Synthesis of a key intermediate of semaglutide

Abstract

The invention discloses a method for synthesizing a key intermediate of semaglutide, and belongs to the field of drug synthesis. The specific synthesis method includes: using Fmoc-Gly-OH, an activation reagent, a reaction solvent, a catalyst and H-Arg(Pbf)-OH as reaction raw materials, performing activation and condensation reactions, washing and recrystallization, and separating to obtain a key intermediate of semaglutide; using 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide as activators and catalysts, a key intermediate of semaglutide is obtained more stably and effectively, and the synthesis of the key intermediate of semaglutide by the present invention has high purity and yield; at the same time, the present invention has the advantages of simple preparation process, stable product quality, high purity and yield, etc.

Description

Technical Field

[0001] The invention belongs to the field of drug synthesis, and specifically relates to a method for synthesizing a key intermediate of semaglutide. Background Art

[0002] Semaglutide is the main active ingredient of the hypoglycemic drug Ozempic (approved for sale in the United States in 2017) and the weight loss drug Wegovy (approved for sale in the United States in 2021) developed by the Danish pharmaceutical giant Novo Nordisk. Semaglutide belongs to the GLP-1 receptor agonist (GLP-1RA) class of hypoglycemic drugs. GLP-1 is an incretin hormone that promotes insulin secretion in a glucose concentration-dependent manner, inhibits glucagon secretion to lower blood sugar, and also has the effect of delaying gastric emptying. And because of this incidental effect of delaying gastric emptying, semaglutide can not only complete its main function of lowering blood sugar, but also has the effect of suppressing appetite, making weight loss its second indication, and therefore it has exploded as a new phenomenal drug. The current synthesis methods of semaglutide are mainly divided into two methods: fermentation and chemical methods. Fmoc-Gly-Arg(Pbf)-OH is the key starting material in the chemical synthesis of semaglutide, as shown in compound 1 below.

[0003]

[0004] Compound 1

[0005] Patent CN105001140B discloses a method for synthesizing fatty diacid derivatives for preparing insulin analogs, obtaining compound 1 with a purity of 99.3% and a yield of 81%, but the reaction temperature, time and yield need to be further optimized. Therefore, the prior art urgently needs a technology that can synthesize key intermediates of semaglutide, so as to provide technical support for better preparation and synthesis of semaglutide. Summary of the invention

[0006] The object of the present invention is to provide a method for synthesizing a key intermediate of semaglutide to solve the problem of high synthesis cost of Fmoc-Gly-Arg(Pbf)-OH.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] A method for synthesizing a key intermediate of semaglutide, comprising:

[0009] Fmoc-Gly-OH and an activation agent undergo an activation reaction to form an active intermediate product, and then the active intermediate product undergoes a condensation reaction with H-Arg(Pbf)-OH to generate Fmoc-Gly-Arg(Pbf)-OH, i.e., a key intermediate of semaglutide; the activation agent is at least one of HONB, HOBT, HOSU and 2-phenoxy-N-(3-pyridyl)acetamide, and the catalyst is at least one of DIEA, EDCI, sodium bicarbonate, DCC, DIC and 1-phenyl-3-(cyclohexylmethylene)carbodiimide. 2-phenoxy-N-(3-pyridyl)acetamide is stable in a reaction solvent, and is used in combination with 1-phenyl-3-(cyclohexylmethylene)carbodiimide to more effectively form a stable amide bond, thereby obtaining a key intermediate of semaglutide with a higher purity and yield.

[0010] Preferably, in the above technical solution, Fmoc-Gly-OH and the activation reagent are mixed in a mass ratio of 1:0.2-0.8.

[0011] Preferably, in the above technical solution, the mass ratio of the amount of the catalyst used to the amount of Fmoc-Gly-OH used is 1:1-5.

[0012] Preferably, in the above technical scheme, the activation reaction and the condensation reaction are both carried out in the presence of a reaction solvent, the reaction solvent is at least one of dichloromethane, N,N-dimethylformamide, tetrahydrofuran / water, and the amount of Fmoc-Gly-OH in the reaction solvent is 0.1-0.18 g / ml.

