as follows: The structure of compound 5-2 is as follows: ; The structure of compound 5-3 is:

[0058] The raw materials used in the following examples, namely the main peptide chain of compound 1, Arg34GLP-1(9-37), were purchased from Chongqing Paijin Biotechnology Co., Ltd., and the side chain of compound 7 and the dipeptide of compound 6 were purchased from Chengdu Pukang Biotechnology Co., Ltd. Unless otherwise specified, the raw materials and reagents used in the following examples can be purchased commercially.

[0059] Beneficial effects of the present invention The present invention provides a method for preparing smegglutinin, which ensures complete reaction of raw materials (≤2.0%) while precisely controlling the proportion and purity of over-modified impurity 1 and over-modified impurity 2, reducing the generation of impurities, and the final product smegglutinin crude product has a purity of 87%~91%.

[0060] In the first aspect, the present invention optimizes the coupling sequence, that is, the main peptide chain compound 1 is first linked to the side chain compound 5 with unprotected carboxyl groups, and then coupled to the dipeptide structure compound 6 with high steric hindrance. This effectively avoids the problem of over-modification caused by the coexistence of multiple active sites in the traditional route.

[0061] Secondly, through the optimization of reaction feed and solvent, the present invention eliminates the need to purify the reaction solution in a multi-step reaction. Instead, the mixture from the previous step is directly added to the next step. For example, the reaction solution containing compound 2 can be directly used for the synthesis of compound 3 without the need to pre-purify compound 2 or perform impurity removal before using it for the synthesis of compound 3. The reaction feed and solvent in each step of the reaction solution will not interfere with each other's reactions or the next step of the reaction.

[0062] Thirdly, specifically addressing the coupling difficulties caused by the Aib structure in compound 6, this invention preferentially uses p-toluenesulfonic anhydride as a condensation reagent to significantly activate its dipeptide carboxyl group, enabling it to efficiently form an amide bond with compound 2 under mild conditions, thereby greatly improving the connection efficiency and selectivity of key steps.

[0063] As shown in the attached figure, both compounds 1 and 5 exhibit severe solution instability and rapidly degrade over time. Therefore, in subsequent synthesis processes, it is essential to ensure the use of freshly prepared or confirmed purity intermediates and to incorporate high-performance liquid chromatography (HPLC) process monitoring to precisely control the reaction progress and effectively suppress the chain reaction of impurities caused by the degradation of raw materials. Therefore, in the fourth aspect, addressing the unstable and continuously decomposing characteristics of the key intermediates (compounds 1 and 5) in solution, this invention establishes a corresponding real-time HPLC monitoring method. This method can simultaneously monitor the conversion rate of raw materials and the formation of key over-modified impurities (impurities 1 and 2) during the reaction process, using "raw material peak area ≤ 2.0% and specific impurity peak area ≤ 5.0% (or 2.0%)" as a scientific and precise reaction termination criterion. This proactive process control strategy suppresses the accumulation of impurities at the source, overcoming the problems of inaccurate feed rates and batch variations caused by intermediate degradation.

[0064] Based on the above-mentioned process optimization and control, this invention successfully overcomes the main defects in existing synthesis methods, specifically in the following aspects: (1) High product purity: Through precise coupling sequence and efficient condensation system, combined with real-time HPLC monitoring, side reactions such as over-modification are effectively suppressed, so that the purity of crude smegglutinin can be stably maintained at 87~91%.

[0065] (2) High production efficiency: The key coupling reactions are all completed within 3 hours. There is no need to purify each step. The overall reaction time is greatly shortened. The conditions are mild and the operation is simple, making it suitable for large-scale industrial production.

