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WO2013042129A1 - Procédé amélioré pour la préparation de bivalirudine - Google Patents

Procédé amélioré pour la préparation de bivalirudine Download PDF

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Publication number
WO2013042129A1
WO2013042129A1 PCT/IN2011/000664 IN2011000664W WO2013042129A1 WO 2013042129 A1 WO2013042129 A1 WO 2013042129A1 IN 2011000664 W IN2011000664 W IN 2011000664W WO 2013042129 A1 WO2013042129 A1 WO 2013042129A1
Authority
WO
WIPO (PCT)
Prior art keywords
bivalirudin
gly
glu
pro
protected
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2011/000664
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English (en)
Inventor
Satyanarayana Kota
Kali Satya Bhujanga Rao Adibhatla
Venkaiah Chowdary Nannapaneni
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Natco Pharma Ltd
Original Assignee
Natco Pharma Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Natco Pharma Ltd filed Critical Natco Pharma Ltd
Priority to PCT/IN2011/000664 priority Critical patent/WO2013042129A1/fr
Publication of WO2013042129A1 publication Critical patent/WO2013042129A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin

Definitions

  • the invention relates to a method of preparing bivalirudin by solid phase peptide synthesis employing Fmoc methodology.
  • Bivalirudin (I) is a polypeptide consisting of 20 amino acid residues. It is used as an anticoagulant in the treatment of unstable angina for patients undergoing percutaneous coronary intervention / percutaneous transluminal coronary angioplasty (PCI/PTCA). Its mechanism action is by direct thrombin inhibition.
  • the amino acid sequence of the peptide is D-Phenyalalanine-L-prolyl-L-argininyl-L-prolyl-glycyl-glycyl-glycyl-glycyl- L-asparginyl-glycyl-L-alpha-aspartylL-phenylsalanyl-L-alpha-glutamyl-L- alphaglutamyl-L-isoleucyl-L-prolyl-L-tyrosyl-L-leucine and drug is used as trifluoroacetate hydrate.
  • Bivalirudin is prepared by three different peptide synthetic methodologies. These are solution phase peptide synthesis, solid phase peptide synthesis and hybrid synthesis involving both the solution and solid phase synthesis.
  • Hybrid synthesis is combination of solid and solution peptide synthesis. In these methodologies a part of synthesis is done in solid phase and other part is done solution phase.
  • Recent patents WO 2007/033383, US20070093423A describe synthesis of bivalirudin by hybrid methodology. The methodology involves synthesis of fragments by solid phase technique, then purifying by HPLC and finally coupling the purified fragments by solution phase peptide synthesis. Overall, the process is laborious and difficult to implement on commercial scale as one needs to facilities for both solution and solid phase syntheses. Creating cGMP production facilities for both solution and solid phase synthesis is highly expensive and not feasible.
  • the main objective of the present invention is to prepare highly pure bivalirudin by fmoc solid phase peptide synthesis.
  • the invention consists of following steps:
  • the nitro protecting group is then cleaved by formic acid involving reduction reaction in presence of Pd/C to yield crude bivalirudin.
  • the crude bivalirudin is purified by preparative HPLC and lyophilized to get bivalirudin with a purity of more than 99%.
  • the present application relates to an improved, commercially viable process for preparation of bivalirudin.
  • the process basically consists of following step:
  • the first step in solid phase synthesis involves anchoring the carboxyl group of last amino acid in the chain to solid support.
  • solid supports such as Wang resin, chlorotrityl chloride resin, TentaGel TGA, TentGel S AC, TentaGel S PHB, ChemMatrix Wang or HMPB Chem Matrix.
  • chlorotrityl chloride resin is preferred due to better and easy anchoring procedures and commercial availability.
  • the first amino acid to anchor is Fmoc-Leu-OH using DIEA as catalyst in DMF solvent.
  • fmoc group is cleaved using 1-50% piperidine in DMF, DMSO, diethyl acetamide or any other suitable aprotic solvent.
  • the fmoc cleaving agent may be selected from other secondary bases such DBU, diethyl amine etc. either as reagent single or in combination form 1-50%, combination of other secondary bases such as DBU, diethyl amine, etc.
  • the free amine group is coupled to next amino acid in the sequence, which is Fmoc-Tyr(tBu)-OH in a solvent.
  • the peptide coupling reagent could be DIC, HBTU, TBTU, PyBoP, HATU, HCTU, with tertiary bases such as DIEA, N-methylmorphiline.
  • the fmoc-amino acids and coupling agent are always taken in excess. It could be 1-8 equivalents to resin substitution.
  • the attached fmoc group is cleaved as above, then next amino acid, Glu(OtBu)-OH in the sequence is coupled and cleaved.
  • other amino acids in the sequence are coupled and cleaved in the following sequence.
  • the amino acids are used in 1-8 equivalents to substitution.
  • the peptide chain is cleaved from the resin by treating with cocktail reagent consisting TFA (trifluoroacetic acid), ethanethiol, TIS (triisopropyl silane), p-cresol, water in various ratios. Preferred rations are TFA, TIS, cresol, water (92.5, 2.5, 2.5, and 2.5) for l-6h at 0-40°C.
  • cocktail reagent consisting TFA (trifluoroacetic acid), ethanethiol, TIS (triisopropyl silane), p-cresol, water in various ratios. Preferred rations are TFA, TIS, cresol, water (92.5, 2.5, 2.5, and 2.5) for l-6h at 0-40°C.
  • the cleaved peptide in TFA is concentrated to about 1/10 th volume, treated with suitable solvent such as diethyl ether, diisopropyl ether, methylisobutyl tertiary ether and
  • the hitherto known processes uses very expensive and commercially not easily available building block Fmoc-Arg(Pmc/Pbf)-OH for synthesis, whereas in the present invention the building block is substituted by cheap and easily available Fmoc-Arg(N02)-OH. This replacement has reduced the overall cost of production of bivalirudin appreciably.
  • the present process also uses environmentally friendly, non-hazardous, industrially convenient nitro cleavage reagent formic acid-Pd/C in place of the normally used hydrogenation at high pressure.
  • the synthesis of peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure staring from chlorotrity chloride resin (lOg, substitution 1.1 meq/g).
  • the resin was transferred to the reaction vessel of the peptide synthesizer (Sonata, Protein technologies, USA) and the synthesis was carried out using 2 molar excess protected amino acids with TBTU/DIEA activator.
  • the first amino acid leucine was attached to resin in presence of 2 molar excess DIEA. After attaching the first amino acid excess chloride groups on chloritryl resin were neutralized by treating with methanol.
  • the assembled peptide on the resin was treated with 300 ml cleaver of cocktail mixture consisting of TFA(95%), TES (2.5), water (2.5%) for 3 h at 25-30°C and filtered to remove resin.
  • the cocktail mixture was subjected to distillation, treated with ether and precipitated nitro protected peptide is filtered and dried (yield 19.0g).
  • Fractions with purity greater than 99% were pooled and loaded again onto prep C-18 column (50x 250mm, 100A 0 ) using trifluoroacetic acid buffer (BufferA; 0.1% trifluoroacetic acid in water, Buffer B; 0.1% trifluroacetic acid in acetonitrile) The fractions with more than 99% purity were pooled and lyophilized to get purified final product (2.4g).
  • BufferA 0.1% trifluoroacetic acid in water
  • Buffer B 0.1% trifluroacetic acid in acetonitrile

