WO2003042393A1 - Preparation enzymatique de mycophenolate-mofetile - Google Patents

Preparation enzymatique de mycophenolate-mofetile Download PDF

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Publication number
WO2003042393A1
WO2003042393A1 PCT/IN2001/000202 IN0100202W WO03042393A1 WO 2003042393 A1 WO2003042393 A1 WO 2003042393A1 IN 0100202 W IN0100202 W IN 0100202W WO 03042393 A1 WO03042393 A1 WO 03042393A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
acid
mycophenolate mofetil
reaction
carried out
Prior art date
Application number
PCT/IN2001/000202
Other languages
English (en)
Inventor
Nitin Patil
Rakesh Mendhe
Anand Khedkar
Ramakrishnan Melarkode
Shrikumar Suryanarayan
Original Assignee
Biocon 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 Biocon Limited filed Critical Biocon Limited
Priority to PCT/IN2001/000202 priority Critical patent/WO2003042393A1/fr
Publication of WO2003042393A1 publication Critical patent/WO2003042393A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid

Definitions

  • the present invention relates to an improved method for synthesis of mycophenolate mofetil using an enzyme as catalyst in a water-free organic solvent and its subsequent purification.
  • Mycophenolate mofetil is an immunosuppressant, which is the morpholinoethyl ester of mycophenolic acid.
  • U.S. Pat. No. 4,753,935 describes a two-step process for synthesis of mycophenolate mofetil
  • mycophenolic acid is reacted with thionyl chloride to make acid chloride.
  • the acid chloride is then reacted with 2- morpholinoethanol to give mycophenolate mofetil and hydrogen chloride.
  • Disadvantages of this process include involvement of corrosive chemicals, formation of a dimeric impurity and involvement of two steps in the process.
  • 5,247,083 teaches a process for mycophenolate mofetil synthesis by re luxing mycophenolic acid with 2-mo holinoethanol in an organic solvent or a mixture of organic solvents capable of azeotropic removal of water.
  • the process involves high reaction temperatures.
  • a process for synthesis of mycophenolate mofetil by reacting mycophenolic acid and 2-morpholinoethanol in a organic solvent containing water using enzyme as a catalyst is described in WO 00/34503. Presence of water, which is also the byproduct of the reaction leads equilibrium limitation, and therefore, results in poor conversion of mycophenolic acid to mycophenolate mofetil.
  • synthesis of mycophenolate mofetil was carried out by reacting mycophenolic acid and 2-morpholinoethanol in an anhydrous organic solvent using enzyme as a catalyst.
  • Use of an anhydrous organic solvent leads to higher conversion of mycophenolic acid to mycophenolate mofetil.
  • Water that is generated in the reaction may also be removed using molecular sieves to further improve conversion of mycophenolic acid to mycophenolate mofetil.
  • the enzymatic esterification of mycophenolic acid with 2-mo ⁇ pholinoethanol in an anhydrous condition in the presence of molecular sieves is not reported.
  • Separation of mycophenolate mofetil is carried out by its selective extraction in aqueous solution of a weak acid. The impurities and unreacted mycophenolic acid, if any, do not get extracted. The acid in the aqueous layer is then neutralized using a base, which allows back extraction of mycophenolate mofetil in an organic solvent and further crystallization.
  • the present invention concerns methods for synthesis and purification of mycophenolate mofetil.
  • the method for synthesis involves reacting mycophenolic acid with 2-morpholinoethanol in an anhydrous organic solvent using an enzyme as a catalyst.
  • the method for purification of mycophenolate mofetil concerns its extraction into aqueous solution of a weak acid followed by neutralization of the acid with a base. Subsequently, mycophenolate mofetil is back extracted in an organic solvent, which is finally crystallized.
