WO2009040828A1 - A process for purification of mycophenolic acid - Google Patents
A process for purification of mycophenolic acid Download PDFInfo
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- WO2009040828A1 WO2009040828A1 PCT/IN2007/000532 IN2007000532W WO2009040828A1 WO 2009040828 A1 WO2009040828 A1 WO 2009040828A1 IN 2007000532 W IN2007000532 W IN 2007000532W WO 2009040828 A1 WO2009040828 A1 WO 2009040828A1
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- mycophenolic acid
- water
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- immiscible solvent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/87—Benzo [c] furans; Hydrogenated benzo [c] furans
- C07D307/88—Benzo [c] furans; Hydrogenated benzo [c] furans with one oxygen atom directly attached in position 1 or 3
Definitions
- the present invention relates to a process for purification of mycophenolic acid.
- Mycophenolic acid was first reported as a secondary metabolite of F 'enicillium glaucum (B. Gosio, Riv. Igiene Sanita Pub. Ann., 7, 825, 1896). Later, it has been isolated from the fermentation broths of various Penicillium Species including P. brevicompactum, P. stoloniferum, P. scarbum, P. griseobrunneum, and P. viridicatum. After discovery of mycophenolic acid, it has been reported to have anti-bacterial, anti-viral, anti-fungal, anti-tumor and immunosuppressive activities.
- Mycophenolic acid is produced by fermentation of Penicillium Species followed by recovery (isolation and purification) of mycophenolic acid from the fermentation broth.
- EP 1624070 disclose a process for production of mycophenolic acid that consisted recovery steps including centrifugation or filtration of biomass, use of an adsorption resin after clarification, elution of the resin using a solvent, precipitation, centrifugation or filtration to remove the supernatant, washing of the precipitate and drying of the precipitate. The process has been reported to yield mycophenolic acid with HPLC purity ⁇ 98%.
- WO06/031665 discloses a process for isolation of mycophenolic acid involving addition of a suitable base to the fermentation broth, separation of the mycelium from the liquid phase, addition of a suitable acid to the liquid phase, separation of mycophenolic acid from the liquid phase, dissolution of separated mycophenolic acid in toluene and crystallization from toluene solution. This process has been reported to yield mycophenolic acid with HPLC purity of 99.2%.
- the invention also relates to a process for purification of mycophenolic acid comprising steps of:
- a extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; b. acidifying the aqueous layer from step (a) to obtain precipitate; c. recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d. extracting the water immiscible layer (organic layer) from step (c) with a second aqueous base to selectively remove polar impurities; e. concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and f. optionally, purifying the mycophenolic acid by recrystallization.
- the principal object of the present invention is to develop a process for purification of mycophenolic acid.
- Another main object of the present invention is to obtain pure form of mycophenolic acid.
- the present invention relates to a process for purification of mycophenolic acid comprising steps of: (a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base to selectively remove polar impurities; (b) extracting the water immiscible layer from step (a) with a second aqueous base; (c) combining the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; (d) acidifying the aqueous layer from step (c) to obtain precipitate; (e) recovering the precipitate by filtration or extraction with a third water-immiscible solvent; (f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and (g) optionally, purifying the mycophenolic acid by recrystallization; and a process for purification of mycophenolic acid comprising steps of:
- the present invention relates to a process for purification of mycophenolic acid comprising steps of: a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base to selectively remove polar impurities; b) extracting the water immiscible layer from step (a) with a second aqueous base; c) combining ,the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; d) acidifying the aqueous layer from step (c) to obtain precipitate; e) recovering the precipitate by filtration or extraction with a third water- immiscible solvent; f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and g) optionally, purifying the mycophenolic acid by recrystallization.
- the first, second and third water- immiscible solvent are selected from a group comprising ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, heptane and/or mixtures thereof.
- the first and second aqueous base are selected from a group comprising sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia.
- the polar impurities are one or more impurities at RRTs 0.52, 0.64 and 0.93.
