The present invention relates to crystalline L-menthyl (2R, 5S)-5-(4-amino-5-fluoro-2-oxo-2H-pyrimidin-1-yl)[1,3]oxathiolan-2-carboxylate and process for preparation thereof.
L-menthyl (2R,5S)-5-(4-amino-5-fluoro-2-oxo-2H-pyrimidin-1-yl)[1,3]oxathiolane-2-
carboxylate is a useful intermediate in the synthesis of the well known antiviral drug emtricitabine.
Emtricitabine of formula I 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,
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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 human immunodeficiency virus (HIV) replication by inhibiting reverse transcription. The said compounds represent an important development in the arsenal of drugs against HIV, particularly HIV-1.
Emtricitabine and its 5-defluorocytosine analogue also known as Lamivudine or 3TC 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 has two chiral centres and therefore four stereoisomers exist namely (2R, 5S) and (2S, 5R) which are the cis isomers and (2R, 5R) and (2S, 5S) which are the trans isomers. The cis (2R, 5S) isomer is found to have profound antiviral activity while the other isomers such as the cis (2S, 5R) and the trans isomers (2S, 5S) and (2R, 5R)
have lower therapeutic activity and are found to be of reduced interest in therapeutics. The synthetic approach adopted for emtricitabine involves introduction of the pyrimidine base in the 1,3-oxathiolane ring. Several stereoselective processes employing chiral auxiliaries have been developed to selectively obtain the cis (2R, 5S) isomer.
European Patent no 515157 (herein after the '157 patent) discloses a process for the preparation of emtricitabine which involves condensation 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.
PCT Application No WO 04/085432 (herein after the '432 PCT) discloses a process for the preparation of emtricitabine by isolation of the intermediate L-menthyl 1,3-oxathiolan-5S-(5-fluorocytosin-1-yl)-2R-carboxylate as the oxalate salt. The L-menthyl group in the oxalate salt is reduced with sodium borohydride to obtain emtricitabine in the crude form which is recrystallized from methanol and isopropyl acetate.
The '432 PCT discloses that L-menthyl ester of emtricitabine is a gel that is inseparable from the mother liquors by simple filtration and has to be isolated by chromatographic purification. As per the disclosure of the '432 PCT, the '157 patent does not report the preparation of menthyl emtricitabine intermediate with desired stereochemistry. The '432 PCT further discloses that in order to obtain emtricitabine which is the cis (2R, 5S) isomer, the corresponding L-menthyl ester having configuration (1'R, 2'S, 5'R) on the menthyl ring and (2R, 5S) configuration on the oxathiolane ring must be employed. Example 18 of the '157 patent refers to the L-menthyl derivative having inverted configuration on the oxathiolane ring whereas Example 19 refers to the derivative having inverted configuration on the menthyl ring.
The present inventors have surprisingly found that the L-menthyl ester of emtricitabine of Formula II (herein after "crystalline L-methyl emtricitabine"), can be isolated in crystalline form by simple crystallization from the reaction mass thereof. The present process does not employ tedious and time-consuming chromatographic purification or salt formation.
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Figure 1: Powder X-ray diffraction pattern of crystalline L-menthyl emtricitabine Figure 2: HPLC chromatogram of crystalline L-menthyl emtricitabine
A first aspect of the present invention provides crystalline L-menthyl emtricitabine of Formula II,
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A second aspect of the present invention provides crystalline L-menthyl emtricitabine which exhibits characteristic powder X-ray diffraction pattern as depicted in Figure 1. Crystalline L-menthyl emtricitabine according to the present invention exhibits a powder X-ray diffraction pattern showing characterisctic 20 values (°) at : 4.38, 7.36, 10.54, 12.00, 12.72, 13.84, 14.44, 14.72, 15.08, 15.74, 16.84, 17.34, 17.60, 18.30, 18.48, 19.44, 19.80, 20.98, 21.78, 22.08, 22.64, 23.12, 24.04, 24.38, 24.88, 25.42, 25.80, 26.06, 26.80, 27.30, 27.88, 28.26, 28.48, 28.82, 29.28, 29.54, 29.90, 30.16, 30.46, 31.12, 31.42, 31.76, 32.30, 32.68, 33.04, 33.32, 33.90, 34.96, 35.66, 36.64, 37.20, 37.50, 38.00, 39.06, 39.46, 39.78.
