A method for synthesizing semaglutide dipeptide fragment

Abstract

The invention provides a method for synthesizing a semaglutide dipeptide fragment, and the synthesis route is as follows: the invention selects chloroformate compounds as new condensation reagents, which can activate the carboxyl group of N-protected histidine, improve the reactivity of N-protected histidine, promote the reaction to proceed in the direction of generating a target product, improve the purity and yield of the product, thereby lowering the impurity content and making the reaction more economical and efficient; and the reaction process using the chloroformate compounds as condensation reagents is milder, and no unnecessary modification of 2-aminoisobutyric acid is required, thus shortening the reaction steps, making the operation more simple, the cost lower, and more conducive to industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical intermediate synthesis, and in particular to a method for synthesizing a semaglutide dipeptide fragment. Background Art

[0002] Semaglutide, a long-acting glucagon-like peptide-1 (GLP-1) analog developed by Novo Nordisk of Denmark, can play a hypoglycemic role through multiple mechanisms such as stimulating pancreatic synthesis, secreting insulin and inhibiting glucagon secretion. In addition, it can inhibit appetite and reduce food intake by inhibiting the feeding center, while acting on the gastrointestinal tract to delay gastric emptying, thereby increasing satiety. Therefore, semaglutide not only has a significant hypoglycemic effect, but also has a significant weight loss effect. The injection for the treatment of type 2 diabetes in adults was launched in the United States in 2017 and was approved for marketing in China in 2021. In 2021, the FDA approved the indication for long-term weight management, which was approved for marketing in China in 2024. In 2023, Novo Nordisk's semaglutide sales reached US$21.055 billion, and sales in the first quarter of 2024 alone reached US$6.085 billion, and market demand remains strong. The sequence structure of semaglutide is as follows: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(PEG-PEG-γ-Glu-OctadecanedioicAicd)-Glu-Phe-Lle-Ala-Tr p-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0003] Semaglutide is composed of a 31-amino acid main chain and a fatty acid side chain, with two amino acids His-□Aib at the main chain end (Aib is a non-natural amino acid). There are few reports on the synthesis of semaglutide terminal dipeptide fragments in the reported synthesis literature. The existing synthesis methods of semaglutide dipeptide fragment R1-His-Aib-OH are mainly divided into two categories: solid phase and liquid phase synthesis, as follows:

[0004] Patents CN106928343A, CN101133082A, CN106478806A, and CN109627317A provide solid-phase synthesis methods for semaglutide dipeptide fragments. The specific resins used in the solid-phase synthesis method are expensive, have low yields, many impurities, and are difficult to purify, resulting in extremely high costs. Patent CN112625087A mentions a liquid-phase synthesis method, but it is necessary to use a more expensive condensing agent and activator, which is costly and has residual by-products, posing risks to product quality and is not conducive to industrial production. Patent CN117659159A has an extremely long synthesis step and involves conversions between different N-protecting groups. The process is cumbersome and not conducive to industrial production. CN113667007A makes unnecessary modifications to the carboxyl group of 2-aminoisobutyric acid, complicating simple problems. CN113667006A uses thionyl chloride and high temperature reflux conditions to activate the His carboxyl group, which easily leads to amino acid racemization and increased impurity content, and is not suitable for the synthesis of amino acids.

[0005] In summary, the existing synthesis methods of semaglutide dipeptide fragments have long synthesis steps, long production cycles, low purity and yield, high production costs, and are not conducive to large-scale production; and the condensing agents and activators used will introduce corresponding impurities, posing risks to product quality. Therefore, it is urgent to develop a new synthesis method of semaglutide dipeptide fragments. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for synthesizing a semaglutide dipeptide fragment, which has both simple operation and high yield, a short synthesis route, mild reaction conditions, low cost, and is suitable for industrial production.

[0007] To achieve the above-mentioned and other related purposes, the present invention provides a method for synthesizing a semaglutide dipeptide fragment (R1-His-Aib-OH), and the synthetic route is as follows:

[0008]

[0009] Wherein, R1 is selected from Fmoc or Cbz, and R2 is selected from C1-C3 alkyl.