[0013] Preferably, in the above technical solution, the mass ratio of the amount of H-Arg(Pbf)-OH used to the amount of Fmoc-Gly-OH used is 1:0.5-1.5.

[0014] Preferably, in the above technical scheme, the active intermediate product or Fmoc-Gly-Arg(Pbf)-OH is separated using a recrystallization solvent, the recrystallization solvent is at least one of dichloromethane / methyl tert-butyl ether, ethyl acetate / methyl tert-butyl ether, dichloromethane / petroleum ether, and ethyl acetate / petroleum ether, and the volume ratio of the amount of the recrystallization solvent used to the amount of the reaction solvent used is 1:1-7.

[0015] Preferably, in the above technical solution, the reaction temperature of the activation reaction is 20-30°C, and the reaction time is 3-6 h; the reaction temperature of the condensation reaction is 20-30°C, and the reaction time is 4-6 h.

[0016] Preferably, in the above technical solution, the activation reagent is 2-phenoxy-N-(3-pyridyl)acetamide, and the catalyst used in the activation reaction and the condensation reaction is 1-phenyl-3-(cyclohexylmethylene)carbodiimide.

[0017] Preferably, in the above technical scheme, after the condensation reaction is completed, that is, before Fmoc-Gly-Arg(Pbf)-OH is recrystallized and separated, it needs to be washed with a saturated sodium bicarbonate solution for 2-3 times, then washed with a 10% citric acid solution for 2-3 times, and washed with saturated brine for 1-2 times. The volume ratio of the washing amount of the saturated sodium bicarbonate solution to the amount of the reaction solvent is 1:0.5-2, the volume ratio of the washing amount of the 10% citric acid solution to the amount of the reaction solvent is 1:0.5-2, and the volume ratio of the washing amount of the saturated brine to the amount of the reaction solvent is 1:0.5-2.

[0018] Preferably, the present invention also provides a key intermediate of semaglutide, which is obtained by the above-mentioned synthesis method.

[0019] Preferably, in a preferred embodiment of the present invention, Fmoc-Gly-ONB is prepared as follows: Fmoc-Gly-OH, HONB and dichloromethane are stirred to obtain a mixture, and then the temperature is lowered to 0-10°C, DIEA is added dropwise to the mixture and stirred for 20-40 min, followed by reaction at 20-30°C for 3-6 h, and after the reaction is completed, the insoluble matter is removed by filtration to obtain a filtrate, the filtrate is concentrated and dried to obtain a dry product, and then dichloromethane is added to the dry product to dissolve it to obtain a dry product liquid, and then petroleum ether is added to the dry product solution, and crystallization is carried out at 0-10°C for 10-15 h to obtain crystals, and the crystals are dried to obtain Fmoc-Gly-ONB.

[0020] Preferably, in the above embodiment, Fmoc-Gly-OH and HONB are mixed in a mass ratio of 1:0.2-0.8, the amount of Fmoc-Gly-OH in dichloromethane is 0.1-0.18 g / ml, the mass ratio of DIEA used to Fmoc-Gly-OH used is 1:1-5, the volume ratio of the amount of dichloromethane added to the dried product to the amount of dichloromethane used in the mixture is 1:0.5-4, and the volume ratio of petroleum ether used to the amount of dichloromethane used in the mixture is 1:0.5-3.

[0021] Preferably, in a preferred embodiment of the present invention, Fmoc-Gly-Arg(Pbf)-OH is prepared as follows: Fmoc-Gly-ONB, H-Arg(Pbf)-OH and dichloromethane are stirred and mixed to obtain a mixture, and then the temperature is reduced to 0-10°C, DIEA is added dropwise to the mixture, and the mixture is reacted at 20-30°C for 4-6 hours, and the reaction is monitored by HPLC. After the reaction is complete, a saturated sodium bicarbonate solution is added to wash 2-3 times, followed by adding 8-12% citric acid solution to wash 2-3 times, and then adding saturated brine to wash 1-2 times, separating the organic phase, adding methyl tert-butyl ether to the organic phase, and crystallizing at -5-0°C for 10-15 hours to obtain crystals, and the crystals are dried to obtain Fmoc-Gly-Arg(Pbf)-OH, which is the key intermediate of semaglutide.

