WO2008003637A2

WO2008003637A2 - Isolation and use of amine salts of mycophenolic acid

Info

Publication number: WO2008003637A2
Application number: PCT/EP2007/056486
Authority: WO
Inventors: De Robertus Mattheus Pater; Van De Emilius Johannes Albertus Xaverius Sandt; Pieter Gijsbert Weber
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2006-07-05
Filing date: 2007-06-28
Publication date: 2008-01-10
Anticipated expiration: 2009-01-05
Also published as: WO2008003637A3; WO2008003637B1

Abstract

The present invention provides a method for the isolation of mycophenolic acid from a fermentation broth comprising the steps of: (a) Adding a water-immiscible solvent to said fermentation broth; (b) Separating the water-immiscible phase from the aqueous phase, and; (c) Adding an amine to the water-immiscible phase obtained in step (b). Furthermore, the present invention provides the use of mycophenolate amines in the preparation of mycophenolate esters, in particular mycophenolate mofetil, and mycophenolate salts, in particular mycophenolate sodium salt.

Description

ISOLATION AND USE OF AMINE SALTS OF MYCOPHENOLIC ACID

Field of the invention

The present invention relates to a method for the isolation of mycophenolic acid derivatives, especially amine salts of mycophenolic acid and the use thereof.

Background of the invention

Mycophenolic acid (MPA, also known as 6-(4-hydroxy-6-methoxy-7-methyl-3- oxo-5-phthalanyl)-4-methyl-4-hexenoic acid, 6-(1 ,3-dihydro-4-hydroxy-6-methoxy-7- methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoic acid, Ci₇H₂o0₆ and CAS 24280-93-1 ) is a compound with various advantageous properties. Next to antibiotic activity, MPA also displays antifungal, antiviral and antitumor properties and the compound has been used in the treatment of psoriasis and recently as immunosuppressant. The chemical structure of MPA is:

MPA is most economically obtainable from microorganisms, such as Penicillium brevicompactum, Penicillium stoloniferum and related species. From the free acid two products are produced, which are commercially being applied as immunosuppressant, namely the salt mycophenolate sodium and the ester mycophenolate mofetil. In order to obtain these latter products in as pure as possible form it is necessary to isolate MPA from the fermentation broth with high purity in a form that can be used in the synthesis of said two final products. Unfortunately, direct crystallization of MPA from a fermentation broth does not lead to a product with a purity that is sufficient for subsequent steps. One way to overcome this problem is to recrystallize said MPA crystals. Another solution is to convert said MPA crystals into crystals of MPA derivatives. It has been established that amine salts of MPA as described in WO 04/20426 and Aust. J. Chem. (1978), 31 , 353-64 are particularly suitable for the latter purpose.

Nevertheless, the approach outlined above introduces additional steps in the overall isolation process, which is clearly unfavorable in view of yield, number of process operations, duration of the overall process, amount of waste streams and use of auxiliary reagents. Hence the current MPA isolation processes leave room for further improvement.

Detailed description of the invention

In a first aspect of the present invention there is provided a method for the isolation of amine salts of mycophenolic acid from a fermentation broth comprising less steps than known from prior art methods, i.e.:

(a) Adding a water-immiscible solvent to said fermentation broth; (b) Separating the water-immiscible phase from the aqueous phase, and;

