Process for the preparation of liraglutide side chain and intermediates thereof

Abstract

The application relates to the technical field of compound synthesis, in particular to a preparation method of a liraglutide side chain and an intermediate thereof. The preparation method of an intermediate 1 forming a liraglutide side chain comprises the following steps: reacting palmitic acid and HOSU to form a reaction crude product, and crystallizing the reaction crude product to form the intermediate 1, wherein the solvent selected for crystallization is a mixed solvent of ester solvents and alkane solvents, the mass ratio of the ester solvents to the alkane solvents is 1:5-1:10, and the structural formula of the intermediate 1 is The intermediate formed by the preparation method has high yield, high purity and low homologous impurity content, so that the liraglutide side chain formed has high purity and low homologous impurity content, thereby a synthesis method suitable for large-scale production of the liraglutide side chain can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compound synthesis, in particular, relates to a preparation method of liraglutide side chain and intermediates thereof. BACKGROUND

[0002] Diabetes is a metabolic disease characterized by high blood sugar, which is caused by insulin secretion defects or impaired biological action, or both. Long-term high blood sugar leads to chronic damage, dysfunction of various tissues, especially eyes, kidneys, heart, blood vessels, and nerves, which seriously endangers human health and life.

[0003] Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue that can increase the number of beta cells to promote cell differentiation and has certain protective effect on the heart. It is used for blood glucose control in adult patients with type 2 diabetes and is suitable for patients whose blood glucose is still not well controlled after treatment with the maximum tolerated dose of metformin or sulfonylurea drugs. It is used in combination with metformin or sulfonylurea drugs. The liraglutide side chain is a long-acting side chain of human glucagon-like peptide-1 (GLP-1) analogue for treating diabetes, and its structure is Fmoc-Lys(Pal-Glu-OtBu)-OH. The chemical name is N2-[fluorenylmethyloxycarbonyl]-N6-[N-(1-oxohexadecyl)-L-GAMMA-glutamyl]-L-lysine 1'-tert-butyl ester. This compound is a long-acting Fmoc-protected lysine that can be directly connected to the polypeptide backbone to form liraglutide.

[0004] Specifically, Fmoc-Lys(Pal-Glu-OtBu)-OH is a protected lysine with a linker, which can be used to directly synthesize the liraglutide backbone, and after the synthesis of the polypeptide, the protection can be removed to obtain the liraglutide target product. However, during the synthesis of liraglutide, impurities in the side chain will directly affect the purity of liraglutide, thereby endangering its safety and effectiveness. Therefore, it is necessary to strictly control the impurities in the side chain, and the most difficult to control impurities in the side chain are isomers and homologues.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application provides a preparation method of liraglutide side chain and intermediates thereof. The present application provides a preparation method of an intermediate of liraglutide side chain, which has high yield, high purity and low homologous impurity content, so that the liraglutide side chain formed has high purity and low homologous impurity content, thereby obtaining a synthesis method suitable for large-scale production of liraglutide side chain.

[0007] The present application is realized as follows:

[0008] In a first aspect, the present application provides a preparation method of intermediate 1 of a liraglutide side chain, comprising: reacting palmitic acid and HOSU to form a reaction crude product, and crystallizing the reaction crude product to form the intermediate 1, wherein the solvent selected for crystallization is a mixed solvent of an ester solvent and an alkane solvent, the mass ratio of the ester solvent and the alkane solvent is 1:5-1:10, and the intermediate 1 has the following structural formula:

[0009]

[0010] In an optional embodiment, the ester solvent is selected from C2-C10 ester solvents, preferably C2-C6 ester solvents, preferably C3-C5 ester solvents, and more preferably any one of ethyl acetate, methyl acetate, methyl formate and ethyl formate;

[0011] Preferably, the alkane solvent is selected from C1-C10 straight-chain alkane solvents, preferably C2-C8 straight-chain alkane solvents, preferably C4-C7 straight-chain alkane solvents, and more preferably n-heptane;

[0012] Preferably, the number of crystallization is 2 or more.

[0013] In an optional embodiment, the step of forming the reaction crude product comprises mixing the palmitic acid, the HOSU, EDCI and a solvent to react;

[0014] Preferably, the step of forming the reaction crude product comprises mixing the palmitic acid, the solvent and the HOSU, then cooling to below 5°C, maintaining the temperature and adding the EDCI in batches, then incubating at 0-5°C for 1-1.5 hours, then naturally warming to 15-25°C for 6-8 hours;

[0015] Preferably, the mass ratio of the palmitic acid, the HOSU and the EDCI is 1:0.4-0.6:0.7-0.9, for example 1:0.472:0.89.

[0016] In an optional embodiment, after forming the reaction crude product, the reaction crude product is subjected to primary treatment before crystallization, wherein the primary treatment comprises: extracting and collecting an organic phase, concentrating, nitrile solvent purification and drying;

[0017] Preferably, the primary treatment comprises mixing the reaction system after the reaction with water to separate into two phases, collecting the organic phase, adding a water removal drying agent to filter and collect the filtrate, and then concentrating the filtrate to form a concentrate;

[0018] Mixing the concentrate with a nitrile solvent, then cooling and stirring, filtering, washing the filter cake with a nitrile solvent, and then drying.

[0019] In a second aspect, the present application provides a preparation method of intermediate 2 forming a side chain of liraglutide, comprising: reacting intermediate 1 formed by the preparation method of intermediate 1 forming a side chain of liraglutide according to the preceding embodiments with 1-OtBu-L-Glu to form intermediate 2, wherein the structural formula of intermediate 2 is as follows:

[0020]

[0021] In an optional embodiment, the method comprises: pre-cooling DIEA.

