O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine and method for producing the same

Abstract

A method for producing O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine. [Solution] A method to obtain intermediate (I) by condensing N-benzyloxycarbonyl-O-tert-butyl-L-serine and O-tert-butyl-L-serine methyl ester, saponifying (I) with an alkali compound to obtain intermediate (II), condensing (II) with glycine benzyl ester p-toluenesulfonate to obtain intermediate (III), and hydrogenating (III) with hydrogen gas to obtain the final product. JPEG2026047297000018.jpg70146

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and particularly to O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine and its manufacturing method.

Background Art

[0002] Tirzepatide is a novel dual receptor agonist of GIP (gastric inhibitory polypeptide) and GLP-1 (glucagon-like peptide-1), which targets the treatment of diabetes, non-alcoholic steatohepatitis (NASH), and chronic weight management, and its half-life is 116.7 hours. Tirzepatide was developed by Eli Lilly and Company in the United States. On September 7, 2022, the sales application in China of the tirzepatide injection used for improving blood glucose control in adult patients with type 2 diabetes was accepted by the NMPA (National Medical Products Administration of China). On November 8, 2023, tirzepatide manufactured by Eli Lilly and Company obtained a sales certification from the FDA (U.S. Food and Drug Administration) and has become the world's first dual-target (GIP / GLP-1) agonist obesity treatment drug among the currently sold products. Therefore, further development of tirzepatide is expected in the future. O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine is an important intermediate in the synthesis of tirzepatide, and tirzepatide is finally obtained through multiple reaction steps.

[0003] Currently, no literature or patent reports regarding the manufacturing method of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine have been found. According to the applicant's research, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, which is the final compound, was obtained using multiple synthesis steps, but this compound contained many impurities, making purification difficult and the molar yield low.

[0004] In light of this, there is a need for the development of a simple process, mild reaction conditions, and low-cost industrial synthesis route for the production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine. [Overview of the project] [Problems that the invention aims to solve]

[0005] The technical problem that the present invention aims to solve is to provide a method for producing o-tert-butyl-L-seryl-o-tert-butyl-L-seryl-glycine that simplifies the process, uses mild reaction conditions, and furthermore, produces o-tert-butyl-L-seryl-o-tert-butyl-L-seryl-glycine with high purity and high yield. [Means for solving the problem]

[0006] Therefore, according to this application, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine represented by the following formula (I) is provided. [ka]

[0007] Furthermore, according to this application, a method for producing O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine is provided. Step S1) involves condensing N-benzyloxycarbonyl-O-tert-butyl-L-serine and O-tert-butyl-L-serine methyl ester under the action of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester represented by the following formula (II), Step S2) involves saponifying the N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an alkali compound to obtain N-benzyloxycarbonyl-O-tert-butyl-L-serine represented by the following formula (III): Step S3) involves condensing the aforementioned N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine and glycine benzyl ester p-toluenesulfonate under the action of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester represented by the following formula (IV): A manufacturing method is provided, characterized by comprising step S4) a hydrogenation reaction of the N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester with hydrogen gas under the action of a catalyst to obtain O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine represented by the following formula (I). [ka]

[0008] Preferably, step S1) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, and an organic solvent of an organic base, then adding a condensing agent to carry out a condensation reaction to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0009] Preferably, in step S1), the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, the condensing agent, and the organic base is 1:(1~2):(1~1.5):(1~1.5).

[0010] Preferably, the coupling agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, DCC / HOSU, HATU, HBTU, and CDI, and the organic base is triethylamine or N,N-diisopropylethylamine.

[0011] Preferably, the condensing agent is added at a temperature of 0 to 5°C, and the condensation reaction is carried out at a temperature of 0 to 50°C for 12 to 24 hours.

[0012] Preferably, the organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, acetonitrile, and tetrahydrofuran.

[0013] Preferably, step S2) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an organic solvent, then adding a sodium hydroxide solution to carry out a saponification reaction to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine.

[0014] Preferably, in step S2), the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester to sodium hydroxide is 1:(1~2).

[0015] Preferably, in step S2), the sodium hydroxide solution is added at a temperature of 0 to 5°C, and the saponification reaction is carried out at a temperature of 0 to 40°C for 1 to 6 hours.

[0016] Preferably, in step S2), the concentration of the sodium hydroxide solution is 5-60%.

[0017] Preferably, in step S2), the organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, and ethanol.

[0018] Preferably, step S3) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, glycine benzyl ester p-toluenesulfonate, and an organic solvent of an organic base, then adding a condensing agent to conduct a condensation reaction, and then obtaining N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester.

[0019] Preferably, in step S3), the molar ratio of the N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, the glycine benzyl ester p-toluenesulfonate, the organic base, and the condensing agent is 1:(1-2):(1-1.5):(1-1.5).

[0020] Preferably, in step S3), the condensing agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, DCC / HOSU, HATU, HBTU, and CDI, and the organic base is triethylamine or N,N-diisopropylethylamine.

[0021] Preferably, in step S3), the condensing agent is added at a temperature of 0-5°C, and the condensation reaction is conducted at a temperature of 0-50°C for 12-24 hours.

