WO2005019226A1

WO2005019226A1 - A process for the recovery of substantially pure tricyclic macrolide

Info

Publication number: WO2005019226A1
Application number: PCT/IN2003/000283
Authority: WO
Inventors: Nitin Sopanrao Patil; Anand Prakash Khedkar; Rakesh Mendhe; Saurabh Garg; Ramakrishnan Melarkode; Ramavana Gururaja; Shrikumar Suryanarayan
Original assignee: Biocon Limited
Priority date: 2003-08-26
Filing date: 2003-08-26
Publication date: 2005-03-03
Also published as: AU2003269473A1

Abstract

Process for the recovery of a macrolide in substantially pure form comprising: a) treating the macrolide with water immiscible solvent followed by concentration, b) mixing with water, water miscible solvent or mixture thereof, c) performing hydrophobic interaction chromatography and collecting the fractions, d)extracting the fraction containing macrolide with water immiscible solvent followed by concentration, e) adding water miscible solvent to effect separation of impurities from the macrolide compound, f) performing silica gel chromatography and collecting the fractions, g) isolating the macrolide compound in substantially pure form. The macrolide is preferably rapamycin, tacrolimus or immunomycin.

Description

A PROCESS FOR THE RECOVERY OF SUBSTANTIALLY PURE TRICYCLIC MACROLIDE FIELD OF THE INVENTION This invention relates to recovery of tricyclic macrolides. BACKGROUND OF THE INVENTION In 1975, Vezina et al., identified (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)- 9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro- 9,27-dihydroxy-3-[(lR)-2-[(lS,3R,4R)-4-hydroxy-3-methoxycyclo hexyl]-l-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26- hexamethyl-23,27-epoxy-3H-pyrido[2,l- c][l,4]oxaazacyclohentriacontine-l,5,ll, 28,29(4H,6H,31H)- pentone, also known by the synonyms rapamycin and sirolimus as an antifungal antibiotic harvested from a Streptomyces hygroscopicus culture, which was isolated from an Easter Island soil sample. (J. Antibiot. 28, 721-726 (1975); and U.S. Pat. No. 3,929,992, was issued to Sehgal, et. al. Dec. 30, 1975). Martel, R. et al. (1977) described the ability of this compound to inhibit the immune response (Can. J. Physiol. Pharmacol., 55, 48-51). More recently, Calne, R. |Y. et al. (1989), has described rapamycin to be immunosuppressive in rats given heterotopic heart allografts. (Lancet vol. 2, p. 227). A compound, 15,19-Epoxy-3H-pyrido[2,l- c][l,4]oxaazacyclotricosine-l,7,20,21(4H,23H)-tetrone, 5,6,8,11,12, 13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro- 5,19-dihydroxy-3-[(lE)-2-[(lR,3R,4R)-4-hydroxy-3- methoxycyclohexyl]-l-methyl ethenyl]-14,16-dimethoxy- 4,10,12,18-tetramethyl-8-(2-propenyl)-,

(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS), also known by the synonyms FK506 and tacrolimus disclosed by EP 184162 and US 4,894,366 is also useful as immunosuppressant. Another compound, 15,19-Epoxy-3H-pyrido[2,l-c][l,4]oxaazacyclotricosine- 1,7,20,21 (4H,23H)-tetrone,8-ethyl-

