A continuous preparation method of semaglutide

Abstract

The present invention discloses a continuous preparation method of semaglutide, which belongs to the field of chemical pharmaceutical technology. Using twenty-nonapeptide (lysine protected by Boc) as the starting material, condensation with Trt-isobutyric acid to obtain a thirty-peptide intermediate; Trt-histidine active ester and de-Trt thirty-peptide intermediate condensation to obtain a thirty-one peptide intermediate; side chain active ester and thirty-one peptide reaction to obtain a semaglutide precursor; semaglutide precursor deprotection to obtain semaglutide. The reaction is prepared by adjusting the pH value without separating the intermediate state. Inorganic acid and alkali are used to adjust the pH, and water is used as a solvent for all reactions to achieve environmental friendliness. The total reaction yield is about 65%, and the HPLC purity is ≥99%.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical pharmacy, and in particular to a continuous preparation method of semaglutide. Background Art

[0002] Semaglutide, chemical name: N ε26 [(S)-(22,40-dicarboxylic acid-10,19,24-trioxo-3,6,12-15-tetraoxa-9,18,23-triazatetracontan-1-yl)] [Aib 8 ,Arg 34 ]GLP-1 (7-37) is used for lowering blood sugar and also for losing weight. The mechanism of action of semaglutide in lowering blood sugar is glucagon-like peptide 1 (GLP-1) receptor agonist. This type of drug is only available in subcutaneous injection form. Glucagon-like peptide is a kind of "incretin" secreted by the gastrointestinal mucosa of the human body. It has the effect of inhibiting glucagon secretion and promoting insulin secretion. Glucagon-like peptide 1 receptor agonist drugs enhance the secretion of insulin in the human body and inhibit the secretion of glucagon in a glucose concentration-dependent manner to achieve the purpose of regulating blood sugar, so they are suitable for blood sugar control in type 2 diabetes.

[0003] At present, the methods for preparing semaglutide mainly include solid phase synthesis and semi-synthesis. In the prior art, the invention applications with patent numbers CN201510459093.9, CN201811653280.0, CN202210698338.3, and CN202211159916.2 all involve methods for preparing semaglutide. The solid phase synthesis method continuously generates a large amount of waste by protection and deprotection during the preparation process, and its cost is relatively high. The semi-synthesis method mainly obtains twenty-nonapeptides by biological methods, and then connects dipeptides and side chains, or first connects side chains and then dipeptides, and then deprotects; organic solvents such as DMF, acetonitrile, and methanol are used in the reaction process, which will have a huge impact on the environment. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a continuous preparation method of semaglutide, which has the advantages of simple operation, environmental friendliness, high yield and high purity.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a continuous preparation method of semaglutide, the preparation method mainly comprising the following steps:

[0006] 1) The twenty-nonadecanopeptide and Trt-isobutyric acid active ester are subjected to condensation reaction using pure water as solvent to obtain a thirty-nonadecanopeptide intermediate;

[0007] 2) removing Trt protection from the thirty-peptide intermediate to obtain a de-Trt thirty-peptide intermediate;

[0008] 3) condensing the Trt-histidine active ester with the de-Trt thirty-peptide intermediate to obtain a thirty-one-peptide intermediate;

[0009] 4) adding a side chain active ester to react with the thirty-one peptide intermediate to obtain a semaglutide precursor;

[0010] 5) deprotecting the semaglutide precursor to obtain a crude semaglutide product;

[0011] Wherein, the nonadecanepeptide is a lysine protected by Boc;

[0012] The specific preparation route is as follows:

[0013]

[0014]

[0015] The structure of the side chain is as follows:

[0016]

[0017] Furthermore, the preparation method is a continuous reaction, and the intermediate is not separated during the reaction process. Semaglutide is prepared by adjusting the pH value of the reaction system.

[0018] Furthermore, the pH adjustment adopts inorganic acid or inorganic base, the inorganic acid includes one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sodium dihydrogen phosphate, and the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonia water, calcium hydroxide, and sodium phosphate.

[0019] Furthermore, the pH value of step 1) is 8-9; the pH value of step 2) is 3-7; the pH value of step 3) is 8-10; in step 4), the pH value is first adjusted to 1-2 and then to 9-11; in step 5), the pH value is first adjusted to 1-2 and then to 5-6.

