2,2 '-Difluoro-2-deoxy-3,5-di-0-protected-l-0-protected pentose intermediates and improved process for p-anomer nucleosides.
Field of Invention:
The present invention relates to 2,2'-Difluoro-2-deoxy-3,5-di-0-protected-l-0-protected pentose derivatives, more particularly 2,2'-Difluoro-2-deoxy-3,5-di-0-protected-l-0-protected-D- ribofiiranose derivatives of formula (Va) for the preparation of therapeutically active enriched p- anomer nucleosides and its pharmaceutically acceptable salts and more particularly high yield and purity of p-anomer gemcitabine and its hydrohalide salt.
Background and Prior art of the Invention:
Nucleosides having nucleobase selected from pyrimidine or purine bases linked to 2,2'-difluoro- 2-deoxy-D-ribofuranose have therapeutic and commercial value, one such example is gemcitabine hydrohalide represented by the formula (I).

US Patent Nos.4,526,988, 4,692,434 and 4,808,614 discloses processes for the preparation of 2,2'-difluoro-2-deoxy-D-ribofuranosyl nucleoside of general formula (II) including gemcitabine hydrochloride. The process involves a mixture of a and p-anomeric hydroxyl protected 2,2'- difluoro-2-deoxy-D-ribofuranose having suitable leaving group at anomeric position as an one of the key intermediates. The leaving group employed were alkyl/aryl methanesulphonate, acetate, halo etc.

Anomeric mixture of intermediate (IV) is purified to obtain the required P-anomer for further use to obtain the required 2,2'-difluoro-2-deoxy-D-ribofuranosyl nucleosides.

Several improvements in the preparation of 2,2'-difluoro-2-deoxy-ribofuranosyl nucleosides and its intermediates have been reported in the prior art.
US Patent No.4,965,374 discloses a process for the preparation of erythro enantiomer of hydroxy protected lactone.
US Patent No.5,223,608 teaches the preparation of gemcitabine hydrochloride or hydrobromide salt in an anomeric purity of about 99% of p-anomer.
US Patent Nos.5,252,756; 5,256,797; 5,256,798; 5,420,266; 5,424,416; 5,426,183; 5,480,992; 5,541,340; 5,453,499; 5,521,345; 5,559,222; 5,608,043; 5,594,124; 5,606,408; 5,633,367; 5,637,688; 5,808,048; 4,965,374; 5,223,608; 4,34,254 and 5,945,457 are considered as relevant prior art in entirety in the present application.
Prior arts referred above are associated with one or more limitations enumerated below:
i) Use of expensive and hazardous reagent.
ii) Use of low temperature in the process steps adapted.
iii) Use of excess nucleobase.
iv) Use of gaseous ammonia under anhydrous condition for deprotecting the groups.
v) Preparation of protected ribofuranosyl derivative comprising of higher quantity of undesired a-anomer.
vi) Tedious purification procedure adapted to obtain desirable enriched p- anomer of nucleosides.
vii) Purification of 1,3 and 5 protected D-ribofuranose derivate is required.
Thus, there is a need for an improved process for the manufacture of enriched P- anomer nucleoside and its hydrohalide salts of genera] formula (la) and more particularly gemcitabine hydrohalide salts of formula (I). The surprising results of the present invention is an improved process by achieving enhanced yield and purity of P-anomer nucleosides and more particularly gemcitabine hydrochloride salt using novel intermediate 2,2'-difluoro-2deoxy-3,5-di-0- protected-1 -trihaloacetimidate of formula (Va) circumventing the limitations of the prior art.
Objects of the Invention:
Main objects of the invention is to provide key intermediate compound as 2,2'-difluoro-2-deoxy - 3,5-di-O-protected-1 -O-protected-D-ribofuranose.(Va).
Another object of the invention is to provide a process for the preparation of key intermediate of formula (Va).
Yet another object of the invention is to provide a key intermediate (Va) for the preparation of gemcitabine hydrochloride salt of formula (I).
Still another object of the invention is to provide a process for the preparation of p-anomer enriched 2-2'-difluoro-2-deoxy-D-ribofuranosyl nucleosides of general formula (II). Yet another object of the invention is to provide a process for the preparation of p-anomer enriched 2-2'-difluoro-2-deoxy-D-ribofuranosyl nucleoside hydrohalide salt of general formula (la).
Still yet another object of the invention is to provide a process for the enhanced yield and purity P-anomer of gemcitabine and its hydrohalide salts of general formula (I).
Further object of the invention is to provide a process which is easy and economical at commercial level of operation.
Summary of the Invention:
The present invention relates to a novel and key intermediate 2,2'-difluoro-2-deoxy-3,5-di-0- protected-l-O-protected-D-ribofuranose of formula (Va) and its use in the preparation of 2,2'- difluoro-2-deoxy-D-ribofuranosyl nucleoside hydrohalide salt of formula (la), more particularly gemcitabine and its hydrochloride salt of formula (I).
Detailed description of the Invention:
In accordance with the object, the present invention provides a novel intermediate 2,2'-diflluoro- 2-deoxy-3,5-di-0-protected-l-0-protected-D-ribofuranose (Va) for the preparation of 2,2'- difluoro-2-deoxy-ribofuranosyl nucleoside of formula (II).
An embodiment of the invention provides 1,3 and 5 protected pentose as a key intermediate for the preparation of required p-anomer enriched therapeutically active ribofurnosyl nucleosides.
Another embodiment of the invention provides 2,2'-difluoro-2-deoxy-l,3-and 5-tri-O-protected- D-ribofuranosyl derivate (Va) for the preparation of enriched p-anomer ribofuranosyl nucleoside, more particularly gemcitabine hydrohalide salt of formula (I).
Yet another embodiment of the invention provides a process for the preparation of enriched p- anomer of 2,2'-difluoro-2-deoxy-ribofuranosyl nucleoside hydrohalide salt of formula (la), the said process comprising steps of:
(a) preparing 2,2'-Difluoro-2-deoxy-3,5-di-0-protected-D-ribofuranose of formula AA
(b) contacting compound of formula (V) and trihaloacetonitrile in an inert solvent under nitrogen atmosphere in presence of a base, preferably organic base at a temperature ranging between -I0°C to + 10°C for a time period of up to 16 h to obtain compound of formula (Va),

