A method for synthesizing the side chain of semaglutide

Abstract

The present invention belongs to the technical field of chemical drugs, and particularly relates to a method for synthesizing the side chain of semaglutide. The present invention uses a liquid-phase method to prepare a polypeptide side-chain analog. By optimizing the carboxyl activating reagent and reaction solvent, the intermediate required for the side chain of semaglutide can be easily prepared, reducing the steps of complex purification methods such as column chromatography in each step, simplifying the preparation process, reducing the consumption of organic reagents, lowering the cost, being more environmentally friendly and suitable for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical drugs, and particularly relates to a method for synthesizing the side chain of semaglutide. Background Art

[0002] Semaglutide is a GLP-1 analogue developed by Novo Nordisk. Semaglutide has a unique structural design. After once-weekly injection, the blood drug concentration is very stable, and it has very excellent blood glucose-lowering effects and also has the curative effect of weight loss. In the past 10 years of progress in hypoglycemic treatment, the emergence of GLP-1 receptor agonists (GLP-1RAs) is one of the most promising diabetes drugs in modern times. GLP-1RAs lower blood glucose by stimulating insulin secretion in pancreatic β-cells in a glucose-dependent manner and inhibiting glucagon secretion in pancreatic α-cells, and at the same time can promote satiety (directly affecting GLP-1 receptors in the brain and delaying gastric emptying), thereby reducing body weight.

[0003] Semaglutide connects an 18-carbon fatty diacid side chain to the lysine position at the 26th position of the peptide chain, which can mediate and promote the strong binding of semaglutide to albumin, reduce renal clearance rate. Compared with liraglutide with a C16 side chain, the affinity of the semaglutide side chain for albumin is increased by 5-6 times. Binding to albumin can increase the molecular weight of this product, which can avoid being rapidly cleared by the kidneys and prevent metabolic degradation, and prolong the in vivo half-life.

[0004] Since the side chain of semaglutide can be regarded as 4 reaction units, it is commonly obtained by coupling 4 times by solid-phase synthesis in industry and then cutting. In the existing patent literature, there are mainly three patents on the synthesis of the semaglutide side chain, namely: [WO 2009022013], [WO 2009115469], [WO 2009083549], all of which adopt the solid-phase synthesis Fmoc strategy and regard the side chain as 4 reaction units, and there are few reports on the liquid-phase synthesis method of the side chain. For the solid-phase synthesis method, the multi-polymer support is expensive, accounting for the vast majority of the cost of processing materials, generating a large amount of polymer carrier waste, which is difficult to scale up and is not conducive to industrial production. Moreover, the atom economy of solid-phase synthesis is poor, and the raw materials for the side chain are relatively expensive. Therefore, developing a new liquid-phase synthesis method for the side chain has outstanding practical value and market prospects.

[0005] In view of this, this patent has developed a liquid-phase polypeptide synthesis technology. According to the property that the reaction products containing certain "specific functional groups" have specific solubilities in specific solvents (soluble in some solvents and insoluble in some solvents), a chemical synthesis method for directly obtaining pure products by washing the crude reaction products with specific solvents or solvent combinations is provided. Summary of the Invention

[0006] To solve the technical problems of high production waste and expensive raw materials caused by the existing solid-phase synthesis method, the present invention proposes a new method for obtaining the side chain of semaglutide through liquid-phase synthesis. The present invention has the advantages of feasible and controllable process, low cost and high yield, and is suitable for the synthesis method of the side chain of semaglutide for large-scale production.

[0007] The specific technical solution is as follows:

[0008] A method for synthesizing the side chain of semaglutide, comprising the following steps:

[0009] (1) The raw materials R-1 and R-2 are subjected to an amide condensation reaction to obtain the intermediate Z-1; the reaction route is as follows:

[0010]

[0011] (2) The carboxyl group of the intermediate Z-1 is activated with R-3 to obtain the intermediate Z-2; the reaction route is as follows:

[0012]

[0013] (3) The intermediate Z-2 is reacted with the raw material X to finally obtain the side chain of semaglutide; the reaction route is as follows:

[0014]

[0015]

[0016] Wherein, the group R of the raw material X is H, phenyl or ethyl; the corresponding raw materials X are respectively named raw material X-1, raw material X-2

[0017]

[0018] or raw material X-3.

[0019]

[0020] Preferably, the raw material X is X-2.

