DESC:The following specification particularly describes the invention and the manner in which it is to be performed.

INTRODUCTION TO THE INVENTION
The present invention relates to a process for the preparation of Gemcitabine hydrochloride and intermediates thereof.

BACKGROUND OF THE INVENTION
Gemcitabine hydrochloride is chemically known as 2´-deoxy-2´, 2´-difluorocytidine monohydrochloride (ß-isomer) (hereinafter referred to by its adopted name gemcitabine hydrochloride) and has the structural Formula-I.

Gemcitabine hydrochloride is a nucleoside analogue useful in the treatment of several types of tumors and is available in the market under the brand name GEMZAR® in the form of injection. A vial of Gemzar contains either 200 mg or 1 g of gemcitabine hydrochloride (expressed as free base).
U.S. Patent No. 4,526,988 discloses gemcitabine hydrochloride and a process for its preparation. The ‘988 patent specifically discloses the use of hydrolysis reagents such as mildly acidic ion exchange resins and more particularly this patent illustrates the use of tertiary-butyldimethylsilyl as a protecting group in the synthesis of gemcitabine.
U.S. Patent No. 5,223,608 discloses a process for the preparation of gemcitabine hydrochloride. The ‘608 patent specifically illustrates the use of hydrolysis reagents such as strong acids for the preparation of 2-deoxy-2, 2-diflouro-D-erythro-pentofuranos-1-ulose-3, 5-dibenzoate of Formula VA and more particularly this patent illustrates the use of benzoyl as a protecting group in the synthesis of gemcitabine.
International Patent Application Publication No. WO 2005/095430 A1 discloses a process for the preparation of gemcitabine hydrochloride. The ‘430 application describes the use of hydrolysis reagents like strong acids for the preparation of 2-deoxy-2, 2-diflouro-D-erythro-pentofuranos-1-ulose-3, 5-dibenzoate.
International Application Publication No. WO 2006/092808 discloses a process for the purification of gemcitabine hydrochloride by using recrystallization with water and acetic acid to get 99.94% of the ß-anomer from the original 95% of the ß-anomer.
These are the major approaches for the preparation of gemcitabine hydrochloride. The above approaches employ hydrolysis reagents such as mildly acidic ion exchange resins and strong acids in the formation of unprotected or protected lactone. Both types of hydrolysis reagents can cause problems. These approaches suffer from the fact that in the formation of lactone by the use of ion exchange resin and strong acids there may be formed undesirable reaction products as impurities and often lactone reverts back to its open chain precursor because of its sensitivity with strong acids and resins.
The present invention relates to a process for the preparation of
intermediates, and improvements in the purification techniques to afford gemcitabine hydrochloride substantially free from impurities.

SUMMARY OF THE INVENTION
In an aspect, the present invention relates to an improved process for the preparation of Gemcitabine hydrochloride of Formula-I and intermediates thereof.

The present invention provides an improved process for the preparation of Gemcitabine Hydrochloride as depicted in Scheme A

Scheme A

The present invention relates to the process for the preparation of gemcitabine hydrochloride of Formula I comprising the steps of:
I. Condensation of (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 and silylated N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Formula 5 to obtain compound of Formula 6,
II. Deprotection of the compound of Formula 6 to obtain Gemcitabine freebase, compound of Formula 7,
III. Conversion of the compound obtained in step 4 to its Hydrochloride salt

In another aspect the present invention relates to the process for the preparation of gemcitabine hydrochloride of Formula I comprising the steps of:

1. Conversion of ((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate of Formula 2 to (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 by reacting with Benzoyl chloride in a suitable solvent and base,
2. Silylation of N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Fomula 4 with Hexa methyl trisilazane in the presence of a suitable catalyst,
3. Condensation of (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 and silylated N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Formula 5 to obtain compound of Formula 6,
4. Deprotection of the compound of Formula 6 to obtain Gemcitabine freebase, compound of Formula 7,
5. Conversion of the compound obtained in step 4 to its Hydrochloride salt.

The process of present invention is simple, improved, efficient, industrially feasible, and ecofriendly. It provides the desired compound of Formula I with desired yield and purity.
DETAILED DESCRIPTION OF THE INVENTION

In an aspect, the present invention relates to an improved process for the preparation of Gemcitabine hydrochloride of Formula-I and intermediates thereof.

