Preparation method and application of long-acting insulin analogue

Abstract

The invention provides a preparation method and application of a long-acting insulin analogue, and the preparation method comprises the following steps: taking eicosandioic acid A as a starting raw material, carrying out unilateral esterification with trimethylsilyl ethanol to obtain an intermediate B, reacting the intermediate B with Hosu to obtain an active intermediate C, reacting the intermediate C with L-glutamic acid-1-trimethylsilyl ethyl ester to obtain an intermediate D, and carrying out condensation reaction on the intermediate D to obtain the long-acting insulin analogue. The intermediate D reacts with Hosu to obtain an active intermediate E, the intermediate E reacts with AEEA-AEA to obtain an intermediate F, the intermediate F reacts with Hosu to obtain an active intermediate G, and trimethylsilylethyl is removed from the intermediate G through tetrabutylammonium fluoride trihydrate to obtain the Ecobainsulin side chain. The route of the method has the following advantages that related raw materials are simple and easy to obtain, the reaction condition is mild, the yield is high, and the method is suitable for industrial production. The preparation method is simple and easy to implement, large-scale production is easy, the prepared side chain of the Ekoinsulin and A14E, B16E, B25H and DesB30 insulin precursors are coupled with lysine at the B29 site under the alkaline condition to obtain a crude Ekoinsulin product, and the crude product is subjected to two-step purification to obtain the Ekoinsulin with the purity of 99.0% or above.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, and in particular to a method for preparing a long-acting insulin analog and its application. Background Technology

[0002] With improved living standards and changes in lifestyle, the incidence of diabetes in my country has been increasing year by year in recent years. Type 2 diabetes is mainly treated with oral or subcutaneous injections. There are many types of drugs for lowering blood sugar in type 2 diabetes, including metformin, sulfonylureas, glucagon-like peptide-1 (GLP-1) receptor agonists, and insulin analogs. Insulin analogs and their fragments are particularly effective in treating type 2 diabetes, offering better treatment satisfaction and adherence, and have become a hot research topic in recent years. Fatty acid derivatives play an important role in the research of third-generation insulin drugs, especially long-acting insulin analogs.

[0003] Insulin analogue Icosin has been a major player in recent years, gaining widespread trust from patients due to its excellent blood sugar-lowering properties and long half-life. Once-weekly subcutaneous injections further reduce the discomfort associated with medication. The molecular structure of Icosin islets is shown in the attached diagram in the product information leaflet. Figure 1 As shown, its characteristic lies in the linking of long-chain fatty acids to lysine residue at position 29 of the B chain of insulin analogs via glutamate and two ethylene glycol compounds, thereby prolonging the drug's release time under the skin and achieving a long-acting effect. Ico insulin continues the technology of adding fatty acid side chains used in degludec and detemir insulin. At position B29, a 20C fatty acid side chain is linked via a "linker-spacer." Tyrosine at position A14 is replaced with glutamate (A14E), tyrosine at position B16 is replaced with histidine (B16H), and phenylalanine at position B25 is replaced with histidine (B25H). These three amino acid substitutions improve molecular stability and reduce enzymatic degradation. The 20C fatty acid side chain can bind firmly and reversibly to albumin, slowing receptor-mediated clearance. Fatty acid acylation refers to altering the biological properties of proteins by adding fatty acid side chains.

[0004] In existing technologies, the preparation of long-chain fatty acid derivatives is difficult. When using solid-phase synthesis methods, excessive feedstock is required, solvent consumption is high, costs are high, and environmental pollution is significant. When the methyl-protected glutamic acid terminal carboxyl group is hydrolyzed under alkaline conditions, it can cause racemization of the product and hydrolysis of the active ester. Therefore, there is an urgent need for a simple, effective, and low-cost method for synthesizing long-chain fatty acid derivatives. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a method for preparing long-acting insulin analogs. This method is simple to operate, low in cost, and has a high yield. The fatty acid derivatives prepared can be used in the development of long-acting hypoglycemic drugs using general protein modification techniques.

[0006] Another objective of this invention is to provide an application of a method for preparing a long-acting insulin analog.

