INTERMEDIATES

Field of the Invention

The present invention relates to a process for the preparation of 3-O-benzyl- 1 ,2-0- isopropylidene-a-D-glucofuranuronic acid (Formula IV) and ester (Formula V) thereof. The process comprises benzylation of l,2:5,6-di-0-isopropylidene-a-D-glucofuranose (Formula I; hereinafter, referred to as diacetone-D-glucose) in the presence of tetrabutylammonium bromide followed by selective hydrolysis of the product formed (Formula II) using sulfuric acid to obtain 3-O-benzyl- 1,2-O-isopropylidene-a-D- glucofuranose (Formula III). Compound of Formula III is then oxidized to compound of Formula IV which upon esterification provides corresponding ester of Formula V. The process of the invention can be used in the production of fondaparinux sodium, a heparin and blood clotting factor Xa inhibitor.

FORMULA I FORMULA II FORMULA III

FORMULA IV FORMULA V

wherein R is optionally branched alkyl group. Background of the Invention

Fondaparinux sodium is indicated for the prophylaxis of deep vein thrombosis and chemically represented as methyl 0-2-deoxy-6-0-sulfo-2-(sulfoamino)-a-D- glucopyranosyl-(l→4)-0- -D-glucopyranuronosyl-(l→4)-0-2-deoxy-3,6-di-0-sulfo-2- (sulfoamino)-a-D-glucopyranosyl-(l→4)-0-2-0-sulfo-a-L-idop yranuronosyl-(l→4)-2- deoxy-6-0-sulfo-2-(sulfoamino)-a-D-glucopyranoside decasodium salt of following Formula VI.

FORMULA VI

There are numerous methods reported in the literature for the preparation of compounds of Formulae I to V.

Methods in Carbohydrate Chemistry, 6, p. 286-291 (1972), provides benzylation of diacetone-D-glucose using xylene, ether or petroleum ether as solvent and sodium metal as base for the preparation of 3-O-benzyl- 1,2:5, 6-di-O-isopropylidene-a-D-glucofuranose (Formula II; hereinafter, referred to as 3-O-benzyl diacetone-D-glucose). The compound of Formula II is then hydrolyzed using acetic acid to get the compound of the Formula III.

Tetrahedron Lett., 17, p. 3535-3536 (1976), describes preparation of the compound of Formula II from diacetone-D-glucose using costly reagents and solvents such as sodium hydride (base), tetrahydrofuran (solvent), benzyl bromide (reagent) and tetrabutyl ammonium iodide. The present inventors have observed that the recovery of

tetrahydrofuran by this process is very difficult and column operation is required to get the desired compound which is not feasible at commercial scale.

Bull. Korean Chem. Soc, 16(9), p. 805-808 (1995), describes the preparation of compounds of Formulae II and III starting from diacetone-D-glucose and the use of dioxane (toxic solvent) for the preparation of compound of Formula II. This process requires chromatographic purification technique to isolate the product in pure form. The compound of Formula II is then converted into compound of Formula III using aqueous acetic acid.

There are numerous methods reported in the literature for oxidation of alcohols to the corresponding aldehydes, ketones or acids. Selective oxidation of primary or secondary alcohols to the corresponding aldehydes and ketones by terminal oxidant such as chromium oxide (J. Org. Chem., 26, p. 4814-4816 (1961)), dichromate (J. Org. Chem., 35, p. 3589-3590 (1970)), manganese oxide (J. Am. Chem. Soc, 77, p. 4399-4401(1955)) and osmium or ruthenium (J. Synth. Org. Chem. Jpn., 46, p. 930-942 (1988)) is known.

J. Org. Chem., 52, p. 2559 (1987), mentions oxidation of primary and secondary alcohols by using sodium hypochlorite as terminal oxidant, 2,2,6,6-tetramethylpiperidine- 1 -oxyl (hereinafter referred as TEMPO) as a catalyst and potassium bromide as a co- catalyst. Tetrahedron Letters, p. 34, 1 181-1 184 (1993) also describes similar types of reactions.

J. Org. Chem., 61, p. 7452-7454 (1996), makes use of TEMPO and N- chlorosuccinimide (hereinafter referred as NCS) in presence of tetrabutylammonium chloride (TBAC1) as a phase transfer agent for the selective oxidation of primary alcohols to corresponding aldehydes with no overoxidation to carboxylic acids.