[0021] The present invention uses a liquid-phase method to prepare polypeptide side-chain analogs. R-1 is monoterbutyl octadecanedioate pentafluorophenyl ester, R-2 is L-glutamic acid 1-tert-butyl ester, R-3 is pentafluorophenyl trifluoroacetate, and the group R of raw material X is a carboxyl protecting group H, phenyl or ethyl; X-1 is 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azapentadecanoic acid. The side chain of semaglutide can be easily prepared from raw material X, reducing the steps of using complex purification methods such as column chromatography in each step, simplifying the preparation process, reducing the usage of organic reagents, lowering costs, being more environmentally friendly and suitable for large-scale production. The research team of the present invention found based on theoretical research and experiments that when the raw material X is X-2, the benzene ring and the ester group form a carboxyl group protected by benzyl ester, which is easier to remove hydrophilic impurities and improve the product purity.

[0022] Preferably, it specifically includes the following steps:

[0023] (S1) Dissolve raw material R-1 in organic solvent A and place it in a reaction vessel. Cool down the temperature, add raw material R-2 and organic base B to the reaction system, and carry out the reaction; after the reaction is completed, extract to obtain intermediate Z-1;

[0024] (S2) Dissolve intermediate Z-1 in organic solvent C and cool down the temperature, add carboxyl activator R-3 and acid-binding agent to the reaction system, and continue the reaction; after the reaction is completed, extract the solid precipitated in the solution to obtain intermediate Z-2;

[0025] (S3) Dissolve intermediate Z-2 in organic solvent D and cool down the temperature, add raw material X and organic base E to the reaction system, and continue the reaction; after the reaction is completed, extract to obtain the final product of the semaglutide side chain.

[0026] Preferably, in steps (S1)-(S3), the reaction temperature is 20-35°C.

[0027] Further, in steps (S1)-(S3), the reaction temperature is 25-30°C.

[0028] Compared with the previously disclosed synthetic routes, the reaction of the present invention has high tolerance and can synthesize the target product without very harsh conditions, and the process is simple. Therefore, to a certain extent, the application of the present invention in industrial production can save time, reduce energy consumption and industrial costs, which is conducive to the industrial production of the semaglutide side chain.

[0029] Preferably, in step (S1), the organic base B is one of DIEA, triethylamine, imidazole, DBU, pyridine or piperazine; the organic solvent A is one or more of ethanol, tetrahydrofuran, toluene, acetonitrile, dichloroethane and chloroform; the molar ratio of R-1 to R-2 is 1:(1.05-3.19).

[0030] The glutamic acid raw material used in the present invention is L-glutamic acid 1-tert-butyl ester without F-moc protection of the amino group. Compared with the glutamic acid raw material with Fmoc protection of the amino group in the existing preparation method, the deprotection step is omitted, making the preparation process simpler and more efficient.

[0031] Preferably, in step (S2), the acid-binding agent is one of pyridine, piperidine, piperazine, triethylamine or trimethylamine; the organic solvent C is one or more of petroleum ether, n-hexane, cyclohexane, ether, acetonitrile, DMF, dichloroethane and chloroform; the molar ratio of Z-1 to R-3 is 1:(1.1 - 1.21).

[0032] Further, in step (S2), the molar ratio of Z-1 to R-3 is 1:(1.15 - 1.21).

[0033] The previously disclosed routes for synthesizing the side chain of semaglutide all use multi-step reaction routes, and intermediate separation and purification are also required during the preparation. The steps are cumbersome and the operation is troublesome. Moreover, the yield and purity of the side chain of semaglutide synthesized by the prior art are not high. By optimizing the carboxyl activating reagent and reaction solvent, the present invention greatly improves the reaction activity while maintaining stability, and the reaction efficiency is almost 100%. The present invention uses pentafluorophenyl trifluoroacetate as an activating group to activate the carboxyl group, which can improve the yield and purity of the final product and reduce the difficulty of subsequent purification.

[0034] Preferably, in step (S3), the organic base E is one of DIEA, triethylamine, imidazole, DBU, pyridine or piperazine; the organic solvent D is one or more of DMF, ethanol, tetrahydrofuran, toluene, acetonitrile, dichloroethane and chloroform; the molar ratio of Z-2 to X is 1:(1.13 - 2.54).

[0035] Further, in step (S3), the molar ratio of Z-2 to X is 1:(1.65 - 2.54).