- the non-polar impurities are one or both impurities at RRTs 1.10 and 1.34.
- the extraction is carried out either in a batch extraction manner or continuous counter-current extraction manner or continuous fractional extraction manner.
- the mycophenolic acid is in a free acid form or a salt form.
- the salt form is sodium salt of mycophenolic acid.
- the present invention also relates to a process for purification of mycophenolic acid comprising steps of: a) extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; b) acidifying the aqueous layer from step (a) to obtain precipitate; c) recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d) extracting the water immiscible layer from step (c) with a second aqueous base to selectively remove polar impurities; e) concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and f) optionally, purifying the mycophenolic acid by recrystallization.
- the first and second water- immiscible solvent are selected from a group comprising ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, and heptane and/or mixtures thereof.
- the first and second aqueous base are selected from a group comprising sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia.
- the polar impurities are one or more impurities at RRTs 0.52, 0.64 and 0.93.
- the non-polar impurities are one or both impurities at RRTs 1.10 and 1.34.
- the extraction is carried out either in a batch extraction manner or continuous counter-current extraction manner or continuous fractional extraction manner.
- the mycophenolic acid is in a free acid form or a salt form.
- the salt form is sodium salt of mycophenolic acid.
- the invention also relates to a process for purification of mycophenolic acid comprising steps of: a) extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; b) acidifying the aqueous layer from step (a) to obtain precipitate; c) recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d) extracting the water immiscible layer (organic layer) from step (c) with a second aqueous base to selectively remove polar impurities; e) concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and f) optionally, purifying the mycophenolic acid by recrystallization.
- the present invention relates to a novel process for purification of mycophenolic acid.
- the novel process of the instant invention comprises: a) extraction of mycophenolic acid solution in a first water-immiscible solvent with a first aqueous base of low molarity to selectively remove polar analog impurities, b) extraction of organic layer from step (a) with a second aqueous base, c) extraction of the mycophenolic acid solution in the aqueous layer from step (b) with a second water-immiscible solvent to selectively remove non-polar analog impurities, d) acidification of the aqueous layer from step (c) to cause precipitation, e) recovery of the precipitate from step (d) by filtration or extraction with a third water-immiscible solvent, f) crystallization, and g) optional, re-crystallization affording mycophenolic acid in substantially pure form.
- the present invention relates to a novel process for purification of mycophenolic acid. a) extraction of mycophenolic acid solution in a first water-immiscible solvent with a first aqueous base of low molarity to selectively remove polar analog impurities, b) extraction of organic layer from step (a) with a second aqueous base, c) extraction of the mycophenolic acid solution in the aqueous layer from step (b) with a second water-immiscible solvent to selectively remove non-polar analog impurities, d) acidification of the aqueous layer from step (c) to cause precipitation, e) recovery of the precipitate from step (d) by filtration or extraction with a third water-immiscible solvent, f) crystallization, and g) optional, re-crystallization affording mycophenolic acid in substantially pure form.
- the mycophenolic acid solution in water-immiscible solvent may be obtained by extraction of fermentation broth with a water-immiscible solvent. Alternately, it may be obtained by dissolution of crude mycophenolic acid consisting of one or more of the impurities at RRTs 0.52, 0.64, 0.93, 1.10 and 1.34.
- the first, second and third water-immiscible solvent in the present invention may be independently selected from ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, and heptane or mixtures thereof.
- the first and second aqueous base may be independently selected from solution of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia in water.
- the polar analog impurities in present invention relate to one or more impurities at RRT 0.52, 0.64 and 0.93, whereas the non-polar analog impurities relate to one or both impurities at RRT 1.10 and 1.34.
- Extraction in step (a) and step (c) may be done in a batch extraction manner, continuous counter-current extraction manner or continuous fractional extraction manner. Crystallization and re-crystallization in present invention may be carried out by decreasing temperature of mycophenolic acid solution or by evaporation of solvent or both together.
