Semi-synthesis preparation method of semeglutide and preparation method of semeglutide side chain

Abstract

The invention relates to the field of biological medicine preparation, and provides a semi-synthesis preparation method of semeglutide and a preparation method of a semeglutide side chain, the semi-synthesis preparation method comprises the following steps: 1, in the presence of a first base catalyst, mixing a compound SMG-1 solution with a compound III solution, reacting to obtain a compound IV, and filtering to obtain a compound III; wherein the addition amount of the first base catalyst is such that the pH value of the SMG-1 solution is 11.2-11.6; 2, in the presence of a coupling agent and a second base catalyst, a compound IV solution and a compound SMG-3 solution are mixed and coupled to obtain a compound V, and the pH value in the step 2 is 9.5-10.0; and 3, carrying out deprotection on the compound V by using a deprotection agent to finally obtain the semeglutide. The method provided by the invention has the advantages of few reaction steps, reduced cost, high semeglutide purity and high yield, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical preparation, and particularly relates to a semi-synthetic preparation method of semaglutide and a preparation method of the side chain of semaglutide. Technical Background

[0002] The English name of semaglutide is Semaglutide. Semaglutide is a GLP-1 analogue and has 94% homology with human GLP-1. It is a long-acting glucagon-like peptide-1 (GLP-1) analogue developed and produced by Novo Nordisk A / S in Denmark. Compared with other marketed antidiabetic drugs, semaglutide shows better clinical therapeutic effects in controlling and maintaining blood glucose levels and has become a new generation of long-acting antidiabetic drug dominating the diabetes drug market. In addition, studies have found that semaglutide has the potential to treat cancer, Alzheimer's disease, and Parkinson's disease and can reduce the risk of cardiovascular diseases, etc. Therefore, it is particularly important to develop an efficient and economical synthesis process for semaglutide.

[0003] The preparation methods of semaglutide API reported currently are mainly solid-phase total synthesis (SPPS) methods. That is, starting from the carboxyl terminus of the polypeptide, after the first amino acid is connected to the resin, the protecting group of the amino group of the amino acid is removed, and according to the peptide sequence, the next amino acid is coupled, the amino protecting group is removed, and then coupled again. The operation of repeating the cycle is carried out until the amino acid assembly is completed. The polypeptide is cleaved from the resin and all protecting groups are removed at the same time to obtain the crude polypeptide, and then the crude polypeptide is purified and freeze-dried to obtain the polypeptide API. The advantages of solid-phase total synthesis are mainly manifested in that the initial reactants and products are both connected to the solid-phase carrier, so all reactions can be carried out in one reaction vessel, which is convenient for automated operation. However, the continuous use of excessive protected amino acids and excessive coupling agents, and the need for sufficient washing operations after the extension of each amino acid, make the material consumption of this method serious and the cost relatively expensive. In addition, the formation of secondary structures during the synthesis of polypeptides by SPPS often leads to a decrease in the efficiency of each synthesis step. The impurities are often deletion peptides that lose one or more amino acids in the final sequence. At the same time, the side chain of the fatty chain is relatively long, resulting in a strong steric hindrance effect, making the coupling reaction difficult to carry out, and easily leading to problems such as low product yield and racemization. Although there are optimized methods based on SPPS, such as the patent CN117986349B previously applied for and authorized by the applicant, which reduces the synthesis steps, due to the inherent characteristics of SPPS, a large amount of solvent is still inevitably required for sufficient washing.

[0004] The fermentation semi-synthesis method uses the semaglutide precursors Arg34GLP-1(9-37) or Arg34GLP-1(11-37) prepared by biological methods as starting materials, modifies the aliphatic side chain at Lyr and completes the modification of the remaining peptide chain at the N-terminus to finally obtain semaglutide. Since the semaglutide precursors are usually prepared by microbial fermentation technology, the main chain of semaglutide is synthesized through the growth and metabolic processes of microorganisms, which has the characteristics of high efficiency, environmental protection, and high flexibility. At the same time, due to the specificity of microbial fermentation, the racemic isomer impurities and deletion peptide contamination of the semaglutide precursors can be greatly reduced. However, the existing fermentation semi-synthesis method still has problems such as cumbersome steps, low purity of the crude product, and low yield of the final product. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a semi-synthesis preparation method of semaglutide with high purity of the crude product, high yield of the final product, simple steps, and is particularly suitable for industrial production, as well as a preparation method of the side chain of semaglutide.

[0006] One aspect of the present invention provides a semi-synthesis preparation method of semaglutide, comprising the following steps: Step 1: Under the condition of the presence of a first base catalyst, mix the compound SMG-1 solution with the compound III solution to react to obtain compound IV, and the reaction formula is as follows: , wherein, R1 is succinimido, benzotriazolyl, 5-norbornene-2,3-dicarboximido or p-toluenesulfonyl, and the addition amount of the first base catalyst is such that the pH of the SMG-1 solution is 11.2 - 11.6; Step 2: Under the condition of the presence of a coupling agent and a second base catalyst, mix the compound IV solution with the compound SMG-3 solution for coupling to obtain compound V, and the reaction formula is as follows:

[0007] wherein, R2 is trityl or H, R3 is tert-butoxycarbonyl or fluorenylmethoxycarbonyl, and the pH of Step 2 is 9.5 - 10.0; Step 3: Use a deprotecting agent to deprotect compound V to obtain semaglutide.

[0008] Preferably, the addition amount of the first base catalyst is such that the pH of the SMG-1 solution is 11.3 ± 0.1.

[0009] Preferably, the solvents of the compound SMG-1 solution, the compound III solution, and the compound 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 first base catalyst is one or more combinations of triethylamine, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine; the coupling agent is COMU, TPTU, Oxyma / EDCI, PyOxim, or p-toluenesulfonic anhydride; the second base catalyst is one or more combinations of triethylamine, N,N-diisopropylethylamine, tetramethylethylenediamine, aniline, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine.

[0010] More preferably, the solvent of the compound SMG-1 solution is water, the solvent of the compound III solution is acetonitrile, and the solvent of the compound SMG-3 solution is N,N-dimethylformamide.

[0011] More preferably, the first base catalyst is triethylamine.

[0012] More preferably, the coupling agent is COMU and the second base catalyst is N,N-diisopropylethylamine.

[0013] More preferably, step 2 includes: Step 2-1A: Add the second base catalyst and the coupling agent to the compound SMG-3 solution to prepare a compound SMG-3 activation solution; Step 2-2A: Add the compound SMG-3 activation solution to the compound IV solution, and supplement the second base catalyst to make the pH 9.5 - 10.0, and react to obtain a compound V solution; Step 2-3A: Dilute the compound V solution with water, and add an acid to adjust the pH to 5.0, precipitate a white solid, filter and vacuum dry to obtain compound V for step 3.

[0014] More preferably, the acid is phosphoric acid.

[0015] More preferably, the deprotecting agent in step 3 is TFA / TIS / DCM with a volume ratio of 90:5:5.

