Method for preparing semeglutide by one-pot method

Abstract

The invention discloses a one-pot preparation method of semeglutide, and relates to the field of polypeptide synthesis. The preparation method comprises the following specific steps: 1) taking Arg34GLP-1 (9-37) as a starting material, controlling the pH value of a reaction solution, enabling activated dipeptide Fmoc-His-Aib-OH to react with the starting material to obtain an acylation reaction solution, and directly carrying out next-step reaction without aftertreatment after the reaction is finished; 2) adjusting the pH value of the acylation reaction liquid I, adding a compound 2-tetrapeptide into the acylation reaction liquid I, reacting to obtain acylation reaction liquid II, and directly carrying out next-step reaction without post-treatment after the reaction is finished; (3) adding an alkaline reagent to remove the Fmoc protecting group, settling, centrifuging and drying to obtain a crude product of the semeglutide; 4) separating and purifying the crude product to obtain a refined semeglutide product; the first three steps are carried out in the same reactor. Compared with a conventional liquid-phase synthesis method, the preparation method disclosed by the invention is simpler and more convenient, short in construction period, low in output of waste liquid and waste water, greatly reduced in cost, higher in yield and suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a one-pot preparation method of semaglutide. Background Art

[0002] Semaglutide is a long-acting GLP-1 analog developed by Novo Nordisk. Human GLP-1 is a peptide hormone secreted by intestinal L cells. The mechanism of action is as follows: 1) It acts on pancreatic β cells, promotes the synthesis and secretion of insulin, stimulates the proliferation and differentiation of pancreatic β cells, inhibits the apoptosis of pancreatic β cells, increases the number of pancreatic β cells, and has the effect of protecting pancreatic function; 2) It acts on pancreatic α cells, inhibits the release of glucagon, and reduces the release of glycogen; 3) It acts on pancreatic δ cells, promotes the secretion of somatostatin, and participates in inhibiting the secretion of glucagon. The combined effect can help patients achieve the effect of weight loss and blood sugar reduction.

[0003] The preparation of semaglutide is usually carried out by polypeptide solid phase synthesis or fragment condensation methods (CN103848910, CN106928343, WO2016046753, etc.). Among them, solid phase synthesis is easy to form secondary structures such as β-folds for some long peptides and highly hydrophobic peptide fragments, which makes coupling difficult and the condensation efficiency low. In order to solve these problems, a fragment condensation method (CN109456401A) was proposed, but some new problems also emerged, such as the solubility of the fragments, the low coupling efficiency between fragments, and the exothermic dissolution of the fragments. In addition, the fragment condensation operation is relatively complicated and difficult to scale up production.

[0004] The original preparation process of semaglutide is a method of combining biological fermentation with chemical synthesis (CN101910193). First, the Arg-GLP-1 (9-37) fragment is obtained by biological fermentation, and then a chemical method is used to convert the fragment to Lys 26 The ε amino group of the semaglutide side chain and the dipeptide (His-Aib) are introduced to obtain the crude semaglutide peptide. The operation steps of this method are relatively complicated, each step requires post-treatment, and the amount of waste liquid and waste water generated during the synthesis process is large, which further increases the cost, and the yield is relatively low.

[0005] Patent application CN118666987A discloses a continuous preparation method of semaglutide, wherein a twenty-nonapeptide is first reacted with Trt-isobutyric acid active ester to obtain a thirty-nine-peptide intermediate, which is then deprotected to obtain a de-Trt thirty-nine-peptide intermediate, which is then reacted with Trt-histidine active ester to obtain a thirty-one-peptide intermediate, which is then added with a side chain active ester to react to obtain a semaglutide precursor, which is then deprotected to obtain a crude semaglutide; this method does not separate the intermediate state, but the overall reaction yield is only about 65%, and the yield needs to be further improved to reduce production costs.

[0006] Based on the above-mentioned difficulties existing in the prior art, there is an urgent need for a preparation method that is simpler in operation, has a shorter production cycle, lower cost, higher yield, and is more suitable for the industrial production of semaglutide. Summary of the invention

[0007] The present invention solves the deficiencies in the existing process and provides a method for preparing semaglutide with mild reaction conditions, simple process, low cost, short cycle, high total yield and applicable to large-scale production.

