A method for the semi-synthetic preparation of semaglutide and a method for the preparation of the side chain of semaglutide

Abstract

The application relates to the field of biological medicine preparation, and provides a semi-synthetic preparation method of semaglutide and a preparation method of a side chain of semaglutide. The semi-synthetic preparation method comprises the following steps: step 1, a solution of compound SMG-1 is mixed with a solution of compound III in the presence of a first base catalyst to obtain compound IV, wherein the first base catalyst is added in an amount that makes the pH of the SMG-1 solution 11.2-11.6; step 2, a solution of compound IV is mixed with a solution of compound SMG-3 in the presence of a coupling agent and a second base catalyst to obtain compound V through coupling, wherein the pH of step 2 is 9.5-10.0; and step 3, compound V is deprotected by using a deprotection agent, and finally semaglutide is obtained. The method has the advantages of fewer reaction steps, reduced cost, high purity and high yield of semaglutide, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical preparation, specifically to a semi-synthetic preparation method of smegglutinin and a method for preparing the side chain of smegglutinin. Technical Background

[0002] Semaglutide, a GLP-1 analogue with 94% homology to human GLP-1, is a long-acting glucagon-like 1 (GLP-1) analog developed and manufactured by Novo Nordisk, Denmark. Compared to other marketed hypoglycemic drugs, semaglutide has demonstrated superior clinical efficacy in controlling and maintaining blood glucose levels, making it a leading long-acting hypoglycemic agent in the diabetes drug market. Furthermore, research has found that semaglutide has the potential to treat cancer, Alzheimer's disease, and Parkinson's disease, and can reduce the risk of cardiovascular disease. Therefore, developing an efficient and economical synthetic process for semaglutide is particularly important.

[0003] The currently reported methods for preparing semaglutide API are mainly solid-phase total synthesis (SPPS). This involves starting from the carboxyl terminus of the peptide, attaching the first amino acid to a resin, removing the protecting group of the amino group, and then coupling the next amino acid according to the peptide sequence, removing the protecting group, and repeating this cycle until amino acid assembly is complete. The peptide is then cleaved from the resin while removing all protecting groups to obtain the crude peptide, which is then purified and lyophilized to obtain the API. The advantages of SPPS are that the initial reactants and products are attached to a solid support, allowing all reactions to be carried out in a single reaction vessel, facilitating automation. However, the continuous use of excessive protected amino acids and coupling agents, as well as the need for thorough washing after each amino acid elongation, results in significant material consumption and relatively high costs. Furthermore, the formation of secondary structures during SPPS synthesis often reduces the efficiency of each synthetic step. Impurities are often deletion peptides that have lost one or more amino acids in the final sequence. Meanwhile, the long side chains of aliphatic chains result in strong steric hindrance, making coupling reactions difficult and easily leading to low product yields and racemic isomerism. Although there are already optimized methods based on SPPS, such as the applicant's previously applied and granted patent CN117986349B, which reduces the number of synthetic steps, the inherent characteristics of SPPS still inevitably require a large amount of solvent for thorough washing.

[0004] The fermentation semi-synthesis method uses Arg34GLP-1(9-37) or Arg34GLP-1(11-37) prepared by biological methods as a starting material. The fatty chain side chains are modified in Lyr, and the remaining peptide chain is modified at the N-terminus to finally obtain smegglutinin. Since smegglutinin precursors are usually prepared using microbial fermentation technology, the smegglutinin backbone is synthesized through the growth and metabolism of microorganisms, which is highly efficient, environmentally friendly, and flexible. At the same time, the specificity of microbial fermentation can greatly reduce racemic isomer impurities and deletion peptide contamination in the smegglutinin precursor. However, existing fermentation semi-synthesis methods still have problems such as cumbersome steps, low purity of crude products, and low yield of the final product. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a semi-synthetic preparation method for smegglutinin with high purity crude product, high yield of final product, and simple steps, which is particularly suitable for industrial production, as well as a method for preparing smegglutinin side chains.

[0006] One aspect of the present invention provides a semi-synthetic preparation method of smegglutinin, comprising the following steps:

[0007] Step 1: In the presence of the first base catalyst, the solution of compound SMG-1 is mixed with the solution of compound III to react and obtain compound IV. The reaction formula is as follows:

[0008] ,

[0009] Wherein, R1 is succinimide, benzotriazolyl, 5-norbornene-2,3-dicarboximide, or p-toluenesulfonyl, and the amount of the first base catalyst added is such that the pH of the SMG-1 solution is 11.2-11.6, and the first base catalyst is triethylamine;

[0010] Step 2: In the presence of a coupling agent and a second base catalyst, the solution of compound IV is mixed with the solution of compound SMG-3 to obtain compound V through coupling, as shown in the following reaction formula:

[0011] ,

[0012] Wherein, R2 is triphenylmethyl or H, R3 is tert-butyloxycarbonyl or fluorenyloxycarbonyl, the pH of step 2 is 9.5-10.0, and the coupling agent is COMU;

[0013] Step 3: Deprotect compound V using a deprotecting agent to obtain smegglutinin.

[0014] Preferably, the amount of the first alkaline catalyst added is such that the pH of the SMG-1 solution is 11.3 ± 0.1.

[0015] Preferably, the solvents of the SMG-1 solution, the SMG-III solution, and the SMG-3 solution are each independently selected from one or more combinations of water, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone; and the second base catalyst is one or more combinations of triethylamine, N,N-diisopropylethylamine, tetramethylethylenediamine, aniline, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine.

[0016] More preferably, the solvent for the SMG-1 solution is water, the solvent for the SMG-III solution is acetonitrile, and the solvent for the SMG-3 solution is N,N-dimethylformamide.

[0017] More preferably, the second base catalyst is N,N-diisopropylethylamine.

[0018] More preferably, step 2 includes:

[0019] Step 2-1A: Add the second base catalyst and the coupling agent to the SMG-3 solution to prepare an activated SMG-3 solution;

[0020] Step 2-2A: Add the activated solution of compound SMG-3 to the solution of compound IV, and add the second base catalyst to adjust the pH to 9.5-10.0, and react to obtain the solution of compound V;

[0021] Step 2-3A: Add water to the solution of compound V to dilute it, and add acid to adjust the pH to 5.0. A white solid precipitates out. Filter and vacuum dry to obtain compound V, which is used in step 3.

[0022] More preferably, the acid is phosphoric acid.

[0023] More preferably, the deprotecting agent in step 3 is a TFA / TIS / DCM in a volume ratio of 90:5:5.

[0024] Preferably, the semi-synthetic preparation method is a continuous reaction, in which intermediates are not separated during the reaction;

[0025] The compound SMG-3 is Boc-His(Trt)-Aib-OH;

[0026] Step 2 includes:

[0027] Step 2-1B: Add the second base catalyst and the coupling agent to the SMG-3 solution to prepare an activated SMG-3 solution;

[0028] Step 2-2B: Add the activated solution of compound SMG-3 to the solution of compound IV, and add the second base catalyst to adjust the pH to 9.5-10.0. The reaction yields the solution of compound V, which is then used directly in step 3.

[0029] Step 3 includes adding the deprotecting agent HCl / MeOH to the solution of compound V for deprotection.