[0022] After mixing the solvent with the intermediate 1 and then mixing the solvent with 1-OtBu-L-Glu to react at a temperature of -5 to 0°C, the temperature is then lowered to -10 to -5°C, the temperature is maintained and the pre-cooled DIEA is added, and then the reaction is carried out at -10 to -5°C for 1-1.5 h, followed by a reaction at -5 to 0°C for 12-14 h, and then the reaction is carried out at room temperature until it is complete.

[0023] The molar ratio of the intermediate 1 to the 1-OtBu-L-Glu is 1:0.98-1:1.05, and the mass ratio of the intermediate 1 to the solvent is 1:7-1:10.

[0024] In an optional embodiment, after the reaction is completed, post-treatment is carried out, wherein the post-treatment comprises adjusting the pH, collecting the organic phase, washing, filtering, concentrating, purifying with a nitrile solvent, recrystallizing, and drying.

[0025] Preferably, the recrystallization is carried out once.

[0026] Preferably, the post-treatment comprises adjusting the pH of the reaction system to 2.8-3.4 after the reaction is completed, allowing the solution to stand and separate, collecting the organic phase, washing the collected organic layer with dilute hydrochloric acid and water, filtering the collected filter cake after adding a water-removing drying agent, and then concentrating the filter liquor to form a concentrate.

[0027] The concentrate is mixed and dissolved with a nitrile solvent by heating, then the temperature is lowered to allow crystallization, the filter cake is collected by filtering, and the filter cake is recrystallized once with a mixed solvent of an ester solvent and an alkane solvent, and the crystals are dried.

[0028] In a third aspect, the present application provides a preparation method of intermediate 3 forming a side chain of liraglutide, comprising: reacting intermediate 2 formed by the preparation method of intermediate 2 forming a side chain of liraglutide according to the preceding embodiments with HOSU and EDCI to form intermediate 3, wherein the structural formula of intermediate 3 is as follows:

[0029]

[0030] In an optional embodiment, the post-treatment comprises: mixing the intermediate 2, the solvent and the HOSU, then adding the EDCI in batches at 5°C or below, and then incubating the reaction for 1.5-2.5 hours, and then allowing the reaction to complete at room temperature, and then performing post-treatment after the reaction is completed;

[0031] The post-treatment comprises: washing the reaction mixture with water and brine, collecting the organic phase, then adding a water-removing drying agent, filtering to collect the filtrate, and then concentrating the filtrate to form a concentrate;

[0032] The concentrate is stirred with an alcohol solvent at 30-35°C for 1-1.5 hours, then cooled to 0-5°C and stirred for 1-1.5 hours, then filtered, washed with an alcohol solvent, and then dried;

[0033] Preferably, the post-treatment does not include recrystallization or crystallization.

[0034] Preferably, the molar ratio of the intermediate 2 to the HOSU is 1:1.1-1.2, and the molar ratio of the intermediate 2 to the EDCI is 1:1.1-1.3, for example 1:1.25.

[0035] In a fourth aspect, the present application provides a preparation method of a side chain of liraglutide, comprising: reacting the intermediate 3 formed by the preparation method of the intermediate 3 of the side chain of liraglutide according to the above-mentioned embodiments with Fmoc-Lys and DIEA.

[0036] Preferably, the method comprises: mixing a halogenated alkane solvent and the Fmoc-Lys, then cooling to 0-10°C, and then adding the DIEA, the intermediate 3 and an alcohol solvent, and incubating the reaction at 0-10°C for 1.5-2.5 hours, and then allowing the reaction to complete at room temperature.

[0037] Preferably, the halogenated alkane solvent is selected from a polyhalogenated C1-C10 alkane solvent, preferably a polyhalogenated C1-C5 alkane solvent, and most preferably a polyhalogenated C1-C3 alkane solvent, and most preferably dichloromethane.

[0038] The alcohol solvent is a C1-C10 monohydric alcohol, preferably a C1-C5 monohydric alcohol, and more preferably a C1-C3 monohydric alcohol.

[0039] Preferably, the mass ratio of the intermediate 3 to the halogenated alkane solvent is 1:7-1:10, the molar ratio of the intermediate 3 to the Fmoc-Lys is 1:1.1-1.2, and the molar ratio of the intermediate 3 to the DIEA is 1:2.5-3, for example 1:2.85.

[0040] Preferably, after the reaction is completed, post-treatment is performed, which includes: pH adjustment of the reaction system, collection of the organic phase, washing, filtration, concentration, nitrile solvent purification and drying.

[0041] The present application has the following beneficial effects: the embodiment of the present application adopts a specific solvent to form a mixed solvent to crystallize the reaction system of intermediate 1, which can effectively reduce the content of C18 homologous impurities while ensuring the yield in the first step reaction, thereby improving the purity of the intermediate, and then effectively improving the purity and yield of the formed liraglutide side chain. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0043] Figure 1 High liquid chromatogram of intermediate 1 provided for the embodiment 1 of the present application;

[0044] Figure 2 High liquid chromatogram of intermediate 2 provided for the embodiment 1 of the present application;

[0045] Figure 3 High liquid chromatogram of intermediate 3 provided for the embodiment 1 of the present application;

[0046] Figure 4 NMR spectrum of liraglutide side chain provided for the embodiment 1 of the present application;

[0047] Figure 5 High liquid chromatogram of liraglutide side chain provided for the embodiment 1 of the present application. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased in the market.

