A method for synthesizing h-aeea-aeea-oh

Abstract

This invention discloses a method for synthesizing H-AEEA-AEEA-OH, in which the side chains of smegglutinin and telpolide both contain the polyethylene glycol-modified H-AEEA-AEEA-OH, chemically named 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid. The method of this invention features a short production cycle, mild reaction conditions, and is suitable for industrial production. The intermediates in each step of the reaction have high purity and high yield, with an overall yield exceeding 85%. The H-AEEA-AEEA-OH obtained by this method contains few impurities, and the specific impurities H-AEEA-OH and H-AEEA-AEEA-AEEA-OH are both small.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis, and specifically relates to a method for synthesizing the side chains of smegglutinin and telpolide. Background Technology

[0002] Polypeptides and proteins, as active substances and the material basis of life activities in organisms, are closely related to the occurrence of various diseases. Polyethylene glycol modification of proteins reduces the immunogenicity of protein drugs and improves many other properties, such as increasing the stability of proteins to enzymes, prolonging the half-life of proteins in plasma, and increasing their solubility in water. Some polyethylene glycol-modified proteins have achieved good results in drug applications.

[0003] Smegglutide, developed by Novo Nordisk, is used to treat type 2 diabetes in adults. It was approved by the FDA on December 6, 2017, but is not yet available in China. It is currently designated as an adjunct to diet and exercise to improve glycemic control in patients with type 2 diabetes.

[0004] Developed by Eli Lilly, telpotetide selectively binds to and activates two natural incretin receptors, GIP and GLP-1, reducing fasting and postprandial blood glucose levels in patients with type 2 diabetes mellitus (T2DM). In May 2022, the FDA approved it for improving glycemic control in adults with type 2 diabetes, making it the world's first GLP-1 / GIP dual-target receptor agonist. In November 2023, the FDA approved telpotetide for improving long-term weight management in obese or overweight adults with at least one comorbidity. Currently, telpotetide has been approved for marketing in several countries, including the EU and Japan, for the treatment of type 2 diabetes.

[0005] Both smegglutinin and telpolide contain the polyethylene glycol-modified compound H-AEEA-AEEA-OH in their side chains, with the chemical name 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid. Their chemical structures are as follows:

[0006]

[0007] Currently, patent CN117185950A discloses a method for preparing compound (I), the route of which is shown below:

[0008]

[0009] The raw material N3-AEEA-OH used in this patent is difficult to obtain, and its synthesis requires sodium azide, which poses safety issues in production and is not suitable for industrial production. Summary of the Invention

[0010] To address the above drawbacks, this invention discloses a novel method for preparing compound (I), the route of which is shown below. The synthetic route of H-AEEA-AEEA-OH is as follows:

[0011]

[0012] When examining the carboxyl activating reagents, it was found that when compound (III) reacted with N-hydroxysuccinimide (HOSu), very little of compound (VI) was obtained, and a large amount of compound (III) remained unreacted. Purification could only be achieved by column chromatography, resulting in a very low yield. However, when reacting with pentafluorophenol (PFP-OH), compound (III) reacted completely, and after post-treatment, compound (IV) was obtained as a solid with higher purity and yield.

[0013]

[0014] According to one aspect of the present invention, the method includes the following steps:

[0015] Compound (II) was reacted with an amino acid protecting agent under alkaline conditions to prepare compound (III).

[0016] According to one aspect of the present invention, the alkali used in the method is selected from sodium carbonate, sodium bicarbonate, potassium carbonate, and preferably sodium carbonate.

[0017] According to one aspect of the present invention, the amino acid protecting agent in the method is selected from benzyloxycarbonyl succinimide (Z-OSu), benzyl chloroformate (Z-Cl), and preferably benzyloxycarbonyl succinimide (Z-OSu).

[0018] According to one aspect of the present invention, the reaction molar ratio of the base and the amino acid protectant in the method is selected from 1.0:1.0, 1.1:1.1, 1.2:1.2, 1.5:1.5, 2.0:2.0, preferably 1.1:1.1.

