Preparation method of semaglutide side chain

Abstract

The invention relates to the technical field of chemical synthesis, and particularly provides a preparation method of a semaglutide side chain, which comprises the following steps: step S1, carrying out reaction on octadecanedioic acid mono-tert-butyl ester and trifluoroacetic acid pentafluorophenyl ester to generate a first intermediate; s2, enabling the first intermediate to react with 1-tert-butyl L-glutamic acid to generate a second intermediate; s3, enabling the second intermediate to react with pentafluorophenyl trifluoroacetate to generate a third intermediate; and S4, carrying out an amidation reaction on the third intermediate and 17-amino-10-oxo-3, 6, 12, 15-tetraoxa-9-aza-heptadecanoic acid, so as to generate a semaglutide side chain, and carrying out an amidation reaction on the third intermediate and 17-amino-10-oxo-3, 6, 12, 15-tetraoxa-9-aza-heptadecanoic acid. According to the preparation method disclosed by the embodiment of the invention, the pentafluorophenyl trifluoroacetate is used as the condensation reagent for two times to activate the carboxylic acid, and the amidation reaction is carried out for two times to construct the amido bond after each activation, so that compared with the traditional carbodiimide and urea positive ion type condensation reagent, the chiral racemization in the condensation process can be effectively avoided, and the yield of the product is improved. The condensation chiral racemization impurity can be lower than 0.3%.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound preparation, and in particular relates to a method for preparing a semaglutide side chain. Background Art

[0002] Semaglutide has a hypoglycemic effect and is a long-acting glucagon-like peptide 1 (GLP-1) analog, an agonist of the GLP-1 receptor, and has potential therapeutic efficacy for type 2 diabetes mellitus (T2DM). Semaglutide is a long-acting formulation developed based on the basic structure of liraglutide, and its effect in treating type 2 diabetes is better. Therefore, it is mainly used in the medical field and clinical research.

[0003] Among them, the semaglutide side chain is an important fragment for synthesizing semaglutide. At present, as a method for preparing the semaglutide side chain, some pharmaceutical companies use solid phase synthesis, Boc solid phase synthesis method, the C-terminus of Boc-AEEA-AEEA is first fixed on the resin, and the deprotected N-terminus of the Boc-AEEA-AEEA is connected to the carboxyl-activated Boc-1-tert-butyl L-glutamic acid through a condensation reaction, and the operation is repeated (condensation → washing → deprotection → washing → next round of condensation with octadecane dioic acid mono-tert-butyl ester), followed by peptide cutting, separation and purification, and finally the target semaglutide side chain is obtained. However, this synthesis method requires a large excess of 1-tert-butyl L-glutamic acid, octadecane dioic acid mono-tert-butyl ester, condensation agent and a very large amount of solvent, and the cost is relatively high.

[0004] The laboratory also uses various activation groups and condensation agents for splicing. For example, HOSU is used to activate mono-tert-butyl octadecane dioate using a carbodiimide condensation agent, and 1-tert-butyl L-glutamic acid is connected under the action of an organic base. The carboxyl end of the intermediate amino acid is activated again with HOSU, and then AEEA-AEEA is connected. There are also reports of using HOOBT to activate mono-tert-butyl octadecane dioate, then splicing 1-tert-butyl L-glutamic acid, and then activating and splicing AEEA-AEEA. However, although these synthetic schemes can synthesize the side chain of semaglutide, the intermediates are difficult to purify, the chiral racemic impurities are relatively large, and it is impossible to purify the high-purity intermediates by simple recrystallization or beating, resulting in the low purity of the synthesized semaglutide side chain. In order to better purify the intermediates, the loss is also large, and the final yield is very low. In addition, the reaction solution after connecting 1-tert-butyl L-glutamic acid and the reaction solution after connecting AEEA-AEEA are easily emulsified after washing, which is not suitable for large-scale production. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing a semaglutide side chain with high yield, which is simple to operate, easy to purify intermediates, and can effectively avoid chiral racemization during the condensation process.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The method for preparing the semaglutide side chain according to an embodiment of the present invention comprises the following steps:

[0008] Step S1, reacting octadecane dioic acid mono-tert-butyl ester with trifluoroacetic acid pentafluorophenyl ester to generate a first intermediate, wherein the first intermediate is 1-(tert-butyl) 18-(pentafluorophenyl) octadecane dioic acid ester;

[0009] Step S2, reacting the first intermediate with 1-tert-butyl L-glutamic acid to generate a second intermediate, wherein the second intermediate is (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanoylamido)-5-oxopentanoic acid;

[0010] Step S3, reacting the second intermediate with pentafluorophenyl trifluoroacetate to generate a third intermediate, wherein the third intermediate is 1-(tert-butyl)-5-(pentafluorophenyl)(18-(tert-butoxy)-18-oxooctadecanoyl)-L-glutamate;

[0011] Step S4, subjecting the third intermediate to an amidation reaction with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid to generate a semaglutide side chain.