[0022] Preferably, in the above embodiment, Fmoc-Gly-ONB and H-Arg(Pbf)-OH are mixed in a mass ratio of 1:0.5-1.5, the amount of Fmoc-Gly-ONB in ​​dichloromethane is 0.1-0.3 g / ml, the mass ratio of the amount of DIEA used to the amount of Fmoc-Gly-ONB used is 1:1-4, the volume ratio of the amount of saturated sodium bicarbonate solvent used for washing to the amount of dichloromethane used is 1:0.5-2, the volume ratio of the amount of 8-12% citric acid solution used for washing to the amount of dichloromethane used is 1:0.5-2, the volume ratio of the amount of saturated salt water used for washing to the amount of dichloromethane used is 1:0.5-2, and the volume ratio of the amount of methyl tert-butyl ether used to the amount of dichloromethane used is 1:0.5-3.

[0023] Preferably, in a preferred embodiment of the present invention, Fmoc-Gly-Arg(Pbf)-OH is prepared as follows: Fmoc-Gly-OH, HOSU and dichloromethane are stirred and mixed to obtain a mixture, and then the temperature is reduced to 0-10°C, EDCI is added dropwise to the mixture, and the mixture is reacted at 20-30°C for 3-6 h to obtain an intermediate, and then H-Arg(Pbf)-OH is added to the intermediate to continue the reaction for 4-6 h, and the reaction is monitored by TLC or HPLC. After the reaction is complete, a saturated sodium bicarbonate solution is added to wash 2-3 times, followed by adding 8-12% citric acid solution to wash 2-3 times, and then adding saturated brine to wash 1-2 times, separating the organic phase, adding methyl tert-butyl ether to the organic phase, and crystallizing at -5-0°C for 10-15 h to obtain a crystal, and the crystal is dried to obtain Fmoc-Gly-Arg(Pbf)-OH, i.e., the key intermediate of semaglutide.

[0024] Preferably, in the above preferred embodiment, Fmoc-Gly-OH and HOSU are mixed in a mass ratio of 1:0.2-0.8, the amount of Fmoc-Gly-OH in dichloromethane is 0.1-0.18 g / ml, the mass ratio of the amount of EDCI used to the amount of Fmoc-Gly-OH used is 1:1-5, the mass ratio of the amount of H-Arg(Pbf)-OH used to the amount of Fmoc-Gly-OH used is 1:0.5-1.5, the volume ratio of the amount of saturated sodium bicarbonate solvent used for washing to the amount of dichloromethane used is 1:0.5-2, the volume ratio of the amount of 8-12% citric acid solution used for washing to the amount of dichloromethane used is 1:0.5-2, the volume ratio of the amount of saturated salt water used for washing to the amount of dichloromethane used is 1:0.5-2, and the volume ratio of the amount of methyl tert-butyl ether used to the amount of dichloromethane used is 1:0.5-3.

[0025] Preferably, in the preparation of the key intermediate of semaglutide in the present invention, when 2-phenoxy-N-(3-pyridyl)acetamide is used as an activation reagent, 1-phenyl-3-(cyclohexylmethylene)carbodiimide is added as a catalyst to carry out the reaction.

[0026] Preferably, in a preferred embodiment of the present invention, Fmoc-Gly-Arg(Pbf)-OH is prepared as follows: Fmoc-Gly-OH, 2-phenoxy-N-(3-pyridyl)acetamide and dichloromethane are stirred and mixed to obtain a mixture, and then the temperature is lowered to 0-10°C, 1-phenyl-3-(cyclohexylmethylene)carbodiimide is added dropwise to the mixture, and the mixture is reacted at 20-30°C for 3-6 h to obtain an intermediate, and then H-Arg(Pbf)-OH is added to the intermediate to continue the reaction for 4-6 h, and the reaction is monitored by TLC or HPLC. After the reaction is complete, a saturated sodium bicarbonate solution is added to wash 2-3 times, and then an 8-12% citric acid solution is added to wash 2-3 times, and then a saturated saline solution is added to wash 1-2 times, and the organic phase is separated, and methyl tert-butyl ether is added to the organic phase, and crystallized at -5-0°C for 10-15 h to obtain crystals, which were then dried to obtain Fmoc-Gly-Arg(Pbf)-OH, a key intermediate of semaglutide.