[0010] The specific preparation steps of the dipeptide fragment R1-His-Aib-OH are as follows:

[0011] (1) adding N-protected histidine (R1-L-His(Trt)-OH) and a chloroformate compound to an organic solvent 1, and reacting under temperature control to obtain an active ester;

[0012] (2) the active ester obtained in step (1) reacts with 2-aminoisobutyric acid in the presence of an organic solvent 2 and a base to obtain an intermediate;

[0013] (3) After removing the Trt protecting group from the intermediate, the target product, i.e., the dipeptide fragment R1-His-Aib-OH, is obtained.

[0014] The preferred embodiment of step (1) is as follows:

[0015] The chloroformate compound includes at least one of methyl chloroformate, ethyl chloroformate, n-propyl chloroformate or isopropyl chloroformate.

[0016] The organic solvent 1 is at least one selected from methyl tert-butyl ether, dichloromethane, chloroform, tetrahydrofuran, and acetonitrile; dichloromethane is particularly preferred.

[0017] The mass volume ratio of N-protected histidine to the organic solvent 1 is 1:1-10.

[0018] The molar ratio of N-protected histidine to chloroformate compound is 1:1.1-2.0, and 1:1.3 is particularly preferred.

[0019] The reaction temperature is controlled at -10°C to 20°C; the reaction time is 1 hour or more; and after the reaction is completed, the reaction is concentrated under reduced pressure.

[0020] The specific steps of step (1) include: adding N-protected histidine to an organic solvent 1, controlling the temperature at -10°C to 20°C, stirring and dissolving, dropping a chloroformate compound, reacting for 1 hour or more after the dropwise addition is completed, and concentrating under reduced pressure.

[0021] The preferred embodiment of step (2) is as follows:

[0022] The molar ratio of N-protected histidine to 2-aminoisobutyric acid is 1:1.0 to 2.0, and 1:1.2 is particularly preferred.

[0023] The base is selected from triethylamine, potassium carbonate or sodium carbonate.

[0024] The molar ratio of N-protected histidine to base is 1:1.2-2.0.

[0025] The organic solvent 2 is selected from one of dichloromethane, chloroform and ethyl acetate; dichloromethane is preferred.

[0026] The mass volume ratio of N-protected histidine to the organic solvent 2 is 1:2-5.

[0027] The specific steps of step (2) include: adding an organic solvent 2, 2-aminoisobutyric acid and a base to the reaction solution of step (1), controlling the temperature at -10°C to 30°C to react until the reaction is completed as monitored by TLC, washing the reaction system once with a 5% citric acid aqueous solution and saturated brine, drying the organic phase over anhydrous sodium sulfate and filtering, and concentrating the filtrate under reduced pressure to obtain an intermediate.

[0028] All methods capable of removing Trt protection in the art are included in the protection scope claimed by the present invention, among which,

[0029] The specific steps of step (3) include: adding the intermediate to an organic solvent 3, controlling the temperature at -10°C to 10°C, adding trifluoroacetic acid, reacting for a period of time, warming the temperature to room temperature and continuing the reaction until the reaction is completed as monitored by HPLC, concentrating under reduced pressure, and beating the concentrate with an organic solvent 4 under temperature control, and drying to obtain the dipeptide fragment R1-His-Aib-OH.

[0030] The organic solvent 3 is selected from tetrahydrofuran, dichloromethane, acetonitrile or N,N-dimethylformamide, wherein tetrahydrofuran is particularly preferred.

[0031] The mass volume ratio of the intermediate to the organic solvent 3 is 1:2-5.

[0032] The mass ratio of the intermediate to trifluoroacetic acid is 1:2.5-5.5.

[0033] The organic solvent 4 is at least one selected from n-hexane, n-heptane, cyclohexane, methyl tert-butyl ether, ethyl acetate and isopropanol.

[0034] The mass volume ratio of the intermediate to the organic solvent 4 is 1:1.5-20.

[0035] The beating temperature is -10℃~30℃.

[0036] The beating times were twice and the beating time was 0.5 hours.