[0049] The embodiment of the present application provides a preparation method of liraglutide side chain, wherein the structural formula of the liraglutide side chain provided by the embodiment of the present application is Fmoc-Lys(Pal-Glu-OtBu)-OH, and the chemical name is N2-[fluorenylmethyloxycarbonyl]-N6-[N-(1-oxohexadecyl)-L-GAMMA-glutamyl]-L-lysine 1'-tert-butyl ester, which comprises:

[0050] S1, forming intermediate 1;

[0051] The structural formula of the intermediate 1 is as follows:

[0052] In the embodiment of the application, palmitic acid is used as a raw material to react with HOSU to obtain palmitic acid-Osu, and C18 homologous impurities are removed to ≤0.2% by crystallization.

[0053] Specifically, after the palmitic acid, HOSU and the solvent are mixed, the temperature is lowered to below 5°C, and then the temperature is maintained and the EDCI is added in batches, followed by incubation at 0-5°C for 1-1.5 hours, and then natural warming to 15-25°C for 6-8 hours. The embodiment of the application can effectively reduce the formation of impurities, but can also ensure the progress of the reaction and improve the yield of the product.

[0054] The solvent can be selected from halogenated alkanes, and the halogenated alkanes are selected from polyhalogenated C1-C10 alkanes, preferably polyhalogenated C1-C5 alkanes, preferably polyhalogenated C1-C3 alkanes, and most preferably dichloromethane.

[0055] It should be noted that the natural warming referred to in the embodiment of the application means that the reaction system is naturally warmed by heat exchange with the ambient temperature without continuous cooling or additional heating (such as water bath or turning on the warming mode of the reaction kettle).

[0056] The mass ratio of the palmitic acid, the HOSU and the EDCI is 1:0.4-0.6:0.7-0.9, for example, 1:0.472:0.89.

[0057] After the reaction is completed by TLC monitoring, the reaction system is mixed with water and stirred thoroughly, and then separated by standing, and the organic phase is collected. Then, a water removal drying agent is added, and after drying, the filtrate is collected by filtration, and the filtrate is concentrated under reduced pressure to form a concentrate.

[0058] The concentrate is washed with a nitrile solvent at room temperature, and then cooled to 0-5°C and stirred thoroughly, and then filtered under suction, and washed with a nitrile solvent, and then dried under vacuum to form a crude product.

[0059] It should be noted that (1) the water removal drying agent used in the embodiment of the application includes but is not limited to anhydrous magnesium sulfate, anhydrous sodium sulfate and the like, and commercially available drying agents.

[0060] (2) The nitrile solvent used in the embodiment of the application includes but is not limited to acetonitrile.

[0061] (3) The room temperature or room temperature provided in the embodiment of the application both refer to 20-30°C.

[0062] Then the crude product is crystallized, wherein the solvent selected for crystallization is a mixed solvent of an ester solvent and an alkane solvent, and the mass ratio of the ester solvent and the alkane solvent is 1:5-1:10. The above mixed solvent is specifically selected as the crystallization solvent in the embodiment of the present application, which can effectively remove impurities, improve purity, and ensure the yield of intermediate 1. If the crystallization solvent is changed, intermediate 1 cannot be crystallized or the content of impurities in intermediate 1 exceeds the standard.

[0063] The ester solvent is selected from C2-C10 ester solvents, preferably C2-C6 ester solvents, preferably C3-C5 ester solvents, and more preferably any one of ethyl acetate, methyl acetate, methyl formate, and ethyl formate; and the alkane solvent is selected from C1-C10 straight-chain alkane solvents, preferably C2-C8 straight-chain alkane solvents, preferably C4-C7 straight-chain alkane solvents, and preferably n-heptane.

[0064] The number of crystallization is 2 or more. If the number of crystallization is 1, the content of C 14 , C 15 , C 17 , C 18 The content of the homologous impurities in intermediate 1 can still be too high. After the above method provided in the embodiment of the present application is used, the content of the homologous impurities in intermediate 1 is less than 0.2%.

[0065] S2, forming intermediate 2;

[0066] The structural formula of intermediate 2 is as follows:

[0067] Intermediate 1 is reacted with 1-OtBu-L-Glu under alkaline conditions. Specifically, DIEA is pre-cooled at 0°C;

[0068] The solvent is mixed with the intermediate 1 at a temperature of -5 to 0°C, and then mixed with 1-OtBu-L-Glu for reaction. Then, the temperature is lowered to -10 to -5°C, the temperature is maintained, and the pre-cooled DIEA is added. Then, the reaction is carried out at -10 to -5°C for 1-1.5 h, and then at -5 to 0°C for 12-14 h. Then, the temperature is naturally increased to room temperature for complete reaction.

[0069] The pre-cooling of DIEA can control the progress of the reaction and reduce the formation of impurities when the DIEA is added. At the same time, the reaction is carried out in stages in the embodiment of the present application, which can further reduce the formation of impurities. If the material is added or the reaction is carried out directly at room temperature, the content of impurities will increase and the purity of the product will decrease.

[0070] The solvent is selected from a mixed solvent of an alcohol solvent and a halogenated alkane solvent, the halogenated alkane solvent is selected from a polyhalogenated C1-C10 alkane solvent, preferably a polyhalogenated C1-C5 alkane solvent, more preferably a polyhalogenated C1-C3 alkane solvent, and most preferably dichloromethane; and the alcohol solvent is a C1-C10 monohydric alcohol, preferably a C1-C5 monohydric alcohol, and more preferably a C1-C3 monohydric alcohol.

[0071] The molar ratio of the intermediate 1 to the 1-OtBu-L-Glu is 1:0.98-1:1.05, and the mass ratio of the intermediate 1 to the solvent is 1:7-1:10.

