WO2009040827A1

WO2009040827A1 - A process for obtaining tetrahydrolipstatin

Info

Publication number: WO2009040827A1
Application number: PCT/IN2007/000531
Authority: WO
Inventors: Pullela Venkata Srinivas; Chandrashekar Aswathanarayanappa; Umesh Sannachikkanna; Rohith Guptha
Original assignee: Biocon Limited
Priority date: 2007-09-24
Filing date: 2007-11-13
Publication date: 2009-04-02

Abstract

The present invention provides a process of preparing tetrahydrolipstatin, comprising catalytical hydrogenation of lipstatin in polyethylene glycol. Optionally polyethylene glycol is mixed with an organic solvent selected from a group of lower alcohols, non polar solvents and ethers. The invention further includes process of purification of the tetrahydrolipstatin thus obtained.

Description

A PROCESS FOR OBTAINING TETRAHYDROLIPSTATIN

FIELD OF THE PRESENT INVENTION The present invention is in relation to hydrogenation reactions. More particularly, the present invention is in relation to catalytic hydrogenation of lipstatin to tetrahydrolipstatin using polyethylene glycol and palladium on carbon as a catalyst. In addition, the present invention also provides a process for purification of tetrahydrolipstatin.

BACKGROUND AND PRIOR ART OF THE INVENTION

Tetrahydrolipstatin [N-formyl-L-leucine ester with (3S,4S)-3-hexyl-4-[(2S)-2- hydroxytridecyl]-2-oxetanone, Orlistat, orlipastat, is a drug designed to treat obesity.

Tetrahydrolipstatin

Tetrahydrolipstatin is the saturated derivative of lipstatin. Lipstatin was produced by fermentation, Production of lipstatin followed by purification and converting lipstatin into tetrahydrolipstatin by hydrogenation are described in US patent number 4,598,089 and US patent number 6, 156,91 1.

The process of production of crude lipstatin is well described also in a European patent number 0 803 576 in which claims the fermentation method with a microorganism Streptomyces toxytricini. The process chromatographic purification of crude lipstatin is well described in US patent number 4,598,089 which is feasible in lab scale but in large scale it is quite expensive and tedious. The US patent number 6,156,911 discusses the purification of crude lipstatin more in detail. The counter current extraction technique which is employed using liquid - liquid extractions method using a non-polar solvent selected from an aliphatic or aromatic hydrocarbon into a polar solvent selected from a carboxylic acid, an alcohol, an O-monosubstituted mono- or oligo- or polyethyleneglycol, a diol or a dipolar aprotic solvent followed by diluting the polar solvent phase with water or changing the phase ratio and re-extraction of lipstatin into a fresh non polar solvent which is selected from an aliphatic or aromatic hydrocarbon. Preferred aliphatic hydrocarbons are C₅ -C₈ aliphatic hydrocarbons, more preferably C₆ -C₇ aliphatic hydrocarbons like hexane or heptane. Aromatic hydrocarbons may be selected from benzene, optionally substituted by 1 to 3 methyl-groups. Preferred aromatic hydrocarbons are benzene and toluene.

Purification of crude lipstatin by distillation is failed as boiling point of the liquid is more and at higher temperature the lactone ring gets opened and lipstatin get degraded. Purification of lipstatin by crystallization at temperature less than -20 ⁰C is quite difficult as stability of crystals is less and gets melted very fast.

Conversion of lipstatin to tetrahydrolipstatin is disclosed in US patent number

4,598,089 by hydrogenation using a catalyst selected from palladium/carbon, platinum oxide, palladium and the like. Suitable solvents are selected, for example, lower alcohols such as methanol and ethanol. These solvents are highly flammable and handling of the catalyst in the presence of these solvents is much difficult and can catch fire at any time. Regeneration of solvents used for hydrogenation is also difficult.

Purification of lipstatin is discussed in US patent number 6,156,911 by two solvent counter current extraction which on hydrogenation yields pure form of tetrahydrolipstatin. US patent number 6,734,314 discusses the preparation of tetrahydrolipstatin by catalytic hydrogenation and preparation of crystalline tetrahydrolipstatin by adopting several methods of crystallization.