[0016] Preferably, the semi-synthetic preparation method is a continuous reaction, and the intermediate is not separated during the reaction process; The compound SMG-3 is Boc-His(Trt)-Aib-OH; Step 2 includes: Step 2-1B: Add the second base catalyst and the coupling agent to the compound SMG-3 solution to prepare an activated compound SMG-3 solution; Step 2-2B: Add the activated compound SMG-3 solution to the compound Ⅳ solution, and supplement the second base catalyst to make the pH 9.5 - 10.0, and react to obtain a compound Ⅴ solution, and the compound Ⅴ solution is directly used in Step 3; Step 3 includes adding a deprotecting agent HCl / MeOH to the compound Ⅴ solution for deprotection.

[0017] Preferably, the semi-synthetic preparation method is a continuous reaction, and the intermediate is 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 compound SMG-3 solution to prepare an activated compound SMG-3 solution; Step 2-2C: Add the activated compound SMG-3 solution to the compound Ⅳ solution, and supplement the second base catalyst to make the pH 9.5 - 10.0, and react to obtain a compound Ⅴ solution, and the compound Ⅴ solution is directly used in Step 3; Step 3 includes adding a deprotecting agent for deprotection to the compound Ⅴ solution, and the deprotecting agent is diethylamine, NaOH, piperidine or DBU.

[0018] More preferably, the deprotecting agent is diethylamine.

[0019] Preferably, the molar ratio of the compound SMG-1 to the compound Ⅲ is 1:1.0 - 2.0, the molar ratio of the compound IV to the compound SMG-3 is 1:3.0, and the dosage ratio of the compound Ⅴ to the deprotecting agent is 1:5 (w / v).

[0020] Preferably, the reaction temperature of Step 1 is 5 - 15 °C, and the reaction temperature of Step 2 is 10 - 20 °C.

[0021] Preferably, the compound Ⅲ is prepared by the following steps: Step A: In the presence of a condensation reagent, mix the compound SMG-2 solution and the compound I solution, and perform dehydration condensation to obtain the compound II. The reaction formula is as follows: , Among them, Compound 1 is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensation reagent is DCC, a protective gas is used to protect during the reaction process, and the reaction temperature is 2-8 °C; Step B: Compound II is deprotected to obtain Compound III, and the reaction formula is as follows: 。

[0022] Preferably, the solvent of the Compound SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, 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.

[0023] Preferably, Step B uses TFA / TIS / DCM with a volume ratio of 90:5:5 or TFA / TIS / PPW with a volume ratio of 95:2.5:2.5 for deprotection.

[0024] Another aspect of the present invention provides a method for preparing the side chain of semaglutide, and the method includes: Step A: In the presence of a condensation reagent, a Compound SMG-2 solution is mixed with a Compound I solution, and dehydration condensation is carried out to obtain Compound II, and the reaction formula is as follows: , Among them, Compound 1 is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensation reagent is DCC, a protective gas is used to protect during the reaction process, and the reaction temperature is 2-8 °C; Step B: Compound II is deprotected, crystallized, washed and then dried to obtain Compound III, and the reaction formula is as follows: 。

[0025] Preferably, the solvent of the Compound SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, 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.

[0026] Preferably, Step B uses TFA / TIS / DCM with a volume ratio of 90:5:5 or TFA / TIS / PPW with a volume ratio of 95:2.5:2.5 for deprotection.

[0027] The beneficial effects of the present invention are: In the semi-synthetic preparation method of the present 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, there are multiple active sites on the GLP-1(9-37) backbone that can react with the side-chain active ester, and the by-products generated not only affect the purity and yield of the final semaglutide, but also affect the reaction process. Through the optimization of the reaction conditions, the present invention enables the chemoselective connection of the activated ester of SMG-2 with the side-chain amino group of Lys in the Arg34GLP-1(9-37) (SMG-1) sequence, achieving site-directed modification of the side-chain fatty acid, and the content of other by-products is less than 1.5%. In addition, by controlling the reaction conditions, the present invention selectively connects Boc-His(Trt)-Aib-OH or Fmoc-His-Aib-OH (SMG-3) after activation to the glutamate residue at the N-terminus of the SMG-1 backbone, successfully solving the side reaction problem between the activated dipeptide and the backbone arginine, and obtaining high-purity semaglutide with protecting groups. Through the screening of deprotecting reagents, a crude product with high purity and high yield is obtained, and a pure semaglutide with a purity greater than 99.2% and a total molar yield greater than 88% can be obtained by only one purification.

[0028] In a preferred embodiment of the present invention, through the optimization of the reagents used in the reaction process, a continuous reaction is carried out, reducing the purification and treatment process of intermediates, reducing the number of processes and the use of a large amount of reagents, and reducing the cost and the impact on the environment.

[0029] In addition, when synthesizing the active ester of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (SMG-2), the active ester of the side chain will undergo ring-opening rearrangement to generate β-alanyl impurities, and react with the side-chain active ester to generate bis-side-chain β-alanyl impurities. Through the optimization of the reaction conditions, the present invention solves the problem of side reaction impurities in the synthesis process of the side-chain active ester, and high-purity side-chain active ester can be obtained.

[0030] In summary, the method of the present invention has few reaction steps, is simple to operate, greatly simplifies the process, reduces the time and material costs, reduces the generation of waste liquid and by-products, and the prepared semaglutide has high purity and high yield, and is particularly suitable for the large-scale industrial production of semaglutide. Description of the Drawings

[0031] Figure 1 It is the HPLC chromatogram of Compound II-A in Example 1; Figure 2 It is the HPLC chromatogram of Compound III-A in Example 2; Figure 3 It is the HPLC chromatogram of Compound III-B in Example 3; Figure 4 HPLC chromatogram of Compound Ⅲ-C in Example 4; Figure 5 is the HPLC chromatogram of Compound Ⅳ in Example 5; Figure 6 is the HPLC chromatogram of Compound V-A in Example 6; Figure 7 is the HPLC chromatogram of the semaglutide crude product in Example 7; Figure 8 is the HPLC chromatogram of the pure semaglutide in Example 7; Figure 9 is the HPLC chromatogram of the semaglutide crude product in Example 8; Figure 10 is the HPLC chromatogram of the pure semaglutide in Example 8; Figure 11 is the HPLC chromatogram of the semaglutide crude product in Example 9; Figure 12 is the HPLC chromatogram of the pure semaglutide in Example 9; Figure 13 is the HPLC chromatogram of Compound Ⅱ-A in Comparative Example 1; Figure 14 is the HPLC chromatogram of Compound Ⅱ-A in Comparative Example 2; Figure 15 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of pH = 11.0 in Comparative Example 3; Figure 16 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of pH = 11.2 in Comparative Example 3; Figure 17 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of pH = 11.4 in Comparative Example 3; Figure 18 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of pH = 11.61 in Comparative Example 3; Figure 19 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of adjusting pH with DMAP in Comparative Example 4; Figure 20 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of adjusting pH with TMP in Comparative Example 4; Figure 21 is the HPLC chromatogram of Compound Ⅳ prepared under the condition of adjusting pH with triethylamine in Comparative Example 4; Figure 22 is the HPLC chromatogram of Compound V in Comparative Example 5; Figure 23It is the HPLC chromatogram of Compound V in Comparative Example 6; Figure 24 It is the HPLC chromatogram of Compound V in Comparative Example 7; Figure 25 It is the HPLC chromatogram of Compound V in Comparative Example 8; Figure 26 It is the HPLC chromatogram of crude semaglutide in Comparative Example 9; Figure 27 It is the HPLC chromatogram of crude semaglutide in Comparative Example 10; Figure 28 It is the HPLC chromatogram of crude semaglutide in Comparative Example 11; Figure 29 It is the mass spectrum of pure semaglutide prepared in Example 7. Detailed implementation manners

