Claims:Claims:
1) A process for the preparation of highly pure Decitabine of Formula (I).

(I)
comprising the steps of:
a) reacting 5-Azacytosine of Formula II

(II)
with hexa methyl disilazane in presence of ammonium sulphate to yield protected 5-Azacytosine, followed by treatment with 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose to provide mixture of compounds of Formula III

(III)
b) reacting compound of formula III with methanolic ammonia in a ratio of 10 to 15% wt/wt to provide Decitabine (I), wherein the reaction comprises the steps of:
i. charge compound of formula III in to a reaction flask containing methanolic ammonia at 10-15°C;
ii. stir the reaction for 6 hours;
iii. cooled to 0-5°C and again stirred for 5 hours; and
iv. precipitated product was filtered and washed with ethylacetate.
c) purifying decitabine, wherein purification of decitabine comprises of:
i. charge decitabine obtained in stage-b in to a reaction flask containing dimethyl sulfoxide;
ii. filter the contents and filtrate was transferred in to another reaction flask;
iii. methanol was added at 25-30°C;
iv. cooled to 0-5°C and filtered the material to obtain wet cake;
v. charge methanol and stir the mixture at reflux temperature;
vi. cooled to 0-5° C, filter the material and washed with methanol; and
vii. isolating crystalline Form SD of Decitabine

2) A process for the preparation of Decitabine according to claim 1, wherein reaction of 5-Azacytosine with hexa methyl disilazane in presence of ammonium sulphate is carried out at 110 to 130°C.

3) A process for the preparation of Decitabine according to claim 1, wherein addition of 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose was carried for 15 minutes to 30 minutes.

4) A process for the preparation of Decitabine according to claim 1, wherein reaction of protected 5-Azacytosine with 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose is carried out at 20 to 30°C.

5) A process for the preparation of Decitabine according to claim 1, wherein methanolic ammonia used in step b) is in a ratio of 10 to 15% wt/wt with respect to compound of Formula (III).

6) A process for the preparation of Decitabine according to claim 1, wherein Decitabine obtained is substantially pure, having a purity of greater than 99.5% by HPLC.

7) A substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC characterized by
a) x-ray powder diffraction(XRPD) pattern having characteristic peaks at 6.6, 12.9, 13.3, 14.2, 19.3 and 23.3 ± 0.2 °2?; and
b) water content greater than 0.2 % and less than 1.5% wt/wt.

8) A substantially pure crystalline Decitabine Form –SDE according to claim 7, further characterized by DSC having at least two endothermic peaks ranging between:
i) peak in between 75 to 95°C; and/or
ii) peak in between 160 to 175°C; and/or
iii) peak in between 190-200°C.

9) A process for the preparation of Decitabine substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC comprises of:
a) stirring decitabine in mixture of solvent selected from DMSO and methanol at reflux temperature;
b) cooled to 0-5° C; and
b) isolating crystalline Form SDE of Decitabine

10) A process for the preparation of Decitabine substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC according to claim 7 or 8, wherein XRPD of crystalline decitabine is similar as depicted in Fig.1 and DSC of crystalline decitabine is similar as depicted in Fig.2.
, Description:IELD OF THE INVENTION

The present invention relates to a process for the preparation of Decitabine of Formula (I).

(I)

The present invention further relates to a stable crystalline Form of Decitabine designated as Form SDE, which commercially viable for preparing stable dosage forms.

BACKGROUND OF THE INVENTION
4-Amino-1-(2-deoxy-ß-D-erythropentofuranosyl)-1,3,5-triazin-2(1H)-one or Decitabine is believed to exert its antineoplastic effects after phosphorylation and direct incorporation into DNA and inhibition of DNA methyltransferase, causing hypomethylation of DNA and cellular differentiation or apoptosis. Decitabine inhibits DNA methylation in vitro, which is achieved at concentrations that do not cause major suppression of DNA synthesis. Decitabine-induced hypomethylation in neoplastic cells may restore normal function to genes that are critical for the control of cellular differentiation and proliferation. In rapidly dividing cells, the cytotoxicity of decitabine may also be attributed to the formation of covalent adducts between DNA methyl transferase and decitabine incorporated into DNA. Non-proliferating cells are relatively insensitive to decitabine.

