Title of Invention

''AN IMPROVED PROCESS FOR THE ENZYMATIC PREPARATION OF (-)-CIS- LAMIVUDINE"

Abstract

The present invention relates to an improved process for the preparation of cis (-) lamivudine. The process of this invention comprises the step of enantio- and stereoselective hydrolysis of a racemic mixture of 1,3-oxathiolane nucleoiside analogues of formula 2 wherein RCO is a substituted carbonyl derivative where the non-carbonyl moiety R of the ester is selected from straight or branch chained alkyl (e.g. methyl, ethyl, n-propyl, n-butyl), alkoxyalkyl (e.g. phenyl optionally substituted by hydroxy, or halogen), and aminoacid esters (e.g. L-valyl, or L-isoleucyl) with enzyme pencillin G acylase at a temperature in the range of 15-40 deg. C for a period of 2-24 hrs, recovering the product by known methods.

Full Text

The present invention relates to an improved process for the preparation of cis (-) lamivudine. The present invention particularly relates to an improved process for the enzymatic preparation of optically pure (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-(1H)-pyrimidin-2-one (Lamivudine) 1. The process of this invention comprises the step of enantio- and stereoselective hydrolysis of a racemic mixture of 1,3-oxathiolane nucleoside analogues of formula 2 wherein RCO is a substituted carbonyl derivative where the non-caibonyl moiety R of the ester is selected from straight or branch chained alkyl (e.g. methyl, ethyl, n-propyl, n-butyl), alkoxyalkyl (e.g. methoxymethyl), arylalkyl (e.g. benzyl), aryloxylalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by hydroxy, or halogen), and aminoacid esters (e.g. L-valyl, or L-isoleucyl). with enzyme penicillin G acylase (EC. 3.5.1.11). (FormulaRemoved) Resolution of racemic 1,3-oxathiolane nucleoside analogues of formula 2 is an industrially important process since the optically pure forms e.g. of (-)-c/s-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one (Lamivudine) is an effective antiviral agent against HIV, HBV, and other viruses replicating in a similar manner. The synthetic routes to racemic 1,3-oxathiolane nucleoside analogues of formula 2 usually provide a mixture of cis- and trans- diastereomers. Reference may be made to the work of the Glaxo Group Research Limited (Int.pat. WO 91/17159; Eur.Pat. EP 0513 917 A1), of Biochem Pharma Inc. Canada (EP 0 515 157 A1), of Number and coworkers (Humber, D; Jones M.F.; Payne.J.J.; Ramsay, M.V.J. Tetrahedron Lett. 1992, 33, 4624), Jeong.L.S.; Alves, A.J.; Carrigan.S.W.; Kim H.O.; Beach.W.; Chu, C.K. Tetrahedron Lett. 1992, 33, 595.) and of Beach and coworkers (Beach, J.W. et al J.Org.Chem. 1992, 57, 2217). The c/s and trans forms are separated by repeated crystallizations. One of the hitherto known enzymatic processes for the preparation of (-)-c/s-4-amino-1 -(2-hydroxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one 1 is based on destructive resolution of racemic cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one. Reference may be made to the work of Mahmodian and coworkers (Mahmoudian.M.; Bains.B.S.; Drake, C.S.; Hale.R.S.; Jones,P.; Piercey.J.E.; Montgomey, D.S.; Purvis,I.J.; Storer, R. Et al. Enzyme Microb. Technol. 1993, 15 , 749) where the unwanted (+)-c/s-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one is destroyed by reaction with enzyme cytidine deaminase. The residual c/s-(-)-1,3-oxathiolane nucleoside analogue of required stereochemistry is then isolated from the reaction mixture. The major drawback of this process is that the enzyme is not commercially available and has to be specially produced by fermentation. Another disadvantage of the process is that the unwanted isomer is completely destroyed and can not be racemized and recycled. This leads to a net loss of expensive raw materials. Other enzymatic routes to optically pure (-)-c/s-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one (Lamivudine) are based on lipase mediated resolution of an optically pure intermediate such as an oxathiolane intermediate (Cousins,R.P.C.; Mahmoudin.M.; Youds.P.M. Tetrahedron: Asymmetry 1995, 6, 393) or an a-acetoxy sulphide which forms the required oxathiolane nucleus, which is then converted to target nucleoside in 4 steps (Milton,J.; Brand,S; Jones.M.F.; Rayner.C.M. Tetrahedron Lett. 1995, 36, 6961). These methods are indirect and the coupling of the intermediate oxathiolane is accompanied by a loss of optical purity which is not acceptable. A methodology to prepare optically pure (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrirnidine-2-one by resolution of racemic mixture of 1,3-oxathiolane nucleoside