(c) Adding an amine to the water-immiscible phase obtained in step (b). Thus, a solution comprising MPA, is mixed with an amine to form a precipitate of the amine salt of MPA. In the context of the present invention, the solution comprising MPA is a solution derived from a bioprocess in which MPA is formed. In a preferred embodiment, the broth resulting from a bioprocess in which MPA is formed is mixed with a water-immiscible solvent, preferably at a pH value ranging from 1 to 7, more preferably ranging from 3 to 6.5, most preferably ranging from 4 to 6. Subsequently, the water-immiscible phase, also referred to as extract, is separated from the aqueous phase and an amine is added to the water-immiscible phase. The amine salt of MPA precipitates and the precipitate can then be isolated with any of the methods available to the person skilled in the art, such as, but not limited to, filtration, sedimentation, decantation and centrifugation or combinations thereof. Preferably the mixture comprising the amine salt of MPA is cooled to a temperature ranging from -20 to 50°C, more preferably ranging from -10 to 10°C, most preferably ranging from 0 to 5°C, which usually results in an improved recovery yield. Examples of suitable amines are primary, secondary and tertiary amines such as terf-butylamine, cyclohexylamine, dibenzylamine, N,N-di/sopropylethylamine, N,N-dimethylisopropyl- amine, N-methylpiperidine, morpholine, terf-octylamine, /so-propylamine, N,N,N',N'-tetramethylethylenediamine, tributylamine, triethylamine and tripropylamine. The most preferred amine is triethylamine. Using triethylamine, the resulting MPA-triethylamine salt crystallizes with one molecule of triethylamine per molecule MPA whereas most other amines form crystals having two molecules amine per molecule MPA. Thus triethylamine has the advantage that less amine is needed, which is advantageous from an economic and a waste-stream point of view. Another advantage is the high crystallization yield when triethylamine is used compared to other amines. Examples of suitable water-immiscible solvents are alcohols, alkanes, benzene and derivatives thereof, esters, ethers and ketones. Particularly suitable alcohols are butanol and /so-butanol. Suitable benzene derivatives are toluene and xylene. Suitable esters are butyl acetate, ethyl acetate, methyl acetate and propyl acetate. Suitable ethers are diethyl ether and methyl tert-butyl ether.

In a second embodiment, the broth obtained from the bioprocess is filtered and/or centrifuged prior to the addition of the water-immiscible solvent. Such a filtration and/or centrifugation step allows for the removal of biomass, which normally results in improved separation of the aqueous phase and the water-immiscible solvent phase. Additionally, adjusting the pH of the aqueous phase with an alkaline solution prior to filtration and/or centrifugation can be advantageous in view of the overall yield, as undissolved MPA is thus dissolved and will no longer be retained by the filter. Preferred pH values are those ranging from 6 to 12, more preferably ranging from 7 to 1 1 , most preferably ranging from 8 to 10.5, still most preferably ranging from 8.5 to 10.

In a third embodiment, the water-immiscible phase is treated with color- adsorbing material that can be removed by means of filtration or centrifugation. Alternatively, treatment with color-adsorbing material is carried out using a means comprising said color-adsorbing material that can be removed and/or replaced, such as for instance a cartridge. Treatment with color-adsorbing material has the advantage that less color is retained in the end-product, which in turn usually leads to a higher purity of the end-product. Carbon is a preferred example of color-adsorbing material. Other suitable examples are alumina, resins and silica. In a fourth embodiment, the water-immiscible phase is concentrated, for instance by evaporation. Concentration of the water-immiscible phase results in a higher yield and usually also facilitates the treatment with color-adsorbing material. In a fifth embodiment, the water-immiscible phase is treated such that the amount of water present is reduced. Suitable methods for reducing the amount of water are treatment with a water-adsorbing agent such as molecular sieves, CaCI₂, MgSO₄, Na₂SO₄, and similar compounds, or by means of evaporation. Lowering the amount of water has the advantage that a higher yield is obtained.

In a second aspect of the present invention MPA amine salts are used in the production of the derivatives of MPA.

In one embodiment, the amine salts are converted to metal salts by dissolving the amine salt in a suitable solvent and then adding a suitable metal salt. Preferably said metal salt is an alkali metal salt such as the sodium salt. A suitable solvent dissolves the amine salt of MPA, but -where the metal in question is for example sodium- does not dissolve the sodium salt of MPA. Suitable solvents can be solvents like alcohols, alkanes, benzene and derivatives thereof, esters, ethers and ketones. Particularly suitable solvents are acetone, ethyl acetate, methyl ethyl ketone and methyl /so-butyl ketone.