The present invention relates to the process for the preparation of gemcitabine hydrochloride of Formula I comprising the steps of:

I. Condensation of (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 and silylated N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Formula 5 to obtain compound of Formula 6,
II. Deprotection of the compound of Formula 6 to obtain Gemcitabine freebase, compound of Formula 7,
III. Conversion of the compound obtained in step II to its Hydrochloride salt.

The suitable solvents used in step (I) is may be selected from suitable hydrocarbons such as toluene, xylene, ethyl benzene, trimethyl benzene, ether solvents such as Anisole, tetrahydrofuran and the like or mixtures thereof, halogenated hydrocarbons solvents such as dichloromethane, dichloroethane and the like or mixtures thereof. Preferably, it is one or more solvents selected from the group consisting of ethers.

The suitable reagent used in step (I) may be selected from SnCl4 and ZnCl2

The reaction mixture in step (I) is maintained at 0 °C to 100 °C. Preferably, the temperature is at 100-110 °C

The reaction of step (I), wherein the duration of the reaction may be from 1 hr to 10 hrs, preferably from 2 hrs to 5 hrs.
The suitable base used in step (II) may be ammonia, monomethylamine and dimethyl amine.
The suitable solvent used in step (II) is suitable organic solvents may be selected from suitable alcohols such C1-C6 lower alcohol, preferably methanol

The suitable solvent used in step (III) is suitable organic solvent may be selected from suitable alcohols such C1-C6 lower alcohol, Ketones such as Acetone and the like or mixtures thereof, preferably Iso Propyl Alcohol.

The reaction mixture in step (III) is maintained at 0 °C to 80 °C. Preferably, the temperature is at 40-60 °C

The compound obtained in step (III) may be purified in suitable solvents. Suitable solvents can be ketones such as acetone, ethyl methyl ketone or mixture thereof, water or a mixture of water and a ketone solvent.

In another aspect the present invention relates to the process for the preparation of gemcitabine hydrochloride of Formula I comprising the steps of:

1. Conversion of ((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate of Formula 2 to (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 by reacting with Benzoyl chloride in a suitable solvent and base.
2. Silylation of N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Fomula 4 with hexamethyldisilazane in the presence of a suitable catalyst.
3. Condensation of (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 and silylated N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Formula 5 to obtain compound of Formula 6.
4. Deprotection of the compound of Formula 6 to obtain Gemcitabine freebase, compound of Formula 7 using a base.
5. Conversion of the compound obtained in step 4 to its Hydrochloride salt.

The suitable bases in step (1) is organic or inorganic bases wherein, the organic base may be selected from C1.-5 alkyl amines, C1-.5 substituted alkyl amines preferably, triethyl amine (TEA), diisopropyl amine, diisopropylethyl amine, heterocyclic saturated or unsaturated amines, preferably morpholine, piperidine, pyrollidine and pyridine; the suitable inorganic bases used is selected from hydroxides such as NaOH, KOH, carbonates such as NaHCO3, Na2CO3, K2CO3 and like.

The reaction mixture in step (1) is maintained at 0 °C to 50 °C. Preferably, the temperature is at 20-30 °C.

The suitable solvents used in step (1) is may be selected from suitable hydrocarbons such as toluene, xylene, ethyl benzene, trimethyl benzene, halogenated hydrocarbons solvents such as dichloromethane, dichloroethane and the like or mixtures thereof, alcohols such as methanol, ethanol, t-butanol and like or mixtures thereof, ketones such as acetone. Preferably, it is one or more solvents selected from the group consisting of halogenated hydrocarbons.

The suitable catalyst in step (2) may be selected from Trimethyl Silyl chloride.

The suitable solvents used in step (2) is may be selected from suitable hydrocarbons such as toluene, xylene, ethyl benzene, trimethyl benzene, ether solvent like anisole, halogenated hydrocarbons solvents such as dichloromethane, dichloroethane and the like or mixtures thereof, alcohols such as methanol, ethanol, t-butanol and like or mixtures thereof, ketones such as acetone. Preferably, it is one or more solvents selected from the group consisting of halogenated hydrocarbons.

The reaction mixture in step (2) is maintained at 0 °C to 150°C or at reflux temperature of the solvent used. Preferably, the temperature is at 100-110 °C.

The suitable solvents used in step (3) is may be selected from suitable hydrocarbons such as toluene, xylene, ethyl benzene, trimethyl benzene, ether solvents such as Anisole, tetrahydrofuran and the like or mixtures thereof, halogenated hydrocarbons solvents such as dichloromethane, dichloroethane and the like or mixtures thereof. Preferably, it is one or more solvents selected from the group consisting of ethers.

