FIELD

The present patent application relates to a process for the preparation gemcitabine or a salt thereof. More particularly it relates to preparation of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose, an intermediate used in the preparation of several active pharmaceutical ingredients including gemcitabine or a salt thereof.

BACKGROUND

3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose is chemically represented by the structural Formula I and is used as intermediate in the preparation of several active pharmaceutical ingredients including gemcitabine and its salts.

Formula I wherein P is a hydroxyl protecting group.

Gemcitabine hydrochloride is chemically known as 2'-deoxy-2',2 ^' - difluorocytidine monohydrochloride (β-anomer) and has structural Formula II.

Gemcitabine is a synthetic nucleoside analogue that has anti-tumor activity and is commercially available in the market under the brand name GEMZAR ^® in the form of an injection. A vial of Gemzar contains gemcitabine hydrochloride equivalent to either 200 mg or 1 g of gemcitabine free base.

U.S. Patent No. 4,808,614 discloses gemcitabine hydrochloride, compositions containing gemcitabine hydrochloride and their use in the treatment of herpes viral infections.

U.S. Patent No. 5,223,608 discloses a process for the preparation of gemcitabine hydrochloride by using hydrolysis reagents such as strong acids in the preparation of 2-deoxy-2,2-diflouro-D-erythro-pentofuranos-1-u!ose-3, 5-dibenzoate.

PCT Application Publication No WO 2005/095430 A1 discloses a process for the preparation of gemcitabine hydrochloride. This 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 and its purification process.

Almost all of the processes hither to known use isopropylidene protecting group, which requires a costly reagent and further it is not reusable.

Journal of Fluorine Chemistry (1992), 57(1-3), 203-7 discloses a highly stereo selective synthesis of α,α-difluoro-β,γ-dihydroxy esters comprising: Reformatsky reaction of bromo- or iododifluoroacetate with D-glyceraldehyde to get (3R, 4R)-2,2-difluoro-4,5-0-cyclohexylidene-3-triethylsiloxypenta noic acid ethyl ester with diastereoselectivity, by asymmetric induction promoted by Cp ₂ TiCb. However this process involves use of relatively expensive Lewis acid catalyst in high quantity, which is not suitable for commercial manufacturing.

Consequently, it would be a contribution to the art to provide a scalable and safe process for the preparation of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1- oxoribose using commercially available raw materials

SUMMARY OF THE INVENTION

In one aspect, the present application provides a process for preparation of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose compound of Formula I

Formula I wherein P is a hydroxy protecting group, comprises of: a) reacting 2,3-O-Cyclohexylidine-D-glyceraldehyde compound of Formula III with alkylbromodifluoroacetae of Formula IV in the presence of zinc reagent to form compound of Formula V;

Formula III Formula IV wherein R is C1-C4 alkyl b) reaction of compound of Formula V with an acid reagent;

Formula V wherein R is C1-C4 alkyl c) lactonizing the obtained intermediate to form 2-desoxy-2, 2-difluoro-1- oxoribose of Formula Vl;

Formula Vl d) protection of the hydroxy groups of the compound of Formula Vl using a suitable hydroxy-protecting reagent to afford 3,5- dihydroxy protected -2-deoxy-2,2- difluoro -1-oxoribose of Formula I.

In this multi step process, each step may be contemplated individually or in combination of two or more steps.

In another aspect the present application relates to use of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose of Formula I prepared according to the process of present application in the synthesis of active pharmaceutical ingredients including gemcitabine or a salt thereof.

In a specific embodiment of the present invention, 2-deoxy-2, 2-diflouro-D- erythro-pentofuranos-i-ulose-3, 5-dibenzoate is prepared by a process comprising the steps of: a) reacting 2,3-O-Cyclohexylidine-D-glyceraldehyde compound of Formula III with ethylbromodifluoroacetae in the presence of zinc to form Ethyl 2,2-difiuoro-4,5- O-cyclohexylidene-3-hydroxypentanoate; b) reaction of Ethyl 2,2-difluoro-4,5-0-cyclohexylidene-3-hydroxypentanoate with trifluoroacetic acid; followed by azeotrophic distillation to form 2-desoxy-2, 2- difluoro- 1-oxoribose of Formula Vl; c) protection of the hydroxy groups of 2-desoxy-2, 2-difluoro-1-oxoribose by reacting with benzoyl chloride in the presence of pyridine.