  • the present invention provides a A process for the preparation of mycophenolate mofetil comprising of:
  • the enzyme catalyst is selected from a microbial, animal or plant source.
  • the enzyme catalyst is immobilized, or non-immobilized.
  • the enzyme catalyst is selected from a lipase, esterase or protease.
  • the inert organic solvent is toluene or xylene.
  • the said enzyme reaction is carried out for 5 to 120 hours at 20 to 65°C.
  • the weak organic acid is citric acid, acetic acid, fumaric acid, malic acid or maleic acid.
  • the water immiscible solvent is ethyl acetate.
  • the charcolization is carried out at 40 to 90°C.
  • the charcolization is carried out
  • the enzyme reaction is solid phase synthesis.
  • PoFractor means a bioreactor for solid state fermentation to produce mycophenolic acid as described in PCT application WO
  • solution phase synthesis means initial mycophenolic acid concentration in the esterification reaction is equal to or less than its solubility in the organic solvent used for the reaction.
  • solid phase synthesis means initial mycophenolic acid concentration in the esterification reaction is more than its solubility in the organic solvent used for the reaction.
  • Enzyme A is a fungal lipase immobilized onto an ion exchange resin.
  • ' ⁇ nzyme B is a lipase derived from porcine pancreas.
  • ' ⁇ nzyme C is a lipase derived from yeast.
  • Enzyme D is an immobilized lipase derived from yeast.
  • Enzyme E is a fungal recombinant lipase.
  • Enzyme F is a fungal lipase immobilized onto a polymer matrix.
  • the invention involves reacting mycophenolic acid with 2-morpholinoethanol to produce mycophenolate mofetil using enzyme as a catalyst.
  • the reaction is carried out in an anhydrous organic solvent or mixture of two or more organic solvents.
  • the organic solvent is a C 6 -C ⁇ 2 alkane, for example, hexane, cycohexane, heptane, octane or iso-octane; an aromatic solvent, for example, benzene, toluene or xylene; a ketone, for example, acetone or methyl ethyl ketone; a nitrile compound, for example, acetonitrile or a halogenated solvent, for example, chloroform.
  • the enzyme that is added to the reaction mixture is a hydrolase, which may be lipase, esterase or protease from microbial, animal or plant origin.
  • the enzyme may be in either free or immobilized form. Esterification reactions are equilibrium limited.
  • molecular sieves may be added to the reaction mixture to remove the reaction by-product (i. e., water). Removal of the by-product leads to liigher conversion of mycophenolic acid to mycophenolate mofetil.
  • Concentration of mycophenolic acid in the reaction is in the range 0.02 to 15% (w/v).
  • 2-morpholinoetl ⁇ anol to mycophenolic acid molar ratio is kept in the range 1 to 15.
  • the reaction is carried out at a temperature in a range of 20 to 65 °C.
  • the reaction time to achieve certain conversion depends on amount of enzyme added to the reaction mixture and the rate at which the enzyme deactivates. Enzyme may be added to the reaction mixture during the reaction in addition to the enzyme added to in the beginning of the reaction.
  • the present invention also concerns separation and purification of mycophenolate mofetil.
  • the reaction mixture is filtered to remove enzyme.
  • the molecular sieves also are removed with the enzyme if they are added to the reaction mixture.
  • the filtrate is concentrated. If water miscible organic solvent is used for the reaction, the organic solvent is completely removed and the residue is dissolved in a water immiscible organic solvent.
  • the filtrate/concentrate/reaction residue re-dissolved in a water immiscible organic solvent is then extracted with aqueous solution of a weak acid such as citric acid, acetic acid, fumaric acid, malic acid or maleic acid.
  • the extraction may be carried out in multiple stages.
  • Mycophenolate mofetil gets selectively extracted in the acid solution.