[0019] According to one aspect of the present invention, the reaction temperature in the method is selected from 0 to 5 degrees Celsius, 5 to 15 degrees Celsius, 15 to 25 degrees Celsius, 25 to 35 degrees Celsius, and preferably 0 to 5 degrees Celsius.

[0020] According to one aspect of the present invention, the reaction time in the method is selected from 8h, 12h, 24h, 36h, preferably 12h.

[0021] According to one aspect of the present invention, the reaction solvent in the method is selected from the acetone-water system, the tetrahydrofuran-water system, the 1,4-dioxane-water system, and preferably the tetrahydrofuran-water system.

[0022] Compound (III) was prepared into compound (IV) in the presence of a condensing agent and a carboxyl activating agent.

[0023] According to one aspect of the present invention, the condensing agent in the method is selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), preferably dicyclohexylcarbodiimide (EDCI).

[0024] According to one aspect of the present invention, the carboxyl activating agent in the method is selected from pentafluorophenol (PFP-OH), N-hydroxysuccinimide (HOSu), and preferably pentafluorophenol (PFP-OH).

[0025] According to one aspect of the present invention, the reaction molar ratio of the condensing agent and the carboxyl activating agent in the method is selected from 1.0:1.0, 1.1:1.1, 1.2:1.2, 1.5:1.5, 2.0:2.0, preferably 1.1:1.1.

[0026] According to one aspect of the present invention, the reaction temperature in the method is selected from -5 to 5 degrees Celsius, 5 to 15 degrees Celsius, 15 to 25 degrees Celsius, 25 to 35 degrees Celsius, preferably -5 to 5 degrees Celsius.

[0027] According to one aspect of the present invention, the reaction time in the method is selected from 8h, 12h, 24h, 36h, preferably 12h.

[0028] According to one aspect of the present invention, the reaction solvent in the method is selected from dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, N-methylpyrrolidone, ethyl acetate, 1,4-dioxane, preferably dichloromethane.

[0029] Compound (IV) and compound (II) were condensed under alkaline conditions to prepare compound (V).

[0030] According to one aspect of the present invention, the base used in the method is selected from triethylamine, diisopropylethylamine, N-methylmorpholine, sodium carbonate, sodium bicarbonate, potassium carbonate, preferably sodium carbonate.

[0031] According to one aspect of the present invention, the equivalent of the alkali in the method is selected from 1 to 5 equivalents, preferably 3 equivalents.

[0032] According to one aspect of the present invention, the equivalent amount of the compound of formula (II) in the method is selected from 1 to 5 equivalents, preferably 2 equivalents.

[0033] According to one aspect of the present invention, the reaction temperature in the method is selected from -5 to 5 degrees Celsius, 5 to 15 degrees Celsius, 15 to 25 degrees Celsius, 25 to 35 degrees Celsius, preferably 15 to 25 degrees Celsius.

[0034] According to one aspect of the present invention, the reaction time in the method is selected from 8h, 12h, 24h, 36h, preferably 12h.

[0035] According to one aspect of the present invention, the reaction solvent in the above method is selected from dichloromethane, tetrahydrofuran, acetone, ethyl acetate, acetonitrile, methyl tert-butyl ether, preferably tetrahydrofuran.

[0036] Compound (V) was deprotected by hydrogen under pressure in the presence of a catalyst to obtain compound (I).

[0037] According to one aspect of the present invention, the catalyst used in the method is selected from Pd / C, Pd(OAc)2, and preferably Pd / C.

[0038] According to one aspect of the invention, the hydrogen pressure in the method is selected from 5 to 15 atm, preferably 8 to 10 atm.

[0039] According to one aspect of the present invention, the reaction temperature in the method is selected from 20 to 35 degrees Celsius, 35 to 45 degrees Celsius, 45 to 55 degrees Celsius, and preferably 35 to 45 degrees Celsius.

[0040] According to one aspect of the present invention, the reaction time in the method is selected from 8h, 12h, 24h, 36h, preferably 12h.

