FORM 2
THE PATENTS ACT 1970
(Act 39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
(SECTION 10 and Rule 13)
TITLE OF THE INVENTION "AN EFFICIENT PROCESS FOR PREPARATION OF FLUDARABINE"
Emcure Pharmaceuticals Limited.,
an Indian Company, registered under the Indian Company's Act 1957
and having its Registered Office at
Emcure House, T-184, M.I.D.C, Bhosari, Pune-411026, India.
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

FIELD OF THE INVENTION
The present invention provides an efficient process for the preparation of fludarabine and its phosphate salt. More specifically, the invention provides an improved process for the preparation of fludarabine free base with pharmaceutically acceptable purity.
BACKGROUND OF THE INVENTION
Fludarabine phosphate of formula (I), chemically known as 9H-Purin-6-amine-2-fluoro-9-(5-Q-phosphono-B-D-arabino-furanosyl) (2-fluoro-ara-AMP). Fludarabine is a fluorinated nucleotide analog of the antiviral agent vidarabine, 9-fl-D-arabinofuranosyladenine (Ara-A) that is relatively resistant to deamination by adenosine deaminase. Intravenous fludarabine is used as a single agent or in combination with other agents to treat adult patients with chronic lymphocytic leukemia (CLL). Fludarabine phosphate is rapidly dephosphorylated to 2-fIuoro-ara-A and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. This metabolite appears to act by inhibiting DNA polymerase alpha, ribonucleotide reductase and DNA primase, thus inhibiting DNA synthesis.

Various researchers have attempted to synthesize the active pharmaceutical ingredient fludarabine phosphate of formula (I).

US 4,188,378 relates to a method of utilizing 9-β-D-arabinofuranosyl-2-fluoroadenine (2-F-AraA) in the treatment of murine leukemia and as an antiviral agent for DNA virus type I and discloses debenzylation reaction by employing palladium or palladised charcoal or sodium /liquid ammonia. However, no information was disclosed about the experimental conditions however, a yield of 34% was obtained. The use of catalytic hydrogenation using hydrogen gas along with palladium/carbon always results in defluorination and gives a mixture of fludarabine and defluorinated fludarabine (Ara-A). Further, the use of sodium/ammonia or catalytic hydrogenation using pressure reactor (autoclave) for debenzylation reaction is quite tedious and produce an unsatisfactory product with respect to quality and yield.
US 4,210,745 and "Journal of Heterocyclic Chemistry, 16, 157(1957)" disclose an improved process for fludarabine, wherein debenzylation was carried out with boron trichloride in dichloromethane. Even though boron trichloride produces minimum defluorinated product; however the method is limited to the laboratory scale, because boron trichloride liberates hydrochloric acid and boric acid in presence of moisture and alcoholic solvent and require stringent anhydrous conditions for implementation on an industrial scale.
US 5,110,919 disclose a process for preparation of 2,6-(2,3,5,-tri-0-benzyl-J3-D-arabinofuranosyl) purine, which is further treated with fluoroboric acid and sodium nitrite in presence of tetrahydrofuran as solvent. Further, debenzylation with palladium chloride/charcoal produces fludarabine. This debenzylation-hydrogenation reaction provides an average yield; however the use of hydrogen gas necessities the utilization of pressure reactor (autoclave) which makes the reaction tedious and unfriendly for commercial application. In addition to the above drawbacks the use of methoxyethanol solvent is not recommended because of its toxic effect on bone marrow and testicles; high exposure causes granulocytopenia macrocytic anemia, oligospermia and others.
US 5,296,589 provide, a process to prepare pure fludarabine phosphate by converting fludarabine phosphate to sodium salt; finally crystallization of sodium salt from water

