WO2009150657A1 - Procédé amélioré de préparation d’eptifibatide par synthèse en phase solide de type fmoc - Google Patents

Procédé amélioré de préparation d’eptifibatide par synthèse en phase solide de type fmoc Download PDF

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Publication number
WO2009150657A1
WO2009150657A1 PCT/IN2008/000360 IN2008000360W WO2009150657A1 WO 2009150657 A1 WO2009150657 A1 WO 2009150657A1 IN 2008000360 W IN2008000360 W IN 2008000360W WO 2009150657 A1 WO2009150657 A1 WO 2009150657A1
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WO
WIPO (PCT)
Prior art keywords
peptide
eptifibatide
acm
resin
group
Prior art date
Application number
PCT/IN2008/000360
Other languages
English (en)
Inventor
Satyanarayana Kota
Venkateswarlu Tallapaneni
Bhujanga Rao Adibhatla Kali Satya
Nannapaneni Venkaiah Chowdary
Original Assignee
Natco Pharma Limited
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 Limited filed Critical Natco Pharma Limited
Priority to PCT/IN2008/000360 priority Critical patent/WO2009150657A1/fr
Publication of WO2009150657A1 publication Critical patent/WO2009150657A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to a novel process for the preparation of Pharmaceutical grade eptifibatide 1 using solid-phase Fmoc-chemistry.
  • the eptifibatide used for the treatment of cardio vascular disease. It is a short acting parenteral antithrombotics drug used for treating Acute Coronary Syndrome (ACS). It is also used for patients undergoing Percutaneous Coronary Interventions (PCI). Eptifibatide is believed to work by inhibiting platelet aggregation, specifically by blocking the platelet receptor glycoprotein Ilb/IIIa.
  • Chemically eptifibatide is a cyclic heptapeptide containing six amino acids and one mercaptopropionyl (des-aminocysteine, Mpa) residue.
  • An interchain disulphide linkage is formed between the cystiene amide and the mercaptopropionyl moieties. It is produced both by solution and solid phase peptide synthesis.
  • solution-phase route synthesis is laborious as compared to the solid-phase route as after each coupling the peptide formed has to be isolated. Whereas in the solid-phase synthesis, the excess reagents and by-products are washed off by simple filtration. In both the methods desired peptide is prepared by the step- wise addition of amino acid moieties to a building peptide chain.
  • Fmoc-chemistry based synthesis utilize procedures under milder process conditions and because of the base labiality of Fmoc-group, acid-labile side-chain protecting groups are employed providing orthogonal protection.
  • Fmoc based solid phase syntheses are descried in WO /01/121164 A, WO 03/093302, CN 1858060-A, CN 1500805, WO 2006045483, WO 2006 119388, WO 2006 0420 WO 2004 092202, WO 03/093302. These reports vary from each other in following aspects: - Nature of solid support used for assemble of peptide
  • Patent CN 1858060 discloses the solid -phase synthesis of eptifibatide, which comprises attaching Fmoc- ⁇ -protected amino acid to the solid support Rink amide MBHA / AM. Thiol moieties of mercaptopropionic acid and cysteine are protected by benzyl ether. Cleavage of peptide from the resin is affected by TFA/HBr/AcOH system. These cleavage conditions create lot of impurities, as the peptide bonds are also susceptible for HBr/AcOH mixture, and thus form small peptide impurities in this process. This process claims oxidation of crude peptide in air at pH 7.5- 10. These conditions also initiate the formation of impurities.
  • Patent WO 2006045483 describes a general concept of on resin cyclization of cysteines with t-butylsulfenyl, dithiopropionyl thiol protecting groups using Siber resin.
  • eptifibatide is chosen as model compound. There are no details regarding the purity.
  • Patents WO 2006 119388, WO 2006 0420 WO 2004 092202 from Novetide/Teva describe the preparation using trityl protected mercaptopropionic acid and on trityl resin.
  • CN 1500805 describes the preparation of eptifibatide on rink amide resin.
  • thiol of mercaptopropionic acid is protected by triphenylmethyl and guanidine of homoargaine by N,Ndi-tert butoxycarbinyl protecting groups respectively.
  • Patent WO 03/093302 discloses the process for the solid-phase synthesis of eptifibatide comprising of attaching an ⁇ - nitrogen protected ⁇ -carboxamide amino acid to the solid support 4-methoxy trityl polystyrene resin through its thiol side chain, followed by removing the ⁇ -nitrogen protecting group and assembling the peptide chain on the ⁇ - nitrogen.
  • Mercaptopropionic acid is protected by trityl or by disulphide of mercaptopropionic acid.
