FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Complete Specification
[See Sections 10 and rule 13]
Title: A Process For Preparation Of Decitabine
Applicant: (a) INTAS Pharmaceuticals Limited
(b) Company Registered under Indian Company ACT (c) 2nd Floor, Chinubhai Centre,
Ashram Road,
Ahmedabad 380009
Gujarat, India
The followingspecification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention relates to preparation of l-(2-deoxy-β-D-erythro-pentofuranosyl)-5-azacytosine, commonly known as decitabine. The process also relates to purification of decitabine.
BACKGROUND OF THE INVENTION
The present application relates to preparation and purification of decitabine (also known as 2'-deoxy-5-azacytidine; 5-aza-2'deoxycytidine; DAC or 4-amino-l-(2-deoxy-β-D-erythropentofuranosyI)-I,3,5-triazin-2(lH)-one). Decitabine is marketed as Dacogen and can be represented by following structural formula

Decitabine is a cytosine nucleoside (cytidine) analogue and act as hypomethylating agent. It hypomethylates DNA by inhibiting DNA methyl transferase and is used for treating patients affected with myelopastic syndrome (MIDS). There are several prior art reports for preparation of decitabine such as US 3,817,980, US 3,350,388, FR 2105468, journal of nucleic acid research, 1978, 4, 109-113, US 2004/0186283, US 2004/0127436, WO 2008/101448, WO 2008/101448, WO 2009/047314, US 2010/0036112, WO 2010/017547, US 20100249394, WO 2010/040056 and WO 2010/129211.

US patent No. 3,817,980 discloses a process that involves reacting bis-silyl compound of 5-azacytosine with 2-deoxy-3,5-di-0-p-tolulyl-ribofuranosyl chloride in presence of tin tetrachloride. This results in mixture of protected l-(2-deoxy-alpha-D-erythro-pentofuranosyl)-5-azacytosine generally known as a anomer and 1-(2-deoxy-beta-D-erythropentofuranosyl)-5-azacytosine generally known as β anomer or decitabine. This mixture of protected a and β anomer was subjected to fractional crystallisation for exclusive obtainment of β anomer.
US 3,350;388 discloses a process for preparation of 2'-deoxy-5-azacytidine which involves treating a suspension of finely powdered l-(3,5-di-0-p-toluyl-2-deoxy-β-D-ribofuransyl)-4-methylmercapto-2-oxo-1,2-dihydro-1,3,5-triazine in methanol and ammonia. The residue was triturated in absolute ether and then crystallized from anhydrous methanol.
Generally, the process for preparation of decitabine includes reacting protected sugar i.e. 2-deoxy-D-erythro-pentofuranosyl derivatives (II) with sillyl protected azacytocine (III) this can be summarized as following scheme:


Deprotection of (IV) is carried out to obtain decitabine (I) which can be purified further. The protected compound and decitabine obtained after deprotection are usually a mixture of a and β anomer. The reported processes for preparation of decitabine involve deprotection under basic conditions. Commonly used bases include methanolic ammonia as reported in US 2010/0222565, WO 2009/047313, US 3,817,980, and US 3,350,388 or alkali alkoxides such as sodium methoxide as reported in WO 2010/017547, US 2010/0017347, US 2010/0249394, US 2010/0036112. WO 2010/012911 discloses that alkali hydroxides such as sodium hydroxide, potassium hydroxide or organic bases such as triethylamine can be used for deprotection of intermediate of formula (IV) however the process of WO 2010/129211 involves subjecting 1,3,5-di-O-acetyl -2-deoxy-D-ribofuianosyl)-5-azacytidine to methanolic ammonia and cooling the reaction mixture after

completion to'0-10 °C. The decitabine thus isolated is again subjected to purification from DMSO wherein pure decitabine is isolated by using a mixture of methanol and ethyl acetate.
Decitabine is unstable in acidic or alkaline medium as discussed in Piskala, A.; Synackova, H. Tomankova; Fiedler, P.; Zizkowasky, V.Nucleic Acids Res. 1978, 4, (s- 109-s -113). Under acidic condition it degrades in 5-azacytosine and 2-deoxy-D-ribose and in basic conditions it undergoes hydrolytic degradation which results into generation of impurities such as open ring formylated derivative (V)

In alkaline medium the degradation is even faster which is also discussed in US 2006/0205685; however alkaline conditions are essential for deprotection reaction.