In a second embodiment, the amine salts are converted into esters of MPA. Several methods for esterification of MPA have been described. For instance, in US 4,753,935 an acid halide condensation route has been described. This is a two-step process requiring toxic reagents for forming the halide of MPA and/or of the alcohol, which in this case was 2-morpholinoethanol. The use of the amines of the present invention circumvents the necessity for acid halide formation as they react with alcohols without any further activation. In the present invention, esterification of MPA is accomplished by reaction of an amine salt of MPA with an alcohol or an activated alcohol. Preferably the alcohol is a primary alcohol having the general formula HOCH₂CH₂R, wherein R is hydrogen, (substituted) alkyl or substituted nitrogen, non-limiting examples of which are butanol, ethanol, /so-butanol, /so-propanol, propanol, and sec-butanol. The most preferred example is 2-morpholinoethanol as one of the most preferred MPA esters is the 2-morpholinoethyl ester of MPA, or mycophenolate mofetil (MPM), as this ester is widely used as pro-drug of MPA. MPM is prepared starting from the amine salt of MPA and 2-morpholino ethanol. Amine salts suitable for the conversion of MPA into esters of MPA are amines as mentioned above, and in particular amines such as dibenzylamine, cyclohexylamine, /so-propylamine, terf-butylamine, terf-octylamine, triethylamine and N,N,N',N'-tetramethylethylene- diamine. Esterification can be carried out in a solvent, preferably an inert solvent. The term "inert solvent" means a solvent inert under the conditions of the reaction being described in conjunction therewith. Examples of inert solvents in the context of the present invention are alkanes, benzene, cycloalkanes, ethers, ethylbenzene, methylene chloride, toluene and xylene.

In a third embodiment, esterification of the amine salt of MPA is carried out in a non-inert solvent. In EP 649,422 B1 , a route was disclosed concerning refluxing MPA with 2-morpholinoethanol in an inert organic solvent capable of azeotropic removal of water, without the use of additional reagents. This method, wherein MPA was used as free acid, is limited to solvents that are inert. The amine salts of MPA of the present invention however also allow for successful esterification in non-inert solvents. The term "non-inert solvent" means a solvent not being inert under the conditions of the reaction being described in conjunction therewith. Examples in respect of the present invention are alcohols, amines and, particularly suitable, ketones such as acetone, methylisobutyl ketone and cyclic ketones such as cyclohexanone and cyclopentanone. Non-inert solvents can be classified in relation to the degree in which the solvent in question suffers degradation during the reaction in question. A suitable parameter for this is the loss of solvent due to reaction with any material (including the solvent itself) in %.h^"1. Typically, a non-inert solvent has a degradation rate of 0.01 %.h^"1 or higher. Hence, the amine of MPA is esterified with a primary alcohol in a non-inert solvent having a degradation rate higher than 0.01 %.h^"1. It has been established that esterification of MPA can be accomplished in non-inert solvents with a degradation rate higher than 0.01 %.h^"1 with results that are similar, or in some cases surprisingly even better, than prior art methods using inert solvents. Advantageously, this results in the fact that a wider range of solvents becomes available for esterification of MPA. Preferably said non-inert solvent has a degradation rate under the reaction conditions of 0.3-0.01 %.h^"1, more preferably of 0.2-0.01 %.h^"1, most preferably 0.1-0.01 %.h^"1, still most preferably 0.05-0.01 %.h^"1. Suitable non-inert solvents in this respect are ketones such as acetone, cyclohexanone, cyclopentanone, dipropyl ketone, methylisobutyl ketone, methylpropyl ketone, N-methylpyrrolidone and mixtures thereof.