U.S. Patent No. 5,821,374 discloses the use of N-chloro compounds such as N- chloro-4-toluenesulfonamide sodium salt as an oxidant in the TEMPO catalyzed oxidation of primary alcohols to aldehydes. The use of TEMPO and hypochlorite for oxidation purpose is also known from WO 95/07303.

U.S. Patent No. 6,310,200 describes preparation of carboxylic acids from their corresponding alcohols comprising oxidation of the alcohols with hydantoin compounds using TEMPO as catalyst.

There are numerous methods reported in the literature for the preparation of esters from their corresponding carboxylic acid compounds, e.g., U.S. Patent No, 4,818,816 mentions esterification of compound of Formula IV to obtain methyl ester thereof (Formula V wherein R is methyl) using diazomethane. An esterification reaction of a glucofuranuronic acid using diazomethane is also described in Carbohydrate Research, 130, p. 221-241 (1984). A process for the esterification of acidic function of a carbohydrate compound by using methyl iodide in presence of base is described in Bioorganic and Medicinal Chemistry Letters, 1(2), p. 95-98 (1991), and Mini-Reviews in Medicinal Chemistry, 4(2), p. 207-233 (2004).

The inventors of the present invention observed that the processes known for the preparation of the compounds of Formula II and Formula III make use of either costly and toxic solvents or the prepared compound is isolated or purified by commercially non- feasible chromatographic technique(s). They also observed that oxidation of compound of Formula III by known oxidation conditions/techniques to obtain compound of Formula IV is time consuming, slow (about 24 hours) and low yielding. Moreover, the rapid oxidation of the compound of Formula III is not reported. The drawback in using diazomethane and methyl iodide for esterification reaction is that diazomethane is toxic and potentially explosive when exposed to rough surfaces or heated to high temperatures whereas methyl iodide fumes may cause lung, liver, kidney and central nervous system damage.

Diazomethane and methyl iodide are expensive reagents.

Therefore, there is a requirement for a simple, cost effective and commercially scalable process for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D- glucofuranuronic acid (Formula IV) and its corresponding ester (Formula V) using compounds of Formulae I, II and III as intermediates. There is also a requirement of achieving quicker and more efficiently oxidation (about 4 hours to 7 hours) for obtaining the compound of Formula IV in high yield from the compound of Formula III.

Summary of the Invention

The present invention relates to a process for the preparation of 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranose (Formula III) comprising benzylation of diacetone-D- glucose (Formula I) in presence of tetrabutylammonium bromide followed by selective hydrolysis of the product formed (Formula II) using sulfuric acid.

The present invention also relates to a process for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranuronic acid represented by Formula IV comprising oxidation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose represented by Formula III with N-bromosuccinamide (NBS) optionally in presence of 2,2,6,6- tetramethylpiperidine- 1 -oxyl (hereinafter, referred as TEMPO).

The present invention also relates to accelerated oxidation of 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranose (Formula III) to obtain 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronic acid (Formula IV) wherein said oxidation is accelerated by addition of at least one co-oxidant to the reaction mixture comprising an oxidant and a catalyst. The present invention contemplates a process for the oxidation of the compound of Formula III with a mixture of oxidant, co-oxidant and catalyst to obtain corresponding carboxylic acid of Formula IV.

The inventors of the present invention observed that the direct oxidation of alcoholic part/group in a given compound (preferably carbohydrate) to the corresponding carboxylic acid group can be achieved or accelerated by the addition of at least one co- oxidant to the reaction mixture comprising an oxidant, a catalyst and the compound.

Therefore, the present invention provides an one-pot process for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranuronic acid (Formula IV) incorporating accelerated oxidation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose (Formula III) by the use of an oxidant, at least one co-oxidant, and a catalyst.

The present invention also provides a simple, easy to scale-up and cost-effective method for the esterification of the compound of Formula IV. The present invention makes use of an alkanol in the presence of an inorganic acid for the said esterification reaction.

The present invention also provides a process for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranuronic acid (Formula IV) and ester (Formula V) thereof, comprising the use of compounds of Formula I, II and III as intermediate.

Detailed Description of the Invention

The following terminology can be used to understand the process of the present invention. These terminology are not intended in any way to limit the scope of the present invention. Several variants of these terminology would be evident to persons ordinarily skilled in the art. The term "about", as used herein, when used along values assigned to certain measurements and parameters means a variation of 10% from such values, or in case of a range of values, means a 10% variation from both the lower and upper limits of such ranges.

The term "ambient temperature", as used herein, is the temperature or temperature range selected from 0°C to 35°C. The temperature of 20°C to 30°C is preferable.

