BACKGROUND OF INVENTION
Clofarabine (1) is a purine nucleoside antimetabolite and it acts as nucleic acid synthesis inhibitor. Chemically it is known as (2R,3R,4S,5R)-5-(6-amino-2-chloro-9i^-purin-9-yl)-4-fluoro-2-(hydroxymethyl) tetrahydrofuran-3 -ol. FDA-approved Clofarabine (1) for treating relapsed or refractory acute lymphoblastic leukemia (ALL) in children after at least two other types of treatment have failed.
Various synthetic routes are available for the synthesis of Clofarabine (1).
US 6,949,640 patent discloses the synthesis of Clofarabine (1) using 2-deoxy-2-fluoro-3,5-di-0-benzoyl-[a]-D-arabinofuranosyl bromide with 2,6-dichloropurine in presence of sodium hydride to form a mixture of a and p isomers. The p isomer was isolated from a mixture of acetonitrile and chloroform and treated with sodium methoxide. The crude was neutralized with Dowex resin and purified using flash chromatography. The reaction mass so obtained was chilled ethanolic ammonia solution for 16-20hrs to obtain Clofarabine (1). Use of pyrophoric sodium hydride, costly cationic resins (Dowex) and chloroform is not advisable at industrial scale.
US 6,680,382 patent discloses the synthesis of Clofarabine (1) by reacting 2-deoxy-2-fluoro-3,5-di-0-benzoyl-[a]-D-arabinofuranosyl bromide with chloroadenine in presence of potassium salt of 1-hydroxybenzotriazole (KOBt) for 16-48hrs to form a mixture of a and P isomers. P anomer was converted to Clofarabine (1) by treating with sodium methoxide.
US 5,661,136 discloses the synthesis of Clofarabine (1) by treating the 2,6-dichloro-9-(3-0-acetyl-5-0-benzoyl-2-deoxy-2-fluoro-P-D-arabinofuranosyl)-9//-purinen with saturated anhydrous ammonia, followed by treatment with lithium hydroxide monohydrate to obtain Clofarabine (1) with 42.3% yield. The purity of the final compound is not disclosed.
2

The main disadvantage with the above processes is the long duration of time for the completion of reaction resulting in low yields and making the process tedious. Also use of highly pyrophoric chemicals like sodium hydride, solvents like acetonitrile and chloroform restricts their use in industrial scale. Also, use of resins makes the process at large scale very costly.
Hence, the present inventors hereby, report an improved process for the preparation of Clofarabine (1) using simple purification process with lesser reaction time and safer chemicals to obtain better yield and purity of Clofarabine (1).
SUMMARY OF THE INVENTION
Accordingly, in one aspect, the present invention to provide an improved process for the preparation of Clofarabine (1)
The steps involved for the preparation of Clofarabine (1) comprises of the following steps:
a) Reacting 2-deoxy-2-P-fiuoro-l, 3, 5-tri-O-benzoyl-l-a-D-ribofuranose (6) with a suitable brominating agent in a suitable solvent and base to obtain 3, 5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranosyl bromide (5)
b) condensing intermediate (5) with 2,6-dichloropurine (4) in an aprotic solvent and base to form 2, 6-dichloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl)-9//-purine (3)
c) treating intermediate (3) with ammonia gas in an aprotic solvent to obtain 2-Chloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fiuoro-beta-D-arabinofuranosyl)-9H-purin-6-amine (2)
d) debenzoylating intermediate (2) using a suitable base to form Clofarabine (1)
e) purifying the Clofarabine (1) from a mixture of protic and aprotic solvents.
3

Another aspect of the invention is to provide process for the purification of crude Clofarabine (1), from aprotic or protic solvents or mixture thereof. Purification process comprises of the following steps:
1. Adding Clofarabine (1) to a mixture of protic and aprotic solvents
2. heating to 65-70 °C
3. treating with neutral charcoal
4. cooling the reaction mixture to 0-5 °C
5. isolating the crystalline solid at 0-5 °C
Clofarabine (1) produced in the above process after purification is having purity greater than 99.5% by HPLC (High-performance liquid chromatography).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: illustrates the X-Ray powder diffraction pattern (XRPD) of Clofarabine (1) Figure 2: illustrates the differential scanning calorimetry (DSC) of Clofarabine (1)
DETAILED DESCRIPTION OF THE INVENTION

...«Br
OBz

(6)

Step a)

OBz

CI
BzO i F
Bz6
(2)