and it's conversion to fludarabine phosphate. The purification by recrystallization using water at high temperature generates impurities which are not easily removed from the fludarabine phosphate.
US 7,547,776 and US 6,046,322 discloses a method to prepare fludarabine phosphate with purity greater than 99.5%, wherein, fludarabine phosphate alkali metal salts are used as intermediates for the production of fludarabine phosphate with purity at least 99.5%. This purification process results only slight improvement in the purity of the fludarabine phosphate.
The present inventors have observed that all the prior art methods lacks consistency in getting desired quality of debenzylated product i.e. fludarabine on a large scale. Thus, there is a need to develop a method which produces desired quality and quantity of fludarabine and its phosphate salt.
Therefore, to over come the prior art drawbacks, the present inventors have developed a method by replacing the catalytic hydrogenation-debenzylation with transfer hydrogenation for debenzylation reaction, which is reproducible, easily implemented industrially, cost-effective and environmental friendly.
OBJECTS OF THE INVENTION
An object of the present invention is to provide an industrial viable method for the preparation of fludarabine and its phosphate salt with pharmaceutically acceptable purity. More specifically, the invention provides efficient and an improved process for debenzylation of 9-(2,3,5-tri-0-benzyl-p-D-arabinoftiranosyl)-2-fluoroadenine intermediate.
Another object of the present invention is to provide a simple cost effective process for the preparation of fludarabine and its phosphate salt with desired purity.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a consistent process for debenzylation of 9-(23,5-tri-0-benzyl-P-D-arabmofuranosyl)-2-fluoroadenine to fludarabine in presence of palladium charcoal and a transfer hydrogenating agent.
Another aspect of the present invention is to provide a process to purify fludarabine free base with purity greater than 99.5% in presence of cation exchange resin.
In another aspect of the present invention is to provide a process for preparation of fludarabine phosphate with purity greater than 99.95%.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a simple, economical and commercial viable process for the preparation of fludarabine phosphate.
In an embodiment of the present invention provides a process for the preparation of fludarabine with purity greater than 99.5% wherein the fludarabine free base is purified using cation exchange resin.
In another embodiment of the invention, wherein debenzylation-hydrogenation is carried out in presence of transfer hydrogenating agent.
The essential feature of the present invention for the preparation of fludarabine phosphate with purity greater than 99.5% comprises of the following steps:
• debenzylation of 9-(2,3,5-tri-0-benzyl-P-D arabinofuranosyl)-2-fluoroadenine with palladium charcoal and transfer hydrogenating agent in presence of organic solvent;
• fludarabine is purified by using cation exchange resin and

• finally fludarabine is phosphorylated to fludarabine phosphate.
The essential starting material 9-(2,3,5-tri-0-benzyl-fi-D-arabinofuranosyl)-2-fluoroadenine (formula II) for the invention is prepared by the method disclosed in US 4,188,378 and Journal of Medicinal Chemistry 12, 498, (11961).

The synthetic sequence for preparation of fludarabine phosphate is represented in the above scheme I:
The present invention relates to a process for the production of fludarabine phosphate wherein the debenzylation reaction is carried out with palladium charcoal and transfer hydrogenation in presence of an organic solvent. Thus, obtained crude fludarabine free base is purified by cation exchange resin and is then phosphorylated to fludarabine phosphate by the methods know in prior art,

In an embodiment, debenzylation reaction is carried out in presence of palladium /charcoal (Pd-C) and transfer hydrogenating agent in presence of an organic solvent. The present inventors have evaluated various grade palladium/charcoal available in the market ranging from 5% to 10% palladium on charcoal; after various experimentation inventors have found that the 10% Pd-C produces best results for debenzylation in presence of transfer hydrogenating agent. The amount of palladium charcoal utilized is in the range of 15 to 20% w/w of the starting material used. The reaction is carried out at temperature range from 70 to 75°C.
The transfer hydrogenating agent is selected from the group ammonium formate and ammonium acetate; and preferably ammonium formate. The solvents used for debenzylation-hydrogenation are selected from alcohol, ester, acetic acid and water or mixture thereof; preferably solvents are selected from alcohol, however best results are obtained in alcohol and water mixture. An alcoholic solvent is selected from methanol, ethanol, isopropyl alcohol and butyl alcohol; preferably methanol. The ratio of methanol and water are in the range of 60:40 to 70:30 and preferably 65:35.
The fludarabine free base thus produced after debenzylation has purity in the range of 90 to 95% and yield 92%. Further, fludarabine free base is purified by using cation exchange resin at 90 to 95°C in presence of water. The resin is used in the ratio of 1:1 with respect to fludarabine. During purification from resin and water impurities are adsorbed to the resin and results in pure fludarabine free base with purity greater than 99.5%, and yield in the range of 91% to 95%. The resin used is a cation exchange resin was procured from the vendor with trade name "DOWEX® 50WX2-200(H)".
The process for preparation of fludarabine free base of the instant invention is illustrated in the following sequences:
i) 9-(2,3,5-tri-0-benzyl-P-D-arabinofuranosyl)-2-fluoroadenine (formula II) is
treated with palladium charcoal and transfer hydrogenating agent in an
organic solvent at temperature range 70 to 75°C;