The term "base", as used herein, is selected from alkali metal or alkaline earth metal carbonates, bicarbonates or hydroxides.

The term "oxidant", as used herein, is the compound that is used to oxidize 3-0- benzyl- 1,2-O-isopropylidene-a-D-glucofuranose of Formula III. Exemplary but non- limiting examples for oxidant compounds are transition metals or oxide(s) thereof (such as chromium oxide, dichromate, manganese oxide, osmium or ruthenium compounds, etc.), alkali metal or alkaline earth metal hypochlorites or hypobromites (such as sodium hypochlorite or sodium hypobromite), alkali metal halites (such as sodium bromite or sodium chlorite), N-halo compounds (such as N-chloro-4-toluenesulfonamide, N- bromosuccinamide, N-chlorosuccinamide), hydantoin compounds (such as 1,3-dibromo- 5,5-dimethylhydantoin, 1 ,3-dichloro-5,5-dimethylhydantoin), etc.

The term "catalyst", as used herein, is selected from the group comprising of hindered nitroxide compound (also referred as hindered iminoxyl or N-oxyl compound) such as 2,2,6,6 (cis and trans)tetrasubstituted piperidine N-oxyl, e.g., 2,2,6,6- tetramethylpiperidine N-oxyl (TEMPO), bis-4,4'-(2,2,6,6-tetramethylpiperidine N- oxyl)oxamide and like compounds.

The term "co-oxidant", as used herein, is compound(s) that can accelerate the oxidation reaction. It can be added with or after the oxidant. These are also known as secondary oxidants and can be selected from oxidant compounds specified hereinabove. More specifically, alkali metal or alkaline earth metal hypochlorites or hypobromites (such as sodium hypochlorite or sodium hypobromite) or alkali metal halites (such as sodium bromite, sodium chlorite) can be used as co-oxidant. The term "alkyl", as used herein, represents hydrocarbon having (C _n H2 _n+ i-) chemical formula wherein n = 1 to 6. Some of the non-limiting examples are methyl, ethyl, propyl, butyl, isopropyl, isobutyl, tert. butyl, etc.

The term "alkanol", as used herein, represents an alcohol having (C _n H2 _n +i-OH) chemical formula wherein n=l to 6. Some examples are methanol, ethanol, propanol, butanol, isopropanol, isobutanol, tert. butanol, etc.

The term "inorganic acid", as used herein, represents mineral acids like hydrochloric acid, sulfuric acid, nitric acide, phosphoric acid, boronic acids and the like acids.

A first aspect of the present invention provides a process for the preparation of 3-

O-benzyl diacetone-D-glucose represented by Formula II;

FORMULA II

comprising benzylation of diacetone-D-glucose represented by Formula I;

FORMULA I

in the presence of tetra-butyl ammonium bromide.

In an embodiment of this aspect, the benzylation can be performed in the presence of toluene, dimethyl formamide (DMF), dichloromethane (DCM), or mixture thereof.

In another embodiment of this aspect, the benzylation can be performed at a temperature range of about 55°C to about reflux (boiling point) temperature of the solvent used. For example, if toluene is used as solvent then the reflux temperature is about 1 1 1°C and if DMF is used as solvent then the reflux temperature is about 153°C.

In another embodiment of this aspect, the benzylation can be performed in the presence of base.

In another embodiment of this aspect, the benzylation can be performed using benzyl chloride.

In another embodiment of this aspect, the compound of Formula II can be obtained without using chromatographic techniques.

Accordingly, tetra-butylammonium bromide and benzyl chloride are added to solution of diacetone-D-glucose in toluene, DMF or mixture thereof in presence of base followed by heating at temperature range of about 55°C to about reflux (boiling point) temperature of the solvent used. The resultant reaction mixture is then cooled to ambient temperature followed by addition of methanol and water under stirring. The solution so formed is allowed to settle and layers are separated. The product (compound of Formula II) is isolated from the organic layer by recovery of solvent.

The present inventors have preferably used toluene or DMF as the solvent and pulverized potassium hydroxide as the base for the preparation of 3-O-benzyl diacetone- D-glucose of Formula II.

In another embodiment of this aspect, fondaparinux or its sodium salt is prepared by making use of said benzylation reaction.

Fondaparinux or its sodium salt can be prepared from 3-O-benzyl-diacetone-D- glucose (Formula II) by following the procedure described in U.S. Patent No. 4,818,816.