[0036] The raw material X-1 used in the present invention, 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azapentadecanoic acid (AEEA-AEEA), is also without F-moc protection; compared with the use of F-moc-AEEA-AEEA in the existing solid-phase synthesis method, the present invention omits the deprotection step, making the preparation process simpler and more economical.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention prepares a polypeptide side chain analog by a liquid phase method. By optimizing the carboxyl activating reagent and reaction solvent, the intermediate required for the side chain of semaglutide can be simply prepared, reducing the steps of complex purification methods such as column chromatography in each step, simplifying the preparation process, reducing the usage of organic reagents, lowering the cost, being more environmentally friendly and suitable for large-scale production.

[0039] 2. The previously disclosed routes for synthesizing the side chain of semaglutide all use multi-step reaction routes, and intermediate separation and purification are also required during the preparation, with cumbersome steps and troublesome operations. Moreover, the yield and purity of the side chain of semaglutide synthesized by the prior art are not high. The present invention uses pentafluorophenol as an activating group to activate the carboxyl group, greatly improving the reaction activity, increasing the yield and purity of the final product, and reducing the difficulty of subsequent purification.

[0040] 3. The glutamic acid raw material used in the present invention is L-glutamic acid 1-tert-butyl ester without F-moc protection of the amino group. Compared with the glutamic acid raw material with Fmoc protection of the amino group in the existing preparation method, the deprotection step is omitted, making the preparation process simpler and more efficient.

[0041] 4. The raw material AEEA-AEEA used in the present invention is also without F-moc protection. Compared with F-moc-AEEA-AEEA used in the existing solid-phase synthesis method, the deprotection step is omitted, making the preparation process simpler and more economical. Description of the Drawings

[0042] Figure 1 It is the HPLC spectrum of the final product tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu in Example 1.

[0043] Figure 2 It is the MS spectrum of the final product tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu in Example 1.

[0044] Figure 3 For the final product tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu in Example 1 1 HNMR spectrum. Detailed Embodiments

[0045] The following further illustrates the content of the present invention with reference to examples, but does not limit the present invention.

[0046] General Example

[0047] A method for synthesizing the side chain of semaglutide, comprising the following steps:

[0048] (1) The raw materials R-1 and R-2 are reacted through an amide condensation reaction to obtain the intermediate Z-1; the reaction route is as follows:

[0049] (2) The carboxyl group of the intermediate Z-1 is activated with R-3 to obtain the intermediate Z-2; the reaction route is as follows:

[0050]

[0051] (3) The intermediate Z-2 is reacted with the raw material X to finally obtain the side chain of semaglutide; the reaction route is as follows:

[0052]

[0053] Among them, the group R of the raw material X is H, phenyl or ethyl; the corresponding raw materials X are named raw material X-1, raw material X-2 or raw material X-3 respectively.

[0054] Preferably, the raw material X is X-2.

[0055] Preferably, it specifically includes the following steps:

[0056] (S1) Dissolve the raw material R-1 with the organic solvent A and place it in a reaction vessel, cool down, add the raw material R-2 and the organic base B to the reaction system, and carry out the reaction; after the reaction is completed, extract to obtain the intermediate Z-1;

[0057] (S2) Dissolve the intermediate Z-1 with the organic solvent C and cool down, add the carboxyl activating agent R-3 and the acid-binding agent to the reaction system, and continue the reaction; after the reaction is completed, extract the solid precipitated in the solution to obtain the intermediate Z-2;

[0058] (S3) Dissolve the intermediate Z-2 with the organic solvent D and cool down, add the raw material X and the organic base E to the reaction system, and continue the reaction; after the reaction is completed, extract to obtain the final product, the side chain of semaglutide.

[0059] Preferably, in steps (S1)-(S3), the temperature of the reaction is 20-35°C.

[0060] Furthermore, in steps (S1)-(S3), the temperature of the reaction is 25-30°C.

[0061] Preferably, in step (S1), the organic base B is one of DIEA, triethylamine, imidazole, DBU, pyridine or piperazine; the organic solvent A is one or several of ethanol, tetrahydrofuran, toluene, acetonitrile, dichloroethane and chloroform; the molar ratio of R-1 to R-2 is 1:(1.05-3.19).

[0062] Preferably, in step (S2), the acid-binding agent is one of pyridine, piperidine, piperazine, triethylamine or trimethylamine; the organic solvent C is one or more of petroleum ether, n-hexane, cyclohexane, ether, acetonitrile, DMF, dichloroethane and chloroform; the molar ratio of Z-1 to R-3 is 1:(1.1 - 1.21).