5,6,8,11,12,13,14,15,16,17,18,19,24,25,26, 26a-hexadecahydro- 5,19-dihydroxy-3-[(lE)-2-[(lR,3R,4R)-4-hydroxy-3- methoxycyclohexyl]-l-methylethenyl]-14,16-dimethoxy-4,10,12,18- tetramethyh, (3S,4R,5S,8R,9E,12S,14S, 15R,16S,18R,19R,26aS)-, also known by the synonyms immunomycin and FK 520, disclosed in EPO Publication No. 0184162 is also useful as an immunosuppressant. Many other derivatives of these compounds as well as structural analogues have immunosuppressant property. US 5,508,398 discloses a process for separating a neutral non-polypeptide macrolide from acidic, basic and non-polar neutral impurities present in a concentrate of fermentation broth extracts or mother liquors containing said neutral macrolide which comprises in any order, extraction step (a) and optionally one or both of steps (b) and (c), wherein (a) involves extraction with aqueous base, (b) involves extraction with aqueous acid and (c) involves treatment with non-aromatic hydrocarbon solvent. US 5,616,595 discloses a process for recovering water insoluble compounds (including FK506, FK520 and rapamycin) from a fermentation broth which includes sequential steps of concentrating, solubilizing and diafiltering the compound of interest, all through a single closed recirculation system to recover the compound for further downstream purification. The prior art methods for the recovery of macrolide compounds are tedious, does not result' in pure product or require special setup for filtration. The instant invention relates to a novel process for recovery of macrolide compounds. SUMMARY OF THE INVENTION The instant invention relates to a novel process for recovery of macrolide compounds. The novel process of the instant invention comprises: a) treatment of an impure macrolide with water immiscible solvent and concentration and/or mixing, the impure macrolide or concentrate, with water, water miscible solvent or mixture thereof, b) performing hydrophobic interaction chromatography, c) extraction into water immiscible solvent and concentration, d) addition of a water miscible solvent to effect separation of impurities present with the macrolide compound e) performing silica gel chromatography f) isolating the macrolide compound in substantially pure form. The product thus obtained is of pharmaceutically acceptable quality. DETAILED DESCRIPTION OF THE INVENTON As mentioned earlier, The instant invention relates to a novel process for the production and recovery of macrolide compounds. The novel process of the instant invention comprises: a) treatment of a macrolide with water immiscible solvent and concentration and/or mixing, the impure macrolide or concentrate with water, water miscible solvent or mixture thereof, b) performing hydrophobic interaction chromatography, c) extraction into water immiscible solvent and concentration, d) addition of a water miscible solvent to effect separation of impurities present with the macrolide compound, e) performing silica gel chromatography f) isolating the macrolide compound in substantially pure form. The macrolide of the present invention can be produced by fermentation, chemical synthesis or combination of both. The broth obtained by fermentation can be directly extracted by water immiscible solvent. Any crude material in solid, semisolid or liquid form obtained either from broth, synthetic process or combination thereof can be treated with water immiscible solvent to effect solubilization of the macrolide into the water immiscible solvent. The water immiscible solvent containing the macrolide can be concentrated. The concentration can be affected by methods known per se. The concentration can be affected by vaporization of the solvent. The vaporization of the solvent can be carried out by heating without or with reduced pressure. The concentrate thus obtained can be diluted with water, water miscible solvent or mixture thereof. The water miscible solvent can be selected from alcohol, ketone or dielectric aprotic solvent. Preferably, the water miscible solvent can be selected from one or more among methanol, ethanol, isopropyl alcohol, acetone or acetonitrile. The mixture can be then subjected to hydrophobic interaction_. chromatography. The hydrophobic interaction chromatography can be carried out on matrix selected from a group comprising polystyrene divinyl benzene, methacrylates, polystyrene, resin linked or coated with hydrocarbons or any other suitable hydrophobic matrix. The fraction containing the macrolide can be collected and extracted with water immiscible solvent. The water immiscible solvent can be selected from a group comprising hydrocarbons, heterocyclic compounds, ethers or esters. Preferably, the water immiscible solvents can be selected from benzene, toluene, hexane, butanol, dichloromethane, chloroform, ethyl acetate, isobutylacetate, n-butyl acetate or t-butyl acetate. The solution can be subjected to charcoalization. The solution can be concentrated by method discussed earlier. The concentrate can be treated with a water miscible solvent to effect separation of impurities present with the macrolide. The impurities may be present in form of solids or liquid, immiscible with the solvent or both. The impurities can be separated out by filtration, phase separation or both. Preferably, the water miscible solvent can be selected from acetone, methanol, or acetonitrile. The filtrate or product containing phase can.be concentrated and the concentrate can be subjected to silica gel chromatography. The elute from the chromatography can be mixed with water immiscible solvent. The water immiscible solvent can be selected from a group comprising hydrocarbons, heterocyclic compounds, ethers or esters. Preferably the solvent can be selected from hydrocarbon, ether or ester. The solvent used can be a mixture of two or more solvents. Pure macrolide can be isolated from the water immiscible solvent or mixture thereof. In particular, the novel process of the instant invention comprises: a) treatment of an impure macrolide in broth with water immiscible solvent, preferably toluene or ethyl acetate and concentration, b) mixing with water, water miscible solvent or mixture thereof, preferably a mixture of acetone and water, c) performing hydrophobic interaction chromatography, d) extraction into water immiscible solvent, preferably ethyl acetate and concentration, e) addition of a water miscible solvent, preferably acetonitrile, to effect separation of impurities from the macrolide compound, f) performing silica gel chromatography, g) crystallization from one or more solvents selected from ether, ethyl acetate and hexane to afford the macrolide compound in substantially pure form. In particular, the novel process of the instant invention also comprises: a) mixing the impure macrolide with water, water miscible solvent or mixture thereof, preferably a mixture of acetone and water, b) performing hydrophobic interaction chromatography, c) extraction into water immiscible solvent and concentration, d) addition of a water miscible solvent to effect separation of impurities from the macrolide compound, e) performing silica gel chromatography, f) crystallization from one or more solvents selected from ether, ethyl acetate and hexane to afford the macrolide compound in substantially pure form. 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: RECOVERY OF RAPAMYCIN The fermentation broth (31Kg) containing rapamycin was twice extracted with 31 L of toluene. The toluene extract was concentrated to obtain 52 g of residue. The residue was dissolved in 5 L of acetone. 15 L of water was added to this. The, solution was passed through a 1.2L column packed with Sepabeads SP 825 resin. The column was washed with 3-column volume of water and 3-column volume of 25% acetone in water. The product was eluted with 75% acetone in water. The product containing fractions were pooled and diluted with water to make 50% acetone in the solution. The diluted solution was extracted with ethyl acetate. 4 g of activated charcoal was added -to the ethyl acetate extract and mixture was stirred for 20 minutes. The mixture was filtered. The filtrate was concentrated to obtain 15 g of oily residue.' 100 ml of acetonitrile was added to the residue. The precipitate obtained after acetonitrile addition was filtered. The filtrate was concentrated to obtain 14 g of oily residue. The residue was applied to a silica gel column. The column was washed with 2 column volumes of 15% acetone in hexane and 2 column volumes of 30% acetone in hexane. The product was eluted with 40% acetone in hexane. The product containing fractions were pooled and concentrated to 'obtain 5.5 g oily residue. To the residue 10 ml of diethyl ether was added and the product was crystallized at 4 °C. The product was filtered and dried. The product yield was 54%. The chromatographic purity was 99.8%