[0012] In some embodiments of the present application, step S1 includes:

[0013] Octadecanedioic acid mono-tert-butyl ester is added to dichloromethane, and then pyridine is added, and then pentafluorophenyl trifluoroacetate is added dropwise to generate the first intermediate.

[0014] In some embodiments of the present application, in step S1, the molar ratio of octadecane dioic acid mono-tert-butyl ester: pyridine: pentafluorophenyl trifluoroacetate is 1: (1.2-1.5): (1-1.5), and the temperature of adding pentafluorophenyl trifluoroacetate is 20-25° C., and the reaction is kept warm for 4-8 hours.

[0015] In some embodiments of the present application, step S1 further includes:

[0016] After the reaction is completed, the temperature is controlled at 5-10°C, and trifluoroacetic acid aqueous solution is added dropwise to the reaction solution. After sufficient stirring, the liquid is separated, and the organic phase is washed with water three times, dried with sodium sulfate, filtered, and concentrated with a water pump at 40°C until solids are precipitated. Petroleum ether is added for pulping, filtered, and washed with petroleum ether by stirring, and dried to obtain the first intermediate.

[0017] In some embodiments of the present application, step S2 includes:

[0018] The first intermediate is added to dichloromethane, and then 1-tert-butyl L-glutamic acid is added, followed by dropwise addition of triethylamine to generate the second intermediate.

[0019] In some embodiments of the present application, the molar ratio of the first intermediate: triethylamine: 1-tert-butyl L-glutamic acid is 1: (1-1.4): (1-1.2), the temperature for dripping triethylamine is 5-10°C, and the reaction is kept warm for 4-8 hours after the addition.

[0020] In some embodiments of the present application, step S2 further includes:

[0021] The reaction is completed by monitoring the liquid phase, and the temperature is controlled at 5-10°C. A trifluoroacetic acid aqueous solution is dripped into the reaction solution, and the mixture is stirred thoroughly and then allowed to stand for separation. The aqueous phase is extracted with dichloromethane, and the combined organic phases are washed with water for 3 times. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated into an oily substance with a 40°C water pump. Petroleum ether is added to cool the mixture to 10°C to precipitate a solid, which is filtered, washed with petroleum ether by stirring, and dried to obtain the second intermediate.

[0022] In some embodiments of the present application, step S3 includes:

[0023] The second intermediate is added to dichloromethane, and then pyridine is added, and then pentafluorophenyl trifluoroacetate is added dropwise to generate the third intermediate, wherein the molar ratio of the second intermediate: pyridine: pentafluorophenyl trifluoroacetate is 1: (1.2-1.5): (1-1.5), the temperature for adding pentafluorophenyl trifluoroacetate is 20-25°C, and the reaction is kept warm for 4-8 hours.

[0024] In some embodiments of the present application, step S3 further includes:

[0025] After the reaction is completed, the temperature is controlled at 5-10°C, and trifluoroacetic acid aqueous solution is added dropwise to the reaction solution. After sufficient stirring, the liquid is separated, and the organic phase is washed with water three times, dried with sodium sulfate, filtered, concentrated with a water pump at 40°C, and petroleum ether is added until a small amount of liquid remains. Petroleum ether is added to slurry at 5°C, filtered, the wet product is recrystallized with n-heptane / ethyl acetate, filtered, washed with petroleum ether, and dried to obtain the third intermediate.

[0026] In some embodiments of the present application, step S4 includes:

[0027] The third intermediate is added to dichloromethane, and then 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid is added, and triethylamine is added dropwise to generate the semaglutide side chain, wherein the molar ratio of the third intermediate: triethylamine: 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid is 1:(1-1.4):(1-1.2), the temperature of adding triethylamine dropwise is 5-10°C, and the reaction is kept warm for 6-12 hours after the addition;

[0028] After the reaction is completed by liquid phase monitoring, the temperature is controlled at 5-10°C, an aqueous solution of trifluoroacetic acid is added dropwise to the reaction solution, the mixture is stirred thoroughly and allowed to stand for separation, the aqueous phase is extracted with dichloromethane, the combined organic phases are washed with water three times, the organic phase is dried over anhydrous sodium sulfate, the organic phase is filtered through silica gel, and eluted with DCM / CH3OH=10:1. The eluate is concentrated at 40°C to an oily substance, and the oil pump is dried at 60°C to obtain the semaglutide side chain.

[0029] The above technical solution of the present invention has at least one of the following beneficial effects:

[0030] According to the preparation method of an embodiment of the present invention, pentafluorophenyl trifluoroacetate is used as a condensation reagent to activate the carboxylic acid, and after each activation, the amide bond is constructed twice using 1-tert-butyl L-glutamic acid and AEEA-AEEA respectively. Compared with traditional carbodiimide and urea cation-type condensation reagents, chiral racemization in the condensation process can be effectively avoided, and the condensation chiral racemic impurities can be less than 0.3%.