[0072] After the reaction is completed, the pH is adjusted to 2.8-3.4 by using hydrochloric acid, and the organic phase is collected by standing and separating, and then the collected organic phase is washed by using dilute hydrochloric acid and water; a water-removing drying agent is added for drying and water removal, and then the filtrate is collected by filtration, and then the filtrate is concentrated under reduced pressure to form a concentrate;

[0073] The concentrate is mixed and dissolved with a nitrile solvent to form a clear solution, and then the solution is naturally cooled to 10-15℃, and then the crystal is precipitated by incubation, and then the crystal is collected by filtration to form a filter cake;

[0074] The filter cake is mixed with a mixed solvent of an ester solvent and an alkane solvent for recrystallization once, and then the crystal is dried. The selection and ratio of the ester solvent and the alkane solvent are consistent with the selection of the recrystallization of the intermediate 1.

[0075] In the process of forming the intermediate 2, only one recrystallization is needed, and no repeated recrystallization is needed. The repeated recrystallization cannot further improve the purity and reduce the impurity content, but can increase the solvent consumption, and is easy to cause the residual organic solvent and environmental pollution.

[0076] S3, forming an intermediate 3;

[0077] The intermediate 3 has the following structural formula:

[0078] The intermediate 2 is activated to an -Osu ester to obtain palmitic acid-L-Glu(1-OtBu)-Osu (intermediate 3), and the C18 homologous impurity is removed to 0.1% or less by crystallization. Specifically,

[0079] The intermediate 2, a solvent and the HOSU are mixed, and then the EDCI is added in batches at 5℃ or below, and then the reaction is incubated for 1.5-2.5 hours, and then the reaction is completed by natural warming to room temperature. After the reaction is completed, the post-treatment is performed. In the embodiment of the present application, the reaction is first performed at low temperature, and then the reaction is performed at room temperature. The proceeding of the normal reaction is promoted, the proceeding of the side reaction is inhibited, and the formation of the homologous impurity is reduced.

[0080] The molar ratio of the intermediate 2 to the HOSU is 1:1.1-1:1.2, and the molar ratio of the intermediate 2 to the EDCI is 1:1.1-1.3, for example, 1:1.25.

[0081] The post-treatment includes: washing the reaction mixture with water and brine, collecting the organic phase, then adding a water-removing drying agent to remove water, filtering to collect the filtrate, and then concentrating the filtrate under reduced pressure to form a concentrate; stirring the concentrate with an alcohol solvent at 30-35℃ for 1-1.5 hours, then cooling to 0-5℃ for 1-1.5 hours, filtering, washing with an alcohol solvent, and then drying. The present embodiment does not perform recrystallization or crystallization, which would cause the Osu to fall off.

[0082] S4, forming a side chain of liraglutide;

[0083] The intermediate 3 is reacted with Fmoc-Lys.HCl, specifically, mixing a halogenated alkane solvent and the Fmoc-Lys, then cooling to 0-10℃, adding DIEA, the intermediate 3, and an alcohol solvent, and reacting at 0-10℃ for 1.5-2.5 hours, and then naturally warming to room temperature for complete reaction.

[0084] The present embodiment adopts a staged reaction to promote the progress of the positive reaction, reduce the progress of the side reaction, and in turn reduce the content of homologous impurities.

[0085] The halogenated alkane solvent is selected from a polyhalogenated C1-C10 alkane solvent, preferably a polyhalogenated C1-C5 alkane solvent, and more preferably a polyhalogenated C1-C3 alkane solvent, and most preferably dichloromethane; and the alcohol solvent is a C1-C10 monohydric alcohol, preferably a C1-C5 monohydric alcohol, and more preferably a C1-C3 monohydric alcohol. The use of the above solvents facilitates subsequent post-treatment.

[0086] The mass ratio of the intermediate 3 to the halogenated alkane solvent is 1:7-1:10, the molar ratio of the intermediate 3 to the Fmoc-Lys is 1:1.1-1.2, and the molar ratio of the intermediate 3 to the DIEA is 1:2.5-3, for example, 1:2.85.

[0087] The post-treatment includes: adjusting the pH of the reaction system, collecting the organic phase, washing, filtering, concentrating, purifying with a nitrile solvent, and drying.

[0088] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0089] Embodiment 1

[0090] The embodiment of the present application provides a preparation method of a liraglutide side chain, comprising:

[0091] S1, forming an intermediate 1;

[0092] A reaction kettle is added with 550 kg of dichloromethane, 50 kg of palmitic acid and 23.6 kg of HOSU, and cooling is started, and when the temperature is 5°C, 44.5 kg of EDCI is added in four batches, the temperature is controlled to be 5°C during the adding process, and after the adding process is completed, the temperature is kept for 1 h, and then the reaction is stirred at 15°C for 6 h, and TLC is used to determine that the reaction is completed.

[0093] 100 kg of water is added to the reaction solution, and the mixture is stirred for 20 min, and then the mixture is left to stand and separated, and the organic phase is collected; 15 kg of anhydrous magnesium sulfate is added to the clarified liquid, and the mixture is dried for more than 30 min, and then the mixture is filtered, and the filtrate is collected. The filtrate is concentrated under reduced pressure at a temperature of 35°C until a large amount of solid is precipitated.

[0094] 225 kg of acetonitrile is added to the concentrate, and the mixture is stirred at room temperature for 30 min, and then the mixture is cooled to 0°C and stirred for 1 h, and then the mixture is filtered, and the filter cake is washed with acetonitrile, and the filter cake is collected. The collected filter cake is dried at 25°C under vacuum for 8-9 h to obtain a crude product.