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La bivalirudine est un polypeptide contenant 20 acides aminés et est utilisé en tant qu'anticoagulant. La présente invention concerne un procédé simple, économique et commode pour la préparation de bivalirudine par synthèse en phase solide en utilisant la chimie Fmoc. Le peptide linéaire est assemblé sur une résine chlorure de chlorotrityle-polystyrène. Le peptide assemblé est clivé par l'acide trifluoroacétique pour obtenir une nouvelle bivalirudine nitro-protégée, la guanidine de l'arginine étant protégée par un groupe nitro. Le groupe nitro dans la bivalirudine nitro-protégée est clivé par de l'acide formique - Pd/C pour obtenir de la bivalirudine brute. Le réactif et les conditions utilisés dans cette réaction sont respectueux de l'environnement. La bivalirudine brute est purifiée par CLHP préparative en trois phases pour obtenir une bivalirudine ayant une pureté supérieure à 99 %.
PCT/IN2011/000664 2011-09-23 2011-09-23 Procédé amélioré pour la préparation de bivalirudine Ceased WO2013042129A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IN2011/000664 WO2013042129A1 (fr) 2011-09-23 2011-09-23 Procédé amélioré pour la préparation de bivalirudine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IN2011/000664 WO2013042129A1 (fr) 2011-09-23 2011-09-23 Procédé amélioré pour la préparation de bivalirudine

Publications (1)

Publication Number Publication Date
WO2013042129A1 true WO2013042129A1 (fr) 2013-03-28