[0031] Furthermore, the organic acids trifluoroacetic acid and pentafluorophenol generated in the reaction process of the condensation agent pentafluorophenyl trifluoroacetate respectively emulsify the reaction solution of breaking the 1-tert-butyl L-glutamic acid and the reaction solution of connecting AEEA-AEEA, and the effect is very ideal. The addition of trifluoroacetic acid aqueous solution to adjust the pH to below 3 can make the two phases clear. In addition, during the water washing process, the pentafluorophenol remaining in the organic phase is weakly acidic and can avoid emulsification.

[0032] In addition, the intermediates are easy to purify, no column is required, there is less waste, the three wastes are less, the cost is low, the loss of intermediates is small, and the operation is simple. It is a method for producing semaglutide side chains with industrial prospects. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0034] The following first describes in detail the method for preparing the semaglutide side chain according to an embodiment of the present invention.

[0035] The method for preparing the semaglutide side chain according to an embodiment of the present invention comprises the following steps:

[0036] Step S1, reacting octadecane dioic acid mono-tert-butyl ester with trifluoroacetic acid pentafluorophenyl ester to generate a first intermediate, wherein the first intermediate is 1-(tert-butyl) 18-(pentafluorophenyl) octadecane dioic acid ester;

[0037] Step S2, reacting the first intermediate with 1-tert-butyl L-glutamic acid to generate a second intermediate, wherein the second intermediate is (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanoylamido)-5-oxopentanoic acid;

[0038] Step S3, reacting the second intermediate with pentafluorophenyl trifluoroacetate to generate a third intermediate, wherein the third intermediate is 1-(tert-butyl)-5-(pentafluorophenyl)(18-(tert-butoxy)-18-oxooctadecanoyl)-L-glutamate;

[0039] Step S4, subjecting the third intermediate to an amidation reaction with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid to generate a semaglutide side chain.

[0040] According to the preparation method of an embodiment of the present invention, pentafluorophenyl trifluoroacetate is used as a condensation reagent to activate the carboxylic acid, and the amide bond is constructed twice in succession by 1-tert-butyl L-glutamic acid and AEEA-AEEA in sequence. Compared with traditional carbodiimide and urea cation-type condensation reagents, chiral racemization in the condensation process can be effectively avoided, and the condensation chiral racemic impurities can be less than 0.3%.

[0041] Below, we will explain each step in detail.

[0042] Step S1: reacting octadecane dioic acid mono-tert-butyl ester with pentafluorophenyl trifluoroacetate to generate a first intermediate, wherein the first intermediate is 1-(tert-butyl) 18-(pentafluorophenyl) octadecane dioic acid ester.

[0043] That is, according to the preparation method of the present invention, first, tert-butyl octadecane dioate is used as a starting material, and it is reacted with pentaphenyl trifluoroacetate, that is, an activation reaction of the carboxylic acid is carried out to generate the first intermediate 1-(tert-butyl) 18-(pentafluorophenyl) octadecane dioate (i.e. tBuO-Ste-PFP).

[0044] Specifically, the chemical reaction formula is shown in the following formula (1):

[0045]

[0046] In some embodiments of the present application, step S1 includes:

[0047] Octadecanedioic acid mono-tert-butyl ester is added to dichloromethane, and then pyridine is added, and then pentafluorophenyl trifluoroacetate is added dropwise to generate the first intermediate, tBuO-Ste-PFP.

[0048] That is to say, the above reaction is carried out under alkaline conditions. Pyridine is beneficial to the dehydrogenation of the terminal hydroxyl group of tert-butyl octadecanoate, thereby promoting its condensation reaction with pentafluorophenyl trifluoroacetate, that is, the activation reaction of carboxylic acid. In addition, the alkalinity of pyridine is weaker than that of other organic bases, and pentafluorophenyl trifluoroacetate is generally stable. The use of mild pyridine can prevent pentafluorophenyl trifluoroacetate from deteriorating during the reaction, and pyridine is easy to remove by acid washing.

[0049] In some embodiments of the present application, in step S1, the molar ratio of octadecane dioic acid mono-tert-butyl ester: pyridine: pentafluorophenyl trifluoroacetate is 1: (1.2-1.5): (1-1.5), preferably 1: 1.4: 1.2. Excess pyridine as an acid binding agent can promote the reaction and improve the yield. Similarly, excess pentafluorophenyl trifluoroacetate is conducive to improving the yield.

[0050] In addition, preferably, the amount of dichloromethane (i.e. DCM) used is eight times the volume of octadecane dioic acid mono-tert-butyl ester. That is to say, in the present application, a large amount of solvent is not required, and dichloromethane can be further used for subsequent production after simple post-treatment, which reduces the three wastes and is conducive to reducing costs.