[0032] The following further describes the present invention in conjunction with the accompanying drawings and embodiments. However, these embodiments are exemplary and are only convenient for those skilled in the art to further understand the characteristics of the present invention, and do not constitute any limitation to the scope of the present invention. The reagents used in the present invention are all conventional commercially available products without special regulations. The percentages appearing in the present invention are all mass percentages without special instructions. The high-performance liquid chromatograph used for purity detection in the following embodiments is Waters 2695 Separations Module, model: UV(ALLLANCE E2695 / ACQUITY Arc), and the chromatographic column is Kinetex 2.6μm C18 100Å 150*4.6mm (column temperature 30°C). The manufacturer model of the high-performance liquid chromatography used for purifying semaglutide is Hanbang Technology MPLC-DAC50, and the specific parameters can be seen in the relevant embodiments. The mass spectrometer is an ultra-high performance liquid chromatography (Waters Technology (Shanghai) Co., Ltd., ACQUIIY UPLC H-CLASS PIUS), and the column is 1.7μm PEPTIDE XB-C18 10CLC Column 150*2.1nm.

[0033] In the present invention, the semi-synthetic preparation method refers to a preparation method that uses semaglutide precursors Arg34GLP-1(9-37) or Arg34GLP-1(11-37) as starting materials, modifies the fatty acid side chain at Lyr and completes the modification of the remaining peptide chain at the N-terminus to finally obtain semaglutide. The semaglutide precursors Arg34GLP-1(9-37) or Arg34GLP-1(11-37) can be prepared by microbial fermentation technology.

[0034] In the present invention, the protective gas refers to a gas with stable chemical properties that can prevent the protected substance from being oxidized by oxygen in the air, and can be nitrogen, noble gas, etc.

[0035] In the present invention, the abbreviations and their corresponding English meanings are shown in Table 1:

[0036] The semi-synthetic preparation method of semaglutide in the present invention includes: Step 1: In the presence of a first base catalyst, a solution of compound SMG-1 is mixed with a solution of compound III, and compound IV is obtained by reaction. The reaction formula is as follows: , wherein, R1 is benzotriazolyl, succinimidyl, 5-norbornene-2,3-dicarboximido or p-toluenesulfonyl, and the addition amount of the first base catalyst is such that the pH of the SMG-1 solution is 11.2 - 11.6; Step 2: In the presence of a coupling agent and a second base catalyst, a solution of compound IV is mixed with a solution of compound SMG-3, and coupling is carried out to obtain compound V. The reaction formula is as follows:

[0037] wherein, R2 is trityl or H, R3 is tert-butoxycarbonyl or fluorenylmethoxycarbonyl, and the pH of Step 2 is 9.5 - 10.0; Step 3: The compound V is deprotected using a deprotecting agent, crystallized, purified, and freeze-dried to finally obtain semaglutide.

[0038] The inventor of the present invention has found that in Step 1, when the pH of the SMG-1 solution is adjusted to 11.2 - 11.6, the activated ester of SMG-2, i.e., Compound III, can chemically selectively connect with the side-chain amino group of Lys in the SMG-1 sequence, reducing the by-products generated by reactions at other sites, and the obtained Compound IV has high purity. Without purifying the obtained Compound IV solution and directly using it for subsequent reactions will not affect the occurrence of subsequent reactions, with fewer side reactions, and the finally obtained crude semaglutide has high purity. Meanwhile, in Step 2, when the pH of the reaction system is adjusted to 9.5 - 10.0, the side reaction between the activated dipeptide and the main-chain arginine is reduced, which is beneficial to improving the purity of the crude semaglutide. In a preferred embodiment of the present invention, the pH of the SMG-1 solution is adjusted to 11.3 ± 0.1. The pH of the SMG-1 solution is adjusted by a first base catalyst, and the first base catalyst can be one or a combination of more of triethylamine, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine, triisopropylamine, N-methylmorpholine, pyridine, 4-dimethylaminopyridine. In a preferred embodiment, the first base catalyst is triethylamine. When triethylamine is used, the occurrence of side reactions can be further inhibited, and the purity of Compound IV can be improved. The pH of the reaction system in Step 2 is adjusted by a second base catalyst, and the second base catalyst can be one or a combination of more of triethylamine, N,N-diisopropylethylamine, tetramethylethylenediamine, aniline, triisopropylamine, N-methylmorpholine, pyridine, 4-dimethylaminopyridine. In a preferred embodiment, the second base catalyst is N,N-diisopropylethylamine.

[0039] The semi-synthetic preparation method of the present invention is a liquid-phase reaction, and the solvent used is a good solvent that can excellently dissolve each reactant, and its dosage can be such that each reactant is completely dissolved. The solvents of the Compound SMG-1 solution, the Compound III solution, and the Compound SMG-3 solution can each independently be selected from one or a combination of more of water, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone. In a preferred embodiment, the solvent of the Compound SMG-1 solution is water, the solvent of the Compound III solution is acetonitrile, and the solvent of the Compound SMG-3 solution is N,N-dimethylformamide. When the solvents are selected as above, not only can each reactant be fully dissolved, and they can be miscible during the reaction, which is beneficial to the reaction of each reactant, but also it is beneficial to the precipitation of the intermediate and / or the crude semaglutide during the subsequent purification process of the intermediate and / or the crude semaglutide, reducing steps such as washing, and will not reduce the purity of the intermediate and / or the crude semaglutide.

[0040] 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, and the resulting semaglutide with a protecting agent has a higher purity.

[0041] 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 compound SMG-3 solution to prepare a compound SMG-3 activated solution; step 2-2A: adding the compound SMG-3 activated solution to the compound Ⅳ solution, and additionally adding the second base catalyst to make the pH 9.5-10.0, and reacting to obtain a compound Ⅴ solution; step 2-3A: diluting the compound V solution with water, and adding an acid to adjust the pH to 5.0 to precipitate a white solid, filtering and vacuum drying to obtain compound V for use in step 3. Among them, the solvent of the compound SMG-1 solution is water, the solvent of the compound Ⅲ solution is acetonitrile, the solvent of the compound 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, the occurrence of side reactions is reduced. In step 2-3A, high-purity semaglutide with a protecting agent can be precipitated only by adjusting the pH of the solution, without multiple washings to remove unwanted by-products, reducing the processing steps, lowering the cost, and reducing wastewater, which is more environmentally friendly. In a preferred embodiment, phosphoric acid is used to adjust the pH of the solution in step 2-3A. In step 3, the compound V obtained by the above step 2 is deprotected, crystallized, purified and freeze-dried to finally obtain semaglutide. In step 3, the known deprotection can be used to deprotect the compound V. In a preferred embodiment, the deprotecting agent is TFA / TIS / DCM with a volume ratio of 90:5:5.