[0066] (3) Strong process robustness: The established HPLC monitoring method provides clear and reliable quality control points for the production process, ensuring consistency and reproducibility between different production batches. Attached Figure Description

[0067] Figure 1 The HPLC analysis result of compound 1 is shown. Figures 2 to 5 This invention demonstrates the HPLC chromatogram of the continuous degradation process of compound 1 in solution every 1 hour, as shown in the experimental results. Figure 6 Show the blank HPLC chromatogram of compound 5; Figures 7 to 9 This invention demonstrates the HPLC chromatogram of the continuous degradation process of compound 5 in solution every 1 hour, as shown in the experimental results. Figure 10 The HPLC chromatogram of overmodified impurity 1 is shown. Figure 11 The mass spectrum of overmodified impurity 1 is shown; Figure 12 The HPLC chromatogram of overmodified impurity 2 is shown. Figure 13 The mass spectrum of overmodified impurity 2 is shown; Figure 14 The HPLC chromatogram shows the results of Compound 2 prepared in Example 1, where the peak area of ​​the starting material, under strict range monitoring, decreased to ≤ 2.0%, and the peak area of ​​the overmodified impurity 1 was ≤ 5.0%, which was the termination condition. Figure 15 The HPLC chromatogram shows that the peak area of ​​the raw material prepared by Compound 3 in Example 2 decreased to ≤ 2.0% and the peak area of ​​the overmodified impurity 2 was ≤ 2.0% as the termination condition. Detailed Implementation

[0068] To better understand the content of this invention, the technical solution of this invention is further described below with reference to specific embodiments, but the following content should not limit the scope of protection claimed in the claims of this invention in any way.

[0069] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0070] Example 1

[0071] Compound 7 (0.319 g, 1.2 eq), N-hydroxy-5-norbornene-2,3-dicarboximide (1.35 eq), and N,N'-dicyclohexylcarbodiimide (1.4 eq) were dissolved in tetrahydrofuran (2.5 mL) at 20 °C and stirred for 1.0–1.5 h. After the reaction was complete, the mixture was filtered to remove insoluble byproducts, and the filtrate was evaporated to dryness at 35–30 °C to obtain the crude product. The crude product was dissolved in a cutting mixture consisting of trifluoroacetic acid (1.9 mL), dichloromethane (1.1 mL), and triisopropylsilane (0.09 mL) and stirred at 20 °C for 2 h. After the reaction was complete, methyl tert-butyl ether (30 mL) was added to the reaction solution to precipitate the product. The resulting white solid was collected by filtration and washed twice with MTBE to obtain compound 5 as a white solid.

[0072] Example 2 Synthesis of Compound 2 Weigh compound 1 (1.0 g, 1.0 eq) into a reaction flask, add 10 mL of pure water to dissolve it, adjust the pH of the system to 11.0 with ammonia solution, and maintain the reaction temperature at 10.0 °C.

[0073] Compound 5 (1.2 eq) was dissolved in 10 mL of N,N-dimethylformamide, and this solution was slowly added dropwise to an alkaline aqueous solution of compound 1 over approximately 30 minutes. After the addition was complete, the reaction was continued at 10.0 °C, with the total reaction time controlled within the range of 2.0–3.0 hours.

[0074] During this period, the reaction progress was monitored by high-performance liquid chromatography (HPLC). The HPLC monitoring termination condition was that the peak area of ​​compound 1 decreased to ≤2.0% and the peak area of ​​overmodified impurity 1 was ≤5.0%. When the reaction solution containing compound 2 met the above conditions, the reaction was stopped, and without purification, it was directly used in the reaction of Example 3.

[0075] Example 3: Synthesis of Compound 3: In another dry reaction flask, compound 6 (0.55 g, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 2.0 eq), p-toluenesulfonic anhydride (Ts2O, 1.5 eq) and tetrahydrofuran (THF, 3.8 mL) were added and stirred at 15 °C for 20 minutes to form the activated intermediate compound 6 pre-reaction solution.

[0076] The pH of the reaction solution containing compound 2 prepared in Example 2 was adjusted to 9.0 with hydrochloric acid or ammonia. Then, the pre-reaction solution containing the activated intermediate compound 6 was added to it, and the reaction was carried out at 23.0 °C. The total reaction time was controlled within the range of 2.0 to 3.0 hours.

[0077] During the reaction, the reaction progress was monitored by HPLC. The HPLC termination condition was that the following conditions were met simultaneously: (i) the peak area of ​​compound 2 decreased to ≤2.0%; and (ii) the peak area of ​​overmodified impurity 2 was ≤2.0%. When the reaction solution containing compound 3 met the above conditions, the reaction was stopped, and it was used directly in Example 4 without purification.