[0008] The technical solution adopted by the present invention is as follows:

[0009] (1) controlling the reaction pH, introducing the activated dipeptide Fmoc-His-Aib-OH into the terminal amino group of Arg34GLP-1(9-37) through an acylation reaction, and obtaining an acylation reaction solution whose main component is compound 1. After the reaction is completed, the next step of the reaction is directly carried out without post-treatment;

[0010] (2) Adjusting the pH of the acylation reaction solution, adding compound 2-tetrapeptide to the acylation reaction solution, and connecting to the Lys of Arg34GLP-1(9-37) through acylation reaction. 26 A second acylation reaction solution with compound 3 as the main component was obtained, and the next step of reaction was directly carried out without post-treatment after the reaction was completed;

[0011] (3) adding an alkaline reagent to the second acylation reaction solution to perform a hydrolysis reaction to remove the Fmoc protecting group, thereby obtaining a crude semaglutide solution;

[0012] (4) post-treating the crude semaglutide solution to obtain a solid crude semaglutide;

[0013] The steps (1), (2) and (3) are carried out in the same reactor;

[0014] The specific preparation route is as follows:

[0015]

[0016] The post-treatment in step (1) and step (2) of the present invention that does not require post-treatment includes, but is not limited to, one or a combination of operations such as liquid or solid transfer, water washing, alkali washing, acid washing, filtration, ultrafiltration, cyclic ultrafiltration, suction filtration, dilution, concentration, drying, recrystallization, and freeze-drying.

[0017] As an embodiment of the present invention, in step (1), the acylation reaction solvent is a combination of an organic solvent and water, and the organic solvent is selected from a combination of one or more of NMP, DMSO, acetonitrile, ethanol, tetrahydrofuran, DMF and water.

[0018] As a specific embodiment of the present invention, in step (1), the volume ratio of the organic solvent to water in the acylation reaction solvent is 0.05 to 0.3: 1. If the proportion of the organic solvent is too high, Arg34GLP-1(9-37) will be difficult to dissolve.

[0019] As a specific embodiment of the present invention, in the step (1), the acylation reaction solvent is a mixed solvent of tetrahydrofuran and water.

[0020] As an embodiment of the present invention, in the step (1), the pH value of the acylation reaction is first adjusted to 11.0-12.0 with a base, such as 11.1-11.9, 11.2-11.8, 11.3-11.7, 11.4-11.6, and more specifically 11.5. The pH value is then adjusted to 8.0-10.0 with an acid, such as 8.0, 8.5, 9.0, 9.5, 10.0, etc. The alkaline reagent used to adjust the pH is selected from an organic base or an inorganic base, such as triethylamine, N,N-diisopropylethylamine (DIEA), sodium hydroxide, etc. The acidic reagent used to adjust the pH is selected from one of acetic acid, hydrochloric acid, and sulfuric acid. Other organic acids will react with the primary acylation product through a condensation reagent to produce new impurities and reduce the yield.

[0021] As an embodiment of the present invention, in the step (1), the reagent used for activating Fmoc-His-Aib-OH is selected from one or more combinations of N, N'-disuccinimidyl carbonate (DSC), N, N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC), benzotriazol-1-yl-oxytripyrrolidone hexafluorophosphate (PyBop), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU).

[0022] As an embodiment of the present invention, the reaction pH value of step (2) is 11.0-12.0, such as 11.1-11.9, 11.2-11.8, 11.3-11.7, 11.4-11.6, more specifically 11.5. pH>12, or pH<11, are not conducive to the secondary acylation reaction.

[0023] As an embodiment of the present invention, in the step (3), the alkaline reagent used to remove the Fmoc protecting group is selected from one or more combinations of concentrated ammonia, cyclohexylamine, morpholine, diethylamine, diazabicyclo (DBU), and piperidine, preferably one of diethylamine, DBU or piperidine.

[0024] As an embodiment of the present invention, the reaction temperature of step (1) or step (2) or step (3) is 35-50°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C, or 36-48°C, 37-45°C, 38-42°C, 39-41°C, more specifically 40°C.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The present invention can realize the conversion from Arg34GLP-1 (9-37) to semaglutide in the same reactor, the reaction is carried out under the same temperature conditions, and each step of the chemical reaction is completed without post-treatment and directly proceeds to the next step of feeding reaction, the operation is simple and convenient, easy to control, and suitable for large-scale production;

[0027] (2) The present invention does not need to process the acylation product I and acylation product II into solids, which greatly shortens the construction period (relative to the increase in post-processing operations, the cycle of each batch is shortened from about 40.5 to 54.5 hours to about 16.5 hours), thereby greatly reducing labor costs; at the same time, the output of each batch of waste liquid (such as wastewater, organic solvents, etc.) is reduced from about 51.2 to 310.7L to about 22.1L, which greatly reduces the cost of waste liquid treatment and is more in line with the concept of sustainable development;

