Title of Invention	NOVEL PROCESS FOR THE PREPARATION, ISOLATION AND PURIFICATION OF GEMCITABINE HYDROCHLORIDE AND INTERMEDIATES THEREOF
Abstract	An improved one pot process for preparing 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose-3,5-dibenzoate of Formula (I) comprising hydrolysis of (erythro:threo) alkyl-3-dioxalan-4-yl-2,2-difluoro-3-hydroxy propionate of Formula (III) where alkyl group is having C1-C4 number of Carbon atoms using a mild acid and selectively isolating 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose- 3,5-dibenzoate from the mixture of erythro & threo enantiomers using ethyl acetate, ethylene dichloride and diisopropyl ether as solvents with improved yield and purity. A process for isolating (B-anomer enriched Gemcitabine hydrochloride by converting Gemcitabine base into Gemcitabine hydrochloride followed by its purification using solvents from the series of water soluble ethers like 1,4-dioxane or Monoglyme.

Title of Invention

NOVEL PROCESS FOR THE PREPARATION, ISOLATION AND PURIFICATION OF GEMCITABINE HYDROCHLORIDE AND INTERMEDIATES THEREOF

Abstract

An improved one pot process for preparing 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose-3,5-dibenzoate of Formula (I) comprising hydrolysis of (erythro:threo) alkyl-3-dioxalan-4-yl-2,2-difluoro-3-hydroxy propionate of Formula (III) where alkyl group is having C1-C4 number of Carbon atoms using a mild acid and selectively isolating 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose- 3,5-dibenzoate from the mixture of erythro & threo enantiomers using ethyl acetate, ethylene dichloride and diisopropyl ether as solvents with improved yield and purity. A process for isolating (B-anomer enriched Gemcitabine hydrochloride by converting Gemcitabine base into Gemcitabine hydrochloride followed by its purification using solvents from the series of water soluble ethers like 1,4-dioxane or Monoglyme.