- the present invention also relates to a process for mycophenolic acid purification in which the steps in the given process are carried out in a different order, for example, such as: a) extraction of the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar analog impurities, b) acidification of the aqueous layer from step (a) to cause precipitation, c) recovery of the precipitate from step (b) by filtration and dissolution in second water-immiscible solvent or extraction with a second water-immiscible solvent, d) extraction of organic layer from step (c) with a second aqueous base of low molarity to selectively remove polar analog impurities, e) crystallization, and f) optional, re-crystallization
- the ethyl acetate layer was washed with water and concentrated. The concentrate was kept at 5 0 C for crystallization. The crystals were then filtered and dried. The yield was about 60%.
- the content of impurities at RRT 0.52, 0.64, 0.93, 1.10 and 1.34 was 0.08, 0.12, 0.08, 0.07 and 0.08, respectively.
- the ethyl acetate layer was then concentrated under reduced pressure.
- the concentrate was cooled to temperature of 0 to 5 0 C.
- the concentrate was held at this temperature for 3h.
- the crystals were filtered and dried.
- the yield was about 50%.
- the content of impurities at RRT 0.52, 0.64, 0.93, 1.10 and 1.34 was 0.10, 0.10, 0.08, 0.07 and 0.13, respectively.
- the mycophenolic acid solution was extracted with 25 mM sodium carbonate solution using 5 stages, wherein sodium carbonate solution was fed at the first stage and ethyl acetate was fed at the fifth stage. Both phases moved in counter-current manner while the mycophenolic acid solution was fed at the third stage.
- the organic layer exiting the first stage was collected and extracted with 1% NaOH solution twice.
- the aqueous extracts were combined.
- the combined aqueous layer was extracted with n-butanol in 5 stages, wherein 1% NaOH solution was fed at the first stage and butanol was fed at the fifth stage.
- Both phases moved in counter-current manner while the combined aqueous layer was fed at the third stage.
- the aqueous layer exiting the fifth stage was collected and acidified to a pH of 2.5 -3.5 using hydrochloric acid.
- the acidified layer was extracted with ethyl acetate.
- the ethyl acetate layer was washed with water and concentrated.
- the concentrate was kept at 5°C for crystallization.
- the crystals were then filtered and dried. The yield was about 80%.
- the content of impurities at RRT 0.52, 0.64, 0.93, 1.10 and 1.34 was 0.06, 0.07, 0.10, 0.10 and 0.13, respectively.
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Abstract
The present invention relates to a process for purification of mycophenolic acid comprising steps of: (a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base to selectively remove polar impurities; (b) extracting the water immiscible layer from step (a) with a second aqueous base; (c) combining the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; (d) acidifying the aqueous layer from step (c) to obtain precipitate; (e) recovering the precipitate by filtration or extraction with a third water-immiscible solvent; (f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and (g) optionally, purifying the mycophenolic acid by recrystallization.
Description
A PROCESS FOR PURIFICATION OF MYCOPHENOLIC ACE) FIELD OF THE INVENTION
The present invention relates to a process for purification of mycophenolic acid.
BACKGROUND OF THE INVENTION AND PRIOR ART
Mycophenolic acid, having chemical formula I
is an immunosuppressant drug used to prevent rejection in organ transplantation. It was initially marketed as the prodrug mycophenolate mofetil (trade name - Cellcept) to improve oral bioavailability. More recently, Novartis has introduced enteric coated, delayed release formulation of sodium salt of mycophenolic acid or sodium mycophenolate under the trade name of Myfortic.
Mycophenolic acid was first reported as a secondary metabolite of F 'enicillium glaucum (B. Gosio, Riv. Igiene Sanita Pub. Ann., 7, 825, 1896). Later, it has been isolated from the fermentation broths of various Penicillium Species including P. brevicompactum, P. stoloniferum, P. scarbum, P. griseobrunneum, and P. viridicatum. After discovery of mycophenolic acid, it has been reported to have anti-bacterial, anti-viral, anti-fungal, anti-tumor and immunosuppressive activities.