[0022] Preferably, in step S3), the organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, acetonitrile, and tetrahydrofuran.

[0023] Preferably, step S4) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester with an organic solvent, adding a catalyst, introducing hydrogen gas, and conducting a hydrogenation reaction, and then obtaining O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine.

[0024] Preferably, in step S4), the mass ratio of the N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester to the catalyst is 1:(0.01 - 0.2).

[0025] Preferably, in step S4), the hydrogenation reaction is carried out at a temperature of 20 - 30 °C for 20 - 24 hours.

[0026] Preferably, the organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran / water, methanol, ethanol, acetone / water, and ethyl acetate / water, and the catalyst is selected from the group consisting of Pd / C, Raney nickel, and Pd / BaSO4.

[0027] According to this application, a method for producing O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine is provided, which includes first condensing N-benzyloxycarbonyl-O-tert-butyl-L-serine with O-tert-butyl-L-serine methyl ester to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester, then saponifying N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an alkali compound, condensing the resulting product with glycine benzyl ester p-toluenesulfonate, and finally hydrogenating the resulting product with hydrogen gas to obtain O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine. In the manufacturing process of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine described above, this application uses N-benzyloxycarbonyl-O-tert-butyl-L-serine as a raw material and sequentially carries out condensation, saponification, condensation, and hydrogenation reactions, synthesizing the tilzepatide intermediate O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine in just these four steps. Therefore, the process is simplified, the operation is easy, and the reaction conditions are mild. Furthermore, in this application, the yield and purity of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine can be improved by adjusting the specific process conditions in the above manufacturing process. [Brief explanation of the drawing]

[0028] [Figure 1] This is the HPLC chromatogram of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine prepared in Example 2 of the present invention. [Figure 2] This is the hydrogen nuclear magnetic resonance spectrum measured in CD3OD of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine prepared in Example 2 of the present invention. [Figure 3]This is the hydrogen nuclear magnetic resonance spectrum measured in D2O of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine prepared in Example 2 of the present invention. [Figure 4] This is the hydrogen nuclear magnetic resonance spectrum of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine prepared in Example 2 of the present invention, measured in DMSO. [Modes for carrying out the invention]

[0029] To further understand the present invention, preferred embodiments will be described below with reference to examples. However, it should be understood that these descriptions are used only to further illustrate the features and advantages of the present invention and do not limit the scope of the claims of the present invention.

[0030] In response to the conventional demand for a simple process, mild reaction conditions, and low-cost industrial synthesis route for the synthesis of tylzepatide intermediates, this application provides O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine and a method for producing the same. This production process uses N-benzyloxycarbonyl-O-tert-butyl-L-serine as a starting material and involves four reaction steps—condensation, saponification, condensation, and hydrogenation—with other reagents to synthesize the tylzepatide intermediate, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine. This production method simplifies the process route, is easy to operate, uses mild reaction conditions, and reduces the difficulty of industrial production. Furthermore, by controlling the process conditions during the manufacturing process, the yield and purity of the tilzepatide intermediate, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, can be further improved.

[0031] According to this application, first, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine represented by the following formula (I) is provided. [ka]

[0032] Furthermore, in the embodiments of the present invention, the method for producing O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine is as follows: Step S1) involves condensing N-benzyloxycarbonyl-O-tert-butyl-L-serine and O-tert-butyl-L-serine methyl ester under the action of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester represented by the following formula (II), Step S2) involves saponifying the N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an alkali compound to obtain N-benzyloxycarbonyl-O-tert-butyl-L-serine represented by the following formula (III): Step S3) involves condensing the aforementioned N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine and glycine benzyl ester p-toluenesulfonate under the action of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester represented by the following formula (IV): A manufacturing method is disclosed, characterized by comprising step S4) a hydrogenation reaction of the N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester with hydrogen gas under the action of a catalyst to obtain O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine represented by the following formula (I). [ka]

[0033] In the process of producing O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, an intermediate of tylzepatide, this application first uses N-benzyloxycarbonyl-O-tert-butyl-L-serine and O-tert-butyl-L-serine methyl ester as raw materials and carries out a condensation reaction under the conditions of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester. Specifically, this process involves uniformly mixing N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester and an organic base in an organic solvent, then adding a condensing agent and stirring to carry out a condensation reaction, followed by post-treatment to obtain the intermediate compound N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester as an oily substance.

[0034] The reaction equation for the above process is specifically as follows: [ka]

