Synthetic method of insulin degludec

Abstract

The invention discloses a synthesis method of insulin degludec, and belongs to the technical field of chemical pharmacy. The preparation method comprises the following steps: adding a reaction solvent into 50-peptide and a side chain serving as raw materials, adjusting the pH value, and reacting to obtain an insulin degludec crude product; the fifty peptide is an insulin degludec main chain; the side chain is a deprotected active ester; and carrying out preparative liquid phase separation and freeze drying on the insulin degludec crude product to obtain the insulin degludec. In the synthesis method provided by the invention, directional conversion is achieved through a reaction solvent and pH value control, the industrial operation difficulty is reduced, and the reaction yield is greatly improved; the total reaction yield is not less than 82%, and the HPLC purity is not less than 99%. The insulin degludec synthesis method provided by the invention has the advantages of simple operation, high directional conversion rate, environmental friendliness, high yield and high purity.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical pharmacy, and in particular to a method for synthesizing insulin degludec. Background Art

[0002] Insulin degludec, chemical name: lysine B29 (N ε -hexadecanediol-γ-glutamic acid) des(B30) human insulin. Recombinant insulin degludec injection developed by Novo Nordisk of Denmark is a new type of long-acting insulin analog. It was launched in Japan in October 2012 and approved by the European Union in January 2013 for the treatment of patients with type I and II diabetes. After subcutaneous injection, it forms multiple hexamers with a half-life of 24 hours and a duration of action of up to 42 hours.

[0003] There are many methods for preparing insulin degludec at home and abroad. Generally, the main chain of insulin degludec is obtained by genetic recombination technology, and then the side chain t-Bu-Pal-Glu(OSu)-O(t-Bu) is connected and the protection is removed to obtain insulin degludec. Since the main chain includes A chain and B chain, its A1 and B1 amine groups will also participate in the reaction, resulting in low yield and difficult separation.

[0004] Therefore, those skilled in the art are committed to developing a method for synthesizing insulin degludec with high yield, simple separation and low difficulty in industrial operation. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for synthesizing degludec insulin, which has the advantages of simple operation, high directional conversion rate, environmental friendliness, high yield and high purity.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for synthesizing degludec insulin, comprising the following steps:

[0007] Step 1), using 50 peptides and side chains as raw materials, adding a reaction solvent, adjusting the pH value, and reacting to obtain a crude product of degludec insulin; the 50 peptides are the main chain of degludec insulin; the side chain is hexadecandioic acid acylated L-glutamic acid-γ-active ester, and the structural formula of the side chain is as follows:

[0008]

[0009] Step 2), separating the crude product of degludec insulin through preparative liquid phase separation and freeze drying to obtain degludec insulin;

[0010] The specific synthetic route is as follows:

[0011]

[0012] Furthermore, the molar ratio of the main chain to the side chain of the insulin degludec is 1:1 to 1:10, preferably 1:3 to 1:5.

[0013] Furthermore, the R substituent group in the side chain active ester includes any one of pentafluorobenzene (PFP), phthalimide (PHT), 5-norbornene-2,3-dicarboximide (NB), 7-azobenzotriazole (At), succinimide (Su), benzotriazole (Bt), benzopyrazole, benzopyrrole, p-nitrobenzene, pyrazole, and sulfonic acid succinimide.

[0014] Furthermore, the reaction solvent is a mixture of one or more of water, ethanol, methanol, isopropanol, butanediol, DMF, dioxane, and THF. The reaction solvent is preferably a mixed solvent, such as water + DMF; water has a better effect of dissolving the main chain of degludec insulin, and DMF has a better solubility for the active esters of the side chain; a mixed solvent composed of the two can take into account the solubility of the main chain and the side chain.

[0015] Furthermore, the pH value during the reaction is adjusted by an inorganic acid or base; the inorganic base includes one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate; the inorganic acid includes one or more of hydrochloric acid, sulfuric acid, and phosphoric acid.

[0016] Furthermore, during the reaction, the reaction system is first adjusted to alkalinity by an inorganic base, and then adjusted to neutrality by an inorganic acid.