[0042] In a preferred embodiment, the semi-synthetic preparation method of the present invention is a continuous reaction, and the intermediate is not separated during the reaction process. Among them, 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 solution of compound SMG-3 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 additionally adding the second base catalyst to make the pH 9.5-10.0, and 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 HCl / MeOH to the solution of compound V for deprotection. In this embodiment, it is not necessary to purify the compound V obtained in Step 2, and directly add HCl / MeOH for deprotection, which reduces the steps of precipitating, purifying, and redissolving the intermediate and the use of related reagents, simplifies the production process, reduces the cost, and the semaglutide finally obtained can still maintain a high yield and purity.

[0043] In another preferred embodiment of the present invention, the semi-synthetic preparation method of the present invention is a continuous reaction, and the intermediate is not separated during the reaction process; among them, 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 solution of compound SMG-3 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 additionally adding the second base catalyst to make the pH 9.5-10.0, and 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, and the deprotecting agent can be diethylamine, inorganic base, piperidine or DBU, preferably diethylamine. This embodiment also reduces the steps of precipitating, purifying, and redissolving the intermediate and the use of related reagents, simplifies the production process, reduces the cost, and the semaglutide finally obtained can still maintain a high yield and purity.

[0044] 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 dosage 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 there are fewer by-products.

[0045] The present invention also relates to a preparation method of the side chain intermediate compound III of semaglutide, which includes step A: under the condition of the presence of a condensation reagent, mixing the solution of compound SMG-2 with the solution of compound I, and performing dehydration condensation to obtain compound II. The reaction formula is as follows: , wherein compound 1 is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensation reagent is DCC, an inert gas is used for protection during the reaction, and the reaction temperature is 2-8°C; Step B: Compound II is deprotected, crystallized, washed and then dried to obtain compound III. The reaction formula is as follows: .

[0046] In a preferred embodiment, the solvent of the compound SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, 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. In a preferred embodiment, deprotection is carried out in step B using TFA / TIS / DCM with a volume ratio of 90:5:5 or TFA / TIS / PPW with a volume ratio of 95:2.5:2.5.

[0047] Through the optimization of the above specific reaction conditions, the method of the present invention reduces the β-alanyl impurities generated by the ring-opening rearrangement of the side chain active ester and the bis-side-chain β-alanyl impurities generated by the reaction between the side chain active esters during the synthesis of the active ester of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (SMG-2), and the obtained side chain active ester has high purity.

[0048] Example 1: Preparation of compound II-A 1) Add dichloromethane (40 mL, 4V), 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 down to an internal temperature of 0-5°C.

[0049] 2) Weigh N,N'-dicyclohexylcarbodiimide (DCC) (3.76 g, 1.5 eq) and add it to reaction flask ②, then add dichloromethane (10 mL, 1V) to dissolve. Slowly drop the prepared N,N'-dicyclohexylcarbodiimide solution into reaction flask ①, protect with nitrogen and control the internal temperature not to exceed 10°C, react at 2-8°C for 5 h to end, filter and concentrate to dryness to obtain 14.37 g of compound II-A, liquid phase purity: 97.33%. The HPLC spectrum is shown in Figure 1 .

[0050]

[0051] Example 2: Preparation of Compound Ⅲ-A 1) Dissolve 14.37 g of Compound Ⅱ-A prepared in Example 1 in dichloromethane (2.5 mL, 0.25V). Under nitrogen protection and with the temperature controlled at about 5 - 10 °C, successively add trifluoroacetic acid (45 mL, 4.5V) and triisopropylsilane (2.5 mL, 0.25V), and react at 2 - 8 °C for 4 hours under nitrogen protection.

[0052] 2) Control the temperature at 2 - 8 °C, and dropwise add methyl tert-butyl ether (300 mL, 30V) under nitrogen protection. After adding, stir at a controlled temperature for 30 minutes. Filter, and wash the filter cake three times with methyl tert-butyl ether (50 mL, 5V). Remove the filter cake and dry it in a vacuum oven to obtain 9.23 g of Compound Ⅲ-A. Molar yield: 87.25%, liquid phase purity: 99.65%. See the HPLC spectrum in Figure 2 .

[0053]

[0054] Example 3: Preparation of Compound Ⅲ-B 1) Add dichloromethane (40 mL, 4V), 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, and cool to an internal temperature of 0 - 5 °C under nitrogen protection.

[0055] 2) Weigh N,N'-dicyclohexylcarbodiimide (DCC) (3.76 g, 1.5 eq) and add it to Reaction Flask ②, and dissolve it with dichloromethane (10 mL, 1V). Slowly add the prepared N,N'-dicyclohexylcarbodiimide solution dropwise to Reaction Flask ①, control the internal temperature not to exceed 10 °C under nitrogen protection, react at 2 - 8 °C for 5 h to completion, filter and concentrate to dryness to obtain Compound Ⅱ-B.

[0056] 3) Dissolve Compound Ⅱ-B in dichloromethane (2.5 mL, 0.25V). Under nitrogen protection and with the temperature controlled at about 5 - 10 °C, successively add trifluoroacetic acid (45 mL, 4.5V) and triisopropylsilane (2.5 mL, 0.25V), and react at 2 - 8 °C for 4 hours under nitrogen protection.

[0057] 4) Control the temperature at 2 - 8°C. Under nitrogen protection, add methyl tert-butyl ether (300 mL, 30V) dropwise. After addition, control the temperature and stir for 30 minutes. Filter, and wash the filter cake three times with methyl tert-butyl ether (50 mL, 5V). Place the filter cake in a vacuum oven to dry to obtain 8.90 g of compound Ⅲ-B, molar yield: 83.71%, liquid phase purity: 98.21%. See the HPLC chromatogram in Figure 3 。

[0058]

[0059] Example 4: Preparation of compound Ⅲ-C 1) Add dichloromethane (40 mL, 4V), 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 down to an internal temperature of 0 - 5°C.

[0060] 2) Weigh N,N'-dicyclohexylcarbodiimide (DCC) (3.76 g, 1.5 eq) and add it to reaction flask ②. Add dichloromethane (10 mL, 1V) to dissolve. Slowly add the prepared N,N'-dicyclohexylcarbodiimide solution dropwise to reaction flask ①, protect with nitrogen and control the internal temperature not exceeding 10°C. React at 2 - 8°C for 5 h to end. After filtration, concentrate to dryness to obtain compound Ⅱ-C.

[0061] 3) Add dichloromethane (2.5 mL, 0.25V) to dissolve compound Ⅱ-C, protect with nitrogen and control the temperature at about 5 - 10°C. Then successively add trifluoroacetic acid (45 mL, 4.5V) and triisopropylsilane (2.5 mL, 0.25V), and react at 2 - 8°C for 4 hours under nitrogen protection.

[0062] 4) Control the temperature at 2 - 8°C. Under nitrogen protection, add methyl tert-butyl ether (300 mL, 30V) dropwise. After addition, control the temperature and stir for 30 minutes. Filter, and wash the filter cake three times with methyl tert-butyl ether (50 mL, 5V). Place the filter cake in a vacuum oven to dry to obtain 8.29 g of compound Ⅲ-C, molar yield: 82.45%, liquid phase purity: 97.99%. See the HPLC chromatogram in Figure 4 。

[0063]

[0064] Example 5: Preparation of compound Ⅳ 1) Add purified water (360 mL, 18V) and triethylamine (6 mL, 0.3V) to reaction flask ①, mix and stir evenly until the pH is 11.30 ± 0.1 and cool down to an internal temperature of 5 - 15°C. Then add SMG-1 (20.23 g, 1.0 eq) and stir to dissolve.