[0007] The objective of this invention is achieved through the following technical solution: a method for preparing a long-acting insulin analog, comprising the following steps:

[0008] (1) Reaction of eicosanoic acid and trimethylsilylethanol yields eicosanoic acid monotrimethylsilylethyl ester as shown in B;

[0009]

[0010] (2) Reaction of eicosanoic acid monotrimethylsilyl ethyl ester and N-hydroxysuccinimide yields eicosanoic acid succinimide trimethylsilyl ethyl ester as shown in C;

[0011]

[0012] (3) Reaction of eicosanoic acid succinimide trimethyl ethyl ester with L-glutamic acid-1-trimethyl ethyl ester yields trimethylsilylethyl eicosanoic acid-L-Glu-1-trimethyl ethyl ester as shown in D;

[0013]

[0014] (4) Trimethylsilylethyl eicosanodiacyl-L-Glu-1-trimethylsilylethyl ester was reacted with N-hydroxysuccinimide to obtain trimethylsilylethyl eicosanodiacyl-L-Glu(OSu)-1-trimethylsilylethyl ester as shown in E;

[0015]

[0016] (5) Trimethylsilylethyl eicosanoyl-L-Glu(OSu)-1-trimethylsilylethyl ester is reacted with 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamino}-ethoxy)-ethoxy]-acetic acid to obtain trimethylsilylethyl eicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH as shown in F. The 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamino}-ethoxy)-ethoxy]-acetic acid is abbreviated as AEEA-AEEA.

[0017]

[0018] (6) Trimethylsilylethyl eicosanodiacyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH is reacted with N-hydroxysuccinimide to obtain trimethylsilylethyl eicosanodiacyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-(OSu) as shown in G;

[0019]

[0020] (7) The trimethylsilyl ethyl group of trimethylsilyl ethyl eicosanoyl-L-Glu-(1-trimethylsilyl ethyl ester)-AEEA-AEEA-(OSu) was removed by tetrabutylammonium fluoride trihydrate to obtain eicosanoyl-L-Glu-AEEA-AEEA-(OSu) as shown in H.

[0021]

[0022] (8) The insulin precursors A14E, B16H, B25H, and DesB30 were coupled with eicosanoyl-L-Glu-AEEA-AEEA-(OSu) to obtain insulin analogs as shown in I.

[0023] ;

[0024] (9) The crude insulin analogue was purified by two-step chromatography to obtain Ico insulin with a purity of over 99.0%.

[0025] Furthermore, the molar ratio of the amount of trimethylsilylethanol used in step (1) to that of compound A is calculated as 1~1.5:1; preferably 1~1.3:1; more preferably 1~1.2:1.

[0026] Furthermore, the molar ratio of the N-hydroxysuccinimide used in step (2) to the molar ratio of compound B is calculated as 1 to 1.5:1; preferably 1 to 1.2:1; more preferably 1 to 1.1:1.

[0027] Furthermore, the amount of L-glutamic acid-1-trimethylsilyl ethyl ester used in step (3) is calculated according to a molar ratio of 1 to 1.5:1 with respect to compound C; preferably 1 to 1.2:1; more preferably 1 to 1.1:1.

[0028] Furthermore, the molar ratio of the N-hydroxysuccinimide used in step (4) to the molar ratio of compound D is calculated to be 1~1.5:1; preferably 1~1.2:1.

[0029] Furthermore, the amount of 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamido}-ethoxy)-ethoxy]-acetic acid used in step (5) is calculated as a molar ratio of 1 to 1.5:1 with respect to compound E; preferably 1 to 1.2:1; more preferably 1 to 1.1:1.

[0030] Furthermore, the molar ratio of the N-hydroxysuccinimide used in step (6) to the compound F is calculated as 1~1.5:1; preferably 1~1.2:1; more preferably 1~1.1:1.

[0031] Furthermore, the molar ratio of the tetrabutylammonium fluoride trihydrate used in step (7) to the compound G is calculated as 3~5:1; preferably 3~4:1; more preferably 3:1.

[0032] In one embodiment of the present invention, step (1) specifically involves dissolving compound A in solvent A, adding trimethylsilylethanol and 4-dimethylaminopyridine (DMAP), and dropwise adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (DIC). The reaction is continued at room temperature for 2-3 hours. The solid obtained by removing the solvent by vacuum distillation is dissolved in solvent B, and the filtrate is washed with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The filtrate is dried with anhydrous sodium sulfate, filtered, and the solvent is removed by vacuum distillation. The solid is then dissolved by heating with solvent C, cooled to 20-30°C, recrystallized, and filtered to obtain compound B.

[0033] In one embodiment of the present invention, step (2) specifically involves dissolving compound B in solvent A, cooling to -20°C to -10°C, adding N-hydroxysuccinimide (HOSu) and dicyclohexylcarbodiimide (DCC), continuing the reaction at -20°C to -10°C for 2 hours, restoring to 15°C to 30°C and continuing the reaction for 3 to 16 hours, filtering to remove the precipitate, removing the solvent by vacuum distillation, dissolving the solid obtained in solvent B, washing the filtrate with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, removing the solvent by vacuum distillation, then heating to dissolve in solvent C, cooling to 20 to 30°C, recrystallizing and filtering to obtain compound C.

[0034] In one embodiment of the present invention, step (3) specifically involves dissolving compound C and L-glutamic acid-1-trimethylsilyl ethyl ester in solvent D, stirring for 2 hours in the presence of an organic base, concentrating under reduced pressure, adding solvent E, washing with dilute hydrochloric acid solution, water and saturated sodium chloride solution, drying with anhydrous sodium sulfate and filtering, and distilling the filtrate under reduced pressure to obtain compound D.