USFDA approved Decitabine in May 2006 and is marketed under the brand name Dacogen®, in the form of a sterile lyophilized powder for injection. Decitabinbe is a pyrimidine nucleoside analog of cytidine, is used for treating patients with myelodysplastic syndromes (MDS) including previously treated and untreated, de novo and secondary MDS of all French-American-British subtypes (refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and chronic myelomonocytic leukemia) and intermediate-1, intermediate-2, and high-risk International Prognostic Scoring System groups.

Decitabine is a fine, white to almost white powdery substance with empirical formula C8H12N4O4 and molecular weight of 228.21. Decitabine is slightly soluble in ethanol/water (50/50), methanol/water (50/50) and methanol; sparingly soluble in water and soluble in dimethylsulfoxide (DMSO).

U.S. Pat. No. 3,350,388 discloses a process for the preparation of 2'-deoxy-5-azacytidine, which involves treating a suspension of finely powdered 1-(3,5-di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosyl)-4-methylmercapto-2-oxo-1,2-dihydro-1,3,5-triazine in absolute methanol, previously saturated at 0°C with dry ammonia, and allowing it to stand with occasional stirring in a closed vessel at room temperature for 24 hours. A small amount of the precipitate was removed by filtration and the filtrate was evaporated under reduced pressure. The residue was triturated with absolute ether and then crystallized from anhydrous methanol.

M. W. Winkley et al., in Journal of Organic Chemistry, 35(2), pp. 491-495, 1970 disclose a process for the preparation of 2'-deoxy-5-azacytidine, which involves the condensation of a trimethylsilyl derivative of 5-azacytosine with 2-deoxy-1,3,5-tri-O-acetyl-D-ribofuranose, dissolved in dry ether containing acetyl chloride in acetonitrile, to produce 1-(3,5-di-O-acetyl-2-deoxy-.alpha.,.beta.-D-ribofuranosyl)-5-azacytosine, followed by removing the protecting groups with ammonia-saturated ethanol. It also discloses the recrystallization of the .beta.-anomer from a mixture of methanol and 2-propanol to give pure 2'-deoxy-5-azacytidine.

U.S. Pat. No. 3,817,980 discloses a process that involves reacting the bis-silyl compound of 5-azacytosine with 2-deoxy-3,5-di-O-p-toluoyl-ribofuranosyl chloride in the presence of tin tetrachloride. The .alpha./.beta.-anomer mixture of protected decitabine obtained was crystallized from toluene and recrystallized from ethanol. Further, the .beta.-anomer of protected decitabine was obtained by fractional crystallization from ethyl acetate. This .beta.-anomer of protected decitabine was dissolved in absolute methanol saturated with ammonia to form decitabine which was crystallized from ethanol.

Piskala et al., in Journal of Nucleic Acid Research, 1978, 4, 109-113 disclose a process for the preparation of 5-aza-2'-deoxycytidine, which involves the condensation of a compound (A) with silylated 5-azacytosine (B) in acetonitrile at room temperature and in the presence of a molecular sieve to produce a mixture of anomeric di-p-toluate (C) and (D) in high yield, in which the .alpha.-anomer strongly predominated. Further, it also discloses a process for the preparation of .beta.-anomer by carrying out the reaction in the presence of a mixture of mercuric oxide and bromide. On methanolysis of protected anomers (C) and (D), the nucleoside 2'-deoxy-5-azacytosine was obtained.

U.S. Pat. No. 4,209,613 discloses a process for preparing a nucleoside in a single step of silylating a nucleoside base, followed by reacting sugar derivative in the presence of a catalyst.

Jean et al., in Journal of Organic Chemistry, 1986, 51, 3211-3213 disclose a process for the preparation of 5-aza-2'-deoxycytidine, which involves the reaction of methyl 2-deoxy-.alpha.,.beta.-D-ribofuranoside with 9-fluorenylmethoxycarbonyl chloride in anhydrous pyridine to give 1-methoxy-3,5-bis(O-Fmoc)-2-deoxyribofuranose. To an ice cold solution of 1-methoxy-3,5-bis(O-Fmoc)-2-deoxyribofuranose, anhydrous HCl gas was passed in dry ether to produce a 1-chloro derivative in situ, which was reacted with the disilylated 5-azacytosine at room temperature in 1,2-dichloroethane, in the presence of catalytic amount of tin chloride. The resulting product mixture contained the two anomers (.alpha. and .beta.) in approximately equimolar amounts (.alpha./.beta.=1:0.9). From this mixture, the fully deprotected anomer was obtained by the reaction with triethylamine in dry pyridine followed by crystallization from methanol.