Full Text	F0RM2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10; rule 13) Title of the invention - "NOVEL PROCESS FOR THE PREPARATION OF GEMCITABINE HYDROCHLORIDE & INTERMEDIATES THEREOF" 2. Applicant(s) (a) NAME : ARCH PHARMALAB LIMITED (b) NATIONALITY : An Indian Company (c) ADDRESS: "H" Wing, 4th floor, Tex Centre, Off Saki Vihar Road, Chandivali, Andheri(E), Mumbai-400 072, India. 3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed: Field of the Invention The present invention relates to an improved one pot process for synthesis of key intermediate of Formula (I) required for the synthesis of Gemcitabine hydrochloride. Formula (I) The present invention further relates to selective isolation of the erythro isomer of the intermediate using novel solvents. The present invention also relates to a novel process for the preparation of Gemcitabine hydrochloride of the formula (II). More particularly the present invention relates to the isolation and purification of Gemcitabine hydrochloride of formula (I) HO NH2 .HCI Formula (II) 2 Background of the Invention Gemcitabine, a pyrimidine analog, is chemically known as 1-(2'-Deoxy-2', 2'-difluoro-D-ribofuranosyl)-4-aminopyrimidin-2-one. Although Gemcitabine is structurally similar to cytarabine, it has a wider spectrum of antitumour activity due to its different cellular pharmacology and mechanism of action. Gemcitabine belongs to the group of medicines called antimetabolites. Gemcitabine is a type of chemotherapy for treating many types of cancers including lung, pancreatic cancers. It can interfere with the growth of rapidly growing cells like cancer cells and cause cell death. 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1 -ulose-3, 5- dibenzoate of formula (I) is a key intermediate used for synthesis of Gemcitabine hydrochloride of Formula (II). NH2 .HCI Formula (II) US Patent 4,526,988, US 4808614, GB 2136425, US5015743 discloses a process for the preparation of 2-deoxy-2,2-difluoro-1-oxo ribose (lactone) shown by the Formula (IV) wherein an alkyl-3-dioxalan-4-yl-2,2-difluoro-3-hydroxy propionate of Formula (VI) where R is C2H5 is hydrolyzed under mild conditions using mild acidic ion exchange resin. 3 Formula (IV) Formula (VI) Where R is Alkyl group having C1-C4 atoms. The above mentioned patents describe the process for the preparation of lactone intermediate of formula (IV), wherein the hydrolysis of hydroxy propionate in either form is carried out under very mild conditions using acidic ion exchange resin like Dowex 50W-X12 to form the lactone form of the carbohydrate but since reaction using resin is heterogeneous, requires more time. Further, it is specifically disclosed that it is possible to carry out the process with other hydrolytic reagents, but the main disadvantage being the formation of larger amounts of by-products. For example, aqueous acetic acid, or other relatively strong acids such as propionic acid, formic acid, chloroacetic acid, oxalic acid and the like, may be used for the hydrolysis but they do not prevent the reversion reactions. EP Patent 0306190, EP 0688782 and EP 0630905 discloses a process for preparing lactone intermediates and 2',2'-difluoronucleosides whereby reversion back to the lactone's open chain analogue (hydroxyl acid) is minimized and the desired erythro enantiomer can be selectively 4 isolated from an enantiomeric mixture of erythro and threo lactones in crystalline form. EP 0306190 describes the use of monobenzoylated derivative for protection of hydroxyl group. The preparation of enantiomeric mixture of D-erythro and D-threo-2-deoxy-2, 2-difluoro-pentafuranos-1-ulose-3, 5-dibenozate from an enantiomeric mixture of D-erythro & D-threo-2-deoxy-2, 2-difluoro pentafuranose-1-ulose-3-benzoate is described. Starting material required for monobenzoate is enantiomeric mixture containing 3R: 3S (3: 1) poethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate. The hydrolytic agent used for the purpose is trifluroacetic acid. The process results into a crystalline lactone which is stable thereby minimizing reversion back of open chain analogue as well as minimizing formation of undesirable reaction products as expected by the use of strong hydrolytic agents. The term strong acid as defined herein is acid with pKa of about -10 to 2 at room temperature (22°C). Examples of strong acid include inorganic acid such as HCI and H2S04and organic acids such as trifluroacetic acid and p-toluene sulphonic acid. Preferred strong acids are those which have pKa value of -7 to 0. Preferred strong acids are trifluroacetic acid and para-toluene sulphonic acid but in most of the cases trifluroacetic acid is used. The acid employed is in an amount sufficient to provide about 0.05 to 0.5 molar equivalents with respect to starting material. The complete removal of isoalkylidene group takes place in about 2 to 8 hours which is then followed by lactonisation with simultaneous azeotropic removal of water, it is cyclised by distilling water/alcohol, water/acetonitrile, water/acetonitrile/ aromatic solvent. Trifluoroacetic acid (TFA) is a strong, non-oxidizing, organic acid. TFA is a reagent used frequently in organic synthesis due to its nature as an organic-soluble strong acid. The present inventors have however found that trifluoroacetic acid when used as hydrolytic agent in manufacturing of Gemcitabine hydrochloride results in reversion to open chain analogue which affects the yield of the product. 5 The process described in EP Patent 0306190, EP 0688782 and EP 0630905 has following steps: 1. Isolation of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate. 2. Reduction of the lactone to lactol of the formula (V). Formula (V) Here the crude lactone containing the erythro and threo isomers was crystallized from the only use of dichloromethane by dissolving it but to increase the yield, isopropanol or hexane are used as counter solvents. EP0306190 also describe about selective isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranose-1-ulose-3,5-dibenzoate with 95% purity. US Patent No. 6001994 and US 5912366 discloses a process to make Gemcitabine hydrochloride wherein the improvement consists essentially of making the lactone intermediate, 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1ulose-3,5-dibenzoate of Formula (I) from D-erythro-2-Deoxy-2,2-difluoro 4,5-0-(1-ethylpropylidene) pentoic acid tertiary butyl ester which is prepared by the process of reacting S-tertiary-butyl difluoroethane thioate with 2,3-0 (l-ethylpropylidene)-D-glyceraldehyde in a solvent and in the presence of a strong base; with the proviso that the process is conducted in the absence of a catalyst and in the absence of a silyl containing compound. However, the preparation of S-tertiary-butyl difluoroethane thioate involves the use of hazardous chemical like oxalyl 6 chloride. Also, due to their unpleasant odour thiols become unfavorable for synthesis. Beside this at various stages chromatographic purification is done which makes the process less economical. PCT Application WO 2005/095430 discloses the use of trifluoroacetic acid as hydrolytic agent followed by cyclisation & benzoylation to get benzoylated lactone, its reduction to get benzoylated lactol. It further teaches the isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranose-1-ulose-3,5-dibenzoate which is achieved with the help of Methylene dichloride : Hexane & Toluene : Hexane as solvent mixture which gives the yield in the range of 20-33%, however Methylene dichloride : Hexane as a solvent for isolation is used in most of the cases described in the prior art. In addition, hexane being highly inflammable makes its use nonviable on industrial scale. Also, it is a known fact that Toluene is a high boiling solvent which requires relative higher temperature during processing contributing to some unfavorable effects on product thereby making it a less preferred solvent. The present inventors have found that when a strong acid like trifluroacetic acid is used as a hydrolyzing agent, lactone formed during later part of reaction being more prone to reversion back it goes to the open chain analogue in presence of this strong acid. Even after applying reduced pressure in the procedure it is difficult to remove the water and also the strong acid present in the reaction mass further enables the reversion reaction and thus decreasing the yield. Beside this use of toluene during reaction, requires high temperature which results in charring of the product thereby affecting the yield of the product. As far as the solvents are concerned, the use of hexane makes this procedure very unlikely to be used at large scale. Hexane being highly inflammable makes it nonviable on industrial scale. Isolation and crystallization of benzoylated lactone is also disclosed in US patent No. 4526988 and US Patent No. 4808614. However, the processes described in the said patents adopt laborious chromatographic 7 purifications in different stages which make the process industrially unviable. Therefore, the processes disclosed in the prior art for the preparation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate which is a key intermediate for the preparation of Gemcitabine hydrochloride has various disadvantages such as • the use of mild acidic resin which prolongs the duration of process to four days thereby rendering to formation of other side products, • the use of strong acids like trifluoroacetic acid for hydrolysis of acetals leads to formation of lactone but often causes reverse back to its open chain analogue because of its sensitivity to water, • the relative strong acids also produce appreciable amount of undesirable side reaction products as a result of which yield of the intermediate produced is very low which affects the final yield of the Gemcitabine hydrochloride. • the processes require isolation of product formed at every stage. Therefore, the need of the hour is an improved process preferably an insitu or one pot process which will help in avoiding isolation of product at individual stage thereby minimizing unit operations as well as handling loss, reversion back to its open chain analogue and increasing the yield and purity significantly. The inventors have found that the use of the presence of salts used for hydrolysis has not affected the later stage of lactone formation.The reversion back to open chain analogue is minimised to large extent unlike the strong organic and inoganic acid reagents. The inventors have found that the use of the pyridinium salts like pyridinium trifluoroacetate or pyridinium para toluene sulphonate as a mild acid for hydrolysis in one pot, has minimized the reversion back to the lactone's open analogue thus resulting into 2-deoxy-2,2-difluoro-D-erythro- 8 pentafuranos-1-ulose-3,5-dibenzoate with improved yield (44% m/m) and high purity (>99.5%) which ultimately improves the over all yield of Gemcitabine hydrochloride. Inventors while working on suitable hydrolytic agents found that using triethyl amine salt of trifluoro acetic acid or triethyl amine salt of para toluene sulphonic acid, hydrolysis could not be completed even after 24 hours. Preparation of Gemcitabine hydrochloride disclosed in US patent No. 4526988 and US Patent No. 4808614, adopts laborious chromatographic purifications in different stages which make the process industrially nonviable. The process disclosed in US Patent No. 5637688 for the isolation and crystallization of Gemcitabine hydrochloride comprises following steps: a) Deblocking of p-1-(2'-deoxy-2',2'-difluoro-3',5'-di-0-benzoyl-D-ribofuranosyl)-4-aminopyrimidin-2-one with a catalytic amount of an alkylamine in the presence of methanol or ethanol in an environment essentially free of water; b) Treating the resulting solution with hydrochloric acid and an antisolvent selected from the series of acetone, acetonitrile, tetrahydrofuran, propanol, butanol, isobutanol, sec-butanol and isopropanol; c) Purifying the resulting Gemcitabine hydrochloride. In EP0630905, isolation involves dissolution of Gemcitabine hydrochloride (1:1) in hot water and then precipitated by acetone to get 80% (3-anomer rich product which is further purified by re-dissolving in hot water and then again precipitated by acetone to get 99% (3-anomer of Gemcitabine hydrochloride. It also describes isolation of Gemcitabine base of 99% purity from Gemcitabine or its addition salt by dissolving in hot water and increasing 9 pH to 7-9 isolating Gemcitabine of 99% and is further converted into Gemcitabine hydrochloride. WO2005/095430 discloses the isolation of Gemcitabine hydrochloride whereby crude 2'-deoxy-2',2'-difluorocytidine base is dissolved in isopropanol and then precipitated by hydrochloric acid and filtered off. The solution so obtained is then further purified by water and acetone as solvent system which is already reported in prior art. The process of purification disclosed in WO2005/095430 includes dissolving Gemcitabine HCI in water to increase the purity and then again dissolving in water and acetone for further purification thereby increasing the unit operation. Therefore there is a long felt need of a process for the isolation and purification of Gemcitabine hydrochloride, which is selective for the beta-anomerof the Gemcitabine hydrochloride giving high yield and purity. Also there is a need of a process of purification which does not require any reversion of hydrochloride to base by adding other bases like alkyl amine and again converting back to hydrochloride salt which increases unit operation without giving appreciable yields or a process of purification which does not require any pH adjustment as disclosed in prior art. Objects of the invention Therefore, it is an object of the present invention to provide an improved process for the synthesis of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate of Formula (I) that overcomes the problems associated with prior art. It is another object of the present invention to provide an improved process for the synthesis of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate which minimizes the reversion back to the lactone's open chain analogue. 10 It is yet another object of the present invention to provide a process for selective isolation of the erythro isomer of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate from the enantiomeric mixture of erythro and threo lactone using novel solvent system including solvent such as ethyl acetate, dichloroethane and diisopropyl ether. Another object of the present invention is to provide better hydrolytic agent required for the synthesis of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate which minimizes the reversion back to the lactone's open chain analogue. Another object of the present invention is to provide an improved process for the synthesis of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate using mild acid which is a salt of organic acid such as Trifluoroacetic acid and an organic base such as Pyridine. Another object of the present invention is to provide a 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate in high yield along with high purity which in turn gives high yield of Gemcitabine hydrochloride of Formula (II). Another object of the present invention is to provide a process for the selective isolation of beta-anomer of Gemcitabine hydrochloride. Another object of the present invention is to provide a process for purification of Gemcitabine hydrochloride. It is further object of the present invention to provide a process for isolation & purification, which employs the use of water miscible ethers like 1,4-dioxane or Monoglyme. Yet another object of the present invention is to provide a process for beta-anomer of Gemcitabine hydrochloride in high yield along with high purity (99.9%). Summary of the Invention Thus according to an aspect of the present invention, there is provided an improved one pot process for the synthesis of a 2-deoxy-2, 2- 11 difluoro-D-erythro-pentafuranose-1-ulose-3, 5-dibenzoate of Formula (I) comprising reaction of hydrolysis of (Erythro:Threo)alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)- 2,2-difluoro-3-hydroxy propionate of Formula (III) where alkyl group is having C1-C4 number of atoms with hydrolyzing agent in organic solvent and water followed by formation of lactone and dibenzoylation of the lactone. More over it is found that invention also works well with other alkyl groups like methyl, isopropyl, n-propyl and butyl (Erythro:Threo)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)- 2,2-difluoro-3-hydroxy propionate of Formula (III) however reaction with ethyl group has found to be better. Formula (I) Formula (III) Where R is Alkyl group having C1-C4 atoms. 