[0035] In the above process, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, coupling agent, and organic base is 1:(1~2):(1~1.5):(1~1.5). Specifically, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, coupling agent, and organic base is 1:(1.2~1.8):(1.1~1.4):(1.1~1.4). More specifically, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, coupling agent, and organic base is 1:1.5:1.2:1.4. The coupling agent is added at a temperature of 0~5°C. Specifically, the coupling agent is added at a temperature of 1 to 3°C. If the temperature at which the coupling agent is added is too high, the amount of impurities may increase. The condensation reaction is carried out at a temperature of 0 to 50°C. Specifically, the condensation reaction is carried out at temperatures of 0 to 10°C, 10 to 20°C, 20 to 25°C, 25 to 30°C, 30 to 35°C, 35 to 40°C, or 40 to 50°C. Preferably, the condensation reaction is carried out at a temperature of 25 to 35°C. If the temperature of the condensation reaction is too low, the reaction will be incomplete and the yield will decrease, while if the temperature of the condensation reaction is too high, the purity may decrease. The condensation reaction is carried out for 12 to 24 hours. Specifically, the condensation reaction is carried out for 18 to 20 hours. The organic base is triethylamine or N,N-diisopropylethylamine. The coupling agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, DCC / HOSU, HATU, HBTU, and CDI. Furthermore, the coupling agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, HATU, and HBTU. When the coupling agent is selected from the above reagents, the yield and purity of the product can be increased. In the above coupling agent, EDCl / HOBT means that EDCl and HOBT are added simultaneously, and similarly, EDCl / HOSU means that EDCl and HOSU are added simultaneously. The organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, acetonitrile, and tetrahydrofuran. Specifically, the organic solvent is ethyl acetate or acetonitrile.Under the same conditions, the selection of the above organic solvent contributes to improving the yield and purity of the product.

[0036] In this application, the intermediate product, N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester, is then subjected to a saponification reaction with an alkali compound to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine. Specifically, this process involves uniformly mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an organic solvent, then adding a sodium hydroxide solution and stirring to carry out the saponification reaction, followed by post-treatment to obtain the oily substance of the intermediate compound, N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine.

[0037] The reaction equation for the above process is specifically as follows: [ka]

[0038] In the above process, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester to sodium hydroxide is 1:(1~2). Specifically, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester to sodium hydroxide is 1:(1.2~1.8). More specifically, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester to sodium hydroxide is 1:1.5. The sodium hydroxide solution is added at a temperature of 0~5°C. Specifically, the sodium hydroxide solution is added at a temperature of 3~4°C. When the sodium hydroxide solution is added, the temperature in the system rises, so if the temperature at which the sodium hydroxide aqueous solution is added is not properly controlled, the reaction temperature may become too high and impurities may increase. The concentration of the sodium hydroxide solution is 5-60%. Specifically, the concentration of the sodium hydroxide solution is 5-30%. More specifically, the concentration of the sodium hydroxide solution is 10%, 20%, or 30%. If the sodium hydroxide concentration is too high, the purity of the product may decrease. The saponification reaction is carried out at a temperature of 0-40°C. Specifically, the saponification reaction is carried out at a temperature of 0-10°C, 10-15°C, 15-20°C, or 20-25°C. Furthermore, the saponification reaction is carried out at a temperature of 15-25°C. More specifically, the saponification reaction is carried out at a temperature of 15-20°C or 20-25°C. If the temperature of the saponification reaction is too low, the reaction time will be longer and the amount of impurities may increase. At the above temperatures, the reaction is fast, impurities are few, and the purity of the product is guaranteed. The saponification reaction is carried out for 1-6 hours. Specifically, the saponification reaction is carried out for 2-4 hours. More specifically, the saponification reaction is carried out for 3 or 4 hours. The organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, and ethanol.

[0039] According to the present invention, the products obtained as described above, N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine and glycine benzyl ester p-toluenesulfonate, are then subjected to a condensation reaction under the action of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester. Specifically, this process involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, glycine benzyl ester p-toluenesulfonate, and an organic solvent of an organic base, then adding a condensing agent and stirring to carry out the condensation reaction, followed by post-treatment to obtain the intermediate compound N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester as an oily substance.

[0040] The reaction equation for the above process is specifically as follows: [ka]

[0041] In the above process, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, glycine benzyl ester p-toluenesulfonate, condensing agent, and organic base is 1:(1~2):(1~1.5):(1~1.5). Specifically, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, glycine benzyl ester p-toluenesulfonate, condensing agent, and organic base is 1:(1.2~1.8):(1.1~1.4):(1.1~1.4). More specifically, the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, glycine benzyl ester p-toluenesulfonate, coupling agent, and organic base is 1:1.5:1.2:1.4. In this application, glycine benzyl ester p-toluenesulfonate is used as the main raw material. If glycine methyl ester hydrochloride or the like is used as a reaction raw material, the number of reaction steps will increase, the final product may contain many impurities, purification will become difficult, and the molar yield may decrease. The coupling agent is added at a temperature of 0 to 5°C. Specifically, the coupling agent is added at a temperature of 2 to 4°C. If the addition temperature of the coupling agent is too high, the amount of impurities may increase. The condensation reaction is carried out at a temperature of 0 to 50°C. Specifically, the condensation reaction is carried out at temperatures of 0-10°C, 10-20°C, 20-25°C, 25-30°C, 30-35°C, 35-40°C, or 40-50°C. Preferably, the condensation reaction is carried out at a temperature of 25-35°C. Furthermore, the condensation reaction is carried out at a temperature of 30-35°C. If the temperature of the condensation reaction is too low, the reaction will be insufficient and the yield will decrease, while if the temperature of the condensation reaction is too high, the purity may decrease. The condensation reaction is carried out for 12-24 hours. Specifically, the condensation reaction is carried out for 18-20 hours. The organic base is trimethylamine or N,N-diisopropylethylamine. The condensing agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, DCC / HOSU, HATU, HBTU, and CDI. Furthermore, the condensing agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, HATU, and HBTU.When the coupling agent is selected from the above reagents, the yield and purity of the product can be increased. In the above coupling agent, EDCl / HOBT means that EDCl and HOBT are added simultaneously, and similarly, EDCl / HOSU means that EDCl and HOSU are added simultaneously. The organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, acetonitrile, and tetrahydrofuran. Specifically, the organic solvent is ethyl acetate or acetonitrile. Under the same conditions, the selection of the organic solvent contributes to improving the yield and purity of the product. In this application, the process conditions for the condensation reaction in this process and the condensation reaction in step S1) may be the same or different, but are not particularly limited in this application.