[0065] 2) Add acetonitrile (140 mL, 7V), DMF (40 mL, 2V), triethylamine (4 mL, 0.2V) and compound Ⅲ-A (8.58 g, 1.5eq) into reaction flask ②, and stir until dissolved and clarified. Slowly add the solution in reaction flask ② into reaction flask ①, control the temperature at 5 - 15 °C until the reaction ends, add 10% phosphoric acid to adjust the pH to 9.5 - 10.5 to obtain the solution of compound Ⅳ. Liquid phase purity: 93.61%, single impurity is less than 1.0% for all, and the HPLC chromatogram is shown in Figure 5 .

[0066]

[0067] Example 6: Preparation of compound Ⅴ-A 1) Synthesize the solution of compound Ⅳ according to the method of Example 3, with the feeding amount of SMG-1 being 20 g, and the dosages of other reagents being adjusted proportionally with reference to the feeding amount of SMG-1.

[0068] 2) Take another reaction flask, add DMF (100 mL, 5V), N,N-diisopropylethylamine (5 mL, 0.25V), and SMG-3A (11.01 g, 3.0eq), and stir to dissolve. Cool down to 10 - 20 °C, add COMU (8.09 g, 3.0eq), and stir for 30 min to obtain the activated solution of SMG-3A.

[0069] 3) Control the internal temperature at 10 - 20 °C, slowly add the activated solution of SMG-3A into the solution of compound Ⅳ in step 1), and control the pH within the range of 9.5 - 10.0 during the reaction process by supplementing DIEA. The reaction ends after 3 - 5 h.

[0070] 4) Add purified water (1 L, 50V), stir and dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0 to precipitate white solid. Filter, take the solid, and dry it under vacuum at 40 °C for no less than 2 hours to obtain 33.16 g of compound Ⅴ-A. Liquid phase purity: 96.47%, and the HPLC chromatogram is shown in Figure 6 , where the single impurity is ≤1.0% for all, and the molar yield of this step is 118.15%.

[0071]

[0072] Example 7: Preparation of semaglutide 1) Add trifluoroacetic acid (144.5 mL, 4.5V), dichloromethane (8 mL, 0.25V), and triisopropylsilane (8 mL, 0.25V) into the reaction flask, cool down to the internal temperature of 0 - 5 °C, and slowly add compound Ⅴ-A (32.10 g, 1.0eq), control the internal temperature at 0 - 5 °C, and react for 4 hours.

[0073] 2) Keep the above reaction flask at 2 - 8 °C, drop pre-cooled ethyl acetate (2 - 8 °C) (289 mL, 9V) into the above reaction flask for dilution and dissolution. After the dropping is completed, add pre-cooled methyl tert-butyl ether (2 - 8 °C) (722 mL, 22.5V) for precipitation, and stir for 10 minutes. After the reaction solution is precipitated, centrifuge it, collect the wet solid, and add methyl tert-butyl ether (160.5 mL * 3, 5V * 3) for slurry washing three times. Filter, collect the wet solid, and dry it under vacuum at 30 °C for at least 1 h to obtain 28.62 g of semaglutide crude product; the molar yield of this step: 93.50%; the liquid phase purity: 89.90%; the HPLC chromatogram is shown in Figure 7 。

[0074] 3) According to the purification method of subsequent Example 10, 22.84 g of semaglutide was obtained by reverse-phase column chromatography purification. The molar yield of this step: 79.80%; the liquid phase purity: 99.54%, and the HPLC chromatogram is shown in Figure 8 , and the mass spectrum is shown in Figure 29 。The total molar yield of semaglutide prepared by the method of this example is 118.15% * 93.50% * 79.80% = 88.17%.

[0075]

[0076] Example 8: Preparation of semaglutide by continuous reaction (one-pot method) 1) Take reaction flask ①, add purified water (180 mL, 18V) and triethylamine (3 mL, 0.3V), mix and stir evenly, cool down to an internal temperature of 5 - 15 °C, and then add SMG-1 (10.0 g, 1.0 eq) and stir to dissolve.

[0077] 2) Take reaction flask 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), and stir until dissolved and clarified. Add the solution in reaction flask ② to reaction flask ①, control the temperature at 10 - 20 °C and react until the end, and add 5% phosphoric acid to adjust the pH to 9.5 - 10.5.

[0078] 3) Take reaction flask ③, add DMF (50 mL, 5V), N,N-diisopropylethylamine (2.5 mL, 0.25V) and SMG-3A (5.51 g, 3.0 eq), stir to dissolve, cool down to 10 - 20 °C, add COMU (4.05 g, 3.0 eq), and stir for 30 min.

[0079] 4) Slowly add the reaction solution in reaction flask ③ to the reaction solution in 2). After adding, 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 h until the reaction is completed to obtain a solution of compound V-A.

[0080] 5) Cool the solution in 4) to 2 - 8 °C, add 1.5 M HCl / MeOH solution (28 mL, 2.8 V), react at 0 - 5 °C for 4 - 6 h. After the reaction is completed, add purified water (650 mL, 65 V) for precipitation, stir for 30 min, filter to obtain a wet solid. Then redissolve it with DMF (100 mL, 9.5 V) / EA (60 mL, 6.5 V), add acetonitrile (550 mL, 55 V) for precipitation, remove the supernatant, filter, and wash three times with MTBE to obtain 14.89 g of crude semaglutide, with a molar yield of 114.94% and a purity of 86.78%. The HPLC chromatogram is shown in Figure 9 .

[0081] 6) According to the purification method in subsequent Example 10, after one-time purification by reverse-phase column chromatography, 11.43 g of pure semaglutide is obtained, with a purity of 99.23%. The HPLC chromatogram is shown in Figure 10 , and the total molar yield of semaglutide prepared through this example is 88.23%.

[0082]

[0083] Example 9: Preparation of semaglutide by continuous reaction (one-pot method) 1) Take reaction flask ①, add purified water (180 mL, 18 V) and triethylamine (3 mL, 0.3 V), mix and stir evenly, cool down to an internal temperature of 5 - 15 °C, and then add SMG-1 (10.0 g, 1.0 eq) and stir to dissolve.

[0084] 2) Take reaction flask ②, add acetonitrile (70 mL, 7 V), DMF (20 ml, 2 V), triethylamine (2 mL, 0.2 V) and compound Ⅲ-A (4.29 g, 1.5 eq), and stir until dissolved and clear. Add the solution in reaction flask ② to reaction flask ①, control the temperature at 10 - 20 °C and react until the reaction is completed, then add 5% phosphoric acid to adjust the pH = 10 - 11.

[0085] 3) Take reaction flask ③, add DMF (50 mL, 5 V), DIEA (1.97 mL, 3.6 eq), Fmoc-His-Aib-OH (4.37 g, 3.0 eq) and stir to dissolve, cool down to 10 - 20 °C, then add COMU (4.05 g, 3.0 eq) and stir for 1 h. Detect the end of the reaction by TLC.

[0086] 4) Add the solution in reaction flask ③ into the reaction solution in 2), control the temperature at 10~25°C, supplement DIEA, adjust the pH to 9.5~10.0, react for 3~5 h until the reaction is completed, and obtain the solution of compound V-B.

[0087] 5) Concentrate the reaction solution in 4) above to about 200 mL (20V), add diethylamine (25 mL, 2.5V), stir for 4~8 h, monitor the end of the reaction, 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 for two times, then filter, wash with MTBE (50 mL, 5V) three times, and obtain 15.14 g of crude semaglutide, with a molar yield of 116.87% and an HPLC purity of 88.65%. The HPLC chromatogram is shown in Figure 11 .

[0088] 6) According to the purification method in Example 10, after reverse-phase column chromatography purification, 11.58 g of pure semaglutide is obtained by calculation, with a purity of 99.28%. The HPLC chromatogram is shown in Figure 12 , and the total molar yield of semaglutide prepared by the method in Example 7 of this example is 89.39%.

[0089]

[0090] Example 10: Purification of Semaglutide Sample preparation: Accurately weigh 28.62 g of the crude semaglutide prepared according to steps 1)-2) of Example 5, dissolve it in 0.1% ammonia water (1144.8 mL), stir for 4 hours until completely dissolved, and then filter with a 0.65 μm filter membrane.

[0091] Purification: Instrument: Hanbang Technology MPLC-DAC50 preparation system; Purification packing: octadecylsilyl-bonded silica gel packing; Mobile phase A: phosphoric acid aqueous solution with a pH of 3.5; Mobile phase B: acetonitrile; Flow rate: 40 mL / min, detection wavelength: 275 nm.

[0092] The elution gradient is as described in Table 2 (equilibration - elution - regeneration - equilibration):

[0093] Loading amount: The loading amount of the crude product is 8 g. After the loading is completed, elute according to the above elution gradient, and collect the fractions with a detected purity of more than 98.0%.

[0094] Afterwards, the collected fractions were desalted through a multi-functional membrane experimental device (Hangzhou Ruina Membrane Engineering Co., Ltd., RNM-18G). The pore size of the filter membrane was selected to be 1-2 nm, the pressure was set at 1.5 Mpa, the concentrated solution was collected and freeze-dried to obtain 22.84 g of semaglutide pure product with a purity of 99.54%. The molar yield was 79.80%. The HPLC chromatogram is shown in Figure 8 。

[0095] Comparative Example 1: 1) Take reaction flask ①, add dichloromethane (40 mL, 4V), SMG-2 (10 g, 1.0 eq) and N-hydroxysuccinimide (2.03 g, 1.5 eq), stir to dissolve, and cool down to an internal temperature of 10-20 °C.

[0096] 2) Weigh N,N'-diisopropylcarbodiimide (DIC) (2.24 g, 1.5 eq) and add it to reaction flask ②. Add dichloromethane (10 mL, 1V) to dissolve it. Slowly add the prepared N,N'-diisopropylcarbodiimide solution dropwise to reaction flask ①, and control the internal temperature not to exceed 25 °C. React at 10-20 °C for 5 h to finish. After filtration, concentrate to dryness to obtain 13.85 g of compound Ⅱ-A. The liquid phase purity is 90.47%. The HPLC chromatogram is shown in Figure 13 。

[0097]

[0098] Comparative Example 2: 1) Take reaction flask ①, add dichloromethane (40 mL, 4V), SMG-2 (10 g, 1.0 eq) and N-hydroxysuccinimide (2.03 g, 1.5 eq), stir to dissolve, and cool down to an internal temperature of 10-20 °C.

[0099] 2) Weigh N,N'-dicyclohexylcarbodiimide (DCC) (3.76 g, 1.5 eq) and add it to reaction flask ②. Add dichloromethane (10 mL, 1V) to dissolve it. Slowly add the prepared N,N'-dicyclohexylcarbodiimide solution dropwise to reaction flask ①, control the internal temperature not to exceed 25 °C, react at 10-20 °C for 5 h to finish. After filtration, concentrate to dryness to obtain 14.02 g of compound Ⅱ-A. The liquid phase purity is 92.44%. The HPLC chromatogram is shown in Figure 14 。

[0100]

[0101] It can be seen from Example 1 and Comparative Examples 1-2 that when synthesizing the active ester of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (SMG-2), using DCC as the solvent produces fewer impurities and higher purity of the target product than using DIC as the solvent. By controlling the reaction temperature at 2-8 °C and using nitrogen protection during the reaction, the purity of the target product can be further significantly improved.

[0102] Comparative Example 3: 1) Take reaction flasks ①-④, add purified water (40 mL, 20V) respectively, and cool down to an internal temperature of 5-15 °C. Then add SMG-1 (2.0 g, 1.0 eq) respectively.

[0103] 2) Add N,N-diisopropylethylamine to reaction flask ① to adjust the pH of the solution to 11.00 and stir to dissolve; Add N,N-diisopropylethylamine to reaction flask ② to adjust the pH of the solution to 11.20 and stir to dissolve; Add N,N-diisopropylethylamine to reaction flask ③ to adjust the pH of the solution to 11.40 and stir to dissolve; Add N,N-diisopropylethylamine to reaction flask ④ to adjust the pH of the solution to 11.61 and stir to dissolve; 3) Take another reaction flasks ⑤-⑧, 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) into each reaction flask, and stir until dissolved and clear. Slowly add the solutions in reaction flasks ⑥-⑩ into reaction flasks ①-④ respectively, control the temperature at 5-15 °C and react until the end, add 10% phosphoric acid to adjust the pH = 9.5-10.5 to obtain the solution of compound Ⅳ. See the HPLC chromatogram in Figures 15 - 18 .

[0104] The specific data are shown in Table 4 below:

[0105] As shown in Table 4, when the active ester synthesized from tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (SMG-2) reacts with the GLP-1(9-37) backbone, when the pH of the SMG-1 solution is 11.2-11.6, compared with when the pH is 11.0, the residue of GLP-1(9-37) is significantly reduced, the content of impurities is significantly reduced, and the purity of the target product compound Ⅳ is significantly improved, proving that when the pH of the SMG-1 solution is 11.2-11.6, it is more conducive to the reaction of the side-chain active ester with the backbone.

[0106] Comparative Example 4: 1) Take reaction flasks ① - ③, add purified water (40 mL, 20 V) respectively, cool down to an internal temperature of 5 - 15 °C, and then add SMG-1 (2.0 g, 1.0 eq) respectively, and stir to dissolve.

[0107] 2) Add triethylamine to reaction flask ① to adjust the pH of the solution to 11.30 ± 0.1, and stir to dissolve; 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; Add 4-dimethylaminopyridine (DMAP) to reaction flask ③ to adjust the pH of the solution to 11.30 ± 0.1, and stir to dissolve; 3) Take another reaction flasks ④ - ⑥, add acetonitrile (140 mL, 7 V), DMF (40 mL, 2 V), triethylamine (4 mL, 0.2 V) and compound Ⅲ-A (8.58 g, 1.5 eq) to each reaction flask, and stir until dissolved and clear. Slowly add the solutions in reaction flasks ④ - ⑥ to reaction flasks ① - ③ respectively, control the temperature at 5 - 15 °C and react until the end, add 10% phosphoric acid to adjust the pH = 9.5 - 10.5 to obtain the compound Ⅳ solution. The HPLC chromatogram is shown in Figures 19 - 21 。

[0108] The specific data are shown in Table 5 below:

[0109] As described in the above table, when the pH is the same, using triethylamine as the base to adjust the pH can further reduce the residual amount of GLP-1(9 - 37) and the content of impurities compared with other bases, and the purity of the target product compound Ⅳ is further significantly improved.