International Application Publication No. WO 2008/101448 A2 ("WO '448") discloses a process for the preparation of 1-(2-deoxy-alpha-D-erythro-pentofuranosyl)-5-azacytosine (.alpha.-anomer of decitabine) by reacting protected 2-deoxy-D-erythro-pentofuranoside (compound A), wherein R.sup.1 represents an alkyl group having 1 to 6 carbon atoms and R.sup.2 represents an alkanoyl group having 1 to 6 carbon atoms with silylated-5-azacytosine (compound B) wherein R.sup.3 represents an alkyl group having 1 to 4 carbon atoms, wherein the substituents R.sup.3 can be identical or different, in an inert organic solvent in the presence of a Lewis acid, to form protected 1-(2-deoxy-alpha-D-erythro-pentofuranosyl)-5-azacytosine (compound C) (.alpha.-anomer of decitabine) and subsequently removing the alkanoyl protecting groups.

According to the disclosure of WO '448, the prior processes produced only mixtures of .alpha.- and .beta.-anomers. Further, the publication discloses that the .alpha.-anomer of the protected 1-(2-deoxy-alpha-D-erythro-pentofuranosyl)-5-azacytosine has a lower solubility when compared to the .beta.-anomer, which can be present in small amounts in the crude product and, therefore, it is possible to easily obtain a quite pure product in high yield by crystallization. After removal of the protecting groups by treatment with sodium methoxide in methanol, the .alpha.-anomer of decitabine is obtained.

Kun-Tsan et al., in Journal of Pharmaceutical Sciences, 1981, 70(11), 1228-1232, disclose the chemical stability of 5-aza-2'-deoxy cytidine in acidic, neutral and alkaline solution as analyzed by HPLC and possible decomposition products, N-(formylamidino)-N'-.beta.-D-2-deoxyribofuranosylurea and 1-.beta.D-2'-deoxyribofu ranosyl-3-guanyl urea.

Michael et al., in Acta Crystallographica, 1991, C47, 1418-1420 disclose monoclinic crystal coordinates of decitabine monohydrate grown from dimethyl sulphoxide.

International Application Publication No. WO 2004/041195 A1 discloses that decitabine, when exposed to humidity, forms a monohydrate that corresponds to 7% moisture at equilibrium. Decitabine monohydrate is also stable at room temperature and the solid form differential scanning calorimetry of decitabine indicates melting at -201.degree. C., followed by decomposition.

U.S. Patent Application Publication No. 2006/0014949 A1 discloses polymorphic form A, a crystalline anhydrate having an orthorhombic crystal lattice, form B, a crystalline monohydrate, and form C, a crystalline hemihydrate of decitabine. Further, the publication discloses methods for the preparation of form A using solvents such as methanol, acetone, 2-butanol, chloroform, dichloromethane, ethyl ether, hexane, methylsulphide, 2-propanol and 1,1,1-trichloroethane; form B using solvents such as dichloromethane and methanol (1:1), 1,2-dimethoxyethane, 1,1,1,3,3,3-hexafluoro-2-propanol, methanol, methanol and 2,2,2-trifluoroethanol (1:1), 2,2,2-trifluoroethanol, 2,2,2-trifluoroethanol and water (9:1), and water; and form C using 2,2,2-trifluoroethanol and water. Also disclosed is a process for the preparation of amorphous decitabine by crystallization from water.

Despite various process disclosures as discussed above, none of them appears to be particularly suitable for reasons such as not being scalable to industrial quantities, products contaminated with metal impurities, e.g., tin, or other impurities, etc. Hence, there remains a need for simple convenient, industrially amenable, and cost effective processes for the preparation of decitabine.

Further the use of ethanol in large scale is very expensive. Parallel, the use of ethanol, which is having high boiling point, requires high energy to distill off the solvent and time taking.

The present inventors has repeated the above process and found the following disadvantages:
? In most of the patent literature, ethanol was used as solvent in coupling stage, which is tedious for the removal.
? Unwanted reactions are observed during the formation of Decitabine, due to the involvement time lagging process and high boiling solvents.
? Incomplete reactions were observed with excessive impurity formation due to incomplete conversion.