[0042] In this application, the intermediate product obtained as described above, N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester, is subjected to a hydrogenation reaction with hydrogen gas under the action of a catalyst to obtain O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. Specifically, this process involves uniformly mixing N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester with an organic solvent, then adding a catalyst and stirring, introducing hydrogen gas to carry out the hydrogenation reaction, and finally performing post-treatment to obtain the target compound, O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine.

[0043] The reaction equation for the above process is specifically as follows: [ka]

[0044] In the above process, the mass ratio of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester to the catalyst is 1:(0.01~0.2). Specifically, the mass ratio of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester to the catalyst is 1:(0.07~0.15). More specifically, the mass ratio of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester to the catalyst is 1:0.1. The hydrogenation reaction is carried out at a temperature of 20~30°C. Specifically, the hydrogenation reaction is carried out at a temperature of 23~27°C. If the temperature of the hydrogenation reaction is too low, the reaction time will be prolonged, while if the reaction temperature is too high, impurities may increase and become difficult to remove. The hydrogenation reaction is carried out for 20 to 24 hours. Specifically, the hydrogenation reaction is carried out for 21 to 23 hours. The organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran / water, methanol, ethanol, acetone / water, and ethyl acetate / water. Specifically, the organic solvent is methanol. The catalyst is selected from the group consisting of Pd / C, Raney nickel, and Pd / BaSO4. Specifically, the catalyst is Pd / C. [Examples]

[0045] To further understand the present invention, the method for producing O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine according to the present invention will be described in detail below with reference to examples. However, the scope of protection of the present invention is not limited to the following examples.

[0046] All of the raw materials used in this invention are industrial raw materials that can be purchased on the market.

[0047] [Example 1] (1) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester: 100 g of N-benzyloxycarbonyl-O-tert-butyl-L-serine was added to 800 g of ethyl acetate and stirred until completely clear. Then, 90 g of O-tert-butyl-L-serine methyl ester and 51.3 g of trimethylamine were added and stirred until homogeneous. After cooling to 3°C, 9.1 g of HOBT and 78 g of EDCl were added and stirred for 30 minutes, then the temperature was raised to 33°C and the reaction was continued for 18 hours. Complete reaction of N-carbobenzoxy-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to separate the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 168 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0048] (2) Manufacture of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine: 168 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester, an oily substance, was added to 150 g of tetrahydrofuran and 300 g of water. The mixture was cooled to 4°C, and 67.6 g of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the temperature was raised to 23°C and the reaction was allowed to proceed for 3 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was confirmed by TLC. Next, 400 g of ethyl acetate was added to the reaction system, the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution, and the ethyl acetate layer was separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 152 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, an oily substance.

[0049] (3) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester: 152 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was added to 1200 g of ethyl acetate and stirred until completely clarified and dissolved. Then, 121 g of glycine benzyl ester p-toluenesulfonate and 48 g of triethylamine were added and stirred until homogeneous. After cooling to 2°C, 9.3 g of HOBT and 79 g of EDCl were added and the reaction was stirred for 30 minutes, then the temperature was raised to 33°C and the reaction was allowed to proceed for 18 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to separate the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 177 g of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-glycine benzyl ester, an oily substance.

[0050] (4) Production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 177 g of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was added to 1060 g of methanol and stirred to dissolve completely. Then 17.7 g of Pd / C was added, the temperature was controlled to 23°C, nitrogen gas was purged, and then hydrogen gas was introduced, and the reaction was allowed to proceed for 23 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was confirmed by TLC. Next, Pd / C was filtered, and the filtrate was concentrated until a large amount of solid precipitated. After cooling to 2°C and crystallization for 3 hours, it was filtered. The filtered cake was washed with a small amount of methanol and air-dried at 50°C to obtain 89 g of O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. The results showed a molar yield of 73%, an HPLC accuracy of 99.48%, and no isomers were detected.