[0095] A mixed solution of ethyl acetate: n-heptane = 1:5 is prepared in advance, and the amount of the mixed solution is 650 kg (10 times the weight of the crude product to be crystallized). 65 kg of the crude product is added to the reaction kettle, and the mixed solution prepared in advance is added, and the external temperature is controlled to be 65°C, and the mixture is stirred to dissolve the mixture. After the mixture is dissolved, the temperature is kept at 43°C to crystallize for 2 h. The temperature in the reaction kettle is kept at 25°C to crystallize for more than 3 h. The mixture is filtered, and the filter cake is collected.

[0096] A mixed solution of ethyl acetate: n-heptane = 1:5 is prepared, and the amount of the mixed solution is 650 kg (10 times the weight of the crude product to be crystallized). The filter cake is put into the reaction kettle, and 650 kg of the mixed solution is added, and the external temperature is controlled to be 65°C, and the mixture is stirred to dissolve the mixture. After the mixture is dissolved, the temperature is kept at 43-48°C to crystallize for 2 h, and the temperature in the reaction kettle is kept at 20-25°C to crystallize for more than 3 h. The mixture is filtered, and the filter cake is collected.

[0097] The collected filter cake is naturally ventilated and dried for more than 4 h, and then dried at 30°C for more than 12 h to form the intermediate 1, and the amount of the intermediate 1 is 56.8 kg.

[0098] The liquid chromatogram of the intermediate 1 is shown in Figure 1 According to Figure 1 , the purity of the intermediate 1 is 99.68%, and the content of the homologous impurities is 0.15%. The mass yield of the intermediate 1 formed in the whole S1 is 113.6%, and the molar yield is 82.4%.

[0099] S2, forming an intermediate 2;

[0100] Pre-cool DIEA at 0°C.

[0101] Into the reactor, add 315 kg of dichloromethane and 67.39 kg of methanol, and start cooling. When the temperature is between 0°C, add 45 kg of intermediate 1 under stirring. After stirring for 5 min, add 27.27 kg of 1-OtBu-L-Glu, and continue cooling to control the temperature between -5°C. Drop 19.77 kg of pre-cooled DIEA while controlling the temperature between -5°C. After the drop is completed, control the internal temperature at -5°C for 1 h, and then maintain the temperature in the reactor at 0°C until the raw material is substantially reacted (about 12 h), and then react until completion.

[0102] After the reaction is completed, adjust the pH of the reaction solution to 3 with 2N hydrochloric acid (the amount of concentrated hydrochloric acid used is 17.58 kg) under stirring, and separate the liquid. Collect the organic layer. Wash the organic layer with 2% dilute hydrochloric acid twice, and with water three times, with a single amount of 87.18 kg each time, and collect the organic layer.

[0103] Add 17.58 kg of anhydrous magnesium sulfate to the organic phase, and dry for more than 30 min. Filter to collect the filtrate, and concentrate the filtrate under reduced pressure at a temperature of 35°C until a large amount of solid is precipitated.

[0104] Add 225 kg of acetonitrile to the concentrate, heat to 60°C to dissolve, and then naturally cool to precipitate most of the solid. Further cool to 10°C, and maintain the temperature for 2 h to crystallize. Filter, and collect the filter cake.

[0105] Add 80.86 kg of ethyl acetate to the filter cake, and then add 40.5 kg of n-heptane. Heat to 55°C to dissolve, and then naturally cool to room temperature. Further cool to about 5°C, and maintain the temperature in the reactor at about 5°C to crystallize for 1.5 h. Filter, and rinse the filter cake with n-heptane once. Dry the filter cake at 50°C for 12 h to obtain intermediate 2.

[0106] The high liquid chromatogram of intermediate 2 is shown in Figure 2 , and according to Figure 2 , the purity is 99.40%, and the homologous impurity is 0.16%. The mass yield of intermediate 2 is 48.5 kg, and the molar yield is 86.3%.

[0107] S3, form intermediate 3;

[0108] Into the reactor, add 315 kg of dichloromethane, and then add 45 kg of intermediate 2 and 13.5 kg of HOSU. Stir to dissolve while controlling the temperature below 5°C. Then add 24.3 kg of EDCI in batches while controlling the temperature below 5°C. After the addition is completed, maintain the temperature for 2 h, and then restore the temperature to room temperature to stir for more than 3 h.

[0109] After the reaction is completed, the reaction solution is washed with water and 10% salt water respectively for 2 times, each time with a single amount of 45 kg, and the organic phase is collected.

[0110] Anhydrous magnesium sulfate 9 kg is added to the organic phase, and dried for more than 2 h, filtered, and the filtrate is collected. The filtrate is concentrated under reduced pressure at a temperature of 35-40°C until a large amount of solid is precipitated.

[0111] Isopropyl alcohol is added to the concentrate, stirred at 30-35°C for 1 h, and then cooled to 0-5°C for 1 h. Filtration is performed, the filter cake is washed with isopropyl alcohol, and the filter cake is collected.

[0112] The filter cake is dried at 30-35°C for more than 24 h, and 46.54 kg of intermediate 3 is obtained. The HPLC chart of intermediate 3 is shown in Figure 3 , and according to Figure 3 , the purity is 99.38%, and the homologous impurity is 0.10%. The mass yield of intermediate 3 is 103.3%, and the molar yield is 84.7%.