[0027] Preferably, in the above preferred embodiment, Fmoc-Gly-OH and 2-phenoxy-N-(3-pyridyl)acetamide are mixed in a mass ratio of 1:0.2-0.8, and the amount of Fmoc-Gly-OH in dichloromethane is 0.1-0.18 g / ml, the mass ratio of 1-phenyl-3-(cyclohexylmethylene)carbodiimide to Fmoc-Gly-OH is 1:1-5, the mass ratio of H-Arg(Pbf)-OH to Fmoc-Gly-OH is 1:0.5-1.5, the volume ratio of saturated sodium bicarbonate solvent for washing to dichloromethane is 1:0.5-2, the volume ratio of 8-12% citric acid solution for washing to dichloromethane is 1:0.5-2, the volume ratio of saturated brine for washing to dichloromethane is 1:0.5-2, and the volume ratio of methyl tert-butyl ether to dichloromethane is 1:0.5-3.

[0028] Preferably, in the present invention, when preparing the key intermediate of semaglutide, 4-amino-N,N-dimethylaniline can be added on the basis of using 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide. The addition of 4-amino-N,N-dimethylaniline can make the reaction between the reactants proceed faster, improve the selectivity between the reactants, and improve the purity of the key intermediate of semaglutide. And make the activation reaction product more stable, so that the yield is also improved.

[0029] Preferably, the mass ratio of the amount of 4-amino-N,N-dimethylaniline used to the amount of Fmoc-Gly-OH used is 1:12-25.

[0030] Preferably, in a preferred embodiment of the present invention, Fmoc-Gly-Arg(Pbf)-OH is prepared as follows: Fmoc-Gly-OH, 2-phenoxy-N-(3-pyridyl)acetamide and dichloromethane are stirred and mixed to obtain a mixture, and then the temperature is lowered to 0-10°C, 1-phenyl-3-(cyclohexylmethylene)carbodiimide is added dropwise to the mixture, and then 4-amino-N,N-dimethylaniline is added to the mixture, and the mixture is reacted at 20-30°C for 3-6 h to obtain an intermediate, and then H-Arg(Pbf)-OH is added to the intermediate to continue the reaction for 4-6 h, and the reaction is monitored by TLC or HPLC. After the reaction is complete, a saturated sodium bicarbonate solution is added to wash 2-3 times, and then an 8-12% citric acid solution is added to wash 2-3 times, and then a saturated saline solution is added to wash 1-2 times, and the organic phase is separated, and methyl tert-butyl ether is added to the organic phase, and crystallized at -5-0°C for 10-15 h to obtain crystals, which were then dried to obtain Fmoc-Gly-Arg(Pbf)-OH, a key intermediate of semaglutide.

[0031] Preferably, in the above preferred embodiment, Fmoc-Gly-OH and 2-phenoxy-N-(3-pyridyl)acetamide are mixed in a mass ratio of 1:0.2-0.8, and the amount of Fmoc-Gly-OH in dichloromethane is 0.1-0.18 g / ml, the mass ratio of 1-phenyl-3-(cyclohexylmethylene)carbodiimide to Fmoc-Gly-OH is 1:1-5, the mass ratio of 4-amino-N,N-dimethylaniline to Fmoc-Gly-OH is 1:12-25, the mass ratio of H-Arg(Pbf)-OH to Fmoc-Gly-OH is 1:0.5-1.5, the volume ratio of saturated sodium bicarbonate solvent for washing to dichloromethane is 1:0.5-2, the volume ratio of 8-12% citric acid solution for washing to dichloromethane is 1:0.5-2, the volume ratio of saturated brine for washing to dichloromethane is 1:0.5-2, and the volume ratio of methyl tert-butyl ether to dichloromethane is 1:0.5-3.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The invention uses Fmoc-Gly-OH, an activation reagent, a reaction solvent, a catalyst and H-Arg(Pbf)-OH as reaction raw materials to carry out a catalytic reaction to obtain a key intermediate of semaglutide; uses 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide as an activator and a catalyst to more stably and effectively obtain the key intermediate of semaglutide with higher purity and yield; further adds 4-amino-N,N-dimethylaniline to accelerate the reaction while reducing the generation of intermediate by-products, thereby improving the production effect; meanwhile, the invention has the advantages of simple preparation process, stable product quality, high purity and yield, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0035] Figure 1 This is a purity determination result diagram of Fmoc-Gly-Arg(Pbf)-OH;

