The present invention relates to a process for the purification of nucleoside analogues which does not employ chromatographic techniques.
Nucleoside analogues such as 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine (DDC), 2',3'-didehydro-3'-deoxythymidine (D4T), 2',3'-dideoxyinosine (DDI), and various 2'-fluoro-derivatives of these nucleosides are known to be effective in halting HIV replication by inhibiting reverse transcriptase. Emtricitabine and Lamivudine are promising antiviral drugs having activity in particular against retroviruses like human immunodeficiency virus (HIV), which is recognized as an etiologic agent of autoimmune deficiency syndrome (AIDS) and hepatitis B virus (HBV). Both emtricitabine and lamivudine exert protective activity against HIV induced cytopathogenicity.
Emtricitabine of Formula la also known as FTC is chemically (-)-p-L-4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone whereas lamivudine or 3TC is the 5-defluoro analogue of Formula lb,
(Formula Removed)
Emtricitabine has two chiral centres and therefore four stereoisomers are possible, namely (2R, 5S) and (2S, 5R) which are the cis isomers and (2R, 5R) and (2S, 5S) which are the trans isomers. The p-anomer having the configuration (2R, 5S) is found to have profound antiviral activity. The known processes for synthesis of emtricitabine and analogous compounds involve introduction of the pyrimidine base in the 1,3-oxathiolane ring which leads to a product which is a mixture of the various stereoisomers. Stereoselective processes have also been developed to obtain the p-anomer. However in such processes the crude compound is purified by chromatographic techniques.
European Patent No 513200 discloses a process for preparation of emtricitabine by reaction of 5-0-protectedhydroxymethyl-2-acetoxy-1,3-oxathiolane with 5-fluorocytosine
followed by deprotection of the O-protected hydroxymethyl group to afford emtricitabine which is isolated and purified by flash chromatography.
European Patent No 574487 discloses a process for preparation of (±)- emtiricitabine which involves separation of (±)-cis- and (±)-trans-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine by silica gel chromatography followed by fluorination of (±)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine with trifluoromethyl hypofluorite to obtain (±)-emtiricitabine.
European Patent No 654031 discloses a process for preparation of cis and trans isomers of emtricitabine by reaction of 5-fluorocytosine with 2,7-dioxa:5-thia-bicyclo[2.2.1]heptan-3-one derivative in the presence of trimethylsilyl iodide to give a mixture of cis and trans isomers in a 10:1 ratio.
European Patent Application No 1155695 discloses a process for preparation of emtricitabine which involves subjecting (±)-cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine-5'-monophosphate to enzymatic hydrolysis with 5'-nucleotidase. The resulting mixture is loaded onto a sephadex column and the eluted fractions are treated with alkaline phosphatase. The product obtained is purified by silica gel column chromatography followed by high performance liquid chromatography to afford (-)-emtricitabine.
European Patent Application No 1439177 discloses a process for the preparation of emtricitabine by enzymatic hydrolysis of the corresponding racemic 5'-0-acyl protected derivative followed by purification by chromatography or salt formation.
European Patent no 515157 discloses a process for the preparation of emtricitabine by reaction of L-menthyl cis-1,3-oxathiolan-5S-acetoxy-2R-carboxylate with 5-fluorocytosine in the presence of iodotrimethylsilane followed by reduction of the L-menthyl ester group of the product so formed with lithium aluminium hydride to obtain a crude compound which is subjected to silica gel column chromatography and eluted with ethyl acetate-hexane-methanol to afford emtricitabine.
European Patent No 1180104 discloses a process for separation of cis and trans isomers of 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane by simulated moving bed chromatography.
European Patent No 984013 discloses a process for preparation of emtricitabine by exposing the racemic mixture thereof to cytidine-deoxycytidine deaminase, which selectively deaminates the cytosine moiety in of the (+)-enantiomer to form a uracil moiety. The (-) enantiomer of emtricitabine is isolated by acidification. Other methods for separation of (±)- emtiricitabine reported therein are HPLC and chromatography using chiral column.
European Patent No 757684 (herein after the '684 patent) discloses a process for preparation of lamivudine by basification of salicylate salt thereof with triethylamine. The salicylate salt is prepared by reduction of L-menthyl ester of lamivudine followed by treatment of the reaction product with salicylic acid.
PCT Application No WO 04/085432 (herein after the '432 PCT) discloses a process for preparation of emtricitabine by salification of L-menthyl 1,3-oxathiolan-5S-(5-fluorocytosin-1-yl)-2R-carboxylate with oxalic acid. The L-menthyl group in the oxalate salt so formed is reduced with sodium borohydride. The crude product is recrystallized from methanol and isopropyl acetate. According to the disclosure of the '432 PCT the process reported in the '684 patent does not work out in case of emtricitabine as L-menthyl ester of emtricitabine is a gummy mass which is difficult to isolate.
The prior art methods for preparation of emtricitabine and analogues thereof employ tedious and time consuming chromatographic purification techniques or enzymatic resolution to separate the racemic mixture in order to obtain the (-)-enantiomer (or the p-anomer) of emtricitabine. The present inventors have found that by following the process reported in the '432 PCT there is formation of about 0.3% or more of emtricitabine sulfoxide impurity of Formula II (herein after emtricitabine sulfoxide),
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In order to develop accurate pharmaceutical formulations the active ingredient must be in a state of high purity. It is thus desirable to develop alternate process for purification of emtricitabine, which substantially eliminates emtricitabine sulfoxide impurity and also does not require chromatographic purification or enzymatic resolution.
The present inventors have developed a novel process for purification of emtricitabine which renders it substantially free of the sulfoxide impurity. In the present process the formation of emtricitabine sulfoxide impurity is greatly reduced due to the presence of an antioxidant. The present process is simple, cost effective and favourable on an industrial scale to obtain emtricitabine of enhanced purity with as low sulfoxide impurity as possible without employing time consuming chromatographic techniques or requiring enzymatic resolution.
The term "substantially free" as used herein signifies sulfoxide impurity content of less than 0.1%.
Figure 1 depicts the HPLC chromatogram of emtricitabine purified according to the present process.
A first aspect of the present invention provides emtricitabine of Formula la substantially free of emtricitabine sulfoxide.