DETAILED DESCRIPTION OF THE INVENTION

The present patent application relates to a process for the preparation of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose, which is used as an intermediate in the preparation of several active pharmaceutical ingredients including gemcitabine or a salt thereof.

All percentages and ratios used herein are by weight of the total composition and all measurements made are at 25°C and normal pressure unless otherwise designated. All temperatures are in Degrees Celsius unless specified otherwise. The present invention can comprise (open ended) or consist essentially of the components of the present invention as well as other ingredients or elements described herein. As used herein, "comprising" means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms "having" and "including" are also to be construed as open ended unless the context suggests otherwise.

Terms such as "about," "generally," "substantially," and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.

In one aspect the present application provides a process for preparation of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose compound of Formula I

Formula I wherein P is a hydroxy protecting group, comprises of: a) reacting 2,3-O-Cyclohexylidine-D-glyceraldehyde compound of Formula III with alkylbromodifluoroacetae of Formula IV in the presence of zinc reagent to form compound of Formula V;

Formula III Formula IV wherein R is C1-C4 alkyl b) reaction of compound of Formula V with an acid reagent;

Formula V wherein R is C1-C4 alkyl c) lactonizing the obtained intermediate to form 2-desoxy-2, 2-difluoro-1- oxoribose of Formula Vl;

Formula Vl d) protection of the hydroxy groups of the compound of Formula Vl using a suitable hydroxy-protecting reagent to afford 3,5- dihydroxy protected -2-deoxy-2,2- difluoro -1-oxoribose of Formula I.

The present application also encompasses the use of 2,3-O-Cyclopentylidine- D-glyceraldehyde instead of 2,3-O-Cyclohexylidine-D-glyceraldehyde following the same reaction sequence with small variations in the reaction conditions, which may be envisaged by one skilled in the art.

Step a) involves reaction of 2,3-O-Cyclohexylidine-D-glyceraldehyde compound of Formula III with alkylbromodifluoroacetae of Formula IV in the presence of zinc reagent to form compound of Formula V;

Alkylbromodifluoroacetae includes C1-C4 alkylbromodifluoroacetae, preferably ethylbromodifluoroacetate.

The reaction is suitably carried out under Reformatsky reaction conditions. Suitable zinc reagent includes zinc powder or any other reagent that may be used in Reformatsky reaction conditions. Suitable solvent that may be used in step a) includes but not limited to: ethers such as diethyl ether, diisopropyl ether, ter-butyl methyl ether, dibutyl ether, tetrahydrofuran, 1,2-Dimethoxyethane, 2-methoxy ethanol, 2-ethoxy ethanol, anisole and the like; aliphatic or alicyclic hydrocarbons such as hexanes, n-heptane, n- pentane, cyclohexane, methylcyclohexane, nitro methane and the like; chlorinated hydrocarbons such as dichloromethane, chloroform, 1,1,2-trichloroethane, 1,2- dichloroethene and the like; aromatic hydrocarbons such as toluene, xylenes, chloro benzene and the like; and mixtures thereof.

Suitable temperatures for conducting the reaction may range from about 25°C to about reflux temperature of the solvent used, preferably reflux temperature of the solvent used. The reaction may be conducted till the completion of the reaction. Typically the reaction time varies from about 1 hour to about 15 hours.

The reaction may optionally be carried out in the presence of a suitable hydroxy protecting group such as trimethyl silyl chloride.

After completion of the reaction, reaction mixture is quenched with suitable acid such as hydrochloric acid and extracted with organic solvent such as ethylacetate. The organic layer containing the product may be separated, washed with water and proceeded to next step directly or it can be distilled to obtain the product as residue.