(c) preparing separately suitably protected silyl derivative of nucleobase by reacting nucleobase and silylating agent in an inert solvent at a temperature ranging between 80°C to 115°C for a time period of 12 h to 15 h,
(d) reacting compound of formula (Va) and silylated nucleobase of step (c) in an inert solvent in presence of lewis acid catalyst to obtain compound of formula (Ha),
(e) dissolving compound of formula (Ha) in methanol, adding ammonia solution at a temperature ranging between 0°C to 10°C, raising the temperature up to 25°C, stirring for about 24 h, removing methanol to obtain mixture of a and p-anomer of 2,2'-Difluoro-2-deoxy-3',5'-dihydroxy-D-ribofuranosyl nucleoside (II),

(f) dissolving compound (II) in methanol, adding activated carbon, stirring, filtering, adding concentrated hydrohaloacid to the filtrate, cooling the mixture to about 0°C, filtering the solid obtained, drying the filtered solid to obtain crude anomer nucleoside hydrohalide salt compound of formula (la), and
(g) dissolving crude compound (la) in water, adding activated charcoal, stirred, filtered, filtrate added to acetic acid, separating the solid by adapting conventional method, washing the solid with acetone diying to obtain pure 0- anomer nucleoside of compound (la).

The above sequential steps adapted for the preparation of ribofuranosyl nucleoside hydrohalide salt of formula (la) is represented by the following flow chart:


Still another embodiment of the invention provides a process for the preparation of gemcitabine hydrochloride when the nucleobase used is cytosine.
2,2'-Difluoro-2-deoxy-D-ribofuranosyl nucleoside of the present invention may exist as either a anomer or P-anomer or mixture thereof.
Trihaloacetonitrile of the present invention is selected from a group consisting of trichloro acetonitrile and tribromoacetonitrile.
Organic base used in the present invention is selected from diethylamine, triethylamine, diisopropylethylamine, pyridine,2,4-dimethylaminopyridine, N-methyl morpholine, cyclohexyl amine and like, preferably triethylamine and diisopropylethylamine but not limited to these amines.
Inert solvents are those solvents which do not participate in the essential reaction. Suitable inert solvents is selected from the group of chlorinated hydrocarbons, e.g. dichloromethane and 1,2- dichloroethane; esters e.g. acetic acid (C1-4) alkyl esters e.g. ethyl acetate; ethers e.g. diisopropylether; aromatic hydrocarbons e.g. toluene, xylenes etc. Preferred chlorinated hydrocarbon solvent are dichloromethane and 1,2-dichloroethane.
Hydroxy protecting group may be selected from formyl, 2-chloroacetyl, benzyl, benzoyl, substituted benzoyl, diphenylmethyl, triphenyl methyl, 4-nitrobenzyl, phenoxycarbonyl, tertiary butyl, methoxymethyl, tetyrahydropyranyl, allyl, tetrahydrothienyl, 2-methoxethoxy methyl, methoxy acetyl, phenoxy acetyl, isobutyryl, benzyloxy carbonyl, ethoxy carbonyl, mesyl, trimethylsilyl, isopropyl dimethylsilyl, triisopropyl silyl, tertiary butyldimethyl silyl or methyldiisopropyl silyl and the like.
Lewis acid catalyst used is selected from the group consisting of aluminium chloride, tintetrachloride, trimethylsilyltrifluoromethanesulphonate, trimethylsilylnona fluorobutyl sulphonate, trimethylsilyl perchlorate, borontrifluoride diethyletherate, trimethylsilyl tetrafluoborate and the like.
:obases are selected from purine or pyrimidine bases represented by the structures as shown below:

Wherein R1 = H, alkyl or halogen; R2 = OH; R3 = H or halogen and R4 = H or nitrogen protective group.
The nucleobases mentioned above forms a part of the representative nucleobases used in the present invention but not limited to these nucleobases.
In the process of present invention the molar ratio of compound (V) to trihaloacetonitrile ranges between 1:5 to 1:12 preferably between 1:5 to 1:10.
In the process of present invention the molar ratio of compound (V) to base ranges between 1:1 to 1:1.5.
In the process of present invention the molar ratio of intermediate (Va) to nucleobase ranges between 1:1.2 to 1:2.0.
In the process of present invention the molar ration of intermediate (Va) to the Lewis acid used ranges between 1:1.5 to 1 : 3.0.
The invention is illustrated with the following examples and should not be construed to limit the scope of the present invention.
Examples
Example 1: Preparation of 2-deoxy-2,2'-difluoro-3, 5-di-O-benzoyl-l-trihaloacetamido-D- ribofuranose (Va);
To a cooled mixture of triholoacetonitrile (215 g) and an organic amine (4.5 g) under nitrogen atmosphere is added 2-deoxy-2,2'-difluoro-3.5-di-0-benzoyl-D-ribofuranose (30 g) dissolved in an inert organic solvent. Stirred the mixture between 0 to 5°C for 16 h to complete the reaction. Worked up by evaporation under vacuum to obtain the title compound (Va). (40 g).
Example 2: Preparation of protected nucleobase;
N-acetyl cytosine (30 g) is taken in ethylene dichloride (82 g). To it trimethylsilyl chloride (2.6 g) and hexamethyldisilazane (37 g) are added and heated to reflux between 80° - 90° for 10 h. Distill off the solvent under reduced pressure to obtain bissilylated cytosine (42 g).
Example 3: Preparation of protected ribonucleoside (Ila);
Compound (Va, 40 g) and suitably protected silylated derivative (40g) of nucleobase in an organic solvent (300 ml) is refluxed in presence of a lewis acid (41.5 g). Reaction mixture is cooled to room temperature, poured onto aqueous HC1, separating the organic layer washing the organic layer with sodium bicarbonate solution and saturated sodium chloride solution. Removed organic solvent to yield title compound (Ila, 38 g).
Example 4: Preparation of deprotected ribonucleoside (II);
Compound (Ila, 38 g) obtained in example 3 is contacted with 25% ammonium hydroxide solution (75 ml) in methanol (190 ml) and the mixture is stirred at room temperature for about 24 h. Remove methanol under reduced pressure to obtain the title compound (II, 22 g).
Example 5: Preparation of crude ribonucleoside hydrochloride (la);
a) Compound (II, 20 g) obtained in example (4) is dissolved in methanol, stirred with activated charcoal and filtered. To the filtrate added concentrated hydrochloric acid, cooled the mixture to 0°C to 5°C for 4 h. Separated the precipitated solid, dried the solid to obtain mixture containing P-anomer ribonucleoside (la, 4 g).
Example 6: Preparation of p-anomer enriched ribonncleoside (la);
Compound (la, 4 g) obtained in example (5) is dissolved in DM water (27 ml) by heating. The solution is treated with activated charcoal (350 mg) and filtered. The filtrate in treated with glacial acetic acid (320 ml), mixture stirred at room temperature, separated the solid, dried under vacuum to obtain P-anomer ribonucleoside (2.8 g) of anomeric purity upto 99.80%.