[0037] As described above, the method for synthesizing a semaglutide dipeptide fragment of the present invention has the following beneficial effects:

[0038] 1. The present invention selects chloroformate compounds as new condensation reagents, which can activate the carboxyl group of N-protected histidine, improve the reactivity of N-protected histidine, promote the reaction to generate the target product, improve the purity and yield of the product, thereby lowering the impurity content and making the reaction more economical and efficient; and the reaction process using chloroformate compounds as condensation reagents is milder, and no unnecessary modification of 2-aminoisobutyric acid is required, thus shortening the reaction steps, making the operation simpler, lowering the cost, and more conducive to industrial production.

[0039] 2. In the process of removing Trt, the present invention uses organic solvent 3 as the reaction medium, which can provide a milder and more efficient reaction environment, accelerate the removal of Trt, and reduce the temperature required for the reaction, thereby improving production efficiency, reducing costs, and achieving higher product purity and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a liquid chromatogram of the product Fmoc-His-Aib-OH prepared in Example 7 of the present invention.

[0041] Figure 2 This is the H NMR spectrum of the product Fmoc-His-Aib-OH prepared in Example 7 of the present invention.

[0042] Figure 3 This is the mass spectrum of the product Fmoc-His-Aib-OH prepared in Example 7 of the present invention. DETAILED DESCRIPTION

[0043] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0044] Example 1

[0045] Preparation of intermediate Fmoc-His(Trt)-Aib-OH:

[0046] Fmoc-L-His(Trt)-OH (10 g, 16.14 mmol) and 50 ml of dichloromethane were added to a 250 ml three-necked flask, and ethyl chloroformate (2.28 g, 21.01 mmol) was added dropwise at a temperature of 5°C to 15°C. After the addition was complete, the mixture was stirred for 1 h and concentrated to dryness under reduced pressure. 30 ml of dichloromethane, 2-aminoisobutyric acid (2.47 g, 23.95 mmol) and triethylamine (3.27 g, 32. 32mmol), the temperature was controlled at 5°C ~ 15°C and stirred for 2h, and the reaction was completed after TLC monitoring. The reaction system was then washed once with 20ml of 5% citric acid aqueous solution and 20ml of saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated to dryness under reduced pressure to obtain 11.21g of an off-white solid, i.e., the intermediate Fmoc-His(Trt)-Aib-OH, with a yield of 98.6% and a purity of 97.71%.

[0047] Example 2

[0048] Preparation of intermediate Fmoc-His(Trt)-Aib-OH:

[0049] Fmoc-L-His(Trt)-OH (10 g, 16.14 mmol) and 30 ml of dichloromethane were added to a 250 ml three-necked flask, and ethyl chloroformate (3.50 g, 32.26 mmol) was added dropwise at a temperature of 0°C to 10°C. After the addition was complete, the mixture was stirred for reaction for 2 h and concentrated to dryness under reduced pressure. 40 ml of ethyl acetate, 2-aminoisobutyric acid (3.32 g, 32.20 mmol) and potassium carbonate (2.68 g, 19.39 mmol) were added, and the mixture was stirred for reaction for 2 h at a temperature of 20°C to 30°C. After the reaction was complete, TLC was monitored, and the system was then washed once with 20 ml of a 5% aqueous citric acid solution and 20 ml of saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated to dryness under reduced pressure to obtain 9.79 g of an off-white solid, i.e., the intermediate Fmoc-His(Trt)-Aib-OH, with a yield of 86.1% and a purity of 96.51%.

[0050] Example 3

[0051] Preparation of intermediate Cbz-His(Trt)-Aib-OH:

[0052] Cbz-L-His(Trt)-OH (5 g, 9.41 mmol) and 50 ml of methyl tert-butyl ether were added into a 250 ml three-necked flask, ethyl chloroformate (1.33 g, 12.23 mmol) was added dropwise at a temperature of -10°C to 5°C, the mixture was stirred for reaction for 1 h, and the mixture was concentrated to dryness under reduced pressure; 10 ml of dichloromethane, 2-aminoisobutyric acid (1.00 g, 9.70 mmol) and triethylamine (1.71 g, 16.90 mmol) were added, the mixture was stirred for reaction for 2 h at a temperature of -10°C to 0°C, and the reaction was complete after TLC monitoring. The system was then washed once with 20 ml of a 5% aqueous citric acid solution and 20 ml of saturated brine, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated to dryness under reduced pressure to obtain 5.38 g of an off-white solid intermediate Cbz-His(Trt)-Aib-OH with a yield of 92.8% and a purity of 95.16%.