Recovery of catalyst is a serious problem after the hydrogenation in homogeneous catalysis. Intensive work is focused on this which resulted in liquid-liquid biphasic catalysis which is most elegant method; in this regard soluble polymer supported catalysis in combination with an organic solvent is attempted. One of the inexpensive solvents polyethylene glycol has been used as an efficient recyclable reaction medium for the recycling of homogeneous catalysts in hydrogenation. However additional procedures such as extraction by ethers or alkanes were necessary to separate catalysts from the reaction mass

OBJECTS OF THE INVENTION: The main object of the present invention is to develop a process for obtaining tetrahydrolipstatin from lipstatin.

Another object of the present invention is to develop a process for obtaining tetrahydrolipstatin from lipstatin by catalytic hydrogenation method. Yet another object of the present invention is to develop a process for obtaining tetrahydrolipstatin from lipstatin by catalytic hydrogenation method using polyethylene glycol and palladium on carbon as a catalyst.

Still another object of the present invention is to develop a process for purification of tetrahydrolipstatin.

STATEMENT OF THE INVENTION:

Accordingly, the present invention provides a process for obtaining tetrahydrolipstatin, said process comprising steps of catalytically hydrogenating lipstatin in polyethylene glycol to obtain tetrahydrolipstatin.

DETAILED DESCRIPTION OF THE INVENTION: The present invention is in relation to a process for obtaining tetrahydrolipstatin, said process comprising steps of catalytically hydrogenating lipstatin in polyethylene glycol to obtain tetrahydrolipstatin.

Another embodiment of the present invention, wherein the catalyst is palladium on carbon. Yet another embodiment of the present invention, wherein the polyethylene glycol is optionally mixed with one or more organic solvents selected from a group comprising

1, 4-dioxane, non polar solvents such as pentane, hexane and heptane, alcohols such as methanol, ethanol, n-propanol and isopropyl alcohol and ethers such as di isopropyl ether. Still another embodiment of the present invention, wherein the size of polyethylene glycol is ranging from 200 monomers to 400 monomers.

Still another embodiment of the present invention, wherein the hydrogenation reaction is carried out at a temperature ranging from 15° to 4O⁰C. Still another embodiment of the present invention, wherein the hydrogenation reaction is carried out at a pH ranging from 1 to 7 and preferably at a pressure of about 5 kg/ cm².

Still another embodiment of the present invention, wherein the tetrahydrolipstatin obtained is purified by steps comprising, a) dissolving crude tetrahydrolipstatin in a non polar solvent; b) extracting dissolved tetrahydrolipstatin into a polar phase; c) adding water to the extract obtained from step (b); d) extracting tetrahydrolipstatin of step (c) into a non-polar solvent; and e) isolating solid tetrahydrolipstatin at low temperature.

Still another embodiment of the present invention, wherein the temperature maintained to isolate solid tetrahydrolipstatin is in the range of 0° to 5° C.

Still another embodiment of the present invention, wherein the non polar solvents are aliphatic hydrocarbons selected from a group comprising pentane, hexane, cyclohexane and heptane.

Still another embodiment of the present invention, wherein the polar phase is a mixture of carboxylic acid, water and an organic solvent.

Still another embodiment of the present invention, wherein the carboxylic acid is acetic acid and the organic solvent is acetonitrile.

Definitions:

As used herein the term 'tetrahydrolipstatin' is also known as Orlistat. 'Lipstatin' refers to a precursor of tetrahydrolipstatin which is having four hydrogen lesser than tetrahydrolipstatin.

As used herein the term 'ambient temperature' is also known as room temperature. As used herein the term 'hydrogenation' refers to the addition of hydrogen to unsaturated hydrocarbon atoms in lipstatin with hydrogen gas in the presence of a catalyst which is having palladium metal.

The analysis of the samples was performed by HPLC, using waters symmetry Ci g type

250X4.6 mm, 5μm column. Detection was done by UV detector at 205 nm wavelength. The mobile phase was a gradient mixture of acetonitrile and 0.1% phosphoric acid in aqueous solution. It is found that the polyethylene glycol and palladium on carbon used for hydrogenation can be reused with out regeneration. After hydrogenation product is extracted to an organic solvent which is not miscible with polyethylene glycol with out filtration to separate the catalyst. The mixture of polyethylene glycol and catalyst can use as such for hydrogenation of fresh lipstatin.