[0051] In addition, the temperature of adding pentafluorophenyl trifluoroacetate is 20-25°C, and the temperature is kept for 4-8 hours after the addition. In other words, the reaction efficiency is high, the reaction conditions are mild and easy to operate, and the production cost is low.

[0052] In some embodiments of the present application, step S1 further includes:

[0053] The reaction is monitored to be complete in the liquid phase (for example, when no or less than 0.1% of mono-tert-butyl octadecane dioate is detected), the temperature is controlled at 5-10° C., an aqueous solution of trifluoroacetic acid (for example, a 5% by mass aqueous solution of trifluoroacetic acid prepared by 0.25 eq of trifluoroacetic acid) is added dropwise to the reaction solution, the mixture is stirred thoroughly and separated, the organic phase is washed with water for 3 times, dried with sodium sulfate, filtered, concentrated with a water pump at 40° C. until solid precipitates, and slurried with petroleum ether (specifically, petroleum ether can be added until a small amount of liquid remains, and slurried with petroleum ether at 20° C.), filtered, washed with petroleum ether by stirring, and dried to obtain a first intermediate with good crystalline purity.

[0054] That is to say, in the preparation method of the present application, the intermediate obtained in the first step, namely tBuO-Ste-PFP, has a certain solubility in petroleum ether. tBuO-Ste-PFP with good purity can be obtained by slurrying and filtering the petroleum ether, and the loss of the intermediate is small, which can effectively avoid chiral racemization in the condensation process.

[0055] Step S2, reacting the first intermediate with 1-tert-butyl L-glutamic acid to generate a second intermediate, wherein the second intermediate is (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanoylamide)-5-oxopentanoic acid.

[0056] That is, after obtaining the first intermediate tBuO-Ste-PFP, it is further subjected to an amidation reaction with 1-tert-butyl L-glutamic acid to generate the second intermediate (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanoylamide)-5-oxopentanoic acid (i.e., tBuO-Ste-Glu-OtBu).

[0057] Specifically, the reaction formula is shown in the following formula (2):

[0058]

[0059] The first intermediate undergoes an amidation reaction with 1-tert-butyl L-glutamic acid to generate pentafluorophenol as a byproduct, and pentafluorophenol can effectively break the emulsification effect of the reaction solution connected with 1-tert-butyl L-glutamic acid.

[0060] In some embodiments of the present invention, step S2 specifically includes:

[0061] The first intermediate is added to dichloromethane, and then 1-tert-butyl L-glutamic acid is added, followed by dropwise addition of triethylamine to generate the second intermediate.

[0062] 1-tert-butyl L-glutamic acid has poor solubility in conventional solvents. The use of triethylamine can effectively break the internal salt balance of the amino acid, so that the carboxyl end of 1-tert-butyl L-glutamic acid reacts with triethylamine to form a triethylamine salt, thereby increasing the solubility and better participating in the reaction to form the second intermediate, namely tBuO-Ste-Glu-OtBu.

[0063] In some embodiments of the present application, the molar ratio of the first intermediate: triethylamine: 1-tert-butyl L-glutamic acid is 1: (1-1.4): (1-1.2), preferably 1: 1.2: 1.1. Excess triethylamine as an acid binding agent can promote the reaction and improve the yield. Similarly, excess 1-tert-butyl L-glutamic acid is conducive to improving the yield.

[0064] In addition, DCM can be set to five times the volume of the first intermediate, for example. Similarly, the amount of solvent used in this step is also controllable, and dichloromethane can be further used in subsequent production after simple post-treatment, which reduces the three wastes and helps reduce costs.

[0065] In some embodiments of the present invention, the temperature of adding triethylamine is 5-10° C., and the reaction is kept warm for about 5 hours.

[0066] That is to say, the reaction efficiency is high, the reaction conditions are mild and easy to operate, and the production cost is low.

[0067] In some embodiments of the present invention, the temperature for dripping triethylamine is 5-10°C, and the temperature is kept for 4-8 hours after the addition to carry out the reaction. The reaction conditions are simple and easy to control, and the cost is low.

[0068] In some embodiments of the present invention, step S2 may further include:

[0069] The liquid phase monitors the reaction to be completed (for example, the first intermediate is monitored to be no residue, or the residue is less than a predetermined amount, such as 1% or less), the temperature is controlled at 5-10°C, a trifluoroacetic acid aqueous solution (for example, 0.3eq trifluoroacetic acid prepared as a 5% mass fraction trifluoroacetic acid aqueous solution) is dripped into the reaction solution, the mixture is stirred thoroughly and then allowed to stand for separation, the aqueous phase is extracted with dichloromethane, the combined organic phases are washed with water 3 times, the organic phases are dried over anhydrous sodium sulfate, filtered, concentrated into an oily substance by a 40°C water pump, petroleum ether is added to cool to 10°C to precipitate a solid, filtered, stirred and washed with petroleum ether, and dried to obtain a second intermediate with good purity. The second intermediate is a crystalline white powder.

