FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rulel3)
l.TITLE OF THE INVENTION
PROCESS FOR PREPARATION OF GEMCITABINE HYDROCHLORIDE
2. APPLICANT (S)
(a) NAME: Prof. Dr. Bandgar B. P.
(b) NATIONALITY: Indian
(c) ADDRESS: Vice-chancellor, Solapur University, Solapur-Pune
Highway, Kegaon, Solapur-413 255, Maharashtra, India.
(a) NAME: Dr. Jaweed Mukarram S. M.
(b) NATIONALITY: Indian
(c) ADDRESS: H.No.-4-8-65, Nawabpura, Aurangabad-431 001,
Maharashtra, India.
(a) NAME: Salman Mukarram S. M.
(b) NATIONALITY: Indian
(c) ADDRESS: School of Chemical Sciences, Solapur University, Solapur-
Pune Highway, Kegaon, Solapur-413 255, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION
The present invention provides process for the preparation of Gemcitabine hydrochloride. The process of preparation relates to the process of preparation of intermediate compounds which results in reduced processing stages, enhanced yield and avoided impurity formation and higher purity.
The following specification particularly describes the invention and the manner in which it is to be performed.

The present invention provides improved process for the preparation of Gemcitabine hydrochloride. The process relates to preparation of intermediate compound 2, 3-0-isopropylidene-D-Glyceralehyde from the starting material D-mannitol by one pot process and further its conversion to Ethyl -3-(2, 2-dimethyl-l, 3-dioxolan-4-yl)-2, 2-difluro-3-hydroxy propionate Erythro: Threo (3:1) and making of Gemcitabine hydrochloride.
Gemcitabine hydrochloride (Formula I) is chemically known as 2'-deoxy-2', 2'-difluorocytidine monohydrochloride (β-isomer). It is a nucleoside metabolic inhibitor that exhibits anti tumor activity.

The process of preparation of Gemcitabine and its pharmaceutically acceptable salts are disclosed in T. S. Chou et. al, Synthesis 1992, 565; C. R. Schimdt et. al., J. O. C. (1991), 56, 4056-4058; R.W. Kierstead et. al., J. M. C. (1983), 26, 1561-1569; Jurczak et. al., Tetrahedron Vol 42, No 2, 447-488 (1986).
US patent No 4,808,614 discloses 2, 2-difluoro-2-desoxycarbohydrate which is used to prepare antiviral nucleosides.

US patent No 5,223,608 discloses a convenient process for obtaining 2'-deoxy-2', 2'-difluoronucleosides having the desired erythro and beta stereochemistry.
US patent No 5,808,048 discloses process for preparing Gemcitabine hydrochloride.
US patent No 7,235,647 discloses intermediate and process for preparing of beta- anomer enriched 2'-deoxy, 21, 21-difluoro-D-ribofuranosyl nucleosides.
The processes for the preparation of intermediates for preparation of Gemcitabine and Gemcitabine hydrochloride are also disclosed in WO005095430 and WO2007049294.
The PCT application WO2007049295 discloses an improved one-pot process for preparing 2-deoxy-D-erythro-2, 2-difluoro-pentafuranose-]-ulose-3, 5-dibenzoate.
The PCT application WO2006095359 discloses the synthesis of 2-deoxy-2, 2-difluoro-D-ribofuranose-3, 5-di-(aroyl) derivative.
The present inventors while developing a process for the preparation of Gemcitabine hydrochloride have developed the one-pot process for the preparation of 2, 3-0-isopropylidene-D-Glyceralehyde (Formula II) from D-mannitol then converting it to Ethyl -3-(2, 2-dimethyl-l; 3-dioxolan-4-yl)-2, 2-difluro-3-hydroxy propionate Erythro: Threo (3:1) (Formula III) in high purity. It is converted to 2-deoxy-D-erythro-2, 2-difluro pentafuranos-l-ulose-3, 5-dibenzoate (Formula IV) and in subsequent steps it is converted in to Gemcitabine hydrochloride.
In one of the aspect of the invention a one pot process for the preparation of 2, 3-0-isopropylidene - D-Glyceralehyde of Formula II