[0035] In one embodiment of the present invention, step (4) specifically involves dissolving compound D in solvent A, cooling to -20°C to -10°C, adding N-hydroxysuccinimide (HOSu) and dicyclohexylcarbodiimide (DCC), continuing the reaction at -20°C to -10°C for 2 hours, restoring to 15°C to 30°C and continuing the reaction for 3 to 16 hours, filtering to remove the precipitate, removing the solvent by vacuum distillation, dissolving the solid obtained in solvent B, washing the filtrate with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, removing the solvent by vacuum distillation, then heating to dissolve in solvent C, cooling to 20 to 30°C, recrystallizing and filtering to obtain compound E.

[0036] In one embodiment of the present invention, step (5) specifically involves dissolving compound E and 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamido}-ethoxy)-ethoxy]-acetic acid in solvent C, stirring overnight for 2 hours in the presence of an organic base, concentrating under reduced pressure, adding solvent E, washing with dilute hydrochloric acid solution, water and saturated sodium chloride solution, drying with anhydrous sodium sulfate and filtering, and distilling the filtrate under reduced pressure to obtain compound F.

[0037] In one embodiment of the present invention, step (6) specifically involves dissolving compound F in solvent A, cooling to -20°C to -10°C, adding N-hydroxysuccinimide (HOSu) and dicyclohexylcarbodiimide (DCC), continuing the reaction at -20°C to -10°C for 2 hours, restoring to 15°C to 30°C and continuing the reaction for 3 to 16 hours, filtering to remove the precipitate, removing the solvent by vacuum distillation, dissolving the solid obtained in solvent B, washing the filtrate with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, and removing the solvent by vacuum distillation to obtain compound G.

[0038] In one embodiment of the present invention, step (7) specifically involves dissolving trimethylsilyl ethyl eicosanoyl-L-Glu-(1-trimethylsilyl ethyl ester)-AEEA-AEEA-(OSu) in solvent D, adding tetrabutylammonium fluoride trihydrate and stirring continuously to carry out the deprotection reaction. After stirring at room temperature for 1-2 hours, solvent F is added and filtered for two hours to obtain the final product H.

[0039] The solvents A to F used in this invention are for dissolving or purifying, and their amount and number of uses are not limited. However, considering the cost and the effect achieved, it is preferable to limit the amount of solvents A to F.

[0040] Solvent A mentioned in step 1) is preferably one or at least two of N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile or dichloromethane, preferably dichloromethane;

[0041] The amount of solvent A used is calculated based on a volume-to-mass ratio of 10-50 mL to 1 g of solvent A to eicosanoic acid; preferably, it is calculated as 19-21 mL to 1 g.

[0042] The amount of trimethylsilane ethanol used in step 1) is 1 to 1.5:1 in molar ratio with the eicosanoic acid, preferably 1.2:1.

[0043] The amount of 4-dimethylaminopyridine (DMAP) used in step 1) is such that its molar ratio with the eicosanoic acid is 0.1 to 0.2:1, preferably 0.2:1.

[0044] The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) used in step 1) is 1 to 1.2:1 in molar ratio with the eicosanoic acid, preferably 1.2:1.

[0045] The preferred reaction time at room temperature after the addition of the substance described in step 1) is 3 hours.

[0046] The solvent B used in step 1) is preferably one or at least two of ethyl acetate, dichloromethane, or n-heptane, preferably ethyl acetate;

[0047] The solvent C used in the recrystallization described in step 1) is one or at least two of n-heptane, petroleum ether, or ethyl acetate, preferably n-heptane.

[0048] Solvent A mentioned in step 2) is preferably one or at least two of N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, or dichloromethane, preferably dichloromethane;

[0049] The amount of solvent A is calculated based on a volume-to-mass ratio of 10-50 mL to 1 g of eicosanoic acid monotrimethylsilyl ethyl ester; preferably 19-21 mL to 1 g.

[0050] The amount of dicyclohexylcarbodiimide used in step 2) is 1 to 1.2:1 in molar ratio with the eicosanoic acid monotrimethylsilyl ethyl ester, preferably 1.1:1.

[0051] The cooling temperature described in step 2) is preferably -20℃ to -15℃, more preferably -20℃.

[0052] The preferred time for the reaction to continue at 15–30°C as described in step 2) is 12–16 hours.

[0053] The solvent B used in step 2) is preferably one or at least two of ethyl acetate, dichloromethane, or n-heptane, preferably ethyl acetate;

[0054] The solvent C used in the recrystallization described in step 2) is one or at least two of n-heptane, petroleum ether, or ethyl acetate, preferably n-heptane.

