WO2003042393A1

WO2003042393A1 - Enzymatic preparation of mycophenolate mofetil

Info

Publication number: WO2003042393A1
Application number: PCT/IN2001/000202
Authority: WO
Inventors: Nitin Patil; Rakesh Mendhe; Anand Khedkar; Ramakrishnan Melarkode; Shrikumar Suryanarayan
Original assignee: Biocon Ltd; Biocon India Ltd
Current assignee: Biocon Ltd
Priority date: 2001-11-16
Filing date: 2001-11-16
Publication date: 2003-05-22
Anticipated expiration: 2004-05-16

Abstract

The present invention relates to an improved method for synthesis of mycophenolate mofetil by reacting mycophenolic acid with an excess of 2-morpholino ethanol using an enzyme as catalyst in a water-free organic solvent and its subsequent purification. The use of an anhydrous organic solvent leads to higher conversion of mycophenolic acid. Water generated in the reaction may also be removed using molecular sieves to further improve conversion of mycophenolic acid to mycophenolate mofetil.

Description

ENZYMATIC PREPARATION OF MYCOPHENOLATE MOFETIL

FIELD OF INVENTION

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.

BACKGROUND INFORMATION

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 In the first step, 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. U.S. Pat. No. 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.

In the present invention, 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-moηpholinoethanol 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.

SUMMARY OF THE INVENTION

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:

(i) reacting mycophenlic acid with 2-morpholino ethanol with an enzyme catalyst in an anhydrous inert organic solvent in the presence of molecular sieves, (ii) filtering to get a clear reaction mixture, (iii) extracting the reaction mixture into a weak organic acid, (iv) adjusting the pH to 7.5 - 9.0,

(v) extracting into a water immiscible solvent,

(vi) charcolization, (vii) filtering to get pure mycophenolate mofetil.

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

10 to 60 minutes.The enzyme reaction is solid phase synthesis.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "PlaFractor" means a bioreactor for solid state fermentation to produce mycophenolic acid as described in PCT application WO

01/64931. As used herein, the term "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.

As used herein, the term "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.

As used herein, the term "Enzyme A" is a fungal lipase immobilized onto an ion exchange resin. As used herein, the term 'Εnzyme B" is a lipase derived from porcine pancreas. As used herein, the term 'Εnzyme C" is a lipase derived from yeast. As used herein, the term "Enzyme D" is an immobilized lipase derived from yeast. As used herein, the term "Enzyme E" is a fungal recombinant lipase.

As used herein, the term "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. Here, the organic solvent is a C₆-Cι₂ 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. Optionally, 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-morpholinoetlιanol 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. Optionally, 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 invention is now illustrated with following examples below which are not intended to be limiting. Example 1

Preparation of mycophenolic acid

The culture Penicillium brevicompactum was fermented on wheat bran for 5 days in the PlaFractor. The koji was extracted using ethyl acetate. After concentration of the ethyl acetate extract, 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.

Example 2

Enzyme-catalyzed solution phase synthesis of mycophenolate mofetil

Different commercially available enzymes were screened for solution phase synthesis of mycophenolate mofetil. In a typical reaction, 0.05 g of mycophenolic acid was dissolved in 10 ml of anhydrous toluene. To this, molar equivalent of 2-morpholino ethanol was added. 0.05 g of various enzymes were added to the reaction mixture. The mixture was incubated in an orbital shaker at 37 °C. The conversions of mycophenolic acid to mycophenolate mofetil observed were quantified using HPLC analysis and are tabulated in Table 1.

Table 1. Solution phase synthesis of mycophenolate mofetil using different enzymes

Example 3

Enzyme-catalyzed solid phase synthesis of mycophenolate mofetil

25.0 g of MPA (purity ~ 97.2%) was taken into a stirred tank reactor. 500 ml of anhydrous toluene was added to the reactor. 25.0 g of molecular sieves, 12.5 g of Enzyme F and molar equivalent of 2-morpholino ethanol were added to the reaction mixture. The reaction was carried out at 25 °C. After 8 h, 94.0% conversion of mycophenolic acid was obtained.

Example 4

Enzyme-catalyzed solid phase synthesis of mycophenolate mofetil

0.5 g of MPA (purity ~ 97.2%) was taken into a flask. 10 ml of anhydrous toluene was added to the flask. 0.5 g of molecular sieves, 0.5 g of Enzyme D and molar equivalent of 2-morpholino ethanol were added to the reaction mixture. The reaction was carried out at 25 °C. After 24 h, 96.1% conversion of mycophenolic acid was obtained.

Example 5

Enzyme-catalyzed synthesis of mycophenolate mofetil in organic solvent: Effect of presence of water

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. For reactions carried out in presence of molecular sieves, molecular sieves were added to the reaction mixture. Here, the weight ratio of mycophenolic acid to molecular sieves was kept 1:1. The conversions obtained in these reactions after 24 h are given in Table 2.

Table 2. Effect of water on enzyme-catalyzed synthesis of mycophenolate mofetil

C₀ - Initial mycophenolic acid concentration

Example 6

Synthesis of mycophenolate mofetil using Enzyme E immobilized on CaC0 powder

15.0 g of Enzyme E 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.

5.0 g of MPA (purity ~ 97.2%) was taken into a flask. 500 ml of anhydrous toluene was added to the flask. 5.0 g of molecular sieves, 25.0 g of Enzyme E bound on CaCΘ₃ and 4 ml of 2-morpholino ethanol were added to the reaction mixture. The reaction was carried out at 25 °C. After 48 h, 90.7% conversion of mycophenolic acid was obtained.

Example 7

Separation and purification of mycophenolate mofetil

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.

Example 8

Separation and purification of mycophenolate mofetil 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.

Claims

1. A process for the preparation of mycophenolate mofetil comprising of: (viii) reacting mycophenlic acid with 2-morpholino ethanol with an enzyme catalyst in an anhydrous inert organic solvent in the presence of molecular sieves, (ix) filtering to get a clear reaction mixture, (x) extracting the reaction mixture into a weak organic acid,

(xi) adjusting the pH to 7.5 - 9.0, (xii) extracting into a water immiscible solvent,

(xiii) charcolization, (xiv) filtering to get pure mycophenolate mofetil.

2. A process as in claim 1, wherein the enzyme catalyst is selected from a microbial, animal or plant source.

3. A process as in claim 1, wherein the enzyme catalyst is immobilized.

4. A process as in claim 1, wherein the enzyme catalyst is non-immobilized.

5. A process as in claim 1, wherein the enzyme catalyst is selected from a lipase, esterase or protease.

6. A process as in claim 1, wherein the inert organic solvent is toluene or xylene.

7. A process as in claim 1, wherein the enzyme reaction is carried out for 5 to 120 hours.

8. A process as in claim 1, wherein the enzyme reaction is carried out at 20 t 65°C.

9. A process as in claim 1, wherein the weak organic acid is citric acid, acetic acid, fumaric acid, malic acid or maleic acid..

10. A process as in claim 1, wherein the water hnmiscible solvent is ethyl acetate.

11. A process as in claim 1, wherein charcolization is carried out at 40 to 90°C.

12. A process as in claim 1, wherein charcolization is carried out 10 to 60 minutes.

13. A process as in claim 1, wherein the enzyme reaction is solid phase synthesis.