[0053] Example 4

[0054] Preparation of intermediate Fmoc-His(Trt)-Aib-OH:

[0055] Fmoc-L-His(Trt)-OH (10 g, 16.14 mmol) and 50 ml of dichloromethane were added into a 250 ml three-necked flask, and methyl chloroformate (1.98 g, 20.95 mmol) was added dropwise at a temperature of 5°C to 15°C. After the addition was completed, the mixture was stirred for 1 h and concentrated to dryness under reduced pressure. 60 ml of dichloromethane, 2-aminoisobutyric acid (2.47 g, 23.95 mmol) and triethylamine (3.27 g, 32.3 2mmol), the temperature was controlled at 0℃~15℃ and stirred for reaction for 1h, and the reaction was completed after TLC monitoring. Then the reaction system was washed once with 30ml of 5% citric acid aqueous solution and 30ml of saturated brine respectively, the organic phase was collected, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 10.98g of off-white solid, i.e., intermediate Fmoc-His(Trt)-Aib-OH, with a yield of 94.65% and a purity of 97.52%.

[0056] Example 5

[0057] Preparation of intermediate Fmoc-His(Trt)-Aib-OH:

[0058] Fmoc-L-His(Trt)-OH (10 g, 16.14 mmol) and 50 ml of dichloromethane were added into a 250 ml three-necked flask, and isopropyl chloroformate (2.57 g, 20.97 mmol) was added dropwise at a temperature of 10°C to 20°C. After the addition was complete, the mixture was stirred for 2 h and concentrated to dryness under reduced pressure. 60 ml of dichloromethane, 2-aminoisobutyric acid (2.47 g, 23.95 mmol) and triethylamine (3.27 g, 32. 32mmol), the temperature was controlled at 5°C ~ 15°C and stirred for 1h. After the reaction was completed by TLC monitoring, the reaction system was washed once with 30ml of 5% citric acid aqueous solution and 30ml of saturated brine respectively. The organic phase was collected, dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated to dryness under reduced pressure to obtain 10.98g of off-white solid, i.e., the intermediate Fmoc-His(Trt)-Aib-OH, with a yield of 91.68% and a purity of 97.05%.

[0059] Example 6

[0060] Preparation of intermediate Fmoc-His(Trt)-Aib-OH:

[0061] Fmoc-L-His(Trt)-OH (10 g, 16.14 mmol) and 50 ml of dichloromethane were added into a 250 ml three-necked flask, and n-propyl chloroformate (2.57 g, 20.97 mmol) was added dropwise at a temperature of 10°C to 20°C. After the addition was complete, the mixture was stirred for 2 h and concentrated to dryness under reduced pressure. 60 ml of dichloromethane, 2-aminoisobutyric acid (2.47 g, 23.95 mmol) and triethylamine (3.27 g, 32. 32mmol), the temperature was controlled at 5°C ~ 15°C and stirred for reaction for 1h. After TLC monitoring, the reaction was complete, and then the reaction system was washed once with 30ml of 5% citric acid aqueous solution and 30ml of saturated brine respectively. The organic phase was collected, dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated to dryness under reduced pressure to obtain 10.98g of off-white solid, i.e., the intermediate Fmoc-His(Trt)-Aib-OH, with a yield of 91.13% and a purity of 96.74%.

[0062] Example 7

[0063] Preparation of Fmoc-His-Aib-OH:

[0064] Take 10g of the intermediate Fmoc-His(Trt)-Aib-OH prepared in Example 1 and 35ml of tetrahydrofuran and add them to a 250ml three-necked flask, cool to -5°C, add 40g of trifluoroacetic acid and react for 1h, warm to room temperature, react for 2h and monitor the reaction by HPLC to complete; concentrate the system to dryness under reduced pressure, add 100ml of methyl tert-butyl ether, cool to -5°C and beat for 0.5h; filter, beat the filter cake with 100ml of methyl tert-butyl ether for 0.5h at -5°C; filter, and vacuum dry the filter cake to obtain 6.15g of white powder, i.e., the product Fmoc-His-Aib-OH, with a yield of 93.7% and a purity of 99.71%. See the attached drawings of the specification for liquid phase purity, nuclear magnetic hydrogen spectrum and mass spectrum.