analogues of formula 2 by using lipases from different sources has been reported by Liotta and co-workers (Liotta; D.C., McDonough.G. A ; Schinazi; R. F. , Decatur, G. A ., Choi, W. B.; US patent: US5892025, 1998 and earlier patents cited therein). However this process has the disadvantage that the enzyme does not discriminate between the c/s and trans isomers and a pure c/s-isomef has to copolymer of acrylamide and N,N' - methylen-bis(acrylamide). In another embodiment of the invention the separation of (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one 1 and 1,3-oxathiolane nucleoside analogues of formula 2 from the reaction mixture may be effected by freeze drying and extraction of the freeze dried residue with organic solvent such as chloroform, ethanol or methanol; evaporation of the solvent and column chromatography of the residue using an eluent comprising of a mixture of chloroform and methanol in the range 5-30%. In yet another embodiment of the invention the separation of (-)-c/s-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one 1 and 1,3-oxathiolane nucleoside analogues of formula 2 from the reaction mixture may be effected by using adsorbant polymeric resins such as polystyrene (Amberlite XAD-4, XAD-7, XAD-16), polyacrylic esters (Amberlite XAD-7) and desorption of the material by extraction with methanol and evaporation of the solvent. Further purification if necessary may be done by and column chromatography of the residue using an eluent comprising of a mixture of chloroform and methanol in the range 5-30%. The invention is described in the following example by way of illustration only and should not be construed to limit the scope of the invention. The example what is provided describes the penicillin G acyalse enzyme mediated enantio- and stereoselective hydrolysis of a racemic mixture of 1,3-oxathiolane nucleoside analogues of formula 2 and an analytical method to determine the course of reaction based on HPLC analysis. Example 1 : (-)-cis-4-amino-1 -(2-hvdroxymethyl-1.3-oxathione-5-yl)-(1 H)-pyrimidine-2-one (Lamiyudine) The (±)-4-amino-1-(2-benzoyloxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one 2 (300 mg) was dissolved in water (600 ml) by warming, cooled to 30° C and its pH was adjusted to 7.5 with 2N ammonia solution. The immobilized enzyme Penicillin G acylase (3 g, 500 units; immobilized on copolymer of methylacrylamide , N,N' -methylen-bis(acrylamide) and a monomer carrying oxirane groups, Eupergit C) was added and the reaction mixture was stirred at 80 rpm. The reaction was followed by copoiymer or acrylamide and N,N' - methylen-bis(acrylamide). In another embodiment of the invention the separation of (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one 1 and 1,3-oxathiolane nucleoside analogues of formula 2 from the reaction mixture may be effected by freeze drying and extraction of the freeze dried residue with organic solvent such as chloroform, ethanol or methanol; evaporation of the solvent and column chromatography of the residue using an eluent comprising of a mixture of chloroform and methanol in the range 5-30%. In yet another embodiment of the invention the separation of (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one 1 and 1,3-oxathiolane nucleoside analogues of formula 2 from the reaction mixture may be effected by using adsorbant polymeric resins such as polystyrene (Amberlite XAD-4, XAD-7, XAD-16), polyacrylic esters (Amberlite XAD-7) and desorption of the material by extraction with methanol and evaporation of the solvent. Further purification if necessary may be done by and column chromatography of the residue using an eluent comprising of a mixture of chloroform and methanol in the range 5-30%. This invention relates to an improved process for the enzymatic preparation of optically pure (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one (Lamivudine) of formula 1 of the drawing accompanying the specification which comprises the step of enantio and stereoselective hydrolysis a racemic mixture of 1,3-oxathiolane nucleoside analogues of formula 2 wherein R is a substituted carbonyl derivative where the nori-carbonyl moiety R of the ester is selected from straight or branch chained alkyl (e.g. methyl, ethyl, n-propyl, n-butyl), alkoxyalkyl (e.g. methoxymethyl), arylalkyl (e.g. benzyl), aryloxylalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen), and aminoacid esters(e.g. L-valyl, or L-isoleucyl) in presence of enzyme penicillin G acylase (EC.3.5.1.11)from Escherichia coli in an aqueous solution at pH in the range of 5-8.5, at a temperature in the range of 15-40° C, for