Formula II the process include of following steps;
a) reacting D-Manitol with a mixture of p-toluene sulfonic acid and 2, 2-dimethoxy propane in the presence of a polar aprotic solvent,
b) treating the reaction mixture with aqueous solution of inorganic base,
c) wash the solution of step b) with suitable solvent,
d) extracting the aqueous layer in halogenated solvent,
e) contacting halogenated layer with sodium periodate,
f) isolating 2, 3-O-isopropylidine-D-Glyceraldehyde from the reaction mixture thereof.
The above one pot process of preparation of 2, 3-O-isopropylidene-D-Glyceralehyde proceeds without isolation of 1, 2, 5, 6 -diisopropylidine. The D-Mannitol is reacted with a mixture of p-toluene sulfonic acid and 2, 2-dimethoxy propane in the presence of a polar aprotic such as one or more of dimethyl sulfoxide, dimethylformamide, dimethyl acetamide and N-methyl-2-pyrrolidone. After completion of reaction the reaction mixture is poured in the aqueous solution of inorganic alkali bases. The inorganic bases may include of carbonates, bicarbonates, hydroxides of sodium and potassium. Finally aqueous alkaline layer is washed with suitable organic solvent such as hexane, petroleum ether, pentane, isopropyl ether and diethyl ether. The intermediate compound is extracted in halogenated solvent like methylene chloride, chloroform and ethylene dichloride. The halogenated layer is treated with sodium periodate which after completion of the reaction; methylene chloride is removed under reduced pressure to yields 2, 3-O-isopropylidene-D-Glyceralehyde in high yield and purity.

In second aspect of the invention the 2, 3-O-isporopylidene-D-Glyceralehyde is converted into pure (Erythro: Threo) ethyl-3-(2, 2-dimethyl-l, 3-dioxolan-4-yl)-2, 2-difluoro propionate the process includes the steps of;
a) contacting mixture of 2,3-O-isopropylidene-D-Glyceralehyde and bromodifluroethyl acetate with zinc dust and trimethyl silyl chloride in ether solvent,
b) extracting the reaction mixture in suitable solvent,
c) isolating pure (Erythro: Threo) ethyl-3-(2,2-dimethyl-l,3-dioxolan-4-yl)-2,2-difluoro propionate by high vacuum distillation (Formula III)

The process of preparation of pure (Erythro: Threo) ethyl-3-(2, 2-dimethyl-l, 3-dioxolan-4-yl)-2, 2-difluoro propionate involves the controlled addition of mixture of 2, 3-0-isopropylidene-Glyceralehyde and bromodifluroethyl acetate in the suspension of zinc dust and trimethyl silyl chloride in ether solvent such as tetrahydrofuran. After addition, the reaction mixture is heated to reflux and then poured into chilled solution of hydrochloric acid in water. Finally product is extracted in suitable solvent. After removal of solvent, the product is distilled at 60°C -80°C temperature and vacuum less than 4mm of Hg to get pure product in colourless oil form in high purity 98.5% or more.

The suitable solvent used for the extraction of product from acidic aqueous layer is limited to the water immiscible solvents such as halogenated, acetate, hydrocarbons, ether solvents e.g. methylene chloride, chloroform, ethyl acetate, Toluene, diethyl ether and the like.
In third aspect of the invention, the process of the purification of 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate includes the steps of;
a) contacting pure (Erythro: Threo) ethyl-3-(2,2-dimethyl-l,3-dioxolan-4-yl)-2,2-difluoro propionate with pyridinium trifluro acetate salt in acetonitrile and water,
b) subjecting step a) to bezoylation,
c) isolating 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate from reaction mixture thereof,
d) crystallizing step c) product with heptane and tetrahydrofuran to get purified 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate (Formula IV)