The present inventors now developed a process for the preparation of Decitabine, which is advantageous in all aspects and yields highly pure Decitabine in good yield on large scale. The process is more convergent than the previous routes and allows a substantial reduction of using toxic and corrosive reagents, which is more commercially feasible.

According to the present invention, Decitabine is developed with a cost effective process and reducing the time, without using any Chromatographic purification procedures.

In view of the above and to overcome the prior-art problems the present inventors had now developed an improved process for the preparation of Decitabine, which yield in the formation of a novel crystalline form of Decitabine, using industrially feasible and viable process, with the use of industrially friendly solvents, which does not include tedious work up and time lagging steps.

OBJECTIVE OF THE INVENTION
The main objective of the invention relates to a process for the preparation of highly pure Decitabine (I).

Yet another objective of the invention relates to a process for the purification of Decitabine (I).

Yet another objective of the present invention further relates to a stable crystalline Form of Decitabine designated as Form SDE, which commercially viable for preparing stable dosage forms.

SUMMARY OF THE INVENTION
The main aspect of the present invention relates to a process for the preparation of highly pure Decitabine of Formula (I).

(I)
comprising the steps of:
a) reacting 5-Azacytosine of Formula II

(II)
with hexa methyl disilazane in presence of ammonium sulphate to yield protected 5-Azacytosine, followed by treatment with 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose to provide mixture of compounds of Formula III

(III)
b) reacting compound of formula III with methanolic ammonia in a ratio of 10 to 15% wt/wt to provide Decitabine (I), wherein the reaction comprises the steps of:
i. charge compound of formula III in to a reaction flask containing methanolic ammonia at 10-15°C;
ii. stir the reaction for 6 hours;
iii. cooled to 0-5°C and again stirred for 5 hours; and
iv. precipitated product was filtered and washed with ethylacetate.
c) purifying decitabine, wherein purification of decitabine comprises of:
i. charge decitabine obtained in stage-b in to a reaction flask containing dimethyl sulfoxide;
ii. filter the contents and filtrate was transferred in to another reaction flask;
iii. methanol was added at 25-30°C;
iv. cooled to 0-5°C and filtered the material to obtain wet cake;
v. charge methanol and stir the mixture at reflux temperature;
vi. cooled to 0-5° C, filter the material and washed with methanol; and
vii. isolating crystalline Form SD of Decitabine

In another aspect of the present invention relates to a substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC characterized by
a) x-ray powder diffraction(XRPD) pattern having characteristic peaks at 6.6, 12.9, 13.3, 14.2, 19.3 and 23.3 ± 0.2 °2?; and
b) water content greater than 0.2 % and less than 1.5% wt/wt.

In another aspect of the present invention relates to a process for the preparation of Decitabine substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC comprises of:
a) stirring decitabine in mixture of solvent selected from DMSO and methanol at reflux temperature;
b) cooled to 0-5° C; and
a) isolating crystalline Form SDE of Decitabine.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is an example of X-ray powder diffraction (“XRPD”) pattern of crystalline Form SDE of Decitabine (I) obtained according the present invention.
Fig. 2 is an example of DSC pattern of crystalline Form SDE of Decitabine (I) obtained according the present invention.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process for the preparation of highly pure Decitabine of Formula (I).

(I)
comprising the steps of:
a) reacting 5-Azacytosine of Formula II

(II)
with hexa methyl disilazane in presence of ammonium sulphate, wherein reaction of 5-Azacytosine with hexa methyl disilazane in presence of ammonium sulphate is carried out at 110 to 130°C, to yield protected 5-Azacytosine, followed by addition of of 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose for 15 minutes to 30 minutes at a temperature ranging from 20°C to 30°C to provide mixture of compounds of Formula III

(III)
b) reacting compound of formula III with methanolic ammonia in a ratio of 10 to 15% wt/wt with respect to compound of Formula (III) to provide Decitabine (I), wherein the reaction comprises the steps of:
i. charge compound of formula III in to a reaction flask containing methanolic ammonia at 10-15°C;
ii. stir the reaction for 6 hours;
iii. cooled to 0-5°C and again stirred for 5 hours; and
iv. precipitated product was filtered and washed with ethylacetate.
c) purifying decitabine, wherein purification of decitabine comprises of:
i. charge decitabine obtained in stage-b in to a reaction flask containing dimethyl sulfoxide;
ii. filter the contents and filtrate was transferred in to another reaction flask;
iii. methanol was added at 25-30°C;
iv. cooled to 0-5°C and filtered the material to obtain wet cake;
v. charge methanol and stir the mixture at reflux temperature;
vi. cooled to 0-5° C, filter the material and washed with methanol; and
vii. isolating crystalline Form SD of Decitabine