[0036] Figure 2 This is a graph showing the statistical calculation results for the yield of Fmoc-Gly-Arg(Pbf)-OH. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0039] The abbreviations used in the specification and claims have the following meanings:

[0040]

[0041] Embodiment 1:

[0042] Preparation of Fmoc-Gly-ONB: Fmoc-Gly-OH, HONB and dichloromethane are stirred to obtain a mixture, and then the temperature is lowered to 8°C, DIEA is added dropwise to the mixture and stirred for 30 min, followed by reaction at 25°C for 4 h. After the reaction is completed, the insoluble matter is removed by filtration to obtain a filtrate, the filtrate is concentrated and dried to obtain a dry product, and then dichloromethane is added to the dry product to dissolve it to obtain a dry product liquid, and then petroleum ether is added to the dry product solution, and crystallization is carried out at 5°C for 12 h to obtain a crystal, and the crystal is dried to obtain Fmoc-Gly-ONB. Fmoc-Gly-OH and HONB were mixed in a mass ratio of 1:0.33, the amount of Fmoc-Gly-OH in dichloromethane was 0.2 g / ml, the mass ratio of DIEA to Fmoc-Gly-OH was 1:4.52, the volume ratio of dichloromethane added to the dried product to the amount of dichloromethane in the mixture was 1:3.33, and the volume ratio of petroleum ether to the amount of dichloromethane in the mixture was 1:1.11.

[0043] Preparation of Fmoc-Gly-Arg(Pbf)-OH: Fmoc-Gly-ONB, H-Arg(Pbf)-OH and dichloromethane were stirred to obtain a mixture, and then the temperature was lowered to 8°C, DIEA was added dropwise to the mixture, and the mixture was reacted at 25°C for 5 h. The reaction was monitored by HPLC. After the reaction was complete, a saturated sodium bicarbonate solution was added to wash twice, followed by a 10% citric acid solution to wash twice, and then a saturated saline solution to wash once, and the organic phase was separated. Methyl tert-butyl ether was added to the organic phase, and crystallized at -2°C for 12 h to obtain a crystal, and the crystal was dried to obtain Fmoc-Gly-Arg(Pbf)-OH, which is the key intermediate of semaglutide. Fmoc-Gly-ONB and H-Arg(Pbf)-OH are mixed in a mass ratio of 1:1.1, the amount of Fmoc-Gly-ONB in ​​dichloromethane is 0.2 g / ml, the mass ratio of DIEA to Fmoc-Gly-ONB is 1:3.57, the volume ratio of saturated sodium bicarbonate solvent for washing to dichloromethane is 1:1, the volume ratio of 10% citric acid solution for washing to dichloromethane is 1:1, the volume ratio of saturated brine for washing to dichloromethane is 1:1, and the volume ratio of methyl tert-butyl ether to dichloromethane is 1:1.2.

[0044] Embodiment 2:

[0045] The preparation of Fmoc-Gly-ONB was the same as in Example 1.