  • the pH of the acid solution is then adjusted with a base to 7.0 - 9.0.
  • the pH-adjusted acid solution is then extracted with ethyl acetate. This extraction may also be carried out in multiple stages.
  • the ethyl acetate extract may be treated with charcoal to improve color of the extract.
  • the ethyl acetate extract is then concentrated and kept at 4 °C.
  • Mycophenolate mofetil is crystallized out of the concentrate. The crystals are filtered and dried. If water miscible organic solvent is used for the reaction, the organic solvent that is used to dissolve the reaction residue is a water immiscible solvent such as ethyl acetate, toluene, or xylene.
  • the culture Penicillium brevicompactum was fermented on wheat bran for 5 days in the PlaFractor.
  • the koji was extracted using ethyl acetate.
  • mycophenolic acid was back extracted into 10% sodium hydroxide.
  • the aqueous layer was acidified using 6 N HC1 to pH 2.5 to 2.8 and re-extracted into ethyl acetate.
  • the ethyl acetate layer was charcoalized, concentrated and the product was crystallized at 0 °C. MPA crystals formed were confirmed to be 97.2% purity by HPLC.
  • Enzyme A Three different enzymes (Enzyme A, Enzyme D and Enzyme F) were used to study effect of water presence in the reaction mixture on conversion of mycophenolic acid to mycophenolate mofetil.
  • the initial mycophenolic acid concentration was 0.5% (w/v) for Enzyme A and Enzyme D whereas the concentration was 5% (w/v) for Enzyme F.
  • To desired amount of mycophenolic acid 10 ml of anhydrous toluene was added.
  • the initial mycophenolic acid to 2- morpholino ethanol molar ratio was 1:1.2.
  • For the reactions carried out in presence of water 0.015 ml of water was added to the reaction mixture.
  • Enzyme E 15.0 g was mixed with 75 ml of acetate buffer (pH 4.5, 50 mM). 30.0 g of anhydrous CaCOs was added to the mixture. The mixture was stirred for 1 h at 26 °C. 20 ml of chilled acetone was added to the mixture, which was subsequently filtered. The solids were allowed to dry at room temperature for 20 minutes. 30.8 g of solid white power was obtained which was used for the reaction.
  • the reaction mixture (500 ml) from reaction done with Enzyme F (Example 3) was filtered to remove enzyme and molecular sieves.
  • the filtrate was twice extracted with 500 ml of 5% citric acid solution in water.
  • the two extracts were pooled together and its pH was adjusted to 8.0.
  • the pH-adjusted mixture was extracted twice with ethyl acetate.
  • the two extracts were pooled together.
  • 5.0 g charcoal was added to the mixture, which was kept at 60 °C for 20 minutes. Charcoal was removed by filtration.
  • the filtrate was concentrated and kept at 4 °C for 16 h.
  • Mycophenolate mofetil crystallized out of the solution, which was filtered and dried at 60 °C for 5 h. 31.0 g of white powder was obtained.
  • Mycophenolate mofetil obtained from Example 4 was purified from reaction mixture as carried out in Example 7 and 5.2 g of white powder was obtained.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un perfectionnement du procédé utilisé pour la synthèse du mycophénolate-mofétile. En l'occurrence, on fait réagir de l'acide mycophénolique sur du 2-morpholino-éthanol en excédent, une enzyme intervenant comme catalyseur dans un solvant organique anhydre. Cette réaction est suivie d'une opération de purification. L'utilisation d'un solvant organique anhydre permet un taux de conversion plus élevé de l'acide mycophénolique. Pour éliminer l'eau dégagée par la réaction on peut avoir recours à un tamis moléculaire, ce qui donne un taux plus élevé de conversion de l'acide mycophénolique en mycophénolate-mofétile.
PCT/IN2001/000202 2001-11-16 2001-11-16 Preparation enzymatique de mycophenolate-mofetile WO2003042393A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/IN2001/000202 WO2003042393A1 (fr) 2001-11-16 2001-11-16 Preparation enzymatique de mycophenolate-mofetile

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Application Number Priority Date Filing Date Title
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006024582A1 (fr) * 2004-09-03 2006-03-09 Poli Industria Chimica Spa Procede de preparation de mycophenolate mofetil par transesterification enzymatique
US7019133B2 (en) * 2003-02-21 2006-03-28 Chunghwa Chemical Synthesis & Biotech Co., Ltd. Process for making mycophenolate mofetil by transesterification
EP1844041A1 (fr) * 2005-01-20 2007-10-17 Apotex Fermentation Inc. Procede ameliore de preparation de mycophenolate mofetil
US7683188B2 (en) 2004-04-26 2010-03-23 TEVA Gyógyszergyár Zártkōrūen Mūkōdō Részvénytársaság Process for preparation of mycophenolic acid and ester derivatives thereof
WO2010041269A1 (fr) * 2008-09-10 2010-04-15 Ipca Laboratories Limited Procédé de préparation d’acide mycophénolique, son sel et ses dérivés esters

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5247083A (en) * 1992-07-10 1993-09-21 Syntex (U.S.A.) Inc. Direct esterification of mycophenolic acid
WO2000034503A2 (fr) * 1998-12-09 2000-06-15 Biocon India Limited Procedes de production d'esters de mycophenolate

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5247083A (en) * 1992-07-10 1993-09-21 Syntex (U.S.A.) Inc. Direct esterification of mycophenolic acid
WO2000034503A2 (fr) * 1998-12-09 2000-06-15 Biocon India Limited Procedes de production d'esters de mycophenolate

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BHIRUD V.S. ET AL.: "Influence of reaction media on esterification catalysed by Mucor miehei lipase", JOURNAL OF THE OIL TECHNOLOGISTS ASSOCIATION OF INDIA, vol. 23, no. 3, 1 July 1991 (1991-07-01), pages 44 - 47, XP002046399, ISSN: 0970-4094 *
GUISAN J.M. ET AL.: "Preparation of new lipases derivatives with high activity-stability in anhydrous media: adsorption on hydrophobic supports plus hydrophobization with polyethylenimine", JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC, vol. 11, no. 4-6, 22 January 2001 (2001-01-22), pages 817 - 824, XP002214923 *
MITTELBACH M.: "Lipase catalyzed alcoholysis of sunflower oil", JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY, XP002076829 *
PERRAUD R. AND LABORET F.: "Optimization of methyl propionate production catalysed by Mucor miehei lipase", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 44, 1995, pages 321 - 326, XP001113037 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7019133B2 (en) * 2003-02-21 2006-03-28 Chunghwa Chemical Synthesis & Biotech Co., Ltd. Process for making mycophenolate mofetil by transesterification
US7683188B2 (en) 2004-04-26 2010-03-23 TEVA Gyógyszergyár Zártkōrūen Mūkōdō Részvénytársaság Process for preparation of mycophenolic acid and ester derivatives thereof
WO2006024582A1 (fr) * 2004-09-03 2006-03-09 Poli Industria Chimica Spa Procede de preparation de mycophenolate mofetil par transesterification enzymatique
US7727751B2 (en) 2004-09-03 2010-06-01 Poli Industria Chimica Spa Method for the preparation of mycophenolate mofetil by enzyme tranesterification
EP1844041A1 (fr) * 2005-01-20 2007-10-17 Apotex Fermentation Inc. Procede ameliore de preparation de mycophenolate mofetil
EP1844041A4 (fr) * 2005-01-20 2009-08-05 Apotex Fermentation Inc Procede ameliore de preparation de mycophenolate mofetil
AU2006207789B2 (en) * 2005-01-20 2012-11-22 Apotex Fermentation Inc. An improved process for the preparation of mycophenolate mofetil
WO2010041269A1 (fr) * 2008-09-10 2010-04-15 Ipca Laboratories Limited Procédé de préparation d’acide mycophénolique, son sel et ses dérivés esters
US20110166347A1 (en) * 2008-09-10 2011-07-07 Ipca Laboratories Ltd. Process for preparation of mycophenolic acid, its salt and ester derivatives

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