A second aspect of the present invention provides a process for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose represented by Formula III;

FORMULA III comprising treating 3-O-benzyl diacetone-D-glucose represented by Formula II;

FORMULA II

with sulphuric acid.

embodiment of this aspect, the treatment of 3-O-benzyl diacetone-D-glucose of Formula II with sulphuric acid can be performed in the presence of methanol and water.

In another embodiment of this aspect, the treatment of Formula II with sulphuric acid can be performed at a temperature of about 35°C to about 45°C.

In another embodiment of this aspect, the compound of Formula III can be obtained without using chromatographic techniques.

Accordingly, sulphuric acid is added to a solution of 3-O-benzyl diacetone-D- glucose of Formula II in methanol and water. The reaction mixture so formed is heated to about 35°C to about 45°C for about 20 hours to 25 hours under stirring. The mixture is then cooled and subjected to pH adjustment until 7.2 to 7.5 using base, followed by stirring. The solvent is recovered under vacuum followed by addition of dichloromethane (DCM) and water. The mixture is allowed to settle and the layers so formed are separated. The product is then isolated from the organic layer as gummy mass.

The present inventors have preferably used sodium hydroxide as base for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose of Formula III.

The compound of Formula II can be prepared by following the process described in the first aspect of the present invention.

In another embodiment of this aspect, fondaparinux or sodium salt thereof can be prepared by making use of the compound of Formula III.

Fondaparinux or sodium salt thereof can be prepared from 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranose (Formula III) by following the procedure described in U.S. Patent No. 4,818,816.

A third aspect of the present invention provides a process for the preparation of 3- O-benzyl- 1 ,2-O-isopropylidene-a-D-glucofuranose represented by Formula III;

FORMULA III

comprising:

a) benzylating diacetone-D-glucose represented by Formula I in the presence of tetra-butyl ammonium bromide to obtain 3-O-benzyl diacetone-D-glucose represented by Formula II; and

FORMULA I FORMULA II

b) treating the compound of Formula II with sulphuric acid.

In an embodiment of this aspect, the benzylation can be performed in the presence of toluene, dimethyl formamide (DMF), dichloromethane (DCM), or a mixture thereof.

In another embodiment of this aspect, benzylation can be performed at temperature range of about 55°C to about reflux temperature of the solvent used. For example, if toluene is used as solvent then the reflux temperature is about 1 11°C and if DMF is used as solvent then the reflux temperature is about 153°C.

In another embodiment of this aspect, benzylation can be performed in the presence of a base.

In another embodiment of this aspect, benzylation can be performed using benzyl chloride.

In another embodiment of this aspect, the compound of Formula II can be obtained without using chromatographic techniques.

In another embodiment of this aspect, the treatment of compound of Formula II with sulphuric acid can be performed in the presence of methanol and water.

In another embodiment of this aspect, the treatment of Formula II with sulphuric acid can be performed at temperature of about 35°C to about 45°C.

In another embodiment of this aspect, the compound of Formula III can be obtained without using chromatographic techniques.

Accordingly, tetra-butylammonium bromide and benzyl chloride are added to solution of diacetone-D-glucose in toluene, DMF or mixture thereof in presence of base followed by heating at temperature range of about 55°C to about reflux (boiling point) temperature of the solvent used. The resultant reaction mixture is then cooled to ambient temperature followed by addition of methanol and water under stirring. The solution so formed is allowed to settle and layers are separated. The product (compound of Formula II) is isolated from the organic layer by recovery of solvent. Sulphuric acid is added to the solution of compound of Formula II in methanol and water. The reaction mixture so formed is heated to about 35°C to about 45°C for about 20 hours to 25 hours under stirring. The mixture is then cooled and subjected to pH adjustment until 7.2 to 7.5 using base, followed by stirring. The solvent is recovered under vacuum followed by addition of dichloromethane (DCM) and water. The mixture is allowed to settle and the layers so formed are separated. The product (compound of Formula III) is then isolated from the organic layer as gummy mass.

The present inventors have preferably used toluene or DMF as solvent and pulverized potassium hydroxide as base for the preparation of 3-O-benzyl diacetone-D- glucose of Formula II, and sodium hydroxide as base for the preparation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose of Formula III.

In another embodiment of this aspect, fondaparinux or sodium salt thereof can be prepared by making use of the compound of Formula III.

Fondaparinux or sodium salt thereof can be prepared from 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranose (Formula III) by following the procedure described in U.S. Patent No. 4,818,816.