[0065] Example 8

[0066] Preparation of Cbz-His-Aib-OH:

[0067] Take 10g of the intermediate Cbz-His(Trt)-Aib-OH prepared in Example 3 and 35ml of tetrahydrofuran, add them into a 250ml three-necked flask, cool to -5℃~5℃, add 40g of trifluoroacetic acid and react for 1h, warm to room temperature, react for 2h and monitor the reaction completion by HPLC; concentrate the system to dryness under reduced pressure, add 100ml of methyl tert-butyl ether, cool to -10℃~-5℃ and beat for 0.5h; filter, beat the filter cake with 100ml of methyl tert-butyl ether for 0.5h at -10℃~-5℃; filter, and vacuum dry the filter cake to obtain 5.69g of white powder product Cbz-His-Aib-OH with a yield of 93.73% and a purity of 98.25%.

[0068] In summary, the present invention selects chloroformate compounds as new condensation reagents, which can activate the carboxyl group of N-protected histidine, improve the reactivity of N-protected histidine, promote the reaction to generate the target product, improve the purity and yield of the product, thereby lowering the impurity content and making the reaction more economical and efficient; and the reaction process of chloroformate compounds as condensation reagents is milder, and no unnecessary modification of 2-aminoisobutyric acid is required, which shortens the reaction steps, makes the operation more simple, and has lower cost, which is more conducive to industrial production. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0069] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for synthesizing a semaglutide dipeptide fragment, characterized in that: The synthetic route is as follows: Wherein, R1 is selected from Fmoc or Cbz, and R2 is selected from C1-C3 alkyl; The specific preparation steps are as follows: (1) adding N-protected histidine (R1-L-His(Trt)-OH) and a chloroformate compound to an organic solvent 1, controlling the temperature at -10°C to 20°C to react for 1 hour or more, and concentrating under reduced pressure after the reaction is completed to obtain an active ester; Wherein: the chloroformate compound is selected from at least one of methyl chloroformate, ethyl chloroformate, n-propyl chloroformate or isopropyl chloroformate; the organic solvent 1 is selected from at least one of methyl tert-butyl ether, dichloromethane, chloroform, tetrahydrofuran and acetonitrile; the mass volume ratio of N-protected histidine to organic solvent 1 is 1:1-10; the molar ratio of N-protected histidine to chloroformate compound is 1:1.1-2.0; (2) the active ester obtained in step (1) reacts with 2-aminoisobutyric acid in the presence of an organic solvent 2 and a base to obtain an intermediate; Wherein: the molar ratio of N-protected histidine to 2-aminoisobutyric acid is 1:1.0-2.0; the base is selected from triethylamine, potassium carbonate or sodium carbonate; the molar ratio of N-protected histidine to the base is 1:1.2-2.0; the organic solvent 2 is selected from one of dichloromethane, chloroform and ethyl acetate; the mass volume ratio of N-protected histidine to organic solvent 2 is 1:2-5; (3) adding the intermediate to organic solvent 3, controlling the temperature at -10°C to 10°C, adding trifluoroacetic acid, reacting for a period of time, warming to room temperature and continuing the reaction until the reaction is completed as monitored by HPLC, concentrating under reduced pressure, and beating the concentrate with organic solvent 4 under temperature control, and drying to obtain the target product, i.e., the dipeptide fragment R1-His-Aib-OH; Wherein: organic solvent 3 is selected from tetrahydrofuran, dichloromethane, acetonitrile or N,N-dimethylformamide; the mass volume ratio of the intermediate to the organic solvent 3 is 1:2-5; the mass ratio of the intermediate to trifluoroacetic acid is 1:2.5-5.5; organic solvent 4 is selected from at least one of n-hexane, n-heptane, cyclohexane, methyl tert-butyl ether, ethyl acetate and isopropanol; the mass volume ratio of the intermediate to the organic solvent 4 is 1:1.5-20.

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