[0020] Furthermore, the molar ratio of the Trt-isobutyric acid active ester to the twenty-nonapeptide is 1 to 2:1; the molar ratio of the Trt-histidine active ester to the de-Trt thirty-peptide intermediate is 1 to 2:1; and the molar ratio of the side chain active ester to the thirty-undeceptide intermediate is 1 to 2:1.

[0021] Furthermore, the molar ratio of the Trt-isobutyric acid active ester to the twenty-nonapeptide is 1 to 1.2:1; the molar ratio of the Trt-histidine active ester to the de-Trt thirty-peptide intermediate is 1 to 1.2:1; and the molar ratio of the side chain active ester to the thirty-undeceptide intermediate is 1 to 1.2:1.

[0022] Furthermore, in the Trt-isobutyric acid active ester, Trt-histidine active ester and side chain active ester, the R substituent group is a combination of any one or more of pentafluorophenoxy, succinimide, phthalimide, N-5-norbornene-2,3-dicarboximide, 1-benzotriazole and 7-azobenzotriazole.

[0023] Furthermore, the mobile phase used in the purification process of the crude semaglutide is alcohol and water, and the alcohol includes methanol, ethanol, and isopropanol.

[0024] The beneficial effects of the present invention are as follows: compared with the prior art, the preparation method of semaglutide provided by the present invention adopts a continuous reaction, and semaglutide is prepared without separating the intermediate state by adjusting the pH; pure water is used as a solvent, which is environmentally friendly; the yield of the prepared semaglutide can reach 65%, and the HPLC purity is greater than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 and 2 This is the mass spectrum of semaglutide prepared in Example 1.

[0026] Figure 3 This is a HPLC chart of semaglutide prepared in Example 1.

[0027] Figure 4 This is the HPLC chart of semaglutide prepared in Example 1. DETAILED DESCRIPTION

[0028] The present invention is further described below by specific examples, but these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0029] Example 1

[0030] At 10-25°C, add 3.27 g (0.001 mol) of 29-neck peptide (lysine protected by Boc) to a 50 ml two-necked bottle, add 20 ml of purified water, stir, add 0.425 g (0.001 mol) of Trt-Aib-OSu, adjust the pH to about 8.5, continue stirring for 1-2 hours, and obtain the 30-neck peptide (intermediate 1).

[0031]

[0032] The pH was adjusted to about 3 with 2N hydrochloric acid, and the reaction was stirred for 1-2 hours. The pH was further adjusted to 7 with sodium carbonate to obtain the Trt-free tridecapeptide (intermediate 2).

[0033]

[0034] 0.72 g (0.001 mol) of Trt-His (Trt)-OSu was added, and the pH was adjusted to about 8.5. Stirring was continued for 2-3 h to obtain a thirty-undecene peptide (intermediate 3).

[0035]

[0036] The pH was adjusted to about 1.5 with hydrochloric acid, and the reaction was carried out for 2 hours to remove the Boc on lysine. The pH was then adjusted to about 10.5 with sodium carbonate, and 0.83 g (0.001 mol) of the side chain -OSu active ester was added. The reaction was continued for 2-3 hours to obtain the semaglutide precursor (intermediate 4).

[0037]

[0038] 2N hydrochloric acid was added to the reaction system, the pH was maintained at about 1.5, and the mixture was stirred for 2 hours. The pH was adjusted to about 5.3, solid was precipitated, and the mixture was centrifuged to obtain crude semaglutide.

[0039]

[0040] The crude semaglutide was used to prepare a liquid phase by an isopropanol / water system to obtain 2.71 g of semaglutide. The yield was 65.9%. The HPLC purity was greater than 99%. MS: m / z=4112.15.

[0041] Example 2

[0042] At 10-25°C, add 3.27g (0.001mol) of 29-neck peptide (lysine protected by Boc) to a 50ml two-necked bottle, add 20ml of purified water, stir, add 0.48g (0.001mol) of Trt-Aib-OPHT, adjust the pH to about 8.5, continue stirring for 1-2h, and obtain 30-neck peptide.

[0043] The pH was adjusted to about 3 with 2N hydrochloric acid, and the reaction was stirred for 1-2 hours. The pH was further adjusted to 7 with sodium carbonate to obtain the Trt-free thirty-peptide.