EXAMPLE 2: RECOVERY OF TACROLIMUS The fermentation broth (5Kg) containing tacrolimus was twice extracted with 5 L of ethyl acetate. The ethyl acetate extract was concentrated to obtain 22 g of residue. The residue was dissolved in 1 L of acetone. 4 L of water was added to this. The solution was passed through a 0.6L column packed with Sepabeads SP 825 resin. The column was washed with 3-column volumes of water and 3-column volumes of 25% acetone in water. The product was eluted with 75% acetone in water. The product containing fractions were pooled and concentrated. The concentrated solution was extracted with ethyl acetate. 1.5 g of activated charcoal was added to the ethyl acetate extract and mixture was stirred for 20 minutes. The mixture was filtered. The filtrate was concentrated to obtain 5.8 g of oily residue. The residue was twice extracted with acetonitrile. The acetonitrile extract was concentrated to obtain 3.9 g of oily residue. The residue was applied to a silica gel column. The column was washed with 3 column volumes of 25% ethyl acetate in hexane and 3 column volumes of 50% ethyl acetate in hexane. The product was eluted with 75% ethyl acetate in hexane. The product containing fractions were pooled and concentrated to obtain 1 g oily residue. To the residue 2.5 ml of ethyl acetate was added. The product was crystallized at 4 °C by slow addition of hexane. The product was filtered and dried. The product yield was 60%. The chromatographic purity was greater than 99.5% EXAMPLE 3: RECOVERY OF IMMUNOMYCIN The fermentation broth (16Kg) containing immunomycin was extracted with 16 L of ethyl acetate. The ethyl acetate extract was concentrated to obtain 36 g of residue. The residue was dissolved in 2 L of acetone. 8 L of water was added to this. The solution was passed through a 1.2L column packed with Sepabeads SP 825 resin. The column was washed with 3-column volumes of water and 3-column volumes of 25% acetone in water. The product was eluted with 75% acetone in water. The product containing fractions were pooled and concentrated. The concentrated solution was extracted with ethyl acetate. 15 g of activated charcoal was added to the ethyl acetate extract and mixture was stirred for 20 minutes. The mixture was filtered. The filtrate was concentrated to obtain 62 g of oily residue. The residue was twice extracted with acetonitrile. The acetonitrile extract was concentrated to obtain 43 g of oily residue. The residue was applied to a silica gel column. The column was washed with 3 column volumes of 25% ethyl acetate in hexane and 3 column volumes of 50% ethyl acetate in hexane. The product was eluted with 75% ethyl acetate in hexane. The product containing fractions were pooled and concentrated to obtain 12 g oily residue. To the residue 12.5 ml of ether was added. The product was crystallized at 4 °C by slow addition of hexane. The product was filtered and dried. The product yield was 56%. The chromatographic purity was greater than 99.6%