[0028] (3) The mass yield of crude semaglutide in the process of the present invention can reach 94.9-105.1%, with strong repeatability, which is at least about 25% higher than the mass yield of crude products in the prior art (48.9-70.1%); the purity of crude semaglutide can reach 81-87%, which is at least about 18% higher than the purity of crude products in the prior art (48.0-62.8); the content of semaglutide can reach 63-69%, which is at least about 20% higher than the content in the prior art (40.4-42.5). The total yield of fine semaglutide obtained by the process of the present invention is 75.4-86.2%, which is at least 10.4% higher than the 65% in the prior art. This greatly saves costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a preparation route map of semaglutide of the present invention;

[0030] Figure 2 is the HPLC chart of semaglutide obtained in Example 1;

[0031] Figure 3 is the HPLC chart of semaglutide obtained in Example 2;

[0032] Figure 4 is the MS graph of semaglutide obtained in Example 2;

[0033] Figure 5 is the HPLC chart of semaglutide obtained in Example 3;

[0034] Figure 6 This is the HPLC chart of semaglutide obtained in Comparative Example 1;

[0035] Figure 7 This is the HPLC chart of semaglutide obtained in Comparative Example 2;

[0036] Figure 8 This is the preparation route of semaglutide in Comparative Example 3;

[0037] Fig. 9 This is the HPLC chart of semaglutide obtained in Comparative Example 3. DETAILED DESCRIPTION

[0038] In order to better understand the content of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection requested by the claims of the present invention in any way.

[0039] The raw materials and reagents used in the following examples, such as compound 2-tetrapeptide and dipeptide Fmoc-His-Aib-OH, can be purchased from commercial sources unless otherwise specified.

[0040] The main peptide chain of Arg34GLP-1 (9-37) was prepared by Nanjing Hanxin Pharmaceutical Technology Co., Ltd. Its preparation process refers to the disclosed invention patent CN202111664146.2 declared by the company, and all its contents can be directly introduced into the present invention.

[0041] Specific synthetic route reference Figure 1 shown.

[0042] Example 1

[0043] Step 1:

[0044] Prepare 4350 mL of a mixed solvent with a volume ratio of water to ethanol of 0.05:1, first add DIEA to adjust the pH to 11.0, then use acetic acid to adjust the pH to 8.0, stir and heat to 35°C. Add 172.6 g of Arg34GLP-1(9-37) with a content of 86.9% and a purity of 93.4% (1.0 eq., 47.2 mmol, equivalent to 100% Arg34GLP-1(9-37) 150.0 g), and stir until dissolved. Dissolve 65.6 g (3 eq., 141.7 mmol) of dipeptide Fmoc-His-Aib-OH and 16.31 g (3 eq., 141.7 mmol) of HOSU / DCC in 1224.6 mL of DMF, stir and dissolve to activate, and then drop into the above-mentioned solution containing Arg34GLP-1(9-37) to carry out acylation reaction. During the dropwise addition, the pH was maintained at 8.0±0.2, the reaction temperature was 35°C, the dropwise addition time was controlled to be 30 min, and the reaction was completed after the dropwise addition was completed to obtain an acylation reaction solution having compound 1 as the main component. After the reaction was completed, the next step of feeding reaction was directly carried out without post-treatment. The operation time of the entire step 1 (including solvent preparation, feeding and reaction time) was 2.5 h, and the solvent volume used was 4.4+1.2=5.6 L.

[0045] Step 2:

[0046] The pH of the acylation reaction liquid obtained in the previous step was adjusted to 11.0 using DIEA. 47.1g (1.2eq., 56.7mmol) of compound 2 was weighed and dissolved in 1111.5mL DMF, and then dropped into the acylation reaction liquid for acylation reaction. During the dropping, the pH was maintained at 11.0±0.2, the reaction temperature was 35°C, and the dropping time was controlled to be 30min. The reaction was completed after the dropping, and the acylation reaction liquid with the main component of compound 3 was obtained. After the reaction was completed, the next step of feeding reaction was directly carried out without post-treatment. The operation time of the entire step 2 (including liquid preparation, feeding and reaction time) was 3h, and the volume of the solvent used was 1.1L.

[0047] Step 3:

[0048] To the acylation reaction solution obtained in the previous step, 10% of the volume of diethylamine (cleavage reagent) was added, and the mixture was stirred at 35° C. for 1 h to perform a hydrolysis reaction to remove the Fmoc protecting group, thereby obtaining a crude semaglutide solution (5.6+1.1+0.67=7.37 L).

