A process for the preparation of a somatostatin side chain

Abstract

The application belongs to the field of pharmaceutical chemical industry, and particularly relates to a liquid-phase preparation method of a side chain of somatuline. The preparation method of the application is that Fmoc-His(Trt)-OH reacts with H-Aib-OtBu under the action of a condensing agent T3P to obtain Fmoc-His-Aib-OH. The preparation method provided by the application simplifies operation steps, can control monomer impurities below 0.05%, can control inferior isomer impurities below 0.01%, is short in a synthesis period, is low in cost, has few by-products, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemical industry, and specifically relates to a method for preparing semaglutide side chains. Background Technology

[0002] Sermaglutide, developed by Novo Nordisk, is a novel long-acting glucagon-like peptide-1 (GLP-1) analogue. It not only has significant hypoglycemic effects but also noticeable weight-loss benefits. This drug is the second GLP-1 hypoglycemic agent in Novo Nordisk's diabetes pipeline to possess both hypoglycemic and weight-loss effects (the other being liraglutide). Sermaglutide exhibits multiple effects, including lowering blood sugar, reducing weight, promoting pancreatic islet cell regeneration, and protecting the cardiovascular system, showing broad clinical application prospects. Current purification techniques for sermaglutide mostly employ chromatographic and solid-liquid methods, which are difficult and cumbersome. Therefore, it is necessary to ensure sufficient purity of the introduced modified fragments during peptide modification to reduce the burden of subsequent separation and purification.

[0003] Fmoc-His-Aib-OH is an important modified fragment of semaglutide. Patent CN108059666B mentions the process of reacting Fmoc-His(Trt)-OH and HOSu under DCC to generate the active ester Fmoc-His(Trt)-OSu, which is then further reacted with H-Aib-OH under alkaline catalysis to generate Fmoc-His(Trt)-Aib-OH. The process is as follows: This method requires a further Trt removal step from Fmoc-His(Trt)-Aib-OH to obtain Fmoc-His-Aib-OH. Repeating this method revealed that the active ester is unstable and readily hydrolyzes in THF-water to form Fmoc-His(Trt)-OH, resulting in cumbersome post-processing and low yield.

[0004] Patent WO2013098191A1 reports that Fmoc-His(Trt)-Aib-OH is obtained by removing Trt under the action of TIPS and TFA, but the purity is not reported.

[0005] Fomc-His-Aib-OH is the last step in the semaglutide process, so the single impurities in Fomc-His-Aib-OH have a significant impact on API quality. CN112625087A discloses a method for preparing Fmoc-His(Trt)-Aib-OH, but the total purity can only reach a maximum of 98.27%, and the content of single impurities is relatively high.

[0006] Patent CN116355042A reports that Fmoc-His-Aib-OH,R1 is directly obtained from Fmoc-His(Trt)-OH through two stages of condensation and deprotection. Tert-butyl ester can be removed simultaneously in the later step of deprotection of Trt, and the target product can be obtained in one pot. However, the overall yield is not high, and the condensing agent HOBT is an explosive compound with a high risk factor.

[0007] In summary, the existing preparation process of semaglutide side chains involves numerous synthetic steps, a long synthesis cycle, low purity and yield, and high production costs, which is not conducive to large-scale production. Summary of the Invention

[0008] To address the problems of numerous steps, long synthesis cycles, and difficult product separation in the synthesis of semaglutide side chains in existing technologies, this invention provides a method for preparing high-purity semaglutide basic side chains. This method features a short reaction route, simple operation, milder reaction, is economical and environmentally friendly, and has a high yield, making it suitable for industrial production.

[0009] This invention is specifically achieved through the following technical solution:

[0010] A method for preparing a semaglutide side chain includes the following steps:

[0011]

[0012] a) Compound H-Aib-OtBu.HCl and compound Fmoc-His(Trt)-OH were added to an organic solvent, T3P solution was added, and pyridine was added to control the temperature for reaction. The mixture was separated, washed, and dried to obtain a concentrate containing intermediate I.

[0013] b) Add the concentrate to an organic solvent, add acid under controlled temperature, stir and heat to deprotect compound I, add crystallization solvent under controlled temperature, stir and crystallize at room temperature, filter and dry to obtain white solid I, i.e., side chain compound.

[0014] Preferably, the molar ratio of Fmoc-His(Trt)-OH, H-Aib-OtBu.HCl, T3P, and pyridine in step a) is 1:1 to 1.5:1.0 to 2.0:3 to 4.

[0015] Preferably, the T3P solution in step a) is a 50% ethyl acetate solution.

[0016] Preferably, the organic solvent in step a) is selected from one or a combination of dichloromethane, ethyl acetate, toluene, and acetonitrile, and more preferably ethyl acetate.

[0017] Preferably, the temperature control temperature in step a) is -10 to 0°C, and more preferably 0°C.

[0018] Preferably, the organic solvent in step b) is selected from one or a combination of dichloromethane, trichloromethane, and 1,2-dichloromethane, and more preferably dichloromethane.

[0019] Preferably, the acid in step b) is one or a combination of trifluoroacetic acid, hydrochloric acid, and sulfuric acid, and more preferably trifluoroacetic acid.

[0020] Preferably, the mass-to-volume ratio of the concentrate to the acid in step b) is 1:3.5 to 4.5, with mass expressed in g and volume expressed in ml.

[0021] Preferably, the temperature control temperature in step b) is 0–5°C.

[0022] Preferably, the heating temperature in step b) is 20°C to 25°C, and more preferably 25°C.

[0023] Preferably, the crystallization solvent in step b) is one or a combination of ethylene glycol dimethyl ether, acetonitrile, chloroform, acetone, and methyl tert-butyl ether, and more preferably a mixed solvent of ethylene glycol dimethyl ether and methyl tert-butyl ether.

[0024] In another embodiment, the separation in step a) is selected from a 5% hydrochloric acid aqueous solution.

[0025] In another embodiment, the washing in step a) is selected from purified water and saturated saline solution.

[0026] Compared with the prior art, the technical effects achieved by the present invention are as follows:

[0027] 1. The present invention directly obtains Fmoc-His-Aib-OH from Fmoc-His(Trt)-OH through two stages of condensation and deprotection. Tert-butyl ester can be removed simultaneously in the later step of deprotection of Trt, and intermediate I does not need to be separated, thus simplifying the operation steps.

[0028] 2. The semaglutide side chain compound prepared by this invention has higher purity. Using T3P condensing agent, it has excellent reaction selectivity and low epimerization effect. It is non-toxic and non-allergenic. By-products can be easily removed by washing with water. Single impurities can be controlled below 0.05%, and inferior isomer impurities can be controlled below 0.01%. The synthesis cycle is short, the cost is low, and there are few by-products, making it suitable for industrial production.

[0029] 3. The preparation method of the present invention can provide high-purity modified fragments for semaglutide and other polypeptide products, reducing the purification pressure after modification. Detailed Implementation

[0030] The present invention will be further illustrated by the following embodiments. It should be understood that the embodiments and other contents of the present invention are only used to illustrate the present invention and are not intended to limit the present invention. Therefore, simple improvements to the present invention under the premise of the method of the present invention are all within the scope of protection of the present invention.

[0031] Example 1

[0032] Ethyl acetate (400 ml), H-Aib-OtBu.HCl (21.53 g, 110 mmol.), and Fmoc-His(Trt)-OH (61.97 g, 100 mmol.) were added to a reaction flask and stirred until homogeneous. Pyridine (27.68 g, 350 mmol.) was added to the reaction system and stirred. The temperature was controlled at 0 °C. T3P solution (50% ethyl acetate solution, 95.45 g, 150 mmol.) was added dropwise over approximately 1.5 h. The reaction was allowed to proceed for 6-8 h, and the reaction was monitored until Fmoc-His(Trt)-OH disappeared, at which point the reaction was stopped. 5% hydrochloric acid aqueous solution (300 ml) was added to the reaction system and stirred for 10 min. The mixture was allowed to stand and separated, and the organic phase was collected. The organic phase was washed successively with purified water (300 ml) and saturated saline (200 ml), dried, and concentrated to obtain intermediate I.

[0033] Add dichloromethane (240 ml) to the concentrate and stir. While maintaining the temperature at 0–5°C, add trifluoroacetic acid (200 ml) dropwise to the reaction system. After the addition is complete, raise the temperature to 25°C and stir for 2.5–3.5 hours. Once intermediate I has reacted completely, maintain the temperature at 0–5°C and begin adding ethylene glycol dimethyl ether / methyl tert-butyl ether (150 ml: 300 ml) dropwise. After the addition is complete, raise the temperature to room temperature and stir to induce crystallization. Crystallization takes 5–7 hours. Filter, dry, and obtain a white solid. Overall yield: 91.98%, HPLC purity: 99.95%, maximum single impurity: 0.03%, inferior isomer: 0.005%.

[0034] Intermediate I: 1 H-NMR (500MHz, CDCl3) δ7.90 (dd, J=7.4, 1.5Hz, 2H), 7.77 (dd, J=7.5, 1.4Hz, 2H), 7.70 (s, 1H), 7.34 (td, J=7.5, 1.5Hz, 2H), 7.31–7. 19(m,18H),5.50–5.33(m,4H),4.69(t,J=5.9Hz,1H),4.46(s,1H),3.32(ddd,J=41.0,12.4,6.6Hz,,2H),1.74(s,6H),1.42(s,9H).

[0035] Example 2

[0036] Add dichloromethane (400 ml), H-Aib-OtBu.HCl (19.57 g, 100 mmol.), and Fmoc-His(Trt)-OH (61.97 g, 100 mmol.) to a reaction flask and stir until homogeneous. Add pyridine (23.73 g, 300 mmol.) to the reaction system and stir. Controlling the temperature at -5°C, add T3P solution (50% ethyl acetate solution, 63.63 g, 100 mmol.) dropwise over approximately 1.5 h. Maintain the reaction temperature for 6-8 h, monitoring the reaction until Fmoc-His(Trt)-OH disappears, at which point the reaction is stopped. Add 5% hydrochloric acid aqueous solution (300 ml) to the reaction system and stir for 10 min. Allow to stand and separate the liquids, collecting the organic phase. Wash the organic phase successively with purified water (300 ml) and saturated saline (200 ml), dry, concentrate, and use directly in the next reaction step.

[0037] Add 240 ml of chloroform to the concentrate and stir. While maintaining the temperature at 0–5 °C, add 145 ml of sulfuric acid dropwise to the reaction system. After the addition is complete, raise the temperature to 20 °C and stir for 2.5–3.5 h. Once intermediate I has reacted completely, lower the temperature to 0–5 °C and begin adding acetonitrile / methyl tert-butyl ether (150 ml; 300 ml). After the addition is complete, raise the temperature to room temperature and stir to induce crystallization. Crystallization takes 5–7 h. Filter, dry, and obtain a white solid. Overall yield: 89.92%, HPLC purity: 99.93%, maximum single impurity: 0.04%, inferior isomer: 0.007%.

[0038] Example 3

[0039] Toluene (400 ml), H-Aib-OtBu.HCl (29.36 g, 150 mmol.), and Fmoc-His(Trt)-OH (61.97 g, 100 mmol.) were added to a reaction flask and stirred until homogeneous. Pyridine (31.64 g, 400 mmol.) was added to the reaction system and stirred. The temperature was controlled at -10 °C. T3P solution (50% ethyl acetate solution, 127.27 g, 200 mmol.) was added dropwise over approximately 1.5 h. The reaction was maintained at this temperature for 6-8 h, and the reaction was stopped when Fmoc-His(Trt)-OH disappeared. 5% hydrochloric acid aqueous solution (300 ml) was added to the reaction system and stirred for 10 min. The mixture was allowed to stand and separated, and the organic phase was collected. The organic phase was washed successively with purified water (300 ml) and saturated saline (200 ml), dried, concentrated, and used directly in the next reaction step.

[0040] Add 240 ml of 1,2-dichloromethane to the concentrate and stir. While maintaining the temperature at 0–5 °C, add 290 ml of hydrochloric acid dropwise to the reaction system. After the addition is complete, raise the temperature to 25 °C and stir for 2.5–3.5 h. Once intermediate I has reacted completely, lower the temperature to 0–5 °C and begin adding acetone / methyl tert-butyl ether (150 ml: 300 ml). After the addition is complete, raise the temperature to room temperature and stir to induce crystallization. Crystallization takes 5–7 h. Filter, dry, and obtain a white solid. Overall yield: 88.53%, HPLC purity: 99.92%, maximum single impurity: 0.04%, inferior isomer: 0.008%.

[0041] Example 4

[0042] Ethyl acetate (400 ml), H-Aib-OtBu.HCl (21.53 g, 110 mmol.), and Fmoc-His(Trt)-OH (61.97 g, 100 mmol.) were added to a reaction flask and stirred until homogeneous. Pyridine (27.68 g, 350 mmol.) was added to the reaction system and stirred. The temperature was maintained at 0 °C. T3P solution (50% ethyl acetate solution, 31.82 g, 50 mmol.) was added dropwise over approximately 0.5 h. The reaction was maintained at this temperature for 6-8 h, and the reaction was stopped when Fmoc-His(Trt)-OH disappeared. 5% hydrochloric acid aqueous solution (300 ml) was added to the reaction system and stirred for 10 min. The mixture was allowed to stand and separated, and the organic phase was collected. The organic phase was washed successively with purified water (300 ml) and saturated saline (200 ml), dried, and concentrated to obtain intermediate I.

[0043] Add 240 ml of dichloromethane to the concentrate and stir. While maintaining the temperature at 0–5 °C, add 200 ml of trifluoroacetic acid dropwise to the reaction system. After the addition is complete, raise the temperature to 25 °C and stir for 2.5–3.5 h. Once intermediate I has reacted completely, lower the temperature to 0–5 °C and begin adding ethylene glycol dimethyl ether / methyl tert-butyl ether (150 ml: 300 ml). After the addition is complete, raise the temperature to room temperature and stir to induce crystallization. Crystallization takes 5–7 h. Filter, dry, and obtain a white solid. Overall yield: 83.41%, HPLC purity: 99.90%, maximum single impurity: 0.06%, inferior isomer: 0.012%.

[0044] Comparative Example

[0045] Add 100.0 g of Fmoc-His(Trt)-OH to DCM, stir until the system is clear, and then cool to 0°C. Add 61.3 g of HBTU and 31.28 g of DIEA, and stir for 30 min. Continue to add 30.97 g of EDC, and stir for 1 h. The reaction is complete when TLC shows no obvious Fmoc-His(Trt)-OH. After the reaction is complete, add 47.37 g of H-Aib-OtBu.HCl. After the addition is complete, react at 0°C for 1 h, then at 20°C for 3 h. After the reaction is complete, quench the reaction solution with water. Separate the liquid and collect the organic phase. Wash the organic phase with saturated sodium bicarbonate solution, separate the liquid and collect the organic phase. Wash the organic phase with water, separate the liquid and collect the organic phase. Concentrate the organic phase to obtain the concentrate, which can be used directly in the next step.

[0046] At room temperature (25°C), TFA (134.86 g) and TIPS (18.73 g) were added to the concentrate obtained in the previous step, and the reaction was maintained at this temperature for 5 hours. After the reaction was completed, the concentrate was concentrated, and n-heptane was added. The mixture was separated, and the lower layer was collected. Acetonitrile was added to the lower layer and concentrated to 2-3 times the weight of Fmoc-His(Trt)-OH. After concentration, acetonitrile and MTBE were added to the concentrate. The mixture was stirred at 15-25°C to induce crystallization. After filtration, washing, and drying, a white solid was obtained, with a total yield of 61.8%, HPLC purity of 99.88%, maximum single impurity of 0.06%, and inferior isomers of 0.04%.

Claims

1. A method for preparing a semaglutide side chain, characterized in that, The preparation method includes the following steps: (1) Compound H-Aib-OtBu.HCl and compound Fmoc-His(Trt)-OH were added to an organic solvent, and T3P solution and pyridine were added to control the temperature of the reaction. The mixture was separated, washed and dried to obtain a concentrate containing intermediate I. (2) Add the concentrate to an organic solvent, add acid under controlled temperature, stir and heat to deprotect compound I, add crystallization solvent under controlled temperature, stir and crystallize at room temperature, filter and dry to obtain white solid I, i.e. side chain compound. The synthetic route is as follows:

2. The preparation method according to claim 1, characterized in that, The molar ratio of Fmoc-His(Trt)-OH, H-Aib-OtBu.HCl, T3P and pyridine in step (1) is 1:1~1.5:1.0~2.0:3~4.

3. The preparation method according to claim 1, characterized in that, The organic solvent mentioned in step (1) is selected from one or a combination of dichloromethane, ethyl acetate, toluene, and acetonitrile.

4. The preparation method according to claim 1, characterized in that, The temperature control temperature mentioned in step (1) is -10 to 0℃.

5. The preparation method according to claim 1, characterized in that... The organic solvent mentioned in step (2) is selected from one or a combination of dichloromethane, trichloromethane, 1,2-dichloroethane.

6. The preparation method according to claim 1, characterized in that, The acid mentioned in step (2) is one or a combination of trifluoroacetic acid, hydrochloric acid, and sulfuric acid.

7. The preparation method according to claim 1, characterized in that... The mass-to-volume ratio of the concentrate to the acid in step (2) is 1:3.5 to 4.5, with mass expressed in g and volume expressed in mL.

8. The preparation method according to claim 1, characterized in that... The temperature control temperature mentioned in step (2) is 0 to 5℃.

9. The preparation method according to claim 1, characterized in that... The heating temperature mentioned in step (2) is 20℃~25℃.

10. The preparation method according to claim 1, characterized in that... The crystallization solvent mentioned in step (2) is one or a combination of ethylene glycol dimethyl ether, acetonitrile, chloroform, acetone, and methyl tert-butyl ether.

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