[0078] Example 4: Synthesis and purification of crude smegglutinin (compound 4);

[0079] The reaction solution containing compound 3 obtained in Example 3 was adjusted to pH 5.0 with acetic acid, and isoelectric point precipitation was performed. The mixture was filtered, and the solid was collected. The solid was then slurried with acetonitrile (ACN, 10 mL) at room temperature for 30 minutes to remove residual trace impurities. The mixture was then filtered, and the slurried solid was collected.

[0080] The pulped solid was placed in a pyrolysis solution (5.0 mL trifluoroacetic acid + 4.7 mL dichloromethane + 0.3 mL triisopropylsilane) and stirred at 20 °C for 1.5 hours.

[0081] After the reaction was completed, 100 mL of methyl tert-butyl ether (MTBE) was added to the lysis buffer to precipitate the product. The product was filtered, washed twice with MTBE (2 × 10 mL), and dried under vacuum to obtain 1.351 g of crude product, with a yield of 95.76% and a purity of 89.27% ​​(compound 4).

[0082] Example 5 Step 1: Replace compound 5 in Example 2 with compounds 5-1 to 5-3 respectively, and carry out the first coupling reaction to obtain different reaction solutions containing compound 2. When the HPLC monitoring conditions of Example 2 are met, stop the reaction, do not purify, and use it directly in Step 2. Step 2: The reaction solution containing compound 2 from Step 1 was subjected to a second coupling reaction according to the method of Example 3. The difference from Example 3 is that the p-toluenesulfonic anhydride was replaced with either pentafluorophenol or HBTU. The resulting reaction containing compound 3 was then used to synthesize and purify crude semaglutide (compound 4) according to the method of Example 4. The yield and purity of semaglutide were determined, as follows: ; Examples 2 and 3 employ a combined feeding scheme of compound 5 and Ts2O, which significantly increases the yield and purity compared to groups 1-5. The feeding combination of compound 5 as the feed for the first coupling reaction and Ts2O as the condensing agent for the second coupling reaction has less interference compared to the schemes of groups 1-5. It is not necessary to purify the reaction solution in each step to remove impurities from the previous reaction process, thus obtaining a higher yield. Group 5 uses HBTU as the condensing agent, and the purity of the crude smegglutinin obtained is about 68%. This is because the ester activity of compound 6 in the formation of compounds 2 to 3 is low, resulting in a reaction time of 6-12 hours, requiring additional feed. Compound 2 degrades in alkaline solution, producing impurities that cannot be removed by pulping.

[0083] Example 6: Preparation of crude semaglutide by condensation with Bs2O using a dual-index monitoring strategy. (1) Compound 2 was prepared according to the steps of Example 2.

[0084] (2) Compound 3 was prepared according to step (2) of Example 3, except that the condensation reagent used was replaced with benzenesulfonic anhydride (Bs2O, 0.56 g, 2.0 eq), and the pre-reaction time was extended to 30 minutes.

[0085] (3) Following the steps (3) of Example 4, subsequent processing and purification were carried out to obtain 1.320g of crude product, with a yield of 93.56% and a purity of 87.42% for crude smegglutide (compound 4).

[0086] Example 7: Detection of parameters by high performance liquid chromatography The HPLC monitoring and purity detection involved in Examples 2 and 3 of this invention both adopted the following chromatographic conditions: Column: C4 (4.6×150 mm, packing particle size 3.5 μm); Mobile phase: Phase A is 20 mM ammonium formate aqueous solution (pH 3~4), Phase B is acetonitrile; Flow rate: 0.8~1.2 mL / min; Detection wavelength UV: 214 nm; In Example 2, high-performance liquid chromatography (HPLC) was used to monitor the reaction process of the reaction solution containing compound 2. The reverse HPLC detection gradient used was: ; In Example 3, high-performance liquid chromatography (HPLC) was used to monitor the reaction process of the reaction solution containing compound 3. The reverse HPLC detection gradient used was: ; The conditions used in the high-performance liquid chromatography method can effectively separate the raw material, target peptide, overmodified impurity 1, and overmodified impurity 2. Example 8 Degradation rate analysis of compounds 1 and 5 The solution stability of compounds 1 and 5 was monitored using a high-performance liquid chromatography (HPLC) system. Detailed data and chromatograms of their degradation processes are shown in Tables 1-2 and the appendix. Figures 1-9 .