[0046] Preparation of Fmoc-Gly-Arg(Pbf)-OH: Fmoc-Gly-ONB, H-Arg(Pbf)-OH and N,N-dimethylformamide were stirred and mixed to obtain a mixture, and then the temperature was lowered to 8°C, EDCI was added dropwise to the mixture, and the mixture was reacted at 25°C for 5 h. The reaction was monitored by HPLC. After the reaction was complete, 10% citric acid solution was added to precipitate, the precipitate was filtered out, and ethyl acetate was added to the precipitate to dissolve to obtain a dissolving solution, saturated sodium bicarbonate solution was added to the dissolving solution to wash twice, then 10% citric acid solution was added to wash twice, and then saturated brine was added to wash once, the organic phase was separated, petroleum ether was added to the organic phase, and crystallized at -2°C for 12 h to obtain crystals, and the crystals were dried to obtain Fmoc-Gly-Arg(Pbf)-OH, which is the key intermediate of semaglutide. Fmoc-Gly-ONB and H-Arg(Pbf)-OH are mixed in a mass ratio of 1:1.1, the amount of Fmoc-Gly-ONB in ​​N,N-dimethylformamide is 0.2 g / ml, the mass ratio of EDCI used to Fmoc-Gly-ONB is 1:2.94, the volume ratio of ethyl acetate used to N,N-dimethylformamide used is 2:1, the volume ratio of saturated sodium bicarbonate solvent used for washing to N,N-dimethylformamide used is 1:1, the volume ratio of 10% citric acid solution used for washing to N,N-dimethylformamide used is 1:1, the volume ratio of saturated brine used for washing to N,N-dimethylformamide used is 1:1, and the volume ratio of petroleum ether used to N,N-dimethylformamide used is 1:1.11.

[0047] Embodiment 3:

[0048] The preparation of Fmoc-Gly-ONB was the same as in Example 1.

[0049] Preparation of Fmoc-Gly-Arg(Pbf)-OH: Fmoc-Gly-ONB, H-Arg(Pbf)-OH and tetrahydrofuran were stirred and mixed to obtain a mixture, and then the temperature was lowered to 8°C, and an aqueous sodium bicarbonate solution was added dropwise to the mixture, and the mixture was reacted at 25°C for 5 h. The reaction was monitored by HPLC. After the reaction was complete, the tetrahydrofuran was removed by concentration to obtain a concentrate, and ethyl acetate was added to the concentrate to separate the organic phase. Subsequently, 10% citric acid solution was added to the organic phase to wash twice, and then saturated brine was added to wash once, and the organic phase was separated again. Methyl tert-butyl ether was added to the organic phase, and crystallized at -2°C for 12 h to obtain crystals, and the crystals were dried to obtain Fmoc-Gly-Arg(Pbf)-OH, which is the key intermediate of semaglutide. Fmoc-Gly-ONB and H-Arg(Pbf)-OH are mixed in a mass ratio of 1:1.1, the amount of Fmoc-Gly-ONB in ​​tetrahydrofuran is 0.2 g / ml, the mass ratio of the amount of sodium bicarbonate aqueous solution to the amount of Fmoc-Gly-ONB is 1:2.94, the volume ratio of the amount of ethyl acetate to the amount of tetrahydrofuran in the mixture is 2:1, the volume ratio of the amount of 10% citric acid solution to the amount of tetrahydrofuran to be washed is 1:1, the volume ratio of the amount of saturated brine to be washed is 1:1, and the volume ratio of methyl tert-butyl ether to tetrahydrofuran to be used is 1:1.2.

[0050] Embodiment 4:

[0051] Preparation of Fmoc-Gly-Arg(Pbf)-OH: Fmoc-Gly-OH, HOSU and dichloromethane were stirred and mixed to obtain a mixture, and then the temperature was lowered to 8°C, EDCI was added dropwise to the mixture, and the mixture was reacted at 25°C for 5 h to obtain an intermediate, and then H-Arg(Pbf)-OH was added to the intermediate to continue the reaction for 3 h. The reaction was monitored by HPLC. After the reaction was complete, a saturated sodium bicarbonate solution was added to wash twice, followed by a 10% citric acid solution to wash twice, and then a saturated saline solution to wash once, and the organic phase was separated. Methyl tert-butyl ether was added to the organic phase, and crystallized at -2°C for 12 h to obtain a crystal, and the crystal was dried to obtain Fmoc-Gly-Arg(Pbf)-OH, which is the key intermediate of semaglutide. Fmoc-Gly-OH and HOSU were mixed in a mass ratio of 1:0.38, the amount of Fmoc-Gly-OH in dichloromethane was 0.13 g / ml, the mass ratio of EDCI used to Fmoc-Gly-OH used was 1:1.55, the mass ratio of H-Arg(Pbf)-OH used to Fmoc-Gly-OH used was 1:0.59, the volume ratio of the amount of saturated sodium bicarbonate solvent used for washing to the amount of dichloromethane used was 1:1, the volume ratio of the amount of 10% citric acid solution used for washing to the amount of dichloromethane used was 1:1, the volume ratio of the amount of saturated brine used for washing to the amount of dichloromethane used was 1:1, and the volume ratio of methyl tert-butyl ether used to dichloromethane used was 1:1.2.