Thus it is crucial to provide alkaline condition which facilitates deprotection reaction till completion yet does not cause degradation of decitabine thus produced.
The bases used in prior art processes are very strong and generates highly alkaline reaction conditions which may affect the stability of decitabine. In case very mild base is used the alkalinity of the reaction medium will not be sufficient enough to support deprotection reaction.
Therefore, it is crucial to provide distinct alkaline condition which- facilitates deprotection reaction and assist it till completion yet does not cause degradation of decitabine thus produced. In addition to this isolation of decitabine from highly alkaline reaction medium also affect its stability; and decitabine thus obtained may be unstable and can go for faster degradation. Therefore it is also critical to isolate decitabine from a near neutral reaction medium.
- Thus the present application provides a process for preparation of decitabine which involves carrying out deprotection of compound of formula (VII) and isolation of decitabine thus prepared in presence of optimal basic conditions which avoids degradation of decitabine and eventually results in better purity and yield. The present application also provides a process for purification of decitabine. The process is simple, efficient and safe to handle on large scale.
OBJECTS OF THE INVENTION
The main objective of the present invention is to provide a process for preparation of pure Decitabine.
Another object of the invention is to provide a simple, efficient and safe to handle process for the preparation of Decitabine.

Another object of the invention is to provide a process for the purification of Decitabine.
SUMMARY OF THE INVENTION
Accordingly in an aspect the present application provides a process for preparation of decitabine comprising:
a. treating protected compound of formula (VII) with a base selected from group
consisting of alkali or alkali earth metal carbonate;
b. adjusting the pH of reaction mixture;
c. isolating decitabine.
In another aspect the process of present application provides process for purification of decitabine. The process comprises steps of:
a. providing a solution or suspension of decitabine in a first solvent;
b. partially removing the first solvent;
c. adding a second solvent;
d. isolating pure decitabine.
The Decitabine prepared by process of present invention has at least 99.8% enantiomeric purity.
DETAILED DESCRIPTION
Unless otherwise specified, the term "decitabine" refers to β anomer of l-(2-deoxy-D-erythro-pentofuranosyl)-5-azacytosine which may or may not have contamination of a anomer.

The term "crude" or "crude decitabine "refers to a mixture of a and β anomers of 1-(2-deoxy-D-erythro-pentofuranosyl)-5-azacytosine in any ratio.
The term a anomer refers to l-(2-deoxy-a-D-erythro-pentofuranosyl)-5-azacytosine. The term "pure" or substantially pure decitabine refers to product which contains at least 99.0% of l-(2-deoxy-P-D-erythro-pentofuranosyl)-5-azacytosine.
Accordingly, in one aspect there is provided a process to prepare crude decitabine. The process involves treating protected compound of formula (VII) with a base in presence of solvent. The pH of the reaction mixture of deprotection is alkaline and it is further adjusted before isolation of crude decitabine.
The process of present application can be depicted as follows:


The protecting group R of compound of formula VII can be selected for aroyl or acyl such as toluoyl, acetyl.
The suitable solvent used for stage 1, i.e. deprotection of decitabine of present application may be selected from any straight or branched chain alcohol. Examples of solvents include methanol, ethanol, isopropanol, n-butanol, isobutanol. The especially preferred solvent is methanol or isopropanol. The solvents used in the process may contain some amount of water as is usually present in commercially available solvents however considering the instability of decitabine in aqueous