[0087] Table 1: Changes in the main peak of compound 1 during degradation. ; Table 2: Changes in the main peak of compound 5 during degradation. ; As shown in Tables 1 and 2, both key intermediates, compound 1 and compound 5, exhibit severe solution instability and rapidly degrade over time. Therefore, in subsequent synthetic processes, it is essential to ensure the use of freshly prepared or confirmed purity intermediates and to incorporate high-performance liquid chromatography (HPLC) process monitoring to precisely control the reaction progress and effectively suppress chain reactions of impurities caused by the degradation of raw materials.

[0088] Comparative Example 1: Preparation of crude smegglutinin using a fixed-time method (1) Compound 2 was prepared according to the steps of Example 2, except that HPLC monitoring was not performed and the reaction was stopped after a total reaction time of 2.5 hours.

[0089] (2) Compound 3 was prepared according to the steps of Example 3, except that HPLC monitoring was not performed and the reaction was stopped after a total reaction time of 2.5 hours.

[0090] (3) Following the steps of Example 4, subsequent processing and purification were carried out to obtain 1.293g of crude product, with a yield of 91.70% and a purity of 81.28% for crude smegglutide (compound 4).

[0091] Comparative Example 2: Preparation of crude semaglutide using a single-index monitoring method (1) Compound 2 was prepared according to the steps of Example 2, except that the termination condition monitored by HPLC was only: the peak area of ​​compound 1 decreased to ≤ 2.0%.

[0092] (2) Compound 3 was prepared according to the steps of Example 3, except that the termination condition monitored by HPLC was only: the peak area of ​​compound 2 decreased to ≤ 2.0%.

[0093] (3) Following the steps of Example 4, subsequent processing and purification were carried out to obtain 1.128g of crude product, with a yield of 79.95% and a purity of 84.83% for crude smegglutide (compound 4).

[0094] Comparative Example 3: Preparation of crude semaglutide using the error index monitoring method (1) Compound 2 was prepared according to the steps of Example 2, except that the termination condition monitored by HPLC was: the peak area of ​​compound 1 was ≥ 2.0%.

[0095] (2) Compound 3 was prepared according to the steps of Example 3, except that the termination condition monitored by HPLC was: the peak area of ​​compound 2 was ≥ 2.0%.

[0096] (3) Following the steps of Example 4, subsequent processing and purification were carried out to obtain 1.250g of crude product, with a yield of 88.60% and a purity of 87.13% for crude smegglutide (compound 4).

[0097] Table 3. Effects of different monitoring strategies on the purity of crude semaglutide. ; As shown in Table 3, the crude product obtained by the fixed reaction time method (Comparative Example 1) had the lowest purity (70%), while the purity of the single index method (Comparative Example 2) which only monitored the raw material conversion rate was improved (80%). However, the dual index monitoring method (Examples 2-4) could stably obtain high-purity (>89%) products. This fully demonstrates the key role of dual index monitoring in controlling impurities and improving product quality.

[0098] In Comparative Example 3, despite using incorrect HPLC-monitored termination conditions (starting material ≥ 2.0%), crude semaglutide with a purity of 87.13% was still obtained. We believe this may be due to early reaction termination, which accidentally avoided potential over-modification side reactions. However, this strategy lacks scientific basis and process robustness: it cannot guarantee complete reaction conversion, potentially leading to yield loss; the result is highly accidental and cannot be reproducible across different batches. In contrast, the dual-indicator monitoring strategy, by ensuring complete reaction (starting material ≤ 2.0%) while actively controlling the formation of key impurities (impurities ≤ 5.0% or 2.0%), achieves precise, proactive, and reliable process control.

[0099] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.

Claims

1. A process for the preparation of semaglutide, characterized in that, The method comprises the steps of: ； S1: mixing compound 1 and water, and then adding a mixture of compound 5 and a first organic solvent to perform a first coupling reaction to obtain a reaction solution containing compound 2, which is directly used in the S2 step; S2: mixing compound 6, a base, a condensation reagent and a second organic solvent to perform a pre-activation reaction to form a pre-reaction solution containing an activated intermediate compound 6; After adjusting the pH of the reaction solution containing compound 2, the pre-reaction solution containing compound 6 is mixed to perform a second coupling reaction to obtain a reaction solution containing compound 3; S3: the reaction solution containing compound 3 is subjected to one or more steps of reaction to obtain compound 4. The structure of the compound 5 is as follows: 。 2. The method of claim 1, wherein the semaglutide is prepared by, The one or more steps of reaction of the reaction solution containing compound 3 are specifically adjusting the pH of the reaction solution containing compound 3, isoelectric point precipitation, filtration, beating, cleavage reaction and purification to obtain compound 4.