[0070] The organic acid trifluoroacetic acid and the byproduct pentafluorophenol can effectively break the emulsification effect of the reaction solution of 1-tert-butyl L-glutamic acid. The addition of trifluoroacetic acid aqueous solution to adjust the pH to below 3 can make the two phases clear. During the water washing process, the pentafluorophenol remaining in the organic phase is weakly acidic and can avoid emulsification.

[0071] Step S3, reacting the second intermediate with pentafluorophenyl trifluoroacetate to generate a third intermediate, wherein the third intermediate is 1-(tert-butyl)-5-(pentafluorophenyl)(18-(tert-butoxy)-18-oxooctadecanoyl)-L-glutamate.

[0072] That is, after obtaining the second intermediate tBuO-Ste-Glu-OtBu, the carboxylic acid activation reaction is further carried out by pentafluorophenyl trifluoroacetate to obtain the third intermediate 1-(tert-butyl)-5-(pentafluorophenyl)(18-(tert-butoxy)-18-oxooctadecanoyl)-L-glutamate (i.e., tBuO-Ste-Glu(PFP)-OtBu).

[0073] Specifically, the reaction formula is shown in the following formula (3):

[0074]

[0075] In addition, the reaction system and reaction conditions of the activation reaction of the carboxylic acid can refer to the above step S1. For example, the second intermediate can be added to a DCM solvent environment, pyridine is added, and then pentafluorophenyl trifluoroacetate is added dropwise.

[0076] In addition, the molar ratio of the second intermediate, pyridine and pentafluorophenyl trifluoroacetate can be set to 1:(1.2-1.5):(1-1.5), preferably 1:1.4:1.2. In addition, preferably, the amount of dichloromethane (ie, DCM) used is eight times the volume of octadecane dioic acid mono-tert-butyl ester.

[0077] Furthermore, the temperature of adding pentafluorophenyl trifluoroacetate dropwise is 20-25° C., and the temperature is kept for 4-8 hours after the addition. In other words, the reaction efficiency is high, the reaction conditions are mild and easy to operate, and the production cost is low.

[0078] In some embodiments of the present application, step S3 further includes:

[0079] The reaction is monitored to be complete in the liquid phase (for example, when no second intermediate is left or less than 0.1% is detected), the temperature is controlled at 5-10° C., an aqueous solution of trifluoroacetic acid (for example, a 5% by mass aqueous solution of trifluoroacetic acid prepared with 0.25 eq of trifluoroacetic acid) is added dropwise to the reaction solution, the mixture is stirred thoroughly and separated, the organic phase is washed with water for 3 times, dried with sodium sulfate, filtered, concentrated with a water pump at 40° C. until solid precipitates, and petroleum ether is added for pulping (specifically, petroleum ether can be added until a small amount of liquid remains, and petroleum ether is added for pulping at 20° C.), filtered, washed with petroleum ether by stirring, and dried to obtain a third intermediate with good crystalline purity.

[0080] That is to say, in the preparation method of the present application, the intermediate tBuO-Ste-Glu(PFP)-OtBu obtained in the third step has a certain solubility in petroleum ether. tBuO-Ste-Glu(PFP)-OtBu with good purity can be obtained by slurrying and filtering the petroleum ether, and the loss of the intermediate is small, which can effectively avoid chiral racemization in the condensation process.

[0081] Step S4, the third intermediate is subjected to an amidation reaction with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid (ie, AEEA-AEEA) to generate a semaglutide side chain (tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu).

[0082] That is to say, after two activations of the carboxylic acid and two amidation reactions to construct the amide bond, the target product, semaglutide side chain (tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu), is generated.

[0083] Specifically, the reaction formula is shown in the following formula (4):

[0084]

[0085] Similarly, the by-product pentafluorophenol in step S4 also plays a good role in demulsifying the reaction solution of AEEA-AEEA.

[0086] In some embodiments of the present application, step S4 includes:

[0087] The third intermediate tBuO-Ste-Glu(PFP)-OtBu is added to dichloromethane, and then AEEA-AEEA is added, and triethylamine is added dropwise to generate the semaglutide side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtB. That is, the amidation reaction is carried out in the presence of an organic base triethylamine.

[0088] In some embodiments of the present invention, the molar ratio of the third intermediate: triethylamine: AEEA-AEEA is 1:(1-1.4):(1-1.2), preferably 1:1.2:1.1.

[0089] In addition, triethylamine is added dropwise at a temperature of 5-10°C, and the reaction is maintained at this temperature for 6-12 hours.

[0090] In addition, DCM can be, for example, five times the volume of the third intermediate. There is not much solvent waste, and it is suitable for industrial-scale production.