ii) the obtained fludarabine is purified by using cation exchange resin at 90 to
95°C in water and iii) finally pure fludarabine free base is phosphorylated with triethylphosphate
and phosphorous oxychloride.
The reaction conditions utilized in the present invention is efficient, environmentally friendly, lead higher purity and compatibility to an industrial scale.
The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however is not to be construed limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art, without departing from the spirit of the invention.
The invention is further explained with the help of following illustrative example, however, in no way these examples should be construed as limiting the scope of the invention.

EXAMPLE:
Example 1:
Preparation of Fludarabine (III):
9-(2,3,5-tri-0-benzyl-β-D-arabinofuranosyl)-2-fluoroadenine (formula II; lOOg) was suspended in water (700 ml), palladium-charcoal (10%; 20 g), ammonium formate (100 g) and methanol (1400 ml) at 25 to 30°C temperature. The reaction mixture was stirred for 2 hours at 70 to 75°C temperature. After completion, reaction mass was cooled to 60 to 65°C; catalyst was removed by filtration. The filtrate was concentrated under reduced pressure. The solid was filtered and to the wet cake water (5000 ml) and cation exchange [100 g, DOWEX® 50WX2-200(H)] was added and heated to 90 to 95°C. The resulting solution was filtered and cooled to 25 to 30°C. A pure fludarabine was isolated after drying at 50 to 55°C under reduced pressure for 12 to 15 hours to give a yield of 41 g of formula (III), Purity: > 99.5%.

We claim,
1. A process for preparation of fludarabine with purity greater than 99.5%
comprising,
a. Treatment of 9-(2,3,5-tri-0-benzyl-beta-D-arabinofuranosyl)-2-
fluoroadenine with palladium charcoal and transfer hydrogenating agent
in an organic solvent and isolation of product,
b. fludarabine obtained in step (a) is optionally purified by resin.
2. The process as claimed in claim 1(a), wherein the palladium charcoal is having strength of 5 to 10%, preferably 10%.
3. The process as claimed in claim 1(a), wherein the palladium charcoal employed is 20% of the 9-(2,3,5-tri-0-benzyl-beta-D-arabinofuranosyl)-2-fluoroadenine.
4. The process as claimed in claim 1(a), wherein the transfer hydrogenating agent is selected from the group of ammonium formate and ammonium acetate, preferably ammonium formate.
5. The process as claimed in claim 1(a), wherein the organic solvent is selected from the group alcohol, ester, acetic acid and water or mixture thereof.
6. The process as claimed in claim 5, wherein the alcoholic solvent is selected from methanol, ethanol, isopropyl alcohol and butyl alcohol; preferably methanol.
7. The process as claimed in claim 5 and clam 6, wherein the organic solvent is optionally mixed with water in the ratio of 60:40 to 70:30 (alcohol to water ratio), more specifically 65:35 ratio.
8. The process as claimed in claim 1(b), wherein the resin is cation exchange.

9. A process for preparation of fludarabine phosphate comprising,
a. Treatment of 9-(2, 3, 5-tri-O-benzyl-beta-D arabinofuranosyl)-2-
fluoroadenine with palladium-charcoal and transfer hydrogenating agent
in an organic solvent.
b. fludarabine obtained in step (a) is optionally purified by cation exchange
resin.
c. fludarabine free base is phosphorylated with phosphorylated with using
triethylphosphate and phosphoroxychloride.
10. A process for purification of fludarabine comprising, treating fludarabine with
resin in water.