[0055] The solvent D mentioned in step 3) is preferably one or both of dichloromethane and trichloromethane, preferably dichloromethane.

[0056] The amount of solvent D used in step 3) is calculated based on a volume-to-mass ratio of 10-50 mL to 1 g of solvent D and the eicosanoic acid succinimide trimethylsilyl ethyl ester, preferably 19-21 mL to 1 g.

[0057] The organic base mentioned in step 3) is preferably one or at least two of diethylamine, triethylamine, and N,N-diisopropylethylamine, with N,N-diisopropylethylamine being the most preferred.

[0058] Step 3) The amount of the organic base is calculated according to a molar ratio of 1 to 2:1 with the eicosanoic acid succinimide trimethylsilyl ethyl ester; preferably 1.2 to 1.5:1; more preferably 1.5:1.

[0059] Step 3) The solvent D is preferably one or at least two of dichloromethane, trichloromethane, and ethyl acetate, with ethyl acetate being the most preferred.

[0060] Step 3) The amount of solvent D is calculated based on a volume-to-mass ratio of 10-50 mL to 1 g of solvent D and the eicosanoic acid succinimide trimethylsilyl ethyl ester, preferably 19-21 mL to 1 g.

[0061] The concentration of the hydrochloric acid solution mentioned in step 3) is 0.1 to 0.2N, preferably 0.2N.

[0062] Solvent A mentioned in step 4) is preferably one or at least two of N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, or dichloromethane, preferably dichloromethane;

[0063] Step 4) The amount of solvent A is calculated based on a volume-to-mass ratio of 10 to 50 mL to 1 g of solvent A to the trimethylsilyl ethyl eicosanoyl-L-Glu-1-trimethylsilyl ethyl ester; preferably, it is calculated based on a volume-to-mass ratio of 19 to 21 mL to 1 g.

[0064] The cooling temperature described in step 4) is preferably -20℃ to -15℃, more preferably -20℃.

[0065] The preferred time for the reaction to continue at 15–30°C as described in step 4) is 12–16 hours.

[0066] The amount of dicyclohexylcarbodiimide used in step 4) is calculated based on a molar ratio of 1 to 1.2:1 between dicyclohexylcarbodiimide and trimethylsilylethyleicosanoyl-L-Glu-1-trimethylsilylethyl ester, preferably 1.1:1.

[0067] The solvent C used in the recrystallization described in step 4) is preferably one or at least two of n-heptane, petroleum ether, or ethyl acetate, with n-heptane being the most preferred.

[0068] The solvent D mentioned in step 5) is one or both of dichloromethane and trichloromethane, preferably dichloromethane.

[0069] The amount of solvent D used in step 5) is calculated based on a volume-to-mass ratio of 10 to 50 mL to 1 g of the solvent D and the trimethylsilyl ethyl eicosanoyl-L-Glu(OSu)-1-trimethylsilyl ethyl ester, preferably 19 to 21 mL to 1 g.

[0070] The organic base mentioned in step 5) is preferably one or at least two of diethylamine, triethylamine, and N,N-diisopropylethylamine, with N,N-diisopropylethylamine being the most preferred.

[0071] The amount of 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamido}-ethoxy)-ethoxy]-acetic acid used in step 5) is calculated based on a molar ratio of 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamido}-ethoxy)-ethoxy]-acetic acid to the trimethylsilyl ethyl eicosanoyl-L-Glu(OSu)-1-trimethylsilyl ethyl ester of that composition is 1 to 1.2:1, preferably 1.1:1.

[0072] Step 5) The amount of the organic base is calculated based on a molar ratio of 1 to 2:1 between the organic base and the trimethylsilylethyleicosanoyl-L-Glu(OSu)-1-trimethylsilylethyl ester; preferably 1.2 to 1.5:1; more preferably 1.5:1.

[0073] Step 5) The solvent E is preferably one or at least two of dichloromethane, trichloromethane, and ethyl acetate, preferably ethyl acetate.

[0074] Step 5) The amount of solvent E is calculated based on a volume-to-mass ratio of 10 to 50 mL to 1 g of the solvent E to the trimethylsilyl ethyl eicosanoyl-L-Glu(OSu)-1-trimethylsilyl ethyl ester, preferably 19 to 21 mL to 1 g.

[0075] The concentration of the hydrochloric acid solution mentioned in step 5) is 0.1 to 0.2N, preferably 0.2N.

[0076] Solvent A mentioned in step 6) is preferably one or at least two of N,N-dimethylformamide, N-methylpyrrolidone, acetonitrile, or dichloromethane, preferably dichloromethane;

[0077] Step 6) The amount of solvent A is calculated based on a volume-to-mass ratio of 10-50 mL to 1 g of solvent A to the trimethylsilyl ethyl eicosanoyl-L-Glu-(1-trimethylsilyl ethyl ester)-AEEA-AEEA-OH; preferably, it is calculated based on a volume-to-mass ratio of 19-21 mL to 1 g.