[0051] [Example 2] (1) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester: 1.7 kg of N-benzyloxycarbonyl-O-tert-butyl-L-serine was added to 13.6 kg of ethyl acetate and stirred until completely clear. Then, 1.62 kg of O-tert-butyl-L-serine methyl ester and 0.87 kg of trimethylamine were added and stirred until homogeneous. After cooling to 1°C, 0.155 kg of HOBT and 1.32 kg of EDCl were added and the mixture was stirred for 30 minutes. The temperature was then raised to 33°C and the reaction was continued for 18 hours. Complete reaction of N-carbobenzoxy-O-tert-butyl-L-serine was confirmed by TLC. Next, 7 kg of water was added to the reaction mixture to quench the reaction, and the mixture was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 2.4 kg of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0052] (2) Manufacture of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine: 2.4 kg of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was added to 4.8 kg of tetrahydrofuran and 12 kg of water. The mixture was cooled to 3°C, and 1 kg of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the temperature was raised to 22°C and the reaction was allowed to proceed for 3 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was confirmed by TLC. Next, 6 kg of ethyl acetate was added to the reaction system, and the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution. The ethyl acetate layer was then separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 2.2 kg of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, an oily substance.

[0053] (3) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester: 2.2 kg of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was added to 17.4 kg of ethyl acetate and stirred until completely clarified and dissolved. Then, 1.927 kg of glycine benzyl ester p-toluenesulfonate and 0.72 kg of triethylamine were added and stirred until homogeneous. After cooling to 4°C, 0.134 kg of HOBT and 1.043 kg of EDCl were added and stirred for 30 minutes, then the temperature was raised to 33°C and the reaction was continued for 18 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was confirmed by TLC. Next, 10 kg of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to separate the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 2.8 kg of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-glycine benzyl ester.

[0054] (4) Production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 2.8 kg of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was added to 12 kg of methanol and stirred to dissolve completely. Then 0.196 kg of Pd / C was added, the temperature was controlled to 23°C, nitrogen gas was purged, and then hydrogen gas was introduced, and the reaction was allowed to proceed for 23 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was confirmed by TLC. Next, Pd / C was filtered, and the filtrate was concentrated until a large amount of solid precipitated. After cooling to 4°C and crystallization for 3 hours, it was filtered. The filtered cake was washed with a small amount of methanol and air-dried at 50°C to obtain 1.477 kg of O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. As a result, the molar yield was 71%, the HPLC accuracy was 99.41% (see Figure 1), and no isomers were detected. The nuclear magnetic resonance spectra are shown in Figures 2, 3, and 4. Figures 2, 3, and 4 are hydrogen nuclear magnetic resonance spectra measured in different solvents for the final product.

[0055] [Example 3] (1) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester: 100 g of N-benzyloxycarbonyl-O-tert-butyl-L-serine was added to 800 g of ethyl acetate and stirred until completely clear. Then, 90 g of O-tert-butyl-L-serine methyl ester and 61.2 g of N,N-diisopropylethylamine were added and stirred until homogeneous. After cooling to 3°C, 9.1 g of HOBT and 78 g of EDCl were added and the reaction was stirred for 30 minutes. The temperature was then raised to 33°C and the reaction was continued for 18 hours. Complete reaction of N-carbobenzoxy-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 164 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0056] (2) Manufacture of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine: 164 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester, an oily substance, was added to 160 g of tetrahydrofuran and 300 g of water. The mixture was cooled to 4°C, and 72.4 g of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the temperature was raised to 23°C and the reaction was allowed to proceed for 3 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was confirmed by TLC. Next, 400 g of ethyl acetate was added to the reaction system, the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution, and the ethyl acetate layer was separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 150 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, an oily substance.

[0057] (3) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester: 150 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was added to 1200 g of ethyl acetate and stirred until completely clarified and dissolved. Then, 133 g of glycine benzyl ester p-toluenesulfonate and 61.8 g of N,N-diisopropylethylamine were added and stirred until homogeneous. After cooling to 2°C, 9.3 g of HOBT and 79 g of EDCl were added and the reaction was stirred for 30 minutes, then the temperature was raised to 33°C and the reaction was allowed to proceed for 18 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 178 g of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-glycine benzyl ester, an oily substance.

[0058] (4) Production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 178 g of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was added to 1060 g of methanol and stirred to dissolve completely until clarified. Then 17.8 g of Pd / C was added, the temperature was controlled to 23°C, nitrogen gas was purged, and then hydrogen gas was introduced, and the reaction was allowed to proceed for 23 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was confirmed by TLC. Next, Pd / C was filtered, and the filtrate was concentrated until a large amount of solid precipitated. After cooling to 2°C and crystallization for 3 hours, it was filtered. The filtered cake was washed with a small amount of methanol and air-dried at 50°C to obtain 86 g of O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. The results showed a molar yield of 70%, an HPLC accuracy of 99.43%, and no isomers were detected.

[0059] [Example 4] (1) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester: 100 g of N-benzyloxycarbonyl-O-tert-butyl-L-serine was added to 800 g of ethyl acetate and stirred until completely clear. Then, 90 g of O-tert-butyl-L-serine methyl ester and 51.3 g of triethylamine were added and stirred until homogeneous. After cooling to 3°C, 9.1 g of HOBT and 78 g of EDCl were added and the mixture was reacted at 0-5°C for 18 hours. TLC confirmed that N-carbobenzoxy-O-tert-butyl-L-serine had not reacted completely. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the mixture was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 79 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0060] (2) Manufacture of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine: 79 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was added to 80 g of tetrahydrofuran and 150 g of water, cooled to 4°C, and 31 g of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the mixture was reacted at 0-5°C for 23 hours. TLC confirmed that the N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester had not yet completely reacted. Next, 400 g of ethyl acetate was added to the reaction system, the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution, and then the ethyl acetate layer was separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 71 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, an oily substance.