[0091] In some embodiments of the present invention, step S4 further includes:

[0092] After the reaction is completed by liquid phase monitoring, the temperature is controlled at 5-10°C, and trifluoroacetic acid aqueous solution is added dropwise to the reaction solution. After sufficient stirring, the mixture is allowed to stand for separation. The aqueous phase is extracted with dichloromethane, and the combined organic phases are washed with water three times. The organic phase is dried over anhydrous sodium sulfate, and the organic phase is filtered on silica gel and eluted with DCM / CH3OH=10:1. The eluate is concentrated at 40°C to an oily substance, and the oil pump is dried at 60°C to obtain a semaglutide side chain with good purity.

[0093] The organic phase is filtered through silica gel, and the residual byproduct pentafluorophenol will be directly eluted by DCM, while the product will be adsorbed on the silica gel and cannot be taken off by DCM. In order to obtain the product, DCM / CH3OH=10:1 is used for elution, and the eluate is concentrated into an oil at 40°C and dried by an oil pump at 60°C to obtain a semaglutide side chain with good purity.

[0094] In addition, trifluoroacetic acid also has a good effect on preventing the reaction solution from emulsifying. Dropping trifluoroacetic acid aqueous solution to adjust the pH to below 3 can make the two phases clear. In addition, during the water washing process, the pentafluorophenol remaining in the organic phase is weakly acidic and can prevent emulsification.

[0095] From the above, it can be seen that according to the preparation method of the present application, the purification of intermediate products and final products is relatively simple, the chiral racemization in the condensation process is effectively avoided, the equivalent ratio of the main raw materials is appropriate, the dosage of the condensation agent and the solvent is normal, the operation of each step is simple, and the yield of each step is normal, which is suitable for industrial production.

[0096] In order to enable technical researchers in the field to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments.

[0097] Example 1

[0098] (1) At room temperature of about 20°C, add DCM (3.2L) and tert-butyl octadecane dioate (400g, 1.08mol, 1.0eq) to a 5L reaction bottle, and then add pyridine (119.5g, 1.51mol, 1.4eq). Cool in an ice bath to an internal temperature of 20°C, and add pentafluorophenyl trifluoroacetate (362.8g, 1.30mol, 1.2eq) dropwise at a temperature of 20-25°C. After the addition is complete, the system becomes a colorless transparent liquid. Keep the reaction warm for 3h to obtain the first intermediate.

[0099] The liquid phase monitors that the mono-tert-butyl octadecane dioate accounts for 0.04% of the intermediate 1, and the temperature is controlled at 5-10°C. A 5% mass fraction trifluoroacetic acid aqueous solution prepared by trifluoroacetic acid (30.8g, 0.27mol, 0.25eq) is added dropwise to the reaction solution, and the mixture is separated after being fully stirred for 15 minutes. The organic phase is washed 3 times with 0.5L water each time, dried with sodium sulfate, filtered, and concentrated to a large amount of solid precipitation by a water pump at 40°C. 0.8L petroleum ether is added to bring a small amount of liquid to the residue, 0.8L petroleum ether is added to pulp at 20°C, filtered, 0.3L petroleum ether is stirred and washed, and the water pump is dried at 40°C and then the oil pump is dried at 45°C to obtain 478.4g of the first intermediate of white flaky crystals, with a HPLC content of 99.7%, and a yield of 82.6% in this step. Because the first intermediate has a small polarity, has a certain solubility in petroleum ether, and has a good crystal form, it can be well pulped and impurized by petroleum ether.

[0100] (2) Take a 5L reaction bottle, add 2.4L DCM, then add intermediate 1-1 (478.4g, 0.89mol, 1.0eq), 1-tert-butyl L-glutamic acid (199.3g, 0.98mol, 1.1eq), cool to 5°C in an ice-salt bath, control the temperature at 5-10°C, and add TEA (86.0g, 0.85mol, 1.05eq) dropwise. The heat release is weak. After the addition is completed, keep the temperature at 5-10°C for 5h to obtain the second intermediate.

[0101] The first intermediate was monitored by liquid phase and the reaction was completed. The temperature was controlled at 5-10°C. A 5% mass fraction trifluoroacetic acid aqueous solution prepared by trifluoroacetic acid (30.5g, 0.27mol, 0.3eq) was added dropwise to the reaction solution. After being fully stirred for 15 minutes, the solution was allowed to stand for separation. The aqueous phase was extracted with 1LDCM. The organic phase was combined and washed with 0.5L water 3 times each time. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated with a 40°C water pump, concentrated to an oily substance without fraction, and 3L petroleum ether was added to slowly cool to 10°C to precipitate solids. The solids were filtered, washed with petroleum ether by stirring, dried with a water pump at 40°C, and then dried with an oil pump at 45°C to obtain 438.0g of a white powder second intermediate. The HPLC content was 97.6% (1.6% pentafluorophenol remained). The yield of this step was 88.4%. The second intermediate was a fine powder and had a large polarity. It could be used directly in the next step without further purification in this step.