[0113] S4, forming a liraglutide side chain;

[0114] Into the reaction kettle, dichloromethane 314.78 kg, Fmoc-Lys.HCl 37.33 kg, and anhydrous ethanol 87.96 kg are added, cooled to 0°C, 12.42 kg of DIEA is added, then 45 kg of intermediate 3 is added, followed by dropwise addition of 18.46 kg of DIEA, and finally anhydrous ethanol 87.96 kg is added. After completion of addition, the reaction is maintained at 0°C for 2 h, and then naturally warmed to room temperature for 1 h. TLC point plate until the reaction is complete.

[0115] The reaction solution is adjusted to pH 3 with 2N hydrochloric acid (about 20.39 kg of hydrochloric acid) while stirring, and then allowed to stand for separation. The organic layer is collected. The organic layer is washed with 2% dilute hydrochloric acid and pure water respectively for 2 times, each time with a single amount of 67.61 kg. The organic layer is collected. (Each time, 45.08 kg of dichloromethane and 18.54 kg of anhydrous ethanol are added.)

[0116] Anhydrous sodium sulfate 26.83 kg is added to the organic phase, and dried for more than 30 min. 10.73 kg of silica gel is added to the funnel, and then filtered. The filter cake is washed with 107.33 kg of dichloromethane, and the filtrate is collected. The filtrate is concentrated under reduced pressure at a temperature of 35°C until a large amount of solid is precipitated.

[0117] 310.17 kg of acetonitrile is added to the concentrate, dissolved at 80°C, and then cooled to most of the solid precipitates. The temperature is then lowered to 15°C, and stirred for 1 h. Centrifugation is performed, and the filter cake is collected. The filter cake is slurried with 224.31 kg of acetonitrile at an internal temperature of 50°C for 1 h, and then cooled to 10-15°C for crystallization for 1 h. Filtration is performed, and the filter cake is collected.

[0118] The filter cake is baked at 50 DEG C for 12 hours, and 59.01 kg of product is obtained. The nuclear magnetic spectrum is shown in Figure 4 , and the high liquid spectrum is shown in Figure 5 .

[0119] According to Figure 4 , the required liraglutide side chain is synthesized in the embodiment, the purity is 99.83%, the homologous impurity is less than or equal to 0.05%, and the molar yield is 89.1%.

[0120] Embodiment 2-Embodiment 3

[0121] Embodiments 2-3 are prepared according to the preparation method provided in Embodiment 1, and the difference is that some operation conditions are different, and the specific conditions are as follows:

[0122] In S1 of Embodiment 2, the starting temperature is 0 DEG C, and the temperature is kept at 0 DEG C for 1.5 hours, and after the temperature is raised, the temperature is 25 DEG C for 8 hours.

[0123] In the post-treatment process after the reaction is completed, the concentration temperature is 40 DEG C; after acetonitrile is added, the temperature is lowered to 5 DEG C, and the drying temperature is 30 DEG C; the ethyl acetate: n-heptane = 1:10; the crystallization temperature is 48 DEG C for 2 hours, and then 20 DEG C for 4 hours;

[0124] In S2, the intermediate 1 is added at-5 DEG C, the temperature is lowered to-10 DEG C after the 1-OtBu-L-Glu is added, the temperature of the process of adding DIEA is-10 DEG C, and after the adding is completed, the temperature is controlled at-10 DEG C, and then the temperature is raised to-5 DEG C for reaction.

[0125] The molar ratio of the intermediate 1 to the 1-OtBu-L-Glu is 1:0.98; the mass ratio of the intermediate 1 to the solvent is 1:10. The pH is adjusted to 2.8 by hydrochloric acid.

[0126] In S3, the molar ratio of the intermediate 2 to the HOSU is 1:1.1, and the molar ratio of the intermediate 2 to the EDCI is 1:1.25.

[0127] In S4, the mass ratio of the intermediate 3 to the halogenated alkane solvent is 1:9; the molar ratio of the intermediate 3 to the Fmoc-Lys is 1:1.15; and the molar ratio of the intermediate 3 to the DIEA is 1:2.85.

[0128] The remaining operations and conditions are consistent with Embodiment 1.

[0129] In S1 of Embodiment 3, the starting temperature is 2 DEG C, and the temperature is kept at 2 DEG C for 1.5 hours, and after the temperature is raised, the temperature is 20 DEG C for 7 hours.

[0130] The concentration temperature during the post-treatment after the reaction was 37℃; the temperature was decreased to 3℃ after adding acetonitrile, and the drying temperature was 28℃; ethyl acetate: n-heptane = 1:7; the crystallization temperature was 45℃ for 2.5 hours, and then 22℃ for 3 hours;

[0131] In S2, intermediate 1 was added at -2℃, the temperature was decreased to -8℃ after adding 1-OtBu-L-Glu, and the temperature during the dropwise addition of DIEA was -8℃; the temperature was controlled at -10℃ after the dropwise addition was completed, and then the temperature was increased to -2℃ for reaction.

[0132] The molar ratio of intermediate 1 to 1-OtBu-L-Glu was 1:1.05; and the mass ratio of intermediate 1 to the solvent was 1:8. The pH was adjusted to 3.4 by hydrochloric acid.

[0133] In S3, the molar ratio of intermediate 2 to HOSU was 1:1.2, and the molar ratio of intermediate 2 to EDCI was 1:1.25.

[0134] In S4, the mass ratio of intermediate 3 to the halogenated alkane solvent was 1:10; the molar ratio of intermediate 3 to Fmoc-Lys was 1:1.2; and the molar ratio of intermediate 3 to DIEA was 1:2.85.

[0135] The remaining operations and conditions were consistent with Example 1.