A third aspect of the present invention provides pure crystalline L-menthyl emtricitabine. As used herein the term "pure" refers to a purity of 98% or more, more preferably to a purity of 99% or more and most preferably to a purity of 99.9% or more.
A fourth aspect of the present invention provides a process for the preparation of crystalline L-menthyl emtricitabine of Formula II,
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wherein the said process comprises of,
adding a first organic solvent to the reaction mass or concentrate or mother liquor comprising L-menthyl emtricitabine,
adding a second organic solvent,
isolating crystalline L-menthyl emtricitabine from the reaction mass thereof.
Crystalline L-menthyl emtricitabine according to the present invention can be isolated directly from the reaction mass thereof. The term "reaction mass" as used herein is understood to mean the reaction mixture comprising L-menthyl emtricitabine obtained by any process or a residue obtained by evaporation of solvent(s) from the said reaction mixture.
A suitable first organic solvent is added to a reaction mass or concentrate or mother liquor comprising L-menthyl emtricitabine. The mixture is refluxed and a second organic solvent is added and the resultant mixture is cooled to about 20°C to about 30°C. The solid so obtained is filtered and dried under vacuum at about 30°C to about 40°C to obtain crystalline L-menthyl emtricitabine.
The first organic solvent can be selected from the group comprising of C3-8 esters such as for example ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate and the like. A mixture of two or more such first organic solvents can also be effectively employed. The second organic solvent can be selected from the group comprising of C-M alkanols such as for example methanol, ethanol, n-propanol, isopropanol and the like. A mixture of two or more such second organic solvents can also be effectively employed.
A fifth aspect of the present invention provides a process for the preparation of crystalline L-menthyl emtricitabine of Formula II,
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wherein the said process comprises of,
a) reacting 5-fluorocytosine derivative of Formula III,
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wherein P is hydrogen or protecting group, with a 1,3-oxathiolane derivative of Formula IV, wherein L is a leaving group,
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to get a compound of Formula V, wherein P is hydrogen or protecting group,
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rotecting the protecting group in the compound of Formula V,
adding a first organic solvent to the reaction mixture obtained in step a) or b),
adding a second organic solvent,
isolating crystalline L-menthyl emtricitabine from the reaction mass thereof.
Compunds of Formula III and IV can be prepared by processes known in the art. A solution of 5-fluorocytosine derivative of Formula III wherein P is hydrogen or protecting group in an organic solvent is reacted with 1,3-oxathiolane derivative of Formula IV wherein L is a leaving group. The reaction mixture is heated to reflux for about 15-20 hours and cooled to about 25°C to about 30°C. To the cooled solution deionized water is added at about 25°C to about 30°C and the mixture stirred for about 0.5-1.0 hours. The organic layer is separated and washed with concentration hydrochloric acid and deionized water and then with aqueous sodium chloride solution. The organic layer is recovered under vacuum. The residue so obtained is treated with a first organic solvent followed by a second organic solvent essentially by the process reported in the fourth aspect of the present invention to obtain crystalline L-menthyl emtricitabine. Suitable protecting groups as well as the method of removal the protecting groups are known to a person of ordinary skills in the art. Crystalline L-menthyl emtricitabine obtained according to the process of the present invention can be converted to emtricitabine by processes known in the art, for example the process reported in European Patent No. 515157.
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.
EXAMPLE 1
Preparation of (5-Fluoro-2-trimethylsilanyloxy-pyrimidin-4-yl)-trimethyl silanyl-amine
5-fluorocytosine (100 g), hexamethyldisilazane (400 ml) and ammonium sulphate (5 g) were charged at 25°C. The mixture was refluxed at 125°C to 135°C for 4 hours when a clear solution was obtained. The solution was cooled 70°C to 80°C and excess hexamethyldisilazne was recovered under vacuum at a pressure of 650-700 mm Hg and 70°C -80°C to get a residue. The residue was cooled to 25°C to 30°C and methylene chloride (1000 ml) was added to it. To this mixture triethylamine (140 ml) was added over 0.5 hours at 20°C to 25°C and the resultant mixture stirred for 0.5 hours at 20°C to 25°C and used as such.