[0030] Preferably, the semi-synthetic preparation method is a continuous reaction, in which intermediates are not separated during the reaction;

[0031] The compound SMG-3 is Fmoc-His-Aib-OH;

[0032] Step 2 includes:

[0033] Step 2-1C: Add the second base catalyst and the coupling agent to the SMG-3 solution to prepare an activated SMG-3 solution;

[0034] Step 2-2C: Add the activated solution of compound SMG-3 to the solution of compound IV, and add the second base catalyst to make the pH 9.5-10.0. The reaction yields the solution of compound V, which is directly used in step 3.

[0035] Step 3 includes adding a deprotecting agent to the solution of compound V for deprotection, wherein the deprotecting agent is diethylamine, NaOH, piperidine, or DBU.

[0036] More preferably, the deprotecting agent is diethylamine.

[0037] Preferably, the molar ratio of compound SMG-1 to compound III is 1:1.0-2.0, the molar ratio of compound IV to compound SMG-3 is 1:3.0, and the molar ratio of compound V to the deprotecting agent is 1:5 (w / v).

[0038] Preferably, the reaction temperature in step 1 is 5-15℃, and the reaction temperature in step 2 is 10-20℃.

[0039] Preferably, compound III is prepared by the following steps:

[0040] Step A: In the presence of a condensing agent, a solution of compound SMG-2 is mixed with a solution of compound I, and dehydration condensation is performed to obtain compound II, as shown in the following reaction formula:

[0041] ,

[0042] Compound I is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensing agent is DCC, a protective gas is used during the reaction, and the reaction temperature is 2-8℃.

[0043] Step B: Compound II undergoes deprotection to yield compound III, as shown in the following reaction formula:

[0044] .

[0045] Preferably, the solvent of the SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, and the solvent of the compound I solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, and the molar ratio of compound SMG-2 to compound I is 1:1.1-2.0.

[0046] Preferably, step B uses a TFA / TIS / DCM with a volume ratio of 90:5:5 or a TFA / TIS / PPW with a volume ratio of 95:2.5:2.5 for deprotection.

[0047] The beneficial effects of this invention are:

[0048] In the semi-synthetic preparation method of this invention, the active ester synthesized from tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (SMG-2) is reacted with the GLP-1 (9-37) backbone. However, the GLP-1 (9-37) backbone has multiple active sites that can react with the side-chain active ester. The resulting byproducts not only affect the purity and yield of the final smegraglutide but also the reaction process. This invention, through optimization of reaction conditions, enables the active ester of SMG-2 to be chemically selectively linked to the side-chain amino group of Lys in the Arg34GLP-1 (9-37) (SMG-1) sequence, achieving site-specific modification of the side-chain fatty acid. The content of other byproducts is less than 1.5%. Furthermore, this invention successfully resolved the side reaction problem between the activated dipeptide and the arginine residue in the main chain of SMG-1 by selectively linking Boc-His(Trt)-Aib-OH or Fmoc-His-Aib-OH (SMG-3) to the N-terminus of the SMG-1 main chain after activation by controlling the reaction conditions, thus obtaining high-purity smegglutide with a protecting group. Through screening of deprotection reagents, a high-purity and high-yield crude product was obtained; pure smegglutide with a purity greater than 99.2% and a total molar yield greater than 88% can be obtained with only one purification step.

[0049] In a preferred embodiment of the present invention, by optimizing the reagents used in the reaction process, a continuous reaction is carried out, which reduces the purification and processing of intermediates, reduces the number of steps and the use of a large number of reagents, thereby reducing costs and environmental impact.

[0050] Furthermore, during the synthesis of active esters from tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu(SMG-2), the active ester in the side chain undergoes ring-opening rearrangement to generate β-alanine impurities, and reacts with the active ester in the side chain to generate di-side-chain β-alanine impurities. This invention solves the problem of side-reaction impurities during the synthesis of side-chain active esters by optimizing the reaction conditions, thus obtaining high-purity side-chain active esters.

[0051] In summary, the method of the present invention has fewer reaction steps and is simple to operate, which greatly simplifies the process, reduces time and material costs, reduces the generation of waste liquid and by-products, and the prepared smegglutinin has high purity and high yield, making it particularly suitable for large-scale industrial production of smegglutinin. Attached Figure Description

[0052] Figure 1 The HPLC chromatogram of compound II-A from Example 1 is shown below.

[0053] Figure 2 The HPLC chromatogram of compound Ⅲ-A in Example 2 is shown below.

[0054] Figure 3 The HPLC chromatogram of compound Ⅲ-B in Example 3 is shown below.

[0055] Figure 4 Example 4: HPLC chromatogram of compound III-C;

[0056] Figure 5 The HPLC chromatogram of compound IV in Example 5 is shown below.

[0057] Figure 6 Here is the HPLC chromatogram of compound VA from Example 6;

[0058] Figure 7 The HPLC chromatogram of crude smegglutinin in Example 7 is shown below.

[0059] Figure 8 The HPLC chromatogram of pure smegglutinin is shown in Example 7.

[0060] Figure 9 The HPLC chromatogram of crude smegglutinin in Example 8 is shown below.

[0061] Figure 10 The HPLC chromatogram of pure smegglutinin is shown in Example 8.

[0062] Figure 11 The HPLC chromatogram of crude smegglutinin in Example 9 is shown below.

[0063] Figure 12 The HPLC chromatogram of pure smegglutinin is shown in Example 9.

[0064] Figure 13 The HPLC chromatogram of compound II-A in Comparative Example 1;

[0065] Figure 14 The HPLC chromatogram of compound II-A in Comparative Example 2;

[0066] Figure 15 The HPLC chromatogram of compound IV prepared under pH 11.0 conditions in Comparative Example 3 is shown below.

[0067] Figure 16 The HPLC chromatogram of compound IV prepared under pH 11.2 conditions in Comparative Example 3 is shown below.

[0068] Figure 17 The HPLC chromatogram of compound IV prepared under pH 11.4 conditions in Comparative Example 3 is shown below.

[0069] Figure 18 The HPLC chromatogram of compound IV prepared under pH 11.61 in Comparative Example 3 is shown below.

[0070] Figure 19 The HPLC chromatogram of compound IV prepared under pH-adjusted conditions using DMAP is shown in Comparative Example 4.

[0071] Figure 20 The HPLC chromatogram of compound IV prepared under pH-adjusted conditions using TMP is shown in Comparative Example 4.

[0072] Figure 21 The HPLC chromatogram of compound IV prepared under pH adjustment conditions using triethylamine is shown in Comparative Example 4.

[0073] Figure 22 The HPLC chromatogram of compound V in Comparative Example 5;

[0074] Figure 23 The HPLC chromatogram of compound V in Comparative Example 6;

[0075] Figure 24 The HPLC chromatogram of compound V in Comparative Example 7;

[0076] Figure 25 The HPLC chromatogram of compound V in Comparative Example 8;

[0077] Figure 26 The HPLC chromatogram of crude smegglutinin from Comparative Example 9 is shown below.

[0078] Figure 27 The HPLC chromatogram of crude smegglutinin from Comparative Example 10 is shown below.

[0079] Figure 28 The HPLC chromatogram of crude smegglutinin from Comparative Example 11 is shown below.