[0052] Embodiment 5:

[0053] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this embodiment is different from that in Example 4, except that HOSU is replaced by HOBT, and EDCI is replaced by DCC. Fmoc-Gly-OH and HOBT are mixed in a mass ratio of 1:0.46, and the mass ratio of the amount of DCC used to the amount of Fmoc-Gly-OH used is 1:1.44. Other conditions and parameters are the same as in Example 4.

[0054] Embodiment 6:

[0055] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this example is different from that in Example 5, except that DCC is replaced by DIC. The mass ratio of the amount of DIC used to the amount of Fmoc-Gly-OH used is 1:2.41, and the other conditions and parameters are the same as in Example 5.

[0056] Embodiment 7:

[0057] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this example is compared with that in Example 4, except that HOSU is replaced by 2-phenoxy-N-(3-pyridyl)acetamide, EDCI is replaced by 1-phenyl-3-(cyclohexylmethylene)carbodiimide, and other conditions and parameters are the same as those in Example 4.

[0058] Embodiment 8:

[0059] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this example is compared with that in Example 7, except that Fmoc-Gly-OH and 2-phenoxy-N-(3-pyridyl)acetamide are mixed in a mass ratio of 1:0.55, and other conditions and parameters are the same as those in Example 7.

[0060] Embodiment 9:

[0061] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this example is compared with that in Example 7, except that the mass ratio of 1-phenyl-3-(cyclohexylmethylene)carbodiimide to Fmoc-Gly-OH is 1:1.12, and the other conditions and parameters are the same as those in Example 7.

[0062] Embodiment 10:

[0063] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this embodiment is compared with that in Example 7, except that 1-phenyl-3-(cyclohexylmethylene)carbodiimide is added dropwise to the mixture, and then 4-amino-N,N-dimethylaniline is added to the mixture, wherein the mass ratio of 4-amino-N,N-dimethylaniline to Fmoc-Gly-OH is 1:20.

[0064] Embodiment 11:

[0065] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this embodiment is compared with that in Example 10, except that 1-phenyl-3-(cyclohexylmethylene)carbodiimide is added dropwise to the mixture, and then 4-amino-N,N-dimethylaniline is added to the mixture, wherein the mass ratio of the amount of 4-amino-N,N-dimethylaniline used to the amount of Fmoc-Gly-OH is 1:16.

[0066] Comparative Example 1:

[0067] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this example is compared with that in Example 7, except that EDCI is not replaced, and other conditions and parameters are the same as those in Example 7.

[0068] Comparative Example 2:

[0069] Preparation of Fmoc-Gly-Arg(Pbf)-OH: The preparation of Fmoc-Gly-Arg(Pbf)-OH in this example is compared with that in Example 7, except that HOSU is not replaced, and other conditions and parameters are the same as those in Example 7.

[0070] Experimental Example 1:

[0071] Purity determination: Fmoc-Gly-Arg(Pbf)-OH prepared in Example 1-11 and Comparative Example 1-2, i.e., the key intermediate of semaglutide, was subjected to HPLC analysis to obtain the purity.

[0072] like Figure 1 As shown, the purity of Fmoc-Gly-Arg(Pbf)-OH prepared in Example 7 of the present invention is higher than that in Example 4, indicating that 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide can be used as key reaction substances to synthesize Fmoc-Gly-Arg(Pbf)-OH, and has a higher purity effect; the purity of Fmoc-Gly-Arg(Pbf)-OH prepared in Example 10 is higher than that in Example 7, indicating that on the basis of using 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide, adding 4-amino-N,N- The reaction product is treated with dimethylaniline to further improve the purity of Fmoc-Gly-Arg(Pbf)-OH. Compared with Comparative Examples 1 and 2, the purity of Fmoc-Gly-Arg(Pbf)-OH prepared in Example 7 is higher than that in Examples 1 and 2, indicating that 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide need to be used simultaneously for reaction, and the use of 2-phenoxy-N-(3-pyridyl)acetamide or 1-phenyl-3-(cyclohexylmethylene)carbodiimide alone has no obvious promoting effect on the synthesis purity of Fmoc-Gly-Arg(Pbf)-OH.