In another embodiment, the present invention further relates to a substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC characterized by
a) x-ray powder diffraction(XRPD) pattern having characteristic peaks at 6.6, 12.9, 13.3, 14.2, 19.3 and 23.3 ± 0.2 °2?; and
b) water content greater than 0.2 % and less than 1.5% wt/wt.

In another embodiment, the present invention further relates to a substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC, further characterized by DSC having at least two endothermic peaks ranging between:
i) peak in between 75 to 95°C; and/or
ii) peak in between 160 to 175°C; and/or
iii) peak in between 190-200°C.

In another embodiment, the present invention further relates a process for the preparation of Decitabine substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC comprises of:
a) stirring decitabine in mixture of solvent selected from DMSO and methanol at reflux temperature;
b) cooled to 0-5° C; and
c) isolating crystalline Form SDE of Decitabine

In another embodiment, the present invention further relates to substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC, wherein XRPD of crystalline decitabine is similar as depicted in Fig.1 and DSC of crystalline decitabine is similar as depicted in Fig.2.

In another particular embodiment of the present invention relates to a process for the purification of Decitabine comprising the steps of
a) stirring decitabine in mixture of solvent selected from DMSO and methanol at reflux temperature;
b) cooled to 0-5° C; and
c) isolating crystalline Form SDE of Decitabine

Isolating pure Decitabine (I) free from process related impurities is one of the critical parameter. If the desired purity is not achieved repeat these steps to get the desired purity. Decitabine obtained after this purification is highly pure and having a purity of greater than 99.9 %. The obtained Decitabine as per the present invention is not only highly pure and also meet the requirements of all ICH guidelines.

Drying may be also be performed by any conventional process not limited to spray drying or distillation to remove the solvent. Drying may be performed under reduced pressure conditions also. Reduced pressure conditions may be suitably utilized by person skilled in the art in order to obtain the dried material. The drying may be performed at a temperature ranging from 40-65°C for a time ranging from 8 to 16 hours depending upon the physical attributes of the end product obtained i.e. Pure Decitabine. Decitabine (I) obtained according to the present invention is highly pure having purity exceeding 99.9%.

Achieving highly pure material was one of the aims of the present invention, as most of the prior disclosed processes resulted in lower purity materials possessing often impurities difficult to remove or involving much purification. While working on ways to find out to get highly pure materials having purity greater than 99.9%, inventors of the present application observed that all the steps of the preparing Decitabine according to the present invention are desirable, however they should not be construed to limit the scope of the invention

The process related impurities that appear in the impurity profile of the Decitabine may be substantially removed by the process of the present invention resulting in the formation of highly pure material, which is having a purity of greater than99.9%. The process of the present invention is as summarized below:

In another embodiment, the Decitabine obtained by the processes of the present application may be formulated as intravenous injection. In these the active product is mixed with one or more pharmaceutically acceptable excipients.

The following examples illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention.

EXAMPLES

Example-1
Process for preparation of 1-Methyl-D-ribose

Methanol (850ml) charged to a reaction vessel and cooled to 0 - 5°C. Acetyl chloride (42.5 ml, 0.53 moles) was added and stirred contents for 15min. Methanol (5100ml) charged to another reaction vessel and cooled to 0-5°C before adding 2-Deoxy-D-ribose (850gm, 6.35moles) under stirring at 0-5°C. Reaction mass content from vessel-1 was added to vessel-2 maintaining reaction mass temp 0-5°C and stirred till complete conversion of 2-Deoxy-D-ribose. Reaction monitored on TLC (mobile phase: DCM: methanol = 8.5:1.5). After completion of reaction, charged 425gm sodium bicarbonate to reaction mass and pH adjusted to 7–8. Contents stirred for 30 min at 10 – 15°C. Rectaion mass ffiltered on buchner funnel through celite bed and wash bed with methanol (100ml). Distill off the combined filtrates completely at below 45°C under vacuum to get oily product which was as such used in next stage.
Yield: 810 - 890 gm
HPLC purity = 95.7%