Mycophenolic acid is produced by fermentation of Penicillium Species followed by recovery (isolation and purification) of mycophenolic acid from the fermentation broth. EP 1624070 disclose a process for production of mycophenolic acid that consisted recovery steps including centrifugation or filtration of biomass, use of an adsorption resin after clarification, elution of the resin using a solvent, precipitation, centrifugation
or filtration to remove the supernatant, washing of the precipitate and drying of the precipitate. The process has been reported to yield mycophenolic acid with HPLC purity ~ 98%.
WO06/031665 discloses a process for isolation of mycophenolic acid involving addition of a suitable base to the fermentation broth, separation of the mycelium from the liquid phase, addition of a suitable acid to the liquid phase, separation of mycophenolic acid from the liquid phase, dissolution of separated mycophenolic acid in toluene and crystallization from toluene solution. This process has been reported to yield mycophenolic acid with HPLC purity of 99.2%.
During fermentation for production of mycophenolic acid, several analog impurities are generated. No prior art information is available for separation of these impurities from mycophenolic acid.
In our invention, we have arrived at a process to obtain substantially pure form of mycophenolic acid as compared to the methods known in the prior art. The series of steps followed in our process is critical for obtaining pure form of mycophenolic acid. The process followed comprises: a. extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base of low molarity to selectively remove polar impurities; b. extracting the water immiscible layer (organic layer) from step (a) with a second aqueous base; c. combining the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; d. acidifying the aqueous layer from step (c) to obtain precipitate; e. recovering the precipitate by filtration or extraction with a third water- immiscible solvent; f. crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and g. optionally, purifying the mycophenolic acid by recrystallization.
The invention also relates to a process for purification of mycophenolic acid comprising steps of:
a. extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; b. acidifying the aqueous layer from step (a) to obtain precipitate; c. recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d. extracting the water immiscible layer (organic layer) from step (c) with a second aqueous base to selectively remove polar impurities; e. concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and f. optionally, purifying the mycophenolic acid by recrystallization.
OBJECTS OF THE INVENTION
The principal object of the present invention is to develop a process for purification of mycophenolic acid.
Another main object of the present invention is to obtain pure form of mycophenolic acid.
STATEMENT OF THE INVENTION:
Accordingly, the present invention relates to a process for purification of mycophenolic acid comprising steps of: (a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base to selectively remove polar impurities; (b) extracting the water immiscible layer from step (a) with a second aqueous base; (c) combining the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; (d) acidifying the aqueous layer from step (c) to obtain precipitate; (e) recovering the precipitate by filtration or extraction with a third water-immiscible solvent; (f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and (g) optionally, purifying the mycophenolic acid by recrystallization; and a process for purification of mycophenolic acid comprising steps of: (a) extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; (b)
acidifying the aqueous layer from step (a) to obtain precipitate; (c) recovering the precipitate by filtration or extraction with a second water-immiscible solvent; (d) extracting the water immiscible layer from step (c) with a second aqueous base to selectively remove polar impurities; (e) concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and (f) optionally, purifying the mycophenolic acid by recrystallization.
DETAILED DESCRIPTION OF THE DVVENTION
The present invention relates to a process for purification of mycophenolic acid comprising steps of: a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base to selectively remove polar impurities; b) extracting the water immiscible layer from step (a) with a second aqueous base; c) combining ,the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; d) acidifying the aqueous layer from step (c) to obtain precipitate; e) recovering the precipitate by filtration or extraction with a third water- immiscible solvent; f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and g) optionally, purifying the mycophenolic acid by recrystallization.
In another embodiment of the present invention, the first, second and third water- immiscible solvent are selected from a group comprising ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, heptane and/or mixtures thereof.
In still another embodiment of the present invention, the first and second aqueous base are selected from a group comprising sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia.
In still another embodiment of the present invention, the polar impurities are one or more impurities at RRTs 0.52, 0.64 and 0.93.
In still another embodiment of the present invention, the non-polar impurities are one or both impurities at RRTs 1.10 and 1.34.