[0017] Furthermore, in step 1), the reaction system is added with 50 peptides and a reaction solvent, and after adjusting the pH value to alkaline, the side chain active ester is added in batches, and after the reaction is maintained for a period of time, the pH value is adjusted to neutral to obtain a crude solution of degludec insulin. The side chain active ester needs to be added in batches. Since the active ester needs to be in excess, adding it all at once will not only fail to promote the reaction, but also waste the side chain active ester; adding in batches can achieve the highest reaction efficiency and optimal cost control.

[0018] Furthermore, in step 2), the mobile phase for purification preparation is alcohol and water.

[0019] The invention has the following beneficial effects: compared with the prior art, the synthesis method of degludec insulin provided by the invention adopts a deprotected side chain active ester to directly react with the main chain of degludec insulin, and achieves directional conversion by means of a reaction solvent and controlling the pH value; a mixed solvent is adopted to take into account the solubility of the main chain and the side chain; the pH value is regulated during the reaction, and in an alkaline environment, the selectivity of the reaction can be greatly improved, so that the side chain active ester mainly reacts with the lysine ε site at the B29 position of the insulin main chain, and rarely reacts with the amino groups at the ends of the two insulin chains AB; in order to prevent the polypeptide from being hydrolyzed, the pH is adjusted back after the reaction is completed; the difficulty of industrial operation is reduced, and the reaction yield is greatly improved; the method has the advantages of simple operation, high directional conversion rate, environmental friendliness, high yield and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the mass spectrum of degludec insulin prepared in Example 1.

[0021] Figure 2 This is the liquid chromatogram of degludec insulin prepared in Example 1. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1

[0024]

[0025] At 10-25°C, under nitrogen protection, in a 500mL three-necked flask, add 5.57g of degludec insulin main chain (50AA, 0.001mol), add 200mL of purified water, add 30mL of THF, stir, add NaOH, adjust the pH to 10.5, add 2.2g of side chain (deprotected active ester, pentafluorophenyl, PFP) (0.004mol) in batches, keep the pH at 10-10.5, and continue stirring for 2-3h. Adjust the pH to neutral to obtain a crude solution of degludec insulin.

[0026] The crude solution of degludec insulin was separated by preparative liquid phase separation with an isopropanol / water system and freeze-dried to obtain 5.11 g of degludec insulin. The yield was 83.6%. The HPLC purity was greater than 99%. The molecular mass ([M+H]) was: 6100.8342. The mass spectrum of the obtained degludec insulin is shown in Figure 1 As shown in the liquid chromatogram Figure 2 The chromatographic peak results are shown in Table 1 below.

[0027] Table 1. Chromatographic peak results Chromatographic peak results

[0028] name Retention time area %area Peak height 1 16.275 23492 0.03 2548 2 16.665 77169824 99.12 2763700 3 17.455 664947 0.85 56948

[0029] Example 2

[0030]

[0031] At 10-25°C, under nitrogen protection, in a 500mL three-necked flask, add 5.57g of degludec insulin main chain (50AA, 0.001mol), add 200mL of purified water, add 20mL of DMF, stir, add NaOH, adjust the pH to 10.5, add 2.05g of side chain (deprotected active ester, succinimide, Su) (0.004mol) in batches, keep the pH at 10-10.5, and continue stirring for 2-3h. Adjust the pH to neutral to obtain a crude solution of degludec insulin.

[0032] The crude insulin degludec solution was separated by preparative liquid phase separation using an isopropanol / water system and freeze-dried to obtain 5.04 g of insulin degludec with a yield of 82.5% and a HPLC purity of more than 99%.

[0033] Example 3

[0034] At 10-25°C, under nitrogen protection, in a 500mL three-necked flask, add 5.57g of degludec insulin main chain (50AA, 0.001mol), add 200mL of purified water, add 30mL of THF, stir, add LiOH, adjust the pH to 10.5, add 2.1g of side chain (deprotected active ester, benzotriazole, Bt) (0.004mol) in batches, keep the pH at 10-10.5, and continue stirring for 2-3h. Adjust the pH to neutral to obtain a crude solution of degludec insulin.

[0035] The crude insulin degludec solution was separated by preparative liquid phase separation using an isopropanol / water system and freeze-dried to obtain 5.10 g of insulin degludec with a yield of 83.4% and a HPLC purity of more than 99%.

