FIELD OF INVENTION:
The present invention relates to improved process for purification of tacrolimus. BACKGROUND OF THE INVENTION:
Tacrolimus was discovered in 1987 by a Japanese team headed by T. Goto, T. Kino and H. Hatanaka; it was the first macrolide immunosuppressant discovered. Like ciclosporin, it was found in a soil fungus, although it is produced by a type of bacteria, Streptomyces tsukubaensis. The name tacrolimus is reportedly derived from Tsukuba macrolide immunosuppressant'.
The drug is owned by Astellas Pharma Inc., and is sold under the tradenames Prograf® and Protopic®. It is sometimes referred to as FK-506, an early name relating to its action. It was first approved by the Food and Drug Administration (FDA) in 1994 for use in liver transplantation, this has been extended to include kidney, heart, small bowel, pancreas, lung, trachea, skin, cornea, bone marrow, and limb transplants.
In EP 0184162 patent, process for isolation and purification of tacrolimus is disclosed, wherein the cultured broth is filtered with an aid of diatomaseous earth. The mycelial cake is extracted with acetone, the combined acetone layer and filtrate is passed through column of a non -ionic adsorption resin " Diaion HP-20" using aqueous acetone as mobile phase, which is concentarted under reduced pressure to give resudual water.
The above obtained residual water is extracted three times with ethyl acetate and concentarted under reduced pressure to give oily residue. The oily residue is adsorbed on the acidic silica gel ( special silica gel grade 12, maker fuji devision Co.) and subjected to column chromatography using the same silica gel, hexane and ethyl acetate is used as a mobile phase. The collected fractions are concentarted under reduced pressure to give oily residue, which is further carried out the same column purification followed by eluents evoporation to give yellowish oily residue.
The above obtained yellow oil residue is purified through column chromatography using silica gel (Merck Co., Ltd 230-400mesh) and mixture of ethyl acetate and n-hexane used as a mobile phase. The first fraction is concentarted under reduced pressure to give white power, which is again performed repeated crystallizations in acetonitrile to obtain pure tacrolimus.
As per the prior art process, crude tacrolimus is purified by using column chromatograpy with different types of silca gel and mixture of solvents and purification method is applied for 4-5 times. Eventhough, the obtained product is containing some impurities,which is further subjected to repeated crystallizations to give desired quality of
The prior art process is involved more number of times column chromatography purifications and repeated recrystallizations in a solvent medium to give the desire quality of the final product. These purification steps demands lot of solvent system, more time cycle and low yield finally it increase raw material cost of the final product.
US 6,492,251 patent revelas purification method of cuude tacrolimus, wherein 1 M aqueous silver nitrate solution is passed through Diaion® RCP 160M (H* type), which is then washed with water to remove an excess amount of silver nitrate. The ion exchange resin is equilibrated with methanol and then a mixture of ethyl acetate and methanol.
The crude tacrolimus is purified through column chromatography by using the above prepared ion exchane resin using ethyl acetate and methanol as a mobile phase followed by evaporation to give tacrolimus.
According to US '251 patent silver ion is used for making ion exchange resin. Tacrolimus is purified by using ion exchange resin, it is a possibility to present silver ions in the final product, which requires repeated crystallization methods in different solvents to give pharmaceutically acceptable quality. This process demands use of ion resin like silver nitrate and repeated crystallization methods in different solvents to give disired quality, it leads yield loss of the final product and increse the cost on final product.
Accoridng to the prior art processes, there is a need to develop simple prification method for tacrolimus to avoide number of purification steps through column purifications and use ion exchange resin.
OBJECT OF THE INVENTION:
The main object of the present invention is to develop a simple purification and isolation method of tacrolimus.
The other object of the present invention is to avoide number of purification and isolation steps through column chromatography.
Yet another object of the present invention is to avoide ion exchange resin eliminate the contamination of silver ion in the final product.
SUMMARY OF THE INVENTION:
The present aspect of the invention is to provide a simple purification and isolation of tacrolimus,wherein tacrolimus is subjected to purification through column chromatograpy using known solvent system, without employing silver ion resin to give semi pure tacrolimus, optionally which is further carried out same purification method followed by crystallization to give pure tacrolimus.
A method for purification of crude tacrolimus which comprising the steps of:
a) passing crude tacrolimus through stationary phase,
b) using solvents or mixture of solvent, water and acid as a mobile phase,
c) evaporating the eluents to give tacrolimus,
d) optionally repeating the step b, & c and
e) treating with organic solvent to give pure tacrolimus.
According to the present invention, crude tacrolimus is treated with Acetonitrile and Di- isopropyl ether and then passing through the stationary phase, the stationary phase is selected from Ci8 Redisep RP column having the length about 22 cm and dia 3.5 cm of Combiflash companion instrument connected with UV detector. The crude tacrolimus means it is containing the impurities around 10-15%.
The crude tacrolimus is passing through Cjg redisep RP column having the length about 22 cm and dia 3.5 cm of Combiflash Companion instrument connected with UV detector, the mobile phase is selected from solvent or a mixture of solvent, water and acid or mixtures thereof. The solvent is selected from acetonitrile, methanol, Ethanol, Dimethyl formamide, dimethyl acetamide, tetrahydrofuran, Dioxane and water miscible solvent, preferred solvent is acetonitrile.