We Claim:
1. A compound of formula (Va)

Wherein, P = Hydroxy protecting group.

2. A compound of claim 1,wherein the hydroxy protective group P is selected from a group consisting offormyl, 2-chloroacetyl, benzyl, diphenylmethyl, triphenyl methyl, 4- nitrobenzyl, phenoxycarbonyl, tertiaiy butyl, methoxymethyl, tetyrahydropyranyl, allyl, tetrahydrothienyl,, 2-methoxethoxy methyl, methoxy acetyl, phenoxy acetyl, isobutyryl, ethoxy carbonyl, benzyloxy carbonyl, mesyl, trimethylsilyl, isopropyl dimethylsilyl, methyldiisopropyl silyl, triisopropyl silyl, or tertiary butyldimethyl silyl.
3. A process for the preparation of p-anomer of nucleoside of formula (la), the said process comprising steps of:
(a) preparing 2,2'-Difluoro-2-deoxy-3',5'-di-0-protected-D-ribofuranose of formula (V),
(b) contacting compound of formula (V) and trihaloacetonitrile in an inert solvent in presence of a base, preferably organic base at a temperature ranging between -10°C to + 10°C for a time period of up to 16 h to obtain compound of formula (Va),
(c) preparing separately silyl derivative of nucleobase by reacting nucleobase and silylating agent in an inert solvent at a temperature ranging between 110°C to 115°C for a time period of 12 h to 15 h,
(d) reacting compound of formula (Va) and silylated nucleobase of step (c) in an inert solvent in presence of lewis acid catalyst to obtain compound of formula (Ha),

(e) dissolving compound of formula (Ha) in methanol, adding ammonia solution at a temperature ranging between 0°C to 10°C raising the temperature up to 25°C stirring for about 24 h, removing methanol to obtain mixture of a anomer and P- anomer of 2,2'-Difluoro-2-deoxy-3,5-dihydroxy-D-ribofuranosyl nucleoside (II),

(f) dissolving compound (II) in methanol, adding activated carbon, stirring, filtering, adding concentrated hydrohaloacid to the filtrate, cooling to 0°C, filtering the solid, drying to obtain enriched crude P-anomer of (la), and
(g) dissolving the enriched crude P-anomer of (la) of step (f) in water, adding activated charcoal, stirred, filtered to filtrate added to acetic acid, separating the solid by conventional method, followed by washing the solid with acetone and drying to obtain pure P-anomer of (la).
4. The process of claim3, wherein trihaloacetonitrile used is selected from a group consisting of trichloroacetonitrile and tribromoacetonitrile.
5. The process of claim3, wherein the base used is selected from a group consisting .of diethyulamine, triethylamine, diisopropylethyllamine, cyclohexylamine, pyridine, 2,4- dimethylamino pyridine, N-methyl morpholine etc. Preferably triethylamine and diisopropylethylamine.
6. The process of claim 3, wherein the inert solvent used is selected from a group consisting of chlorinated hydrocarbons, e.g. dichloromethane and 1,2-dichloroethane; esters e.g. acetic acid (Cj-4) alkyl esters e.g. ethyl acetate; ethers e.g. diisopropylether; aromatic hydrocarbons eg. toluene, xylenes etc. Preferred chlorinated hydrocarbon solvent are dichloromethane and 1,2-dichloroethane.
7. The process of claim3, wherein the nucleobase is pyrimidine or purine base.
8. The process of claim3, wherein the lewis acid used is selected from a group consisting of tintetrachloride, trimethylsilyltrifluoromethanesulphonate, trimethylsilyl nonafluorobutyl sulphonate, trimethylsilyl perchlorate, borontrifluoride diethyletherate, trimethylsilyl tetrafluoborate and the like.
9. The process of claim 7, wherein the pyrimidine base is cytosine.
10. The process of claim3, wherein the molar ratio of compound (V) to trihaloacetonitrile ranges between 1:5 to 1:12 preferably between 1:5 to 1:10.
11. The process of claim 3, wherein the molar ratio of compound (V) to base ranges between 1:1 to 1:1.5.
12. The process of claim3, wherein the molar ratio of intermediate (Va) to nucleobase ranges from 1:1.2 to 1:2.0.
13. The process of claim 3 wherein the molar ration of intermediate (Va) to lewis acid ranges from 1 : 1.5 to 1 : 3.0.