12 According to another aspect of the present invention, there is provided a process for the selective isolation of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranose-1-ulose-3, 5-dibenzoate from the enantiomeric mixture of erythro and threo isomers of 2-deoxy-2, 2-difluoro-pentafuranose-1-ulose-3, 5-dibenzoate using novel solvents such as ethyl acetate, ethylene dichloride and Diisopropyl ether. Thus according to an aspect of the present invention there is provided a process for the isolation of P-anomer enriched Gemcitabine hydrochloride of Formula (II) comprising conversion of Gemcitabine base having a and p anomers into p-Gemcitabine hydrochloride using organic solvent chosen from the series of water miscible ethers like 1,4-dioxane or Monoglyme. According to another aspect of the present invention there is also provided a process for the purification of p-Gemcitabine hydrochloride of Formula (II) comprising dissolving of Gemcitabine hydrochloride (P-anomer enriched) in water and selectively crystallizing using organic solvent chosen from water miscible ethers like 1,4-dioxane or Monoglyme. Brief Description of Figures: Figure 1: DSC of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate shows an endotherm at 123.69°C Figure 2 DSC of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate shows an endotherm at 123.15°C Figure 3: DSC of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate shows an endotherm at 123.19°C DSC stands for Differential Scanning Calorimetry^ Detailed Description of the Invention The present invention addresses the need of a one pot process for the synthesis of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate of Formula (I) and loss due to reversion of lactone by 13 minimizing the reversion back to the lactone's open chain analogue formed which affects the overall yield of the Gemcitabine hydrochloride. Formula (I) Also the present invention provides a process for exclusively isolating 2-deoxy-2, 2-difluoro-D-erythro-pentafuranose-1-ulose-3, 5-dibenzoate from the mixture of erythro and threo isomers of 2-deoxy-2, 2-difluoro- pentafuranose-1-ulose-3, 5-dibenzoate. The inventors of the present invention have surprisingly found that use of mild acid which is salt of organic acid such as Trifluoroacetic acid and organic base such as Pyridine as a hydrolytic agent minimizes the reversion back to the lactone's open chain analogue and thus gives good yield as compared to the prior art. The preferred mild acid according to the present invention is a Lewis acid having pKa value around 4.00 to 6.00, more preferably between 5.00-5.5. The preferred Lewis acid according to the present invention is pyridinium trifluoroacetate. The present invention provides an improved one pot process for the hydrolysis cum lactonisation of alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate where alkyl group is having C1-C4 number of carbon atoms using acetonitrile as solvent in presence of water and 14 pyridinium salts like pyridinium trifluoroacetate or pyridinium para toluene sulphonate as mild hydrolytic agent which also could be supporting in lactonisation to provide the required lactone in a quantitative yield , which is then taken for insitu benzoylation. However use of pyridinium para toluene sulphonate although resulted in good yield and purity but duration required for the same is more. Alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate of required R isomer was charged into a round bottom flask, and acetonitrile and water and pyridinium salts like pyridinium trifluoroacetate or pyridinium para toluene sulphonate were added to it and the reaction mixture was heated to reflux. The reaction mixture was stirred at reflux temperature for 2- 15 preferably 3-12 hours. Mixture of Acetonitrile and water was azeotropically distilled completely to give oil. Ethyl acetate was added in the reaction mass and subjected to distillation at NTP. The obtained oil was dissolved in ethyl acetate; dry pyridine was added followed by 4,4'-dimethyl aminopyridine. The mixture was heated to 60-65°C under N2 atmosphere. Solution of benzoyl chloride in ethyl acetate for benzoylation was added drop wise over 3 hours at 60-65°C. The reaction was stirred at 60-65X for another 3 hours and then cooled to room temperature (25-30°C) and stirred for another 3 hours. The reaction mass was cooled to 0-5°C and stirred for another 1 hour and filtered. The filtrate was concentrated under vacuum at 35°-40 °C to yield oil. The preferred solvents according to present invention for the selective isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate from the mixture of erythro and threo isomers to get selective erythro isomer are ethyl acetate, ethylene dichloride and diisopropylether. Thus, the oil obtained above was taken into ethyl acetate or ethylene dichloride followed by distillation to get a thick mass to which ethyl acetate or ethylene dichloride was then added under stirring and 15 contents were cooled to 0-5°C and maintained for half an hour. Diisopropyl ether was then added to the contents and continued further for 10-15 min. Product so obtained is filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. The selective erythro isomer of 2-deoxy-2,2-difluoro-D-erythro-pentafuronose-1-ulose-3,5-dibenzoate is obtained in high yield which is about 44% (m/m) along with purity >99.5%. Entire Schematic representation for the preparation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate is shown as follows: Step -1: Acetal formation of D-mannitol OH OH D-Mannitol 2,2-dimethoxypropane l,2:5,6-diisopropylidine- D-mannitol Step -2: Preparation of 2,3-isopropylidene-D-Glyceraldehyde Methylene dichloride HX, -CH* l,2:5,6-diisopropylidine-D-mannitol 2,3-O-isopropylidene-D-glyceraldehyde Step -3: Preparation of (Erythro:Threo)Ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxypropionate 16 O. XH o OH CH, P H3C Bromodifluoro ethylacetate *» Zn, Tetrahydrofuran H3C O 2,3-O-isopropylidene-D-glyceraldehyde (Erythro:Threo)Ethyl-3-(2,2-dimethyl-l,3-dioxolan -4-yl)-2,2-difluoro-3-hydroxy propionate Erythro:Threo(3:l) Step -4: a) Insitu preparation and benzoylation of 2-deoxy-2,2-difluoro-pentafuranose -1-ulose-to get 3,5-dibenzoate derivative. For the present invention insitu schematic representation for the conversion of (erythro:threo)ethyl-3-(2,2-dimethyl-1 -3-dioxalane-4-yl)-2,2- difluoro-3-hydroxy propionate into 2-deoxy-2,2-difluoro-pentafuranos-1- ulose-3,5-dibenzoate is as follows:- ^, + 1 X H 1. Acetonitrile, 2. Benzoyl chloride, pyridne DMAP in ethyl acetate (Erythro:Threo)Ethyl-3-(2,2-dimethyl-l,3-dioxolan -4-yl)-2,2-difluoro-3-hydroxy propionate Erythro:Threo(3:l) 2-deoxy-2,2-difluoro-D-pentafuranose-l-ulose-3,5-dibenzoate (Mixture of Erythro & Threo) x" Trifluoroacetate / Para toluene sulfonate b) Isolation of erythro isomer selectively using solvents like Ethyl acetate or Ethylene dichloride with Diisopropyl ether as solvent system. athe use of novel solvents such as water miscible ethers like 1,4-dioxane or Monoglyme. The ratio of isolated Gemcitabine hydrochloride is in the range 80-90% (3 and 4-12% a anomer. The process for the purification of Gemcitabine hydrochloride containing 80-90% p-anomer further uses the concept of solvent and antisolvent wherein water is used as so Ivent and antisolvent is chosen from the series of water soluble ethers resulting into selective crystallization of Gemcitabine hydrochloride with high isomeric purity of 99.9% and higher yields. Entire Schematic representation for the isolation and purification of Gemcitabine hydrochloride is shown as follows: 5) Reduction of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate: 2-deoxy-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate was reduced to benzoylated lactol (2-Deoxy-2,2-difluoro-D-ribofuranose-3,5-dibenzoate) using Vitride which is sodium bis (2-methoxyethoxy) aluminium hydride and commercially available as a 70% w/w solution in toluene as reducing agent and Tetrahydrofuran as solvent in temperature range of -40 to -70°C. The duration of the reaction was around 30 -120 minutes. 2-deoxy-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate was isolated by usual method resulting in the yield in the range of 90-95% and having purity of 80-85%. C6H5OCO 0. Vitride, Tetrahydrofuran \ \/ »• F C6H5OCO 2-deoxy-2,2-difluoro-D-erythro-pentafuranos- 2-deoxy-2,2-difluoro-D-riboforanose-3,5-dibenZoate l-ulose-3,5-dibenzoate Mixture of a & (3 6) Methyl sulfonationa of 2-deoxy-2,2-difluoro-D-ribofuranose— 3,5-dibenzoate: Benzoylated lactol (2-Deoxy-2,2-difluoro-D-ribofuranose-3,5- dibenzoate) so obtained was subjected to reaction with methanesulfonyl chloride in methylene dichloride as solvent and triethylamine as base at temperature of -25 to +10°C, preferably at -20 to 5°C, more preferably at -20 to 0°C to form 2-deoxy-2,2-difluoro-D-ribofuranose-3,5-dibenzoate-1 -methane sulfonate 90-95 yield & HPLC purity 80-85%. C,H,OCO- Methanesulfonyl chloride \( N^tfOMs Triethyl amine , Methylene dichloride C6H5OCO 2-deoxy-2,2-difluoro-D-ribofuranose-3,5-dibenzoate 2-deoxy-2,2-difluoro-D-ribofuranose-3,5- dibenzoate-1-methane sulfonate Mixture ofa& P 7) Condensation of 2-deoxy-2,2-difluoro-D-ribofuranose— 3,5-dibenzoate-1-methanesulfonate with N -acetyl bis trimethylsilyl cytosine derivative followed by preparation of Gemcitabine base: The bistrimethylsilyl derivative of acetyl cytosine was prepared by treatment of acetyl cytosine with hexamethyldisilazane and trimethylsilyl chloride in ethylene dichloride. The product from step 2 was then subjected to coupling with bistrimethylsilyl derivative of acetyl cytosine in presence of trimethylsilyl triflate at 80-82X, resulting into 2'-deoxy-2',2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate (1.2-1.3w/w). The 2'-deoxy-2\2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate was hydrolyzed in methanol 1:8 to 15 w/v using ammonia at a temperature in the range of -10 to +10°C preferably at -5 to +5°C for 5-20 hours preferably 8-10 hours. After the hydrolysis, the methanolic solution was concentrated to get a thick mass which was then dissolved in water and extracted with ethyl acetate followed with Diisopropyl ether. Aqueous layer so obtained was concentrated under vacuum at 30-60°C preferably at 40-45°C Isolation of Gemcitabine hydrochloride: The Gemcitabine base so obtained was converted into its hydrochloride by adding solvents like 1,4-dioxane or Monoglyme in ratio of 1:4-12(w/v) preferably 1:5-8(w/v) and heating the contents at 40-90°C preferably at 50-70X more preferably at 60-68°C followed by cooling the contents to 10-30°C preferably 10-15°C to give crude (solid) Gemcitabine hydrochloride having 80-90% of B-anomer & 4-12% of a-anomer. HO OH Mixture of a & P Monoglyme+Con.HCI or dioxane+Con.HCI »- P anomer enriched (80-90)% a anomer(4-12)% Purification of Gemcitabine hydrochloride: The isolated product obtained in the above reaction has a-anomer (4-12%) and B-anomer (80-90%) which is further purified to 99.9% by dissolving crude Gemcitabine hydrochloride in water 1:4-8(w/v) preferably 1:5-7(w/v) and heating the contents at 40-90°C preferably at 50-70°C more preferably 60-68°C to get a solution to which solvents like 1,4-dioxane or Monoglyme in the ratio of 1:40-100(w/v) preferably 1:50-70(w/v) are added followed by heating the contents at 40-90°C preferably at 50-70°C followed by cooling to 10-30°C preferably 10-15°C to get Gemcitabine hydrochloride of purity 99.9%. The overall yield at this stage with respect to 2'-deoxy-2',2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate is 15 to 16%. 21 The preferred solvent is water and the preferred anti solvent is selected from the series of water soluble ethers preferably 1,4-dioxane and Monoglyme with improved yield and high purity of 99.9%. The most preferred solvents for the isolation of p-anomer of Gemcitabine hydrochloride and purification of Gemcitabine hydrochloride rich in p-anomer are the water miscible ethers like 1,4-dioxane or Monoglyme. The purity & assay of the product thus obtained is analyzed using HPLC. Column specification: 4.6 mm X 25 cm column, that contains 5\|_i packing L7. Wave length selected: 275 nm Mobile phase: Prepare a filtered and degassed solution containing 13.8 g of monobasic sodium phosphate and 2.5 ml of phosphoric acid in 1000 ml of water. Flow rate: 1.2 ml/min Diluent: Filtered and degassed water for HPLC The details of the invention, its objects and advantages are explained hereunder in greater details in relation to non-limiting exemplary illustrations. The examples are merely illustrative and do not limit the teaching of this invention and it would be obvious that various modifications or changes in the procedural steps by those skilled in the art 22 without departing from the scope of the invention and shall be consequently encompassed within the ambit and spirit of this approach and scope thereof. Examples: Example 1; Preparation of 1,2 : 5,6-diisopropylidene-D-mannitol: To a 5L four neck round bottom flask fitted with Thermometer pocket, condenser with CaCI2 guard tube and overhead stirrer were added 600 ml of 1,2-dimethyoxy ethane, 250gm of D-mannitol and 400 ml of 2,2-dimethoxy propane. The mixture was stirred at room temperatures and 375 mg of anhydrous stannous chloride was added to the reaction mixture. The slurry was heated to reflux for approximately 50 to 90 min until mixture becomes clear. The solution was cooled below reflux temperature & pyridine (0.5ml) was added to the reaction mixture and the same was stirred for 10 min. The solution was concentrated below 40 to 45°C to give thick solid mass. The mixture was allowed to cool to room temperature (25°C). Methylene dichloride (500 ml) was added to it and mixture was stirred at room temperature for 30 min.Hexane(2. 5L) was added to this mixture and cooled to 0-10°C and stirred for more 30 min and filtered off. The white solid was dried in vacuum at 35-40°C for 4 hrs. 200gm white solid obtained has GC purity 99%. Example 2: Preparation of 2,3-O-lsopropylidene-D-glyceraldehyde: The solid (200g) 1,2:5,6-diisopropylidene-D-mannitol obtained as in example 1 was added to 2000ml of Methylene dichloride. The mixture was warmed to 30-32°C and stirred for 30 min. The resulting solution was filtered. The filtrate obtained was cooled to 15°C and 10% aqueous Sodium bicarbonate solution (80ml) was added to the reaction mass. Sodium periodate (317 gm) was added to reaction mixture and the resulting slurry is stirred at temperature between 20-25°C for another 2 hours. 