A fourth aspect of the present invention provides a process for the preparation of 3- 0-benzyl-l,2-0-isopropylidene-a-D-glucofuranuronic acid represented by Formula IV;

FORMULA IV

comprising oxidation of 3-0-benzyl-l,2-0-isopropylidene-a-D-glucofuranose represented by Formula III;

FORMULA III

with N-bromosuccinamide (NBS) optionally in presence of 2,2,6,6-tetramethylpiperidine- 1-oxyl (TEMPO).

In an embodiment of this aspect, the oxidation reaction can be performed in the presence of tetrahydrofuran, ether, water, or mixture thereof.

In another embodiment of this aspect, the oxidation reaction can be performed at ambient temperature.

In another embodiment of this aspect, the oxidation of compound of Formula III can be performed with NBS in the presence of TEMPO.

In another embodiment of this aspect, the oxidation of compound of Formula III can be performed with NBS in the presence of TEMPO and base. Accordingly, base, TEMPO and NBS are added to a solution of compound of Formula III in THF and water followed by stirring at ambient temperature. Upon completion of reaction, the reaction mixture is quenched with sodium bicarbonate and/or sodium thiosulphate solution followed by washing with dichloromethane (DCM). The pH of the resultant aqueous phase is then adjusted to 1 by diluted hydrochloric acid followed by extraction of product in dichloromethane. The product (compound of Formula IV) is isolated by removal of solvent.

The present inventors preferably selected sodium bicarbonate as base.

In another embodiment of this aspect, fondaparinux or sodium salt thereof is prepared by making use of said oxidation reaction.

In another embodiment of this aspect, 3-O-benzyl- 1,2-O-isopropylidene-a-D- glucofuranuronic acid of Formula IV can be further used to prepare fondaparinux or sodium salt thereof.

3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose (Formula III) can be prepared by any process known in the prior art. The present inventors have followed the process described in Methods in Carbohydrate Chemistry, 6, p. 286-291 (1972), for the preparation of the compound of Formula III.

Fondaparinux or sodium salt thereof can be prepared from 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronic acid (Formula IV) by following the procedure described in U.S. Patent No. 4,818,816.

A fifth aspect of the present invention provides highly pure 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronic acid (Formula IV).

In an embodiment of this aspect, 3-O-benzyl- 1,2-O-isopropylidene-a-D- glucofuranuronic acid (Formula IV) having purity of 90% or more by HPLC is provided.

A sixth aspect of the present invention provides process for oxidation of 3-0- benzyl-l,2-0-isopropylidene-a-D-glucofuranose represented by Formula III;

FORMULA III

comprising use of oxidant, co-oxidant, catalyst or mixture thereof.

In an embodiment of this aspect, the oxidation reaction produces 3-0-benzyl-l,2- O-isopropylidene-a-D-glucofuranuronic acid represented by Formula IV.

FORMULA IV

In another embodiment of this aspect, the oxidation of the compound of Formula III is performed at ambient temperature.

In another embodiment of this aspect, the oxidation of the compound of Formula III is performed in presence of base.

In another embodiment of this aspect, the oxidation of the compound of Formula III comprises use of oxidant, catalyst and co-oxidant.

Accordingly, oxidant, co-oxidant and catalyst are added to the stirred solution of 3- O-benzyl- 1 ,2-O-isopropylidene-a-D-glucofuranose (Formula III) at ambient temperature. The solution of compound of Formula III is prepared by mixing THF (or ether), water and base to the compound of Formula III. Upon completion of reaction, the product (3-0- benzyl-l,2-0-isopropylidene-a-D-glucofuranuronic acid) of Formula IV is isolated.

In another embodiment of this aspect, preferably, NBS as oxidant, TEMPO as catalyst, sodium chlorite as co-oxidant, and sodium bicarbonate as base can be used for said oxidation. In another embodiment of this aspect, fondaparinux or sodium salt thereof is prepared by making use of said oxidation reaction.

3-0-benzyl- l,2-0-isopropylidene-a-D-glucofuranose (Formula III) can be prepared by any process known in the prior art. The present inventors have followed the process described in Methods in Carbohydrate Chemistry, 6, p. 286-291 (1972), for the preparation of the compound of Formula III.

Fondaparinux or sodium salt thereof can be prepared from 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronic acid (Formula IV) by following the procedure described in U.S. Patent No. 4,818,816.