a period in the range of 2-24 h, monitoring the extent of hydrolysis by HPLC up to 20 -25 % hydrolysis ; recovering the enzyme by removal of the reactants; and recovering the optically pure (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one (Lamivudine) 1 by known methods. The invention is described in the following example by way of illustration only 'and should not be construed to limit the scope of the invention. The example what is provided describes the penicillin G acyalse enzyme mediated enantio- and stereoselective hydrolysis of a racemic mixture of 1,3-oxathiolane nucleoside analogues of formula 2 and an analytical method to determine the course of reaction based on HPLC analysis. Example 1 : (-)-c/'s-4-amino-1 -(2-hydroxymethyl-1 -3-oxathione-5-yl)-(1 H)-pyrimidine-2-one (Lamivudine) The (±)-4-amino-1 -(2-benzoyloxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one 2 (300 mg) was dissolved in water (600 ml) by warming, cooled to 30° C and its pH was adjusted to 7.5 with 2N ammonia solution. The immobilized enzyme Penicillin G acylase (3 g, 500 units; immobilized on copolymer of methylacrylamide , N,N' -methylen-bis(acrylamide) and a monomer carrying oxirane groups, Eupergit C) was added and the reaction mixture was stirred at 80 rpm. The reaction was followed by HPLC which showed the disappearance of the (±)-cis-4-amino-1-(2-benzoyloxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one and formation of Lamivudine. The enzymatic reaction was 25% complete in 2 h and virtually stopped. The enzyme was then filtered, the reaction mixture was stirred with polyacrylic ester (Amberlite XAD-7) resin (10g, Fluka AG, Switzerland) to adsorb the unreacted material for 2 h. Complete adsorption of the (±)-cis-4-amino-1 -(2-benzoyloxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one was checked by HPLC analysis. The resin was then filtered, pH of the filtrate was adjusted to 10 with ammonia solution and evaporated to dryness by freeze drying. The residue was chromatographed over silica gel with 5% methanol in chloroform. The first fraction of benzoic acid was discarded. The fraction eluting with rf 0.52 was collected as a gummy material (33 mg, 68 %) which was crystallized from boiling ethanol to obtain cis-(-)-Lamivudine. M.p. 128-130° C; [α]D25 -120°( c 1, MeOH). Determination of course of reaction: The disappearance of (±)-cis-4-amino-1-(2-benzoyloxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one during enzymatic hydrolysis was monitored by HPLC Column : C-8, 5x250 mm, Chrompack, The Netherlands; Detection wavelength 282 nm.; solvent 40% acetonitrile-water with 0.16% perchloric acid; flow rate 1 ml/min; retention times: cis-7.3 and trans-6.8; product (Lamivudine) 4.1 min. Example 2 5-(4-amino-2-oxo-1,2-dihydro-1 -pyrimidinyl)-1,3-oxathiolan-2-ylmethyl 2-phenylacetate 2b (700 mg) was dissolved in tris(hydroxymethyl)aminomethane buffer (0.05 M, 1L), its pH was adjusted to 5.5 with 1N hydrochloric acid solution and immobilized enzyme Penicillin G acylase (3 g, 500 units) was added. The reaction mixture was stirred at 80 rpm for 24 h at 25° C. The enzyme was then filtered, the reaction mixture stirred with polyacrylic ester (XAD-7) resin (10g, Fluka AG, Switzerland) to adsorb the unreacted 5-(4-amino-2-oxo-1,2-dihydro-1 -pyrimidinyl)-1,3-oxathiolan-2-ylmethyl 2-phenylacetate for 10 h. The resin was then filtered, pH of the filtrate was adjusted to 10 with ammonia solution and evaporated to dryness by freeze drying. The residue was chromatographed over silica gel with 5% methanol in chloroform. The fraction eluting with rf 0.52 was collected as a gummy material (33 mg, 68 %) which was crystallized from boiling ethanol to obtain cis-(-)-Lamivudine. M.p. 128-130° C; [a]D 25 - 121° ( c 1, MeOH). Example 3 : 5-(4-amino-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxathiolan-2-ylmethyl acetate 2 b (542 mg) was dissolved in water (600 ml) by warming, cooled to 20° C and its pH was adjusted to 8.5 with 2N ammonia solution. Penicillin G acylase (2 ml, 400 units; suppliers Fluka AG, Switzerland) was added and the reaction mixture was stirred at 100 rpm at 20° C for 12 h. The reaction mixture was freeze dried and the residue was chromatographed over silica gel with 5% methanol in chloroform and the fraction eluting with rf 0.53 was collected (43 mg, 80 %) which was crystallized from boiling ethanol to obtain cis-(-)-Lamivudine. M.p. 122-125° C; [α]D25 -112° ( c 1, MeOH). Example 4: 5-(4-amino-2-oxo-1,2-dihydro-1 -pyrimidinyl)-1,3-oxathiolan-2-ylmethyl 2-phenylacetate 2b (700 mg) was dissolved in water, pH was adjusted to 7.5 with 2N ammonia