Suitably the compound of Formula V may be mixture of anti and syn forms. The ratio of anti and syn forms may range from about 50: 50 to about 98:2. It is preferable to have the anti form more than the syn form.

Step b) involves reaction of compound of Formula V with a suitable acid reagent.

Acid reagents used in the process of step b) include acids and acidic ion- exchange resins. Suitable acids that may be used include hydrochloric acid, sulphuric acid, per chloric acid, acetic acid, trifluoroacetic acid, formic acid and the like.

Suitable acidic ion-exchange resins include styrene-divinyl benzene, acrylic- divinyl benzene resins such as Dowex 50, Amberlite, acidic Wang resin and like. Suitable solvent that may be used in step b) includes but not limited to: water, alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, ter-butyl alcohol, 1-pentanol, 2-pentanol, neo pentyl alcohol, amyl alcohol, 2-methoxy ethanol, 2-ethoxy ethanol, ethylene glycol, glycerol and the like; ketones such as acetone, butanone; 2-pentanone, 3-pentanone, methyl butyl ketone, methyl iso-butyl ketone and the like; esters such as ethyl formate, methyl acetate, ethyl acetate, propyl acetate, ter-butyl acetate, iso-butyl acetate, methyl propanoate, ethyl proponoate, methyl butanoate, ethyl butanoate and the like; nitriles such as acetonitrile, propionitrile and the like; polar and non polar aprotic solvents such as N,N-dimethyl formamide.Toluene, N,N-dimethyl acetamide, N-methy! pyrrolidone, pyridine, dimethyl sulphoxide, sulpholane, formamide, acetamide, propanamide and the like; and mixtures thereof.

Suitable temperatures for conducting the reaction range from about 20 ⁰ C to about reflux temperature of the solvent used, preferably reflux temperature of the solvent used. The reaction can be conducted till the completion of the reaction. Typically the reaction time varies from about 1 hour to about 10 hours.

After completion of the reaction the open chain intermediate compound may be isolated or the same can proceeded to the next reaction in-situ.

Step c) involves lactonization of the intermediate obtained in step b) to form 2- desoxy-2, 2-difluoro-1-oxoribose of Formula Vl;

A water immiscible solvent is added to the reaction mixture and subjected to azeotropic distillation to remove water and water miscible solvent. The distillation can be conducted at temperatures of about 50 ⁰ C to about 150 ⁰ C.

Water immiscible solvents that can be used include hydrocarbons and halogenated hydrocarbons such as n-hexane, cyclohexane, n-heptane, toluene, chlorobenzene, 1 ,2-dichlorobenzene, xylenes and the like

The reaction mixture comprising the product may be used directly in the subsequent reaction step or suitably it can be concentrated to obtain a residue.

Step d) involves protection of the hydroxy groups of the compound of Formula Vl using a suitable hydroxy-protecting reagent to afford 3,5- dihydroxy protected -2- deoxy-2,2-difluoro -1-oxoribose of Formula I. Suitable hydroxyl protecting reagent that may be used include a) benzoyl chloride or substituted benzoyl chloride represented by the structural Formula VII,

Formula VII wherein Xi, X2 are independently selected from H, F, Cl, Br, I and NO ₂ ; preferably substituted at 3-position or 5-position; b) substituted silyl chloride represented by the structural Formula VIII,

Formula VIII wherein Ra, Rb and Rc are independently selected from H, Ci-C ₆ alkyl, phenyl, substituted phenyl with a proviso that all Ra, Rb and Rc are not H.

Suitable bases which can be used include but are not limited to organic bases such as pyridine, triethylamine, imidazole, 2,6-lutidine, 2,3-lutidine, 3,5-lutidine and the like.