100 gm of sodium sulfate was added to the reaction & the slurry is stirred for another 15 min. The reaction mass was filtered and the solid 23 residue was washed twice with 200 ml of methylene dichloride. The resulting filtrate is collected and solvent was distilled at 40-45X to obtain oil which was heated up to 50-52°C under vigorous stirring to remove traces of solvent. The oil weight is 180 gm with 95% (GC purity). Example 3: Preparation of (Erythro:Threo) ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate : To the 5L round bottom flask fitted with overhead stirrer, condenser, pressure equalizing dropping funnel 1.8L of tetrahydrofuran was added. Zn dust (180 gm) and trimethylsilyl chloride (28.76ml) were added to it under stirring. The mixture was stirred at room temperature for 15 min and than heated to reflux and mixture of 2,3-O-lsopropylidene-D-glyceraldehyde (180 gm) and Bromodifluoroethyl acetate (248 ml) was added through dropping funnel to the reaction mixture slowly over a period of 30-40 min. The reaction was stirred with reflux for another 2hours. After 2 hours the reaction mass was cooled to room temperature and poured in mixture of 143 ml of Cone. HCI and 1100 gm ice under stirring. The mixture was stirred for 15 min and the organic layer was separated. The aqueous layer was further extracted with ethyl acetate (200mlX 3). The collective organic layer was washed with brine followed by 5% aq. Sodium bicarbonate solution. The organic layer was dried over sodium sulfate and concentrated under vacuum to yield the product (250 gm) with GC purity of desired isomer 75%. Example 4: Preparation & isolation of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate: To a 3L four-necked round bottom flask 82g of ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate having 75% of required R isomer was charged, and acetonitrile (820ml) and water (26ml) and pyridinium trifluoroacetate (14.76g) were added to it and the reaction mixture was heated to reflux. The reaction mixture was stirred at reflux temperature for 3 hours. Mixture of Acetonitrile and water was azeotropically distilled completely to give oil. Ethyl acetate (90ml) was 24 added in the reaction mass and subjected to distillation at NTP. The obtained oil was dissolved in ethyl acetate (328ml), dry pyridine (96ml) was added followed by 4, 4'-dimethyl aminopyridine (9.52g). The mixture was heated to 65°C under N2 atmosphere. Solution of benzoyl chloride (96ml) in ethyl acetate (255ml) was added drop wise over 3 hours at 65°C. The reaction was stirred at 65°C for another 3 hours and then cooled to room temperature (25-30°C) and stirred for another 3 hours. The reaction mass was cooled to 0-5°C and stirred for another 1 hour and filtered. The filtrate was concentrated under vacuum at 35°C to yield 160 g oil. Oil so obtained was taken into 164ml ethyl acetate followed by distillation to get a thick mass to which 61.5 ml ethyl acetate was then added under stirring and contents were cooled to 0-5°C followed by keeping for half an hour. Diisopropyl ether (205ml) was then added to the contents and continued further for 10-15 min. Product so obtained is filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. Yield (53.3g), HPLC purity of +99.5%, DSC has shown the endotherm in the range of 121-125X (peak at 123.69°), Figure 1. Example 6: Preparation & isolation of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1 -ulose-3,5-dibenzoate: To a 3L four-necked round bottom flask 90g of ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate having 75% of required R isomer was charged, and acetonitrile (900ml) and water (29ml) and pyridinium para toluene sulphonate (19.6g) were added to it and the reaction mixture was heated to reflux. The reaction mixture was stirred at reflux temperature for 12 hours. Mixture of Acetonitrile and water was azeotropically distilled completely to give oil. Ethyl acetate (100ml) was added in the reaction mass and subjected to distillation at NTP. The obtained oil was dissolved in ethyl acetate (360ml), dry pyridine (105ml) was added followed by 4, 4'-dimethyl aminopyridine (10.44g). The mixture was heated to 65°C under N2 atmosphere. Solution of benzoyl chloride (105ml) in ethyl acetate (260ml) was added drop wise over 3 hours at 25 65°C. The reaction was stirred at 65°C for another 3 hours and then cooled to room temperature (25-30°C) and stirred for another 3 hours. The reaction mass was cooled to 0-5°C and stirred for another 1 hour and filtered. The filtrate was concentrated under vacuum at 35°C to yield 160 g oil. Oil so obtained was taken into 170ml ethyl acetate followed by distillation to get a thick mass to which 61.5 ml ethyl acetate was then added under stirring and contents were cooled to 0-5°C followed by keeping for half an hour. Diisopropyl ether (210ml) was then added to the contents and continued further for 10-15 min. Product so obtained is filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. Yield (59g), HPLC purity of +99.5%. DSC has shown endotherm in the range of 121-125X (peak at 123.15°) Figure 2. Example 7: Procedure in example E was repeated with 162 g (Erythro:Threo) ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate resulting in to 290 g oil. Thick oil so obtained is taken into 300ml ethylene dichloride followed by distillation under vacuum at 55-60°C to get a thick mass to which 122 ml ethylene dichloride was then added under stirring and contents were cooled to 0-5°C followed by keeping for half an hour. Diisopropyl ether (400ml) was then added to the contents and continued further fori0-15 min. Product so obtained was filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. Yield (105.3g), HPLC purity of +99.5 % & more, DSC has shown endotherm in the range of 121-125°C (peak at 123.19°) Figure 3. Example 8: Preparation of 2-deoxy-D-erythro-2,2-difluoro- ribofuranose-3, 5-dibenzoate: 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate (200g) was dissolved in (2 L) of tetrahydrofuran in a 5L round bottom flask. (0.2L) of vitride (70% solution in toluene) was added between -50 to -70°C. The reaction was continued for 60 minutes. On completion, 26 reaction was arrested by adding methanol (0.24L) followed by hydrolysis with 7% HCI (1.3L). After stirring for ~30 min the aqueous layer was separated at RT. The aqueous layer was repeatedly extracted with Ethyl acetate (0.8 L X 2). The collected organic layer was washed with brine (0.5 L X 2) and 5% aqueous NaHC03 (0. 5 L X2) followed by brine (0. 5 L X 2). The organic layer was concentrated under vacuum to get thick oil (0. 2kg). Example 9: Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methanesulfonate: 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate (0.2kg) oil was dissolved in (2L) Methylene dichloride. The reaction mixture was cooled to 0 to -20°C and (0.12 L) triethylamine was added. To this stirred mixture was added (0. 06L) Methane sulfonylchloride drop wise between -20°C to -15°C. The reaction was stirred at same temperature till completion of reaction (in ~3hours). Temperature was brought to room temperature & 5% HCI (0.9 L) was added to reaction. The aqueous layer was separated and extracted with Methylene dichloride (0.4 L X 2). The organic layer was washed with brine (0.5 L X2) & by 5% aqueous sodium bicarbonate followed by brine (0.5 L X 2). The organic layer was collected and concentrated to give oil (0.24kg). Example 10: Preparation of Gemcitabine base: Acetyl cytosine (200g) was added to 1,2-dichloroethane (4L) in 10L round bottom flask. Hexamethyldisilazane (0.34L) was added to the slurry. The resulting mixture was heated to distill out ~1 L mixture. The reaction mass cooled to 50°C under nitrogen and trimethyl silyl chloride (0.015 L) was added to the mixture. The resulting mixture was refluxed at 82-85°C for 3 hours. The solution was cooled to 45°C under nitrogen and distilled at 45-50°C to obtain solid. Fresh ethylene dichloride (2.6 L) was added to the solid so obtained followed by addition of Trimethyl silyl triflate (0.26 L). To this solution of mesylate (0.24 L) obtained from example no 2 dissolved in ethylene dichloride (1.2 L) was added. The resulting mixture was heated 27 to reflux at 86-92°C for 18 hours till 2-deoxy-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methnesulfonate was consumed. The reaction mixture was cooled at room temperature and 5% HCI (1.8 L) was added to the reaction mixture followed by separation of aqueous layer and extraction with ethylene dichloride (0.4 L X 2). The combined organic layer was washed with brine (1 L X 2). The collected organic layer was treated with sodium bicarbonate (0.2 Kg) to neutralize acidity. The dried organic layer was concentrated to give oil (0.26kg). The resulting oil obtained in previous example (0.26kg) was dissolved in methanol (3.4L). The resulting solution was cooled to -5 to +5°C and ammonia gas purged through the mixture under stirring for 8-10 hours between -5 to +5°C till complete consumption of 2',2'-difluoro-2'-deoxy-N-acetyl cytidine-3',5'-dibenzoate. The temperature was allowed to increase to room temperature. The solution was concentrated under vacuum to obtain oil which was dissolved in water (0.8 L). Ethyl acetate (0.2 L) was added to it and the mixture was stirred for 15 min. The aqueous layer separated was extracted with ethyl acetate (0.2 L). The combined ethyl acetate layer was extracted with (2.6 L) water. The aqueous layer was filtered and concentrated under vacuum at 40-45°C to give oil (0.1 Kg). Example 11: Isolation of Gemcitabine hydrochloride: To the 20g of thick oil of Gemcitabine base obtained as in example 10 was added 1,4-Dioxane (120 ml) and the mixture was heated to 60°-68°C and stirred for 15 minutes. Cone. HCI (10ml) was then added to the mixture and the contents were heated at same temperature for one hour. Reaction mass was then cooled to 10-15°C and maintained for 30 minutes. The solid thus obtained was filtered off and washed with 1,4-Dioxane and dried under vacuum at 55-60 °C to get 8g material with Specific Optical Rotation i.e.[a]D = +40° and HPLC purity of 85% B-anomer and 8% a-anomer. Example 12: Purification of Gemcitabine hydrochloride: 28 10g material obtained using protocol of example 11 was taken in water (60 ml) and the mixture was heated to 55-60°C to get a clear solution. 1,4-Dioxane (600ml) was added to the solution under stirring and continued for one hour at 60-68°C. The contents were cooled to 10-15°C & the mixture was stirred at this temperature for 30 min and filtered, washed with 1,4-Dioxane and dried at 55-60°C under vacuum to get 7.5g compound of HPLC purity 99.9% p-anomer with Specific Optical Rotation i.e.[a]D = +47°. Example 13: Isolation of Gemcitabine hydrochloride: To 20g of thick oil of Gemcitabine base obtained in example 10 was added Monoglyme (120 ml) and the mixture was heated at 60 - 68°C and stirred for 15 minutes. Cone. HCI (10 ml) was then added to the mixture and the contents were heated at same temperature for one hour. Reaction mass was then cooled to 10-15°C and maintained for 30 minutes. The solid thus obtained is filtered off and washed with Monoglyme and dried under vacuum at 55-60°C to get 8g material having HPLC purity with 85% P-anomer & 9% a-anomer, Specific Optical Rotation i.e.[a]o= +40° Example 14: Purification of Gemcitabine hydrochloride: 10 g material obtained as in example 13 was taken in water (60 ml) and the mixture was heated to 60-65°C to get a clear solution. Monoglyme (600ml) was added to the solution under stirring and continued for one hour at 60-65°C. The contents were cooled to 10-15°C. The mixture was stirred at this temperature for 30 minutes and filtered, washed with Monoglyme and dried at 55-60°C under vacuum to get 7.3 g product with HPLC purity 99.9% P-anomer with Specific Optical Rotation i.e.[a]D= +47°. The present invention has following advantages: • The process according to the present invention minimizes the reversion back to the lactone's open chain analogue thereby 29 increasing the yield of lactone intermediate & overall yield of Gemcitabine hydrochloride effectively. • Time required in the present invention for the process of hydrolysis during synthesis of 2-deoxy-2,2-difluoro-D erythro-pentafuronose-1-ulose-3,5-dibenzoate is reduced from four days to 3-12 hours. • An Insitu operation, one of the essential features of the present invention has minimized unit operations as well as handling loss also, as isolation of individual stages is avoided. • 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1 -ulose-3,5-dibenzoate is obtained in high yield which is about 44% (m/m) and 65% (w/w). • Selective isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate from the enantiomeric mixture of erythro and threo isomers with good purity and higher yield could be achieved with the help of solvents like ethyl acetate or ethylene dichloride with diisopropylether which have not been reported earlier. • Gemcitabine hydrochloride isolated is not only pure but also higher in yield than reported earlier. 30 Claims: .1. A process for the preparation of 2-deoxy-2,2-difluoro-D erythro-pentafuronose-1-ulose-3,5-dibenzoate of the formula (I) FORMULA (I) comprising a) hydrolysis of (Erythro:Threo)alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)- 2,2-difluoro-3-hydroxy propionate of formula (III) comprising the use of mild acid, CH3 Formula (III) Where R = Alkyl group having C1-C4 atoms b) cyclisation to form lactone and c) benzoylation of lactone d) Isolation of erythro isomer using novel solvents The process according to claim 1 wherein said alkyl group of formula (III) is ethyl. The process according to claim 1 wherein the mild acid is a salt of organic acid such as trifluroacetic acid or para toluene sulphonic acid and organic base such as Pyridine. 31 4. The process according to claim 1 wherein the mild acid is selected from pyridinium trifluoroacetate or pyridinium para toluene sulphonate. 5. The process according to claim 1 wherein the preferred mild acid is pyridinium trifluoroacetate 6. The process according to claim 1 wherein the mild acid has the pKa value around 4.00 to 6.00, more preferably between 5.00-5.5. 7. A process for selective isolation of erythro isomer of 2-deoxy-2,2-difluoro-D erythro-pentafuronose-1 -ulose-3,5-dibenzoate comprising dissolving the enantiomeric mixture of erythro and threo isomers in ethyl acetate or ethylene dichloride, cooling the solution and adding Diisopropyl ether followed by collecting the precipitated erythro enantiomer. 8. The process according to claim 7 wherein the selective erythro isomer of 2-deoxy-2,2-difluoro-D-erythro-pentafuronose-1-ulose-3,5-dibenzoate is obtained in yield of about 44% (m/m) along with purity >99.5%. 9. A process of isolating (3-anomer enriched Gemcitabine hydrochloride from the mixture of a & p anomers of Gemcitabine base the steps comprising of a) adding water soluble ether to Gemcitabine base b) heating the mixture to dissolve the base (oil) and adding thereto hydrochloric acid; c) cooling the mixture and d) isolating the precipitated (3-anomer enriched Gemcitabine hydrochloride. 10. The process according to claim 9 wherein the said water soluble ether is selected from the group comprising of 1,4-dioxane or Monoglyme. 11. The process according to claim 9 wherein the ratio of Gemcitabine base to said water soluble ether is in the range of 1 : 4 -12 (w/v) 32 12.The process according to claim 11 wherein the ratio of Gemcitabine base to said water soluble ether is in the range of 1 : 5 - 8 (w/v). 13. The process according to claim 9 wherein the Gemcitabine base along with the water soluble ether is heated at a temperature of 40-90°C. 14. The process according to claim 13 wherein the Gemcitabine base along with the water soluble ether is heated at a temperature of 50-70°C. 15. The process according to claim 13 wherein the Gemcitabine base along with the water soluble ether is heated at a temperature of 60-68°C. 16. The process according to claim 9 wherein the reaction mixture is cooled to a temperature of 10-30°C. 17. The process according to claim 16 wherein the mixture is cooled to a temperature of 10-15°C. 18. The process according to claim 9 wherein the said P-anomer enriched Gemcitabine hydrochloride has 80-90% of p-anomer and 4-12% of a-anomer. 19. A process for purification of P-anomer enriched Gemcitabine hydrochloride comprising the steps of a) dissolving p-anomer enriched Gemcitabine hydrochloride in water by heating the contents ; b) addition of a water soluble ether and cooling the mixture; and c) crystallizing the purified P-anomer enriched Gemcitabine hydrochloride. 20. The process according to claim 19 wherein the ratio of Gemcitabine hydrochloride to water is in the range of 1 : 4 - 8 (w/v) 21. The process according to claim 20 wherein the ratio of Gemcitabine hydrochloride to water is in the range of 1: 5 - 7 (w/v). 33 22. The process according to claim 19 wherein the Gemcitabine hydrochloride and water are heated at a temperature of 40-90°C. 23. The process according to claim 22 wherein the Gemcitabine hydrochloride and water are heated at a temperature of 50-70°C. 24. The process according to claim 22 wherein the Gemcitabine hydrochloride and water are heated at a temperature of 60-68°C. 25. The process according to claim 19 wherein the ratio of solution of Gemcitabine hydrochloride to said water soluble ether in the range of 1 :40-100(w/v) 26. The process according to claim 25 wherein the ratio of solution of Gemcitabine hydrochloride to said water soluble ether is in the range of 1 : 50 - 70 (w/v ) 27. The process according to claim 19 wherein the said water soluble ether is selected from the group consisting of 1,4-dioxane and Monoglyme. 28. The process according to claim 19 wherein the reaction mixture is cooled to a temperature of 10-30°C. 29. The process according to claim 28 wherein the reaction mixture is cooled to a temperature of 10-15°C. 30. The process according to claim 19 wherein the said (3-anomer enriched Gemcitabine hydrochloride has 99.9% of p-anomer. 30. The process according to claim 19 wherein the said purified (3- Gemcitabine hydrochloride has yield in the range of 15-16% with respect to 2'-deoxy-2',2'-difluoro-N-acetyl cytidine-3',5'-dibenzoate. Dated this 10th day of August 2006 Abstract An improved one pot process for preparing 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose-3,5-dibenzoate of Formula (I) comprising hydrolysis of (erythro:threo) alkyl-3-dioxalan-4-yl-2,2-difluoro-3-hydroxy propionate of Formula (III) where alkyl group is having C1-C4 number of Carbon atoms using a mild acid and selectively isolating 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose-3,5-dibenzoate from the mixture of erythro & threo enantiomers using ethyl acetate, ethylene dichloride and diisopropyl ether as solvents with improved yield and purity. A process for isolating (3-anomer enriched Gemcitabine hydrochloride by converting Gemcitabine base into Gemcitabine hydrochloride followed by its purification using solvents from the series of water soluble ethers like 1,4-dioxane or Monoglyme. 35