[0041] According to one aspect of the present invention, the reaction solvent in the method is selected from methanol, ethanol, and preferably methanol.

[0042] According to one aspect of the present invention, the crystallization solvent in the method is selected from ethanol, isopropanol, and preferably ethanol.

[0043] The raw materials used in this method are readily available, the synthesis route is short, the conditions are mild, the yield is high, the product quality is good, and it is suitable for industrial production.

[0044] This invention uses compound (II) as a starting material, and after protecting the amino group with Cbz, prepares compound (III). Compound (III) is then activated with pentafluorophenol to prepare compound (IV). Compound (IV) and compound (II) are condensed to prepare compound (V). Compound (V) is then deprotected by hydrogenation to prepare compound (I).

[0045] This invention provides a method for synthesizing the polyethylene glycol-modified compound H-AEEA-AEEA-OH. This method has a short production cycle, mild reaction conditions, and is suitable for industrial production. The intermediates in each step of the reaction have high purity and high yield, with an overall yield exceeding 85%. The H-AEEA-AEEA-OH obtained by this method has a low number of impurities, and the specific impurities H-AEEA-OH and H-AEEA-AEEA-AEEA-OH are both small. Attached Figure Description

[0046] Figure 1 The liquid phase detection spectrum of intermediate (III);

[0047] Figure 2 The liquid phase detection spectrum of intermediate (IV);

[0048] Figure 3 The liquid phase detection spectrum of intermediate (V);

[0049] Figure 4 The liquid phase detection spectrum of intermediate (I);

[0050] Figure 5 The liquid phase detection spectrum is for intermediate (VI). Detailed Implementation

[0051] The following embodiments are for further illustration of some preferred embodiments of the present invention and are not all embodiments. Other embodiments based on the present invention made by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0052] In this invention, unless otherwise specified, all abbreviations have the conventional meanings understood by those skilled in the art.

[0053] Example 1 Preparation of compound (III)

[0054]

[0055] Take 81.6 g of compound (II), 58.3 g of sodium carbonate, and 400 mL of water. Stir and cool to 0–5°C. Add 137.1 g of Z-OSu and 400 mL of tetrahydrofuran solution dropwise. After the addition is complete, react at 0–5°C for 12 h. Concentrate the reaction solution. Add 400 mL of dichloromethane and 200 mL of water to the concentrate and stir to separate the layers. Adjust the pH of the aqueous layer to 2 with hydrochloric acid, add 400 mL of dichloromethane, and extract three times. Combine the dichloromethane layers, wash with water, dry, and concentrate to obtain a pale yellow oily substance of compound (III) with a purity of 99.47% and a yield of 98%. Its HPLC chromatogram is shown below. Figure 1 As shown.

[0056] Example 2 Preparation of compound (III)

[0057] Take 30 g of compound (II), 21.4 g of sodium carbonate, and 150 mL of water. Stir and cool to 0–5°C. Add 50.4 g of Z-OSu and 150 mL of tetrahydrofuran solution dropwise. After the addition is complete, react at 0–5°C for 12 h. Concentrate the reaction solution. Add 150 mL of dichloromethane and 100 mL of water to the concentrate and stir to separate the layers. Adjust the pH of the aqueous layer to 2 with hydrochloric acid, add 150 mL of dichloromethane, and extract three times. Combine the dichloromethane layers, wash with water, dry, and concentrate to obtain a pale yellow oily substance of compound (III) with a purity of 99.36% and a yield of 97%.

[0058] Example 3 Preparation of compound (IV)

[0059]

[0060] Take 99.1 g of compound (III) and 990 mL of dichloromethane, cool to 0–5 °C, add 67.5 g of pentafluorophenol and 70.3 g of EDCI, and react at 0–5 °C for 12 h. The reaction solution is washed with acid, alkali, and saturated brine, then dried, filtered, and concentrated. The concentrate is crystallized in 900 mL of n-heptane, filtered, and dried to obtain a white solid of compound (IV) with a purity of 97.90% and a yield of 97%. Its HPLC chromatogram is shown below. Figure 2 As shown.