Scheme-1
4

In one embodiment, this invention provides an improved process for the synthesis of Clofarabine (1) with more than 99.5% purity.
Step a) involves reaction of 2-deoxy-2-P-fluoro-l,3,5-tri-0-benzoyl-l-a-D-ribofuranose (6) with a suitable brominating agent in presence of an aprotic solvent and base to obtain 3, 5-di-0-benzoyl-2-deoxy-2-fluoro-a-D-arabinofuranosyl bromide (5).2-deoxy-2-(3-fluoro-l,3,5-tri-0-benzoyl-l-a-D-ribofuranose (6) may be dissolved in an aprotic solvent and cooled to -10 to 5 °C, preferably -5 to 0 °C, with the addition of a suitable brominating agent.
In one embodiment, the suitable brominating agent may be selected from a group comprising of triphenylphosphonium dibromide, hydrobromic gas, hydrobromic acid (48% & 62%) in combination with glacial acetic acid (33%), or the like, preferably hydrobromic acid in glacial acetic acid (33%), was used in the present invention.
In another embodiment, the suitable base used in step a) may be selected from a group of alkali metal bicarbonates comprising of sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, cesium bicarbonate or the like, preferably sodium bicarbonate was used in the present invention.
Step b) proceeds with condensation of intermediate (5) with 2,6-dichloropurine (4) to form 2, 6-dichloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl)-9//-purine (3). Intermediate (5) may be dissolved in a suitable aprotic solvent and reacted with 2,6-dichloropurine (4) in presence of strong base and heated. The base may be selected from alkaline metal alkoxide which helps in dehydrohalogenation. The said reaction may be heated at 40-100 °C, preferably 50-55 °C. The reaction mass so obtained may be purified from different protic and aprotic solvents to obtain 2, 6-dichloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl)-9//-purine (3).
The bases used in step b) may be selected from a group of metal alkoxides comprising of sodium methoxide, sodium ethoxide; potassium methoxide, potassium ethoxide, sodium butoxide, potassium butoxide, potassium-t-butoxide, sodium tertiary butoxide,
5

lithium methoxide, or the like. Preferably potassium-t-butoxide was used in the present invention.
Step c) involves the amination of chloro group of intermediate (3). Prior arts use
ammonia dissolved in protic solvent, while the present invention uses ammonia gas
directly being passed into the reaction mixture. Intermediate (3) may be dissolved in a
suitable aprotic solvent at 25-30 °C and treated with an aminating agent preferably
ammonia gas. On completion of reaction, the reaction solvent may be distilled off to
isolate crude 2-Chloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fluoro-beta-D-
arabinofuranosyl)-9H-purin-6-amine (2).
Step d) this involves debenzoylation of intermediate (2). Intermediate (2) may be dissolved in a suitable protic solvent at 25-30 °C and treated with suitable base which acts as deprotecting agent. On completion of reaction, a suitable organic acid preferably acetic acid may be added to the reaction mass and stirred with an additional base. The solvent may be distilled, and the solid mass so obtained may be dissolved in an aprotic solvent. The said reaction may be then heated at 30-80 °C, preferably at 40-60 °C and filtered under vacuum. The filtrate may be treated with decolorizing agent and filtered. The filtrate may be distilled, and the crude isolated from a mixture of suitable protic and aprotic solvents to obtain crude Clofarabine (1).
The bases selected in step d) may be selected from a group of alkali metal alkoxide or alkali metal carbonates. Alkali metal alkoxide may be selected from a group comprising of sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium tertiary butoxide, potassium tertiary butoxide or the like and the alkali metal carbonates may be selected form a group comprising sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate or the like. Preferably, sodium methoxide and sodium carbonate were used in step d) in the present invention.
Step e) proceeds with the purification of crude Clofarabine (1). Crude Clofarabine (1) may be dissolved in a mixture of suitable protic and aprotic solvent and heated to 50-
6

100 °C, preferably 65-70 °C. The reaction mixture may be treated with neutral charcoal and cooled to 0-25 °C, preferably 0-5 °C to obtain pure Clofarabine (1).
The suitable protic solvents used in the above step a), step b), step d) and step e) may be selected from a group comprising of water, methanol, ethanol, butanol, isopropyl alcohol or mixtures thereof. Preferably water, methanol and isopropyl alcohol were used in the present invention.
The suitable aprotic solvents used in the above steps step a), step b), step c), step d) and step e) may be selected from a group comprising of dichloromethane, dichloroethane, acetonitrile, ethyl acetate, acetone, diethyl ether, 1,4-dioxane, diethyl ether, hexane, cyclohexane, toluene, tetrahydrofuran or mixtures thereof, preferably dichloromethane, acetonitrile, ethyl acetate, acetone were used in the present invention.
The pure Clofarabine (1) obtained after purification by HPLC is having purity greater than 99.5% and total impurities less than 1.0 % (w/w) and any single unknown impurity less than 0.10 % (w/w), forms another embodiment of the invention.
The possible impurities identified in clofarabine are alpha anomer of Clofarabine (1) and 2-chloro adenine.
In another embodiment, the 2-deoxy-2-P-fluoro-l, 3, 5-tri-O-benzoyl-l-a-D-ribofuranose (6) used in the present invention is prepared according to the synthesis shown in scheme -2.
7