[0036] Example 4

[0037] At 10-25°C, under nitrogen protection, in a 500mL three-necked flask, add 5.57g of degludec insulin main chain (50AA, 0.001mol), add 200mL of purified water, add 30mL of THF, stir, add NaOH, adjust the pH to 10.5, add 2.2g of side chain (deprotected active ester, 5-norbornene-2,3-dicarboximide, NB) (0.004mol) in batches, keep the pH at 10-10.5, and continue stirring for 2-3h. Adjust the pH to neutral to obtain a crude solution of degludec insulin.

[0038] The crude insulin degludec solution was separated by preparative liquid phase separation using an isopropanol / water system and freeze-dried to obtain 5.06 g of insulin degludec with a yield of 82.8% and an HPLC purity of more than 99%.

[0039] Comparative Example 1

[0040] At 10-25°C, under nitrogen protection, in a 500mL three-necked flask, add 5.57g of degludec insulin main chain (50AA, 0.001mol), add 200mL of purified water, add 2.05g of side chain (deprotected active ester, succinimide, Su) (0.004mol) in batches, keep pH at 7.0-7.5, and continue stirring for 2-3h. HPLC shows that the reaction is almost impossible to proceed.

[0041] The synthetic route of the side chain (deprotected active ester) used in the above Examples 1 to 4 and Comparative Example 1 is as follows:

[0042]

[0043] Among them, the R substituent groups include: pentafluorobenzene (PFP), phthalimide (PHT), 5-norbornene-2,3-dicarboximide (NB), 7-azobenzotriazole (At), succinimide (Su), benzopyrazole, benzopyrrole, p-nitrobenzene, pyrazole, and sulfonic acid succinimide.

[0044] 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 method for synthesizing insulin degludec, characterized in that: The steps include: Step 1), using 50 peptides and side chains as raw materials, adding a reaction solvent, adjusting the pH value, and reacting to obtain a crude product of degludec insulin; the 50 peptides are the main chain of degludec insulin; the side chain is hexadecandioic acid acylated L-glutamic acid-γ-active ester, and the structural formula of the side chain is as follows: Step 2), separating the crude product of degludec insulin through preparative liquid phase separation and freeze drying to obtain degludec insulin; The specific synthetic route is as follows:

2. A method for synthesizing insulin degludec as claimed in claim 1, characterized in that: The molar ratio of the main chain to the side chain of the insulin degludec is 1:1 to 1:

10.

3. A method for synthesizing insulin degludec as claimed in claim 1, characterized in that: The R substituent group in the side chain active ester includes any one of pentafluorobenzene (PFP), phthalimide (PHT), 5-norbornene-2,3-dicarboximide (NB), 7-azobenzotriazole (At), succinimide (Su), benzotriazole (Bt), benzopyrazole, benzopyrrole, p-nitrobenzene, pyrazole, and sulfonic acid succinimide.

4. A method for synthesizing insulin degludec as claimed in claim 1, characterized in that: The reaction solvent is a mixture of one or more of water, ethanol, methanol, isopropanol, butanediol, DMF, dioxane, and THF.

5. A method for synthesizing insulin degludec as claimed in claim 1, characterized in that: The pH value during the reaction is adjusted by inorganic acids and bases; the inorganic bases include one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate; the inorganic acids include one or more of hydrochloric acid, sulfuric acid, and phosphoric acid.

6. A method for synthesizing insulin degludec as claimed in claim 1 or 2, characterized in that: During the reaction, the reaction system is first adjusted to alkalinity by an inorganic base, and then adjusted to neutrality by an inorganic acid.

7. A method for synthesizing insulin degludec as claimed in claim 1, characterized in that: In step 1), pentapeptide and reaction solvent are added to the reaction system, the pH value is adjusted to be alkaline, and then the side chain active ester is added in batches. After the reaction is maintained for a period of time, the pH value is adjusted to neutral to obtain a crude solution of degludec insulin.

8. A method for synthesizing insulin degludec as claimed in claim 1, characterized in that: In step 2), the mobile phase for purification preparation is alcohol and water.

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