In still another embodiment of the present invention, the extraction is carried out either in a batch extraction manner or continuous counter-current extraction manner or continuous fractional extraction manner.
In still another embodiment of the present invention, the mycophenolic acid is in a free acid form or a salt form. -1
In still another embodiment of the present invention, the salt form is sodium salt of mycophenolic acid.
The present invention also relates to a process for purification of mycophenolic acid comprising steps of: a) extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; b) acidifying the aqueous layer from step (a) to obtain precipitate; c) recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d) extracting the water immiscible layer from step (c) with a second aqueous base to selectively remove polar impurities; e) concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and f) optionally, purifying the mycophenolic acid by recrystallization.
In still another embodiment of the present invention, the first and second water- immiscible solvent are selected from a group comprising ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, and heptane and/or mixtures thereof.
In still another embodiment of the present invention, the first and second aqueous base are selected from a group comprising sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia.
In still another embodiment of the present invention, the polar impurities are one or more impurities at RRTs 0.52, 0.64 and 0.93.
In still another embodiment of the present invention, the non-polar impurities are one or both impurities at RRTs 1.10 and 1.34.
In still another embodiment of the present invention, the extraction is carried out either in a batch extraction manner or continuous counter-current extraction manner or continuous fractional extraction manner.
In still another embodiment of the present invention, the mycophenolic acid is in a free acid form or a salt form.
In still another embodiment of the present invention, the salt form is sodium salt of mycophenolic acid.
In our invention, we have arrived at a process to obtain substantially pure form of mycophenolic acid as compared to the methods known in the prior art. The series of steps followed in our process is critical for obtaining pure form of mycophenolic acid. The process followed comprises: a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base of low molarity to selectively remove polar impurities; b) extracting the water immiscible layer (organic layer) from step (a) with a second aqueous base; c) combining the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; d) acidifying the aqueous layer from step (c) to obtain precipitate; e) recovering the precipitate by filtration or extraction with a third water- immiscible solvent;
f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and g) optionally, purifying the mycophenolic acid by recrystallization.
The invention also relates to a process for purification of mycophenolic acid comprising steps of: a) extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar impurities; b) acidifying the aqueous layer from step (a) to obtain precipitate; c) recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d) extracting the water immiscible layer (organic layer) from step (c) with a second aqueous base to selectively remove polar impurities; e) concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and f) optionally, purifying the mycophenolic acid by recrystallization.
The present invention relates to a novel process for purification of mycophenolic acid.
The novel process of the instant invention comprises: a) extraction of mycophenolic acid solution in a first water-immiscible solvent with a first aqueous base of low molarity to selectively remove polar analog impurities, b) extraction of organic layer from step (a) with a second aqueous base, c) extraction of the mycophenolic acid solution in the aqueous layer from step (b) with a second water-immiscible solvent to selectively remove non-polar analog impurities, d) acidification of the aqueous layer from step (c) to cause precipitation, e) recovery of the precipitate from step (d) by filtration or extraction with a third water-immiscible solvent, f) crystallization, and g) optional, re-crystallization affording mycophenolic acid in substantially pure form.
During production of mycophenolic acid by fermentation, several analog impurities are generated. The HPLC method used herein for analysis of mycophenolic acid is: Column: Waters, Symmetry, C8, 5 μm, diameter - 4.6 mm, length - 250 mm Flow rate: 1.5 ml/min Detection wavelength: 254 nm Injection volume: 10 μL Diluent: Methanol Temperature: 25°C
Approximate retention time of mycophenolic acid: 8 - 10 min Mobile phase: Buffer A - acetonitrile; Buffer B -water with 0.1% v/v phosphoric acid. The gradient was as:
Time (min) Buffer A (%) Buffer B (%)
0 40 60
14 40 60
20 75 25
22 40 60 25 40 60
Mycophenolic acid isolated using known methods in literature was analyzed by the given HPLC method, which showed polar impurities at RRTs (relative retention times) 0.52, 0.64 and 0.93 as well as non-polar impurities at RRTs 1.10 and 1.34. Depending on variations in HPLC systems, the RRTs of the impurities may vary in range from - 0.05 to + 0.05 of the given values.