[0080] Figure 29 The mass spectrum of the pure smegglutinin prepared in Example 7 is shown. Detailed Implementation

[0081] The present invention will be further described below with reference to the accompanying drawings and embodiments. However, these embodiments are exemplary and are only intended to help those skilled in the art to further understand the features of the present invention, and do not constitute any limitation on the scope of the present invention. Unless otherwise specified, all reagents used in the present invention are conventional commercially available products. Unless otherwise specified, all percentages appearing in the present invention are mass percentages. In the following embodiments, the high-performance liquid chromatograph used for purity detection is a Waters 2695 Separations Module, model: UV (ALLLANCE E2695 / ACQUITY Arc), and the chromatographic column is a Kinetex 2.6μm C18 100Å 150*4.6mm (column temperature 30℃). The high-performance liquid chromatograph used for the purification of smegglutinin is a Hanbang Technology MPLC-DAC50; specific parameters can be found in the relevant embodiments. The mass spectrometer was an ultra-high performance liquid chromatography system (Waters Technology (Shanghai) Co., Ltd., ACQUIIY UPLC H-CLASS PIUS), and the column was a 1.7μm PEPTIDE XB-C18 10CLC Column 150*2.1nm.

[0082] In this invention, the semi-synthetic preparation method refers to a method for preparing smegglutinin by using the smegglutinin precursor Arg34GLP-1(9-37) or Arg34GLP-1(11-37) as a starting material, modifying the fatty chain side chains in Lyr, and modifying the remaining peptide chain at the N-terminus, ultimately obtaining smegglutinin. The smegglutinin precursor Arg34GLP-1(9-37) or Arg34GLP-1(11-37) can be prepared using microbial fermentation technology.

[0083] In this invention, the protective gas refers to a chemically stable gas that prevents the protected substance from being oxidized by oxygen in the air, and can be nitrogen, rare gases, etc.

[0084] In this invention, the meanings of the abbreviations and their corresponding English translations are shown in Table 1:

[0085]

[0086] The semi-synthetic preparation method of smegglutinin of the present invention includes: Step 1: In the presence of a first base catalyst, a solution of compound SMG-1 and a solution of compound III are mixed to react and obtain compound IV, as shown in the following reaction formula:

[0087] ,

[0088] Wherein, R1 is benzotriazolyl, succinimide, 5-norbornene-2,3-dicarboximide, or p-toluenesulfonyl, and the amount of the first base catalyst added is such that the pH of the SMG-1 solution is 11.2-11.6;

[0089] Step 2: In the presence of a coupling agent and a second base catalyst, the solution of compound IV is mixed with the solution of compound SMG-3 to obtain compound V through coupling, as shown in the following reaction formula:

[0090] ,

[0091] Wherein, R2 is triphenylmethyl or H, R3 is tert-butyloxycarbonyl or fluorenemethyloxycarbonyl, and the pH of step 2 is 9.5-10.0;

[0092] Step 3: Deprotect compound V using a deprotecting agent, crystallize, purify, and freeze-dry to finally obtain smegglutinin.

[0093] The inventors discovered that adjusting the pH of the SMG-1 solution to 11.2-11.6 in step 1 enables the activated ester of SMG-2, namely compound III, to chemically selectively link with the side-chain amino group of Lys in the SMG-1 sequence, reducing byproducts generated from reactions at other sites and resulting in high purity of compound IV. The obtained compound IV solution is used directly in subsequent reactions without purification, without affecting the occurrence of subsequent reactions, resulting in fewer side reactions and high purity of the final crude smegglutinin. Simultaneously, adjusting the pH of the reaction system to 9.5-10.0 in step 2 reduces the side reaction between the activated dipeptide and the main chain arginine, which is beneficial for improving the purity of the crude smegglutinin. In a preferred embodiment of the invention, the pH of the SMG-1 solution is adjusted to 11.3 ± 0.1. The pH of the SMG-1 solution is adjusted using a first base catalyst. The first base catalyst can be one or more combinations of triethylamine, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine. In a preferred embodiment, the first base catalyst is triethylamine. Using triethylamine further suppresses side reactions and improves the purity of compound IV. The pH of the reaction system in step 2 is adjusted using a second base catalyst. The second base catalyst can be one or more combinations of triethylamine, N,N-diisopropylethylamine, tetramethylethylenediamine, aniline, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine. In a preferred embodiment, the second base catalyst is N,N-diisopropylethylamine.

[0094] The semi-synthetic preparation method of this invention is a liquid-phase reaction, wherein the solvent used is a good solvent capable of effectively dissolving each reactant, and the amount used is sufficient to completely dissolve each reactant. The solvents for the SMG-1 solution, the compound III solution, and the SMG-3 solution can each be independently selected from one or more combinations of water, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone. In a preferred embodiment, the solvent for the SMG-1 solution is water, the solvent for the compound III solution is acetonitrile, and the solvent for the SMG-3 solution is N,N-dimethylformamide. When the solvent is selected as described above, not only can each reactant be fully dissolved, but the miscibility during the reaction is also beneficial to the reaction of each reactant. Furthermore, it facilitates the precipitation of the intermediate and / or crude smegglutide during the subsequent purification process, reducing washing steps, and does not reduce the purity of the intermediate and / or crude smegglutide.

[0095] In a preferred embodiment of the present invention, the coupling agent in step 2 can be COMU, TPTU, Oxyma / EDCI, PyOxim, or p-toluenesulfonic anhydride, preferably COMU. Compared with other coupling agents, COMU can further reduce the side reaction between the activated dipeptide and the main chain arginine, resulting in a higher purity smegglutide with the protective agent.

[0096] In a preferred embodiment of the present invention, step 2 includes: step 2-1A: adding the second base catalyst and the coupling agent to the SMG-3 solution to prepare an activated SMG-3 solution; step 2-2A: adding the activated SMG-3 solution to the compound IV solution, and adding the second base catalyst to adjust the pH to 9.5-10.0, reacting to obtain a compound V solution; step 2-3A: diluting the compound V solution with water, adding acid to adjust the pH to 5.0, precipitating a white solid, filtering, and vacuum drying to obtain compound V, which is used in step 3. Wherein, the solvent of the SMG-1 solution is water, the solvent of the compound III solution is acetonitrile, the solvent of the SMG-3 solution is N,N-dimethylformamide, the first base catalyst is triethylamine, the second base catalyst is N,N-diisopropylethylamine, and the coupling agent is COMU. By optimizing the above experimental conditions, side reactions were reduced. In steps 2-3A, high-purity smegraglutide with the protective agent can be precipitated simply by adjusting the solution pH, eliminating the need for multiple washings to remove unwanted byproducts. This reduces processing steps, lowers costs, and reduces wastewater, making it more environmentally friendly. In a preferred embodiment, phosphoric acid is used to adjust the solution pH in steps 2-3A. In step 3, compound V obtained in step 2 is deprotected, crystallized, purified, and lyophilized to finally obtain smegraglutide. Known deprotection agents can be used to deprotect compound V in step 3. In a preferred embodiment, the deprotecting agent is a TFA / TIS / DCM mixture with a volume ratio of 90:5:5.

[0097] In a preferred embodiment, the semi-synthetic preparation method of the present invention is a continuous reaction, in which intermediates are not separated during the reaction, wherein compound SMG-3 is Boc-His(Trt)-Aib-OH; step 2 includes: step 2-1B: adding the second base catalyst and the coupling agent to the compound SMG-3 solution to prepare an activated solution of compound SMG-3; step 2-2B: adding the activated solution of compound SMG-3 to the solution of compound IV, and adding the second base catalyst to adjust the pH to 9.5-10.0, reacting to obtain a solution of compound V, which is directly used in step 3; step 3 includes adding the deprotecting agent HCl / MeOH to the solution of compound V for deprotection. In this embodiment, there is no need to purify compound V obtained in step 2, and HCl / MeOH is directly added for deprotection, reducing the steps of precipitation, purification, and redissolution of intermediates and the use of related reagents, simplifying the production process, reducing costs, and the final obtained smegglutinin can still maintain high yield and purity.