[0136] Process optimization

[0137] (1) Selection of crystallization solvent in S1

[0138] The crystallization solvents, dissolution temperatures, and solid precipitation temperatures were as shown in Table 1, and all were used for crystallization of the crude product provided in Example 1.

[0139] Table 1 Crystallization conditions

[0140]

[0141]

[0142] The purity and impurity content of the product after crystallization were as shown in Table 2.

[0143] Table 2 Crystallization results

[0144]

[0145] According to Table 1 and Table 2, using single solvent acetone, dichloromethane, n-hexane and methyl tert-butyl ether cannot be precipitated to obtain solid, and then crystallization cannot be realized. While using single solvent n-heptane and acetonitrile, the content of homologous impurities is high, and the product purity is low. The intermediate 1 obtained by using ethyl acetate is too little, and the yield is low. In summary, the mixed solvent of ester solvent and alkane solvent is used as the crystallization solvent.

[0146] (2) Recrystallization of intermediate 2;

[0147] The intermediate 2 provided by Example 1 is recrystallized, and the recrystallization solvent is shown in Table 3.

[0148] Table 3 recrystallization and results

[0149]

[0150] It can be seen that the recrystallization of intermediate 2 has no effect on removing homologous impurities, so the intermediate is not recrystallized.

[0151] (3) Crystallization of intermediate 3;

[0152] The intermediate 3 provided by Example 1 is recrystallized, and the crystallization conditions and results are shown in Table 4 and Table 5.

[0153] Table 4 crystallization conditions

[0154]

[0155] Table 5 -Osu results after crystallization

[0156]

[0157] According to Table 4 and Table 5, the crystallization of intermediate 3 has a certain effect on removing impurities, but the -OSu protection is removed during crystallization, that is, the product is decomposed, so it is not suitable.

[0158] The above (2) and (3) recrystallization or crystallization process is consistent with the operation of crystallizing intermediate 1 in Example 1, which will not be described in detail here.

[0159] (4) Synthesis parameters of intermediate 2 in S2

[0160] The specific parameters are shown in Table 6.

[0161] Table 6 synthesis parameters

[0162]

[0163]

[0164] From table 6, the impurity conversion rate can be reduced from 106% to 50% after optimization, the optimal conditions are that the mass of dichloromethane is 7-10 times of the mass of intermediate 1, the molar amount of 1-OtBu-L-Glu is 0.98-1.05 eq of the molar amount of intermediate 1, and the reaction temperature is-10-0℃.

[0165] (5) Synthesis parameters of intermediate 3 in S3

[0166] The specific parameters are shown in table 7.

[0167] Table 7 Synthesis parameters

[0168]

[0169] From table 7, it can be seen that changing the solvent amount, feeding ratio and reaction temperature has no effect on reducing the conversion rate.

[0170] (6) Synthesis parameters of liraglutide side chain in S4

[0171] The specific parameters are shown in table 8.

[0172] Table 8 Synthesis parameters

[0173]

[0174]

[0175] From table 8, the impurity conversion rate can be reduced from 102% to 62% by using the conditions of the embodiment of the application.

[0176] (7) Solvent selection for synthesis of liraglutide side chain in S4

[0177] The specific selection is shown in table 9.

[0178] Table 9 Solvent

[0179]

[0180] It is found in the experiment that when a single solvent is used in the post-treatment process, the product is not easy to be discharged, but the use of alcohol solvents such as methanol and anhydrous ethanol can solve this problem, and the purity of the obtained product has no obvious influence.

[0181] In summary, the embodiment of the present application uses palmitic acid (containing C18 stearic acid impurities 0.5%, other C14, C15, C17 impurities 0.1-0.3% not the same) as raw material, through the process control of intermediate 1, finished product synthesis step, and the crystallization purification of intermediate 1, finally control C14, C15, C17, C18 homologous impurities in the side chain of liraglutide ≤0.1%, obtain high purity product; the operation is simple, the process condition is mild, the raw material is easy to obtain; the operation safety is high, suitable for large-scale production of liraglutide side chain.

[0182] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A process for the preparation of a side chain of liraglutide, characterized in that, The method comprises the following steps: palmitic acid and HOSU are reacted to form a reaction crude product, the reaction crude product is crystallized to form intermediate 1, The solvent selected for crystallization is a mixture of an ester solvent and an alkane solvent, the mass ratio of the ester solvent to the alkane solvent is 1:5-1:10, the number of crystallization is 2 or more, and the intermediate 1 has the structural formula ; reacting the intermediate 1 with 1-OtBu-L-Glu to form intermediate 2, wherein intermediate 2 has the following structural formula: ; the molar ratio of the intermediate 1 to the 1-OtBu-L-Glu is 1:0.98-1:1.05; the mass ratio of the intermediate 1 to the solvent for forming the intermediate 2 is 1:7-1:10; the reaction temperature is -10 to 0°C; the intermediate 2 is mixed with HOSU and EDCI to form intermediate 3, and after the reaction is completed, the following post-treatment is performed; the reaction mixture is washed with water and brine, and the organic phase is collected, then water is removed by adding a water-removing drying agent, and after drying, the filtrate is collected by filtration, and then the filtrate is concentrated under reduced pressure to form a concentrate; the concentrate and an alcohol solvent are stirred at 30-35 DEG C for 1-1.5 hours, then the temperature is lowered to 0-5 DEG C and stirred for 1-1.5 hours, then filtered, washed with an alcohol solvent, and then dried; and the post-treatment does not include recrystallization or crystallization; In the formula, the structure of the intermediate 3 is as follows: ; halogenated alkane solvent and Fmoc-Lys are mixed, then the temperature is lowered to 0-10 DEG C, DIEA, the intermediate 3 and an alcohol solvent are added, and the reaction is performed at 0-10 DEG C for 1.5-2.5 hours, and then the temperature is naturally increased to 20-30 DEG C until the reaction is completed. The intermediate 3 and the halogenated alkane solvent are in a mass ratio of 1:7-1:10; the intermediate 3 and the Fmoc-Lys are in a molar ratio of 1:1.1-1.2; and the intermediate 3 and the DIEA are in a molar ratio of 1:2.5-3.