[0061] (3) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester: 71 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was mixed with 560 g of ethyl acetate and stirred until completely clarified. Then, 56 g of glycine benzyl ester p-toluenesulfonate and 22 g of triethylamine were added and stirred until homogeneous. After cooling to 2°C, 4.3 g of HOBT and 37 g of EDCl were added and the mixture was reacted at 0-5°C for 18 hours. TLC confirmed that N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine had not reacted completely. Next, 200 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 41 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-glycine benzyl ester.

[0062] (4) Production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 41 g of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was added to 250 g of methanol, and after stirring to completely clarify and dissolve, 4.1 g of Pd / C was added, the temperature was controlled to 23°C, nitrogen gas was purged, and then hydrogen gas was introduced, and the reaction was allowed to proceed for 23 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester was confirmed by TLC. Next, Pd / C was filtered, and the filtrate was concentrated until a large amount of solid precipitated. After cooling to 2°C and crystallization for 3 hours, it was filtered. The filtered cake was washed with a small amount of methanol and air-dried at 50°C to obtain 20 g of O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. The results showed a molar yield of 16%, an HPLC accuracy of 99.28%, and no isomers were detected.

[0063] [Comparative Example 1] This comparative example uses N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, and glycine methyl ester hydrochloride as main raw materials and requires multiple synthesis steps. Moreover, the resulting final compound, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, contains many impurities, making purification difficult and resulting in a low molar yield. Furthermore, this comparative example involves one more reaction step than the example of the present invention.

[0064] (1) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester: 100 g of N-benzyloxycarbonyl-O-tert-butyl-L-serine was added to 800 g of ethyl acetate and stirred until completely clear. Then, 90 g of O-tert-butyl-L-serine methyl ester and 51.3 g of triethylamine were added and stirred until homogeneous. The mixture was then cooled to 3°C, 9.1 g of HOBT and 78 g of EDCl were added, and the mixture was stirred for 30 minutes. The temperature was then raised to 33°C and the reaction was continued for 18 hours. The complete reaction of N-carbobenzoxy-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and the mixture was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 167 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0065] (2) Manufacture of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine: 167 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester, an oily substance, was added to 150 g of tetrahydrofuran and 300 g of water. The mixture was cooled to 4°C, and 67.6 g of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the temperature was raised to 23°C and the reaction was allowed to proceed for 3 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was confirmed by TLC. Next, 400 g of ethyl acetate was added to the reaction system, the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution, and the ethyl acetate layer was separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 153 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, an oily substance.

[0066] (3) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine methyl ester: 153 g of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was added to 1200 g of ethyl acetate and stirred until completely clarified and dissolved. Then, 52.5 g of glycine methyl hydrochloride and 52.8 g of triethylamine were added and stirred until homogeneous. After cooling to 2°C, 9.4 g of HOBT and 79.9 g of EDCl were added and stirred for 30 minutes, then the temperature was raised to 33°C and the reaction was continued for 18 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to separate the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 157 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-glycine methyl ester.

[0067] (4) Preparation of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine: 157 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine methyl ester was added to 160 g of tetrahydrofuran and 300 g of water. The mixture was cooled to 4°C, and 61.7 g of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the mixture was heated to 23°C and reacted for 3 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine methyl ester was confirmed by TLC. Next, 400 g of ethyl acetate was added to the reaction system, and the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution. The ethyl acetate layer was then separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 138 g of the oily substance N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine.

[0068] (5) Production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 138 g of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine was added to 828 g of methanol and stirred to dissolve completely. Then 13.8 g of Pd / C was added, the temperature was controlled to 23°C, nitrogen gas was purged, and then hydrogen gas was introduced, and the reaction was allowed to proceed for 23 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine was confirmed by TLC. Next, Pd / C was filtered, and the filtrate was concentrated until a large amount of solid precipitated. After cooling to 2°C and crystallization for 3 hours, it was filtered. The filtered cake was washed with a small amount of methanol and air-dried at 50°C to obtain 45 g of O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. The results showed a molar yield of 37%, an HPLC yield of 93.6%, and no isomers were detected.

[0069] [Comparative Example 2] This comparative example uses N-tert-butoxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, and glycine methyl ester hydrochloride as the main raw materials and requires multiple synthesis steps. Moreover, the resulting final compound, O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, contains many impurities, making purification difficult and resulting in a low molar yield. Furthermore, this comparative example involves one more reaction step than the example of the present invention.

[0070] (1) Preparation of N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester: 100 g of N-tert-butoxycarbonyl-O-tert-butyl-L-serine was mixed with 800 g of ethyl acetate and stirred until completely cleared and dissolved. Then, 100 g of O-tert-butyl-L-serine methyl ester and 54.2 g of trimethylamine were added and stirred until homogeneous. After cooling to 3°C, 7.5 g of HOBT and 88 g of EDCl were added and stirred for 30 minutes. The temperature was then raised to 33°C and the reaction was allowed to proceed for 18 hours. Complete reaction of N-tert-butoxycarbonyl-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and then the reaction was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 157 g of the oily substance N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

[0071] (2) Manufacturing of N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine: 157 g of the oily substance N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was added to 150 g of tetrahydrofuran and 300 g of water. The mixture was cooled to 4°C, and 75 g of 30% sodium hydroxide aqueous solution was slowly added. After stirring until homogeneous, the temperature was raised to 23°C and the reaction was allowed to proceed for 3 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester was confirmed by TLC. Next, 400 g of ethyl acetate was added to the reaction system, the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution, and the ethyl acetate layer was separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 136 g of the oily substance N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine.