[0098] In another preferred embodiment of the present invention, the semi-synthetic preparation method of the present invention is a continuous reaction, and the intermediates are not separated during the reaction; wherein compound SMG-3 is Fmoc-His-Aib-OH; step 2 includes: step 2-1C: adding the second base catalyst and the coupling agent to the compound SMG-3 solution to prepare an activated solution of compound SMG-3; step 2-2C: adding the activated solution of compound SMG-3 to the solution of compound IV, and adding the second base catalyst to make the pH 9.5-10.0, reacting to obtain a solution of compound V, and the solution of compound V is directly used in step 3; step 3 includes adding a deprotecting agent to the solution of compound V for deprotection, the deprotecting agent can be diethylamine, inorganic base, piperidine or DBU, preferably diethylamine. This embodiment also reduces the steps of precipitation, purification, and redissolution of intermediates and the use of related reagents, simplifies the production process, reduces costs, and the final obtained smegglutinin can still maintain high yield and purity.

[0099] In a preferred embodiment of the present invention, the molar ratio of compound SMG-1 to compound III is 1:1.0-2.0, the molar ratio of compound IV to compound SMG-3 is 1:3.0, and the molar ratio of compound V to the deprotecting agent is 1:5 (w / v). The reaction temperature of step 1 is 5-15°C, and the reaction temperature of step 2 is 10-20°C. At the above ratios and temperatures, the reaction is more complete and produces fewer byproducts.

[0100] This invention also relates to a method for preparing compound III, an intermediate of the smegglutinin side chain, comprising step A: mixing a solution of compound SMG-2 with a solution of compound I in the presence of a condensing agent, and then performing dehydration condensation to obtain compound II, as shown in the following reaction formula:

[0101] ,

[0102] Compound I is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensing agent is DCC, an inert gas is used for protection during the reaction, and the reaction temperature is 2-8℃.

[0103] Step B: Compound II undergoes deprotection, crystallization, washing, and drying to obtain compound III, as shown in the following reaction formula:

[0104] .

[0105] In a preferred embodiment, the solvent for the SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, and the solvent for the compound I solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran. The molar ratio of compound SMG-2 to compound I is 1:1.1-2.0. In a preferred embodiment, step B uses a TFA / TIS / DCM with a volume ratio of 90:5:5 or a TFA / TIS / PPW with a volume ratio of 95:2.5:2.5 for deprotection.

[0106] The method of the present invention, through optimization of the specific reaction conditions described above, reduces the β-alanine impurities generated by the ring-opening rearrangement of the side-chain active esters and the di-side-chain β-alanine impurities generated by the reaction between the side-chain active esters during the synthesis of active esters from tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu(SMG-2), resulting in high purity of the obtained side-chain active esters.

[0107] Example 1: Preparation of compound II-A

[0108] 1) Add dichloromethane (40 mL, 4 V), SMG-2 (10 g, 1.0 eq) and N-hydroxysuccinimide (2.03 g, 1.5 eq) to reaction flask ①, stir to dissolve, protect with nitrogen and cool to an internal temperature of 0~5℃.

[0109] 2) Weigh 3.76 g (1.5 eq) of N,N'-dicyclohexylcarbonimide (DCC) into reaction flask ② and dissolve it in dichloromethane (10 mL, 1V). Slowly add the prepared N,N'-dicyclohexylcarbonimide solution dropwise into reaction flask ① under nitrogen protection and with the internal temperature controlled not exceeding 10℃. React at 2-8℃ for 5 h. After filtration, concentrate to dryness to obtain 14.37 g of compound II-A, with a liquid phase purity of 97.33%. See HPLC chromatogram for details. Figure 1 .

[0110]

[0111] Example 2: Preparation of compound III-A

[0112] 1) Add dichloromethane (2.5 mL, 0.25 V) to dissolve 14.37 g of compound II-A prepared in Example 1. Under nitrogen protection and temperature control of about 5~10 °C, add trifluoroacetic acid (45 mL, 4.5 V) and triisopropylsilane (2.5 mL, 0.25 V) in sequence. React at 2~8 °C for 4 hours under nitrogen protection.

[0113] 2) Under nitrogen protection, add methyl tert-butyl ether (300 mL, 30V) dropwise at a controlled temperature of 2-8℃. After addition, stir for 30 minutes while maintaining the temperature. Filter, and wash the filter cake three times with methyl tert-butyl ether (50 mL, 5V). Remove the filter cake and dry in a vacuum oven to obtain compound III-A 9.23 g, molar yield: 87.25%, liquid chromatography purity: 99.65%. See HPLC chromatogram for reference. Figure 2 .

[0114]

[0115] Example 3: Preparation of compound III-B

[0116] 1) Add dichloromethane (40 mL, 4 V), SMG-2 (10 g, 1.0 eq) and N-hydroxy-5-norbornene-2,3-dicarboximide (3.17 g, 1.5 eq) to reaction flask ①, stir to dissolve, protect with nitrogen and cool to an internal temperature of 0~5℃.

[0117] 2) Weigh 3.76 g (1.5 eq) of N,N'-dicyclohexylcarbonimide (DCC) into reaction flask ② and dissolve it in dichloromethane (10 mL, 1V). Slowly add the prepared N,N'-dicyclohexylcarbonimide solution dropwise into reaction flask ① under nitrogen protection and with the internal temperature controlled not exceeding 10 °C. React at 2–8 °C for 5 h until the reaction is complete. After filtration, concentrate to dryness to obtain compound II-B.

[0118] 3) Add dichloromethane (2.5 mL, 0.25 V) to dissolve compound II-B, protect with nitrogen and control the temperature at about 5~10℃, then add trifluoroacetic acid (45 mL, 4.5 V) and triisopropylsilane (2.5 mL, 0.25 V) in sequence, and react at 2~8℃ for 4 hours under nitrogen protection.

[0119] 4) Under nitrogen protection, add methyl tert-butyl ether (300 mL, 30V) dropwise at a controlled temperature of 2-8℃. After addition, stir for 30 minutes while maintaining the temperature. Filter, and wash the filter cake three times with methyl tert-butyl ether (50 mL, 5V). Remove the filter cake and dry in a vacuum oven to obtain 8.90 g of compound III-B, molar yield: 83.71%, liquid chromatography purity: 98.21%. See HPLC chromatogram for reference. Figure 3 .

[0120]

[0121] Example 4: Preparation of compound III-C

[0122] 1) Add dichloromethane (40 mL, 4 V), SMG-2 (10 g, 1.0 eq) and 1-hydroxybenzotriazole (2.39 g, 1.5 eq) to reaction flask ①, stir to dissolve, protect with nitrogen and cool to an internal temperature of 0~5℃.

[0123] 2) Weigh 3.76 g (1.5 eq) of N,N'-dicyclohexylcarbonimide (DCC) and add it to reaction flask ②. Dissolve the solution in dichloromethane (10 mL, 1V). Slowly add the prepared N,N'-dicyclohexylcarbonimide solution dropwise to reaction flask ①. Under nitrogen protection and with the internal temperature controlled not exceeding 10 °C, react for 5 h at 2-8 °C. After filtration, concentrate to dryness to obtain compound II-C.