2. The production method according to claim 1, characterized by, The ester solvent is selected from C2-C10 ester solvents; and the alkane solvent is selected from C1-C10 straight-chain alkane solvents.

3. The preparation method according to claim 1, characterized in that, The ester solvent is a C2-C6 ester solvent; and the alkane solvent is a C2-C8 straight-chain alkane solvent.

4. The method of claim 1, wherein, The ester solvent is a C3-C5 ester solvent; and the alkane solvent is a C4-C7 straight-chain alkane solvent.

5. The preparation method according to claim 1, characterized in that, The ester solvent is any one of ethyl acetate, methyl acetate, methyl formate and ethyl formate. The alkane solvent is n-heptane.

6. The method of claim 1, wherein, The step of forming the reaction crude product comprises mixing the palmitic acid, the HOSU, EDCI and a solvent to react.

7. The preparation method according to claim 1, characterized in that, The step of forming the reaction crude product comprises mixing the palmitic acid, the solvent for forming the reaction crude product and the HOSU, then lowering the temperature to below 5 DEG C, then maintaining the temperature and adding the EDCI in batches, then maintaining the temperature at 0-5 DEG C for 1-1.5 hours, then naturally increasing the temperature to 15-25 DEG C and reacting for 6-8 hours.

8. The production method according to claim 6 or 7, characterized by, The palmitic acid, the HOSU and the EDCI are in a mass ratio of 1:0.4-0.6:0.7-0.

9.

9. The method of claim 1, wherein, After the reaction crude product is formed, the reaction crude product is subjected to primary treatment before crystallization, wherein the primary treatment comprises extracting to collect the organic phase, concentrating, purifying with a nitrile solvent and drying.

10. The method of claim 9, wherein, The primary treatment comprises mixing the reaction system after the reaction is completed with water, separating the phases, collecting the organic phase, adding a water-removing drying agent, collecting the filtrate by filtration, and then concentrating the filtrate to form a concentrate; The concentrate is mixed with a nitrile solvent, then the temperature is lowered and stirred, the filter cake is washed with the nitrile solvent after filtration, and then dried.

11. The method of claim 1, wherein, The step of forming the intermediate 2 of the side chain of liraglutide comprises pre-cooling the DIEA. The reaction is carried out by mixing the solvent with the intermediate 1 at a temperature of -5 to 0°C, then mixing with 1-OtBu-L-Glu, then cooling to -10 to -5°C, maintaining the temperature and adding pre-cooled DIEA, then reacting at -10 to -5°C for 1-1.5 h, then reacting at -5 to 0°C for 12-14 h, then naturally warming to 20-30°C until the reaction is complete.

12. The method of claim 11, wherein, The step of forming the intermediate 2 of the liraglutide side chain further comprises: after the reaction is completed, post-treatment is carried out, wherein the post-treatment comprises adjusting the pH, collecting the organic phase, washing, filtering, concentrating, nitrile solvent purification, recrystallization and drying.

13. The method of claim 12, wherein, The post-treatment comprises: After the reaction is completed, the pH of the reaction system is adjusted to 2.8-3.4, and the organic phase is collected by standing and separating. The organic layer is collected by washing with dilute hydrochloric acid and water; after adding a water-removing drying agent, the filtrate is collected by filtering, and then the filtrate is concentrated to form a concentrate; The concentrate is mixed and dissolved with a nitrile solvent by heating, and then the filter cake is collected by filtering after cooling and crystallization, The filter cake is recrystallized once by mixing with a mixed solvent of an ester solvent and an alkane solvent, and the crystals are dried.

14. The method of claim 1, wherein, The step of forming the intermediate 3 comprises: mixing the intermediate 2, a solvent and HOSU below 5°C, then adding EDCI in batches, then incubating for 1.5-2.5 hours, naturally warming to 20-30°C until the reaction is complete, and then post-treatment after the reaction is completed.

15. The preparation method according to claim 14, characterized in that, The molar ratio of the intermediate 2 to the HOSU is 1:1.1-1:1.2, and the molar ratio of the intermediate 2 to the EDCI is 1:1.1-1.

3.

16. The method of claim 1, wherein, The halogenated alkane solvent is selected from a polyhalogenated C1-C10 alkane solvent, and the alcohol solvent is a C1-C10 monohydric alcohol.

17. The method of claim 1, wherein, The halogenated alkane solvent is selected from a polyhalogenated C1-C5 alkane solvent, and the alcohol solvent is a C1-C5 monohydric alcohol.

18. The method of claim 1, wherein, The halogenated alkane solvent is selected from a polyhalogenated C1-C3 alkane solvent, and the alcohol solvent is a C1-C3 monohydric alcohol.

19. The method of claim 1, wherein, The halogenated alkane solvent is dichloromethane.

20. The method of claim 1, wherein, After the reaction of the intermediate 3 is completed, post-treatment is carried out, and the post-treatment comprises: adjusting the pH of the reaction system, collecting the organic phase, washing, filtering, concentrating, nitrile solvent purification and drying.

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