In a fourth embodiment, esterification of the amine salt of MPA is carried out under non-boiling conditions. EP 649,422 B1 mentioned above limits the esterification of MPA to reaction at reflux. The amine salts of MPA of the present invention however also allow for successful esterification under non-boiling conditions, i.e. in a non-boiling mixture. The term "non-boiling mixture" refers to a given conversion or process that is carried out at a temperature below the boiling point that the mixture in question has at that specific pressure. In respect of the present invention, the term "mixture" comprises at least one solvent. Thus, the boiling point of a mixture refers to the boiling point of said solvent per se or to the boiling point of the solvent as changed by the presence of other components, i.e. through the formation of one or more azeotropes, whichever is lower. If there are components present, other than the solvent, which have lower boiling points than the solvent and which do not form an azeotrope with the solvent, the mixture will boil first at the temperature of that or those components. This or these first boiling points are not included in the term "non-boiling mixture". In general, such components, which may be catalysts, reactants, reaction products or even co-solvents and the like, are usually present in minor amounts compared to the solvent, for instance 0.01-10% (w/w), 0.1-5% (w/w) or 1-3% (w/w). Hence, the amine of MPA is esterified in a conversion with a primary alcohol in a solvent at a temperature that is at least 0.2% below the boiling point of the mixture comprising MPA, primary alcohol and solvent. This temperature is maintained at the value of 0.2% below the boiling point of the mixture, or even below that value, throughout the duration of the conversion. In other words, the time during which the temperature is maintained at said value is, for example, 30 min for a conversion that takes 30 min, 1 h for a conversion that takes 1 h, 5 h for a conversion that takes 5 h, 10 h for a conversion that takes 10 h, 2O h for a conversion that takes 20 h, 40 h for a conversion that takes 40 h, 80 h for a conversion that takes 80 h, and so on and so forth. The advantages of esterification at a temperature below the boiling point are already manifest at temperatures only slightly below the boiling point: equipment for condensing solvent vapors and returning these condensed vapors are no longer required and the energy input required to reach and maintain the boiling point, which normally is substantial, can be circumvented. Furthermore, formation of unwanted by-products generally is lower at lower reaction temperatures. On the one hand, typical temperatures below the boiling point are temperatures that are 0.2-10% below the boiling point, preferably 0.5-5% below the boiling point.

In a fifth embodiment of the present invention, esterification of amines of MPA can be positively influenced (i.e. reduction of reaction time, increase of maximum conversion) by the addition of substances that are capable of adsorbing water. These substances can be present in the mixture of MPA, solvent and primary alcohol. Substances that are capable of adsorbing water are for instance salts of alkali and earth alkali metals and usually these salts are carbonates, halides or sulfates. Suitable examples are CaCI₂, CaSO₄, K₂CO₃, K₂SO₄, MgSO₄, Na₂CO₃, Na₂SO₄ and the like. Preferred other substances are molecular sieves, preferably those with pore sizes ranging from 0.1-0.6 nm, more preferably ranging from 0.2-0.5 nm, most preferably ranging from 0.3-0.4 nm.

In a sixth embodiment of the present invention, it was found that impurities, for instance the so-called impurity B (as defined in the European Pharmacopoeia) in the case of MPM, can be efficiently removed to levels below 1 %, even below 0.3% and even below 0.2 and 0.1 %, by treating a solution of MPM with adsorbents, such as silica, alumina, aluminosilicates or carbon. It is known that impurity B, which is the esterification product of a mycophenolate impurity produced during fermentation, is difficult to remove. However, we now surprisingly found a very effective means to reduce the level for impurity B in MPM in an efficient and economic manner. For instance, treatment with silica can either be done using column chromatography or by addition of silica to a solution of MPM followed by removal of the silica by means known to those skilled in the art. Treatment can be repeated to reduce the impurity levels even further. Preferably said treatment is performed using a column, where an MPM solution, for example a solution of MPM in ethyl acetate but other solvents may also be used, is treated with silica, for example Silica gel 60 but other silica's may also be used. Treatment with adsorbents such as silica can be done at various stages of the esterification process. The silica can be regenerated using suitable solvents, for example alcohols such as methanol or ethanol, to remove the adsorbed impurities from the silica and therewith regenerating the silica to be able to reuse the silica.

In a third aspect of the present invention, the MPA esters obtainable from the MPA amines of the present invention can be used in pharmaceutical compositions, for instance in antifungal, antiviral and/or antitumor compositions, but also in compositions useful in the treatment of psoriasis and as immunosuppressant. Particularly suitable is a pharmaceutical composition comprising MPM. EXAMPLES

General Methods

HPLC analysis was performed on a Waters Atlantis dC-iβ column (5μm; 4.6x150 mm; W 32371 X 12), using as mobile phase A MiIIiQ water with 0.1 vol% formic acid and as mobile phase B CH₃CN with 0.1 vol% HCO₂H. The run time was 13 minutes and the flow 1.5 mL. min^"1. Detection wavelength was at 251 nm. The following gradient was applied:

Time (min) Mobile phase A (%) Mobile phase B (%)

As dilution buffer 400 mg ammonium formate was dissolved in 700 mL water and the pH was adjusted to 3.5 with HCO₂H and next 1300 mL acetonitrile was added. In the above system, the retention time of MPM was 1.4 min and that of MPA 4.9 min. TLC analysis was performed on Silica gel 60 F254 TLC plates (Merck 1.05715). As eluent CHCI3/CH3OH 10/1 (v/v) was used. Detection of components was performed using UV visualization at 254 nm and/or with iodine vapor. Sample dilution was done with HPLC dilution buffer. In this system the following Rf-values were observed: MPA -0.4 and MPM -0.55.