The suitable reagent used in step (3) may be selected from SnCl4 and ZnCl2

The reaction mixture in step (3) is maintained at 0 °C to 100 °C. Preferably, the temperature is at 100-110 °C

The reaction of step (3), wherein the duration of the reaction may be from 1 hr to 10 hrs, preferably from 2 hrs to 5 hrs.

The suitable base used in step (4) may be ammonia, monomethylamine and dimethyl amine.

The suitable solvent used in step (4) is suitable organic solvents may be selected from suitable alcohols such C1-C6 lower alcohol, preferably methanol

The suitable solvent used in step (5) is suitable organic solvent may be selected from suitable alcohols such C1-C6 lower alcohol, Ketones such as Acetone and the like or mixtures thereof, preferably Iso Propyl Alcohol.

The reaction mixture in step (5) is maintained at 0 °C to 80 °C. Preferably, the temperature is at 40-60 °C

The compound obtained in step 5 may be purified in suitable solvents. Suitable solvents can be ketones such as acetone, ethyl methyl ketone or mixture thereof, water or a mixture of water and a ketone solvent.

The solvents that are used at various stages may be removed by using the conventional techniques known in the art. Those techniques may be distillation, evaporation, filtration or other techniques with or without application of a vacuum. The distillation is frequently carried out at about 650 to 800 mm Hg, or 400 to 750 mm Hg.

The filtration technique used during various stages of the present invention may be pressure filtration, gravity filtration, or vacuum filtration. Other solid-liquid separation techniques, such as centrifugation or decantation, may be used instead of filtration.

Thus the process described in the present invention has significant advantages over the existing processes in terms of less number of steps, milder reactions, inexpensive reagents, and simple crystallization methods to obtain high purity compounds at each step. Accordingly the present invention provides an efficient process for the preparation of gemcitabine hydrochloride on a commercial scale.

Certain specific aspects and embodiments of the present invention will be explained in more detail with reference to the following examples, which are provided by way of illustration only and should not be construed as limiting the scope of the invention in any manner.

EXAMPLES
EXAMPLE 1: Benzoylation of 2- Deoxy-2, 2-difluoro-D-ribofuranose-3, 5-dibenzoate.
In a round bottom flask 500 ml of Dichloromethane and 50 gms of 2- Deoxy-2, 2-difluoro-D-ribofuranose-3, 5-dibenzoate were charged and stirred for clear solution under an inert atmosphere. The reaction mass was then cooled to about 3°C and added 18gms of triethylamine. 22.75 gms of Benzoyl chloride was added dropwise to the reaction mixture at about 5°C and then the reaction mixture was stirred at room temperature for about 2 hours. The reaction mixture was quenched in Hydrochloric acid solution (20ml of Con. HCl in 180 ml of water) and stirred for 10 minutes. The organic layer was then separated and washed with 132ml of water. The organic layer was then treated with NaHCO3 solution (12gms in 175 ml of water), separated and distilled the organic layer to obtain the titled compound.
Yield: 60gms
Purity by HPLC: 93%

EXAMPLE 2: Benzoylation of 2- Deoxy-2, 2-difluoro-D-ribofuranose-3, 5-dibenzoate.
In a round bottom flask 275 ml of Toluene and 50 gms of 2- Deoxy-2, 2-difluoro-D-ribofuranose-3, 5-dibenzoate were charged and stirred for clear solution under an inert atmosphere. The reaction mass was then cooled to about 3°C and charged 18gms of triethylamine. 22.75 gms of Benzoyl chloride was added dropwise to the reaction mixture at below 5°C and then the reaction mixture was stirred for about 1 hour and further stirred at room temperature for 1 hour. The reaction mixture was quenched in Hydrochloric acid solution (20ml of Con. HCl in 180 ml of water) and stirred for 10 minutes. The organic layer was then separated and washed with 132ml of water. The organic layer was then treated with NaHCO3 solution (12gms in 175 ml of water), separated and distilled to obtain the titled compound.
Yield: 58.6gms
Purity by HPLC: 92%