conditions it is advisable to use anhydrous solvent or solvent with a water content which does not facilitate degradation of decitabine.
The base used for the stage 1, can be selected from a group consisting of alkali carbonate or alkaline earth metal carbonate. Exemplary base can be sodium carbonate, potassium carbonate or calcium carbonate. Base in the present application of the may be used in amount ranging between 0.1 mole to 5 mole, preferably between about 0.1 to 2 moles with respect to intermediate (IV). Since decitabine can degrade in aqueous medium it is-preferable to use anhydrous base.
Thus, the process of stage. 1, involves preparing a mixture of protected compound of formula(VII) in a solvent followed by addition of base. The reaction may be carried out at suitable temperature. Although the reaction can be carried out under heating preferably the reaction of stage 1, is carried out at about ambient temperature i.e. 5°C to 40°C.
After completion of deprotection the reaction mixture can be filtered and pH of reaction mixture is checked. If pH of filtrate is more than 9 it may be adjusted to lower than 9. The pH can be adjusted by addition of any suitable organic or inorganic acid. Examples of acids include acetic acid, formic acid, succinic acid, hydrochloric acid. Since decitabine is unstable under highly acidic conditions so preferably the pH is adjusted to 6.5 to 8.5. Once the pH is adjusted; if desired the reaction mixture can be subjected to charcoal treatment followed by distillation of solvent. The solvent used in deprotection reaction can be removed completely or partially. A second solvent may be added to the residue or to the slurry obtained by removal of first solvent.

Second solvent can be selected from any alcohol other than the alcohol used as first solvent. Preferably the second solvent can be selected from ethanol, propanol, isopropanol, butanol or iso-butanol. To avoid the degradation of decitabine preferably anhydrous alcohols are used as second solvent. The second solvent is preferably added at a temperature of distillation of first solvent and the mixture containing residual reaction solvent, crude decitabine and second solvent is stirred at a temperature in range of 40 - 55 °C for about 2-8 hours. Crude decitabine can be separated from the reaction mixture by cooling the reaction mixture at or below 35 °C.
According to another aspect, there is provided a- process for purification of decitabine to obtain decitabine substantially free of a anomer, the process comprises steps of:
a. providing a solution or suspension of crude decitabine in a first solvent;
b. partially removing the first solvent of the step a);
c. adding a second solvent to the reaction mixture obtained in step b);
d. removing the solvent from reaction mixture obtained in step c);
e. isolating pure decitabine.
In one embodiment, the solvent used in stage 2, step (a), is selected from alcohol. Specifically, the solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol or iso-butanol.
In one embodiment, the crude decitabine is dissolved in the solvent at a temperature of about ambient temperature to about 70 °C specifically at about 35 - 65 °C and more specifically at about 45 - 65 °C. The reaction mixture thus obtained may be stirred for 2 - 8 hours and then the first solvent is removed partially by distillation.

The term partially removing the first solvent in step b) refers to specifically 60%; more specifically greater than 85% or may be complete (100%) removal of the solvent.
Second solvent is added to the slurry obtained from step a) and the reaction mixture may be stirred for 30 minutes to 5 hours. Removal of solvent in step d) is accomplished by substantially or complete evaporation of the solvent or distillation of the solvent. Pure decitabine can be isolated from reaction mixture of step d) at a temperature from 0-40 °C. Preferably pure decitabine is isolated at 10 - 30 °C. The isolation as mentioned in step e) is done by methods such as filtration, filtration under vacuum, centrifugation or combination thereof. The decitabine obtained by above process may further be dried in, for example under reduced pressure until the residual solvent content reduces to the desired amount:
The decitabine prepared by process of present application has characteristic 29 values corresponding to 6.64, 12.27, 13.89, 18.35, 18.87, 19.36, 19.60, 23.78, 26.64, 24.73, 26.61, 27.98, 29.45, 29.63, 30.39, 31.28 (±0.29).
Another embodiment of present application provides enantiomerically pure decitabine containing less than about 0.2% of a anomer, more preferably less than about 0.1% and most preferably less than about 0.05%.
Protected compound (VII) which can also be called as protected precursor of decitabine used in the process of present application can be prepared by following any method known from the art including the references disclosed in the background section of this application or else if desired it can also be prepared by the process described in example 1 of present application.