The present invention provides less usage of palladium on carbon which in turn reduces the cost of production and minimizes the risk in handling palladium on charcoal. The present invention provides the process for the preparation of tetrahydrolipstatin, comprising, the catalytically hydrogenating lipstatin in polyethylene glycol. Optionally polyethylene glycol is mixed with one or more organic solvents. Organic solvent is selected from a group consisting of lower alcohols, non polar solvents, ethers and 1 ,4- dioxane.

Preferably, the catalyst is selected from the group consisting of palladium. The catalyst is preferably palladium on charcoal which is 50% wet with water. Preferably the hydrogenation step is performed at the temperature maintained between 15 to 40 ⁰C. Preferably the pH of the reaction mass during hydrogenation is maintained in the range of 1 and 7. Preferably the hydrogenation is performed at the reaction pressure less than 5 kg/cm². The present invention provides preparation of tetrahydrolipstatin in polyethylene glycol optionally mixed with lower alcohols which is selected from group of methanol, ethanol, isopropyl alcohol and n-propanol, most preferably the lower alcohol selected is methanol and ethanol.

The present invention provides preparation of tetrahydrolipstatin in polyethylene glycol optionally mixed with non polar solvents selected one among pentane, hexane, cyclohexane and n-heptane; most preferred non polar solvents are hexane and heptane. The present invention provides the preparation of tetrahydrolipstatin in polyethylene glycol optionally mixed with ether which is preferentially diisopropyl ether. The present invention provides the preparation of tetrahydrolipstatin in polyethylene glycol optionally mixed with 1,4-dioxane. The present invention provides the re-use of polyethylene glycol and palladium on carbon for many more reactions with out filtrating the reaction mass. Present invention provides the use of celite as filter aid for easy filtration as the product is waxy in nature. The present invention provides the purification of tetrahydrolipstatin with the process comprising the steps of: a) dissolving crude tetrahydrolipstatin in a non polar solvent; b) extracting tetrahydrolipstatin into a polar phase; c) adding water to the extract obtained from step (b); and d) extracting tetrahydrolipstatin into a non-polar solvent; e) isolating solid tetrahydrolipstatin at low temperature.

Present invention provides a novel method for the purification of tetrahydrolipstatin. First crude tetrahydrolipstatin is dissolved in first non polar solvent wherein the first non polar solvent is selected from aliphatic hydrocarbon, extracting tetrahydrolipstatin into polar phase wherein the polar phase is prepared by mixing carboxylic acid, water and organic solvent. The product is extracted into a second non-polar solvent by diluting polar phase with water wherein the second non polar solvent is selected from aliphatic hydrocarbon. Tetrahydrolipstatin is isolated by chilling the non-polar solvent to a low temperature.

First non-polar solvent used for the dissolution of crude tetrahydrolipstatin is selected from aliphatic hydrocarbons which is preferably from a group comprising of heptane, hexane and cyclohexane. The polar phase is the mixture of carboxylic acid, water and organic solvent, which is preferably a mixture of acetic acid, water and acetonitrile. The ratio of the mixture is; Carboxylic acid: water: Organic solvent :: 77.5%:2.5%:20%.

Second non-polar solvent used for the extraction of tetrahydrolipstatin is selected from aliphatic hydrocarbons which are preferably from a group consists of heptane, hexane and cyclohexane. Isolation of purified tetrahydrolipstatin is done by chilling second non-polar solvent extract at low temperature, which is preferably ranging from 0 to 5 ⁰C.

The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the invention.

Example-1:

To a solution of crude Lipstatin (30 g) in Poly ethylene glycol-200 (150 ml) Palladium on charcoal (2.2 g, 50% wet with water) was added, followed by hydrogenation ( hydrogen pressure is about 5 kg/cm²) at temperature 15-40⁰C for about 24 hours. Product was extracted by di-isopropyl ether which washed with water and dried over sodium sulphate anhydrous and concentrated to residue to yield crude tetrahydrolipstatin (25g, purity 96.1%). Note: The Polyethylene glycol-200 and Palladium on charcoal mixture was preserved for re-use.