EXAMPLE 3: Preparation of Gemcitabine Hydrochloride
Step 1: Silylation of N-acetyl Cytosine
In a round bottom flask under stirring 1000ml of toluene, 47.61 gms of N-acetyl Cytosine and 64.73 gms of hexamethyl disilazane were charged in the presence of nitrogen atmosphere. 34ml of the solvent is distilled off under reduced pressure and the temperature of the reaction mass is reduced to 35°C. 0.41gms of Trimethyl silyl chloride was charged to the reaction mass and stirred the reaction mass at reflux temperature for two hours. Cooled the reaction mass to about 60°C and distill the solvent under the reduced pressure to obtain Bis silylated N-acetyl Cytosine.
Step 2: Condensation of Bis silylated N-acetyl Cytosine and 2- Deoxy-2, 2-difluoro-D-ribofuranose-1, 3, 5-dibenzoate.
To the compound obtained in step 1 charged 850ml of Anisole and stirred the reaction mass for 10 – 15 min. at room temperature under nitrogen atmosphere. The reaction mass was cooled to 10°C and added 189.1gms of Stannic Chloride slowly at below 15°C. Slowly raise the temperature of the reaction mass to 105°C and then add a solution of 50 gms of 2- Deoxy-2, 2-difluoro-D-ribofuranose-1, 3, 5-dibenzoate in 250 ml of Anisole. The reaction mass was the stirred for 6 hours at the same temperature, cooled to room temperature and charged 284.7gms of sodium bicarbonate. Reaction mass was further cooled to 5°C and added 105ml of water. Temperature of the reaction mass was raised to 30°C, stirred for one hour filtered the inorganic salts and separated the organic layers. Extracted the aqueous layer twice with 350 ml and 150ml of dichloromethane. The organic layer was combined and distilled under reduced pressure. To the obtained crude charged 50ml of methanol and distilled under vaccum.
Step 3: Deprotection
To the compound obtained in step 2, 600ml of methanol was added and stirred for complete dissolution of the compound. 100ml of aqueous ammonia was charged to the reaction mass and stirred for 12 hours. Temperature of the reaction mass was raised to 43°C and 25gms of activated carbon was charged and stirred for one hour at the same temperature. Reaction mass was then filtered through hyflo bed and the filterate was distilled under vaccum at 50°C. Charged 50ml of acetone to the crude and distilled the solvent to obtain Gemcitabine freebase.
Step 4: Preparation of 2'-Deoxy-2', 2’-diflourocytidine monohydrochloride crude from 2’-Deoxy-2', 2’-diflourocytidine.

Charged 625ml of acetone and 2.5ml Iso Propyl Alcohol to the crude obtained in step 3 and raised the temperature of the reaction mass to 50°C. 19gm of Hydrochloric acid was added slowly to the reaction mass and stirred for about 30 minutes at the same temperature. The reaction mass was then cooled to 10°C and stirred for about 30 minutes, filtered, washed and dried.
Yield: 19.2gms
Step 5: Purification of Gemcitabine HCl
The dried compound obtained in step 4 and 24.96 ml of water were charged in a round bottom flask under stirring and temperature of the reaction mass was raised to 50°C. 195.8 ml of acetone was charged to the reaction mixture and continued stirring for 30 minutes. It is then cooled to 20°C and stirred for 30 minutes. The separated solid was filtered, washed and dried.
Yield: 16.1gms
Purity by HPLC: 99.62%
,CLAIMS:We Claim:
1. A process for the preparation of gemcitabine hydrochloride of Formula I,

comprising the steps of:
I. Condensation of (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3,

and silylated N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Formula 5

to obtain compound of Formula 6,

II. Deprotection of the compound of Formula 6 to obtain Gemcitabine freebase, compound of Formula 7,

III. Conversion of the compound obtained in step II to its Hydrochloride salt.

2. A process for the preparation of gemcitabine hydrochloride of Formula I comprising the steps of:
I. Conversion of ((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methyl benzoate of Formula 2

to (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3

by reacting with Benzoyl chloride in a suitable solvent and base;
II. Silylation of N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Fomula 4

with Hexa methyl trisilazane in the presence of a suitable catalyst;
III. Condensation of (4R,5R)-5-((benzoyloxy)methyl)-3,3-difluoro tetra hydrofuran-2,4-diyl dibenzoate of Formula 3 and silylated N-(2-oxo-1,2-dihydropyrimidin-4-yl)acetamide of Formula 5;

to obtain compound of Formula 6;

IV. Deprotection of the compound of Formula 6 to obtain Gemcitabine freebase, compound of Formula 7;

V. Conversion of the compound obtained in step 4 to its Hydrochloride salt.