OBz
OBz lH-imidazolc
,„i\V\\OBz
BzO
/

s?
OBz
BzO

OH

S^
OBz
BzO
V
N O

Ethyl Acetate Et3N.3IIF

OBz

Alcohol

OH //
O I N

,„n^0Bz;
BzO
OBz

(6)
Scheme- 2
In another embodiment, the pure Clofarabine (1) obtained after purification is having X-Ray powder diffraction (XRPD) pattern as shown in figure 1 and the 2 theta values were provided in table 1.
Tablel:

2(9) deg. Relative Intensity (%)
7.97 23.27
9.14 2.01
11.46 43.94
14.51 25.97
16.48 26.97
16.95 24.71
17.93 100
19.38 7.11
20.36 3.07
20.96 4.69
22.96 18.67

23.93 19.00
24.45 47.38
25.34 50.68
25.91 35.21
26.64 2.36
26.96 2.44
27.89 2.80
28.99 11.6
29.55 7.97
30.47 10.92
30.75 30.46
30.97 23.64
32.06 21.99
32.81 8.51
33.16 8.73
34.24 9.82
34.69 6.73
36.25 8.32
In another embodiment, the crystalline Clofarabine (1) obtained after purification is having sharp endothermic peak at 234.83 °C in the Differential Scanning Calorimetry thermogram as illustrated in figure 2.
The following examples further illustrate the present invention, but should not be construed in anyway, as to limit its scope.
EXAMPLES
EXAMPLE-1: Preparation of 3,5-di-0-benzoyl-2-deoxy-2-fluoro-o>D-arabino furanosyl bromide (5)
lOOg of 2-deoxy-2-P-fluoro-l,3,5-tri-0-benzoyl-l-a-D-ribofuranose (6) was dissolved in lOOOmL of dichloromethane and cooled to -5 to 0 °C. 580mL of hydrobromic acid solution in acetic acid was added to the reaction mass and stirred at -5 to 0 °C. The temperature of the reaction mass was raised to 25-30 °C and stirred for 12-14hrs. On completion of reaction, the reaction mass was washed with water at 25-30 °C and layers separated. The organic layer was washed with 10% sodium bicarbonate. The organic
9

layer was separated and distilled off to obtain crude 3,5-di-0-benzoyl-2-deoxy-2-fluoro-a-D-arabino furanosyl bromide (5), which is directly used in the next step.
EXAMPLE-2: Preparation of 2,6-dichloro-9-(3,5-di-0-benzoyl-2-deoxy-2-fluoro-D-arabino furanosyI)-9/Z-purine (3)
39.69g of 2,6-dichloropurine (4) and 25.80g of potassium-t-butoxide were dissolved in 1200mL of acetonitrile at 25-30 °C. The reaction mixture was heated to 50-55 °C. To this, a solution of intermediate (5) obtained in example-1 in acetonitrile was added and stirred for lhr at 50-55 °C. The reaction mixture was cooled to 25-30 °C and stirred for 6-7hrs. On completion of reaction, the reaction mass was quenched with ice water at 25-30 °C. Further, the reaction mass was extracted with dichloromethane and treated with neutral charcoal and filtered. The filtrate was distilled off under vacuum and the crude solid was dissolved in isopropyl alcohol at 25-30 °C and cooled to 0-5 °C. The reaction mixture was then heated to 85-90 °C, then cooled to room temperature. The solid so obtained, was filtered and dried under vacuum below 40 °C. The dried solid was then dissolved in a mixture of methanol and ethyl acetate at 25-30 °C, heated to 65-70 °C for 12hrs and cooled to 0-5 °C. The solid obtained was filtered washed with chilled methanol and dried under vacuum at 35-40 °C to obtain 2,6-dichloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fluoro-D-arabino furanosyl)-9//-purine (3). Yield: 45 %; Purity: 97%
EXAMPLE-3: Preparation of crude 2-Chloro-9-(3, 5-di-0-benzoyI-2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-9H-purin-6-amine (2)
lOOg of intermediate (3) was dissolved in 3000mL of acetonitrile and ammonia gas was passed for 12hrs at 25-30 °C to form 2-Chloro-9-(3, 5-di-0-benzoyl-2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-9H-purin-6-amine (2). On completion of reaction, the solvent of the reaction mixture was distilled off completely under vacuum and cooled
10