Example-2
Process for preparation of 1-Methyl-3,5-di-O-acetyl-D-ribose

1-Methyl-D-ribose (850gm, 5.73moles) and Dichloromethane (8500ml) charged to reaction vessel equipped thermo-well and cooling bath at 20-30°C. Contents cooled to 0- 5°C and charged pyridine (2155gm, 27.3) under stirring. Acetyl Chloride (2132gn, 27.16) charged to reaction mass at 0-5 °C and stirred contents for 2hrs to get complete conversion of starting material. Reaction monitored on TLC basis (mobile phase: DCM:methanol = 9:1). Reaction mass quenched with 5% HCl (4250ml) at 0-5°C and stirred for 30 min. Organic layer separated and washed with 5% HCl (4250ml) once again. The organic layer washed with 5% sodium carbonate solution for twice and (4250ml x 2 times) and with purified water three times (4250ml x 3 times). Organic layer dried on sodium sulfate, filtered through celite bed and distilled off completely at below 40°C under vacuum to get 1-Methyl-3,5-di-O-acetyl-D-ribose as an oily residue.
Yield = 1100 - 1190 gm;
HPLC purity = 95.0%.

Example-3
Process for preparation of mixture of alpha and beta Decitabine

5-Azacytosine (500gm, 4.46moles), ammonium sulphate (30gm, 0.23moles) and HMDS (3lit) charged to reaction vessel equipped with water condenser, and heating system. Reaction mass heated to 115-125 °C and stirred for 5-6hrs to get clear solution. It is further maintained for 2hrs at same temperature. Organic volatiles are distilled off from reaction mass at 65-70°C temp. under vacuum to get residue. DCM (11.5 lit) charged to contents and stirred at 25-35°C for 30min. 1-Methyl-3,5-di-O-acetyl-D-ribose (1.10Kg, 4.73moles) charged under stirring and stirred for 20min. Reaction mass cooled to 0-5°C and added TMS-Triflate (994.0gm, 4.47moles) maintaining temp 0 - 5°C over a period of 30-40 min. Allowed reaction mass temp to raise 20-30°C and stirred for 2 hrs to get complete conversion of starting material. Prepared sodium carbonate solution using sodium carbonate (236gm) and purified water (11800mL) and charged to reaction mass slowly at 20-30°C under stirring. Separated aqueous and organic layer Organic layer. Aqueous layer extracted with DCM (5lit). The collective organic layer was washed with saturated sodium bicarbonate (12.0lit) and separated organic layer. It was dried over sodium sulphate and distilled off under vacuum completely to get oily residue. Charged IPA (4.0 lit) under stirring to the residue and stirred for 2hrs. Cooled contents to 0-5°C and stirred for 3hr. The precipitated product filtered on buchner funnel and washed wet cake with IPA (500ml). The isolated product dried at 35-40°C for 6-8hrs to get coupled product.
Yield = 500 - 550gm,
HPLC purity: Alpha isomer = 44.07%, Beta isomer = 42.49%

Example-4
Process for preparation of crude Decitabine

The coupled product (500gm, 1.60moles) charged to methanolic ammonia (10-15%) at 10-15°C. Reaction mass stirred 25-30°C for 6 hrs to get complete conversion. Contents cooled to 0-5°C and maintained under stirring for 5hrs. Precipitated product filtered on buchner funnel and wet cake washed with ethyl acetate (250ml). The isolated product dried at 35-45°C for 5 hrs to get crude Decitabine.
Yield = 90 - 110 gm,
HPLC purity = 96.64%

Example-5
Purification of crude Decitabine

Decitabine (100gm, 1.75moles) dissolved in DMSO (300ml) in reaction vessel to get clear solution and filtered through the membrane filter. The filtrate transferred to reaction vessel and charged methanol (2.0lit) slowly at 25-30°C. It was cooled to 0 - 5°C and maintained under stirring for 5hr. The precipitated product filtered on buchner funnel to get crude Decitabine. The above process optionally repeated with wet cake product to get purified crude Decitabine. To the wet cake product, charged methanol (6.0 lit) and contents heated to reflux to get clear solution and maintained for 30min. Added activated carbon (5gm) and maintained under stirring for 30min. Reaction mass filtered at above 50°C though hyflow bed. Filtered MLs transferred to reaction vessel and cooled to 0-5°C and maintained at same temperature for 4hr. The precipitated product filtered on buchner funnel and wet cake washed with methanol (100ml). The isolated product dried in VTD at 45-50° for 10-12hrs to get pure Decitabine.
Yield = 50 - 60 gm,
HPLC purity = 99.8%