Example 1 Preparation of MPA extract

After adjusting the pH to 8.5 using NaOH, a broth resulting from a fed-batch Penicillium brevicompactum-baseό fermentation process (2000 L containing 1.6 g/L MPA) was filtered using membrane filtration. The mycelium cake was diafiltrated with water (3000 L), resulting in 4000 L total permeate. After membrane filtration ethyl acetate (1000 L) was added and the pH was adjusted to 5 using H₂SO₄. To remove dissolved water and to increase the MPA concentration the organic layer was concentrated under vacuum. Treatment with active carbon was used to decolorize the extract. The obtained extract (235 L) contains 1 1 g/L MPA (yield 80%). Example 2 Crystallization of MPA-isopropylamine salt (MPA-IPA) from MPA extract From an MPA ethyl acetate extract (200 ml_, containing 8.12 mmol MPA) obtained analogous to Example 1 , 1 10 ml. were distilled off over a period of 1 h at 79°C. The residue was cooled to 55°C and a mixture of ethyl acetate (10 ml.) and isopropyl amine (6 ml.) were added. During cooling at ca 48°C crystallization started, further cooled to 1 1 °C and stirred for 1 h. The precipitate was filtered off and washed/replaced with ethyl acetate (15 ml_). The wet cake was dried under vacuum at room temperature to give 2.788 g of the title compound with a purity of 83%; yield: 75.1 %.

Example 3 Crystallization of MPA-t-butylamine salt (MPA-TBA) from MPA extract Example 3.1 : From an MPA ethyl acetate extract (200 ml_, containing 8.12 mmol MPA) obtained analogous to Example 1 , 1 18 ml. were distilled off over a period of 1 h at 80°C. The residue was cooled to 60°C and a mixture of ethyl acetate (18 ml.) and t-butyl amine (12 ml.) were added. During the addition crystallization started. The suspension was further cooled to 22°C and stirred for 1 h. The precipitate was filtered off and washed/replaced with ethyl acetate (20 ml_). The wet cake was dried under vacuum at room temperature to give 3.422 g of the title compound with a purity of 80%; yield: 85.6%.

Example 3.2: As Example 3.1 , however with 170 ml. extract (containing 6.90 mmol MPA) and 4 ml. tert-butyl amine to give 2.952 g of the title compound with a purity of 80%; yield: 86.8%.

Example 4 Crystallization of MPA-triethylamine salt (MPA-TEA) from MPA extract

Example 4.1 : From an MPA ethyl acetate extract (200 ml_, containing 8.12 mmol MPA) obtained analogous to Example 1 , 100 ml. were distilled off over a period of 1 h at 79°C. The residue was cooled to 60°C and a mixture of ethyl acetate (10 ml.) and triethyl amine (6 ml.) were added. During cooling at ca 42°C crystallization started, further cooled to 10°C and stirred for 1 h. The precipitate was filtered off and washed/replaced with ethyl acetate (15 ml_). The wet cake was dried under vacuum at room temperature to give 2.843 g of the title compound with a purity of 95%; yield: 78.7%. Example 4.2: As Example 4.1 , however with 163 ml. extract (containing 6.62 mmol MPA) and 7 ml. triethyl amine to give 2.676 g of the title compound with a purity of 92%; yield: 88.4%.