  • the process consists of number steps and uses expensive raw materials.
  • the present process does not involve the isolation of thiol peptide, susceptible to aerial oxidation leading to the formation of impurities, which hampers the purification of the final product and yield.
  • Homoarginine moiety is obtained from Fmoc-Har (Pbf)-OH. Protection of guanidine of homoargaine by Pbf group affords hydrophobicity for highly hydrophilic homoargnine, which facilitates smooth coupling.
  • Disulfide loop in the peptide amide is introduced through an efficient process by the deprotection and oxidation of thiol moieties in the single step without isolation of -SH peptide. 4.
  • the present process yields pharmaceutical grade eptifibatide with >99% purity of isolated product.
  • the present invention relates to a novel process for the preparation of Pharmaceutical grade eptifibatide 1, which involves, (i) assembling amino acid residues and mercaptopropionic acid with an appropriate thiol protecting groups on a solid phase resin to yield resin bound peptide 2, (ii) cleaving the peptide thus obtained from the resin with concomitant removal of side chain protecting groups except acm protecting group of thiol moiety to obtain crude peptide 3. (iii) crude peptide 3 having protected thiol groups, is deprotected and oxidized with iodine in water/acetic acid to obtain crude eptifibatide. (iv) and finally subjecting to chromatographic purification.
  • the procedure described in the present invention is simple, efficient and cost effective and adoptable for large-scale production.
  • Figure 1 HPLC Chromatogram of peptide 3
  • Figure 2 HPLC Chromatogram of crude eptifibatide 1
  • Figure 3 HPLC chromatogram of pooled fractions of primary purification
  • Figure 4 HPLC chromatogram of pooled fractions of secondary purification
  • Figure 5 HPLC chromatogram of lyophilized eptifibatide
  • the purity and yield of the peptides are important aspects of any route of synthesis. Purity is represented by the degree of presence of pharmacologically active related impurities. The trace amounts of impurities present in the peptide may disturb and adversely affect the beneficial action of the peptide when used as a therapeutic agent. In solution phase synthesis repeated purifications at each step invariable lead to low yield of the final peptide.
  • the present invention is a novel process to achieve high purity with enhanced yield of the target peptide eptifibatide through solid phase methodology.
  • the step- wise introduction of N ⁇ -protected amino acids in solid phase peptide syntesis normally involves the carboxyl group activation of the incoming amino acid or the use of pre-formed activated amino acid derivatives.
  • the reagents HOBt/ DIC, HBTU, TBTU, Py Boc and HATU are the preferred tools for in situ carboxyl activation.
  • HBTU, TBTU, Py Boc and HATU are expensive hence inexpensive HOBt/DIC is used for coupling.
  • simple alpha amino protected Fmoc-homoargaine without side chain guanidine protection is used initially. In this case coupling did not take place efficiently, hence Fmoc-Har (Pbf)-OH is used.
  • Oxidative cyclization of protected or non-protected sulfhydryl groups with formation of disulfide structures is usually carried out at the final synthetic step. In few cases it is also carried out before cleavage of the peptide molecule from the solid support.
  • the oxidation of open-chain peptide containing free and / or certain types of protected sulfhydryl groups with iodine in methanol or acetic acid, acetic acid/water is a convenient and safe process.
  • the deprotection and oxidation of acm protecting groups of cysteine and mercaptopropionic acid is effected by iodine.
  • Synthesis of the peptide was carried out by a regular stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Rink amide resin.
  • the resin was transferred to the reaction vessel of the peptide synthesizer (Endeavor 90, AAPPTEC peptide synthesizer) and the linear peptide was assembled on it using 6.0 times mole excess amino acid derivatives, on the peptide synthesizer.
  • the first amino acid Fmoc-Cys (Acm)-0H was coupled to the resin by deprotection the Fmoc-group on the resin followed by activation of Fmoc-Cys (Acm)-OH.
  • Buffer A 0.5% AcOH in water + acetonitrile (95+5)
  • Buffer B 0.5% AcOH in water + acetonitrile (50+50)
  • Buffer A 0.5% AcOH in water + acetonitrile (95+5)
  • Buffer B 0.5% AcOH in water + acetonitrile (50+50)
  • Buffer C 0.1 M Ammonium acetate in water + acetonitrile (95+5), pH 6.5

Abstract

L’eptifibatide est un heptapeptide cyclique contenant six acides aminés et un résidu mercaptopropionyle (désaminocystéine, Mpa). Une liaison disulfure interchaîne est formée entre les fractions cystéine amide et mercaptopropionyle. C’est un médicament antithrombotique administré par voie parentérale à courte durée d’action utilisé pour traiter le syndrome coronarien aigu (SCA). La présente invention concerne un procédé simple, pratique et économique de préparation d’eptifibatide par synthèse peptidique en phase solide de type Fmoc. Le peptide linéaire est assemblé sur une résine Rink amide, dans laquelle les groupes thiol de la cystéine et l’acide mercaptopropionique sont protégés par un groupe acm et la guanidine de l’homoarginine par un groupe pbf. Le peptide assemblé est coupé de la résine pour donner un peptide linéaire dans lequel les thiols de la cystéine et l’acide mercaptopropionique sont protégés par le groupe acm. Le peptide linéaire est oxydé par de l’iode réactif à oxydation légère pour donner de l’eptifibatide. Une purification par CLHP en trois étapes donne de l’eptifibatide de qualité pharmaceutique de pureté supérieure à 99 %.
PCT/IN2008/000360 2008-06-09 2008-06-09 Procédé amélioré de préparation d’eptifibatide par synthèse en phase solide de type fmoc WO2009150657A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IN2008/000360 WO2009150657A1 (fr) 2008-06-09 2008-06-09 Procédé amélioré de préparation d’eptifibatide par synthèse en phase solide de type fmoc

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IN2008/000360 WO2009150657A1 (fr) 2008-06-09 2008-06-09 Procédé amélioré de préparation d’eptifibatide par synthèse en phase solide de type fmoc

Publications (1)

Publication Number Publication Date
WO2009150657A1 true WO2009150657A1 (fr) 2009-12-17

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PCT/IN2008/000360 WO2009150657A1 (fr) 2008-06-09 2008-06-09 Procédé amélioré de préparation d’eptifibatide par synthèse en phase solide de type fmoc

Country Status (1)

Country Link
WO (1) WO2009150657A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013057736A2 (fr) * 2011-06-07 2013-04-25 Emcure Pharmaceuticals Limited Préparation du peptide eptifibatide
CN110894212A (zh) * 2018-08-24 2020-03-20 翰宇药业（武汉）有限公司 一种合成依替巴肽硫醚的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003093302A2 (fr) * 2002-05-03 2003-11-13 Avecia Limited Procede de synthese de peptides
CN1500805A (zh) * 2002-11-14 2004-06-02 吉尔生化（上海）有限公司 一种制备依非巴特的新工艺
US20040249121A1 (en) * 2003-04-07 2004-12-09 Avi Tovi Process for production of cyclic peptides
WO2005100381A2 (fr) * 2004-04-08 2005-10-27 Millennium Pharmaceuticals, Inc. Procedes de preparation de l'eptifibatide
WO2006041945A2 (fr) * 2004-10-04 2006-04-20 Novetide, Ltd. Processus d'echange de contre-ion pour peptides
CN1858060A (zh) * 2005-05-08 2006-11-08 周达明 固相多肽合成依非巴特的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003093302A2 (fr) * 2002-05-03 2003-11-13 Avecia Limited Procede de synthese de peptides
CN1500805A (zh) * 2002-11-14 2004-06-02 吉尔生化（上海）有限公司 一种制备依非巴特的新工艺
US20040249121A1 (en) * 2003-04-07 2004-12-09 Avi Tovi Process for production of cyclic peptides
WO2005100381A2 (fr) * 2004-04-08 2005-10-27 Millennium Pharmaceuticals, Inc. Procedes de preparation de l'eptifibatide
WO2006041945A2 (fr) * 2004-10-04 2006-04-20 Novetide, Ltd. Processus d'echange de contre-ion pour peptides
CN1858060A (zh) * 2005-05-08 2006-11-08 周达明 固相多肽合成依非巴特的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIONG Y ET AL.: "Fmoc solid-phase synthesis and purification of eptifibatide", XIAMEN DAXUE XUEBAO, vol. 46, no. 1, January 2007 (2007-01-01), pages 100 - 103, XP008100026, ISSN: 0438-0479 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013057736A2 (fr) * 2011-06-07 2013-04-25 Emcure Pharmaceuticals Limited Préparation du peptide eptifibatide
WO2013057736A3 (fr) * 2011-06-07 2013-07-04 Emcure Pharmaceuticals Limited Préparation du peptide eptifibatide
CN110894212A (zh) * 2018-08-24 2020-03-20 翰宇药业（武汉）有限公司 一种合成依替巴肽硫醚的方法
CN110894212B (zh) * 2018-08-24 2021-06-04 翰宇药业（武汉）有限公司 一种合成依替巴肽硫醚的方法

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