[0061] Example 4 Preparation of compound (IV)

[0062] Take 50 g of compound (III) and 500 mL of dichloromethane, cool to 0–5 °C, add 34.1 g of pentafluorophenol and 35.5 g of EDCI, and react at 0–5 °C for 12 h. The reaction solution is washed with acid, alkali, and saturated brine, then dried, filtered, and concentrated. The concentrate is crystallized in 500 mL of n-heptane, filtered, and dried to obtain compound (IV) as an off-white solid with a purity of 97.85% and a yield of 97%.

[0063] Example 5 Preparation of compound (V)

[0064]

[0065] Take 81.5 g of compound (II), 79.5 g of sodium carbonate, and 600 mL of water. Stir and cool to 0–5°C. Add 115.8 g of compound (IV) and 600 mL of tetrahydrofuran solution dropwise. After the addition is complete, raise the temperature to 15–25°C and react overnight. Concentrate the reaction solution, add 600 mL of dichloromethane to the concentrate, and stir to separate the layers. Adjust the pH of the aqueous layer to 2 with hydrochloric acid, add 600 mL of dichloromethane, and extract three times. Combine the dichloromethane layers, wash with water, dry, and concentrate to obtain a pale yellow oily substance of compound (V) with a purity of 98.13% and a yield of 98%. Its HPLC chromatogram is shown below. Figure 3 As shown.

[0066] Example 6 Preparation of compound (V)

[0067] Take 32.6 g of compound (II), 31.8 g of sodium carbonate, and 300 mL of water. Stir and cool to 0–5°C. Add 46.3 g of compound (IV) and 300 mL of tetrahydrofuran solution dropwise. After the addition is complete, raise the temperature to 15–25°C and react overnight. Concentrate the reaction solution, and add 300 mL of dichloromethane to the concentrate while stirring to separate the layers. Adjust the pH of the aqueous layer to 2 with hydrochloric acid, and extract three times with 300 mL of dichloromethane. Combine the dichloromethane layers, wash with water, dry, and concentrate to obtain a pale yellow oily substance of compound (V) with a purity of 98.16% and a yield of 98%.

[0068] Example 7 Synthesis of compound (I)

[0069]

[0070] Take 110.6 g of compound (V), 1500 mL of methanol, Pd / C: 11 g, purge three times with nitrogen, and react overnight at 35–45°C with hydrogen at 8–10 atm. Filter the reaction solution, concentrate, add 800 mL of ethanol to the concentrate to crystallize, filter, and dry to obtain compound (I) as an off-white solid with a purity of 99.66%. Impurity AEEA was not detected, and impurity tri-AEEA: 0.07%. The yield was 95%. Its HPLC chromatogram is shown below. Figure 4 As shown.

[0071] Example 8 Synthesis of compound (I)

[0072] Take 44.2 g of compound (V), 700 mL of methanol, Pd / C: 4.4 g, purge three times with nitrogen, and react overnight at 35–45°C with hydrogen at 8–10 atm. Filter the reaction solution, concentrate it, add 400 mL of ethanol to the concentrate to induce crystallization, filter, and dry to obtain compound (I) as an off-white solid with a purity of 99.59%. Impurity AEEA was undetectable, and impurity tri-AEEA: 0.08%. Yield: 96%.

[0073] Example 9 Synthesis of compound (VI)

[0074]

[0075] Take 38g of compound (III), 61.8g of EDCI, 34.4g of HOSu, and 450mL of dichloromethane. React at 0-5°C for 12 hours. Wash the reaction solution with 10% sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated brine, respectively. Dry the dichloromethane layer, filter, and concentrate to obtain a light yellow oily substance of compound (VI) with a purity of 42.31% and compound (III) with a purity of 30.30%. Its HPLC chromatogram is shown below. Figure 5 As shown.