[0078] The cooling temperature described in step 6) is preferably -20℃ to -15℃, more preferably -20℃;

[0079] The preferred reaction time after restoring the temperature to 15–30°C in step 6) is 12–16 hours.

[0080] The amount of dicyclohexylcarbodiimide used in step 6) is calculated based on a molar ratio of 1 to 1.2:1 between dicyclohexylcarbodiimide and trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH, preferably 1.1:1.

[0081] The solvent F mentioned in step 7) is one or at least two of methyl ether, n-heptane, and petroleum ether, preferably methyl ether;

[0082] Step 7) m(trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-(OSu):V(dichloromethane)=1:10, preferably 1:8, more preferably 1:5

[0083] Step 7) n(tetrabutylammonium fluoride trihydrate) / n(trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-(OSu)) = 3~5:1, preferably a molar ratio of 3~4:1; more preferably 3:1;

[0084] Step 7) The reaction temperature is 0-30°C, preferably 10-30°C, and more preferably 20-30°C;

[0085] Step 7) The reaction time is 1 to 3 hours, more preferably 2 hours.

[0086] Step 8) The concentration of the reactant is 10~30 mg / mL, more preferably 10~20 mg / mL;

[0087] Step 8) The reaction pH is 10.0~12.0, more preferably 11.5~12.0;

[0088] Step 8) The reaction temperature is 0–15°C, more preferably 10–15°C;

[0089] Step 8) n(A14E,B16E,B25H,DesB30): n(eicosanodiacyl-L-Glu-AEEA-AEEA-(OSu))=2~3:1, more preferably eq=2.5:1;

[0090] Step 8) The concentration of A14E, B16E, B25H, and DesB30 protein precursors is 10-30 mg / mL, more preferably 10-20 mg / mL;

[0091] Step 8) The reaction solvent is a 0.1M triethylamine aqueous solution, a 0.1M Na2CO3 solution, or a 0.1M Na2CO3 / ACN=5:5 (v / v) mixed solution, preferably a 0.1M Na2CO3 / ACN=5:5 (v / v) mixed solution.

[0092] The method described above for preparing a long-acting insulin analog is particularly suitable for preparing long-acting insulin analogs.

[0093] Step 9) Specifically, the purification process consists of two steps: The first step is reverse-phase chromatography purification, in which the synthesis reaction solution is purified by reverse chromatography using C18 silica gel resin to obtain the first step reverse-phase chromatography solution; The second step is reverse-phase chromatography purification, in which the first step reverse-phase chromatography solution is purified by reverse chromatography using C18 silica gel resin to obtain a reverse-phase chromatography solution containing insulin with a purity of over 99.0%.

[0094] The present invention has the following advantages and effects compared with the prior art:

[0095] (i) The preparation method provided by the present invention is simple to operate, has controllable quality, and is suitable for industrial scale-up production.

[0096] (ii) In the second step of the synthesis of intermediate E in this invention, N-hydroxysuccinimide / dicyclohexylcarbodiimide is used instead of 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate (TSTU), which is also economical and feasible.

[0097] (iii) The present invention has a four-step synthesis using the same method of activating carboxyl groups and the same three-step condensation method with amines, using as few raw materials as possible and the simplest and cheapest raw materials, resulting in lower cost and fewer impurities.

[0098] (iv) The two-step reversed phase chromatography of this invention uses environmentally friendly and simple, inexpensive raw materials, resulting in lower cost and higher purity. Attached Figure Description

[0099] Figure 1 The molecular structure of the islet of Ico mentioned in the background art;

[0100] Figure 2 ESI-MS plot;

[0101] Figure 3 This is the HPLC chromatogram after the first step of reversed-phase chromatography purification;

[0102] Figure 4 This is the HPLC chromatogram after the second step of reversed-phase chromatography purification. Detailed Implementation

[0103] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments, but the embodiments of this invention are not limited thereto.

[0104] Example 1

[0105] 1. Preparation of ethidium trimethylsilyl ethyl eicosanoate

[0106]

[0107] Eicosanoic acid (8.5 g, 24.8 mmol) was suspended in dichloromethane (170 mL). Trimethylsilylethanol (3.5 g, 29.7 mmol) and 4-dimethylaminopyridine (0.6 g, 4.97 mmol) were added. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (4.6 g, 29.7 mmol) was added dropwise at room temperature. After the addition was complete, the mixture was reacted at room temperature for 3 hours. The mixture was concentrated under reduced pressure, and 200 mL of ethyl acetate was added to the concentrated residue. The residue was washed successively with 200 mL of saturated sodium bicarbonate, 200 mL of water, and 200 mL of saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to remove ethyl acetate, yielding a white solid residue. The residue was recrystallized by adding 200 mL of n-heptane, filtered, and the filter cake was collected. The filter cake was washed with n-heptane (100 mL) and the filter cake was collected. Eicosanoic acid monotrimethylsilyl ethyl ester, dried under reduced pressure to constant weight, 9.3 g, yield: 85%.