A seventh aspect of the present invention provides a process for preparation of 3-

0-benzyl-l,2-0-isopropylidene-a-D-glucofuranuronic acid represented by Formula IV;

FORMULA IV

by accelerated oxidation of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose represented by Formula III;

FORMULA III

that comprises addition of a co-oxidant in the reaction mixture comprising oxidant, catalyst and the compound of Formula III.

In another embodiment of this aspect, the said accelerated oxidation is performed at ambient temperature. In another embodiment of this aspect, the said accelerated oxidation is performed in the presence of base.

Accordingly, the oxidant and catalyst are added to a stirred solution of 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose (Formula III) at ambient temperature followed by addition of co-oxidant. The solution of the compound of Formula III is prepared by mixing it with THF (or ether), water and base. Upon completion of reaction, the product (3-0-benzyl- l,2-0-isopropylidene-a-D-glucofuranuronic acid) of Formula II is isolated.

The term "reaction mixture", as used in this aspect, is the mixture comprising the compound of Formula III, THF (or ether), base, water, oxidant and catalyst. The term "reaction mixture" for this aspect excludes co-oxidant.

In another embodiment of this aspect, preferably, NBS as oxidant, TEMPO as catalyst, sodium chlorite as co-oxidant, and sodium bicarbonate as base can be used for the said accelerated oxidation.

In another embodiment of this aspect, fondaparinux or sodium salt thereof is prepared by making use of said accelerated oxidation reaction.

3-0-benzyl- l,2-0-isopropylidene-a-D-glucofuranose (Formula III) can be prepared by any process known in the prior art. The present inventors have followed the process described in Methods in Carbohydrate Chemistry, 6, p. 286-291 (1972), for the preparation of the compound of Formula III.

Fondaparinux or sodium salt thereof can be prepared from 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronic acid (Formula IV) by following the procedure described in U.S. Patent No. 4,818,816.

An eighth aspect of the present invention provides a process for the preparation of ester represented by Formula V;

FORMULA V

wherein R is optionally branched alkyl group; from 3-0-benzyl-l,2-0-isopropylidene-a- D-glucofuranuronic acid represented by Formula IV;

FORMULA IV

comprising the use of alkanol.

In an embodiment of this aspect, the process can be performed in the presence of inorganic acid.

In another embodiment of this aspect, the process can be performed at ambient temperature.

Accordingly, the alkanol and inorganic acid are added to the compound of Formula IV followed by stirring at ambient temperature. The resultant reaction mixture is cooled (temperature reduced by 10°C from ambient temperature) and the product (ester) is isolated from the reaction mixture as gummy mass.

The present inventors preferably selected methanol as the alkanol, hydrochloric acid as the inorganic acid and 20°C to 30°C as ambient temperature.

The compound of Formula IV is a known compound and can be prepared by following methods known in the prior art (for example, U.S. Patent No. 4,818,816). The present inventors have prepared the compound of Formula IV by oxidizing the corresponding alcohol precursor using oxidant (such as NBS) in presence of catalyst (such as TEMPO) and adding co-oxidant (such as sodium chlorite) into the reaction mixture comprising the oxidant and catalyst.

In another embodiment of this aspect, the process can be used for the preparation of fondaparinux or sodium salt thereof.

Fondaparinux or sodium salt thereof can be prepared from alkyl 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronate (Formula V) by following the procedure described in U.S. Patent No. 4,818,816.

In another embodiment of this aspect, the R in Formula V can be selected from methyl, ethyl, propyl and isopropyl.

A ninth aspect of the present invention provides highly pure methyl 3-O-benzyl-

1,2-O-isopropylidene-a-D-glucofuranuronate (Formula V wherein R is methyl).

In an embodiment of this aspect, methyl 3-0-benzyl-l,2-0-isopropylidene-a-D- glucofuranuronate (Formula V wherein R is methyl) having purity of 90% or more by HPLC is provided.

A tenth aspect of the present invention provides a process for the preparation of ester represented by Formula V;

FORMULA V

wherein R is optionally branched alkyl group;

comprising:

a) benzylating diacetone-D-glucose represented by Formula I in presence of tetra- butyl ammonium bromide to obtain 3-O-benzyl diacetone-D-glucose represented by Formula II;

FORMULA I FORMULA II

b) treating the compound of Formula II with sulphuric acid to obtain 3-O-benzyl- 1,2-O-isopropylidene-a-D-glucofuranose represented by Formula III;

FORMULA III

c) oxidizing the compound of Formula III with oxidant, optionally in presence of catalyst and/or co-oxidant, to obtain 3-O-benzyl- 1,2-O-isopropylidene-a-D- glucofuranuronic acid represented by Formula IV; and

FORMULA IV

d) treating the compound of Formula IV with alkanol in presence of inorganic acid.