[0063] Further, in step (S2), the molar ratio of Z-1 to R-3 is 1:(1.15 - 1.21).

[0064] Preferably, in step (S3), the organic base E is one of DIEA, triethylamine, imidazole, DBU, pyridine or piperazine; the organic solvent D is one or more of DMF, ethanol, tetrahydrofuran, toluene, acetonitrile, dichloroethane and chloroform; the molar ratio of Z-2 to X is 1:(1.13 - 2.54).

[0065] Further, in step (S3), the molar ratio of Z-2 to X is 1:(1.65 - 2.54).

[0066] Example 1

[0067]

[0068] (1) Dissolve the raw material R-1 (20 g, 37.27 mmol) in 400 mL of ethanol solution, place it in a flask under nitrogen protection. After cooling the reaction solution to 0 - 10 °C, add the raw material R-2 (7.95 g, 39.13 mmol) and DIEA (5.06 g, 39.13 mmol) to the reaction system under nitrogen protection, and react at 30 °C overnight. After TLC shows that the raw materials are consumed, cool down to crystallize, filter by suction, wash the filter cake with 400 mL of ethanol in two portions, and dry to obtain the intermediate Z-1 (18.5 g, yield = 89.31%).

[0069]

[0070] (2) Dissolve the intermediate Z-1 (18.5 g, 33.29 mmol) in 200 mL of petroleum ether, cool it to 0 - 10 °C, and then add R-3 (10.25 g, 36.61 mmol) and pyridine (2.9 g, 36.61 mmol) to the reaction system under nitrogen protection, and continue to react at 30 °C. After reacting for 1 h, a large amount of solid precipitates in the solution. After TLC shows that the raw materials are completely consumed, filter by suction, wash the filter cake with 300 mL of petroleum ether in two portions, and dry to obtain the intermediate Z-2 (22.7 g, yield = 94.48%).

[0071]

[0072] (3) Dissolve the intermediate Z-2 (22.7 g, 33.29 mmol) in 200 mL of DMF. After cooling to 0 - 10 °C, add X-1 (11.64 g, 37.74 mmol) and DIEA (4.88 g, 37.74 mmol) to the reaction system under nitrogen protection, and then return to 30 °C to continue the reaction. After reacting overnight, TLC shows that the raw materials are completely consumed. Wash the reaction solution twice with 200 mL of a mixed solution of 0.5 N HCl / 10% NaCl, and then wash it once with saturated brine. Dry the organic phase with anhydrous MgSO4, filter and concentrate to obtain the crude product. The crude product is separated and purified by a silica gel column with 100 - 200 mesh (DCM:MeOH = 50:1 - 30:1 - 20:1), collect the target eluate, concentrate and dry to obtain the final product semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (18.7 g, yield 70.28%), as Figure 1 shown in the HPLC chromatogram, with a purity of 98.2%.

[0073] The mass spectrometry data of the final product is as Figure 2 shown, [M + H+] = 846.7 g / mol.

[0074] The 1H NMR data of the final product is as Figure 3 shown, 1 1H NMR (400 MHz, DMSO) δ (ppm): 12.60 (s, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.90 (t, J = 5.6 Hz, 1H), 7.67 (t, J = 5.7 Hz, 1H), 4.09 - 3.99 (m, 3H), 3.88 (s, 2H), 3.55 (tdd, J = 9.1, 6.0, 3.3 Hz, 8H), 3.43 (dt, J = 11.8, 5.9 Hz, 4H), 3.24 (dq, J = 28.1, 5.8 Hz, 4H), 2.20 - 2.06 (m, 6H), 1.89 (td, J = 13.5, 7.5 Hz, 1H), 1.80 - 1.69 (m, 1H), 1.54 - 1.43 (m, 4H), 1.39 (brs, 18H), 1.23 (brs, 24H).

[0075] Example 2

[0076]

[0077] (1) Dissolve raw material R-1 (6.33 g, 12.27 mmol) in 150 mL of ethanol solution, place it in a flask under nitrogen protection. After cooling the reaction solution to 0 - 10 °C, add raw material R-2 (2.64 g, 39.13 mmol) and DIEA (1.68 g, 13.12 mmol) to the reaction system under nitrogen protection, and react at 30 °C overnight. After TLC shows that the raw materials are consumed completely, cool down to crystallize, filter by suction, wash the filter cake twice with 400 mL of ethanol in two portions, and dry to obtain intermediate Z-1 (5.7 g, yield = 89.31%).

