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Solid-phase synthesis method of ziconotide

A technology of solid-phase synthesis of ziconotide, applied in peptide preparation methods, chemical instruments and methods, peptides, etc., can solve the difficulties of separation and purification, low accuracy of disulfide bond connection, low yield, etc. problems, to achieve the effect of easy preparation and purification, the same number of steps, and simplified steps

Active Publication Date: 2013-04-17
NANTONG SHIMEIKANG PHARMA CHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The synthetic route II of ziconotide has the disadvantages of low accuracy and low yield of disulfide bonds, and the total yield is only 30%.
The final separation and purification is also very difficult

Method used

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  • Solid-phase synthesis method of ziconotide
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  • Solid-phase synthesis method of ziconotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of ziconotide linear peptide resin.

[0041] Resin swelling: Weigh 1g of Fmoc-Rink-Amide-MBHA resin into a 20ml BD syringe with sieve plate (common glass peptide reactor can also be used), swell with 3 times the volume of the resin in DCM for 1-4 hours, and wait until the resin swells completely Then, the Fmoc protective group was removed twice with a 20% (v / v) piperidine / DMF solution for 5 min and 20 min, respectively, and washed with DCM and DMF 6 times alternately.

[0042] Amino acid connection: Dissolve 2mmol of Fmoc protected amino acid, 2mmol of HoBt, 2mmol of HBTU, 4mmol of DIPEA in DMF, pre-activate for 5 minutes, then add the swollen resin (resin quality * substitution value = 0.5mmol), shake at room temperature The reaction time is 1.5-2.5 hours. The dosage of each amino acid and condensing agent and the specific reaction time are shown in Table 1. Fmoc removal before connecting the next amino acid. Use piperidine in N, N dimethylformamide ...

Embodiment 2

[0050] Example 2: Cutting of the resin and removal of part of the side chain protecting groups.

[0051] Mix trifluoroacetic acid, anisole, dimercaptoethane and anisole in a volume ratio of 90:5:2.5:2.5, or combine trifluoroacetic acid, methanol, water and triisopropylsilane in a volume ratio 88:5:5:2 was mixed, and the linear fully protected peptide resin prepared in Example 1 was added to any of the above two mixtures. The volume of the mixed solution used per 1g of linear fully protected peptide resin was 10 mL, and shake After 2 hours of reaction, the reaction solution was poured into cold ether at -20°C, precipitated, and the white precipitate was collected after centrifugation, and air-dried to obtain a linear crude peptide containing Acm and tBu in the side chain of Cys, weighing 1.4g, and the crude yield was 94%.

Embodiment 3

[0052] Example 3: Formation of Ziconotide Bicyclic Peptide.

[0053] 1.0 g of the linear peptide prepared in Example 2 was dissolved in acetic acid to make the concentration of the linear peptide 0.5 mg / mL, and then 10 times the molar amount of I was added 2 (Dissolved in methanol), stir for 1 hour at room temperature, then add 20% water of the reaction solution volume, check whether the reaction is complete or not by HPLC, after the reaction is complete, add 0.1M ascorbic acid until the solution becomes colorless, the reaction mixture is frozen After drying, washing the precipitate with ether, the crude bicyclic peptide of Cys side chain containing tBu was obtained, weighing 0.91g, and the crude yield of this step was 91%.

[0054] The analytical HPLC detection method is:

[0055] Equipment: C18 analytical column: 4.6×150mm;

[0056] Eluent A: 0.1% (v / v) TFA / H 2 O;

[0057] Eluent B: 0.08% (v / v) TFA / acetonitrile;

[0058] Flow rate: 1ml / min;

[0059] Detection wavelength: 220nm;

[0060]...

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Abstract

The invention discloses a solid-phase synthesis method of ziconotide, and the method comprises the following steps: with Fmoc(9-fluorenylmethyloxycarbonyl)-amino resin as a solid-phase carrier, successively carrying out condensation reaction for connecting 25 protected amino acids to obtain linear fully-protected peptide resin, wherein three groups of Cys (cysteine) with disulfide bond formed areseparately connected with Trt (triphenylmethyl), Acm (acetamidomethyl), or tBu (t-butyl) protecting group; cutting resin, and simultaneously removing all amino acid protecting groups except for Acm and tBu to obtain a linear peptide containing Acm and tBu; oxidizing the linear peptide to form a first pair of disulfide bonds, and simultaneously removing Acm and forming a second pair of disulfide bonds to obtain bicyclic peptide resin; and removing tBu of the bicyclic peptide resin, and simultaneously carrying out cyclization to form a third pair of disulfide bonds and to obtain ziconotide. In the method disclosed by the invention, Trt, Acm and tBu are selected to protect the three groups of Cys, thereby improving the formation accuracy of the disulfide bonds; and after resin is cut off, three pairs of disulfide bonds are sequentially formed through two-step reaction, thereby simplifying steps and improving productivity.

Description

Technical field [0001] The invention relates to a solid phase synthesis method of ziconotide. Background technique [0002] Ziconotide, with the trade name Prialt, was developed and marketed by Elan Corporation of the United States. It was first listed in the United States in January 2005 and was approved by the European Union in February 2005. The main indications are: strong analgesic effect on rodent acute, chronic and neuropathic pain models, and can safely and effectively relieve the pain of patients with malignant and non-malignant pain, including patients who are ineffective in opioid therapy, and are tolerated It has good sex and is also used for severe chronic pain patients with poor efficacy of other analgesic treatments. The drug effect is better than other analgesic products. The analgesic effect of intrathecal injection is 1000 times stronger than that of morphine, and there will be no side effects when morphine is used, such as respiratory depression and drug resis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/06C07K1/04C07K1/02
CPCY02P20/55
Inventor 董守良李梅兴
Owner NANTONG SHIMEIKANG PHARMA CHEM
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