In an embodiment of this aspect, the benzylation can be performed in the presence of toluene, dimethyl formamide (DMF), Dichloromethane, or mixture thereof.

In another embodiment of this aspect, the benzylation can be performed at temperature range of about 55°C to about reflux (boiling point) temperature of the solvent used. For example, if toluene is used as solvent then the reflux temperature is about 1 1 1°C and if DMF is used as solvent then the reflux temperature is about 153°C.

In another embodiment of this aspect, the benzylation can be performed in the presence of base.

In another embodiment of this aspect, the benzylation can be performed in the presence of pulverized potassium hydroxide.

In another embodiment of this aspect, the benzylation can be performed using benzyl chloride.

In another embodiment of this aspect, the compound of Formula II can be obtained without using chromatographic techniques.

In another embodiment of this aspect, the treatment of the compound of Formula

II with sulphuric acid can be performed in the presence of methanol and water.

In another embodiment of this aspect, the treatment of Formula II with sulphuric acid can be performed at temperature of about 35°C to about 45°C.

In another embodiment of this aspect, the compound of Formula III can be obtained without using chromatographic techniques.

In another embodiment of this aspect, the oxidation of the compound of Formula

III is performed at ambient temperature.

In another embodiment of this aspect, the oxidation of the compound of Formula III is performed in the presence of base.

In another embodiment of this aspect, the oxidation of the compound of Formula III is performed in the presence of tetrahydrofuran (THF), ether, water or mixture thereof.

In another embodiment of this aspect, the oxidation of the compound of Formula III comprises use of oxidant, catalyst and co-oxidant.

In another embodiment of this aspect, preferably, NBS as oxidant, TEMPO as catalyst, sodium chlorite as co-oxidant, and sodium bicarbonate as base can be used for the said oxidation. In another embodiment of this aspect, step d) can be performed at ambient temperature.

The present inventors preferably selected methanol as alkanol and hydrochloric acid as inorganic acid.

In another embodiment of this aspect, the R in Formula V can be selected from methyl, ethyl, propyl and isopropyl.

In another embodiment of this aspect, the process can be used for the preparation of fondaparinux or sodium salt thereof.

Fondaparinux or sodium salt thereof can be prepared from alkyl 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronate (Formula V) by following the procedure described in U.S. Patent No. 4,818,816.

Although first to ninth aspects and embodiments thereof mentioned hereinabove describe process for preparation of various intermediate compounds of 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronate (Formula V), the same embodiments and process details can be employed herein in this aspect to understand the process of the invention.

Diacetone-D-glucose of Formula I used hereinabove in various aspects can be prepared by any process known in prior art. It is generally known from the prior art that monosaccharides which contain two sterically adjacent OH groups in the cis-position can be reacted with aldehydes or ketones in the presence of sulphuric acid, zinc chloride or phosphorus oxide to obtain the corresponding acetals. Thus, the diacetone-D-glucose can be obtained by reacting D-glucose with acetone in the presence of sulphuric acid. In order to achieve high conversions, the water resulting from the ketalization must be bound or eliminated from the reaction mixture.

While the present invention has been described in terms of its specific aspects, 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.

In the following section, aspects are described by way of examples to illustrate the processes of the invention. However, the examples mentioned below are not intended in any way to limit the scope of the present invention. Several variants of the examples would be evident to persons ordinarily skilled in the art. Examples:

Example 1 : Process for the Preparation of 3-O-Benzyl diacetone-d-glucose

To a solution of diacetone-D-glucose (100 g) in toluene (500 ml) at ambient temperature, pulverized potassium hydroxide (43.11 g), tetrabutyl ammonium bromide (12.4 g) and benzyl chloride (58.4 g) were added. The reaction mixture was slowly heated to 80°C to 85°C. At the same temperature, the reaction mixture was stirred for 2 hours to 3 hours and then cooled to 25°C to 30°C. Methanol (20 ml) was added to the mixture and the resultant reaction mixture was stirred for 30 minutes. Water (500 ml) was added to the reaction mixture under stirring and the mixture was allowed to settle. The organic layer so formed was separated and washed twice with water (500 ml x 2). The solvent was recovered under vacuum at 60°C from the resultant organic layer to obtain the titled compound.