Full Text

F0RM2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
Title of the invention - "NOVEL PROCESS FOR THE PREPARATION OF GEMCITABINE HYDROCHLORIDE & INTERMEDIATES THEREOF"
2. Applicant(s)
(a) NAME : ARCH PHARMALAB LIMITED
(b) NATIONALITY : An Indian Company
(c) ADDRESS: "H" Wing, 4th floor, Tex Centre, Off Saki Vihar Road, Chandivali, Andheri(E),
Mumbai-400 072, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be
performed:

Field of the Invention
The present invention relates to an improved one pot process for
synthesis of key intermediate of Formula (I) required for the synthesis of Gemcitabine hydrochloride.

Formula (I)
The present invention further relates to selective isolation of the erythro isomer of the intermediate using novel solvents.
The present invention also relates to a novel process for the preparation of Gemcitabine hydrochloride of the formula (II). More particularly the present invention relates to the isolation and purification of Gemcitabine hydrochloride of formula (I)
HO
NH2 .HCI
Formula (II)
2

Background of the Invention
Gemcitabine, a pyrimidine analog, is chemically known as 1-(2'-Deoxy-2', 2'-difluoro-D-ribofuranosyl)-4-aminopyrimidin-2-one. Although Gemcitabine is structurally similar to cytarabine, it has a wider spectrum of antitumour activity due to its different cellular pharmacology and mechanism of action. Gemcitabine belongs to the group of medicines called antimetabolites. Gemcitabine is a type of chemotherapy for treating many types of cancers including lung, pancreatic cancers. It can interfere with the growth of rapidly growing cells like cancer cells and cause cell death.
2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1 -ulose-3, 5-
dibenzoate of formula (I) is a key intermediate used for synthesis of Gemcitabine hydrochloride of Formula (II).
NH2 .HCI

Formula (II)
US Patent 4,526,988, US 4808614, GB 2136425, US5015743 discloses a process for the preparation of 2-deoxy-2,2-difluoro-1-oxo ribose (lactone) shown by the Formula (IV) wherein an alkyl-3-dioxalan-4-yl-2,2-difluoro-3-hydroxy propionate of Formula (VI) where R is C2H5 is hydrolyzed under mild conditions using mild acidic ion exchange resin.
3

Formula (IV)

Formula (VI)
Where R is Alkyl group having C1-C4 atoms.
The above mentioned patents describe the process for the preparation of lactone intermediate of formula (IV), wherein the hydrolysis of hydroxy propionate in either form is carried out under very mild conditions using acidic ion exchange resin like Dowex 50W-X12 to form the lactone form of the carbohydrate but since reaction using resin is heterogeneous, requires more time. Further, it is specifically disclosed that it is possible to carry out the process with other hydrolytic reagents, but the main disadvantage being the formation of larger amounts of by-products. For example, aqueous acetic acid, or other relatively strong acids such as propionic acid, formic acid, chloroacetic acid, oxalic acid and the like, may be used for the hydrolysis but they do not prevent the reversion reactions.
EP Patent 0306190, EP 0688782 and EP 0630905 discloses a process for preparing lactone intermediates and 2',2'-difluoronucleosides whereby reversion back to the lactone's open chain analogue (hydroxyl acid) is minimized and the desired erythro enantiomer can be selectively
4

isolated from an enantiomeric mixture of erythro and threo lactones in crystalline form.
EP 0306190 describes the use of monobenzoylated derivative for protection of hydroxyl group. The preparation of enantiomeric mixture of D-erythro and D-threo-2-deoxy-2, 2-difluoro-pentafuranos-1-ulose-3, 5-dibenozate from an enantiomeric mixture of D-erythro & D-threo-2-deoxy-2, 2-difluoro pentafuranose-1-ulose-3-benzoate is described. Starting material required for monobenzoate is enantiomeric mixture containing 3R: 3S (3: 1) poethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate. The hydrolytic agent used for the purpose is trifluroacetic acid. The process results into a crystalline lactone which is stable thereby minimizing reversion back of open chain analogue as well as minimizing formation of undesirable reaction products as expected by the use of strong hydrolytic agents. The term strong acid as defined herein is acid with pKa of about -10 to 2 at room temperature (22°C). Examples of strong acid include inorganic acid such as HCI and H2S04and organic acids such as trifluroacetic acid and p-toluene sulphonic acid. Preferred strong acids are those which have pKa value of -7 to 0. Preferred strong acids are trifluroacetic acid and para-toluene sulphonic acid but in most of the cases trifluroacetic acid is used. The acid employed is in an amount sufficient to provide about 0.05 to 0.5 molar equivalents with respect to starting material. The complete removal of isoalkylidene group takes place in about 2 to 8 hours which is then followed by lactonisation with simultaneous azeotropic removal of water, it is cyclised by distilling water/alcohol, water/acetonitrile, water/acetonitrile/ aromatic solvent.
Trifluoroacetic acid (TFA) is a strong, non-oxidizing, organic acid. TFA is a reagent used frequently in organic synthesis due to its nature as an organic-soluble strong acid. The present inventors have however found that trifluoroacetic acid when used as hydrolytic agent in manufacturing of Gemcitabine hydrochloride results in reversion to open chain analogue which affects the yield of the product.
5

The process described in EP Patent 0306190, EP 0688782 and EP 0630905 has following steps:
1. Isolation of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate.
2. Reduction of the lactone to lactol of the formula (V).

Formula (V)
Here the crude lactone containing the erythro and threo isomers was crystallized from the only use of dichloromethane by dissolving it but to increase the yield, isopropanol or hexane are used as counter solvents. EP0306190 also describe about selective isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranose-1-ulose-3,5-dibenzoate with 95% purity.
US Patent No. 6001994 and US 5912366 discloses a process to make Gemcitabine hydrochloride wherein the improvement consists essentially of making the lactone intermediate, 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1ulose-3,5-dibenzoate of Formula (I) from D-erythro-2-Deoxy-2,2-difluoro 4,5-0-(1-ethylpropylidene) pentoic acid tertiary butyl ester which is prepared by the process of reacting S-tertiary-butyl difluoroethane thioate with 2,3-0 (l-ethylpropylidene)-D-glyceraldehyde in a solvent and in the presence of a strong base; with the proviso that the process is conducted in the absence of a catalyst and in the absence of a silyl containing compound. However, the preparation of S-tertiary-butyl difluoroethane thioate involves the use of hazardous chemical like oxalyl
6

chloride. Also, due to their unpleasant odour thiols become unfavorable for synthesis. Beside this at various stages chromatographic purification is done which makes the process less economical.
PCT Application WO 2005/095430 discloses the use of trifluoroacetic acid as hydrolytic agent followed by cyclisation & benzoylation to get benzoylated lactone, its reduction to get benzoylated lactol. It further teaches the isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranose-1-ulose-3,5-dibenzoate which is achieved with the help of Methylene dichloride : Hexane & Toluene : Hexane as solvent mixture which gives the yield in the range of 20-33%, however Methylene dichloride : Hexane as a solvent for isolation is used in most of the cases described in the prior art. In addition, hexane being highly inflammable makes its use nonviable on industrial scale. Also, it is a known fact that Toluene is a high boiling solvent which requires relative higher temperature during processing contributing to some unfavorable effects on product thereby making it a less preferred solvent.
The present inventors have found that when a strong acid like trifluroacetic acid is used as a hydrolyzing agent, lactone formed during later part of reaction being more prone to reversion back it goes to the open chain analogue in presence of this strong acid. Even after applying reduced pressure in the procedure it is difficult to remove the water and also the strong acid present in the reaction mass further enables the reversion reaction and thus decreasing the yield. Beside this use of toluene during reaction, requires high temperature which results in charring of the product thereby affecting the yield of the product. As far as the solvents are concerned, the use of hexane makes this procedure very unlikely to be used at large scale. Hexane being highly inflammable makes it nonviable on industrial scale.
Isolation and crystallization of benzoylated lactone is also disclosed in US patent No. 4526988 and US Patent No. 4808614. However, the processes described in the said patents adopt laborious chromatographic
7

purifications in different stages which make the process industrially unviable.
Therefore, the processes disclosed in the prior art for the preparation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate which is a key intermediate for the preparation of Gemcitabine hydrochloride has various disadvantages such as
• the use of mild acidic resin which prolongs the duration of
process to four days thereby rendering to formation of other side
products,
• the use of strong acids like trifluoroacetic acid for hydrolysis of
acetals leads to formation of lactone but often causes reverse
back to its open chain analogue because of its sensitivity to
water,
• the relative strong acids also produce appreciable amount of
undesirable side reaction products as a result of which yield of
the intermediate produced is very low which affects the final yield
of the Gemcitabine hydrochloride.
• the processes require isolation of product formed at every stage.
Therefore, the need of the hour is an improved process preferably
an insitu or one pot process which will help in avoiding isolation of product at individual stage thereby minimizing unit operations as well as handling loss, reversion back to its open chain analogue and increasing the yield and purity significantly.
The inventors have found that the use of the presence of salts used for hydrolysis has not affected the later stage of lactone formation.The reversion back to open chain analogue is minimised to large extent unlike the strong organic and inoganic acid reagents.
The inventors have found that the use of the pyridinium salts like pyridinium trifluoroacetate or pyridinium para toluene sulphonate as a mild acid for hydrolysis in one pot, has minimized the reversion back to the lactone's open analogue thus resulting into 2-deoxy-2,2-difluoro-D-erythro-
8

pentafuranos-1-ulose-3,5-dibenzoate with improved yield (44% m/m) and high purity (>99.5%) which ultimately improves the over all yield of Gemcitabine hydrochloride.
Inventors while working on suitable hydrolytic agents found that using triethyl amine salt of trifluoro acetic acid or triethyl amine salt of para toluene sulphonic acid, hydrolysis could not be completed even after 24 hours.
Preparation of Gemcitabine hydrochloride disclosed in US patent No. 4526988 and US Patent No. 4808614, adopts laborious chromatographic purifications in different stages which make the process industrially nonviable.
The process disclosed in US Patent No. 5637688 for the isolation and crystallization of Gemcitabine hydrochloride comprises following steps:
a) Deblocking of p-1-(2'-deoxy-2',2'-difluoro-3',5'-di-0-benzoyl-D-ribofuranosyl)-4-aminopyrimidin-2-one with a catalytic amount of an alkylamine in the presence of methanol or ethanol in an environment essentially free of water;
b) Treating the resulting solution with hydrochloric acid and an antisolvent selected from the series of acetone, acetonitrile, tetrahydrofuran, propanol, butanol, isobutanol, sec-butanol and isopropanol;
c) Purifying the resulting Gemcitabine hydrochloride.
In EP0630905, isolation involves dissolution of Gemcitabine hydrochloride (1:1) in hot water and then precipitated by acetone to get 80% (3-anomer rich product which is further purified by re-dissolving in hot water and then again precipitated by acetone to get 99% (3-anomer of Gemcitabine hydrochloride.
It also describes isolation of Gemcitabine base of 99% purity from Gemcitabine or its addition salt by dissolving in hot water and increasing
9

pH to 7-9 isolating Gemcitabine of 99% and is further converted into Gemcitabine hydrochloride.
WO2005/095430 discloses the isolation of Gemcitabine hydrochloride whereby crude 2'-deoxy-2',2'-difluorocytidine base is dissolved in isopropanol and then precipitated by hydrochloric acid and filtered off. The solution so obtained is then further purified by water and acetone as solvent system which is already reported in prior art. The process of purification disclosed in WO2005/095430 includes dissolving Gemcitabine HCI in water to increase the purity and then again dissolving in water and acetone for further purification thereby increasing the unit operation.
Therefore there is a long felt need of a process for the isolation and purification of Gemcitabine hydrochloride, which is selective for the beta-anomerof the Gemcitabine hydrochloride giving high yield and purity.
Also there is a need of a process of purification which does not require any reversion of hydrochloride to base by adding other bases like alkyl amine and again converting back to hydrochloride salt which increases unit operation without giving appreciable yields or a process of purification which does not require any pH adjustment as disclosed in prior art.
Objects of the invention
Therefore, it is an object of the present invention to provide an improved process for the synthesis of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate of Formula (I) that overcomes the problems associated with prior art.
It is another object of the present invention to provide an improved process for the synthesis of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate which minimizes the reversion back to the lactone's open chain analogue.
10

It is yet another object of the present invention to provide a process for selective isolation of the erythro isomer of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate from the enantiomeric mixture of erythro and threo lactone using novel solvent system including solvent such as ethyl acetate, dichloroethane and diisopropyl ether.
Another object of the present invention is to provide better hydrolytic agent required for the synthesis of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate which minimizes the reversion back to the lactone's open chain analogue.
Another object of the present invention is to provide an improved process for the synthesis of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate using mild acid which is a salt of organic acid such as Trifluoroacetic acid and an organic base such as Pyridine.
Another object of the present invention is to provide a 2-deoxy-2, 2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate in high yield along with high purity which in turn gives high yield of Gemcitabine hydrochloride of Formula (II).
Another object of the present invention is to provide a process for the selective isolation of beta-anomer of Gemcitabine hydrochloride.
Another object of the present invention is to provide a process for purification of Gemcitabine hydrochloride.
It is further object of the present invention to provide a process for isolation & purification, which employs the use of water miscible ethers like 1,4-dioxane or Monoglyme.
Yet another object of the present invention is to provide a process for beta-anomer of Gemcitabine hydrochloride in high yield along with high purity (99.9%).
Summary of the Invention
Thus according to an aspect of the present invention, there is provided an improved one pot process for the synthesis of a 2-deoxy-2, 2-
11

difluoro-D-erythro-pentafuranose-1-ulose-3, 5-dibenzoate of Formula (I) comprising reaction of hydrolysis of (Erythro:Threo)alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)- 2,2-difluoro-3-hydroxy propionate of Formula (III) where alkyl group is having C1-C4 number of atoms with hydrolyzing agent in organic solvent and water followed by formation of lactone and dibenzoylation of the lactone. More over it is found that invention also works well with other alkyl groups like methyl, isopropyl, n-propyl and butyl (Erythro:Threo)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)- 2,2-difluoro-3-hydroxy propionate of Formula (III) however reaction with ethyl group has found to be better.