[0049] Step 4:

[0050] After the reaction, 14.7 L of tetrahydrofuran (2 times the volume of the crude semaglutide solution) was added to precipitate solids, which were centrifuged and dried to obtain 219.95 g of crude semaglutide with a mass yield of 98.2% (mass yield = crude mass * content / 150). Figure 2 As shown, the content is 67% and the purity is 85.6%.

[0051] The operation time of step 3 and step 4 (including the time for liquid preparation, material addition, reaction, precipitation, centrifugation and drying) is 11 hours, and the volume of solvent used is 0.67+14.7=15.37L.

[0052] The crude semaglutide obtained in step 4 is purified (the purification method refers to Example 4 in the disclosed patent CN202310571825.8) to obtain semaglutide with a purity of 99.8%, and the yield of the purification step can reach 80%. Therefore, the total yield is 98.2%*80%=78.56%.

[0053] Example 2

[0054] Step 1:

[0055] Prepare 4350 mL of a mixed solvent of water and tetrahydrofuran in a volume ratio of 0.2:1, first add triethylamine to adjust the pH to 11.5, then use hydrochloric acid to adjust the pH to 9.0, stir and heat to 40°C. Add 172.6 g of Arg34GLP-1(9-37) with a content of 86.9% and a purity of 93.4% (1.0 eq., 47.2 mmol, equivalent to 100% Arg34GLP-1(9-37) 150.0 g), and stir until dissolved. Dissolve 65.6 g (3 eq., 141.7 mmol) of dipeptide Fmoc-His-Aib-OH and 73.8 g (3 eq., 141.7 mmol) of PyBop in 1224.6 mL of DMF, stir and dissolve to activate, and then drop into the above-mentioned solution containing Arg34GLP-1(9-37) to carry out acylation reaction. During the addition, the pH was maintained at 9.0±0.2, the reaction temperature was 40°C, and the addition time of the activated dipeptide was controlled to be 30 min. The reaction was completed when the addition was completed, and an acylation reaction solution with the main component being compound 1 was obtained. After the reaction was completed, the next step of feeding reaction was directly carried out without post-treatment. The operation time of the entire step 1 (including the preparation of solvent, addition of materials and reaction time) was 2.5 h, and the volume of the solvent used was 4.4+1.2=5.6 L.

[0056] Step 2:

[0057] The pH of the acylation reaction liquid obtained in the previous step was adjusted to 11.5 using triethylamine. Acylation reaction was carried out in the acylation reaction liquid, and the pH was maintained at 11.5±0.2 during the dropwise addition. The reaction temperature was 40°C, and the dropwise addition time was controlled to be 30 min. The reaction was completed after the dropwise addition, and the acylation reaction liquid with the main component of compound 3 was obtained. After the reaction was completed, the next step of feeding reaction was directly carried out without post-treatment. The operation time of the entire step 2 (including liquid preparation, feeding and reaction time) was 3 h, and the volume of the solvent used was 1.1 L.

[0058] Step 3:

[0059] Concentrated aqueous ammonia (0.67 L) with a volume of 10% of the acylation reaction solution was added to the acylation reaction solution obtained in the previous step, and the mixture was stirred at 40° C. for 1 h to perform a hydrolysis reaction to remove the Fmoc protecting group, thereby obtaining a crude semaglutide solution (5.6+1.1+0.67=7.37 L).

[0060] Step 4:

[0061] After the reaction, 14.7 L of tetrahydrofuran (2 times the volume of the crude semaglutide solution) was added to precipitate solids, which were centrifuged and dried to obtain 228.5 g of crude semaglutide with a mass yield of 105.1%. The HPLC test results are as follows: Figure 3 As shown, the content is 69%, the purity is 87.02%, and the MS of the target product is as follows Figure 4 shown.

[0062] The operation time of step 3 and step 4 (including the time for liquid preparation, material addition, reaction, precipitation, centrifugation and drying) is 11 hours, and the volume of solvent used is 0.67+14.7=15.37L.

[0063] The crude semaglutide obtained in step 4 is purified (the purification method refers to Example 4 in the disclosed patent CN202310571825.8) to obtain semaglutide with a purity of 99.8%, and the yield of the purification step can reach 82%. Therefore, the total yield is 105.1%*82%=86.2%.