[0072] (3) Preparation of N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine methyl ester: 136 g of N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was mixed with 1100 g of ethyl acetate and stirred until completely clarified. Then, 50.7 g of glycine methyl hydrochloride and 47.6 g of triethylamine were added and stirred until homogeneous. The mixture was then cooled to 2°C, 9 g of HOBT and 77.9 g of EDCl were added, and the mixture was stirred for 30 minutes. The temperature was then raised to 33°C and the reaction was allowed to proceed for 18 hours. Complete reaction of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine was confirmed by TLC. Next, 400 g of water was added to the reaction mixture to quench the reaction, and the mixture was separated to collect the ethyl acetate layer. The ethyl acetate layer was washed with 6N hydrochloric acid aqueous solution, 5% sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 148 g of the oily substance N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-glycine methyl ester.

[0073] (4) Preparation of N-tert-butoxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 148 g of the oily substance N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine methyl ester was added to 150 g of tetrahydrofuran and 300 g of water. The mixture was cooled to 4°C, 62.3 g of 30% sodium hydroxide aqueous solution was slowly added, and the mixture was stirred until homogeneous. The temperature was then raised to 23°C and the reaction was allowed to proceed for 3 hours. Complete reaction of N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine methyl ester was confirmed by TLC. Next, 400 g of ethyl acetate was added to the reaction system, the pH was adjusted to 2-3 with 6N hydrochloric acid aqueous solution, and the ethyl acetate layer was separated by liquid-liquid extraction. The ethyl acetate layer was washed three times with saturated sodium chloride. After washing, the ethyl acetate layer was dried with sodium sulfate, filtered, and concentrated to dryness to obtain 132 g of the oily substance N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine.

[0074] (5) Production of O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine: 132 g of N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine was added to 660 g of dichloromethane, stirred to completely clarify and dissolve, then 65.3 g of trifluoroacetic acid was added, and the reaction was carried out for 4 hours at a controlled temperature of 23°C. Complete reaction of N-tert-butoxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine was confirmed by TLC. Next, the dichloromethane and trifluoroacetic acid were concentrated to dryness, 500 g of methanol was added, and the mixture was cooled to 7°C. The pH was adjusted to 6-7 using 35 g of triethylamine, then cooled to 2°C, crystallized for 3 hours, and filtered. The filtered cake was washed with a small amount of methanol and air-dried at 50°C to obtain 34 g of O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine. The results showed a molar yield of 28%, an HPLC accuracy of 95.3%, and no isomers were detected.

[0075] Table 1 shows the yield and purity data for Example 1, Comparative Example 1, and Comparative Example 2, in which different raw materials were selected.

[0076] [Table 1]

[0077] [Example 5] In this example, the effect of the condensing agent on the target product was investigated. Specifically, the same manufacturing method as in Example 1 was used, except that the type of condensing agent in steps (1) and (3) was changed. The yield and purity of the target product obtained from the experiment are shown in Table 2 below.

[0078] [Table 2]

[0079] [Example 6] In this example, the effect of the condensation reaction temperature on the target product was investigated. Specifically, the same manufacturing method as in Example 1 was used, except that the condensation reaction temperature was changed. The yield and purity of the target product obtained from the experiment are shown in Table 3 below.

[0080] [Table 3]

[0081] [Example 7] In this example, the effect of organic solvents on the target product was investigated. Specifically, the same production method as in Example 1 was used, except that the organic solvent options in steps (1) and (3) were changed. The yield and purity of the target product obtained from the experiment are shown in Table 4 below.

[0082] [Table 4]

[0083] [Example 8] In this example, the effect of sodium hydroxide concentration on the target product was investigated. Specifically, the same manufacturing method as in Example 1 was used, except that the concentration of the sodium hydroxide solution in step (2) was changed. The experimental results, reaction time (reaction time in step (2)) and the purity of the obtained target product are shown in Table 5 below.

[0084] [Table 5]

[0085] [Example 9] In this example, the effect of the saponification reaction temperature on the target product was investigated. Specifically, the same manufacturing method as in Example 1 was used, except that the temperature of the saponification reaction in step (2) was changed. The experimental results, including the reaction time (reaction time in step (2)) and the purity of the obtained target product, are shown in Table 6 below.

[0086] [Table 6]

[0087] Thus, the present invention provides a method for producing O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, a tylzepatide intermediate, which is suitable for scale-up production. This synthesis method requires simple equipment, has good reaction efficiency, low production costs, and the purity of the final product can reach 99.0% or higher.

[0088] The above description of the embodiments is merely for the purpose of understanding the method and spirit of the present invention. Those skilled in the art will understand that various improvements and modifications can be made to the present invention without departing from the principles of the present invention, and that these improvements and modifications are also covered within the scope of protection of the claims of the present invention.