Claims

We claim:

1. A novel process for the recovery of a macrolide in substantially pure form comprising: a) treating the macrolide with water immiscible solvent followed by concentration, b) mixing with water, water miscible solvent or mixture thereof, c) performing hydrophobic interaction chromatography and collecting the fractions, d) extracting the fraction containing macrolide with water immiscible solvent followed by concentration, e) adding water miscible solvent to effect separation of impurities from the macrolide compound, f) performing silica gel chromatography and collecting the fractions, g) isolating the macrolide compound in substantially pure form.

2. A process as in claim 1, wherein the macrolide is selected from rapamycin, tacrolimus or immunomycin.

3. A process as in claim 1, wherein the water immiscible solvent is selected from a group comprising hydrocarbons, heterocyclic compounds, ethers, esters or a mixture of two or more of these.

4. A process as in claim 3, wherein the water immiscible solvents is selected from benzene, toluene, hexane, butanol, dichloromethane-, chloroform, ethyl acetate, isobutylacetate, n-butyl acetate, t-butyl acetate or a mixture of two or more of these.

5. A process as in claim 1, wherein the water miscible solvent is selected from one or more among alcohol, ketone or dielectric aprotic solvent.

6. A process as in claim 5, wherein the water miscible solvent is selected one or more among methanol, ethanol, isopropyl alcohol, acetone or acetonitrile.

7. A process as in claim 1, wherein the hydrophobic interaction chromatography is performed using matrix selected from polystyrene divinyl benzene, methacrylates, polystyrene or matrix linked or coated with hydrophobic groups.

8. A process as in claim 1 step (e), wherein the solvent is selected from acetone, methanol or acetonitrile.

9. A process as in claim 1, wherein the macrolide compound is isolated by crystallization.

10. A process as in claim 9, wherein the crystallization is carried out in one or more solvents selected from ethyl acetate, diethyl ether or hexane.

11. A process as in claim 1, wherein the macrolide is obtained by fermentation.

12. A process as in claim 1, wherein the macrolide is obtained by synthetic process.

13. A novel process for the recovery of the macrolide in substantially pure form as in claim 1 from crude macrolide comprising: a) mixing with water, water miscible solvent or mixture thereof, b) performing hydrophobic interaction chromatography and collecting the fractions, c) extracting the fraction containing macrolide with water immiscible solvent followed by concentration, d) adding water miscible solvent to effect separation of impurities from the macrolide compound, e) performing silica gel chromatography and collecting the fractions, f) isolating the macrolide compound in substantially pure form.

14. A process as in claim 13, wherein the macrolide is selected from rapamycin, tacrolimus or immunomycin.

15. A process as in claim 13, wherein the water immiscible solvent is selected from a group comprising hydrocarbons, heterocyclic compounds, ethers or esters.

16. A process as in claim 15, wherein the water immiscible solvents is selected from benzene, toluene, hexane, butanol, dichloromethane, chloroform, ethyl acetate, isobutylacetate or butyl acetate.

17. A process as in claim 13, wherein the water miscible solvent is selected from one or more among alcohol, ketone or dielectric aprotic solvent.

18. A process as in claim 17, wherein the water miscible solvent is selected one or more among methanol, ethanol, isopropyl alcohol, acetone or acetonitrile.

19. A process as in claim 13, wherein the hydrophobic interaction chromatography is performed using matrix selected from polystyrene divinyl benzene, methacrylates, polystyrene or matrix linked or coated with hydrophobic groups.

20 A process as in claim 13 step (d), wherein the solvent is selected from acetone, methanol or acetonitrile.

21. A process as in claim 13, wherein the macrolide compound is isolated by crystallization.

22. A process as in claim 21, wherein the crystallization is carried out in one or more solvents selected from ethyl acetate, diethyl ether or hexane.

23. A process as in claim 13, wherein the macrolide is obtained by fermentation.

24. A process as in claim 13, wherein the macrolide is obtained by synthetic process.