[0064] Example 3

[0065] Step 1:

[0066] Prepare 4350 mL of a mixed solvent with a volume ratio of water to DMF of 0.3:1, first add sodium hydroxide solution to adjust the pH to 12.0, then use dilute sulfuric acid to adjust the pH to 10.0, stir and heat to 50°C. Add 172.6 g of Arg34GLP-1(9-37) with a content of 86.9% and a purity of 93.4% (1.0 eq., 47.2 mmol, equivalent to 150.0 g of Arg34GLP-1(9-37) with a content of 100%), and stir until dissolved. Dissolve 65.6 g (3 eq., 141.7 mmol) of dipeptide Fmoc-His-Aib-OH and 16.3 g (3 eq., 141.7 mmol) of EDC in 1224.6 mL of DMF, stir and dissolve to activate, and then drop into the above-mentioned solution containing Arg34GLP-1(9-37) to carry out acylation reaction. During the addition, the pH was maintained at 10.0±0.2, the reaction temperature was 50°C, the addition time was controlled to be 30min, and the reaction was completed after the addition was completed to obtain an acylation reaction solution with the main component being compound 1. After the reaction was completed, the next step of feeding reaction was directly carried out without post-treatment. The operation time of the entire step 1 (including solvent preparation, feeding and reaction time) was 2.5h, and the solvent volume used was 4.4+1.2=5.6L.

[0067] Step 2:

[0068] The pH of the acylation reaction liquid obtained in the previous step was adjusted to 12.0 using sulfuric acid. 47.1g (1.2eq., 56.7mmol) of compound 1 was weighed and dissolved in 1111.5mL DMF, and then dropped into the acylation reaction liquid for acylation reaction. During the dropwise addition, the pH was maintained at 12.0±0.2, the reaction temperature was 50°C, and the dropwise addition time was controlled to be 30min. The reaction was completed after the dropwise addition, and the acylation reaction liquid with the main component being compound 3 was obtained. After the reaction was completed, the next step of feeding reaction was directly carried out without post-treatment. The operation time of the entire step 2 (including liquid preparation, feeding and reaction time) was 3h, and the volume of the solvent used was 1.1L.

[0069] Step 3:

[0070] 10% of the volume of DBU was added to the acylation reaction solution obtained in the previous step, and the mixture was stirred at 50° C. for 1 h to perform a hydrolysis reaction to remove the Fmoc protecting group, thereby obtaining a crude semaglutide solution (5.6+1.1+0.67=7.37 L).

[0071] Step 4:

[0072] After the reaction, acetic acid was added to adjust to the isoelectric point, and 14.7 L of water, twice the volume of the crude semaglutide solution, was added to precipitate the solid, which was centrifuged and dried to obtain 225.92 g of crude semaglutide with a mass yield of 94.9%. The HPLC test results are as follows Figure 5As shown, the content is 63% and the purity is 80.99%.

[0073] The operation time of step 3 and step 4 (including the time for liquid preparation, material addition, reaction, precipitation, centrifugation and drying) is 11 hours, and the volume of solvent used is 0.67+14.7=15.37L.

[0074] The crude semaglutide obtained in step 4 is purified in two steps (the purification method refers to Example 4 in the disclosed patent CN202310571825.8) to obtain semaglutide with a purity of 99.8%, and the yield of the purification step can reach 79.5%.

[0075] The crude semaglutide obtained in step 4 is purified (the purification method refers to Example 4 in the disclosed patent CN202310571825.8) to obtain semaglutide with a purity of 99.6%, and the yield of the purification step can reach 81%. Therefore, the total yield is 94.9%*79.5%=75.4%.

[0076] Comparative Example 1

[0077] Compared with Example 2, post-processing operation is added to step 1 and step 2 of Comparative Example 1, and other operations and parameters are the same as those of Example 2.

[0078] Step 1:

[0079] Prepare 4350 mL of a mixed solvent of water and tetrahydrofuran in a volume ratio of 0.2:1, add triethylamine dropwise to adjust the pH to 11.5, then use hydrochloric acid to adjust the pH to 9.0, stir and heat to 40°C. Add 172.6 g of Arg34GLP-1(9-37) with a content of 86.9% and a purity of 93.4% (1.0 eq., 47.2 mmol, equivalent to 150.0 g of Arg34GLP-1(9-37) with a content of 100%), and stir until dissolved. Dissolve 73.8 g (3 eq., 141.7 mmol) of PyBop in 1224.6 mL of DMF, stir and dissolve to activate, and then drop into the above-mentioned solution containing Arg34GLP-1(9-37) to carry out acylation reaction. During the addition, the pH was maintained at 9.0±0.2, the reaction temperature was 40°C, the addition time was controlled to 30min, and the reaction was terminated when the addition was completed. The pH of the reaction solution was adjusted to the isoelectric point, 17.4L of water was added to precipitate, centrifuged, and dried to obtain solid compound 1. The operation time of the entire step 1 (including the time for preparing the solvent, adding materials, reacting, precipitating the solid, centrifuging, and drying) was 14.5h, and the solvent volume was 4.4+1.2+17.4=23L.