The process of present application provides highly pure decitabine by using ideal alkaline conditions which avoids degradation of decitabine. The purification of decitabine as disclosed in present application enables selective isolation of β anomer from a mixture of a and β anomer. The process is simple efficient and can be easily performed on plant scale and is also economically viable.
The pure decitabine obtained by process of present application may be formulated into a dosage form by combining with pharmaceutically acceptable excipient by using. known techniques.
In the following section embodiments are described by a way of examples to illustrate the process of invention. However, these are not intended in any way to limit the scope of present invention. Several variants of these examples would be evident to present person ordinarily skilled in the art.
Examples-Example 1 - Preparation of protected decitabine
To 5-azacytosine (50g), hexamethyl disilazane (250 ml) and ammonium sulphate (5g) were added. The reaction mixture was heated to 140 - 150 °C and maintained at the same for 3 hrs under stirring. The reaction mass was further cooled to 100 - 105 °C followed by complete distillation of hexamethyl disilazane to obtain an oily residue. The reaction temperature was cooled to 30 - 35 °C followed by addition of 1000 ml methylene chloride. The reaction mixture was cooled to -5 - 0 °C and to this 25 ml of trimethylsilyltriflate, 140g of l-chloro-2-deoxy-3,5-di-0-p-tolouyl-D-ribofuranose and 250 ml triethyl amine were added. Reaction mixture was stirred at -

5 - 0 °C for 2 hours and then the temperature was increased to 25 - 30 °C. Water (600ml) was added to reaction mixture and layers were separated.
The pH of organic layer was adjusted to 7 - 8 by sodium carbonate, layers separated and 1000ml toluene was added to organic layer followed by complete distillation of solvent. Toluene (500ml) was added to residue and the resultant slurry was heated to 50 - 55 °C for 30 minutes, reaction mixture was fdtered and the product thus obtained was dried under vacuum.
Example 2 - Preparation of 2'-deoxy -5-azacytidine i.e. decitabine
4000 ml methyl alcohol and 100g of compound of formula (VII) were charged in a round bottom flask. The temperature was maintained at 20 - 25 °C. Anhydrous sodium carbonate (11.4g) was added and reaction mixture was stirred at 20 - 25 °C for 7 - 8 hrs. After completion of reaction the reaction mixture was filtered and the pH of the filtrate was checked. If pH was more than 8 then it was adjusted to 6.5 -8.5 by using acetic acid. Organic layer was treated with 60g charcoal and filtered; solvent was distilled off till about 200ml of solvent remain in flask followed by addition of anhydrous ethanol (1400ml). The solvent was distilled and the reaction mixture was cooled to 30 - 35 °C. The reaction mixture was filtered and the solid thus obtained was dried to obtain the title compound.
Example 3 - Preparation of β anomer enriched decitabine
Decitabine (119g); 18Lit methanol were charged in a round bottom flask. Temperature of reaction mixture was increased to 50 - 60 °C and it was stirred at the same for 2 hrs followed by distillation of methanol. 16.7Lit of methanol was distilled and anhydrous ethanol 10.4L.it was added to the reaction mass. The solvents were

distilled off, and reaction mixture was cooled to 30 - 35 °C followed by filtration. The solid thus obtained was dried to obtain 92g of title compound.
Purity = 99.97%

We Claim:

a. treating compound of formula (VII )
1. A process for preparing decitabine of formula (I) comprising:

wherein R is protecting group with a base selected from group consisting of alkali-metal carbonate or alkali earth metal carbonate;
b. adjusting the pH of reaction mixture;
c. isolating decitabine (I).

2. A process as claimed in claim 1, wherein R can be selected from a group consisting of aroyl or acyl protecting group such as toluolyl or acetyl.
3. A process as claimed in claim 1, wherein the reaction is in presence of a solvent.
4. A process as claimed in claim 1, step b, wherein the pH of reaction medium is adjusted to 6.5 - 8.5.
5. A process as claimed in claim 1, wherein the pH is adjusted by addition of acid selected from organic or inorganic acid.
6. A process as claimed in claim 4, wherein pH is adjusted by addition of acid selected from acetic acid, formic acid, succinic acid or hydrochloric acid.
7. A process for purification of decitabine comprising steps of:

a) providing a solution of decitabine as prepared in claim 1) in a first solvent;
b) substantially distilling off the solvent of step a);
c) adding second solvent;
d) isolating pure decitabine.

8. A process as claimed in claim 1 or 7, wherein solvent is selected form linear or branched chain alcohol.
9. A process as claimed in claim 7, wherein the solvent is selected from methanol, ethanol, propanol, isopropanol, butanol, isobutanol or mixture thereof.