[0044] 0.77 g (0.001 mol) of Trt-His (Trt)-OPHT was added, and the pH was adjusted to about 8.5. Stirring was continued for 2-3 h to obtain thirty-one peptides.

[0045] The pH was adjusted to about 1.5 with 2N hydrochloric acid, and the reaction was carried out for 2 hours to remove the Boc on lysine. The pH was then adjusted to about 10.5 with sodium carbonate, and 0.83 g (0.001 mol) of the side chain -OSu active ester was added. The reaction was continued for 2-3 hours to obtain the semaglutide precursor.

[0046] 2N hydrochloric acid was added to the reaction system, the pH was maintained at about 2, and the mixture was stirred for 2 h. The pH was adjusted to about 5.3, solid was precipitated, and the mixture was centrifuged to obtain crude semaglutide.

[0047] The crude semaglutide was used to prepare a liquid phase through an isopropanol / water system to obtain 2.68 g of semaglutide with a yield of 65.0% and a HPLC purity of more than 99%.

[0048] Example 3

[0049] At 10-25°C, add 3.27g (0.001mol) of 29-neck peptide (lysine protected by Boc) to a 50ml two-necked bottle, add 20ml of purified water, stir, add 0.52g (0.001mol) of Trt-Aib-OPFP, adjust the pH to about 9.0, continue stirring for 1-2h, and obtain 30-neck peptide.

[0050] The pH was adjusted to about 3 with 2N hydrochloric acid, and the reaction was stirred for 1-2 hours. The pH was further adjusted to 7 with sodium carbonate to obtain the Trt-free thirty-peptide.

[0051] Add 0.81 g (0.001 mol) of Trt-His (Trt)-OPFP, adjust the pH to about 9.5, and continue stirring for 2-3 hours to obtain thirty-one peptides.

[0052] The pH was adjusted to about 1.5 with 2N hydrochloric acid, and the reaction was carried out for 2 hours to remove the Boc on lysine. The pH was then adjusted to about 10.5 with sodium carbonate, and 0.83 g (0.001 mol) of the side chain -OSu active ester was added. The reaction was continued for 2-3 hours to obtain the semaglutide precursor.

[0053] 2N hydrochloric acid was added to the reaction system, the pH was maintained at about 2, and the mixture was stirred for 2 h. The pH was adjusted to about 5.3, solid was precipitated, and the mixture was centrifuged to obtain crude semaglutide.

[0054] The crude semaglutide was used to prepare a liquid phase by an isopropanol / water system to obtain 2.71 g of semaglutide with a yield of 65.7% and a HPLC purity of more than 99%.

[0055] Example 4

[0056] At 10-25°C, add 3.27g (0.001mol) of 29-neck peptide (lysine protected by Boc) to a 50ml two-necked bottle, add 20ml of purified water, stir, add 0.49g (0.001mol) of Trt-Aib-ONB, adjust the pH to about 8.0, continue stirring for 1-2h, and obtain 30-neck peptide.

[0057] The pH was adjusted to about 3 with 2N hydrochloric acid, and the reaction was stirred for 1-2 hours. The pH was further adjusted to 7 with sodium carbonate to obtain the Trt-free thirty-peptide.

[0058] Add 0.79 g (0.001 mol) of Trt-His (Trt)-ONB, adjust the pH to about 8.5, and continue stirring for 2-3 hours to obtain thirty-one peptides.

[0059] The pH was adjusted to about 1.5 with 2N hydrochloric acid, and the reaction was carried out for 2 hours to remove the Boc on lysine. The pH was then adjusted to about 10.5 with sodium carbonate, and 0.83 g (0.001 mol) of the side chain -OSu active ester was added. The reaction was continued for 2-3 hours to obtain the semaglutide precursor.

[0060] 2N hydrochloric acid was added to the reaction system, the pH was maintained at about 2, and the mixture was stirred for 2 h. The pH was adjusted to about 5.3, solid was precipitated, and the mixture was centrifuged to obtain crude semaglutide.

[0061] The crude semaglutide was used to prepare a liquid phase through an isopropanol / water system to obtain 2.69 g of semaglutide with a yield of 65.5% and a HPLC purity of more than 99%.

[0062] Example 5

[0063] At 10-25°C, add 3.27g (0.001mol) of 29-neck peptide (lysine protected by Boc) to a 50ml two-necked bottle, add 20ml of purified water, stir, add 0.425g (0.001mol) of Trt-Aib-OSu, adjust the pH to about 8.5, continue stirring for 1-2h, and obtain 30-neck peptide.