to 25-30 °C to obtain 2-Chloro-9-(3,5-di-0-benzoyl-2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-9H-purin-6-amine (2).
EXAMPLE-4: Preparation of crude Clofarabine (1)
Intermediate (2) was dissolved in 1300mL of methanol and 500mL of sodium methoxide in methanol was added to it at 25-30 °C. The reaction mass was stirred for 15-20 min. On completion of reaction, 25.2g of acetic acid and 5.0g of sodium carbonate were added to the reaction mass at 25-30 °C. The solvent was removed by distillation under vacuum below 55 °C and the crude so obtained was dissolved in 3200mL of acetone. The reaction mass was heated to 45-50 °C, stirred for lhr and filtered under vacuum. The solid so obtained was dissolved in 1200mL of acetone at 50-55 °C, treated with neutral charcoal at 25-30 °C and filtered. The filtrate was distilled off under vacuum below 55 °C. The crude so obtained was dissolved in 1500mL of dichloromethane and stirred for 30 min at 40 °C. The reaction mixture was then cooled to 25-30 °C, filtered and dried under vacuum at 20-25 °C. The solid so obtained was dissolved in a mixture of 14 volumes of methanol and 4 volumes of ethyl acetate at 25-30 °C. The reaction mixture was heated with addition of neutral charcoal, at 65-70 °C and filtered. The filtrate was collected, cooled to 25-30 °C and stirred for 10-12hrs. Further the solution was cooled to 0-5 °C, filtered under vacuum below 0°C. The solid so obtained was dried under vacuum below 55 °C to obtain crude Clofarabine (1). Yield: 60%; Purity: 99.2%. EXAMPLE-5: Purification of Clofarabine (1)
The crude Clofarabine (1) was dissolved in a mixture of 1500mL of methanol and 500mL of acetonitrile at 25-30 °C. The reaction mass was heated at 65-70 °C and 2.0 g of neutral charcoal was added. The reaction mass was then filtered through Hyflo and the filtrate cooled to 25-30 °C for 10-12hrs.The reaction mass was further cooled to 0-5 °C and filtered under vacuum. The solid so obtained was washed with chilled methanol and dried under vacuum below 55 °C to obtain pure Clofarabine (1). Yield: 70%; Purity: 99.85%. XRD: figure 1; DSC: figure 2.
11

We claim:
1) A process for the preparation of Clofarabine (1), comprising:
Jk/
CI
HO J F
H<3
Clofarabine
(1)
a) bromination of 2-deoxy-2-P-fluoro-l, 3, 5-tri-O-benzoyl-l-a-D-ribofuranose (6)


OBz

>OBz

(6)
with hydrobromic acid in combination with glacial acetic acid in presence of base to obtain 3, 5-di-0-benzoyl-2-deoxy-2-fIuoro-alpha-D-arabinofuranosyl bromide (5);

.. »Br
BzCT
OBz

b) condensing intermediate (5) with 2,6-dichloropurine (4); ci
< J
(4)
in an aprotic solvent and base to form 2, 6-dichloro-9-(3, 5-di-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl)-9//-purine (3);
12


BzO

c) treating intermediate (3) with an ammonia gas in an aprotic solvent to obtain 2-Chloro-9-(3,5-di-0-benzoyl-2-deoxy-2-fiuoro-beta-D-arabino furanosyl)-9H-purin-6-amine (2);

BzO

BzO

NH,

(2)
d) debenzylating intermediate (2) in presence of base to form
Clofarabine (1); and
e) purification of Clofarabine (1) from a mixture of methanol and
acetonitrile.
2) The process as claimed in claim 1, wherein the suitable base used in step a), b) and d) is selected from sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, cesium bicarbonate, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium tertiary butoxide, potassium tertiary butoxide, sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate or combination thereof.
3) A process for the purification of Clofarabine (1) comprising:
a. adding Clofarabine (1) to a mixture of protic and aprotic solvents;
b. heating the reaction mixture to 65-70 °C;
c. treating the reaction mixture with neutral charcoal;
13

d. cooling the reaction mixture to 0-5 °C; and
e. isolating the crystalline pure Clofarabine (1).
4) The process as claimed in claim 3, wherein the protic solvent is selected from water, methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol; aprotic solvent is selected from a group comprising of acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N, N-dimethylformamide, methyl tertiary butyl ether, hexane, cyclohexane, toulene, tetrahydrofuran or mixtures thereof.
5) A process for the preparation of crystalline Clofarabine (1) of purity more than 99.5%(w/w) from a mixture of methanol and acetonitrile having X-ray diffractogram as shown in figure 1.