3. The method of preparing semaglutide according to claim 1, characterized in that, In the S1 step, 80-120 g of compound 1 is added per 1 L of water; and / or, in the S1 step, after mixing compound 1 and water, a mixed solution is obtained, and the pH of the mixed solution is adjusted to 10-12; and / or, in the S1 step, after mixing compound 1 and water, a mixed solution is obtained, and after adjusting the pH of the mixed solution, the temperature is stabilized at 8-12℃; and / or, in the S1 step, the first organic solvent is selected from any one or a mixture of N,N-dimethylformamide, toluene, xylene, methanol, ethanol and acetonitrile; and / or, in the S1 step, the mixture of compound 5 and the first organic solvent is added dropwise; and / or, in the S1 step, the first coupling reaction is performed at 8-15℃; and / or, in the S2 step, the condensation reagent of the pre-activation reaction is selected from any one or a combination of p-toluenesulfonic anhydride, methylsulfonic anhydride and benzenesulfonic anhydride, or a combination consisting of HONB and DIC, or a combination consisting of Sulfo-NHS and EDC·HCl; and / or, in the S2 step, the base is selected from one or more of N,N-diisopropylethylamine, sodium hydroxide and ammonia.

4. The method of claim 1, wherein the semaglutide is prepared by, In the S2 step, the second organic solvent is selected from one or more of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane and acetone; and / or, in the S2 step, the pre-activation reaction is performed for 10-30 minutes; and / or, in the second coupling reaction, the temperature is 15-25.0℃; and / or, in the S2 step, the pH of the reaction solution containing compound 2 is adjusted to 8-10.

5. The method of claim 2, wherein the semaglutide is prepared by, The pH is 4.0-6.0; and / or, the solvent for beating is selected from one or more of acetonitrile, methyl tert-butyl ether, tetrahydrofuran, acetone, dichloromethane and N,N-dimethylformamide; and / or, the cleavage solution used in the cleavage reaction comprises trifluoroacetic acid, an organic solvent and at least one cation scavenger; the organic solvent is selected from dichloromethane, methyl tert-butyl ether or a mixture thereof; and / or, the cation scavenger is selected from triisopropylsilane, water or a combination thereof.

6. The method of claim 5, wherein the semaglutide is prepared by, The volume ratio of trifluoroacetic acid, dichloromethane and triisopropylsilane in the cleavage solution is 1: (0.8-1.2): (0.05-0.1).

7. The method of claim 5, wherein the semaglutide is prepared by, The mass-volume ratio of the compound 3 to the cleavage solution is 1g: 8-10ml; and / or, the temperature of the cleavage reaction is 15-25.0 ℃; and / or, the time of the cleavage reaction is 1-2.0h.

8. The method of claim 1, wherein, The over-modification impurity 1 produced in the step S1 has the following structure: ; the step S1 monitors the reaction progress by liquid chromatography, and the termination condition of the step S1 is that the peak area of compound 1 is reduced to ≤ 2.0% and the peak area of over-modification impurity 1 is ≤ 5.0% simultaneously.

9. The method of claim 1, wherein, The over-modification impurity 2 produced in the step S2 has the following structure: ； The step S2 is monitored by liquid chromatography, and the termination condition of the step S2 is that (i) the peak area of the compound 2 is reduced to ≤2.0%; and (ii) the peak area of the over-modification impurity 2 is ≤2.0% are simultaneously met.

10. The method of claim 1, wherein, In the step S1, the compound 5 is prepared by a method comprising the following steps: mixing the compound 7, N-hydroxy-5-norbornene-2,3-dicarboximide, an organic solvent and a dehydration condensation agent, and reacting to obtain, wherein the compound 7 has the following structure: 。

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