[0080] Step 2:

[0081] Solid compound 1 was dissolved in 2722.5 mL of mixed solvent (water and tetrahydrofuran volume ratio of 0.2:1), the pH was adjusted to 9.0 with triethylamine, and the temperature was stirred and raised to 40°C. 47.1 g (1.2 eq., 56.7 mmol) of compound 2 was weighed and dissolved in 1111.5 mL of DMF, and added to the tetrahydrofuran / water solution containing compound 1 for acylation reaction. During the addition, the pH was maintained at 9.0 ± 0.2, the reaction temperature was 40°C, and the addition time was controlled to 30 min. The reaction was terminated when the addition was completed. The pH of the reaction solution was adjusted to the isoelectric point, 10.9 L of water was added to precipitate, and the solid compound 3 was obtained by centrifugation and drying. The operation time of the entire step 2 (including the time for liquid preparation, addition, reaction, precipitation, centrifugation and drying) was 15 h, and the volume of the solvent used was 2.7 + 1.1 + 10.9 = 14.7 L.

[0082] Step 3:

[0083] Solid compound 3 was dissolved in 2722.5 mL of a mixed solvent of water and tetrahydrofuran in a volume ratio of 0.2:1, and then 283.0 mL of concentrated aqueous ammonia was added and stirred at 25°C for 1 h to perform a hydrolysis reaction to remove the Fmoc protecting group to obtain a crude semaglutide solution (2.72+0.28=3 L).

[0084] Step 4:

[0085] After the reaction, 10.5 L of tetrahydrofuran (3.5 times the volume of the crude semaglutide solution) was added to precipitate solids, centrifuged, and dried to obtain 178.0 g of crude semaglutide with a mass yield of 48.9%. The HPLC test results are as follows: Figure 3 As shown, the content is 41.2% and the purity is 62.81%.

[0086] The operation time of step 3 and step 4 (including the time for liquid preparation, material addition, reaction, precipitation, centrifugation and drying) is 11 hours, and the volume of solvent used is 13.5L.

[0087] Comparative Example 2 (Raw material was enlarged to 150g target propeptide)

[0088] Refer to the synthetic routes of Example 4, Example 16 and Example 23 in patent CN111378028A.

[0089] Step 1: DIPEA was added to water and the pH was adjusted to 11.5. Then, 172.6 g of Arg34GLP-1 (9-37) with a content of 86.9% and a purity of 93.4% (150.0 g of Arg34GLP-1 (9-37) with a converted content of 100%) was added to 28.3 L of this solution in an ice-water bath. Then, 117.5 g of DMF solution of compound 1 (1.2 L) was dripped into the reaction solution dropwise, and the reaction was carried out in an ice-water bath for 1 h. The pH of the reaction solution was adjusted to the isoelectric point, 15.0 L of water was added to precipitate solids, and the mixture was centrifuged and dried to obtain compound 2. The operation time of the entire step 1 (including the time for liquid preparation, addition of materials, reaction, precipitation of solids, centrifugation and drying) was 16.5 h, and the solvent volume was 28.3 + 1.2 + 15.0 = 44.5 L.

[0090] Step 2: Dissolve 80.0 g of Boc-His(Trt)-Aib-OH and 23.7 g of N-hydroxysuccinimide in 846.6 mL of DCM, then add dropwise a DCM (423 mL) solution containing 42.3 g of DCC. After reacting for 16 hours, filter out the white solid, evaporate the filtrate to dryness, and then slurry with 1269.8 mL of acetonitrile, filter out the white solid, evaporate the filtrate to dryness again, and use the activated crude product directly for the reaction.

[0091] The above 80.77g crude product and 150g compound 2 were dissolved in DMF (12.3L), 230.8mL DIPEA was added, and the mixture was stirred at 25°C overnight to obtain compound 4 (compound 4 in patent CN111378028A). The operation time of the entire step 2 (including liquid preparation, material addition, and reaction) was 24h, and the volume of the solvent used was 0.847+0.423+1.27+12.3+0.231=15.1L.

[0092] Step 3: 150.0 g of compound 4 with a purity of about 70% was added to 1687.5 mL of freshly prepared lysate (TFA / PhSMe / PhOMe / EDT=90:5:2:3), stirred for 1.5 h, 9.4 L of ether was added, a white solid was precipitated, and ether was added for slurrying (9.4 L) after centrifugation. The mixture was repeated three times and dried to obtain crude semaglutide. The HPLC test results were as follows: Figure 7 As shown, the content is 40.4%, the purity is 47.98%, and the mass yield is 61.0%.