The present invention relates to a novel process for purification of mycophenolic acid. a) extraction of mycophenolic acid solution in a first water-immiscible solvent with a first aqueous base of low molarity to selectively remove polar analog impurities, b) extraction of organic layer from step (a) with a second aqueous base, c) extraction of the mycophenolic acid solution in the aqueous layer from step (b) with a second water-immiscible solvent to selectively remove non-polar analog impurities, d) acidification of the aqueous layer from step (c) to cause precipitation,
e) recovery of the precipitate from step (d) by filtration or extraction with a third water-immiscible solvent, f) crystallization, and g) optional, re-crystallization affording mycophenolic acid in substantially pure form.
In the present invention, the mycophenolic acid solution in water-immiscible solvent may be obtained by extraction of fermentation broth with a water-immiscible solvent. Alternately, it may be obtained by dissolution of crude mycophenolic acid consisting of one or more of the impurities at RRTs 0.52, 0.64, 0.93, 1.10 and 1.34. The first, second and third water-immiscible solvent in the present invention may be independently selected from ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, and heptane or mixtures thereof. The first and second aqueous base may be independently selected from solution of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia in water.
The polar analog impurities in present invention relate to one or more impurities at RRT 0.52, 0.64 and 0.93, whereas the non-polar analog impurities relate to one or both impurities at RRT 1.10 and 1.34. Extraction in step (a) and step (c) may be done in a batch extraction manner, continuous counter-current extraction manner or continuous fractional extraction manner. Crystallization and re-crystallization in present invention may be carried out by decreasing temperature of mycophenolic acid solution or by evaporation of solvent or both together.
The present invention also relates to a process for mycophenolic acid purification in which the steps in the given process are carried out in a different order, for example, such as: a) extraction of the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non-polar analog impurities, b) acidification of the aqueous layer from step (a) to cause precipitation, c) recovery of the precipitate from step (b) by filtration and dissolution in second water-immiscible solvent or extraction with a second water-immiscible solvent,
d) extraction of organic layer from step (c) with a second aqueous base of low molarity to selectively remove polar analog impurities, e) crystallization, and f) optional, re-crystallization
The following examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein.
EXAMPLES
Example 1
20 g mycophenolic acid; impurities at RRTs 0.52, 0.64, 0.93, 1.10 and 1.34 containing 0.40, 0.53, 0.13, 0.18 and 0.32% respectively, was dissolved in 400 ml of ethyl acetate. The mycophenolic acid solution was extracted with 200 ml of 25 mM sodium carbonate solution. The organic layer was then extracted with 200 ml of 1% NaOH solution twice. The aqueous extracts were combined. The combined aqueous layer was extracted with n-butanol twice with a phase ratio of 1: 0. 2. The aqueous layer was acidified to a pH of 2.5 -3.5 using hydrochloric acid. The acidified layer was extracted with ethyl acetate. The ethyl acetate layer was washed with water and concentrated. The concentrate was kept at 50C for crystallization. The crystals were then filtered and dried. The yield was about 60%. The content of impurities at RRT 0.52, 0.64, 0.93, 1.10 and 1.34 was 0.08, 0.12, 0.08, 0.07 and 0.08, respectively.