Formula (I)

Formula (III)
Where R is Alkyl group having C1-C4 atoms.
12

According to another aspect of the present invention, there is provided a process for the selective isolation of 2-deoxy-2, 2-difluoro-D-erythro-pentafuranose-1-ulose-3, 5-dibenzoate from the enantiomeric mixture of erythro and threo isomers of 2-deoxy-2, 2-difluoro-pentafuranose-1-ulose-3, 5-dibenzoate using novel solvents such as ethyl acetate, ethylene dichloride and Diisopropyl ether.
Thus according to an aspect of the present invention there is provided a process for the isolation of P-anomer enriched Gemcitabine hydrochloride of Formula (II) comprising conversion of Gemcitabine base having a and p anomers into p-Gemcitabine hydrochloride using organic solvent chosen from the series of water miscible ethers like 1,4-dioxane or Monoglyme.
According to another aspect of the present invention there is also provided a process for the purification of p-Gemcitabine hydrochloride of Formula (II) comprising dissolving of Gemcitabine hydrochloride (P-anomer enriched) in water and selectively crystallizing using organic solvent chosen from water miscible ethers like 1,4-dioxane or Monoglyme.
Brief Description of Figures:
Figure 1: DSC of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate shows an endotherm at 123.69°C Figure 2 DSC of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate shows an endotherm at 123.15°C Figure 3: DSC of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate shows an endotherm at 123.19°C DSC stands for Differential Scanning Calorimetry^
Detailed Description of the Invention
The present invention addresses the need of a one pot process for the synthesis of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate of Formula (I) and loss due to reversion of lactone by
13

minimizing the reversion back to the lactone's open chain analogue formed which affects the overall yield of the Gemcitabine hydrochloride.

Formula (I)
Also the present invention provides a process for exclusively isolating 2-deoxy-2, 2-difluoro-D-erythro-pentafuranose-1-ulose-3, 5-dibenzoate from the mixture of erythro and threo isomers of 2-deoxy-2, 2-difluoro- pentafuranose-1-ulose-3, 5-dibenzoate.
The inventors of the present invention have surprisingly found that use of mild acid which is salt of organic acid such as Trifluoroacetic acid and organic base such as Pyridine as a hydrolytic agent minimizes the reversion back to the lactone's open chain analogue and thus gives good yield as compared to the prior art.
The preferred mild acid according to the present invention is a Lewis acid having pKa value around 4.00 to 6.00, more preferably between 5.00-5.5. The preferred Lewis acid according to the present invention is pyridinium trifluoroacetate.
The present invention provides an improved one pot process for the hydrolysis cum lactonisation of alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate where alkyl group is having C1-C4 number of carbon atoms using acetonitrile as solvent in presence of water and
14

pyridinium salts like pyridinium trifluoroacetate or pyridinium para toluene sulphonate as mild hydrolytic agent which also could be supporting in lactonisation to provide the required lactone in a quantitative yield , which is then taken for insitu benzoylation. However use of pyridinium para toluene sulphonate although resulted in good yield and purity but duration required for the same is more.
Alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate of required R isomer was charged into a round bottom flask, and acetonitrile and water and pyridinium salts like pyridinium trifluoroacetate or pyridinium para toluene sulphonate were added to it and the reaction mixture was heated to reflux. The reaction mixture was stirred at reflux temperature for 2- 15 preferably 3-12 hours. Mixture of Acetonitrile and water was azeotropically distilled completely to give oil. Ethyl acetate was added in the reaction mass and subjected to distillation at NTP. The obtained oil was dissolved in ethyl acetate; dry pyridine was added followed by 4,4'-dimethyl aminopyridine. The mixture was heated to 60-65°C under N2 atmosphere. Solution of benzoyl chloride in ethyl acetate for benzoylation was added drop wise over 3 hours at 60-65°C. The reaction was stirred at 60-65X for another 3 hours and then cooled to room temperature (25-30°C) and stirred for another 3 hours. The reaction mass was cooled to 0-5°C and stirred for another 1 hour and filtered. The filtrate was concentrated under vacuum at 35°-40 °C to yield oil.
The preferred solvents according to present invention for the selective isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate from the mixture of erythro and threo isomers to get selective erythro isomer are ethyl acetate, ethylene dichloride and diisopropylether.
Thus, the oil obtained above was taken into ethyl acetate or ethylene dichloride followed by distillation to get a thick mass to which ethyl acetate or ethylene dichloride was then added under stirring and
15

contents were cooled to 0-5°C and maintained for half an hour. Diisopropyl ether was then added to the contents and continued further for 10-15 min. Product so obtained is filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C.
The selective erythro isomer of 2-deoxy-2,2-difluoro-D-erythro-pentafuronose-1-ulose-3,5-dibenzoate is obtained in high yield which is about 44% (m/m) along with purity >99.5%.
Entire Schematic representation for the preparation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3, 5-dibenzoate is shown as follows:
Step -1: Acetal formation of D-mannitol

OH OH

D-Mannitol 2,2-dimethoxypropane

l,2:5,6-diisopropylidine- D-mannitol

Step -2: Preparation of 2,3-isopropylidene-D-Glyceraldehyde

Methylene dichloride

HX, -CH*

l,2:5,6-diisopropylidine-D-mannitol 2,3-O-isopropylidene-D-glyceraldehyde
Step -3: Preparation of (Erythro:Threo)Ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxypropionate
16

O. XH
o
OH

CH, P
H3C

Bromodifluoro ethylacetate
*»
Zn, Tetrahydrofuran

H3C

O

2,3-O-isopropylidene-D-glyceraldehyde

(Erythro:Threo)Ethyl-3-(2,2-dimethyl-l,3-dioxolan -4-yl)-2,2-difluoro-3-hydroxy propionate Erythro:Threo(3:l)

Step -4: a) Insitu preparation and benzoylation of 2-deoxy-2,2-difluoro-pentafuranose -1-ulose-to get 3,5-dibenzoate derivative.
For the present invention insitu schematic representation for the
conversion of (erythro:threo)ethyl-3-(2,2-dimethyl-1 -3-dioxalane-4-yl)-2,2-
difluoro-3-hydroxy propionate into 2-deoxy-2,2-difluoro-pentafuranos-1-
ulose-3,5-dibenzoate is as follows:-

^,
+ 1 X
H
1. Acetonitrile,
2. Benzoyl chloride, pyridne DMAP in ethyl acetate

(Erythro:Threo)Ethyl-3-(2,2-dimethyl-l,3-dioxolan -4-yl)-2,2-difluoro-3-hydroxy propionate Erythro:Threo(3:l)

2-deoxy-2,2-difluoro-D-pentafuranose-l-ulose-3,5-dibenzoate
(Mixture of Erythro & Threo)

x" Trifluoroacetate / Para toluene sulfonate
b) Isolation of erythro isomer selectively using solvents like Ethyl acetate or Ethylene dichloride with Diisopropyl ether as solvent system.

athe use of novel solvents such as water miscible ethers like 1,4-dioxane or Monoglyme. The ratio of isolated Gemcitabine hydrochloride is in the range 80-90% (3 and 4-12% a anomer.
The process for the purification of Gemcitabine hydrochloride containing 80-90% p-anomer further uses the concept of solvent and antisolvent wherein water is used as so Ivent and antisolvent is chosen from the series of water soluble ethers resulting into selective crystallization of Gemcitabine hydrochloride with high isomeric purity of 99.9% and higher yields.
Entire Schematic representation for the isolation and purification of
Gemcitabine hydrochloride is shown as follows:
5) Reduction of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate:
2-deoxy-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate was
reduced to benzoylated lactol (2-Deoxy-2,2-difluoro-D-ribofuranose-3,5-dibenzoate) using Vitride which is sodium bis (2-methoxyethoxy) aluminium hydride and commercially available as a 70% w/w solution in toluene as reducing agent and Tetrahydrofuran as solvent in temperature range of -40 to -70°C. The duration of the

reaction was around 30 -120 minutes. 2-deoxy-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate was isolated by usual method resulting in the yield in the range of 90-95% and having purity of 80-85%.

C6H5OCO
0.

Vitride, Tetrahydrofuran
\ \/ »•
F
C6H5OCO
2-deoxy-2,2-difluoro-D-erythro-pentafuranos- 2-deoxy-2,2-difluoro-D-riboforanose-3,5-dibenZoate
l-ulose-3,5-dibenzoate
Mixture of a & (3
6) Methyl sulfonationa of 2-deoxy-2,2-difluoro-D-ribofuranose— 3,5-dibenzoate:
Benzoylated lactol (2-Deoxy-2,2-difluoro-D-ribofuranose-3,5-
dibenzoate) so obtained was subjected to reaction with methanesulfonyl chloride in methylene dichloride as solvent and triethylamine as base at temperature of -25 to +10°C, preferably at -20 to 5°C, more preferably at -20 to 0°C to form 2-deoxy-2,2-difluoro-D-ribofuranose-3,5-dibenzoate-1 -methane sulfonate 90-95 yield & HPLC purity 80-85%.

C,H,OCO-
Methanesulfonyl chloride \( N^tfOMs
Triethyl amine , Methylene dichloride
C6H5OCO
2-deoxy-2,2-difluoro-D-ribofuranose-3,5-dibenzoate 2-deoxy-2,2-difluoro-D-ribofuranose-3,5-
dibenzoate-1-methane sulfonate
Mixture ofa& P
7) Condensation of 2-deoxy-2,2-difluoro-D-ribofuranose— 3,5-dibenzoate-1-methanesulfonate with N -acetyl bis trimethylsilyl cytosine derivative followed by preparation of Gemcitabine base:
The bistrimethylsilyl derivative of acetyl cytosine was prepared by treatment of acetyl cytosine with hexamethyldisilazane and trimethylsilyl chloride in ethylene dichloride. The product from step 2 was then subjected to coupling with bistrimethylsilyl derivative of acetyl cytosine in presence of trimethylsilyl triflate at 80-82X, resulting into 2'-deoxy-2',2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate (1.2-1.3w/w).
The 2'-deoxy-2\2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate was hydrolyzed in methanol 1:8 to 15 w/v using ammonia at a temperature in the range of -10 to +10°C preferably at -5 to +5°C for 5-20 hours preferably 8-10 hours. After the hydrolysis, the methanolic solution was concentrated to get a thick mass which was then dissolved in water and extracted with ethyl acetate followed with Diisopropyl ether. Aqueous layer so obtained was concentrated under vacuum at 30-60°C preferably at 40-45°C
Isolation of Gemcitabine hydrochloride:
The Gemcitabine base so obtained was converted into its hydrochloride by adding solvents like 1,4-dioxane or Monoglyme in ratio of 1:4-12(w/v) preferably 1:5-8(w/v) and heating the contents at 40-90°C preferably at 50-70X more preferably at 60-68°C

followed by cooling the contents to 10-30°C preferably 10-15°C to give crude (solid) Gemcitabine hydrochloride having 80-90% of B-anomer & 4-12% of a-anomer.