and Penicillin G acylase immobilized in copolymer of acrylamide and N,N'-methylen-bis(acrylamide) (3 g, 500 units) was added. The reaction mixture was stirred at 80 rpm for 24 h at 15° C. The enzyme was then filtered, the reaction mixture stirred with polyacrylic ester (XAD-7 ) resin (10g, Fluka AG, Switzerland) to adsorb the unreacted 5-(4-amino-2-oxo-1,2-dihydro-1 -pyrimidinyl)-1,3-oxathiolan-2-ylmethyl 2-phenylacetate for 10 h. The resin was then filtered, the filtrate was then treated with Amberlite polystyrene resin XAD-16 (10 g) for 6 h to adsorb the product Lamivudine. The resin was washed with distilled water and then stirred with methanol (200 ml) at 40° C for 1 h. The methanolic solution was evaporated to dryness and the residue was crystallized from boiling ethanol to obtain cis-(-)-Lamivudine. M.p. 128-130° C; [α]D 25 - 121° ( c 1, MeOH). Advantages: 1. Present invention utilizes the enzyme Penicillin G acylase, which is a cheap and commercially available enzyme and has not been utilized before for the preparation of optically pure Lamivudine. 2. The enzyme can be used in immobilized form and recycled several times, thus reducing the cost of the enzyme. 3. The enzyme specifically hydrolyzes only the cis-derivative and it is not necessary to start with a pure cis-diastereomer. Thus a diastereomeric mixture of 1,3-oxathiolane nucleoside analogues of formula 2 can be used for the enzymatic reaction. 4. The unreacted 1,3-oxathiolane nucleoside analogues of formula 2 can be easily removed from reaction mixture by adsorption to polyacrylic ester (Amberlite XAD-7) resin and can be recovered from the resin by simple stirring with methanol in quantitative yields. The resin can again be reused. 5. The product (-)-cis-4-amino-1-(2-hydroxymethyl-1I3-oxathione-5-yl)-(1H)-pyrimidine- 2-one (Lamivudine) in the reaction mixture can be easily removed from reaction mixture by adsorption to polystyrene resin (Amberlite XAD-16) and can be recovered from the resin by simple stirring with methanol in quantitative yields. The resin can again be reused. 6. The process produces very little effluent and is environmentally friendly. We claim: 1. An improved process for the enzymatic preparation of optically pure (-)-cis-4-amino- 1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1 H)-pyrimidine-2-one (Lamivudine) of formula 1 of the drawing accompanying the specification which comprises; hydrolysing a racemic mixture of 1,3-oxathiolane nucleoside analogues of formula 2 wherein R is a substituted carbonyl derivative where the non-carbonyl moiety R of the ester is selected from straight or branch chained alkyl, alkoxyalkyl, arylalkyl, aryloxylalkyl, aryl, and aminoacid esters in presence of enzyme penicillin G acylase in an aqueous solution at pH In the range of 5-8.5 at a temperature In the range of 15-40° C, for a period in the range of 2-24 h, recovering the optically pure (-)-c/s-4-amino-1-(2- hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidlne-2-one (Lamivudine) by known methods. 2. An improved process as claimed in claim 1 wherein the reaction is carried out with enzyme dissolved in aqueous buffer such as tris (hydroxymethyl) aminomethane or alternatively the enzyme may be immobilized on a solid support such as copolymer of methylacrylamide , N,N' - methylen-bis(acrylamide) and a monomer carrying oxirane groups (Eupergit C), copolymer of acrylamide and N,N' - methylen-bis(acrylamide). 3 An Improved process as claimed In claims 1-2 wherein the unreacted 1,3-oxathiolane nucleoside analogue is removed from reaction mixture by treatment with polyacrylic ester ( XAD-7) resin and is recovered by extraction of the resin with methanol. 4. An improved process as claimed in claims 1-3 wherein the reaction product (-)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathione-5-yl)-(1H)-pyrimidine-2-one is recovered from reaction mixture by treatment with polystyrene (XAD-16) resin and is recovered by extraction of the resin with methanol. 5. An improved process for the enzymatic preparation of optically pure (-)-cis-4-amino-1-(2-hydroxymethyl-,3-oxathione-5-yl)-(1 H)-pyrimidlne-2-one substantially as described with reference to the example and drawing accompanying this specification.

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268-del-2000-abstract.pdf

268-del-2000-claims.pdf

268-del-2000-complete specification (granted).pdf

268-del-2000-correspondence-others.pdf

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268-del-2000-description (complete).pdf

268-del-2000-drawings.pdf

268-del-2000-form-1.pdf

268-del-2000-form-19.pdf

268-del-2000-form-2.pdf