Yield: 149 g

Example 2: Process for the Preparation of 3-O-Benzyl- 1,2-O-isopropylidene-a -D- glucofuranose

To a solution of 3-O-benzyl diacetone-D-glucose (134 g; obtained in Example 1) in methanol (550 ml), water (170 ml) and sulphuric acid (1.7 g) were added at ambient temperature and the reaction mixture was heated to 38°C to 43°C. At the same temperature, the reaction mixture was stirred for 22 hours to 24 hours and then cooled to 25°C to 30°C. The pH of the reaction mixture was adjusted to 7.2 to 7.5 by adding 10% sodium hydroxide solution (14 ml) to it. The reaction mixture was then stirred for 15 minutes to 20 minutes followed by complete recovery of solvent under vacuum at 45°C to 50°C. To the resultant mass, dichloromethane (500 ml) and water (200 ml) were added, followed by stirring. The resultant mixture was then allowed to settle and the organic layer was separated. The aqueous layer was washed with dichloromethane (100 ml). The combined organic layer was again washed with water (50 ml) and the titled product (as gummy mass) was obtained from the organic layer by complete recovery of solvent. Example 3 : Process for the Preparation of 3-O-Benzyl- 1 ,2-O-isopropyridene-a-D- glucofuranuronic Acid

To a solution of 3-0-benzyl-l,2-0-isopropylidene-a-D-glucofuranose (1 g) in tetrahydrofuran (THF, 15 ml) and water (4 ml), sodium bicarbonate (1.35 g), 2,2,6,6- tetramehtyl- 1 -piperidnyloxy free radical (TEMPO, 7 mg) and N-bromosuccinimide (NBS, 1.4 g) were added. The mixture was stirred and the reaction progress was monitored with thin layer chromatographic (TLC) technique. Upon completion, the reaction was quenched with saturated sodium bicarbonate solution (17 ml) and 10% sodium thiosulphate (6 ml) followed by dichloromethane (30 ml) washing. The pH of the resultant aqueous phase was adjusted to 1.0 using diluted IN hydrochloric acid and the product was extracted with dichloromethane (DCM, 15 ml). The titled product was obtained after evaporating the solvent.

Yield: 0.65 g

Example 4: Process for the Preparation of S-O-Benzyl-l^-O-isopropyridene-a-D- glucofuranuronic Acid

To a solution of 3-0-benzyl-l,2-0-isopropylidene-a-D-glucofuranose (0.38 mole) in tetrahydrofuran (THF; 590 ml), water (236 ml) and sodium bicarbonate (1.9 mole) at ambient temperature (20°C to 30°C), N-bromosuccinamide (NBS; 1.5 mole) and 2,2,6,6- tetramethylpiperidine- 1 -oxyl (TEMPO; 0.0024 mole) were added. The resulting reaction mixture was stirred for 2 hours followed by addition of sodium chlorite (0.076 mole). The mixture was stirred until completion of reaction at ambient temperature (20°C to 30°C). To the resulting reaction mixture, 4% sodium thiosulphate (1180 ml), water (590 ml) and dichloromethane (590 ml) were added, followed by stirring for 30 minutes. The reaction mixture was allowed to settle. The layers were separated. The aqueous layer was washed with dichloromethane (59 ml). The pH of the aqueous phase was adjusted to 1.0 using 5N HC1 and product was extracted twice using dichloromethane (590 ml x 2). The organic layers were combined and concentrated under vacuum to obtain 3-O-benzyl- 1,2-0- isopropylidene-a-D-glucofuranuronic acid as gummy mass.

Yield: 80% to 95% Example 5: Process for the Preparation of Methyl ester of 3-O-Benzyl- 1,2-0- isopropylidene-g-D-glucofuranuronic Acid

To 3-0-benzyl-l,2-0-isopropylidene-a-D-glucofuranuronic acid (0.237 mole), 0.1% (w/v) methanolic hydrochloric acid (385 ml) was added followed by stirring at 20°C to 25°C. The resultant reaction mixture was stirred until completion of the reaction at 20°C to 25°C. After completion, the reaction mixture was cooled to 10°C to 15°C. The pH of the reaction mixture was adjusted to 7.5-7.7 by addition of 10% sodium hydroxide solution. Methanol was recovered under vacuum at 45°C to 50°C. Ethyl acetate (380 ml) and water (150 ml) were added followed by stirring. The layers were separated and the ethyl acetate layer was washed with 10% brine solution (50 ml). The solvent was recovered under vacuum to obtain gummy mass (compound of Formula V wherein R is methyl).

Yield (w/w): 74%