Formula IV
The process for the purification of 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate involve the contacting pure (Erythro: Threo) ethyl-3-(2, 2-dimethyl-l, 3-dioxolan-4-yl)-2, 2-difluoro propionate with pyridinium trifluro acetate salt in acetonitrile and water. The reaction mixture is heated to reflux. After completion of reaction, the reaction mixture is subjected to azeotropic removal of

solvent yields the compound 2-deoxy-2,2-difluro-l-oxo ribose which subsequently undergoes bezoylation reaction in ethyl acetate and pyridine with 4,4,dimethyl amino pyridine and benzoyl chloride. After completion of reaction, ethyl acetate layer is removed and concentrated under vacuum to yield oily mass. Oily mass is dissolved in toluene and precipitated out by adding hexane. Isolated product is further dissolved in n-heptane and crystallized out by addition of tetrahydrofuran in high purity 99.5% when measured by HPLC and melting point in range of 120°C -124°C.
In fourth aspect of the invention the process of preparation of compound of Formula-V,

the process includes steps of;
a) contacting 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 -dibenzoate with vitride in tetrahydrofuran at -5°C to 10°C temperature.
b) isolating compound of Formula-V from the reaction mixture thereof.
The process involves the reducing of 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-1-ulose-3, 5 - dibenzoate with vitride at temperature -5°C to 100C to get the compound of formula-V.
The compound of Formula V is converted to Gemcitabine hydrochloride (Formula I) by the process known to skilled artisan through WO2006095359 and WO2005095430.

One embodiment of the process in which intermediates prepared by the process of invention is used to make Gemcitabine hydrochloride by the process known in the art.
The present invention is further illustrated by the following examples, which are provided merely to be exemplary of the invention and do not limit the scope of the invention. Certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
Example 1: Preparation of 2, 3-O-isopropylidene-D Glyceraldehyde (Formula II)
Charged mixture of D-mannitol (250 g), p-toluene sulfonic acid (1.25 g), and 2, 2-dimethoxy propane (400 ml) in dimethyl sulfoxide (425 ml) and mixture was stirred at room temperature 25°C -35°C for 16 hour. After completion of the reaction the reaction, mixture was poured into 5% sodum bi carbonate (4.5 L). The aqueous layer was washed with petroleum ether. Then aqueous layer was extracted with methylene chloride and washed with 5% sodium bicarbonate solution.
In methylene chloride added saturated aqueous sodium bi carbonate (80 ml) and sodium periodate (300 gm) under stirring at 20°C -25°C. Stirred the reaction mixture for 2 more hours at same temperature for the completion. After completion of the reaction, the reaction mixture was filtered and subsequent distillation under high vacuum at temperature 45 -55°C to yield the titled compound. Yield: 180 g.
Example 2: Preparation of (Erythro: Threo) ethyl-3-(2,2-dimethyl-l,3-dioxolan-4-vl)-2,2-difluoro propionate (Formula III)
Charged 1.8 L of tetrahydrofuran (THF), zinc dust 180 g and trimethylsilyl hydrochloride (29 mL) under stirring in a flask. The mixture was heated to reflux and mixture of 2, 3-0-isopropylidene-D-glyceraldehyde (180 g) and bromodifluroethyl acetate (248 mL) was added through dropping funnel slowly over a period of 30-40 minutes. The reaction mixture was further stirred under reflux for 2 hours. Then poured in a mixture of concentrated HC1 (143 ml) and chilled water (1.1 L) under stirring, organic layer was

separated and aqueous layer was further extracted with ethyl acetate. The collective organic layer was washed with 5% aqueous sodium bicarbonate solution. The organic layer was dried over sodium sulfate and concentrated to obtain the product as yellow oil. This was distilled at 2 mm Hg mercury at 75 -80°C to give colorless pure product. Yield: 250 g
Example 3: Preparation and isolation of 2-deoxv-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate (Formula IV)
Charge ethy 1-3-(2,2-dimethyl-1,3-dioxolan-4-yi)-2,2-difluro-3-hydroxypropionate (250 gm) and methyl cyanide (2.5 L) and water (80ml) and pyridinium trifluroacetate (45 g ) under stirring, reaction mixture was heated to reflux for 3 hours. After completion of reaction, the methyl cyanide and water was azeometrically distilled out. In the residue obtained ethyl acetate (900 ml), dry pyridine (300 ml) and 4, 4-dimethylaminopyridine (30 gm) was added. The reaction mixture was heated to 65°C under nitrogen atmosphere. Then solution of benzoyl chloride (300 ml) in ethyl acetate (500 ml) was added drop wise over 3 hours at 65°C. The reaction was further stirred at 65°C for 3 hours and then cooled to 0-5°C and filtered. The filtrate was concentrated at 35°C under reduced pressure. The oily mass obtained under stirring was dissolved in toluene (380 ml) and then cooled to 0-5°C. The hexane (750 ml) was then added in portion to precipitate the product. The precipitated product was isolated and recrystallized with from heptane: THF (9:1) to give titled product.
Yield: 130 g HPLC Purity: 99.5%,
DSC endotherm: 121-125°C.

