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(1 H)-pyrimidinone whereas lamivudine or 3TC is the 5-defluoro analogue of Formula Ib,
(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-O-protectedhydroxymethyl-2-acetoxy-1,3-oxathiolane with 5-fluorocytosine
followed by deprotection of the 0-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 trimethyisilyl 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),
(Formula Removed) FORMULA II
Our co-pending Indian Patent Application No. 3195/DEL/2005 discloses a process for the purification of emtricitabine by heating a mixture comprising emtricitabine, an organic solvent and antioxidant such as triphenyl phosphine or butylated hydroxyl anisole. Presence of antioxidants reduces the content of emtricitabine sulfoxide by conversion of emtricitabine sulfoxide to emtricitabine and also controls further oxidation of emtricitabine to its sulfoxide.
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 addressed this problem and a solution in the co-pending patent application No. 3195/DEL/2005.
The present inventors have observed that organic solvents like isopropanol used during purification of emtricitabine are contaminated with peroxides which lead to formation of emtricitabine sulfoxide as an undesired impurity. The present inventors have developed a novel process for purification of emtricitabine which renders it substantially free of the sulfoxide impurity which involves pretreatment of the peroxide containing organic solvent with an agent which destroys the peroxides. Use of pretreated organic solvent for purification of emtricitabine enables preparation of emtricitabine substantially devoid of the sulfoxide impurity. 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 and without employing time consuming chromatographic techniques or requiring enzymatic resolution.
The term "peroxide containing organic solvent" as used herein signifies an organic solvent which is suitable for purification of emtricitabine but is contaminated with
peroxides. The term "organic solvent" as used herein signifies an organic solvent which is suitable for purification of emtricitabine and has been rendered devoid of peroxides.
The term "substantially free" as used herein signifies sulfoxide impurity content of less than 0.06%.
A first aspect of the present invention provides emtricitabine of Formula la containing 0.06% or less 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) refluxing peroxide containing organic solvent with peroxide inhibitor,
(b) separating the organic solvent from the mixture obtained in step a)
(c) treating emtricitabine of Formula la with the organic solvent obtained in step b)
(d) 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 peroxide containing organic solvent and peroxide inhibitor is refluxed for about 1-3 hours and the organic solvent is distilled out. Emtricitabine of Formula la and freshly distilled organic solvent are refluxed for about 0.5 hours. To the resultant solution, activated carbon is charged and the mixture is refluxed for about 0.5-1 hour. The resultant mixture
is filtered and the filtrate concentrated under vacuum at about 30-50°C to obtain a residue. The residue is suitably worked up to obtain emtricitabine of Formula la substantially free of emtricitabine sulfoxide.
The organic solvent suitable for purification of emtricitabine is preferably isopropanol and the peroxide inhibitor is preferably hydroquinone.
EXAMPLE 1
Step 1: Pre-treatment of Isopropyl alcohol:
Isopropyl alcohol (500 ml) and hydroquinone (15 g) were charged at room temperature. The resultant mixture was heated to 78-80°C and then refluxed for 2 hours. Isopropanol was distilled out at atmospheric pressure at 78-80°C.
Step 2: Preparation of emtricitabine substantially free of emtricitabine sulfoxide
Freshly distilled isopropanol (250 ml) obtained in step 1 and crude emtricitabine (25 g) were charged at room temperature. The temperature of the mixture was raised to 78-80°C and the mixture refluxed for 10 minutes. The resultant solution was charged with activated carbon (2.5 g) and then refluxed for 20 minutes. The resultant mixture was filtered while hot and washed with isopropanol (50 ml) preheated to 70-75°C. The filtrate and washing were combined and concentrated under vacuum at 40-45°C to get a residue. The residue was charged with absolute ethanol (125 ml) and the mixture was first heated to 78-80°C and then refluxed under stirring for 10-15 minutes to get a clear solution. The clear solution was cooled to room temperature slowly and stirred for 1 hour. The solution was further cooled to 5-10°C and stirred for 2 hours. The cooled solution was filtered and washed with ethyl acetate (50 ml) and dried under vacuum at 40-45°C to afford the title compound. Yield: 20 g (80%) Purity: 99.95% (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 containing 0.06% or less 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) refluxing peroxide containing organic solvent with peroxide inhibitor,
(b) separating the organic solvent from the mixture obtained in step a)
(c) treating emtricitabine of Formula la with the organic solvent obtained in step b)
(d) isolating emtricitabine of Formula la substantially free of emtricitabine sulfoxide from
the reaction mass thereof.

3. A process according to claim 1 step a) wherein the peroxide containing organic solvent
is isopropanol.
4. A process according to claim 1 step a) wherein the peroxide inhibitor is hydroquinone.