The acid is selected from acetic acid, trifluoroacetic acid, preferred acid is trifluoroacetic acid. The amount of the acid is for the preparation of mobile phase is catalytic amount. The mobile phase is preferably a mixture of acetonitrile and water with catalytic amount of trifluoroacitic acid. The column is run through the mobile phase and collected die eluent, the combined eluents pH is adjusted to 5.0 to 8.0, preferred pH is 6.0 to 7.0.
The resulting solution is subjected to distilliation to remove the organic solvent under reduced pressure to obtain concentrated aqueous layer, which is extracted with water immiscible organic solvents selected from toluene, ethyl acetate, dichloromethane, dichloroethane. The preferable water immiscible organic solvent is ethyl acetate. The concentrated aqueous layer is extracted with ethyl acetate and combined organic layer is concentrated under reduced pressure to give oily tacrolimus.
The above obtained oily layer is subjected to same column purification as mentioned above to get the oily residue, which is treated with organic solvent is selected from diisopropyl ether to separate the solid, which is filtered and washed with diisopropyl ether to give tacrolimus.
The above obtained tacrolimus is subjecting to purification which comprising the steps of:
a) dissolving tacrolimus in a solvent to give the clear solution,
b) precipitating the solid by adding an anti solvent and
c) isolating pure tacrolimus
\ccording to the present invention, tacrolimus is dissolved in a solvent selected from \cetonitrile, Methanol, THF, DMF, Dioxane, preferred solvent is acetonitrile, tacrolimus s dissolved in acetonitrile to obtain clear solution. Precipitate the material by adding an inti solvent water. The separated solid is filtered and dried to give pure tacrolimus.
According to the present invention, the following advantageous are listed below:
i) Simple purification method in less time cycle as compared to the prior art
ii) Increased the productivity
iii) Improved the yield as compared to the prior art
iv) Eliminated the use of silver nitrate (AgNOa)
v) Avoided silver ion contamination in the final product
The invention is further illustrated with a few non-limiting examples as follows.
Process for purification of Tacrolimus, Example-1
Crude tacrolimus (lg) was dissolved in 5 ml acetonitrile and to this clear solution added carbon. Stirred solution for 30 min and filtered. Acetonitrile was distilled under vacuum to give an oil. Added 3 ml Diisopropyl ether to give solid and filtered solid loaded on column. Thus, obtained Tacrolimus was dissolved in 3 ml acetonitrile and 1.5 ml water The solution was loaded on C-18 RediSep 130 gm RP column with length 22 cm and dia 3.5 cm of Combiflash Companion instrument, connected with UV detector. Product was eluted with water (containing 0.01% TFA) & acetonitrile (1:1) mixture on a flow rate 65 ml/min. The fractions were monitored by UV detector. The Product rich fractions were collected separately and pooled. The pH of pooled fraction was adjusted to 6.5 to 7.0. The resulting solution was distilled acetonitrile under reduced pressure at temperature below 40° C. The product was extracted from concentrated aqueous layer with ethyl acetate. The resulting ethyl acetate layer was washed with brine solution. Ethyl acetate was distilled under vacuum at temperature below 40°C. Thus obtained oily tacrolimus (0.7 g),
This oily tacrolimus was further reloaded on C-18 RediSep 130 gm RP column with length 22 cm and dia 3.5 cm of Combiflash Companion instrument, connected with UV detector. Product was eluted with water (containing 0.01% TFA) & acetonitrile (1:1)
mixture on a flow rate 65 ml/min. The fractions were monitored by UV detector. The
\ %
Product rich fractions were collected separately and pooled. The pH of pooled fractions was adjusted to 6.5 to 7.0. The resulted solution was distilled under reduced pressure at temperature below 40° C. The product was extracted from concentrated aqueous layer with ethyl acetate. The resulting ethyl acetate layer was washed with brine solution. Ethyl
acetate was distilled under vacuum at temperature below 40° C to get an oily residue.

Claims:
1. A method of purification of tacrolimus comprising the steps of;
a) passing crude tacrolimus, which is containing impurities aroundlO-15%, through stationary phase
b) using solvents or mixture of solvent, water and acid as a mobile phase
c) running the column through mobile phase and simultaneously collecting the eluent
d) evaporating the eluents and further extracting the product with water immiscible organic solvents to give tacrolimus
e) optionally repeating the step b & c and
f) treating with organic solvent to give tacrolimus.
2. The process according to claim la, wherein the stationary phase is selected from CI8 Redisep RP column having the length about 22 cm and dia 3.5 cm of Combiflash companion instrument connected with UV detector.
3. The process according to claim lb, wherein the solvent is selected from acetonitrile, methanol, ethanol, dimethylformamide, dimethyl acetamide, tetrahydrofuran, dioxane, water or mixtures thereof preferable solvent is acetonitrile.
4. The process according to claim lb, wherein the acid is selected from acetic acid, trifluroacetic acid preferable solvent is trifluoroacetic acid.
5. The process according to claim lc, where collecting the eluent and adjusting the pH to 5.0 to 8.0 preferably 6.0 to 7.0.
6. The process according to claim Id, wherein the water immiscible organic solvent is selected from toluene, ethyl acetate, dichloromethane, dichloroethane preferably ethyl acetate.
7. The process according to claim If, wherein the organic solvent is selected from diisopropyl ether.
8. Purification of the tacrolimus, which comprising the steps of,
a) dissolving tacrolimus in a solvent to give clear solution
b) precipitating the solvent by adding antisolvent
c) and isolating pure tacrolimus.
9. The process according to claim 8a, wherein the solvent is selected from acetonitrile, methanol, tetrahydrofuran, dimethylformamide, dioxane preferably acetonitrile.