[0093] The operation time of step three (including the time for liquid preparation, material addition, reaction, solid precipitation, centrifugation and drying) is 14 h, and the volume of solvent used is 1.7+9.4*4=39.3 L.

[0094] Comparative Example 3 (Raw material was enlarged to 150g target propeptide)

[0095] Refer to the process route in patent CN202210698338.3 (a method for synthesizing semaglutide, which our company has applied for a patent), as follows Figure 8 shown.

[0096] Step 1: Modification of Lys at position 26 by Arg34GLP-1(9-37)

[0097] DIEA was added to 45 L of a mixture of acetonitrile and water (acetonitrile: water = 1:2), cooled to 0-5°C, adjusted to pH 11.5, and 172.6 g of Arg34 GLP-1 (9-37) with a content of 86.9% and a purity of 93.4% was added to form a mixture; 50 g of compound 1 was dissolved in 5.0 L of acetonitrile, and then added dropwise to the above mixture, reacted at 0-5°C for 3 hours, the pH of the obtained reaction solution was adjusted to 5.4 with acetic acid, and then 180 L of purified water was added to precipitate solids, centrifuged, and dried to obtain solid compound 2. The operation time of the entire step 1 (including the time for preparing the solvent, adding materials, reacting, precipitating and centrifuging) was 12.5 h, and the solvent volume was 45+5+180=230 L.

[0098] Step 2: Modification of the terminal amino group

[0099] Dissolve 150 g of Boc-His(Trt)-Aib-OH in 1.5 L of dichloromethane, add 55.5 g of DCC, cool to 0°C, stir for 30 min, add 33 g of HOSu, stir and react at this temperature for 12 hours, filter, collect the filtrate, and spin dry to obtain crude Boc-His(Trt)-Aib-OSu.

[0100] 50 g of the crude Boc-His(Trt)-Aib-OSu and 150 g of compound 2 were dissolved in 3.0 L of DMF, and 300 mL of DIEA was added. The mixture was reacted at room temperature for 8 h. The pH of the reaction solution was adjusted to the isoelectric point. 14.4 L of water was added to precipitate the solution. The solution was centrifuged and dried to obtain compound 4 with a purity of 76.57%. The operation time of the entire step 2 (including liquid preparation, material addition, and reaction) was 24 h, and the volume of the solvent used was 1.5+3+0.3+14.4=19.2 L.

[0101] Step 3: Synthesis of semaglutide

[0102] 150g of compound 4 was added to 1.5L of freshly prepared cleavage solution (trifluoroacetic acid: thioanisole: triisopropylsilane = 95:2:3) and reacted at room temperature for 3h. 15L of icy tert-methyl ether was added to precipitate a white solid. After centrifugation, the product was washed three times with tert-methyl ether and dried to obtain a crude semaglutide with a content of 42.5% and a purity of 59.98% (such as Fig. 9 ), with a mass yield of 70.1%. The crude semaglutide was then further purified by reverse phase HPLC to obtain 1.3 g of semaglutide with a purity of 98.54%.

[0103] The operation time of step three (including the time for liquid preparation, material addition, reaction, solid precipitation, centrifugation and drying) is 15 h, and the volume of solvent used is 1.5+15+15*3=61.5 L.

[0104] Comparative Example 4

[0105] Referring to the continuous preparation method disclosed in patent application CN118666987A, the twenty-nonapeptide is first reacted with Trt-isobutyric acid active ester to obtain a thirty-nine-peptide intermediate, then Trt protection is removed to obtain a de-Trt thirty-nine-peptide intermediate, and then reacted with Trt-histidine active ester to obtain a thirty-one-peptide intermediate, and side chain active ester is added to react to obtain a semaglutide precursor, and finally deprotected to obtain a crude semaglutide; the total reaction yield is about 65%, and the HPLC purity is ≥99%.

[0106] The Lys in the twenty-nonapeptide used in this patented method is protected by Boc and cannot be obtained by biological fermentation, which is costly. Secondly, the terminal amino modification is achieved through two-step liquid phase synthesis and one-step deprotection, which is more complicated to operate and will inevitably produce more impurities, adding pressure to the subsequent purification steps.

[0107] The data comparison between the embodiment and the comparative example (both using 172.6 g of the key raw material Arg34GLP-1 (9-37) from the same source, with a content of 86.9% and a purity of 93.4%) are shown in Tables 1 and 2 below.