[0076] Synthesis of Compound (VI) in Example 10

[0077] Take 26g of compound (III), 25.7g of EDCI, 15.5g of HOSu, and 260mL of dichloromethane. React at 20-25 degrees Celsius for 12 hours. Wash the reaction solution with 10% sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated brine, respectively. Dry the dichloromethane layer, filter, and concentrate to obtain a light yellow oily substance of compound (VI) with a purity of 29.42% and compound (III) with a purity of 26.90%.

[0078] It should be noted that the above preferred embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for synthesizing H-AEEA-AEEA-OH, characterized in that, The synthesis route is as follows: The carboxyl activator of compound (III) is pentafluorophenol (PFP-OH), and the reaction is post-treated to obtain compound (IV).

2. The method for synthesizing H-AEEA-AEEA-OH according to claim 1, characterized in that, Compound (II) was reacted with an amino acid protectant under alkaline conditions to prepare compound (III).

3. The method for synthesizing H-AEEA-AEEA-OH according to claim 2, characterized in that, The amino acid protecting agent is benzooxycarbonyl succinimide (Z-OSu) or benzyl chloroformate (Z-Cl).

4. The method for synthesizing H-AEEA-AEEA-OH according to claim 3, characterized in that, The molar ratio of the base to the amino acid protecting agent is 1.0:1.0, 1.1:1.1, 1.2:1.2, 1.5:1.5, or 2.0:2.0; the reaction temperature is 0–5°C, 5–15°C, 15–25°C, or 25–35°C; the reaction time is 8 h, 12 h, 24 h, or 36 h; and the reaction solvent is an acetone-water system, a tetrahydrofuran-water system, or a 1,4-dioxane-water system.

5. The method for synthesizing H-AEEA-AEEA-OH according to claim 1, characterized in that, Compound (III) was prepared into compound (IV) in the presence of a condensing agent and a carboxyl activating agent, wherein the condensing agent was dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl) or carbodiimide hydrochloride (EDCI).

6. The method for synthesizing H-AEEA-AEEA-OH according to claim 5, characterized in that, The molar ratio of the condensing reagent to the carboxyl activating reagent is 1.0:1.0, 1.1:1.1, 1.2:1.2, 1.5:1.5, or 2.0:2.0; the reaction temperature is selected from -5 to 5 degrees Celsius, 5 to 15 degrees Celsius, 15 to 25 degrees Celsius, or 25 to 35 degrees Celsius; the reaction time is selected from 8 h, 12 h, 24 h, or 36 h; and the reaction solvent is dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, N-methylpyrrolidone, ethyl acetate, or 1,4-dioxane.

7. The method for synthesizing H-AEEA-AEEA-OH according to claim 1, characterized in that, Compound (IV) and compound (II) are condensed under basic conditions to obtain compound (V), wherein the base is selected from triethylamine, diisopropylethylamine, N-methylmorpholine, sodium carbonate, sodium bicarbonate, or potassium carbonate, the equivalent of the base is 1 to 5 equivalents, the equivalent of compound (II) is 1 to 5 equivalents, the reaction temperature is -5 to 5 degrees Celsius, 5 to 15 degrees Celsius, 15 to 25 degrees Celsius, or 25 to 35 degrees Celsius, and the reaction solvent is dichloromethane, tetrahydrofuran, acetone, ethyl acetate, acetonitrile, or methyl tert-butyl ether.

8. The method for synthesizing H-AEEA-AEEA-OH according to claim 1, characterized in that, Compound (V) is deprotected by hydrogen under pressure in the presence of a catalyst to obtain compound (I), wherein the catalyst is Pd / C or Pd(OAc)2.

9. The method for synthesizing H-AEEA-AEEA-OH according to claim 8, characterized in that, The hydrogen pressure is 5–15 atm, the reaction temperature is 20–35 degrees Celsius, 35–45 degrees Celsius, and 45–55 degrees Celsius, the reaction time is 8 h, 12 h, 24 h, and 36 h, and the reaction solvent is methanol or ethanol.

10. The method for synthesizing H-AEEA-AEEA-OH according to claim 1, characterized in that, The crystallization solvent in the method is ethanol or isopropanol.

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