HO
OH
Mixture of a & P

Monoglyme+Con.HCI or dioxane+Con.HCI
»-

P anomer enriched (80-90)% a anomer(4-12)%

Purification of Gemcitabine hydrochloride:
The isolated product obtained in the above reaction has a-anomer (4-12%) and B-anomer (80-90%) which is further purified to 99.9% by dissolving crude Gemcitabine hydrochloride in water 1:4-8(w/v) preferably 1:5-7(w/v) and heating the contents at 40-90°C preferably at 50-70°C more preferably 60-68°C to get a solution to which solvents like 1,4-dioxane or Monoglyme in the ratio of 1:40-100(w/v) preferably 1:50-70(w/v) are added followed by heating the contents at 40-90°C preferably at 50-70°C followed by cooling to 10-30°C preferably 10-15°C to get Gemcitabine hydrochloride of purity 99.9%. The overall yield at this stage with respect to 2'-deoxy-2',2'-difluoro-N-1-acetyl cytidine-3',5'-dibenzoate is 15 to 16%.
21

The preferred solvent is water and the preferred anti solvent is selected from the series of water soluble ethers preferably 1,4-dioxane and Monoglyme with improved yield and high purity of 99.9%. The most preferred solvents for the isolation of p-anomer of Gemcitabine hydrochloride and purification of Gemcitabine hydrochloride rich in p-anomer are the water miscible ethers like 1,4-dioxane or Monoglyme.
The purity & assay of the product thus obtained is analyzed using HPLC.
Column specification: 4.6 mm X 25 cm column, that contains 5|_i packing L7.
Wave length selected: 275 nm
Mobile phase: Prepare a filtered and degassed solution containing 13.8 g of monobasic sodium phosphate and 2.5 ml of phosphoric acid in 1000 ml of water.
Flow rate: 1.2 ml/min
Diluent: Filtered and degassed water for HPLC
The details of the invention, its objects and advantages are explained hereunder in greater details in relation to non-limiting exemplary illustrations. The examples are merely illustrative and do not limit the teaching of this invention and it would be obvious that various modifications or changes in the procedural steps by those skilled in the art
22

without departing from the scope of the invention and shall be consequently encompassed within the ambit and spirit of this approach and scope thereof.
Examples:
Example 1; Preparation of 1,2 : 5,6-diisopropylidene-D-mannitol:
To a 5L four neck round bottom flask fitted with Thermometer pocket, condenser with CaCI2 guard tube and overhead stirrer were added 600 ml of 1,2-dimethyoxy ethane, 250gm of D-mannitol and 400 ml of 2,2-dimethoxy propane. The mixture was stirred at room temperatures and 375 mg of anhydrous stannous chloride was added to the reaction mixture. The slurry was heated to reflux for approximately 50 to 90 min until mixture becomes clear. The solution was cooled below reflux temperature & pyridine (0.5ml) was added to the reaction mixture and the same was stirred for 10 min. The solution was concentrated below 40 to 45°C to give thick solid mass. The mixture was allowed to cool to room temperature (25°C). Methylene dichloride (500 ml) was added to it and mixture was stirred at room temperature for 30 min.Hexane(2. 5L) was added to this mixture and cooled to 0-10°C and stirred for more 30 min and filtered off. The white solid was dried in vacuum at 35-40°C for 4 hrs. 200gm white solid obtained has GC purity 99%.
Example 2: Preparation of 2,3-O-lsopropylidene-D-glyceraldehyde:
The solid (200g) 1,2:5,6-diisopropylidene-D-mannitol obtained as in example 1 was added to 2000ml of Methylene dichloride. The mixture was warmed to 30-32°C and stirred for 30 min. The resulting solution was filtered. The filtrate obtained was cooled to 15°C and 10% aqueous Sodium bicarbonate solution (80ml) was added to the reaction mass. Sodium periodate (317 gm) was added to reaction mixture and the resulting slurry is stirred at temperature between 20-25°C for another 2 hours. 100 gm of sodium sulfate was added to the reaction & the slurry is stirred for another 15 min. The reaction mass was filtered and the solid
23

residue was washed twice with 200 ml of methylene dichloride. The resulting filtrate is collected and solvent was distilled at 40-45X to obtain oil which was heated up to 50-52°C under vigorous stirring to remove traces of solvent. The oil weight is 180 gm with 95% (GC purity).
Example 3: Preparation of (Erythro:Threo) ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate :
To the 5L round bottom flask fitted with overhead stirrer, condenser, pressure equalizing dropping funnel 1.8L of tetrahydrofuran was added. Zn dust (180 gm) and trimethylsilyl chloride (28.76ml) were added to it under stirring. The mixture was stirred at room temperature for 15 min and than heated to reflux and mixture of 2,3-O-lsopropylidene-D-glyceraldehyde (180 gm) and Bromodifluoroethyl acetate (248 ml) was added through dropping funnel to the reaction mixture slowly over a period of 30-40 min. The reaction was stirred with reflux for another 2hours. After 2 hours the reaction mass was cooled to room temperature and poured in mixture of 143 ml of Cone. HCI and 1100 gm ice under stirring. The mixture was stirred for 15 min and the organic layer was separated. The aqueous layer was further extracted with ethyl acetate (200mlX 3). The collective organic layer was washed with brine followed by 5% aq. Sodium bicarbonate solution. The organic layer was dried over sodium sulfate and concentrated under vacuum to yield the product (250 gm) with GC purity of desired isomer 75%.
Example 4: Preparation & isolation of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate:
To a 3L four-necked round bottom flask 82g of ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate having 75% of required R isomer was charged, and acetonitrile (820ml) and water (26ml) and pyridinium trifluoroacetate (14.76g) were added to it and the reaction mixture was heated to reflux. The reaction mixture was stirred at reflux temperature for 3 hours. Mixture of Acetonitrile and water was azeotropically distilled completely to give oil. Ethyl acetate (90ml) was
24

added in the reaction mass and subjected to distillation at NTP. The obtained oil was dissolved in ethyl acetate (328ml), dry pyridine (96ml) was added followed by 4, 4'-dimethyl aminopyridine (9.52g). The mixture was heated to 65°C under N2 atmosphere. Solution of benzoyl chloride (96ml) in ethyl acetate (255ml) was added drop wise over 3 hours at 65°C. The reaction was stirred at 65°C for another 3 hours and then cooled to room temperature (25-30°C) and stirred for another 3 hours. The reaction mass was cooled to 0-5°C and stirred for another 1 hour and filtered. The filtrate was concentrated under vacuum at 35°C to yield 160 g oil. Oil so obtained was taken into 164ml ethyl acetate followed by distillation to get a thick mass to which 61.5 ml ethyl acetate was then added under stirring and contents were cooled to 0-5°C followed by keeping for half an hour. Diisopropyl ether (205ml) was then added to the contents and continued further for 10-15 min. Product so obtained is filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. Yield (53.3g), HPLC purity of +99.5%, DSC has shown the endotherm in the range of 121-125X (peak at 123.69°), Figure 1.
Example 6: Preparation & isolation of 2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1 -ulose-3,5-dibenzoate:
To a 3L four-necked round bottom flask 90g of ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate having 75% of required R isomer was charged, and acetonitrile (900ml) and water (29ml) and pyridinium para toluene sulphonate (19.6g) were added to it and the reaction mixture was heated to reflux. The reaction mixture was stirred at reflux temperature for 12 hours. Mixture of Acetonitrile and water was azeotropically distilled completely to give oil. Ethyl acetate (100ml) was added in the reaction mass and subjected to distillation at NTP. The obtained oil was dissolved in ethyl acetate (360ml), dry pyridine (105ml) was added followed by 4, 4'-dimethyl aminopyridine (10.44g). The mixture was heated to 65°C under N2 atmosphere. Solution of benzoyl chloride (105ml) in ethyl acetate (260ml) was added drop wise over 3 hours at
25

65°C. The reaction was stirred at 65°C for another 3 hours and then cooled to room temperature (25-30°C) and stirred for another 3 hours. The reaction mass was cooled to 0-5°C and stirred for another 1 hour and filtered. The filtrate was concentrated under vacuum at 35°C to yield 160 g oil. Oil so obtained was taken into 170ml ethyl acetate followed by distillation to get a thick mass to which 61.5 ml ethyl acetate was then added under stirring and contents were cooled to 0-5°C followed by keeping for half an hour. Diisopropyl ether (210ml) was then added to the contents and continued further for 10-15 min. Product so obtained is filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. Yield (59g), HPLC purity of +99.5%. DSC has shown endotherm in the range of 121-125X (peak at 123.15°) Figure 2.
Example 7:
Procedure in example E was repeated with 162 g (Erythro:Threo) ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-3-hydroxy propionate resulting in to 290 g oil. Thick oil so obtained is taken into 300ml ethylene dichloride followed by distillation under vacuum at 55-60°C to get a thick mass to which 122 ml ethylene dichloride was then added under stirring and contents were cooled to 0-5°C followed by keeping for half an hour. Diisopropyl ether (400ml) was then added to the contents and continued further fori0-15 min. Product so obtained was filtered off washed with Diisopropyl ether and dried under vacuum at 40-45°C. Yield (105.3g), HPLC purity of +99.5 % & more, DSC has shown endotherm in the range of 121-125°C (peak at 123.19°) Figure 3.
Example 8: Preparation of 2-deoxy-D-erythro-2,2-difluoro-
ribofuranose-3, 5-dibenzoate:
2-deoxy-D-erythro-2,2-difluoro-pentafuranos-1-ulose-3,5-dibenzoate (200g) was dissolved in (2 L) of tetrahydrofuran in a 5L round bottom flask. (0.2L) of vitride (70% solution in toluene) was added between -50 to -70°C. The reaction was continued for 60 minutes. On completion,
26

reaction was arrested by adding methanol (0.24L) followed by hydrolysis with 7% HCI (1.3L). After stirring for ~30 min the aqueous layer was separated at RT. The aqueous layer was repeatedly extracted with Ethyl acetate (0.8 L X 2). The collected organic layer was washed with brine (0.5 L X 2) and 5% aqueous NaHC03 (0. 5 L X2) followed by brine (0. 5 L X 2). The organic layer was concentrated under vacuum to get thick oil (0. 2kg).
Example 9: Preparation of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methanesulfonate:
2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate (0.2kg) oil was dissolved in (2L) Methylene dichloride. The reaction mixture was cooled to 0 to -20°C and (0.12 L) triethylamine was added. To this stirred mixture was added (0. 06L) Methane sulfonylchloride drop wise between -20°C to -15°C. The reaction was stirred at same temperature till completion of reaction (in ~3hours). Temperature was brought to room temperature & 5% HCI (0.9 L) was added to reaction. The aqueous layer was separated and extracted with Methylene dichloride (0.4 L X 2). The organic layer was washed with brine (0.5 L X2) & by 5% aqueous sodium bicarbonate followed by brine (0.5 L X 2). The organic layer was collected and concentrated to give oil (0.24kg).
Example 10: Preparation of Gemcitabine base:
Acetyl cytosine (200g) was added to 1,2-dichloroethane (4L) in 10L round bottom flask. Hexamethyldisilazane (0.34L) was added to the slurry. The resulting mixture was heated to distill out ~1 L mixture. The reaction mass cooled to 50°C under nitrogen and trimethyl silyl chloride (0.015 L) was added to the mixture. The resulting mixture was refluxed at 82-85°C for 3 hours. The solution was cooled to 45°C under nitrogen and distilled at 45-50°C to obtain solid. Fresh ethylene dichloride (2.6 L) was added to the solid so obtained followed by addition of Trimethyl silyl triflate (0.26 L). To this solution of mesylate (0.24 L) obtained from example no 2 dissolved in ethylene dichloride (1.2 L) was added. The resulting mixture was heated
27