[0110] Comparative Example 5: 1) Synthesize the compound Ⅳ solution according to the method of Example 3, with the feeding amount of SMG-1 being 2 g, and the feeding amounts of other reagents being adjusted proportionally according to the feeding amount of SMG-1.

[0111] 2) Take another reaction flask, add ACN (10 mL, 5 V), DIEA (0.39 mL, 3.6 eq), SMG-3A (1.10 g, 3.0 eq) and stir to dissolve, cool down to 10 - 20 °C, and then add p-toluenesulfonic anhydride (0.62 g, 3.0 eq) and stir for 30 min to obtain the SMG-3A activation solution.

[0112] 3) Control the internal temperature at 10 - 20 °C, slowly add the SMG-3A activation solution to the compound Ⅳ solution, and control the pH within the range of 9.5 - 10.0 during the reaction process by supplementing DIEA, and the reaction ends after 3 - 5 h.

[0113] 4) Add purified water (1 L, 50 V), stir and dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing white solid to precipitate from the solution. Filter, collect the solid, and dry it under vacuum at 40 °C for no less than 2 hours to obtain 3.04 g of Compound V-A. The liquid phase purity is 67.43%. The HPLC chromatogram is shown in Figure 22 , and the molar yield of this step is 108.32%.

[0114] Comparative Example 6: 1) Synthesize the solution of Compound IV according to the method of Example 3, with the feeding amount of SMG-1 being 2 g, and the feeding amounts of other reagents being adjusted proportionally according to the feeding amount of SMG-1.

[0115] 2) Take a reaction flask, add DMF (10 mL, 5 V), DIEA (0.39 mL, 3.6 eq), and SMG-3A (1.10 g, 3.0 eq), stir to dissolve, cool down to 10 - 20 °C, then add TPTU (0.56 g, 3.0 eq) and stir for 30 min to obtain the activated solution of SMG-3A.

[0116] 3) Control the internal temperature at 10 - 20 °C, slowly add the activated solution of SMG-3A to the solution of Compound IV, and control the pH within the range of 9.5 - 10.0 during the reaction process by adding DIEA in a supplementary manner. The reaction ends after 3 - 5 h.

[0117] 4) Add purified water (1 L, 50 V), stir and dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0, causing white solid to precipitate from the solution. Filter, collect the solid, and dry it under vacuum at 40 °C for no less than 2 hours to obtain 3.12 g of Compound V-A. The liquid phase purity is 84.01%. The HPLC chromatogram is shown in Figure 23 , and the molar yield of this step is: 111.17%.

[0118] Comparative Example 7: 1) Synthesize the solution of Compound IV according to the method of Example 3, with the feeding amount of SMG-1 being 2 g, and the feeding amounts of other reagents being adjusted proportionally according to the feeding amount of SMG-1.

[0119] 2) Take a reaction flask, add DMF (12 mL, 6 V), DCM (4 mL, 2 V), DIEA (0.47 mL, 4.3 eq), and SMG-3A (1.10 g, 3.0 eq), stir to dissolve, cool down to 10 - 20 °C, then add Oxyma (0.32 g, 3.6 eq) and EDCI (0.43 g, 3.6 eq), and stir for 1 h to obtain the activated solution of SMG-3A.

[0120] 3) Control the internal temperature at 10 - 20 °C, slowly add the SMG-3A activation solution to the solution of Compound Ⅳ, and control the pH within the range of 9.5 - 10.0 during the reaction process by adding DIEA additionally. The reaction ends after 3 - 5 h.

[0121] 4) Add purified water (1 L, 50 V), stir and dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0 to precipitate white solid from the solution. Filter, collect the solid, and dry it under vacuum at 40 °C for no less than 2 hours to obtain 2.93 g of Compound Ⅴ-A. The liquid phase purity is 87.57%. The HPLC chromatogram is shown in Figure 24 , and the molar yield of this step is 104.40%.

[0122] Comparative Example 8: 1) Synthesize the solution of Compound Ⅳ according to the method of Example 3, with the feeding amount of SMG-1 being 2 g, and the feeding amounts of other reagents being adjusted proportionally according to the feeding amount of SMG-1.

[0123] 2) Take a reaction flask, 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, cool down to 10 - 20 °C, and then add Pyoxim (1.00 g, 3.0 eq) and stir for 1 h to obtain the SMG-3A activation solution.

[0124] 3) Control the internal temperature at 10 - 20 °C, slowly add the SMG-3A activation solution to the solution of Compound Ⅳ, and control the pH within the range of 9.5 - 10.0 during the reaction process by adding DIEA additionally. The reaction ends after 3 - 5 h.

[0125] 4) Add purified water (1 L, 50 V), stir and dilute, then add 10% phosphoric acid aqueous solution to adjust the pH of the solution to 5.0 to precipitate white solid from the solution. Filter, collect the solid, and dry it under vacuum at 40 °C for no less than 2 hours to obtain 3.18 g of Compound Ⅴ-B. The liquid phase purity is 89.53%. The HPLC chromatogram is shown in Figure 25 , and the molar yield of this step is 113.31%.

[0126] The results of using different coupling agents are shown in Table 6 below:

[0127] As shown in Table 6, when SMG-3 is activated and then linked to the glutamic acid residue at the N-terminus of the main chain of SMG-1, using COMU significantly further reduces the content of impurities compared with other coupling agents, improves the purity of the target product Compound V, successfully solves the side reaction problem between the activated dipeptide and the main chain arginine, and obtains highly pure semaglutide with protecting groups.

[0128] Comparative Example 9: 1) Prepare the solution of Compound V-B according to Steps 1)-3) of Example 7, with the feeding amount of SMG-1 being 1.0 g, and the feeding amounts of other reagents being adjusted proportionally according to the feeding amount of SMG-1.

[0129] 2) Add 2M NaOH aq. (20 mL, 10V) to the solution of Compound V-B, stir for 3 h, monitor the end of the reaction, add purified water (100 mL, 50V), 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 perform slurry washing twice, then filter, and wash three times with MTBE (50 mL, 5V) to obtain 14.02 g of crude semaglutide, with a molar yield of 108.23% and an HPLC purity of 71.43%. The HPLC chromatogram is shown in Figure 26 .

[0130] Comparative Example 10: 1) Prepare the solution of Compound V-B according to the synthesis method of Example 7, with the feeding amount of SMG-1 being 1.0 g.

[0131] 2) Add piperidine (4 ml, 4V) to the solution of Compound V-B, stir for 3 h, monitor the end of the reaction, add purified water (100 mL, 50V), 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 perform slurry washing twice, then filter, and wash three times with MTBE (50 mL, 5V) to obtain 13.68 g of crude semaglutide, with a molar yield of 105.60% and an HPLC purity of 81.43%. The HPLC chromatogram is shown in Figure 27 .

[0132] Comparative Example 11: 1) Prepare the solution of Compound V-B according to the synthesis method of Example 7, with the feeding amount of SMG-1 being 1.0 g.