[0108] 2. Preparation of eicosanoic acid succinimide trimethylsilyl ethyl ester

[0109]

[0110] Eicosanoic acid monotrimethylsilyl ethyl ester (9.3 g, 21.0 mmol) was suspended in dichloromethane (190 ml), and N-hydroxysuccinimide (2.9 g, 25.2 mmol) and dicyclohexylcarbodiimide (5.2 g, 25.2 mmol) were added at -10 °C. The reaction was continued at -10 °C for 2 hours, and then stirred overnight at room temperature (25 °C). The mixture was filtered, and the filtrate was concentrated under reduced pressure. 200 mL of ethyl acetate was added to the concentrated residue, followed by washing with 200 mL of saturated sodium bicarbonate, 200 mL of water, and 200 mL of saturated brine. The residue was dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to remove ethyl acetate, yielding a white solid residue. 200 mL of n-heptane was added to the residue for recrystallization. The residue was filtered, and the filter cake was collected. The filter cake was washed with 100 mL of n-heptane and collected (eicosanoic acid succinimide trimethylsilyl ethyl ester). The residue was dried under reduced pressure to constant weight, yielding 9.8 g, with a yield of 87%.

[0111] 3. Preparation of trimethylsilylethyleicosanoyl-L-Glu-1-trimethylsilylethyl ester

[0112]

[0113] Dissolve 9.5 g (17.6 mmol) of eicosanoic acid succinimide trimethylsilyl ethyl ester in dichloromethane (100 ml), then add 4.75 g (19.3 mmol) of L-glutamic acid α-tert-butyl ester and 2.5 g (19.3 mmol) of N,N-diisopropylethylamine, and stir at room temperature for 2 hours. Evaporate the dichloromethane solvent under reduced pressure, and dissolve the residual oil in ethyl acetate (100 ml). Wash successively with 0.1 N hydrochloric acid, purified water, and saturated brine, dry to anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a viscous oily substance (i.e., trimethylsilylethyl eicosanoic acid diacyl-L-Glu-1-trimethylsilyl ethyl ester), in 100% yield.

[0114] 4. Preparation of trimethylsilylethyleicosanoyl-L-Glu(OSu)-1-trimethylsilylethyl ester

[0115]

[0116] Trimethylsilylethyleicosanoyl-L-Glu-1-trimethylsilylethyl ester (11.4 g, 17.1 mmol) was dissolved in dichloromethane (100 ml) in an ice bath. N-hydroxysuccinimide (2.35 g, 20.5 mmol) and dicyclohexylcarbodiimide (4.22 g, 20.5 mmol) were added separately at -20 °C, and the reaction was continued at -20 °C for 2 hours. The mixture was then brought to room temperature and stirred overnight. The mixture was filtered, and the solvent was evaporated under reduced pressure to obtain an oily substance. Add 100 mL of ethyl acetate to the concentrated residue, wash successively with 100 mL of saturated sodium bicarbonate, 200 mL of water, and 200 mL of saturated brine, dry with anhydrous sodium sulfate, filter, and distill the filtrate under reduced pressure to remove ethyl acetate, giving a white solid residue. Add 100 mL of n-heptane to recrystallize, filter and collect the filter cake, wash the filter cake with 50 mL of n-heptane, collect the filter cake (which is trimethylsilylethyleicosanodiacyl-L-Glu(OSu)-1-trimethylsilylethyl ester), dry under reduced pressure to constant weight, to give trimethylsilylethyleicosanodiacyl-L-Glu(OSu)-1-trimethylsilylethyl ester, weight 11.7 g, yield: 89%.

[0117] 5. Preparation of trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH

[0118]

[0119] Dissolve 2.0 g (2.6 mmol) of trimethylsilylethyleicosanoyl-L-Glu(OSu)-1-trimethylsilylethyl ester in dichloromethane (20 mL), then add 0.88 g (2.86 mmol) and N,N-diisopropylethylamine (0.37 g (2.86 mmol) sequentially. Stir overnight at room temperature. Evaporate the dichloromethane solvent under reduced pressure, and dissolve the residual oil in ethyl acetate (100 mL). Wash successively with 0.1 N hydrochloric acid, purified water, and saturated brine, dry to anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain a pale yellow viscous oil (i.e., trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH), yield 100%.