Example-2: To the mixture of Poly ethylene glycol-400 (150 ml) and Methanol (30 ml) crude Lipstatin (30 g) was added and stirred well and to the above solution Palladium on charcoal (2.2 g, 50% wet with water) was added and hydrogenated ( hydrogen pressure is about 5 kg/cm²) at temperature 15-40⁰C for about 24 hours. Thus obtained tetrahydrolipstatin was extracted by di-isopropyl ether (100ml X 2 times) which was washed with water and dried over sodium sulphate anhydrous and concentrated to residue to yield crude tetrahydrolipstatin (24.5g, purity 96.5%). Note: The Polyethylene glycol-400 and Palladium on charcoal mixture was preserved for re-use.

Example-3:

To a solution of crude Lipstatin (30 g) in Poly ethylene glycol-200 (150 ml) Palladium on charcoal (2.2 g, 50% wet with water) was added, followed by hydrogenation ( hydrogen pressure is about 5 kg /cm²) at temperature 15-40⁰C for about 24 hours. Product was extracted by n-heptane (100 ml X 2 times) which was washed with water and dried over sodium sulphate anhydrous and concentrated to residue to yield crude tetrahydrolipstatin (23g, purity 95.9%).

Note: The Polyethylene glycol-200 and Palladium on charcoal mixture was preserved for re-use.

Example 4:

To the mixture of Poly ethylene glycol-400 (150 ml) and di-isopropyl ether (30 ml) crude Lipstatin (30 g) was added and stirred well and to the above solution Palladium on charcoal (2.2 g, 50% wet with water) was added and subjected for hydrogenation (hydrogen pressure is about 5 kg/cm²) at temperature 15-40⁰C for about 16 hours. Tetrahydrolipstatin in the reaction mixture was extracted with di-isopropyl ether { 100ml X 2 times) which was washed with water and dried over sodium sulphate anhydrous and concentrated to residue to yield crude tetrahydrolipstatin (25.1g, purity 96.5%).

Note: The Polyethylene glycol-400 and Palladium on charcoal mixture was preserved for re-use.

Example-S:

Crude Lipstatin (30 g) was added to a mixture of Poly ethylene glycol-300 (150 ml) and n-Heptane (30 ml) followed by the addition of Palladium on charcoal (2.2 g, 50% wet with water). The resulting reaction mixture was subjected to hydrogenation (5 kg/cm² pressure) at 15-40⁰C for about 24 hours. Product was extracted into n-Heptane

(100 ml X two times). The n-heptane extract was washed with water and dried over

Na₂SO₄ and evaporated to reside under vacuum to give crude tetrahydrolipstatin (22.5 g, Purity: 96.3%).

Note: The Polyethylene glycol-300 and Palladium on charcoal mixture after product extraction was preserved for re-use.

Example-6: To the mixture of Poly ethylene glycol-400 (150 ml) and n-hexane (30 ml) crude Lipstatin (30 g) was added and stirred well and to the above solution Palladium on charcoal (2.2 g, 50% wet with water) was added and hydrogenated ( hydrogen pressure is about 5 kg/cm²) at temperature 15-40⁰C for about 24 hours. Thus obtained tetrahydrolipstatin was extracted with n-hexane (100ml X 2 times) which was washed with water and dried over sodium sulphate anhydrous and concentrated to residue to yield crude tetrahydrolipstatin (23.1g, purity 96.5%).

Example-7:

To the mixture of Poly ethylene glycol-400 (150 ml) ,1,4 dioxane (30 ml) and n- heptane (120 ml) crude Lipstatin (30g) was added and stirred well and to the above solution Palladium on charcoal (2.2 g, 50% wet with water) was added and hydrogenated ( hydrogen pressure is about 5 kg/cm²) at temperature 15-40⁰C for about 16 hours. Organic layer was separated and tetrahydrolipstatin was further •extracted with n-heptane (100ml X 2 times), the combined organic extract was washed with water and dried over sodium sulphate anhydrous and concentrated to residue to yield crude tetrahydrolipstatin (24.5g, purity 94.5%).