EXAMPLE 2
Preparation of 5S-Chloro-[1,3]oxathiolane-2R-carboxylic acid 2-(1'R, 2'S, 5'R)-isopropyl-5-methyl-cyclohexyl ester
Methylene chloride (3000 ml), 5R-Hydroxy-[1,3]oxathiolane-2S-carboxylic acid 2-isopropyl-5-methyl-cyclohexyl ester (300 g), methanesulphonic acid (1 ml) and dimethylformamide (85 ml) were charged at 25°C and the mixture stirred for 0.5 hours at 25°C. The mixture was cooled to 5°C to 10°C and thionyl chloride (80 ml) was added at 5°C to 10°C over 0.5 hours. The reaction mixture was stirred 10°C to 15°C for 2 hours. Excess methylene chloride was recovered under vacuum at a pressure of 650-700 mm Hg and 40°C to 45°C to get a residue. To the residue methylene chloride (600 ml) was added and the excess methylene chloride was recovered under vacuum at a pressure of 650-700 mm Hg and 40°C to 45°C to get a residue. To the residue methylene chloride (600 ml) was added and the mixture used as such.
EXAMPLE 3
Preparation of crystalline L-menthyl emtricitabine
The mixture obtained in Example 1 was refluxed at 42°C to 45°C. The mixture obtained in Example 2 was slowly charged over 1 hour at refluxing temperature. The combined reaction mixture was refluxed for 18 hours at 42°C to 45°C and the reaction was monitored by high performance liquid chromatography. The mixture was cooled to 25°C
and deionized water (500 ml) was added at 25°C. The resultant mixture was stirred for 0.5 hours at 25°C. The organic layer was separated and washed twice with a concentrated hydrochloric acid solution (1 ml) and deionized water (500 ml) and then with aqueous sodium chloride solution (10%, 500 ml) at 25°C. The organic layer was recovered under vacuum at a pressure of 650-700 mm at 40°C to 45°C to get a residue. To the residue ethyl acetate (1000 ml) was charged at 25°C and the mixture refluxed at 78°C. To the mixture methanol (200 ml) was charged slowly and the resultant mixture slowly cooled to 25°C over 2 hours. The product so obtained was filtered and dried under vacuum at 30°C to 35°C to obtain the title compound. Yield: 250 g XRD as per Figure 1 Purity: 100%byHPLC

WE CLAIM:
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Crystalline L-menthylemtricitabine of Formula II,
Crystalline L-menthyl emtricitabine exhibiting characteristic powder X-ray diffraction pattern as depicted in Figure 1.
Crystalline L-menthyl emtricitabine exhibiting a powder X-ray diffraction pattern with characterisctic 20 values (°) at : 4.38, 7.36, 10.54, 12.00, 12.72, 13.84, 14.44, 14.72, 15.08, 15.74, 16.84, 17.34, 17.60, 18.30, 18.48, 19.44, 19.80, 20.98, 21.78, 22.08, 22.64, 23.12, 24.04, 24.38, 24.88, 25.42, 25.80, 26.06, 26.80, 27.30, 27.88, 28.26, 28.48, 28.82, 29.28, 29.54, 29.90, 30.16, 30.46, 31.12, 31.42, 31.76, 32.30, 32.68, 33.04, 33.32, 33.90, 34.96, 35.66, 36.64, 37.20, 37.50, 38.00, 39.06, 39.46, 39.78.
Pure crystalline L-menthyl emtricitabine.
Pure crystalline L-menthyl emtricitabine according to-claim 4 having a purity of 98% or more.
Pure crystalline L-menthyl emtricitabine according to claim 4 having a purity of 99.9% or more.
7. A process for the preparation of crystalline L-menthyl emtricitabine of Formula II,
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wherein the said process comprises of,
adding a first organic solvent to the reaction mass or concentrate or mother liquor comprising L-menthyl emtricitabine,
adding a second organic solvent,
isolating crystalline L-menthyl emtricitabine from the reaction mass thereof.
8. A process for the preparation of crystalline L-menthyl emtricitabine of Formula II,
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wherein the said process comprises of, a) reacting 5-fluorocytosine derivative of Formula III,
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wherein P is hydrogen or protecting group, with a 1,3-oxathiolane derivative of Formula IV, wherein L is a leaving group,
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to get a compound of Formula V, wherein P is hydrogen or protecting group,
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if required, deprotecting the protecting group in the compound of Formula V,
adding a first organic solvent to the reaction mixture obtained in step a) or b),
adding a second organic solvent,
isolating crystalline L-menthyl emtricitabine from the reaction mass thereof.
9. A process according to claim 7 or 8 wherein the first organic solvent is selected from
the group comprising of C3-8 esters or mixtures thereof.
10. A process according to claim 7 or 8 wherein the second organic solvent is selected
from the group comprising of C1-4 alkanols or mixtures thereof.