[0120] 6. Trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-

[0121] Preparation of (OSu)

[0122]

[0123] Trimethylsilyl ethyl eicosanoyl-L-Glu-(1-trimethylsilyl ethyl ester)-AEEA-

[0124] AEEA-OH (2.7 g, 2.8 mmol) was dissolved in dichloromethane (270 mL) in an ice bath. N-hydroxysuccinimide (0.48 g, 4.2 mmol) and dicyclohexylcarbodiimide (0.865 g, 4.2 mmol) were added separately at -20 °C, and the reaction was continued at -20 °C for 2 hours, then brought to room temperature and stirred overnight. The mixture was filtered, and the solvent was evaporated under reduced pressure to obtain an oily substance. 270 mL of ethyl acetate was added to the concentrated residue, followed by washing with 270 mL of saturated sodium bicarbonate, 270 mL of water, and 270 mL of saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to remove ethyl acetate, yielding a pale yellow oily substance, trimethylsilylethyleicosanoic acid-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-(OSu), weighing 2.9 g.

[0125] 7. Preparation of eicosanodiacyl-L-Glu-AEEA-AEEA-OSu

[0126]

[0127] Trimethylsilylethyleicosanoyl-L-Glu-(1-trimethylsilyl)-AEEA-AEEA-(OSu)

[0128] (2.0 g, 1.89 mmol) was dissolved in 10 mL of dichloromethane, and tetrabutylammonium fluoride trihydrate (1.58 g, 5.67 mmol) was added. After stirring at 20-30 °C for two hours, 100 mL of methyl ether was added to the reaction solution, and stirring was continued for 2 hours. The mixture was then filtered, and the filter cake was washed with methyl ether and dried under reduced pressure to constant weight to give a white solid, eicosanodiacyl-L-Glu-AEEA-AEEA-OSu, weighing 1.2 g, with a yield of 74%.

[0129] Dissolve A14E, B16E, B25H, and DesB30 in a 0.1M Na2CO3 / CAN mixture of 5:5 (v / v), maintaining the concentration of A14E, B16E, B25H, and DesB30 protein precursors at 10–20 mg / mL. Place the solution in an ice-water bath at 0–15°C. Weigh eicosanoic acid-L-Glu-AEEA-AEEA-(OSu) at a ratio of 2.5:1, dissolve it in DMF, and slowly add it dropwise to the solution. Maintain the pH of the reaction at 11.5–12.0 during the addition process. Allow the reaction to proceed for 30 minutes after the addition is complete.

[0130] The ESI-MS result is 6375.9942, which is consistent with the theoretical value. (See...) Figure 2 .

[0131] Example 2

[0132] The acylated crude icoinsulin was purified in two steps to obtain icoinsulin with a purity of over 99.0%.

[0133] Step 1: Reverse phase chromatography purification

[0134] The synthesized sample was diluted, filtered, and purified using C18 silica gel resin. The chromatography column was packed to a height of 25 cm, with a medium loading of 10 g / L, and a flow rate not exceeding 350 cm / h. Buffer A was a 50 mmol / L Tris + 2% ammonium sulfate solution with a pH of 7.5-8.5; Buffer B was a 90% ethanol solution. Equilibration was performed using 10% buffer A, and 10% acetonitrile solution was added during sample loading. The elution gradient was 10-70% B linear wash for 30-40 CV, and elution peaks with a purity higher than 97.0% were collected. The HPLC chromatogram after the first step of reversed-phase chromatography purification is shown below. Figure 3 .

[0135] The second step is reverse-phase chromatography purification.

[0136] The synthesized sample was diluted and the pH was adjusted to 2.5. After filtration, it was purified using C18 silica gel resin. The chromatography column was packed to a height of 25 cm, with a medium loading of 8 g / L and a flow rate not exceeding 350 cm / h. Buffer A was a 0.2% TFA + 1% ammonium sulfate solution at pH 2.5; Buffer B was a 90% ethanol solution; equilibration was performed using 10% Buffer A. The elution gradient was 10-70% B, with linear elution at 40-50 CV. Elution peaks with a purity higher than 99.0% were collected. The HPLC chromatogram after the second step of reversed-phase chromatography purification is shown below. Figure 4 .

[0137] Sedimentation Collection

[0138] The sample obtained from reverse-phase chromatography was added to twice the volume of purified water to adjust the pH to 5.5-6.0, precipitating the sample. The precipitate was stirred thoroughly and centrifuged at 2-8°C. The supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with twice the volume of purified water, with a centrifugal force ≥7500g, a centrifugation time ≥20min, and a temperature of 2-8°C. The supernatant was discarded, and the precipitate was collected. The precipitate was evenly spread in a freeze-drying tray and freeze-dried to collect insulin. The acylation and purification yields of this process in three 1g-scale preparations are shown in Table 1 below.