Suitable solvents which can be used in the process of step c) include but are not limited to: esters such as ethyl formate, methyl acetate, ethyl acetate, propyl acetate, ter-butyl acetate, iso-butyl acetate, methyl propanoate, ethyl proponoate, methyl butanoate, ethyl butanoate and the like; ethers such as tetrahydrofuran, 1,4- dioxane, diethyl ether, 1,2- dimethoxy ethane and the like; hydrocarbons such as n- hexane, cyclohexane, n-heptane, toluene, xylenes and the like; halogenated hydrocarbons such as chlorobenzene, 1 ,2-dichlorobenzene, and the like; aprotic polar solvents such as N,N-dimethyl formamide (DMF), dimethylsulfoxide, dimethylacetamide, acetonitrile and the like; ketones such as acetone, methyl isobutyl ketone, methyl tertiary butyl ketone, and the like; and mixtures thereof. The hydroxy-protecting reagent is added slowly to the reaction mixture. Suitable temperatures for conducting the reaction range from about 5°C to about 100 ⁰ C, preferably about 50 ⁰ C to 70 ⁰ C. Suitably the reaction is performed under inert atmosphere such as under nitrogen or argon.

After completion of the reaction, the reaction mixture is treated with activated charcoal and processed using suitable techniques for precipitation of the solid.

The solid product thus obtained may be further purified by methods such as precipitation, crystallization or slurrying in a solvent. Solvents that may be used for such purposes include, but are not limited to alcohols such as methanol, ethanol, isopropanol and the like; esters such as ethyl acetate, n-propylacetate, isopropyl acetate and the like; ketones such as acetone, ethyl methyl ketone and the like; acetonitrile, chlorohydrocarbons such as dichloromethane, chloroform ; water or mixtures thereof; preferably mixture of dichloromehtname and isopropanol are used.

The solid product is recovered by suitable techniques such as decantation, filtration by gravity or by suction, centrifugation, and the like. Other techniques for separating the solids from the reaction mixtures are also within the scope of this invention. The process may include further drying of the product obtained with or without vacuum and in presence or absence of inert atmosphere.

In a specific embodiment of the present invention, 2-deoxy-2, 2-diflouro-D- ^; erythro-pentofuranos-1-ulose-3, 5-dibenzoate is prepared by a process comprising the steps of: a) reacting 2,3-O-Cyclohexylidine-D-glyceraldehyde compound of Formula III with ethylbromodifluoroacetae in the presence of zinc to form Ethyl 2,2-difluoro-4,5- O-cyclohexylidene-3-hydroxypentanoate; b) reaction of Ethyl 2,2-difluoro-4,5-O-cyclohexylidene-3-hydroxypentanoate with hydrochloric acid; followed by azeotrophic distillation to form 2-desoxy-2, 2- difluoro-1-oxoribose of Formula Vl; c) protection of the hydroxy groups of 2-desoxy-2, 2-difluoro-1-oxoribose by reacting with benzoyl chloride in the presence of pyridine.

In this multi step process, each step may be contemplated individually or in combination of two or more steps. 2-deoxy-2, 2-diflouro-D-erythro-pentofuranos-1-ulose-3, 5-dibenzoate is prepared with improved yield and purity.

In another aspect the present application relates to use of 3,5- dihydroxy protected -2-deoxy-2,2-difluoro -1-oxoribose of Formula I prepared according to the process of present application in the synthesis of active pharmaceutical ingredients including gmcitabine or a salt thereof.

The use of cyclohexylidine protecting group is advantages that cyclohexanone is easily available and cheap reagent. Moreover, during reaction of step b) cylohexanone is formed in the process, which can be easily separated from the reaction mixture by extracting with toluene. The recovered cyclohexanone may be reused after simple purification.

This process of the present application involves the usage of inexpensive, less hazardous and easily available raw materials and makes the process suitable for commercial manufacturing.