[0124] 3) Add dichloromethane (2.5 mL, 0.25 V) to dissolve compound II-C, protect with nitrogen and control the temperature at about 5~10℃, then add trifluoroacetic acid (45 mL, 4.5 V) and triisopropylsilane (2.5 mL, 0.25 V) in sequence, and react at 2~8℃ for 4 hours under nitrogen protection.

[0125] 4) Under nitrogen protection, add methyl tert-butyl ether (300 mL, 30V) dropwise at a controlled temperature of 2-8℃. After addition, stir for 30 minutes while maintaining the temperature. Filter, and wash the filter cake three times with methyl tert-butyl ether (50 mL, 5V). Remove the filter cake and dry in a vacuum oven to obtain 8.29 g of compound III-C, molar yield: 82.45%, liquid chromatography purity: 97.99%. See HPLC chromatogram for reference. Figure 4 .

[0126]

[0127] Example 5: Preparation of Compound IV

[0128] 1) Add purified water (360 mL, 18 V) and triethylamine (6 mL, 0.3 V) to reaction flask ①, mix and stir until homogeneous, pH 11.30 ± 0.1, and cool to an internal temperature of 5-15℃. Then add SMG-1 (20.23 g, 1.0 eq) and stir to dissolve.

[0129] 2) Add acetonitrile (140 mL, 7V), DMF (40 mL, 2V), triethylamine (4 mL, 0.2V), and compound III-A (8.58 g, 1.5 eq) to reaction flask ②, and stir until dissolved and clear. Slowly add the solution from reaction flask ② dropwise to reaction flask ①, and react at 5-15℃ until completion. Add 10% phosphoric acid to adjust the pH to 9.5-10.5 to obtain a solution of compound IV. Liquid chromatography purity: 93.61%, all single impurities less than 1.0%. See HPLC chromatogram for details. Figure 5 .

[0130]

[0131] Example 6: Preparation of compound V-A

[0132] 1) The compound IV solution was synthesized according to the method of Example 5. The amount of SMG-1 was 20 g, and the amounts of other reagents were adjusted proportionally with respect to the amount of SMG-1.

[0133] 2) In a separate reaction flask, add DMF (100 mL, 5V), N,N-diisopropylethylamine (5 mL, 0.25V), and SMG-3A (11.01 g, 3.0 eq) and stir to dissolve. Cool to 10~20℃, add COMU (8.09 g, 3.0 eq), and stir for 30 min to obtain an activated SMG-3A solution.

[0134] 3) Control the internal temperature to 10-20°C, slowly add the SMG-3A activation solution to the compound IV solution in step 1), and control the pH to 9.5-10.0 by adding DIEA during the reaction process. The reaction is completed in 3-5 hours.

[0135] 4) Add purified water (1L, 50V), stir to dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing a white solid to precipitate. Filter, collect the solid, and dry it under vacuum at 40℃ for at least 2 hours to obtain compound V-A 33.16g, liquid phase purity: 96.47%. HPLC chromatogram is shown below. Figure 6 The content of single impurities was ≤1.0%, and the molar yield of this step was 118.15%.

[0136]

[0137] Example 7: Preparation of smegglutinin

[0138] 1) Add trifluoroacetic acid (144.5 mL, 4.5 V), dichloromethane (8 mL, 0.25 V), and triisopropylsilane (8 mL, 0.25 V) to a reaction flask, cool to an internal temperature of 0~5℃, slowly add compound V-A (32.10 g, 1.0 eq), control the internal temperature at 0~5℃, and react for 4 hours.

[0139] 2) Keep the above reaction flask at 2-8℃. Add pre-cooled ethyl acetate (2-8℃) (289mL, 9V) dropwise to the reaction flask for dilution and dissolution. After the addition is complete, add pre-cooled methyl tert-butyl ether (2-8℃) (722mL, 22.5V) to precipitate the product, stirring for 10 minutes. Centrifuge the precipitated reaction solution, collect the solid wet product, add methyl tert-butyl ether (160.5mL*3, 5V*3) for slurry washing three times. Filter, collect the solid wet product, and dry it under vacuum at 30℃ for at least 1 hour to obtain 28.62g of crude smegglutinin; molar yield of this step: 93.50%; liquid phase purity: 89.90%; HPLC chromatogram is shown below. Figure 7 .

[0140] 3) Following the purification method described in Example 10, 22.84 g of smegglutinin was obtained by reversed-phase column chromatography. The molar yield for this step was 79.80%; the liquid chromatography purity was 99.54%. The HPLC chromatogram is shown below. Figure 8 Mass spectrum (see) Figure 29 The total molar yield of smegglutinin prepared by the method in this embodiment is 118.15% * 93.50% * 79.80% = 88.17%.

[0141]

[0142] Example 8: Preparation of smegglutinin by continuous reaction (one-pot method)

[0143] 1) Take reaction flask ①, add purified water (180mL, 18V) and triethylamine (3mL, 0.3V), mix and stir evenly, and cool down to an internal temperature of 5-15℃. Then add SMG-1 (10.0g, 1.0eq) and stir to dissolve.

[0144] 2) Add acetonitrile (70 mL, 7V), DMF (20 mL, 2V), triethylamine (2 mL, 0.2V), and compound III-A (4.29 g, 1.5 eq) to reaction flask ②, and stir until dissolved and clear. Add the solution from reaction flask ② to reaction flask ①, and control the temperature at 10-20℃ until the reaction is complete. Add 5% phosphoric acid to adjust the pH to 9.5-10.5.

[0145] 3) Take reaction flask ③ and add DMF (50mL, 5V), N,N-diisopropylethylamine (2.5mL, 0.25V) and SMG-3A (5.51g, 3.0eq), stir to dissolve, cool to 10~20℃, add COMU (4.05g, 3.0eq), and stir for 30min.

[0146] 4) Slowly add the reaction solution in reaction flask ③ to the reaction solution in 2). After adding the solution, add N,N-diisopropylethylamine to adjust the pH to 9.5~10.0, control the internal temperature at 10-20°C, and react for 3~5 hours until the reaction is complete to obtain a solution of compound VA.

[0147] 5) Cool the solution from step 4) to 2-8℃, add 28 mL of 1.5 M HCl / MeOH solution (2.8 V), and react at 0-5℃ for 4-6 h. After the reaction is complete, add purified water (650 mL, 65 V) to precipitate the product. Stir for 30 min, filter to obtain a solid wet product, redissolve it with DMF (100 mL, 9.5 V) / EA (60 mL, 6.5 V), add acetonitrile (550 mL, 55 V) to precipitate the product, remove the supernatant, filter, and wash three times with MTBE to obtain 14.89 g of crude smegglutinin, with a molar yield of 114.94% and a purity of 86.78%. The HPLC chromatogram is shown below. Figure 9 .

[0148] 6) Following the purification method described in Example 10, 11.43 g of pure smegglutinin was obtained after one purification by reversed-phase column chromatography, with a purity of 99.23%. The HPLC chromatogram is shown below. Figure 10 The total molar yield of smegglutinin prepared in this example was 88.23%.

[0149]

[0150] Example 9: Preparation of smegglutinin by continuous reaction (one-pot method)

[0151] 1) Take reaction flask ①, add purified water (180mL, 18V) and triethylamine (3mL, 0.3V), mix and stir evenly, and cool down to an internal temperature of 5-15℃. Then add SMG-1 (10.0g, 1.0eq) and stir to dissolve.

