The present invention relates to a process for purification of macrolide from analogous compounds by using silver impregnated silica gel. The term "macrolide" as used herein refers to neutral non-polyeptide macrolides, which are lactone containing high molecular weight compounds.
A variety of macrolide compounds such as tacrolimus, rapamycin, ascomycin and their analogs find use as immunosuppressive agents for prevention of graft rejection in bone marrow and organ transplants and in the treatment of various auto-immune diseases. Tacrolimus and its derivatives have been shown to be effective in treating a number of diseases like asthma (PCT Application No WO 90/14826), inflammatory and hyperproliferative skin disease and cutaneous manifestations of immunologically induced illness (European Patent No 315,978).
Macrolides are usually produced by microbial fermentation whereby there is formation of analogous compounds from which the desired component has to be separated and purified. The separation and purification of macrolide from analogous compounds produced by fermentation methods is often complicated as they have same or almost the same number of carbon atoms and are similar to each other in physical properties such as solubility and affinity to solvents.
Use of silver for separating cis-trans isomers of an unsaturated aliphatic acid having same number of carbon atoms is known through J. Chromatography. 149(1978) 417-.
US Patent No 4,894,366 discloses a method for production of tacrolimus by fermentation using Streptomyces species wherein tacrolimus is isolated and purified from the fermentation broth by subjecting the treated broth to silica gel column chromatography.
US Patent No 5, 508, 398 discloses a process for recovering a neutral macrolide antibiotic such as rapamycin from concentrates of fermentation broth extracts or mother liquors whereby acidic and/or basic components formed in the fermentation process are removed by means of aqueous base or acid extraction from a water-immiscible solution of said concentrate and non-polar components are separated from the neutral macrolide by selective solubility.
US Patent No 5,616,595 discloses a process for recovering tacrolimus within single apparatus from fermentation broth by tangential filtration of raw fermentation broth across a solvent compatible porous filtration membrane.
US Patent No 6,492,513 discloses a method for separating a lactone containing high molecular weight compound having a alkyl group as its side chain from a lactone containing high molecular weight compound having an alkenyl group as its side chain, by using a sulphonic acid group containing cation exchange resin treated with silver ions.
US Patent No 6,881,341 discloses a method for separating a lactone-containing high molecular weight compound from a mixture with its analogues by adsorbing the mixture onto basic active alumina and eluting with an organic solvent to separate each of the compounds.
US Patent No 6,576,135 discloses a method for separating a macrolide from a mixture of macrolide and its analogous compounds by adsorbing the mixture onto a nonionic adsorption resin and eluting with an aqueous solvent containing silver ions, or adsorbing the mixture onto basic active alumina and eluting with an organic solvent or carrying out both the steps.
US Patent Application No 2004/0226501 discloses a method of crystallizing a macrolide from a macrolide starting material and from a concentrate residue from whole-broth extraction of macrolide-containing biomatter by combining the macrolide, a polar solvent like ethyl acetate, a hydrocarbon solvent like n-hexane and water and sodium hydroxide to attain a pH of 7 or above.
PCT Application No WO05/010015 discloses a method of purifying tacrolimus from impurities by loading onto a bed of sorption resin or a macroreticular resin and eluting with a combination of water and tetrahydrofuran.
PCT Application No WO05/019226 discloses a process for recovery of a macrolide by treating the macrolide with water immiscible solvent, followed by mixing with water
miscible solvent, performing hydrophobic interaction chromatography, extracting the fraction containing macrolide with water immiscible solvent, adding water miscible solvent to effect separation of impurities from the macrolide compound and then performing silica gel chromatography to obtain the microlide.
The present inventors have surprisingly found that macrolides can be purified from fermentation broth or mother liquors thereof by using silver impregnated silica gel chromatography. The present process shows unexpected efficiency in separating the macrolide from analogous compounds and enables isolation of pure macrolide. The present process is easily scalable and thus industrially advantageous.
The macrolides that can be purified by the present process include lactone containing high molecular weight compounds having molecular weight of 400 or more. They may be monocyclic, bicyclic or tricyclic macrolides or the like. The monocyclic macrolides include erythromycin, leucomycin, methymycins and the like. The tricyclic lactone containing compounds include for example those disclosed in European Patent No EP0184162, EP0427680, EP0532088 or PCT Application No WO 93/04680. Few examples of macrolides that can be purified by the present process include but not limited to tacrolimus (FK-506) of Formula I, dihydro FK-506, FK-520, ascomycin, rapamycin, 32-desmethylrapamycin, immunomycin, everolimus, pimecrolimus and homlogs, analogs or isomers therof.