[0078]

[0079] (2) Dissolve intermediate Z-1 (5.7 g, 10.08 mmol) in 100 mL of petroleum ether, cool it to 0 - 10 °C, and then add R-3 (3.62 g, 12.21 mmol) and pyridine (1.1 g, 11.811 mmol) to the reaction system under nitrogen protection. Return to 30 °C and continue the reaction. After reacting for 1 h, a large amount of solid precipitates in the solution. After TLC shows that the raw materials are completely consumed, filter by suction, wash the filter cake twice with 300 mL of petroleum ether in two portions, and dry to obtain intermediate Z-2 (5.4 g, yield = 94.77%).

[0080]

[0081] (3) Dissolve intermediate Z-2 (5.4 g, 7.5 mmol) in 150 mL of DMF, cool it to 0 - 10 °C, and then add X-2 (4.11 g, 12.37 mmol) and pyridine (4.6 ml, 38.2 mmol) to the reaction system under nitrogen protection. Return to 25 °C and continue the reaction. After reacting overnight, TLC shows that the raw materials are completely consumed. After the reaction, carry out a debenzylation reaction under catalytic hydrogenation conditions using H2, Pd - C, and EtOH to restore the original hydroxyl group. Wash the reaction solution twice with 200 mL of saturated citric acid and 1 M NaOH solution respectively, and then wash it once with saturated brine. Dry the organic phase with anhydrous MgSO4, filter and concentrate to obtain the crude product. Purify the crude product by silica gel column chromatography with 100 - 200 mesh (DCM:MeOH = 50:1 - 30:1 - 20:1), collect the target eluate, concentrate and dry to obtain the final product semaglutide side chain tBuO - Ste - Glu(AEEA - AEEA - OH) - OtBu (6.23 g, yield 73.17%).

[0082]

[0083] Example 3

[0084]

[0085] (1) Dissolve raw material R-1 (10 g, 18.64 mmol) in 400 mL of ethanol solution, place it in a flask under nitrogen protection. After cooling the reaction solution to 0 - 10 °C, add raw material R-2 (3.98 g, 19.57 mmol) and DIEA (2.53 g, 19.57 mmol) to the reaction system under nitrogen protection, and react at 30 °C overnight. After TLC shows that the raw materials are consumed completely, cool down to crystallize, filter by suction, wash the filter cake twice with 400 mL of ethanol in two portions, and dry to obtain intermediate Z-1 (9.5 g, yield = 90.14%).

[0086]

[0087] (2) Dissolve intermediate Z-1 (9.57 g, 17.13 mmol) in 100 mL of petroleum ether, cool it to 0 - 10 °C, and then add R-3 (5.75 g, 19.66 mmol) and pyridine (1.8 g, 17.55 mmol) to the reaction system under nitrogen protection. Return to 30 °C and continue the reaction. After reacting for 1 h, a large amount of solid precipitates in the solution. After TLC shows that the raw materials are consumed completely, filter by suction, wash the filter cake twice with 300 mL of petroleum ether in two portions, and dry to obtain intermediate Z-2 (11.6 g, yield = 94.26%).

[0088]

[0089] (3) Dissolve intermediate Z-2 (4.8 g, 6.4 mmol) in 150 mL of DMF, cool it to 0 - 10 °C, and then add X-3 (5.21 g, 16.28 mmol) and pyridine (3.9 ml, 32.7 mmol) to the reaction system under nitrogen protection. Return to 25 °C and continue the reaction. After reacting overnight, TLC shows that the raw materials are consumed completely. Cut the reaction system with HFIP / DCM solution for 1 h, filter out the reaction solution using a sintered glass funnel. Wash the reaction solution twice with 200 mL of saturated citric acid and 1 M NaOH solution respectively, and then wash it once with saturated brine. Dry the organic phase with anhydrous MgSO4, filter and concentrate to obtain the crude product. Purify the crude product by silica gel column chromatography (200 - 100 mesh, DCM:MeOH = 50:1 - 30:1 - 20:1), collect the target eluate, concentrate and dry to obtain the final product semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (4.43 g, yield 77.24%).