[0152] 2) Add acetonitrile (70 mL, 7V), DMF (20 mL, 2V), triethylamine (2 mL, 0.2V), and compound III-A (4.29 g, 1.5 eq) to reaction flask ②, and stir until dissolved and clear. Add the solution from reaction flask ② to reaction flask ①, and maintain the temperature at 10-20℃ until the reaction is complete. Add 5% phosphoric acid to adjust the pH to 10-11.

[0153] 3) Take reaction flask ③ and add DMF (50mL, 5V), DIEA (1.97mL, 3.6eq), and Fmoc-His-Aib-OH (4.37g, 3.0eq). Stir to dissolve, cool to 10~20℃, then add COMU (4.05g, 3.0eq) and stir for 1h. The reaction is complete when detected by TLC.

[0154] 4) Add the solution in reaction flask ③ to the reaction solution in 2), control the temperature at 10~25℃, add DIEA, adjust the pH to 9.5~10.0, react for 3~5 hours, and then react for 3~5 hours until the reaction is complete to obtain compound VB solution.

[0155] 5) Concentrate the reaction solution from 4) above to approximately 200 mL (20V), add diethylamine (25 mL, 2.5V), and stir for 4–8 h. Once the reaction is complete, add purified water (650 mL, 65V), then add phosphoric acid to adjust the pH to 5.0. The product precipitates; remove the supernatant, add acetonitrile (150 mL * 2, 15V * 2), and stir twice. Filter and wash three times with MTBE (50 mL, 5V) to obtain 15.14 g of crude smegglutinin, with a molar yield of 116.87% and an HPLC purity of 88.65%. The HPLC chromatogram is shown below. Figure 11 .

[0156] 6) Following the purification method of Example 10, 11.58 g of pure smegglutinide was obtained after reversed-phase column chromatography purification, with a purity of 99.28%. The HPLC chromatogram is shown below. Figure 12 The total molar yield of smegglutinin prepared using the method of Example 7 was 89.39%.

[0157]

[0158] Example 10: Purification of smegglutinin

[0159] Sample preparation:

[0160] Accurately weigh 28.62 g of crude smegglutinin prepared according to steps 1)-2) of Example 7, dissolve it in 0.1% ammonia water (1144.8 mL), stir for 4 hours until completely dissolved, and then filter through a 0.65 μm filter membrane.

[0161] purification:

[0162] Instrument: Hanbang Technology MPLC-DAC50 preparation system;

[0163] Purification packing material: Octylsilane-bonded silica gel packing material;

[0164] Mobile phase A: Aqueous solution of phosphoric acid at pH 3.5;

[0165] Mobile phase B: Acetonitrile;

[0166] Flow rate: 40 mL / min, detection wavelength: 275 nm.

[0167] The elution gradient is shown in Table 2 (equilibrium-elution-regeneration-equilibrium):

[0168]

[0169] Loading capacity: The crude sample loading amount is 8g. After loading, elute according to the above elution gradient and collect the fraction with a purity of 98.0% or higher.

[0170] The collected fraction was then subjected to salt exchange using a multifunctional membrane experimental device (Hangzhou Ruina Membrane Engineering Co., Ltd., RNM-18G). The filter membrane pore size was selected as 1-2 nm, and the pressure was set to 1.5 MPa. The concentrate was collected, lyophilized, and 22.84 g of pure smegglutinin with a purity of 99.54% was obtained, with a molar yield of 79.80%. See HPLC chromatogram for details. Figure 8 .

[0171] Comparative Example 1:

[0172] 1) Take reaction flask ① and add dichloromethane (40mL, 4V), SMG-2 (10g, 1.0eq) and N-hydroxysuccinimide (2.03g, 1.5eq), stir to dissolve, and cool to an internal temperature of 10~20℃.

[0173] 2) Weigh 2.24 g (1.5 eq) of N,N'-diisopropylcarbodiimide (DIC) and add it to reaction flask ②. Dissolve the DIC in dichloromethane (10 mL, 1V). Slowly add the prepared N,N'-diisopropylcarbodiimide solution dropwise to reaction flask ①, keeping the internal temperature below 25°C. React at 10-20°C for 5 hours. After filtration, concentrate to dryness to obtain compound II-A1 3.85 g. Liquid chromatography purity: 90.47%. See HPLC chromatogram for details. Figure 13 .

[0174]

[0175] Comparative Example 2:

[0176] 1) Take reaction flask ① and add dichloromethane (40mL, 4V), SMG-2 (10g, 1.0eq) and N-hydroxysuccinimide (2.03g, 1.5eq), stir to dissolve, and cool to an internal temperature of 10~20℃.

[0177] 2) Weigh 3.76 g (1.5 eq) of N,N'-dicyclohexylcarbonimide (DCC) into reaction flask ②, add dichloromethane (10 mL, 1V) to dissolve it, and slowly add the prepared N,N'-dicyclohexylcarbonimide solution dropwise into reaction flask ①, controlling the internal temperature to not exceed 25℃. React at 10~20℃ for 5 h, then filter and concentrate to dryness to obtain compound II-A1 4.02 g, liquid phase purity: 92.44%. HPLC chromatogram is shown below. Figure 14 .

[0178]

[0179] As can be seen from Example 1 and Comparative Examples 1-2, when synthesizing the active ester from tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu(SMG-2), using DCC as a solvent produces fewer impurities and results in higher purity of the target product compared to using DIC as a solvent. Furthermore, controlling the reaction temperature to 2-8°C and using nitrogen protection during the reaction process can further significantly improve the purity of the target product.

[0180] Comparative Example 3:

[0181] 1) Take reaction flasks ①-④ and add purified water (40mL, 20V) to each flask, and cool them down to an internal temperature of 5-15℃. Then add SMG-1 (2.0g, 1.0eq) to each flask.

[0182] 2) Add N,N-diisopropylethylamine to reaction flask ① to adjust the pH of the solution to 11.00, and stir to dissolve;

[0183] Add N,N-diisopropylethylamine to reaction flask ② to adjust the pH of the solution to 11.20, and stir to dissolve;

[0184] Add N,N-diisopropylethylamine to reaction flask ③ to adjust the pH of the solution to 11.40, and stir to dissolve;

[0185] Add N,N-diisopropylethylamine to reaction flask ④ to adjust the pH of the solution to 11.61, and stir to dissolve;

[0186] 3) Take reaction flasks ⑤-⑧, and add acetonitrile (140 mL, 7V), DMF (40 mL, 2V), N,N-diisopropylethylamine (4 mL, 0.2V), and compound III-A (8.58 g, 1.5 eq) to each flask, stirring until dissolved and clear. Slowly add the solution from reaction flasks ⑥-⑩ to reaction flasks ①-④, controlling the temperature at 5-15℃ until completion. Adjust the pH to 9.5-10.5 by adding 10% phosphoric acid to obtain the solution of compound IV. See the HPLC chromatogram for details. Figures 15-18 .

[0187] The specific data is shown in Table 4 below:

[0188]

[0189] As shown in Table 4, when the active ester synthesized by tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (SMG-2) reacts with the main chain of GLP-1 (9-37), the residual GLP-1 (9-37) is significantly reduced and the content of impurities is significantly reduced when the pH of the SMG-1 solution is 11.2-11.6 compared with the pH of 11.0. The purity of the target product compound IV is significantly improved, which proves that the pH of the SMG-1 solution is more favorable for the reaction between the side chain active ester and the main chain.