Example 2
22.04g of mycophenolic acid; impurities at RRTs 0.52, 0.64, 0.93, 1.10 and 1.34 containing 0.40, 0.53, 0.13, 0.18 and 0.32% respectively, was dissolved in 500ml of 1% sodium hydroxide solution. This solution was twice extracted with n-butanol in the ratio 1:0.2. The aqueous layer was then acidified using hydrochloric acid to a pH of 2.9. The acidified solution was extracted with ethyl acetate. The ethyl acetate layer was thrice extracted with 12.5mM sodium carbonate solution in the ratio 1 :0.5. The ethyl acetate layer was then washed with water in the ratio of 6: 1. The ethyl acetate layer was then concentrated under reduced pressure. The concentrate was cooled to temperature of 0 to 5 0C. The concentrate was held at this temperature for 3h. The crystals were
filtered and dried. The yield was about 50%. The content of impurities at RRT 0.52, 0.64, 0.93, 1.10 and 1.34 was 0.10, 0.10, 0.08, 0.07 and 0.13, respectively.
Example 3
Mycophenolic acid; impurities at RRTs 0.52, 0.64, 0.93, 1.10 and 1.34 containing 0.40,
0.53, 0.13, 0.18 and 0.32% respectively, was dissolved in ethyl acetate. The mycophenolic acid solution was extracted with 25 mM sodium carbonate solution using 5 stages, wherein sodium carbonate solution was fed at the first stage and ethyl acetate was fed at the fifth stage. Both phases moved in counter-current manner while the mycophenolic acid solution was fed at the third stage. The organic layer exiting the first stage was collected and extracted with 1% NaOH solution twice. The aqueous extracts were combined. The combined aqueous layer was extracted with n-butanol in 5 stages, wherein 1% NaOH solution was fed at the first stage and butanol was fed at the fifth stage. Both phases moved in counter-current manner while the combined aqueous layer was fed at the third stage. The aqueous layer exiting the fifth stage was collected and acidified to a pH of 2.5 -3.5 using hydrochloric acid. The acidified layer was extracted with ethyl acetate. The ethyl acetate layer was washed with water and concentrated. The concentrate was kept at 5°C for crystallization. The crystals were then filtered and dried. The yield was about 80%. The content of impurities at RRT 0.52, 0.64, 0.93, 1.10 and 1.34 was 0.06, 0.07, 0.10, 0.10 and 0.13, respectively.
Claims
We claim;
1) A process for purification of mycophenolic acid comprising steps of: a) extracting mycophenolic acid solution in a first water immiscible solvent with a first aqueous base to selectively remove polar impurities; b) extracting the water immiscible layer from step (a) with a second aqueous base; c) combining the aqueous layers from step (a) and step (b) and extracting mycophenolic acid solution with a second water-immiscible solvent to selectively remove non-polar impurities; d) acidifying the aqueous layer from step (c) to obtain precipitate; e) recovering the precipitate by filtration or extraction with a third water- immiscible solvent; f) crystallizing the recovered precipitate to obtain mycophenolic acid in substantially pure form; and g) optionally, purifying the mycophenolic acid by recrystallization.
2) The process as claimed in claim 1, wherein the first, second and third water- immiscible solvent are selected from a group comprising ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, heptane and/or mixtures thereof.
3) The process as claimed in claim 1, wherein the first and second aqueous base are selected from a group comprising sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia.
4) The process as claimed in claim 1, wherein the polar impurities are one or more impurities at RRTs 0.52, 0.64 and 0.93.
5) The process as claimed in claim 1, wherein the non-polar impurities are one or both impurities at RRTs 1.10 and 1.34.
6) The process as claimed in claim 1, wherein the extraction is carried out either in a batch extraction manner or continuous counter-current extraction manner or continuous fractional extraction manner.
7) The process as claimed in claim 1, wherein the mycophenolic acid is in a free acid form or a salt form.
8) The process as claimed in claim 7, wherein the salt form is sodium salt of mycophenolic acid.
9) A process for purification of mycophenolic acid comprising steps of: a) extracting the mycophenolic acid solution in a first aqueous base solution with a first water-immiscible solvent to selectively remove non- polar impurities; b) acidifying the aqueous layer from step (a) to obtain precipitate; c) recovering the precipitate by filtration or extraction with a second water- immiscible solvent; d) extracting the water immiscible layer from step (c) with a second aqueous base to selectively remove polar impurities; e) concentrating the water immiscible layer from step (d) followed by crystallization to obtain mycophenolic acid in substantially pure form; and . f) optionally, purifying the mycophenolic acid by recrystallization.