[0089] The above description of the embodiments disclosed herein will enable those skilled in the art to carry out or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of this application. Accordingly, the present invention is not limited to these embodiments shown herein, but should conform to the broadest scope that is consistent with the principles and novel features disclosed herein.

Claims

【Request Item 1】 【Chemistry 1】 O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine, represented by the above formula (I).

2. Step S1) involves condensing N-benzyloxycarbonyl-O-tert-butyl-L-serine and O-tert-butyl-L-serine methyl ester under the action of a coupling agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester represented by the following formula (II), Step S2) involves saponifying the N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an alkali compound to obtain N-benzyloxycarbonyl-O-tert-butyl-L-serine represented by the following formula (III), Step S3) involves condensing the aforementioned N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine and glycine benzyl ester p-toluenesulfonate under the action of a condensing agent and an organic base to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester represented by the following formula (IV), A method for producing o-tert-butyl-L-ceryl-o-tert-butyl-L-ceryl-glycine, comprising step S4) a hydrogenation reaction of the N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester with hydrogen gas under the action of a catalyst to obtain o-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine represented by the following formula (I). 【Chemistry 2】

3. The manufacturing method according to claim 2, characterized in that step S1) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester and an organic solvent of an organic base, then adding a condensing agent to carry out a condensation reaction to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester.

4. The manufacturing method according to claim 2 or 3, characterized in that in step S1), the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-serine, O-tert-butyl-L-serine methyl ester, the condensing agent, and the organic base is 1:(1-2):(1-1.5):(1-1.5).

5. The production method according to claim 2 or 3, characterized in that the coupling agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, DCC / HOSU, HATU, HBTU, and CDI, and the organic base is triethylamine or N,N-diisopropylethylamine.

6. The manufacturing method according to claim 3, characterized in that the condensing agent is added at a temperature of 0 to 5°C, and the condensation reaction is carried out at a temperature of 0 to 50°C for 12 to 24 hours.

7. The production method according to claim 3, characterized in that the organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, acetonitrile, and tetrahydrofuran.

8. The manufacturing method according to claim 2, characterized in that step S2) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester with an organic solvent, then adding a sodium hydroxide solution to carry out a saponification reaction, and finally obtaining N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine.

9. The manufacturing method according to claim 8, characterized in that in step S2), the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine methyl ester to sodium hydroxide is 1:(1-2).

10. The manufacturing method according to claim 8, characterized in that in step S2), the sodium hydroxide solution is added at a temperature of 0 to 5°C, and the saponification reaction is carried out at a temperature of 0 to 40°C for 1 to 6 hours.

11. The manufacturing method according to claim 8, characterized in that in step S2), the concentration of the sodium hydroxide solution is 5 to 60%.

12. The method for producing the product according to claim 8, characterized in that in step S2), the organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, and ethanol.

13. The manufacturing method according to claim 2, characterized in that step S3) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, glycine benzyl ester p-toluenesulfonate, and an organic solvent of an organic base, then adding a condensing agent to carry out a condensation reaction to obtain N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-seryl-glycine benzyl ester.

14. The manufacturing method according to claim 2 or 13, characterized in that in step S3), the molar ratio of N-benzyloxycarbonyl-O-tert-butyl-L-seryl-O-tert-butyl-L-serine, the glycine benzyl ester p-toluenesulfonate, the organic base, and the condensing agent is 1:(1-2):(1-1.5):(1-1.5).

15. The production method according to claim 2 or 13, characterized in that in step S3), the coupling agent is selected from the group consisting of EDCl / HOBT, EDCl / HOSU, DCC / HOSU, HATU, HBTU, and CDI, and the organic base is triethylamine or N,N-diisopropylethylamine.

16. The manufacturing method according to claim 13, characterized in that in step S3), the condensing agent is added at a temperature of 0 to 5°C, and the condensation reaction is carried out at a temperature of 0 to 50°C for 12 to 24 hours.

17. The manufacturing method according to claim 13, characterized in that in step S3), the organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, acetonitrile, and tetrahydrofuran.

18. The manufacturing method according to claim 2, characterized in that step S4) specifically involves mixing N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester with an organic solvent, adding a catalyst, then introducing hydrogen gas to carry out a hydrogenation reaction to obtain O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine.

19. The manufacturing method according to claim 2 or 18, characterized in that in step S4), the mass ratio of the N-benzyloxycarbonyl-O-tert-butyl-L-ceryl-O-tert-butyl-L-ceryl-glycine benzyl ester to the catalyst is 1:(0.01 to 0.2).

20. The manufacturing method according to claim 2 or 18, characterized in that in step S4), the hydrogenation reaction is carried out at a temperature of 20 to 30°C for 20 to 24 hours.

21. The organic solvent is selected from the group consisting of acetonitrile, tetrahydrofuran / water, methanol, ethanol, acetone / water, and ethyl acetate / water, and the catalyst is Pd / C, Raney nickel, and Pd / BaSO4. ４ The manufacturing method according to claim 18, characterized in that it is selected from the group consisting of the following.

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