Example 5 Conversion of MPA-triethylamine salt (MPA-TEA) into MPM

3.0 g MPA-TEA (96 HPLC area%; 6.8 mmol) and 1.3 ml. 2-morpholinoethanol (10.6 mmol) were stirred in orf/70-xylene (9 ml_, boiling point 144°C) at 120°C under nitrogen for 54 hours. The conversion was monitored in time with HPLC:

Time (h) Conversion (%) Time (h) Conversion (%)

Purity MPM + MPA in solution -99 area% (corrected for ort/70-xylene)

The reaction mixture was diluted with 25 mL ort/70-xylene and with water and the pH was adjusted to 1.8 with 2M H₂SO₄. The phases were separated. Ethyl acetate was added to the aqueous phase and under stirring the pH was adjusted to 8 with diluted NaOH. The phases were separated and the ethyl acetate phase was washed twice with water and then concentrated under vacuum. The concentrate was decolorized with 1.5 g SX-ultra carbon. After filtration the filtrate was evaporated under vacuum to dryness. The residue was dissolved in 4 mL ethyl acetate and 20 mL 2-propanol at 45°C. The solution was gradually cooled to 0°C and left in the refrigerator overnight. The crystals were successively filtered off, washed with cold (0°C) 2-propanol, and dried under vacuum, yielding 2.23 g MPM with a HPLC-purity of 99.2 area% (overall yield from MPA-TEA to MPM: 75%). Example 6 Conversion of MPA amine salts into MPM

Various amine salts of MPA were converted into MPM according to the procedure of Example 5 (until measurement of the conversion at 48 h), i.e. at 120°C, in 7 separate runs with the following results:

Example 7 Determination of solvent degradation rate (D) of cyclohexanone 150 ml (143 g) of cyclohexanone was heated under reflux (155°C) for 44 h. The mixture was evaporated under vacuum at 70°C, yielding 6.2 g of oil and thus the remaining solvent was 136.8 g. The solvent degradation rate (D) of cyclohexanone at 155⁰C is:

Dcyc_lo_hexanone 155 = 100% X ((143 - 136.8)/(143 X 44) = 0.1 %.h^"1

The oil obtained above was analyzed by HPLC using the system outlined under 'General Methods'. HPLC analysis of pure cyclohexanone revealed one peak at

2.3 min. HPLC analysis of cyclohexanone after heating for 44 h at 155⁰C revealed an additional peak at 8.0-8.1 min, which was exactly the same as the peak obtained upon analysis of the residual oil obtained in this experiment. Furthermore, HPLC analysis of an esterification reaction mixture consisting of cyclohexanone, MPA and 2-morpholinoethanol after heating for 68 h at 155⁰C revealed the same peak at

8.0-8.1 min.

In a similar experiment, 100 ml (95 g) of cyclohexanone was heated at 125°C for 46 h. The mixture was evaporated under vacuum at 70°C, yielding 2.3 g of oil and thus the remaining solvent was 92.7 g. The solvent degradation rate (D) of cyclohexanone at 125⁰C is: Dcydohβxanonβ 125 = 100% x ((95 - 92.7)/(95 x 46) = 0.05%.h^"1. Example 8

Conversion of MPA-triethylamine salt (MPA-TEA) into MPM in non-boiling cyclohexanone

2.5 g MPA-TEA (96 HPLC area%; 5.7 mmol) was suspended in 25 ml water. Cyclohexanone (25 ml, boiling point 155-156°C) was added and the pH was adjusted under stirring to 3.1 with 4M HCI. The phases were separated, giving 28 ml of aqueous phase and 40 ml of organic phase. The aqueous phase was extracted once more with 25 ml cyclohexanone at pH 3.1. The organic phases were combined and evaporated under vacuum at 90°C. The residue was dissolved in 7.5 ml cyclohexanone to give a final concentration of approximately 250 g MPA per I cyclohexanone. 2-Morpholino- ethanol (1.4 ml; 11.4 mmol) and molecular sieves of 0.3 nm (4 g) were added and the mixture was stirred under nitrogen for 1 10 hours at 120°C. The conversion was monitored in time with HPLC:

Time (h) Conversion (%) Time (h) Conversion (%)

Purity MPM + MPA in solution -93 area% (corrected for cyclohexanone)