[0190] Comparative Example 4:

[0191] 1) Take reaction flasks ①-③, add purified water (40mL, 20V) to each, cool to an internal temperature of 5-15℃, and then add SMG-1 (2.0g, 1.0eq) to each, and stir to dissolve.

[0192] 2) Add triethylamine to reaction flask ① to adjust the pH of the solution to 11.30±0.1, and stir to dissolve;

[0193] Add 2,4,6-trimethylpyridine (TMP) to reaction flask ② to adjust the pH of the solution to 11.30±0.1, and stir to dissolve;

[0194] Add 4-dimethylaminopyridine (DMAP) to reaction flask ③ to adjust the pH of the solution to 11.30±0.1, and stir to dissolve;

[0195] 3) Take reaction flasks ④-⑥ separately, and add acetonitrile (140 mL, 7V), DMF (40 mL, 2V), triethylamine (4 mL, 0.2V), and compound III-A (8.58 g, 1.5 eq) to each flask, stirring until dissolved and clear. Slowly add the solution from reaction flasks ④-⑥ dropwise to reaction flasks ①-③, controlling the temperature at 5-15℃ until completion. Add 10% phosphoric acid to adjust the pH to 9.5-10.5 to obtain the solution of compound IV. See the HPLC chromatogram for details. Figures 19-21 .

[0196] The specific data is shown in Table 5 below:

[0197]

[0198] As shown in the table above, at the same pH, using triethylamine as a base to adjust the pH can further reduce the content of GLP-1(9-37) residue and impurities compared with other bases, and the purity of target product compound IV is further significantly improved.

[0199] Comparative Example 5:

[0200] 1) The compound IV solution was synthesized according to the method of Example 5. The amount of SMG-1 added was 2 g, and the amount of other reagents added was adjusted proportionally according to the amount of SMG-1 added.

[0201] 2) In a separate reaction flask, add ACN (10 mL, 5V), DIEA (0.39 mL, 3.6 eq), and SMG-3A (1.10 g, 3.0 eq), stir to dissolve, cool to 10~20℃, then add p-toluenesulfonic anhydride (0.62 g, 3.0 eq) and stir for 30 min to obtain an activated SMG-3A solution.

[0202] 3) Control the internal temperature to 10-20°C, slowly add the SMG-3A activation solution to the compound IV solution, and control the pH to 9.5-10.0 by adding DIEA during the reaction process. The reaction is completed in 3-5 hours.

[0203] 4) Add purified water (1L, 50V), stir to dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing a white solid to precipitate. Filter, collect the solid, and dry it under vacuum at 40℃ for at least 2 hours to obtain compound V-A 3.04g, liquid phase purity: 67.43%. HPLC chromatogram is shown below. Figure 22 The molar yield of this step was 108.32%.

[0204] Comparative Example 6:

[0205] 1) The compound IV solution was synthesized according to the method of Example 5. The amount of SMG-1 added was 2 g, and the amount of other reagents added was adjusted proportionally according to the amount of SMG-1 added.

[0206] 2) Take a reaction flask and add DMF (10 mL, 5V), DIEA (0.39 mL, 3.6 eq), and SMG-3A (1.10 g, 3.0 eq). Stir to dissolve and cool to 10~20℃. Then add TPTU (0.56 g, 3.0 eq) and stir for 30 min to obtain an activated SMG-3A solution.

[0207] 3) Control the internal temperature to 10-20°C, slowly add the SMG-3A activation solution to the compound IV solution, and control the pH to 9.5-10.0 by adding DIEA during the reaction process. The reaction is completed in 3-5 hours.

[0208] 4) Add purified water (1L, 50V), stir to dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing a white solid to precipitate. Filter, collect the solid, and dry it under vacuum at 40℃ for at least 2 hours to obtain compound V-A 3.12g, liquid phase purity: 84.01%. HPLC chromatogram is shown below. Figure 23 The molar yield of this step was 111.17%.

[0209] Comparative Example 7:

[0210] 1) The compound IV solution was synthesized according to the method of Example 5. The amount of SMG-1 added was 2 g, and the amount of other reagents added was adjusted proportionally according to the amount of SMG-1 added.

[0211] 2) Take a reaction flask and add DMF (12mL, 6V), DCM (4mL, 2V), DIEA (0.47mL, 4.3eq), and SMG-3A (1.10g, 3.0eq). Stir to dissolve and cool to 10~20℃. Then add Oxyma (0.32g, 3.6eq) and EDCI (0.43g, 3.6eq) and stir for 1h to obtain an activated SMG-3A solution.

[0212] 3) Control the internal temperature to 10-20°C, slowly add the SMG-3A activation solution to the compound IV solution, and control the pH to 9.5-10.0 by adding DIEA during the reaction process. The reaction is completed in 3-5 hours.

[0213] 4) Add purified water (1L, 50V), stir to dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing a white solid to precipitate. Filter, collect the solid, and dry it under vacuum at 40℃ for at least 2 hours to obtain compound V-A 2.93g, liquid phase purity: 87.57%. HPLC chromatogram is shown below. Figure 24 The molar yield of this step was 104.40%.

[0214] Comparative Example 8:

[0215] 1) The compound IV solution was synthesized according to the method of Example 5. The amount of SMG-1 added was 2 g, and the amount of other reagents added was adjusted proportionally according to the amount of SMG-1 added.

[0216] 2) Take a reaction flask and add DMF (12 mL, 6 V), DIEA (0.39 mL, 3.6 eq), and SMG-3A (1.10 g, 3.0 eq). Stir to dissolve and cool to 10~20℃. Then add Pyoxim (1.00 g, 3.0 eq) and stir for 1 h to obtain an activated SMG-3A solution.

[0217] 3) Control the internal temperature to 10-20°C, slowly add the SMG-3A activation solution to the compound IV solution, and control the pH to 9.5-10.0 by adding DIEA during the reaction process. The reaction is completed in 3-5 hours.

[0218] 4) Add purified water (1L, 50V), stir to dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing a white solid to precipitate. Filter, collect the solid, and dry it under vacuum at 40℃ for at least 2 hours to obtain compound V-B 3.18g, liquid phase purity: 89.53%. HPLC chromatogram is shown below. Figure 25 The molar yield of this step was 113.31%.

[0219] The results of using different coupling agents are shown in Table 6 below:

[0220]

[0221] As shown in Table 6, when SMG-3 was activated and linked to the glutamic acid residue at the N-terminus of the SMG-1 main chain, COMU was used. Compared with other coupling agents, COMU significantly reduced the impurity content and improved the purity of the target product compound V. It successfully solved the side reaction problem between the activated dipeptide and the main chain arginine, and obtained high-purity smegglutide with a protecting group.

[0222] Comparative Example 9:

[0223] 1) Prepare compound V-B solution according to steps 1)-4) of Example 9. The amount of SMG-1 added is 1.0g, and the amount of other reagents added is adjusted proportionally according to the amount of SMG-1 added.