Certain specific aspects and embodiments of the present application 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 Preparation of 2,3-O-Cyclohexylidine-D-glyceraldehyde (Formula III)

50 gm of D-mannitol and 112 ml of dimethylsulfoxide(DMSO) were charged in a flask and stirred for about 5 minutes. 87 ml of cyclohexanone was charged to the reaction mixture followed by charging 46.5 ml of triethylorthoformate at room temperature. 2.7 ml of borontrifluride diethylether was added and stirred the contents for about 18 hours at room temperature. The reaction mixture was quenched with 200 ml of 7 % aqueous sodium bicarbonate solution and stirred for 10 minutes. Reaction mixture was extracted with ethylacetate (450 ml) and the organic layer was washed with water followed by saturated NaCI solution. The final organic layer was distilled completely under vacuum to obtain a residue. 900 ml of n-hexane was added to the residue and stirred for 5 hours at 25-35 ⁰ C for solid precipitation. Filtered the solid and dried. Dry weight: 55 gm

The above obtained solid was dissolved in 550 ml of dichloromethane in a flask and 730 mg of tetrabutyl ammonium fluoride was added. 300 ml of water was added to the reaction mixture and 47 gm of sodium metaperiodate was added in portions. The reaction mixture was stirred at RT for 10 hours and then diluted with 800 ml of water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (300 ml). Total organic layer was washed with aqueous sodium bicarbonate followed by water and then with saturated NaCI solution. The organic layer was distilled completely to obtain 41 gm of the title compound as solid.

Example 2

Preparation of ethyl 2,2-dif1uoro-4,5-O-cyclohexylidene-3-hydroxypentanoate (Formula V)

28.4 gm of zinc powder was charge into a flask containing 400 ml of THF followed by addition of 5.8 ml of trimethyl silyl chloride under stirring. The reaction mixture was heated to reflux and a mixture of 40 gm of compound of Formula III (prepared as per example 1) and 64 ml of ethyl bromodifluoroacetae was added. The reaction contents were stirred at 60 - 65°C for about 2 hours. The reaction mixture was quenched with aqueous hydrochloric acid and extracted with ethylacetate(500 ml). The organic layer was washed with aqueous sodium bicarbonate solution followed by with saturated NaCI solution and then dried on sodium sulfate. The organic layer was distilled completely to obtain 58 gm of title compound as residue.

Example 3 Preparation of 2-desoxy-2, 2-difluoro-1-oxoribose (Formula Vl)

10 gm of compound obtained in Example 2 was charged into a flask containing 50 ml water, 5 ml hydrochloric acid and 50 ml of toluene was added to the flask and stirred for 5 minutes. The reaction mixture was heated to 70-80 ⁰ C and maintained for 4 hours. The tolune layer containing cyclohexanone was separated.

30 ml of Toluene was added to the aqueous reaction mixture and stirred at 70-80 ⁰ C for 30 minutes. The tolune layer containing cyclohexanone was separated. The aqueous layer pH was adjusted to about 4 with 7 ml pyridine. 50 ml toluene was added and 18 ml distilled from the reaction mass at atmospheric pressure. 18 ml of toluene was added to the reaction mixture again. The process of distilling solvent followed by addition of same quantity of toluene was repeated for 8 more times. Finally solvent was distilled completely under vacuum to obtain residue.

Example 4

Preparation of 2-deoxy-2, 2-diflouro-D-erythro-pentofuranos-1-ulose-3, 5- dibenzoate (Formula I)

The residue obtained in Example 3 was dissolved in 50 ml of ethylacetate at 25-35°C and 8 ml of pyridine was added to it. The reaction mass was heated to 60 ⁰ C and then a mixture of 10 ml of benzoyl chloride and 50 ml of ethylacetate was added slowly for 3 hours. The reaction mixture was maintained at about 60 ⁰ C for about 3 hours under nitrogen atmosphere. The reaction mixture cooled to 25-30 ⁰ C and stirred for 3 hours. 2 gm of activated charcoal was added to the mixture and cooled the contents to about 5 °C. The charcoal was removed by filtration and the filtrate was passed over anhydrous sodium sulfate. The final organic layer was distilled completely to obtain a semi solid material.

10 ml of dichloromethane and 50 ml of isopropyl alcohol was added to the above obtained material and cooled to about 10 ⁰ C. The solution was stirred for 2 hours for solid precipitation. The solid was filtered and dried to obtain 6 gm of the title compound.

Purity HPLC: 99.0% (Beta isomer)