[0102] (3) Take a 5L reaction bottle, add 3.5L DCM, add the second intermediate (438.0g, 0.79mol, 1.0eq) obtained in (2) above and pyridine (87.3g, 1.10mol, 1.4eq) at room temperature (23°C). Cool in an ice bath to 20°C, control the temperature at 20-25°C, and drop pentafluorophenyl trifluoroacetate (264.9g, 0.95mol, 1.2eq). After the dropwise addition, the system is a light yellow transparent liquid. Keep the temperature at 20-25°C for 5h to generate the third intermediate.

[0103] After the reaction of the second intermediate was monitored by the liquid phase, the temperature was controlled at 5-10°C in an ice-salt bath, and a 5% mass fraction trifluoroacetic acid aqueous solution prepared by trifluoroacetic acid (22.5 g, 0.20 mol, 0.25 eq) was added dropwise to the reaction solution. After sufficient stirring for 15 minutes, the liquid was separated, and the organic phase was washed three times with 0.5 L of water respectively. The organic phase was dried with sodium sulfate, filtered, and concentrated with a water pump at 40°C until a large amount of solid precipitated. 0.8 L of petroleum ether was added until a small amount of liquid remained, and 0.8 L of petroleum ether was added to cool to 5°C for pulping, filtered, and washed with 0.3 L of 5°C petroleum ether to obtain a white flaky crystal intermediate 2-1 crude product with a wet weight of 586.3 g.

[0104] The wet product was recrystallized with 0.8L (n-heptane: ethyl acetate = 10:1), cooled to 5°C, filtered, and washed with 0.3L 5°C petroleum ether by stirring. Drying at 45°C gave 464.8g of white flaky crystals of intermediate 2-1, HPLC 99.8%, and a yield of 81.7% in this step. The third intermediate has a smaller polarity than the first intermediate, has a certain solubility in petroleum ether and n-heptane, and has a good crystal form. Recrystallization with n-heptane / ethyl acetate can remove a small amount of impurities.

[0105] (4) Take a 5L reaction bottle, add 2.3L of DCM at room temperature 21°C, then add the third intermediate (464.8g, 0.64mol, 1.0eq) obtained in (3), AEEA-AEEA (218.4g, 0.71mol, 1.1eq), cool to 5°C in an ice-salt bath, and add TEA (78.2g, 0.77mol, 1.2eq) dropwise at 5-10°C. The heat release is weak. After the addition is completed, keep the temperature at 5-10°C for 7h to obtain the target product.

[0106] Liquid phase monitoring of the third intermediate reaction. Control the temperature at 5-10°C, add trifluoroacetic acid (22.0g, 0.19mol, 0.3eq) to the reaction solution, add 5% mass fraction trifluoroacetic acid aqueous solution, stir well for 15 minutes and then stand for separation, extract the aqueous phase with 1L DCM, wash the combined organic phases with 0.5L water 3 times, and dry the organic phase with anhydrous sodium sulfate. The organic phase is filtered on about 4cm silica gel in a 200mm diameter Buchner funnel, and the silica gel is soaked and washed three times with 1L DCM, and the residual by-product pentafluorophenol is directly eluted by DCM. Then soak and wash the silica gel five times with 1L DCM / CH3OH=10:1 mixed solvent. The eluent is concentrated into an oil at 40°C, and the oil pump is pulled dry at 60°C to obtain 418.4g of gel product, HPLC99.2%, chiral racemic impurities 0.16%, and the yield of this step is 76.8%.

[0107] The total yield of the two steps is 45.8%.

[0108] The reactants were subjected to nuclear magnetic resonance experiments to confirm the product structure, and the data are as follows:

[0109] 1H NMR (400MHz, CDCl3) δ = 7.28 (t, J = 5.3, 1H), 6.98 (t, J = 5.3, 1H), 6.67 (d, J = 7.8, 1H), 4.36 (td, J = 9.0 ,4.2,1H),4.08(s,2H),3.95(s,2H),3.67(dd,J=5.5,2.4,2H),3.60(dd,J=12.5,5.3,6H),3.55–3. 48(m,4H),3.48–3.30(m,4H),2.25(dt,J=7.6,6.5,2H),2.20–2.00(m,5H),1.87(dt,J=9.0,7.0,1H),1.51(dd,J=16.1,8.5,4H),1.38(d,J=7.1,18H),1.19(d,J=11.3,24H). The detection results were consistent with the structure of the side chain of the target product semaglutide.

[0110] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a semaglutide side chain, characterized in that: The steps include: Step S1, reacting octadecane dioic acid mono-tert-butyl ester with trifluoroacetic acid pentafluorophenyl ester to generate a first intermediate, wherein the first intermediate is 1-(tert-butyl) 18-(pentafluorophenyl) octadecane dioic acid ester; Step S2, reacting the first intermediate with 1-tert-butyl L-glutamic acid to generate a second intermediate, wherein the second intermediate is (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanoylamido)-5-oxopentanoic acid; Step S3, reacting the second intermediate with pentafluorophenyl trifluoroacetate to generate a third intermediate, wherein the third intermediate is 1-(tert-butyl)-5-(pentafluorophenyl)(18-(tert-butoxy)-18-oxooctadecanoyl)-L-glutamate; Step S4, subjecting the third intermediate to an amidation reaction with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid to generate a semaglutide side chain.