(Formula Removed)
A second aspect of the present invention provides a process for the preparation of emtricitabine of Formula la substantially free of emtricitabine sulfoxide,
(Formula Removed)
wherein the said process comprises of,
(a) heating a mixture of emtricitabine of Formula la, an organic solvent and an antioxidant,
(b) isolating emtricitabine of Formula la substantially free of emtricitabine sulfoxide from the reaction mass thereof.
Emtricitabine of Formula la can be prepared by methods known in the art. A mixture of the emtricitabine of Formula la, an organic solvent and an antioxidant is refluxed for about 0.5 hour. To this mixture activated carbon is added and the resultant mixture is again refluxed for about 0.5-1.0 hour. The resultant solution is filtered and concentrated at about 40-60°C. The residue is taken up in ethanol and heated to reflux. The resultant solution is cooled to about 20-30°C and the solid so formed is suitably worked up to obtain emtricitabine of Formula la substantially free of emtricitabine sulfoxide.
The organic solvent is selected from the group comprising of C1.4 alkanol or mixtures thereof. Preferably the organic solvent is selected from ethanol and isopropanol or mixtures thereof.
The antioxidant is selected from the group comprising of triarylphosphine, butylated hydroxyl anisole, butylated hydroxyl toluene and sodium metabisulphite and the like. Preferably the antioxidant is selected from the group comprising of triarylphosphine and butylated hydroxyanisole. Preferably the triarylphosphine is triphenylphosphine.
EXAMPLE 1 PREPARATION OF EMTRICITABINE SUBSTANTIALLY FREE OF EMTRICITABINE SULFOXIDE
Emtricitabine (50 g), isopropanol (600 ml) and triphenyl phosphine (1.5 g) were charged at room temperature and heated at 80°C for 15 minutes. To the resultant solution activated carbon (5 g) was added and the mixture was heated at 80°C for 30 minutes. The solution was filtered while hot and washed with isopropanol (100 ml). The filtrate was concentrated under vacuum at 45 to 50°C. The residue was taken up in hot absolute ethanol (250 ml) (heated to about 70°C) and refluxed for 10 minutes. The resultant solution was slowly cooled to 25°C and stirred for 2 hours. The solid so formed was filtered, washed with ethyl acetate (100 ml) at room temperature and dried under vacuum at 40-45°C to afford the title compound. Yield : 40 g (80%) Purity : 99.8% (by HPLC)
EXAMPLE 2 PREPARATION OF EMTRICITABINE SUBSTANTIALLY FREE OF EMTRICITABINE SULFOXIDE
Emtricitabine (10 g), ethanol (100 ml) and butylated hydroxyl anisole (0.4 g) were charged at room temperature and heated to 75°C and stirred at 75-80°C for 15 minutes. To the resultant solution activated carbon (0.5 g) was added and the resultant mixture was stirred for 20 minutes at 75-80°C, filtered through hyflo bed while hot and washed with hot absolute ethanol (20 ml) (heated to about 70°C). The filtrate was concentrated under vacuum at 40 to 45°C to a resultant volume of 40-50 ml. The concentrated solution was slowly cooled to 25-30°C in 1 hour and stirred at the same temperature for 2 hours. This solution was again cooled to 5°C and stirred at the 5-10°C for 2 hours. The solid so formed was filtered, washed with prechilled (~5°C) absolute ethanol (10 ml) and dried under vacuum at 40-45°C to afford the title compound. Yield : 7.7 g (77%) Purity : 99.8% (by HPLC)
While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

WE CLAIM :
1. Emtricitabine of Formula la substantially free of emtricitabine sulfoxide.
(Formula Removed)
2. A process for the preparation of emtricitabine of Formula la substantially free of emtricitabine sulfoxide,
(Formula Removed)
wherein the said process comprises of,
(a) heating a mixture of emtricitabine of Formula la, an organic solvent and an antioxidant,
(b) isolating emtricitabine of Formula la substantially free of emtricitabine sulfoxide from the reaction mass thereof.

3. A process according to claim 2 wherein the antioxidant is selected from the group comprising of triarylphosphine, butylated hydroxyl anisole, butylated hydroxyl toluene and sodium metabisulphite.
4. A process according to claim 3 wherein the antioxidant is triarylphosphine.
5. A process according to claim 4 wherein the antioxidant is triphenylphosphine.
6. A process according to claim 3 wherein the antioxidant is butylated hydroxyanisole.
7. A process according to claim 2 wherein the organic solvent is selected from the group comprising of C1-4 alkanol or mixtures thereof.
8. A process according to claim 7 wherein the organic solvent is selected from ethanol and isopropanol or mixtures thereof.
9. A process according to claim 8 wherein the organic solvent is isopropanol.
10. A process according to claim 2 wherein the mixture is refluxed.