[0139] Table 1 Results of three 1g-scale preparations

[0140] batch Acylation yield First step reverse phase chromatography purification yield The second step, reverse-phase chromatography purification, yields... 1 73.2% 87.7% 83.4% 2 75.1% 85.8% 90.0% 3 71.9% 88.1% 84.3%

[0141] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a long-acting insulin analog, characterized in that, The features include the following specific steps: (1) Reaction of eicosanoic acid and trimethylsilylethanol yields eicosanoic acid monotrimethylsilylethyl ester as shown in B; ； (2) Reaction of eicosanoic acid monotrimethylsilyl ethyl ester and N-hydroxysuccinimide yields eicosanoic acid succinimide trimethylsilyl ethyl ester as shown in C; ； (3) Reaction of eicosanoic acid succinimide trimethyl ethyl ester with L-glutamic acid-1-trimethyl ethyl ester yields trimethylsilylethyl eicosanoic acid-L-Glu-1-trimethyl ethyl ester as shown in D; ； (4) Trimethylsilylethyl eicosanodiacyl-L-Glu-1-trimethylsilylethyl ester was reacted with N-hydroxysuccinimide to obtain trimethylsilylethyl eicosanodiacyl-L-Glu(OSu)-1-trimethylsilylethyl ester as shown in E; ； (5) Trimethylsilylethyl eicosanoyl-L-Glu(OSu)-1-trimethylsilylethyl ester is reacted with 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamino}-ethoxy)-ethoxy]-acetic acid to obtain trimethylsilylethyl eicosanoyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH as shown in F. The 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamino}-ethoxy)-ethoxy]-acetic acid is abbreviated as AEEA-AEEA. ； (6) Trimethylsilylethyl eicosanodiacyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH is reacted with N-hydroxysuccinimide to obtain trimethylsilylethyl eicosanodiacyl-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-(OSu) as shown in G; ； (7) The trimethylsilyl ethyl group of trimethylsilyl ethyl eicosanoyl-L-Glu-(1-trimethylsilyl ethyl ester)-AEEA-AEEA-(OSu) was removed by tetrabutylammonium fluoride trihydrate to obtain eicosanoyl-L-Glu-AEEA-AEEA-(OSu) as shown in H. ； (8) The insulin precursors A14E, B16H, B25H, and DesB30 were coupled with eicosanoyl-L-Glu-AEEA-AEEA-(OSu) to obtain insulin analogs as shown in I. ； (9) The crude insulin analogue was purified by two-step chromatography to obtain Ico insulin with a purity of over 99.0%.

2. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The molar ratio of the amount of trimethylsilylethanol used in step (1) to the molar ratio of the eicosanoic acid is 1 to 1.5:

1.

3. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The molar ratio of the amount of N-hydroxysuccinimide used in step (2) to the molar ratio of the eicosanoic acid monotrimethylsilyl ethyl ester of formula B is 1 to 1.5:

1.

4. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The molar ratio of L-glutamic acid-1-trimethylsilyl ethyl ester used in step (3) to that of eicosanoic acid succinimide trimethylsilyl ethyl ester shown in formula C is 1 to 1.5:

1.

5. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The molar ratio of the amount of N-hydroxysuccinimide used in step (4) to the molar ratio of trimethylsilylethyleicosanoic acid-L-Glu-1-trimethylsilylethyl ester shown in formula D is 1 to 1.5:

1.

6. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The amount of 2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-acetamido}-ethoxy)-ethoxy]-acetic acid used in step (5) is in a molar ratio of 1 to 1.5 to 1.5 of the amount of trimethylsilylethyl eicosanoyl-L-Glu(OSu)-1-trimethylsilylethyl ester shown in formula E.

7. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The molar ratio of the amount of N-hydroxysuccinimide used in step (6) to that of trimethylsilylethyleicosanoic acid-L-Glu-(1-trimethylsilylethyl ester)-AEEA-AEEA-OH shown in formula F is 1 to 1.5:

1.

8. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, The amount of tetrabutylammonium fluoride trihydrate used in step (7) is 2~10:1 with the amount of tert-butyleicosanoyl-L-Glu(OtBu)-AEEA-AEEA-(OSu) by volume / mass.

9. The method for preparing a long-acting insulin analog according to claim 1, characterized in that, In step (8), A14E, B16E, B25H, and DesB30 are dissolved in an alkaline aqueous solvent or a mixture of water and organic solvent. Eicosanodiacyl-L-Glu-AEEA-AEEA-(OSu) is slowly added dropwise for reaction. The ratio of n(A14E,B16E,B25H,DesB30):eicosanodiacyl-L-Glu-AEEA-AEEA-(OSu)) is 2~3:

1. The reaction temperature is 0~15℃, the reaction pH is 10.0~12.0, the reaction concentration is 10~30mg / mL, and the reaction solvent is 0.1M triethylamine aqueous solution, 0.1M Na2CO3 solution, or a 0.1M Na2CO3 / CAN=5:5 (v / v) mixed solution.

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