[0090]

[0091] Example 4

[0092]

[0093] (1) Dissolve raw material R-1 (20 g, 37.27 mmol) in 400 mL of ethanol solution, place it in a flask under nitrogen protection. After cooling the reaction solution to 0 - 10 °C, add raw material R-2 (7.95 g, 39.13 mmol) and DIEA (5.06 g, 39.13 mmol) to the reaction system under nitrogen protection, and react at 30 °C overnight. After TLC shows that the raw materials are consumed completely, cool down to crystallize, filter by suction, wash the filter cake twice with 400 mL of ethanol in two portions, and dry to obtain intermediate Z-1 (18.5 g, yield = 89.31%).

[0094]

[0095] (2) Dissolve intermediate Z-1 (18.5 g, 33.29 mmol) in 200 mL of petroleum ether. After cooling to 0 - 10 °C, add R-3 (10.25 g, 36.61 mmol) and pyridine (2.9 g, 36.61 mmol) to the reaction system under nitrogen protection, and then return to 30 °C to continue the reaction. After reacting for 1 h, a large amount of solid precipitates in the solution. After TLC shows that the raw materials are consumed completely, filter by suction, wash the filter cake twice with 300 mL of petroleum ether in two portions, and dry to obtain intermediate Z-2 (22.7 g, yield = 94.48%).

[0096]

[0097] (3) Dissolve intermediate Z-2 (22.7 g, 33.29 mmol) in 200 mL of DMF. After cooling to 0 - 10 °C, add X-2 (12.54 g, 37.74 mmol) and DIEA (4.88 g, 37.74 mmol) to the reaction system under nitrogen protection, and then return to 30 °C to continue the reaction. After reacting overnight, TLC shows that the raw materials are consumed completely. After the reaction, carry out a debenzylation reaction under catalytic hydrogenation conditions using H2, Pd-C, and EtOH to restore the original hydroxyl group. Wash the reaction solution twice with 200 mL of saturated citric acid and 1 M NaOH solution respectively, and then wash once with saturated brine. Dry the organic phase with anhydrous MgSO4, filter and rotary evaporate to obtain the crude product. Purify the crude product by silica gel column chromatography (100 - 200 mesh, DCM:MeOH = 50:1 - 30:1 - 20:1), collect the target eluate, concentrate and dry to obtain the final product semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (19.27 g, yield 72.41%).

[0098]

[0099] Comparing with Comparative Example 1, it can be seen that when the intermediate Z-2 undergoes a condensation reaction with the raw material X-2, the yield of the product of semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu increases. Based on theoretical research and experiments, the research team of the present invention found that when the raw material X is X-2, the benzene ring and the ester group form a carboxyl group protected by benzyl ester, which is easier to remove hydrophilic impurities and improve the product purity.

[0100] Example 5

[0101]

[0102] (1) Dissolve the raw material R-1 (10 g, 18.64 mmol) in 400 mL of ethanol solution, place it in a flask under nitrogen protection. After cooling the reaction solution to 0-10 °C, add the raw material R-2 (3.98 g, 19.57 mmol) and DIEA (2.53 g, 19.57 mmol) to the reaction system under nitrogen protection, and react at 30 °C overnight. After TLC shows that the raw materials are consumed, cool down to crystallize, filter by suction, wash the filter cake with 400 mL of ethanol in two portions, and dry to obtain the intermediate Z-1 (9.5 g, yield = 90.14%).

[0103]

[0104] (2) Dissolve the intermediate Z-1 (9.57 g, 17.13 mmol) in 100 mL of petroleum ether, cool it to 0-10 °C, and add R-3 (5.75 g, 19.66 mmol) and pyridine (1.8 g, 17.55 mmol) to the reaction system under nitrogen protection, then return to 30 °C to continue the reaction. After reacting for 1 h, a large amount of solid precipitates in the solution. After TLC shows that the raw materials are completely consumed, filter by suction, wash the filter cake with 300 mL of petroleum ether in two portions, and dry to obtain the intermediate Z-2 (11.6 g, yield = 94.26%).