Note: The Polyethylene glycol-400 and Palladium on charcoal mixture was preserved for. re-use.

Example-8

The crude tetrahydrolipstatin (20 g, obtained from example- 1) was dissolved in n- Heptane (200 ml) and the solution was cooled to 0⁰C and tetrahydrolipstatin was precipated, filter aid (celite, 30 g) was added, stirred for 30 minutes at 0 to 5 ⁰C and filtered. The wet cake was dried under vacuum at room temperature to remove the solvents. The resulted solids were slurried with Methanol (200 ml) and filtered to remove filter aid. The filtrate obtained was cooled to 0- 5°C, water (30 ml) was added to precipitate and stirred for 2 hrs at 0-5 ⁰C. The product was filtered and dried under vacuum at room temperature to obtain pure tetrahydrolipstatin (10.4g, Purity: 97.5 %).

Example-9

The crude tetrahydrolipstatin (20 g, obtained from example-2) was dissolved in Acetic acid (50 ml) and precipitated by the addition of water (200 ml). The mass was stirred at ambient temperature for about 2 h. the product was collected by filtration. The wet cake was slurried with water and dried under vacuum at room temperature. The resulting product was dissolved in n-heptane (150 ml) and cooled to 0⁰C and stirred at 0-5⁰C for 2 hrs. The product was filtered and dried at room temperature (H g, Purity: 97.4 %).

ExampIe-10

The crude tetrahydrolipstatin (obtained from example-4) was dissolved in n-heptane (200 ml). The product was extracted to the polar phase which is a mixture of acetic acid (77.5 ml), water (2.5ml) and Acetonitrile (20ml). Water (200 ml) was added to the polar phase and the product was extracted to n-Heptane (100 ml X two times), n- heptane extract was washed with water and dried over Na₂S0₄and cooled to 0⁰C and stirred at 0-5⁰C for 2 h. The product was filtered and the wet cake was dried under vacuum at room temperature to obtain pure tetrahydrolipstatin (11.8 g, Purity: 97.9).

Claims

♦We Claim:

1. A process for obtaining tetrahydrolipstatin, said process comprising steps of catalytically hydrogenating lipstatin in polyethylene glycol to obtain tetrahydrolipstatin.

2. The process as claimed in claim 1, wherein the catalyst is palladium on carbon.

3. The process as claimed in claim 1, wherein the polyethylene glycol is optionally mixed with one or more organic solvents selected from a group comprising 1, 4- dioxane, non polar solvents such as pentane, hexane and heptane, alcohols such as methanol, ethanol, n-propanol and isopropyl alcohol and ethers such as di isopropyl ether.

4. The process as claimed in claim 1, wherein the size of polyethylene glycol is ranging from 200 monomers to 400 monomers.

5. The process as claimed in claim 1, wherein the hydrogenation reaction is carried out at a temperature ranging from 15° to 40⁰C.

6. The process as claimed in claim 1, wherein the hydrogenation reaction is carried out at a pH ranging from 1 to 7 and preferably at a pressure of about 5 kg/ cm².

7. The process as claimed in claim 1, wherein the tetrahydrolipstatin obtained is purified by steps comprising, a) dissolving crude tetrahydrolipstatin in a non polar solvent; b) extracting dissolved tetrahydrolipstatin into a polar phase; c) adding water to the extract obtained from step (b); d) extracting tetrahydrolipstatin of step (c) into a non-polar solvent; and e) isolating solid tetrahydrolipstatin at low temperature.

8. The process as claimed in claim 10, wherein the temperature maintained to isolate solid tetrahydrolipstatin is in the range of 0 to 5°C.

9. The process as claimed in claim 10, wherein the non polar solvents are aliphatic hydrocarbons selected from a group comprising pentane, hexane, cyclohexane and heptane.

10. The process as claimed in claim 10, wherein the polar phase is a mixture of carboxylic acid, water and an organic solvent.

11. The process as claimed in claim 13, wherein the carboxylic acid is acetic acid and the organic solvent is acetonitrile.