2. The preparation method according to claim 1, characterized in that: The step S1 comprises: Octadecanedioic acid mono-tert-butyl ester is added to dichloromethane, and then pyridine is added, and then pentafluorophenyl trifluoroacetate is added dropwise to generate the first intermediate.

3. The preparation method according to claim 2, characterized in that: In the step S1, the molar ratio of octadecane dioic acid mono-tert-butyl ester: pyridine: pentafluorophenyl trifluoroacetate is 1: (1.2-1.5): (1-1.5), pentafluorophenyl trifluoroacetate is added dropwise at a temperature of 20-25° C., and the reaction is kept warm for 4-8 hours.

4. The preparation method according to claim 2, characterized in that: The step S1 further comprises: After the reaction is completed, the temperature is controlled at 5-10°C, and trifluoroacetic acid aqueous solution is added dropwise to the reaction solution. After sufficient stirring, the liquid is separated, and the organic phase is washed with water three times, dried with sodium sulfate, filtered, and concentrated with a water pump at 40°C until solids are precipitated. Petroleum ether is added for pulping, filtered, and washed with petroleum ether by stirring, and dried to obtain the first intermediate.

5. The preparation method according to claim 1, characterized in that: The step S2 comprises: The first intermediate is added to dichloromethane, and then 1-tert-butyl L-glutamic acid is added, followed by dropwise addition of triethylamine to generate the second intermediate.

6. The preparation method according to claim 5, characterized in that: The molar ratio of the first intermediate: triethylamine: 1-tert-butyl L-glutamic acid is 1: (1-1.4): (1-1.2), the temperature for dripping triethylamine is 5-10° C., and the reaction is carried out by keeping the temperature for 4-8 hours after the addition.

7. The preparation method according to claim 5, characterized in that: The step S2 further comprises: The reaction is completed by monitoring the liquid phase, and the temperature is controlled at 5-10°C. A trifluoroacetic acid aqueous solution is dripped into the reaction solution, and the mixture is stirred thoroughly and then allowed to stand for separation. The aqueous phase is extracted with dichloromethane, and the combined organic phases are washed with water for 3 times. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated into an oily substance with a 40°C water pump. Petroleum ether is added to cool the mixture to 10°C to precipitate a solid, which is filtered, washed with petroleum ether by stirring, and dried to obtain the second intermediate.

8. The preparation method according to claim 1, characterized in that: The step S3 comprises: The second intermediate is added to dichloromethane, and then pyridine is added, and then pentafluorophenyl trifluoroacetate is added dropwise to generate the third intermediate, wherein the molar ratio of the second intermediate: pyridine: pentafluorophenyl trifluoroacetate is 1: (1.2-1.5): (1-1.5), the temperature for adding pentafluorophenyl trifluoroacetate is 20-25°C, and the reaction is kept warm for 4-8 hours.

9. The preparation method according to claim 8, characterized in that: The step S3 further comprises: After the reaction is completed, the temperature is controlled at 5-10°C, and trifluoroacetic acid aqueous solution is added dropwise to the reaction solution. After sufficient stirring, the liquid is separated, and the organic phase is washed with water three times, dried with sodium sulfate, filtered, concentrated with a water pump at 40°C, and petroleum ether is added until a small amount of liquid remains. Petroleum ether is added to slurry at 5°C, filtered, the wet product is recrystallized with n-heptane / ethyl acetate, filtered, washed with petroleum ether, and dried to obtain the third intermediate.

10. The preparation method according to claim 8, characterized in that: The step S4 comprises: The third intermediate is added to dichloromethane, and then 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid is added, and triethylamine is added dropwise to generate the semaglutide side chain, wherein the molar ratio of the third intermediate: triethylamine: 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid is 1:(1-1.4):(1-1.2), the temperature of adding triethylamine dropwise is 5-10°C, and the reaction is kept warm for 6-12 hours after the addition; After the reaction is completed by liquid phase monitoring, the temperature is controlled at 5-10°C, an aqueous solution of trifluoroacetic acid is added dropwise to the reaction solution, the mixture is stirred thoroughly and allowed to stand for separation, the aqueous phase is extracted with dichloromethane, the combined organic phases are washed with water three times, the organic phase is dried over anhydrous sodium sulfate, the organic phase is filtered through silica gel, and eluted with DCM / CH3OH=10:

1. The eluate is concentrated at 40°C to an oily substance, and the oil pump is dried at 60°C to obtain the semaglutide side chain.

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