[0133] 2) Add DBU (1 ml, 1V) to the solution of Compound V-B, stir for 3 h, monitor the end of the reaction, add purified water (100 mL, 50V), 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 perform slurry washing twice, then filter, and wash three times with MTBE (50 mL, 5V) to obtain 13.54 g of crude semaglutide, with a molar yield of 104.52% and an HPLC purity of 76.69%. The HPLC chromatogram is shown in Figure 28 .

[0134]

[0135] As shown in the above table, in the method of the present invention, by using diethylamine to deprotect semaglutide containing Fmoc protecting agent, compared with other deprotecting agents, crude semaglutide with higher purity can be obtained, achieving better results.

[0136] It can be seen from the above examples and comparative examples that the method of the present invention reduces the occurrence of side reactions, reduces the impurity content, and improves the purity of crude semaglutide by optimizing the reaction conditions. The prepared semaglutide has high purity and yield, and reduces the processing steps of intermediates, reduces the use of organic reagents, reduces costs and is more environmentally friendly. In particular, the continuous preparation method of the present invention does not require the treatment of intermediates, reducing at least 4 processes and materials, and the cost is reduced by at least about 20%. In summary, the method of the present invention has fewer reaction steps, is simple to operate, reduces costs, and the prepared semaglutide has high purity and high yield, and is particularly suitable for the large-scale industrial production of semaglutide.

Claims

1. A semi-synthetic preparation method of semaglutide, characterized in that, It includes the following steps: Step 1: In the presence of a first base catalyst, mix the compound SMG-1 solution with the compound III solution, and react to obtain compound IV. The reaction formula is as follows: ， Wherein, R1 is succinimido, benzotriazolyl, 5-norbornene-2,3-dicarboximido or p-toluenesulfonyl, and the addition amount of the first base catalyst is such that the pH of the SMG-1 solution is 11.2 - 11.6; Step 2: In the presence of a coupling agent and a second base catalyst, mix the compound IV solution with the compound SMG-3 solution, and couple to obtain compound V. The reaction formula is as follows: Wherein, R2 is trityl or H, R3 is tert-butoxycarbonyl or fluorenylmethoxycarbonyl, and the pH of Step 2 is 9.5 - 10.0; Step 3: Use a deprotecting agent to deprotect compound V to obtain semaglutide.

2. The semi-synthetic preparation method according to claim 1, characterized in that, The addition amount of the first base catalyst 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 compound SMG-1 solution, the compound III solution, and the compound 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 first base catalyst is one or more combinations of triethylamine, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine, triisopropylamine, N-methylmorpholine, pyridine, and 4-dimethylaminopyridine; the coupling agent is COMU, TPTU, Oxyma / EDCI, PyOxim, or p-toluenesulfonic anhydride; 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 of the compound SMG-1 solution is water, the solvent of the compound III solution is acetonitrile, and the solvent of the compound SMG-3 solution is N,N-dimethylformamide.

5. The semi-synthetic preparation method according to claim 4, characterized in that, The first base catalyst is triethylamine.

6. The semi-synthetic preparation method according to claim 5, characterized in that, The coupling agent is COMU, and the second base catalyst is N,N-diisopropylethylamine.

7. The semi-synthetic preparation method according to claim 6, characterized in that, Step 2 includes: Step 2-1A: Add the second base catalyst and the coupling agent to the compound SMG-3 solution to prepare a compound SMG-3 activated solution; Step 2-2A: Add the compound SMG-3 activated solution to the compound IV solution, and supplement the addition of the second base catalyst to make the pH 9.5 - 10.0, and react to obtain a compound V solution; Step 2-3A: Add water to dilute the compound V solution, and add an acid to adjust the pH to 5.0 to precipitate a white solid, filter and vacuum dry to obtain compound V for Step 3.

8. The semi-synthetic preparation method according to claim 7, wherein, The acid is phosphoric acid.

9. The semi-synthetic preparation method according to claim 7, characterized in that, The deprotecting agent in Step 3 is TFA / TIS / DCM with a volume ratio of 90:5:

5.

10. The semi-synthetic preparation method according to claim 6, characterized in that, The semi-synthetic preparation method is a continuous reaction, and the intermediate is not separated during the reaction process; The compound SMG-3 is Boc-His(Trt)-Aib-OH; Step 2 includes: Step 2-1B: Add the second base catalyst and the coupling agent to the compound SMG-3 solution to prepare an activated compound SMG-3 solution; Step 2-2B: Add the activated compound SMG-3 solution to the compound Ⅳ solution, and supplement the second base catalyst to make the pH 9.5 - 10.0, and react to obtain a compound Ⅴ solution, and the compound V solution is directly used in Step 3; Step 3 includes adding a deprotecting agent HCl / MeOH to the compound V solution for deprotection.

11. The semi-synthetic preparation method according to claim 6, wherein The semi-synthetic preparation method is a continuous reaction, and the intermediate is 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 compound SMG-3 solution to prepare an activated compound SMG-3 solution; Step 2-2C: Add the activated compound SMG-3 solution to the compound Ⅳ solution, and supplement the second base catalyst to make the pH 9.5 - 10.0, and react to obtain a compound Ⅴ solution, and the compound V solution is directly used in Step 3; Step 3 includes adding a deprotecting agent to the compound V solution for deprotection, and the deprotecting agent is diethylamine, NaOH, piperidine or DBU.

12. The semi-synthetic preparation method according to claim 11, wherein, The deprotecting agent is diethylamine.

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

14. The semi-synthetic preparation method according to claim 1, characterized in that, The reaction temperature of Step 1 is 5 - 15 °C, and the reaction temperature of Step 2 is 10 - 20 °C.

15. The semi-synthetic preparation method according to any one of claims 1-14, characterized in that, The compound Ⅲ is prepared by the following steps: Step A: In the presence of a condensation reagent, mix the compound SMG-2 solution with the compound I solution, and dehydrate and condense to obtain compound II. The reaction formula is as follows: ， Wherein compound 1 is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensation reagent is DCC, and the reaction process is protected by an inert gas, and the reaction temperature is 2 - 8 °C; Step B: Deprotect compound II to obtain compound Ⅲ. The reaction formula is as follows: 。 16. The semi-synthetic preparation method according to claim 15, characterized in that, The solvent of the compound SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, 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.

17. The semi-synthetic preparation method according to claim 15, wherein, Step B uses TFA / TIS / DCM with a volume ratio of 90:5:5 or TFA / TIS / PPW with a volume ratio of 95:2.5:2.5 for deprotection.

18. A preparation method of semaglutide side chain compound III, characterized in that, The method includes: Step A: In the presence of a condensation reagent, mix the compound SMG-2 solution with the compound I solution, and dehydrate and condense to obtain compound II. The reaction formula is as follows: ， Among them, Compound 1 is 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or p-toluenesulfonic anhydride, the condensation reagent is DCC, a protective gas is used for protection during the reaction, and the reaction temperature is 2-8 °C; Step B: Compound II is deprotected to obtain Compound III, and the reaction formula is as follows: 。 19. The preparation method according to claim 18, characterized in that, The solvent of the Compound SMG-2 solution is selected from one or a combination of two of dichloromethane and tetrahydrofuran, 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.

20. The preparation method according to claim 18, characterized in that, In Step B, deprotection is carried out using TFA / TIS / DCM with a volume ratio of 90:5:5 or TFA / TIS / PPW with a volume ratio of 95:2.5:2.5.

Images