While the foregoing pages provide a detailed description of the preferred embodiments of the invention, it is to be understood that the description and examples are illustrative only of the principles of the invention and not limiting. Furthermore, as many changes can be made to the invention without departing from the scope of the invention, it is intended that all material contained herein be interpreted as illustrative of the invention and not in a limiting sense.

Claims:
1) A process for the preparation of highly pure Decitabine of Formula (I).

(I)
comprising the steps of:
a) reacting 5-Azacytosine of Formula II

(II)
with hexa methyl disilazane in presence of ammonium sulphate to yield protected 5-Azacytosine, followed by treatment with 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose to provide mixture of compounds of Formula III

(III)
b) reacting compound of formula III with methanolic ammonia in a ratio of 10 to 15% wt/wt to provide Decitabine (I), wherein the reaction comprises the steps of:
i. charge compound of formula III in to a reaction flask containing methanolic ammonia at 10-15°C;
ii. stir the reaction for 6 hours;
iii. cooled to 0-5°C and again stirred for 5 hours; and
iv. precipitated product was filtered and washed with ethylacetate.
c) purifying decitabine, wherein purification of decitabine comprises of:
i. charge decitabine obtained in stage-b in to a reaction flask containing dimethyl sulfoxide;
ii. filter the contents and filtrate was transferred in to another reaction flask;
iii. methanol was added at 25-30°C;
iv. cooled to 0-5°C and filtered the material to obtain wet cake;
v. charge methanol and stir the mixture at reflux temperature;
vi. cooled to 0-5° C, filter the material and washed with methanol; and
vii. isolating crystalline Form SD of Decitabine

2) A process for the preparation of Decitabine according to claim 1, wherein reaction of 5-Azacytosine with hexa methyl disilazane in presence of ammonium sulphate is carried out at 110 to 130°C.

3) A process for the preparation of Decitabine according to claim 1, wherein addition of 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose was carried for 15 minutes to 30 minutes.

4) A process for the preparation of Decitabine according to claim 1, wherein reaction of protected 5-Azacytosine with 1-methoxy-3,5-di-O-acetyl-2-deoxy ribose is carried out at 20 to 30°C.

5) A process for the preparation of Decitabine according to claim 1, wherein methanolic ammonia used in step b) is in a ratio of 10 to 15% wt/wt with respect to compound of Formula (III).

6) A process for the preparation of Decitabine according to claim 1, wherein Decitabine obtained is substantially pure, having a purity of greater than 99.5% by HPLC.

7) A substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC characterized by
a) x-ray powder diffraction(XRPD) pattern having characteristic peaks at 6.6, 12.9, 13.3, 14.2, 19.3 and 23.3 ± 0.2 °2?; and
b) water content greater than 0.2 % and less than 1.5% wt/wt.

8) A substantially pure crystalline Decitabine Form –SDE according to claim 7, further characterized by DSC having at least two endothermic peaks ranging between:
i) peak in between 75 to 95°C; and/or
ii) peak in between 160 to 175°C; and/or
iii) peak in between 190-200°C.

9) A process for the preparation of Decitabine substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC comprises of:
a) stirring decitabine in mixture of solvent selected from DMSO and methanol at reflux temperature;
b) cooled to 0-5° C; and
b) isolating crystalline Form SDE of Decitabine

10) A process for the preparation of Decitabine substantially pure crystalline Decitabine Form -SDE, having a purity of greater than 99.5% by HPLC according to claim 7 or 8, wherein XRPD of crystalline decitabine is similar as depicted in Fig.1 and DSC of crystalline decitabine is similar as depicted in Fig.2.

Dated this 14th day of August 2015 Signature:
Dr. A.K.CHATURVEDI

DECITABINE PROCESS

ABSTRACT
The present invention relates to a process for the preparation of Decitabine of Formula (I).

(I)
The present invention further relates to a stable crystalline Form of Decitabine designated as Form SDE, which commercially viable for preparing stable dosage forms.