10) The process as claimed in claim 9, wherein the first and second water- immiscible solvent are selected from a group comprising ethyl acetate, butyl acetate, amyl acetate, butanol, tert-butyl methyl ether, toluene, benzene, dichloromethane, chloroform, hexane, and heptane and/or mixtures thereof.
11) The process as claimed in claim 9, wherein the first and second aqueous base are selected from a group comprising sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate, potassium bicarbonate, potassium hydroxide and ammonia.
12) The process as claimed in claim 9, wherein the polar impurities are one or more impurities at RRTs 0.52, 0.64 and 0.93.
13) The process as claimed in claim 9, wherein the non-polar impurities are one or both impurities at RRTs 1.10 and 1.34.
14) The process as claimed in claim 9, wherein the extraction is carried out either in a batch extraction manner or continuous counter-current extraction manner or continuous fractional extraction manner.
15) The process as claimed in claim 9, wherein the mycophenolic acid is in a free acid form or a salt form.
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| IN2156CH2007 | 2007-09-25 | ||
| IN02156/CHE/2007 | 2007-09-25 |
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| PCT/IN2007/000532 WO2009040828A1 (en) | 2007-09-25 | 2007-11-13 | A process for purification of mycophenolic acid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838363A (en) * | 2023-01-09 | 2023-03-24 | 广东蓝宝制药有限公司 | Method for purifying mycophenolic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1158387A (en) * | 1967-06-13 | 1969-07-16 | Ici Ltd | Procedure for Isolation of Mycophenolic Acid |
| WO2005023791A2 (en) * | 2003-09-11 | 2005-03-17 | Sandoz Ag | Process for the production of mycophenolate mofetil |
| WO2005105768A2 (en) * | 2004-04-26 | 2005-11-10 | Teva Gyógyszergyár Zàrtköruen Muködo Rèszvènytàrsasàg | Process for preparation of mycophenolic acid and ester derivatives thereof |
| WO2006031665A1 (en) * | 2004-09-10 | 2006-03-23 | Ivax Pharmaceuticals S.R.O. | Process for isolation of mycophenolic acid |
| WO2006038218A1 (en) * | 2004-10-05 | 2006-04-13 | Biocon Limited | Process for producing mycophenolic acid using penicillium arenicola bicc7673 |
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2007
- 2007-11-13 WO PCT/IN2007/000532 patent/WO2009040828A1/en active Search and Examination
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1158387A (en) * | 1967-06-13 | 1969-07-16 | Ici Ltd | Procedure for Isolation of Mycophenolic Acid |
| WO2005023791A2 (en) * | 2003-09-11 | 2005-03-17 | Sandoz Ag | Process for the production of mycophenolate mofetil |
| WO2005105768A2 (en) * | 2004-04-26 | 2005-11-10 | Teva Gyógyszergyár Zàrtköruen Muködo Rèszvènytàrsasàg | Process for preparation of mycophenolic acid and ester derivatives thereof |
| WO2006031665A1 (en) * | 2004-09-10 | 2006-03-23 | Ivax Pharmaceuticals S.R.O. | Process for isolation of mycophenolic acid |
| WO2006038218A1 (en) * | 2004-10-05 | 2006-04-13 | Biocon Limited | Process for producing mycophenolic acid using penicillium arenicola bicc7673 |
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| Title |
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| WILLIAMS, R. H. ET AL.: "Fermentation, isolation, and biological properties of mycophenolic acid", ANTIMICROB. AGENTS CHEMOTHER., vol. 8, 1968, pages 229 - 233, XP000956424 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838363A (en) * | 2023-01-09 | 2023-03-24 | 广东蓝宝制药有限公司 | Method for purifying mycophenolic acid |
| CN115838363B (en) * | 2023-01-09 | 2024-04-23 | 广东蓝宝制药有限公司 | Purification method of mycophenolic acid |
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