to reflux at 86-92°C for 18 hours till 2-deoxy-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methnesulfonate was consumed. The reaction mixture was cooled at room temperature and 5% HCI (1.8 L) was added to the reaction mixture followed by separation of aqueous layer and extraction with ethylene dichloride (0.4 L X 2). The combined organic layer was washed with brine (1 L X 2). The collected organic layer was treated with sodium bicarbonate (0.2 Kg) to neutralize acidity. The dried organic layer was concentrated to give oil (0.26kg). The resulting oil obtained in previous example (0.26kg) was dissolved in methanol (3.4L). The resulting solution was cooled to -5 to +5°C and ammonia gas purged through the mixture under stirring for 8-10 hours between -5 to +5°C till complete consumption of 2',2'-difluoro-2'-deoxy-N-acetyl cytidine-3',5'-dibenzoate. The temperature was allowed to increase to room temperature. The solution was concentrated under vacuum to obtain oil which was dissolved in water (0.8 L). Ethyl acetate (0.2 L) was added to it and the mixture was stirred for 15 min. The aqueous layer separated was extracted with ethyl acetate (0.2 L). The combined ethyl acetate layer was extracted with (2.6 L) water. The aqueous layer was filtered and concentrated under vacuum at 40-45°C to give oil (0.1 Kg).
Example 11: Isolation of Gemcitabine hydrochloride:
To the 20g of thick oil of Gemcitabine base obtained as in example 10 was added 1,4-Dioxane (120 ml) and the mixture was heated to 60°-68°C and stirred for 15 minutes. Cone. HCI (10ml) was then added to the mixture and the contents were heated at same temperature for one hour. Reaction mass was then cooled to 10-15°C and maintained for 30 minutes. The solid thus obtained was filtered off and washed with 1,4-Dioxane and dried under vacuum at 55-60 °C to get 8g material with Specific Optical Rotation i.e.[a]D = +40° and HPLC purity of 85% B-anomer and 8% a-anomer.
Example 12: Purification of Gemcitabine hydrochloride:
28

10g material obtained using protocol of example 11 was taken in water (60 ml) and the mixture was heated to 55-60°C to get a clear solution. 1,4-Dioxane (600ml) was added to the solution under stirring and continued for one hour at 60-68°C. The contents were cooled to 10-15°C & the mixture was stirred at this temperature for 30 min and filtered, washed with 1,4-Dioxane and dried at 55-60°C under vacuum to get 7.5g compound of HPLC purity 99.9% p-anomer with Specific Optical Rotation i.e.[a]D = +47°.
Example 13: Isolation of Gemcitabine hydrochloride:
To 20g of thick oil of Gemcitabine base obtained in example 10 was added Monoglyme (120 ml) and the mixture was heated at 60 - 68°C and stirred for 15 minutes. Cone. HCI (10 ml) was then added to the mixture and the contents were heated at same temperature for one hour. Reaction mass was then cooled to 10-15°C and maintained for 30 minutes. The solid thus obtained is filtered off and washed with Monoglyme and dried under vacuum at 55-60°C to get 8g material having HPLC purity with 85% P-anomer & 9% a-anomer, Specific Optical Rotation i.e.[a]o= +40°
Example 14: Purification of Gemcitabine hydrochloride:
10 g material obtained as in example 13 was taken in water (60 ml) and the mixture was heated to 60-65°C to get a clear solution. Monoglyme (600ml) was added to the solution under stirring and continued for one hour at 60-65°C. The contents were cooled to 10-15°C. The mixture was stirred at this temperature for 30 minutes and filtered, washed with Monoglyme and dried at 55-60°C under vacuum to get 7.3 g product with HPLC purity 99.9% P-anomer with Specific Optical Rotation i.e.[a]D= +47°.
The present invention has following advantages:
• The process according to the present invention minimizes the reversion back to the lactone's open chain analogue thereby
29

increasing the yield of lactone intermediate & overall yield of Gemcitabine hydrochloride effectively.
• Time required in the present invention for the process of hydrolysis during synthesis of 2-deoxy-2,2-difluoro-D erythro-pentafuronose-1-ulose-3,5-dibenzoate is reduced from four days to 3-12 hours.
• An Insitu operation, one of the essential features of the present invention has minimized unit operations as well as handling loss also, as isolation of individual stages is avoided.
• 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1 -ulose-3,5-dibenzoate is obtained in high yield which is about 44% (m/m) and 65% (w/w).
• Selective isolation of 2-deoxy-2,2-difluoro-D-erythro-pentafuranos-1-ulose-3,5-dibenzoate from the enantiomeric mixture of erythro and threo isomers with good purity and higher yield could be achieved with the help of solvents like ethyl acetate or ethylene dichloride with diisopropylether which have not been reported earlier.
• Gemcitabine hydrochloride isolated is not only pure but also higher in yield than reported earlier.
30

Claims:
.1. A process for the preparation of 2-deoxy-2,2-difluoro-D erythro-pentafuronose-1-ulose-3,5-dibenzoate of the formula (I)

FORMULA (I)
comprising
a) hydrolysis of (Erythro:Threo)alkyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)- 2,2-difluoro-3-hydroxy propionate of formula (III) comprising the use of mild acid,
CH3

Formula (III)
Where R = Alkyl group having C1-C4 atoms
b) cyclisation to form lactone and
c) benzoylation of lactone
d) Isolation of erythro isomer using novel solvents
The process according to claim 1 wherein said alkyl group of formula (III) is ethyl.
The process according to claim 1 wherein the mild acid is a salt of organic acid such as trifluroacetic acid or para toluene sulphonic acid and organic base such as Pyridine.
31

4. The process according to claim 1 wherein the mild acid is selected from pyridinium trifluoroacetate or pyridinium para toluene sulphonate.
5. The process according to claim 1 wherein the preferred mild acid is pyridinium trifluoroacetate
6. The process according to claim 1 wherein the mild acid has the pKa value around 4.00 to 6.00, more preferably between 5.00-5.5.
7. A process for selective isolation of erythro isomer of 2-deoxy-2,2-difluoro-D erythro-pentafuronose-1 -ulose-3,5-dibenzoate comprising dissolving the enantiomeric mixture of erythro and threo isomers in ethyl acetate or ethylene dichloride, cooling the solution and adding Diisopropyl ether followed by collecting the precipitated erythro enantiomer.
8. The process according to claim 7 wherein the selective erythro isomer of 2-deoxy-2,2-difluoro-D-erythro-pentafuronose-1-ulose-3,5-dibenzoate is obtained in yield of about 44% (m/m) along with purity >99.5%.
9. A process of isolating (3-anomer enriched Gemcitabine hydrochloride from the mixture of a & p anomers of Gemcitabine base the steps comprising of
a) adding water soluble ether to Gemcitabine base
b) heating the mixture to dissolve the base (oil) and adding thereto hydrochloric acid;
c) cooling the mixture and
d) isolating the precipitated (3-anomer enriched Gemcitabine hydrochloride.

10. The process according to claim 9 wherein the said water soluble ether is selected from the group comprising of 1,4-dioxane or Monoglyme.
11. The process according to claim 9 wherein the ratio of Gemcitabine base to said water soluble ether is in the range of 1 : 4 -12 (w/v)
32

12.The process according to claim 11 wherein the ratio of Gemcitabine base to said water soluble ether is in the range of 1 : 5 - 8 (w/v).
13. The process according to claim 9 wherein the Gemcitabine base along with the water soluble ether is heated at a temperature of 40-90°C.
14. The process according to claim 13 wherein the Gemcitabine base along with the water soluble ether is heated at a temperature of 50-70°C.
15. The process according to claim 13 wherein the Gemcitabine base along with the water soluble ether is heated at a temperature of 60-68°C.
16. The process according to claim 9 wherein the reaction mixture is cooled to a temperature of 10-30°C.
17. The process according to claim 16 wherein the mixture is cooled to a temperature of 10-15°C.

18. The process according to claim 9 wherein the said P-anomer enriched Gemcitabine hydrochloride has 80-90% of p-anomer and 4-12% of a-anomer.
19. A process for purification of P-anomer enriched Gemcitabine hydrochloride comprising the steps of

a) dissolving p-anomer enriched Gemcitabine hydrochloride in water by heating the contents ;
b) addition of a water soluble ether and cooling the mixture; and
c) crystallizing the purified P-anomer enriched Gemcitabine hydrochloride.

20. The process according to claim 19 wherein the ratio of Gemcitabine hydrochloride to water is in the range of 1 : 4 - 8 (w/v)
21. The process according to claim 20 wherein the ratio of Gemcitabine hydrochloride to water is in the range of 1: 5 - 7 (w/v).
33

22. The process according to claim 19 wherein the Gemcitabine hydrochloride and water are heated at a temperature of 40-90°C.
23. The process according to claim 22 wherein the Gemcitabine hydrochloride and water are heated at a temperature of 50-70°C.
24. The process according to claim 22 wherein the Gemcitabine hydrochloride and water are heated at a temperature of 60-68°C.
25. The process according to claim 19 wherein the ratio of solution of Gemcitabine hydrochloride to said water soluble ether in the range of 1 :40-100(w/v)
26. The process according to claim 25 wherein the ratio of solution of Gemcitabine hydrochloride to said water soluble ether is in the range of 1 : 50 - 70 (w/v )
27. The process according to claim 19 wherein the said water soluble ether is selected from the group consisting of 1,4-dioxane and Monoglyme.
28. The process according to claim 19 wherein the reaction mixture is cooled to a temperature of 10-30°C.
29. The process according to claim 28 wherein the reaction mixture is cooled to a temperature of 10-15°C.
30. The process according to claim 19 wherein the said (3-anomer
enriched Gemcitabine hydrochloride has 99.9% of p-anomer.
30. The process according to claim 19 wherein the said purified (3-
Gemcitabine hydrochloride has yield in the range of 15-16% with
respect to 2'-deoxy-2',2'-difluoro-N-acetyl cytidine-3',5'-dibenzoate.
Dated this 10th day of August 2006

Abstract
An improved one pot process for preparing 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose-3,5-dibenzoate of Formula (I) comprising hydrolysis of (erythro:threo) alkyl-3-dioxalan-4-yl-2,2-difluoro-3-hydroxy propionate of Formula (III) where alkyl group is having C1-C4 number of Carbon atoms using a mild acid and selectively isolating 2-deoxy-D-erythro-2,2-difluoro-pentafuranose-1-ulose-3,5-dibenzoate from the mixture of erythro & threo enantiomers using ethyl acetate, ethylene dichloride and diisopropyl ether as solvents with improved yield and purity.
A process for isolating (3-anomer enriched Gemcitabine hydrochloride by converting Gemcitabine base into Gemcitabine hydrochloride followed by its purification using solvents from the series of water soluble ethers like 1,4-dioxane or Monoglyme.

35

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1362-mum-2005-annexure to form 3(14-8-2006).pdf

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Patent Number

247883

Indian Patent Application Number

1362/MUM/2005

PG Journal Number

22/2011

Publication Date

03-Jun-2011

Grant Date

30-May-2011

Date of Filing

28-Oct-2005

Name of Patentee

ARCH PHARMALAB LIMITED

Applicant Address

"H" WING, 4TH FLOOR, TEX CENTER, OFF SAKI VIHAR ROAD,CHANDIVALI, ANDHERI (E), MUMBAI

Inventors:

#	Inventor's Name	Inventor's Address
1	ASHISH UJAGARE	"H" WING, 4TH FLOOR, TEX CENTER, OFF SAKI VIHAR ROAD,CHANDIVALI, ANDHERI (E), MUMBAI-400072
2	D. A. KOCHREKAR	"H" WING, 4TH FLOOR, TEX CENTER, OFF SAKI VIHAR ROAD,CHANDIVALI, ANDHERI (E), MUMBAI-400072
3	MATHEW C. UZAGARE	"H" WING, 4TH FLOOR, TEX CENTER, OFF SAKI VIHAR ROAD,CHANDIVALI, ANDHERI (E), MUMBAI-400072

PCT International Classification Number

C07D307/26

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA