Synthesis method of semaglutide

A synthesis method and technology of semaglutide, applied in the field of polypeptide drug production, can solve the problems of high separation and purification, limited coupling site, purification of polypeptide fragments, etc., and achieve the solution of purification difficulty, alleviation of difficulty and rapid transformation. Effect

Pending Publication Date: 2022-07-01
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since all polypeptide fragments are fully protected, poor solubility becomes the most difficult problem encountered in this method, and fully protected polypeptide fragments cannot be purified by high performance liquid chromatography, and impurities in the final semaglutide crude peptide There are many, and the technical requirements for separation and purification are also very high
In addition, because the amino acid residues at the end of the fragment may racemize during the coupling process, the coupling sites can only be limited to the Gly and Pro sites that cannot be racemized or the sites with a relatively small risk of racemization

Method used

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  • Synthesis method of semaglutide
  • Synthesis method of semaglutide
  • Synthesis method of semaglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Preparation of Intermediate a

[0088] Weigh Boc-Ala-OH (9.5 g, 50 mmol), HBTU (19.0 g, 55 mmol) and HOBT (8.1 g, 50 mmol) and dissolve in 250 mL of acetonitrile, add DIPEA (13.5 mL, 100 mmol), and activate at room temperature for 5 mins. Methyl thioglycolate (5.4 mL, 60 mmol) was added, and the mixture was reacted at room temperature for 16 hours. After the reaction, the acetonitrile was removed by rotary evaporation, an appropriate amount of ethyl acetate was added to dissolve the product, and 4% NaHCO was used for each. 3 , 1N HCl and saturated brine for three times, combine the organic phases, add anhydrous sodium sulfate, dry and filter, and remove the solvent. 150 mL of 2.0 mol / L hydrogen chloride ethyl acetate solution was added to react for 2 h. After the reaction, the solvent was removed by rotary evaporation, and then washed three times with ethyl acetate to obtain compound a, which was directly used in the next step without purification.

Embodiment 2

[0089] Example 2: Preparation of peptide c peptide resin

[0090] 46.6 g of Rink Amide resin with a substitution degree of 0.43 mmol / g was weighed into a solid-phase reaction column, and swollen with DMF / DCM (1:1) for 30 minutes. Deprotect with 200mLx2DBLK for 5min+7min, and wash with 200mLx5DMF. Weigh Fmoc-Glu-OAllyl (40.9 g, 100.0 mmol) and HOBT (16.2 g, 110.0 mmol) and dissolve them in 150 mL of DMF, add DIC (20.4 mL, 120.0 mmol) under ice bath for activation for 5 min, and add the mixture to the reaction In the column, the reaction was carried out at room temperature for 2 hours. At the end of the reaction, wash the resin with 200mLx3DMF, add 200mL DBLKx2 for deprotection 5min+7min, wash the resin with 200mLx5DMF, and continue to couple Fmoc-Gly-OH, Boc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc in sequence according to the peptide sequence -Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tB...

Embodiment 3

[0091] Example 3: Preparation of Peptide Fragment I

[0092] 44.3 g of the c-peptide resin obtained in Example 2 was weighed into a solid-phase reaction column and swollen with DMF for 30 minutes. Weigh HOBT (8.1 g, 55.0 mmol), DIC (10.2 ml, 60 mmol) and ethyl 3-mercaptopropionate (25.2 ml, 200 mmol), dissolve them in 150 mL of DMF, and add them to the reaction column. Repeat. After the reaction, the resin was washed with 150 mL x 5DMF, 150 mL x 3 MeOH was shrunk, and drained. Add the drained resin to a 1L round-bottomed flask, add the pre-configured lysis solution TFA / H 2 O / PhOMe / PhSMe (90:5:4:1, v / v) 500 mL, magnetically stirred at room temperature for 2 hours, filter the resin under reduced pressure, and collect the filtrate. The resin was washed with a small amount of TFA and the filtrates were combined. The filtrate was slowly added to 2.5L of ice ether for precipitation, centrifuged, washed with 2.5L×3 ice ether, and dried with nitrogen to obtain 21.5g of peptide fra...

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Abstract

The invention discloses a synthesis method of semaglutide. The synthesis method comprises the following steps: synthesis of a peptide fragment I; synthesis of a peptide fragment III: Cys18-Gly31; the preparation method comprises the following steps: synthesizing an intermediate I: His1-Gln17-Cys18-Gly31; synthesizing a crude peptide of the semaglutide; or synthesis of the peptide fragment II: synthesis of the peptide fragment IV: Cys19-Gly31; synthesis of an intermediate II: His1-Ala18-Cys19-Gly31, and synthesis of an intermediate II: His1-Ala18-Cys19-Gly31; and synthesizing the crude semaglutide. According to the method disclosed by the invention, two fragment peptides of solid-phase synthesis can be purified, so that impurities generated by racemization, oxidation and hydrolysis of a final semaglutide product are greatly reduced. Meanwhile, the two segments can be synthesized at the same time, so that the synthesis efficiency is improved. According to the method disclosed by the invention, the crude semaglutide is prepared by carrying out liquid-phase one-step reaction on two segments of fragment peptides in a liquid phase, in the final liquid chromatography purification step, impurities are not defective peptides lacking one or more amino acids, but are uncondensed partial fragments, the purification difficulty is not caused, in addition, the uncondensed partial peptide fragments can be recycled, and the cost is saved.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drug production, and relates to a method for synthesizing semaglutide. Background technique [0002] Semaglutide, a long-acting GLP-1 analog developed by Nove Nordisk for subcutaneous injection once a week, was approved by the US FDA on December 5, 2017. Structurally, semaglutide is a GLP-1 (7-37) chain where Ala at position 8 is replaced by Aib, Lys at position 34 is replaced by Arg, and Lys at position 26 is connected to an octadecanoic acid fatty chain. Compared with liraglutide, semaglutide has a longer aliphatic chain and increased hydrophobicity, but semaglutide is modified with a short chain of PEG, and its hydrophilicity is greatly enhanced. After PEG modification, it can not only bind closely to albumin, cover the enzymatic hydrolysis site of DPP-4, but also reduce renal excretion, prolong the biological half-life, and achieve the effect of long circulation. [0003] Semaglutide che...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/04C07K1/06C07K1/10C07K1/02C07K1/20
CPCC07K14/605
Inventor 唐仁锦尹传龙唐洋明余品香
Owner HYBIO PHARMA
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