[0224] 2) Add 2M NaOH aq. (20mL, 10V) to the solution of compound V-B and stir for 3h. After the reaction is complete, add purified water (100mL, 50V), then add phosphoric acid to adjust the pH to 5.0. The product precipitates, the supernatant is removed, and acetonitrile (150mL*2, 15V*2) is added and stirred twice. After filtration, the product is washed three times with MTBE (50mL, 5V) to obtain 14.02g of crude smegglutinin, with a molar yield of 108.23% and an HPLC purity of 71.43%. The HPLC chromatogram is shown in [reference needed]. Figure 26 .

[0225] Comparative Example 10:

[0226] 1) Prepare compound V-B solution according to the synthesis method of Example 9, with SMG-1 feed amount of 1.0g.

[0227] 2) Add piperidine (4 mL, 4 V) to the solution of compound V-B and stir for 3 h. After the reaction is complete, add purified water (100 mL, 50 V), then add phosphoric acid to adjust the pH to 5.0. The product precipitates. Remove the supernatant, add acetonitrile (150 mL * 2, 15 V * 2), and stir twice. Filter and wash three times with MTBE (50 mL, 5 V) to obtain 13.68 g of crude smegglutinin, with a molar yield of 105.60% and an HPLC purity of 81.43%. The HPLC chromatogram is shown below. Figure 27 .

[0228] Comparative Example 11:

[0229] 1) Prepare compound V-B solution according to the synthesis method of Example 9, with SMG-1 feed amount of 1.0g.

[0230] 2) Add DBU (1 mL, 1 V) to the solution of compound V-B and stir for 3 h. After the reaction is complete, add purified water (100 mL, 50 V), then add phosphoric acid to adjust the pH to 5.0. The product precipitates, the supernatant is removed, and acetonitrile (150 mL * 2, 15 V * 2) is added and stirred twice. After filtration, the product is washed three times with MTBE (50 mL, 5 V) to obtain 13.54 g of crude smegglutinin, with a molar yield of 104.52% and an HPLC purity of 76.69%. The HPLC chromatogram is shown in [reference needed]. Figure 28 .

[0231]

[0232] As shown in the table above, in the method of the present invention, by using diethylamine to deprotect smegglutide containing the Fmoc protectant, a higher purity crude smegglutide can be obtained compared with other deprotecting agents, achieving better results.

[0233] As can be seen from the above examples and comparative examples, the method of the present invention, by optimizing reaction conditions, reduces the occurrence of side reactions, lowers the impurity content, and improves the purity of crude semaglutide. The resulting semaglutide has high purity and yield, and the method also reduces intermediate processing steps and the use of organic reagents, thus lowering costs and being more environmentally friendly. In particular, the continuous preparation method of the present invention eliminates the need for intermediate processing, reducing at least four steps and materials, resulting in a cost reduction of at least approximately 20%. In summary, the method of the present invention has fewer reaction steps, is simpler to operate, lowers costs, and produces semaglutide with high purity and high yield, making it particularly suitable for large-scale industrial production of semaglutide.

Claims

1. A semi-synthetic preparation method for smegglutinin, characterized in that, Includes the following steps: Step 1: In the presence of the first base catalyst, the solution of compound SMG-1 is mixed with the solution of compound III to react and obtain compound IV. The reaction formula is as follows: ， Wherein, R1 is succinimide, benzotriazolyl, 5-norbornene-2,3-dicarboximide, or p-toluenesulfonyl, and the amount of the first base catalyst added is such that the pH of the SMG-1 solution is 11.2-11.6, and the first base catalyst is triethylamine; Step 2: In the presence of a coupling agent and a second base catalyst, the solution of compound IV is mixed with the solution of compound SMG-3 to obtain compound V through coupling, as shown in the following reaction formula: ， Wherein, R2 is H, R3 is fluorenemethyloxycarbonyl, the pH of step 2 is 9.5-10.0, and the coupling agent is COMU; Step 3: Deprotect compound V using a deprotecting agent to obtain smegglutinin, wherein the deprotecting agent is diethylamine.

2. The semi-synthetic preparation method according to claim 1, characterized in that, The amount of the first alkaline catalyst added is such that the pH of the SMG-1 solution is 11.3 ± 0.

1.

3. The semi-synthetic preparation method according to claim 1, characterized in that, The solvents of the SMG-1 solution, the SMG-III solution, and the SMG-3 solution are each independently selected from one or more combinations of water, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone; the second base catalyst is one or more combinations of triethylamine, N,N-diisopropylethylamine, tetramethylethylenediamine, aniline, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine.

4. The semi-synthetic preparation method according to claim 3, characterized in that, The solvent for the SMG-1 solution is water, the solvent for the SMG-III solution is acetonitrile, and the solvent for the SMG-3 solution is N,N-dimethylformamide.

5. The semi-synthetic preparation method according to claim 1, characterized in that, The second base catalyst is N,N-diisopropylethylamine.

6. The semi-synthetic preparation method according to claim 5, characterized in that, Step 2 includes: Step 2-1A: Add the second base catalyst and the coupling agent to the SMG-3 solution to prepare an activated SMG-3 solution; Step 2-2A: Add the activated solution of compound SMG-3 to the solution of compound IV, and add the second base catalyst to adjust the pH to 9.5-10.0, and react to obtain the solution of compound V; Step 2-3A: Add water to the solution of compound V to dilute it, and add acid to adjust the pH to 5.

0. A white solid precipitates out. Filter and vacuum dry to obtain compound V, which is used in step 3.

7. The semi-synthetic preparation method according to claim 6, characterized in that, The acid is phosphoric acid.

8. The semi-synthetic preparation method according to claim 5, characterized in that, The semi-synthetic preparation method is a continuous reaction, and intermediates are not separated during the reaction process; The compound SMG-3 is Fmoc-His-Aib-OH; Step 2 includes: Step 2-1C: Add the second base catalyst and the coupling agent to the SMG-3 solution to prepare an activated SMG-3 solution; Step 2-2C: Add the activated solution of compound SMG-3 to the solution of compound IV, and add the second base catalyst to make the pH 9.5-10.

0. The reaction yields the solution of compound V, which is directly used in step 3. Step 3 includes adding a deprotecting agent, namely diethylamine, to the solution of compound V for deprotection.

9. The semi-synthetic preparation method according to claim 1, characterized in that, The molar ratio of compound SMG-1 to compound III is 1:1.0-2.0, the molar ratio of compound IV to compound SMG-3 is 1:3.0, and the molar ratio of compound V to the deprotecting agent is 1:5 (w / v).

10. The semi-synthetic preparation method according to claim 1, characterized in that, The reaction temperature in step 1 is 5-15℃, and the reaction temperature in step 2 is 10-20℃.

11. The semi-synthetic preparation method according to any one of claims 1-10, characterized in that, Compound III was prepared by the following steps: Step A: In the presence of a condensing agent, a solution of compound SMG-2 is mixed with a solution of compound I, and dehydration condensation is performed to obtain compound II, as shown in the following reaction formula: ， Compound I is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensing agent is DCC, a protective gas is used during the reaction, and the reaction temperature is 2-8℃. Step B: Compound II undergoes deprotection to yield compound III, as shown in the following reaction formula: 。 12. The semi-synthetic preparation method according to claim 11, characterized in that, The solvent for the SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, and the solvent for the compound I solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran. The molar ratio of compound SMG-2 to compound I is 1:1.1-2.

0.

13. The semi-synthetic preparation method according to claim 11, characterized in that, Step B uses a TFA / TIS / DCM with a volume ratio of 90:5:5 or a TFA / TIS / PPW with a volume ratio of 95:2.5:2.5 for deprotection.

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