We Claim:
1 A process for the preparation of Gemcitabine hydrochloride; the process
comprises
a) preparation of 2, 3-O-isopropylidine-D-Glyceraldehyde (Formula II)

by one pot process,
b) the conversion of 2, 3-O-isopropylidine-D-Glyceraldehyde (Formula II)

to ethyl-3-(2, 2-dimethyl-l, 3-dioxolan-4-yl)-2, 2-difluoro propionate (Formula III)


by distillation,
c) the purification of 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 -

from Formula III by crystallization,
dibenzoate (Formula IV)


Formula III d) isolation of Formula V

e) conversion of Formula V
by reduction of 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate (Formula IV),



to Gemcitabine hydrochloride by known process.
2 The process of claim 1 wherein the one pot process of preparation of compound
of Formula II

comprises the steps of;
a) reacting D-Manitol with a mixture of p-toluene sulfonic acid and 2, 2-dimethoxy propane in the presence of a polar aprotic solvent,
b) treating the reaction mixture with aqueous solution of inorganic base,
c) wash the solution of step b) with suitable solvent,
d) extracting the aqueous layer in halogenated solvent,
e) contacting halogenated layer with sodium periodate,
f) isolating 2, 3-O-isopropylidine-D-Glyceraldehyde from the reaction mixture thereof.

3 The process of claim 1 wherein preparation of compound ethyl-3-(2, 2-dimethyl-
1, 3-dioxolan-4-yI)-2, 2-difluoro propionate (Formula III)

by distillation comprises the steps of;
a) contacting mixture of 2,3-O-isopropylidene-Glyceralehyde and bromodifluroethyl acetate with zinc dust and trimethyl silyl chloride in ether solvent,
b) extracting the reaction mixture in suitable solvent,
c) isolating pure (Erythro: Threo) ethyl-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro propionate by high vacuum distillation (Formula III)
4 The process of claim 1 wherein the purification of 2-deoxy-D-erythro-2, 2-
difluoro pentafuranos-l-ulose-3, 5 - dibenzoate (Formula IV)


Formula IV by crystallization comprises the steps of;
a) contacting pure (Erythro: Threo) ethyl-3-(2,2-dimethyl-l,3-dioxolan-4-yl)-2,2-difluoro propionate with pyridinium trifluro acetate salt in acetonitrile and water,
b) subjecting step a) to bezoylation,
c) isolating 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 - dibenzoate from reaction mixture thereof,
d) crystallizing step c) product with heptane and tetrahydrofuran to get purified 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-l-ulose-3, 5 -dibenzoate
5 The process of claim 1 wherein the preparation of compound of Formula V

by reduction comprises the steps of;
a) contacting 2-deoxy-D-erythro-2, 2-difluoro pentafuranos-1 -ulose-3, 5 -dibenzoate with vitride in tetrahydrofuran at -5°C to 10°C temperature,
b) isolating compound of Formula-V from the reaction mixture thereof.
6 The process of claim 2 wherein the suitable solvent is organic solvent.

7 The process of claim 6 wherein the organic solvent is hexane, petroleum ether, pentane, isopropyl ether and diethyl ether.
8 The process of claim 3 wherein the solvent is water immiscible solvents such as halogenated, acetate, hydrocarbons, ether solvents; the ether solvents are methylene chloride, chloroform, ethyl acetate, Toluene and diethyl ether.
9 The compound of Formula IV having HPLC purity 99.5% or more when measured by HPLC.
10 The compound of formula III having HPLC purity 98.5%