The reaction mixture was filtered with dicalite 4108 and the filter cake was washed with cyclohexanone. The filtrate was extracted three times with water at pH 1.8 (acidification with 2M H₂SO₄). The aqueous phases were combined. Ethyl acetate was added and under stirring the pH was adjusted to 8 with diluted NaOH. The phases were separated and the ethyl acetate phase was washed twice with water and then evaporated under vacuum. The purple residue was purified over 20 g silica gel 60 using ethyl acetate/ethanol 20/1 (v/v). The eluate was evaporated under vacuum. The concentrate was dissolved in 3.6 ml ethyl acetate and 20 ml 2-propanol at 40°C. The solution was gradually cooled to 0°C and left in the refrigerator overnight. The crystals were successively filtered off, washed with cold (0°C) 2-propanol, and dried under vacuum, yielding 1.75g MPM with an HPLC-purity of 99.8 area% (overall yield from MPA-TEA to MPM: 71 %). Example 9 Conversion of MPA-IPA into MPA sodium salt

A solution of sodium ethyl hexanoate (0.65 g; 3.9 mmoles) in ethyl acetate (1 OmL) was added over a period of 10 min to a stirred solution of MPA.IPA (1.00 g) in ethyl acetate (60 ml.) at 62°C. During the addition a suspension formed. Stirring was continued at ca 54°C for 1 h. The suspension was cooled to 20°C. After stirring for ca 0.75 h the solids were filtered off and washed with ethyl acetate (15 mL). The cake was dried to give 0.575 g of MPA sodium salt.

Example 10

Conversion of MPA-TBA into MPA sodium salt

A solution of sodium ethyl hexanoate (0.40 g; 2.4 mmoles) in ethyl acetate (1 OmL) was added over a period of 10 min to a stirred suspension of MPA.TBA (1.00 g) in ethyl acetate (60 mL) at 52°C. During the addition the suspension became thinner and almost dissolved, some lumps from the TBA.MPA were present. Stirring was continued at ca 50°C for 1 h. A suspension formed. The suspension was cooled to 24°C. After stirring for ca 0.75 h the solids were filtered off and washed with ethyl acetate (10 mL). The cake was dried to give 0.569 g of MPA sodium salt.

Example 11

Conversion of MPA-TEA into MPA sodium salt

A solution of sodium ethyl hexanoate (0.65 g; 3.9 mmoles) in ethyl acetate (1 OmL) was added over a period of 10 min to a stirred solution of MPA.TEA (1.00 g) in ethyl acetate (60 mL) at 54°C. During the addition a suspension formed. Stirring was continued at ca 54°C for 1 h. The suspension was cooled to 20°C. After stirring for ca 2 h the solids were filtered off and washed with ethyl acetate (15 mL). The cake was dried to give 0.743 g of MPA sodium salt.

Claims

1. Method for the isolation of an amine salt of mycophenolic acid from a fermentation broth comprising the steps of: (a) adding a water-immiscible solvent to said fermentation broth;

(b) separating the water-immiscible phase from the aqueous phase, and;

(c) adding an amine to the water-immiscible phase obtained in step (b).

2. Method according to claim 1 further comprising filtration and/or centrifugation prior to step (a).

3. Method according to any one of claims 1 to 2 further comprising treatment of the water-immiscible phase obtained in step (b) with color-adsorbing material.

4. Method according to any one of claims 1 to 3 further comprising isolation of the amine salt of mycophenolic acid obtained in step (c).

5. Method according to any one of claims 1 to 4 wherein said amine is triethylamine.

6. Method for the production of a mycophenolic acid ester comprising the steps of:

(a) isolating an amine salt of mycophenolic acid from a fermentation broth by adding a water-immiscible solvent to said fermentation broth;

(b) separating the water-immiscible phase from the aqueous phase; (c) adding an amine to the water-immiscible phase obtained in step (b), and

(d) adding an alcohol to the amine salt of mycophenolic acid obtained in step (c).

7. Method according to claim 6 wherein said alcohol is 2-morpholinoethanol.

8. Method according to any one of claims 6 to 7 wherein step (d) is carried out in a solvent having a degradation rate higher than 0.01 %.h^"1 under the conditions of said esterification.

9. Method according to any one of claims 6 to 8 wherein step (d) is carried out at a temperature that is at least 0.2% below the boiling point of the mixture of components.

10. Method according to any one of claims 6 to 9 further comprising treatment with an adsorbent.

1 1. Use of the triethylamine salt of mycophenolic acid in the preparation of a metal salt or an ester of mycophenolic acid.

12. A pharmaceutical composition comprising mycophenolate mofetil obtained according to claim 7.