[0105]

[0106] (3) Dissolve the intermediate Z-2 (4.8 g, 6.4 mmol) in 150 mL of DMF. After cooling to 0 - 10 °C, add X-2 (5.41 g, 16.28 mmol) and pyridine (3.9 mL, 32.7 mmol) to the reaction system under nitrogen protection, and then return to 25 °C to continue the reaction. After reacting overnight, TLC shows that the raw materials are completely consumed. After the reaction, use H2, Pd-C, and EtOH to restore the original hydroxyl group through debenzylation under catalytic hydrogenation conditions. Wash the reaction solution twice with 200 mL of saturated citric acid and 1 M NaOH solution respectively, and then wash it once with saturated brine. Dry the organic phase with anhydrous MgSO4, filter and concentrate to obtain the crude product. The crude product is separated and purified by a silica gel column with 100 - 200 mesh (DCM:MeOH = 50:1 - 30:1 - 20:1), collect the target eluent, concentrate and dry to obtain the final product semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu (4.56 g, yield = 79.43%).

[0107]

[0108] Comparing with Comparative Example 3, it can be seen that the condensation reaction of the intermediate Z-2 and the raw material X-2 results in an increase in the product of the semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu and an increase in the yield. Based on theoretical research and experiments, the research team of the present invention found that when the raw material X is X-2, the benzene ring and the ester group form a benzylester-protected carboxyl group, which is easier to remove hydrophilic impurities and improve the product purity.

[0109] The raw materials and equipment used in the present invention are common raw materials and equipment in the art without special instructions; the methods used in the present invention are conventional methods in the art without special instructions.

[0110] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing the side chain of semaglutide, characterized in that, It includes the following steps: (1) The raw materials R-1 and R-2 undergo an amide condensation reaction to obtain the intermediate Z-1; the reaction route is as follows: (2) Activate the carboxyl group of the intermediate Z-1 with R-3 to obtain the intermediate Z-2; the reaction route is as follows: (3) React the intermediate Z-2 with the raw material X to finally obtain the side chain of semaglutide; the reaction route is as follows: Among them, the group R in the raw material X is H, phenyl or ethyl; the corresponding raw materials X are named raw material X-1, raw material X-2 or raw material X-3 respectively.

2. The synthesis method according to claim 1, characterized in that, The raw material X is X-2.

3. The synthesis method according to claim 1, characterized in that, Specifically, it includes the following steps: (S1) Dissolve the raw material R-1 with the organic solvent A and place it in a reaction vessel, cool down, add the raw material R-2 and the organic base B to the reaction system, and carry out the reaction; after the reaction is completed, extract to obtain the intermediate Z-1; (S2) Dissolve the intermediate Z-1 with the organic solvent C and cool down, add the carboxyl activator R-3 and the acid-binding agent to the reaction system, and continue the reaction; after the reaction is completed, extract the solid precipitated in the solution to obtain the intermediate Z-2; (S3) Dissolve the intermediate Z-2 with the organic solvent D and cool down, add the raw material X and the organic base E to the reaction system, and continue the reaction; after the reaction is completed, extract to obtain the final product, the side chain of semaglutide.

4. The synthesis method according to claim 3, characterized in that, In steps (S1)-(S3), the temperature of the reaction is 20-35 °C.

5. The synthesis method according to claim 4, characterized in that, In steps (S1)-(S3), the temperature of the reaction is 25-30 °C.

6. The synthesis method according to claim 3, characterized in that, In step (S1), the organic base B is one of DIEA, triethylamine, imidazole, DBU, pyridine or piperazine; the organic solvent A is one or several of ethanol, tetrahydrofuran, toluene, acetonitrile, dichloroethane and chloroform; the molar ratio of R-1 to R-2 is 1:(1.05-3.19).

7. The synthesis method according to claim 3, characterized in that, In step (S2), the acid-binding agent is one of pyridine, piperidine, piperazine, triethylamine or trimethylamine; the organic solvent C is one or several of petroleum ether, n-hexane, cyclohexane, ether, acetonitrile, DMF, dichloroethane and chloroform; the molar ratio of Z-1 to R-3 is 1:(1.1-1.21).

8. The synthesis method according to claim 7, characterized in that, In step (S2), the molar ratio of Z-1 to R-3 is 1:(1.15-1.21).

9. The synthesis method according to claim 3, characterized in that, In step (S3), the organic base E is one of DIEA, triethylamine, imidazole, DBU, pyridine or piperazine; the organic solvent D is one or several of DMF, ethanol, tetrahydrofuran, toluene, acetonitrile, dichloroethane and chloroform; the molar ratio of Z-2 to X is 1:(1.13-2.54).

10. The synthesis method according to claim 9, characterized in that, The molar ratio of Z-2 to X is 1:(1.65-2.54).

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