Method for preparing AMG416 by combination of solid and liquid phase

A solid-liquid phase and solid-phase carrier technology, applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problems of reduced product purity and yield, cumbersome post-processing, easy cleavage of disulfide bonds, etc. To achieve the effect of reducing the increase of by-products, avoiding cumbersome post-processing, and reducing adverse factors

Active Publication Date: 2018-06-29
SICHUAN KELUN PHARMA RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method forms disulfide bonds in a solid-phase manner, and the reaction time is too long, usually more than 24 hours, which is not conducive to large-scale production
[0008] The above-mentioned existing AMG416 preparation method mainly has the following deficiencies: 1. When the liquid-phase synthesis method is oxidized to form disulfide bonds, the amount of solvent used is large, which not only increases the cost but also pollutes the environment; post-processing is tedious, and the formed disulfide bonds are easily broken Generate impurities, require multiple purifications and reduce product purity and yield
2. The solid-phase synthesis method requires a long reaction time to form disulfide bonds by oxidation, which increases the cost and is not conducive to large-scale production
[0009] Based on the above technical application background, the inventor of the present invention intends to solve the shortcomings of the existing preparation method such as cumbersome disulfide bond formation process, long reaction time, and large amount of solvent used to pollute the environment, and provide a new method for preparing AMG416

Method used

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  • Method for preparing AMG416 by combination of solid and liquid phase
  • Method for preparing AMG416 by combination of solid and liquid phase
  • Method for preparing AMG416 by combination of solid and liquid phase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) Synthesis of peptide backbone

[0049] Weigh Sieber resin (5g, substitution value 0.69mmol / g resin) and join in the polypeptide reactor, add 50ml of DCM to wash and swell the resin; Use 20% piperidine / DMF solution to remove the Fmoc protecting group on the resin; DMF Wash the resin, remove Fmoc by-products and residual piperidine, ninhydrin detection (Kaiser Test), resin blue, complete deprotection;

[0050] Weigh Fmco-D-Arg(pbf)-OH (3eq, 6.7g), Cl-HoBt (3eq, 1.8g), HBTU (2.9eq, 3.8g) dissolved in 35ml DMF, then add DIEA (3.3eq, 1.9ml) was stirred, the reaction was detected by ninhydrin, filtered, washed with DMF, and detected by ninhydrin, the resin was colorless and transparent, and the condensation reaction was complete. Add 20% piperidine / DMF solution to remove the Fmoc protecting group on the resin, wash the resin with DMF, remove Fmoc by-products and residual piperidine, detect ninhydrin, the resin is blue, and the deprotection is complete.

[0051] Accordin...

Embodiment 2

[0067] (1) Synthesis of peptide backbone

[0068] Take Sieber resin (5.0g, substitution value 0.69mmol / g resin) and join in the polypeptide reactor, add 50ml DCM to wash and swell the resin, add 20% piperidine / DMF solution after filtering to remove the Fmoc protecting group; then add DMF washing resin, ninhydrin detection (Kaiser Test), resin blue, deprotection is complete.

[0069] Weigh Fmco-D-Arg(pbf)-OH (3eq, 6.7g), Oxyma (4.5eq, 2.2g), dissolve in 35ml DMF, then add DIC (6eq, 3.2ml), stir and add to the above polypeptide In the reactor, react at room temperature, take a small sample during the reaction, use ninhydrin to monitor the completion of the condensation, filter, wash the resin with DMF, and detect the ninhydrin, the resin is colorless and transparent, and the condensation reaction is complete. Add 20% piperidine / DMF solution to remove the Fmoc protecting group, wash the resin with DMF, detect ninhydrin, the resin is blue, and the deprotection is complete.

[00...

Embodiment 3

[0085] (1) Synthesis of peptide backbone

[0086] Take Sieber resin (2.0g, substitution value 0.69mmol / g resin) and join in the polypeptide reactor, add 20mlDCM to wash and swell the resin, add 20% piperidine / DMF solution after filtering to remove the Fmoc protecting group; then add DMF Wash the resin, remove Fmoc by-products and residual piperidine, ninhydrin detection (Kaiser Test), resin blue, complete deprotection;

[0087] Dissolve Fmco-D-Arg(pbf)-OH (3eq, 2.7g), Cl-HoBt (3eq, 0.7g) in 7ml DMF, then add DIC (3eq, 0.7ml), stir and add to the above polypeptide In the reactor, react at room temperature, take a small sample during the reaction, use ninhydrin to monitor the completion of the condensation, filter the resin, wash the resin with DMF, and detect the ninhydrin, the resin is colorless and transparent, and the condensation reaction is complete. Add 20% piperidine / DMF solution to remove the Fmoc protecting group, then use DMF to wash the resin to remove Fmoc by-produ...

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PUM

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Abstract

The invention provides a method for preparing AMG416 by combination of solid and liquid phase, and belongs to the field of polypeptide synthesis. The method is characterized by comprising the following steps: firstly, preparing completely-protected main chain peptide resin by using acid-sensitive amino resin as a solid phase carrier; then, directly adding diluted acid lysate to the peptide resin,and simultaneously, adding Y-Cys(X)-OH to form a disulfide bond to obtain a completely-protected target peptide; and finally, performing cracking and purification to obtain the AMG416. By adopting themethod provided by the invention for preparing the AMG416 by combination of the solid and liquid phase, the reaction time is shortened, the increase of by-products caused by long-time reaction is reduced, the purity of the target peptide is improved, the cost is reduced, the pollution to the environment is reduced, and large-scale production is facilitated.

Description

technical field [0001] The invention belongs to the field of polypeptide synthesis, and in particular relates to a method for preparing AMG416 by solid-liquid phase combination. Background technique [0002] AMG416, also known as Velcalcetide, is a polypeptide compound consisting of 8 amino acids, the C-terminus ends with amide, and the N-terminus is modified by acetylation. The sequence is as follows: [0003] [0004] AMG416 was developed by Amgen Corporation of the United States. It has been declared to the FDA and approved in the European Union. It is mainly used for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease undergoing dialysis. Phase III clinical research shows that AMG416 is superior to the currently marketed similar product cinacalcet in the treatment and safety of secondary hyperparathyroidism. The industry expects that after the product is launched, its annual sales will exceed US$1 billion , has broad market prospect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/04C07K1/06
CPCC07K7/06Y02P20/55
Inventor 薛宏祥潘钧铸杨燕苹康艳萍李长兵庹世川王利春王晶翼
Owner SICHUAN KELUN PHARMA RES INST CO LTD
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