(Formula Removed)
1(a) R = -CH2CH=CH2 (FK-506)
1(b) R = -CH2CH2CH3 (dihydro FK-506)
1(c) R = -CH2CH2CH3
Tacrolimus (FK-506) when subjected to purification by the present process was found to contain 1% or less of dihydro FK-506 and FK-520.
Figure 1 is elution profile of tacrolimus (FK-506) purified from the analogous compounds FK-520 and dihydro FK-506 obtained by silver impregnated silica gel chromatography.
A first aspect of the present invention provides a process for separation of a macrolide from homologs, analogs or isomers thereof, wherein the said process comprises of,
a) adsorbing a mixture comprising a macrolide onto silver impregnated silica gel,
b) eluting the mixture of step a) using water immiscible organic solvent.
The mixture comprising the macrolide along with analogs, homologs or isomers thereof used for separation of the macrolide can be the fermentation broth or mother liquors comprising the macrolide or it can be an isolated crude mixture of the macrolide obtained by the methods known in the art. The mixture of macrolide and analogous compounds is first dissolved in a suitable solvent and subjected column chromatography using silver impregnated silica gel having a mesh size of 60-400. The mixture adsorbed on the column was successively eluted with water immiscible organic solvent(s) at a temperature of about 10°C to 30°C. The fractions comprising the macrolide were pooled to recover the macrolide. The water immiscible organic solvent for elution can be selected from the group comprising of aliphatic hydrocarbons, C2-10 esters, chlorinated hydrocarbons, C4-8 ethers or mixtures thereof. Elution can also be carried out using a gradient of organic solvents.
EXAMPLE 1
PURIFICATION OF TACROLIMUS (FK-506) BY SILVER IMPREGNATED SILICA GEL CHROMATOGRAPHY
Silver impregnated silica gel column (26 X 1.3 cm; bed volume 30 ml) was prepared in hexanes. A crude mixture (100 mg) having tacrolimus (84.23%), ascomycin (10.85%) and dihydrotacrolimus (4.11%) was dissolved in ethyl acetate (10 ml). To the above mixture silver impregnated silica gel (1 g) was added and concentrated under vacuum at 40°C. To the concentrated mixture, hexanes (20 ml) was added and again concentrated under vacuum at 40°C to get a dry slurry. The dry slurry was loaded onto the column at 20°C at a loading of 5 g per litre of silica gel. The elution was carried out with a gradient mobile phase of ethyl acetate and hexanes (100% n-hexanes 30 ml; 40% ethylacetate-60% hexanes 60 ml; 50%ethylacetate-50% hexanes 60 ml; 60%ethylacetate-40% hexanes 120 ml; 70%ethylacetate-30% hexanes 120 ml). A flow rate of 1.0 bed volume per hour (0.5ml per minute) was maintained and fractions of 15 ml each were collected. The collected fractions were pooled and concentrated under vacuum at 40°C and the residue so obtained was crystallized from diisopropyl alcohol to afford the title compound. Purity: 96% by HPLC
FK-520 and Dihydro FK506 content: < 1.0 % by HPLC Elution profile as in Figure 1.

WE CLAIM:
1. A process for the separation of a macrolide from homologs, analogs or isomers
thereof, wherein the said process comprises of,
a) adsorbing a mixture comprising a macrolide onto silver impregnated silica gel,
b) eluting the mixture of step a) using water immiscible organic solvent.
2. A process according to claim 1 wherein the silica gel used has a mesh size of 60-400.
3. A process according to claim 2 wherein the silica gel used has a mesh size of 100-400.
4. A process according to claim 1 wherein the water immiscible organic solvent is selected from the group comprising of aliphatic hydrocarbons, C2-10 esters, chlorinated hydrocarbons, C4-8 ethers or mixtures thereof.
5. A process according to claim 4 wherein the organic solvent is n-hexane-ethyl acetate mixture.
6. A process according to claim 1 wherein the flow rate during elution in step b) is maintained at 1 bed volume per hour.
7. A process according to claim 1 wherein step b) is carried out at a temperature of about 10°C to 30°C.
8. A process according to claim 1 wherein the macrolide is a tricyclic macrolide.
9. A process according to claim 8 wherein the macrolide is tacrolimus, rapamycin or a homolog, analog or isomer thereof.
10. A process according to claim 1 wherein the said process further comprises of crystallizing the macrolide.