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2011/000664 Ceased WO2013042129A1 (fr) 2011-09-23 2011-09-23 Procédé amélioré pour la préparation de bivalirudine

Country Status (1)

Country Link
WO (1) WO2013042129A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017019174A1 (fr) * 2015-07-30 2017-02-02 Ambiopharm, Inc. Procédé de production de bivalirudine
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
CN108663439A (zh) * 2017-03-31 2018-10-16 江苏汉邦科技有限公司 一种使用高效液相色谱纯化比伐卢定的方法
US10407464B2 (en) 2014-04-03 2019-09-10 Amgen Inc. Method for preparing AMG 416
CN115073587A (zh) * 2022-07-08 2022-09-20 厦门胜泽泰医药科技有限公司 一种半连续液相合成比伐芦定的合成工艺

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5196404A (en) 1989-08-18 1993-03-23 Biogen, Inc. Inhibitors of thrombin
WO2006045503A1 (fr) 2004-10-19 2006-05-04 Lonza Ag Procede de synthese de peptides en phase solide
WO2007033383A2 (fr) 2005-09-14 2007-03-22 Novetide, Ltd. Procédé destiné à la production de bivalirudine
WO2008109079A2 (fr) 2007-03-01 2008-09-12 Novetide, Ltd. Peptides à pureté élevée
US20090062511A1 (en) 2007-09-05 2009-03-05 Raghavendracharyulu Venkata Palle Process for the preparation of bivalirudin and its pharmaceutical compositions
CN101475631A (zh) 2009-01-08 2009-07-08 苏州中科天马肽工程中心有限公司 比伐卢定的液相合成方法
CN101538317A (zh) 2007-02-08 2009-09-23 中国人民解放军总医院 凝血酶直接抑制剂多肽水合盐及合成方法
CN101555274A (zh) 2009-05-15 2009-10-14 上海昂博生物技术有限公司 一种多肽固相合成比法卢定粗品的制备方法
US20100056755A1 (en) 2008-09-03 2010-03-04 Scinopharm Taiwan Ltd. Process for Making Bivalirudin

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5196404B1 (en) 1989-08-18 1996-09-10 Biogen Inc Inhibitors of thrombin
US5196404A (en) 1989-08-18 1993-03-23 Biogen, Inc. Inhibitors of thrombin
WO2006045503A1 (fr) 2004-10-19 2006-05-04 Lonza Ag Procede de synthese de peptides en phase solide
WO2007033383A2 (fr) 2005-09-14 2007-03-22 Novetide, Ltd. Procédé destiné à la production de bivalirudine
US20070093423A1 (en) 2005-09-14 2007-04-26 Avi Tovi Process for production of Bivalirudin
CN101538317A (zh) 2007-02-08 2009-09-23 中国人民解放军总医院 凝血酶直接抑制剂多肽水合盐及合成方法
WO2008109079A2 (fr) 2007-03-01 2008-09-12 Novetide, Ltd. Peptides à pureté élevée
US20090062511A1 (en) 2007-09-05 2009-03-05 Raghavendracharyulu Venkata Palle Process for the preparation of bivalirudin and its pharmaceutical compositions
US20100056755A1 (en) 2008-09-03 2010-03-04 Scinopharm Taiwan Ltd. Process for Making Bivalirudin
WO2010028122A1 (fr) * 2008-09-03 2010-03-11 Scinopharm Taiwan Ltd. Procédé de fabrication de bivalirudine
CN101475631A (zh) 2009-01-08 2009-07-08 苏州中科天马肽工程中心有限公司 比伐卢定的液相合成方法
CN101555274A (zh) 2009-05-15 2009-10-14 上海昂博生物技术有限公司 一种多肽固相合成比法卢定粗品的制备方法
US20100292436A1 (en) 2009-05-15 2010-11-18 Shanghai Ambiopharm, Inc. Method for producing bivalirudin

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
US10407464B2 (en) 2014-04-03 2019-09-10 Amgen Inc. Method for preparing AMG 416
US11377474B2 (en) 2014-04-03 2022-07-05 Amgen Inc. Method for preparing AMG 416 (etelcalcetide)
WO2017019174A1 (fr) * 2015-07-30 2017-02-02 Ambiopharm, Inc. Procédé de production de bivalirudine
CN108663439A (zh) * 2017-03-31 2018-10-16 江苏汉邦科技有限公司 一种使用高效液相色谱纯化比伐卢定的方法
CN115073587A (zh) * 2022-07-08 2022-09-20 厦门胜泽泰医药科技有限公司 一种半连续液相合成比伐芦定的合成工艺

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