[0073] Experimental Example 2:

[0074] Determination of yield: The Fmoc-Gly-Arg(Pbf)-OH prepared in Example 1-11 and Comparative Example 1-2, i.e., the key intermediate of semaglutide, was statistically calculated to obtain the yield.

[0075] like Figure 2 As shown, the yield of Fmoc-Gly-Arg(Pbf)-OH prepared in Example 7 of the present invention is higher than that in Example 4, indicating that 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide as the key reaction substances can synthesize more Fmoc-Gly-Arg(Pbf)-OH, that is, it has a higher yield; the yield of Fmoc-Gly-Arg(Pbf)-OH prepared in Example 10 is higher than that in Example 7, indicating that on the basis of using 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide, 4-amino-N,N-dimethylaniline is added to the reactants to react. The reaction between the reactants can be promoted to a certain extent, and the yield of Fmoc-Gly-Arg(Pbf)-OH can be further improved; compared with Comparative Examples 1 and 2, the yield of Fmoc-Gly-Arg(Pbf)-OH prepared in Example 7 is higher than that in Examples 1 and 2, indicating that 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide need to be used simultaneously for the reaction, and the use of 2-phenoxy-N-(3-pyridyl)acetamide or 1-phenyl-3-(cyclohexylmethylene)carbodiimide alone has no obvious promoting effect on the synthesis efficiency of Fmoc-Gly-Arg(Pbf)-OH, that is, the yield.

[0076] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0077] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for synthesizing a key intermediate of semaglutide, comprising: Fmoc-Gly-OH is activated by an activation reaction with an activation reagent to form an active intermediate product, and then the active intermediate product is condensed with H-Arg(Pbf)-OH to generate Fmoc-Gly-Arg(Pbf)-OH, which is a key intermediate of semaglutide; the activation reagent is 2-phenoxy-N-(3-pyridyl)acetamide, and the catalyst used in the activation reaction and the condensation reaction is 1-phenyl-3-(cyclohexylmethylene)carbodiimide; The Fmoc-Gly-OH and the activation reagent are mixed in a mass ratio of 1:0.2-0.8; the mass ratio of the added amount of the catalyst to the amount of Fmoc-Gly-OH is 1:1-5; the mass ratio of the used amount of the H-Arg(Pbf)-OH to the used amount of Fmoc-Gly-OH is 1:0.5-1.

5.

2. The method for synthesizing a key intermediate of semaglutide according to claim 1, characterized in that: The activation reaction and the condensation reaction are both carried out in the presence of a reaction solvent, the reaction solvent is at least one of dichloromethane, N,N-dimethylformamide, and tetrahydrofuran / water, and the amount of Fmoc-Gly-OH in the reaction solvent is 0.1-0.18 g / ml.

3. The method for synthesizing a key intermediate of semaglutide according to claim 1, characterized in that: The active intermediate product or Fmoc-Gly-Arg(Pbf)-OH is separated by using a recrystallization solvent, the recrystallization solvent is at least one of dichloromethane / methyl tert-butyl ether, ethyl acetate / methyl tert-butyl ether, dichloromethane / petroleum ether, and ethyl acetate / petroleum ether, and the volume ratio of the amount of the recrystallization solvent used to the amount of the reaction solvent used is 1:1-7.

4. The method for synthesizing a key intermediate of semaglutide according to claim 1, characterized in that: The reaction temperature of the activation reaction is 20-30°C, and the reaction time is 3-6 h. The reaction temperature of the condensation reaction is 20-30°C, and the reaction time is 4-6 h. After the condensation reaction is completed, washing and recrystallization separation are performed.

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