[0108] Table 1 Data comparison between Example 2 and Comparative Examples 1 to 3

[0109]

[0110] Note: The operation time of Comparative Examples 1 to 3 Acylation 1 (Step 1) in the table is the sum of the reaction time and post-treatment time of the corresponding steps; similarly, the time of Comparative Examples 1 to 3 Acylation 2 (Step 2) is the sum of the reaction time and post-treatment time of the corresponding steps.

[0111] The operation time and solvent volume of the hydrolysis and post-treatment in Comparative Examples 2 and 3 correspond only to step 3 (without step 4).

[0112] Table 2 Data comparison of Examples 1 to 3 and Comparative Examples 1 to 4

[0113]

[0114] Note: Total waste liquid output = volume of acylation solvent 1 + volume of acylation solvent 2 + volume of hydrolysis and post-treatment solvent.

[0115] By comparing the data of Examples 1 to 3 and Comparative Examples 1 to 4, it can be seen that the present invention does not need to process the acylation product 1 and the acylation product 2 into solids, which greatly shortens the construction period (relative to the increase in post-processing operations, the cycle of each batch is shortened from about 40.5 to 54.5 hours to about 16.5 hours), thereby greatly reducing labor costs; at the same time, the output of each batch of waste liquid (such as wastewater, organic solvent, etc.) is reduced from about 51.2 to 310.7L to about 22.1L, which greatly reduces the cost of waste liquid treatment and is more in line with the concept of sustainable development; the crude product mass yield of Examples 1 to 3 is 94.9 to 105.1%, and the total mass yield is 75.4 to 86.2%, which is 65% of the total mass yield of Comparative Example 4, and the total yield is at least 10% higher, which greatly saves production costs.

[0116] The above descriptions are only embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent results obtained by utilizing the contents of the present invention specification through equivalent process transformation, or direct or indirect application in other related technical fields, should be included in the protection scope of this patent.

Claims

1. A one-pot method for preparing semaglutide, characterized in that: The process comprises the following reaction steps: (1) controlling the reaction pH, introducing the activated dipeptide Fmoc-His-Aib-OH into the terminal amino group of Arg34GLP-1(9-37) through an acylation reaction, and obtaining an acylation reaction solution whose main component is compound 1. After the reaction is completed, the next step of the reaction is directly carried out without post-treatment; (2) Adjusting the pH of the acylation reaction solution, adding compound 2-tetrapeptide to the acylation reaction solution, and connecting to the Lys of Arg34GLP-1(9-37) through acylation reaction. 26 A second acylation reaction solution with compound 3 as the main component was obtained, and the next step of reaction was directly carried out without post-treatment after the reaction was completed; (3) adding an alkaline reagent to the second acylation reaction solution to perform a hydrolysis reaction to remove the Fmoc protecting group, thereby obtaining a crude semaglutide; (4) post-treating the crude semaglutide to obtain a refined semaglutide product; The steps (1), (2) and (3) are carried out in the same reactor; The specific preparation route is as follows:

2. The synthesis method according to claim 1, characterized in that In step (1), the solvent used in the acylation reaction is selected from a combination of an organic solvent and water, and the organic solvent is selected from one or more of NMP, DMSO, acetonitrile, ethanol, tetrahydrofuran, and DMF.

3. The synthesis method according to claim 2, characterized in that The volume ratio of the organic solvent to water is 0.05-0.3:

1.

4. The synthesis method according to claim 1, characterized in that The reaction temperature of step (1) is 35-50°C.

5. The synthesis method according to claim 1, characterized in that The reaction in step (1) is first adjusted to a pH value of 11.0 to 12.0 using a base, and then adjusted to a pH value of 8.0 to 10.0 using an acid.

6. The synthesis method according to claim 1, characterized in that In step (1), the activation reagent used to activate Fmoc-His-Aib-OH is selected from one or more combinations of N,N'-disuccinimidyl carbonate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidone hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and O-benzotriazole-tetramethyluronium hexafluorophosphate.

7. The synthesis method according to claim 1, characterized in that The reaction temperature of step (2) is 35-50°C.

8. The synthesis method according to claim 1, characterized in that The reaction pH value of step (2) is 11.0-12.

0.

9. The synthesis method according to claim 1, characterized in that In step (3), the alkaline reagent is selected from one or more combinations of concentrated aqueous ammonia, cyclohexylamine, morpholine, diethylamine, diazabicyclo, and piperidine.

10. The synthesis method according to claim 1, characterized in that The reaction temperature of step (3) is 35-50°C.

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