[0064] The pH was adjusted to about 3 with 2N hydrochloric acid, and the reaction was stirred for 1-2 hours. The pH was further adjusted to 7 with sodium carbonate to obtain the Trt-free thirty-peptide.

[0065] Add 0.72 g (0.001 mol) of Trt-His (Trt)-OSu, adjust the pH to about 8.5, and continue stirring for 2-3 hours to obtain thirty-one peptides.

[0066] The pH was adjusted to about 1.5 with 2N hydrochloric acid, and the reaction was carried out for 2 hours to remove the Boc on lysine. The pH was then adjusted to about 9.5 with sodium carbonate, and 0.84 g (0.001 mol) of the side chain -OBt active ester was added. The reaction was continued for 2-3 hours to obtain the semaglutide precursor.

[0067] 2N hydrochloric acid was added to the reaction system, the pH was maintained at about 1.5, and the mixture was stirred for 2 hours. The pH was adjusted to about 5.3, solid was precipitated, and the mixture was centrifuged to obtain crude semaglutide.

[0068] The crude semaglutide was used to prepare a liquid phase through an isopropanol / water system to obtain 2.68 g of semaglutide with a yield of 65.0% and a HPLC purity of more than 99%.

[0069] The above implementation modes are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention. The patent protection scope of the present invention should be defined by the claims.

Claims

1. A continuous preparation method of semaglutide, characterized in that: The preparation method mainly comprises the following steps: 1) The twenty-nonadecanopeptide and Trt-isobutyric acid active ester are subjected to condensation reaction using pure water as solvent to obtain a thirty-nonadecanopeptide intermediate; 2) removing Trt protection from the thirty-peptide intermediate to obtain a Trt-free thirty-peptide intermediate; 3) condensing the Trt-histidine active ester with the de-Trt thirty-peptide intermediate to obtain a thirty-one-peptide intermediate; 4) adding a side chain active ester to react with the thirty-one peptide intermediate to obtain a semaglutide precursor; 5) deprotecting the semaglutide precursor to obtain a crude semaglutide product; Wherein, the lysine of the twenty nonapeptide is protected by Boc; The specific preparation route is as follows: The structure of the side chain is as follows: The preparation method is a continuous reaction, and the intermediate is not separated during the reaction process. Semaglutide is prepared by adjusting the pH value of the reaction system; the pH value of step 1) is 8-9; the pH value of step 2) is 3-7; the pH value of step 3) is 8-10; in step 4), the pH value is first adjusted to 1-2, and then adjusted to 9-11; in step 5), the pH value is first adjusted to 1-2, and then adjusted to 5-6; In the Trt-isobutyric acid active ester, Trt-histidine active ester and side chain active ester, the R substituent group is any one or more of a pentafluorophenoxy group, a succinimide group, a phthalimide group and a N-5-norbornene-2,3-dicarboximide group.

2. The continuous preparation method of semaglutide according to claim 1, characterized in that: The pH is adjusted by using an inorganic acid or an inorganic base, wherein the inorganic acid includes one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sodium dihydrogen phosphate; and the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonia water, calcium hydroxide, and sodium phosphate.

3. The continuous preparation method of semaglutide according to claim 1, characterized in that: The molar ratio of the Trt-isobutyric acid active ester to the twenty-nonapeptide is 1 to 2:1; the molar ratio of the Trt-histidine active ester to the de-Trt thirty-peptide intermediate is 1 to 2:1; and the molar ratio of the side chain active ester to the thirty-undeceptide intermediate is 1 to 2:

1.

4. The continuous preparation method of semaglutide according to claim 3, characterized in that: The molar ratio of the Trt-isobutyric acid active ester to the twenty-nonapeptide is 1 to 1.2:1; the molar ratio of the Trt-histidine active ester to the de-Trt thirty-peptide intermediate is 1 to 1.2:1; and the molar ratio of the side chain active ester to the thirty-undeceptide intermediate is 1 to 1.2:

1.

5. The continuous preparation method of semaglutide according to claim 1, characterized in that: The mobile phases used in the purification process of the crude semaglutide are alcohol and water, and the alcohol includes methanol, ethanol, and isopropanol.

Images