DESC:FIELD OF THE INVENTION:
The present invention relates to process for removal of degradation impurity N-Formyl Azacitidine (NFA) from Azacitidine. Over and above that, the present invention relates to providing an economical and technically facile process for removal of degradation impurity from Azacitidine.
BACKGROUND OF THE INVENTION:
Azacitidine (U-18496 and CC-486) is indicated for the treatment of myelodysplastic syndrome, myeloid leukemia and juvenile myelomonocytic leukemia. Azacitidine, sold under the brand name Vidaza™ among others and it was developed by Celgene and its parent organization is Bristol Myers Squibb.
Azacitidine (CAS Number: 320-67-2) is chemically known as 4-amino-1-ß-D-ribofuranosyl-s-triazin-2(1H)-one and is structurally represented as below:

Azacitidine is a chemical analog of cytidine, a nucleoside in DNA and RNA. Azacitidine is a ribonucleoside, so it is incorporated into RNA to a larger extent than into DNA. Azacitidine's incorporation into RNA leads to the disassembly of polyribosomes, defective methylation and acceptor function of transfer RNA, and inhibition of the production of proteins. Its incorporation into DNA leads to covalent binding with DNA methyltransferases, which prevents DNA synthesis and subsequently leads to cytotoxicity. It has been shown effective against human immunodeficiency virus in vitro and human T-lymphotropic virus.
A. Piskala, F. Sorm, Collect. Czech. Chem. Commun. 29, 2060 (1964) is a first disclosure of chemical synthesis of Azacitidine prepared via a multi-step synthesis starting from peracetylated 1-glycosyl isocyanate.
US7038038 reports the synthetic route of Azacitidine. It also reports purification of crude Azacitidine from dimethyl sulfoxide and Methanol. However, they have not mentioned the purity of Azacitidine. The purification may be schematically represented as following:

SCHEME-1
WO2004/082619 discloses fast recrystallization or purification of Azacitidine from dimethyl sulfoxide/methanol, dimethyl sulfoxide/toluene and dimethyl sulfoxide/chloroform. However, they have not mentioned the percentage yield and purity of Azacitidine.
R. E. Notari and J. L. DeYoung in Pharmaceutical Science, Vol. 64, No. 7, July 1975, p 1148-1157 analyzed stability of Azacitidine in aqueous media and determined that it was relatively instable, when compared with cytidine. The hydrolytic degradation of Azacitidine was studied as a function of pH, temperature, and concentration of deliberate. In particular, at pH <1 the main degradation products were 5-azacytosine and 5-azauracil, while at higher pH values, at higher pH values, drug was lost to products which were essentially nonchromophoric if examined in acidic solutions. Above pH 2.6, first-order plots for drug degradation are biphasic. At 50°, the drug exhibited maximum stability at pH 6.5 in dilute phosphate buffer. The degradation impurities may be schematically represented as following:

SCHEME-2
J. A. Beisler, Journal of Medicinal Chemistry, Vol. 21, No. 27, 1978, p 204-208 studied and mentioned that during the prolonged intravenous infusion time of Azacitidine, facile drug decomposition occurs in aqueous composition giving rise to products of unknown toxicity. Thus, HPLC analysis of 24 hours old aqueous solutions of Azacitidine revealed that the main degradation impurity is N-(formylamidino)-N'-ß-D-ribofuranosylurea (N-Formyl Azacitidine and RGU-CHO) The degradation impurity may be schematically represented as following:

SCHEME-3
ARGEMI, A., & SAURINA, J. (2007) - Study of the degradation of Azacitidine as a model of unstable drugs using a stopped-flow method and further data analysis with multivariate curve resolution, 74(2), 0–182. Particularly; Talanta 74 (2007) 176–182 discloses that in the case of Azacitidine, two successive reactions occur, first with the formation of RGU-CHO as an intermediate product, which is transformed progressively into RGU. In the acid range, reaction rates increase moderately with decreasing pH from 6 to 2. In basic media, the instability becomes more marked with increasing pH. Despite being unstable in the whole range of pH, pH values around 6 could be recommendable for the preparation of Azacitidine solutions.
WO2009/016617 discloses methods of purifying Azacitidine and reports use of N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), or a mixture thereof to purify Azacitidine.
CN102850418 discloses method can reduce the generation of hydrolysis impurity, the obtained product has high purity Azacitidine and reports use of methyl alcohol and water mixed solvent crystallization, the volume ratio of methyl alcohol and water is 5:1~7:1 to purify Azacitidine. However, the said invention is not considered as demonstrative, due to a high level of instability in the presence of water.
Therefore, it is proven from the prior-art that Azacitidine is not stable in aqueous media and its degrades in aqueous formulation. As high level of instability of Azacitidine in the presence of water, purification of Azacitidine in the presence of water or a mixture of solvent with water will be not effective. Use of water for purification of Azacitidine may generate high levels of degradation impurities.
Hence, there is a necessitate to find the improved methods of purifying Azacitidine. A highly pure Azacitidine, which contains minimal amounts of degradation impurity, such as N-Formyl Azacitidine, particularly on a commercial scale. Because, impurities may reduce the effectiveness of drug and generate the side effects.
The present invention provides an improved and large-scale industrial process for removal of degradation impurity N-formyl Azacitidine from Azacitidine. The present invention also avoids use of water and provides an economical and technically facile process for removal of degradation impurity from Azacitidine. The main advantage of the process for purification of Azacitidine as per the present invention is that it provides higher yield of product with higher purity at lower cost final product.
The present invention also provides a method of analyzing the impurity profile of Azacitidine by using chromatography; Preferably by using High Pressure Liquid Chromatography (HPLC). such as liquid or gas chromatography. Methods of liquid chromatography include, for example, Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC), and/or Liquid Chromatography /Mass spectrometry (LC-MS).
OBJECTIVE OF THE INVENTION:
The principal objective of present invention is to provide an industrially advantageous and cost-effective process for the purification of Azacitidine.
The prime object of the present invention is to provide an economical and technically facile process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine.
Another object of the present invention is to provide an industrially advantageous process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine which circumvent the use of water.
Another object of the present invention is to provide a highly pure Azacitidine, which contains minimal amounts of degradation impurity, such as N-Formyl Azacitidine.
Yet another object of present invention is to provide an efficient process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine with yield of product and high purity.
Another object of the present invention is to provide large-scale advantageous process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine to produce low-cost final product.
Yet an another object of present invention is to provide process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine using alcohol as a solvent may include methanol, ethanol, propanol, n-butanol, iso-propanol, 2-butanol and mixture of; Preferably by using methanol.
One more object of present invention is to provide process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine using first reagents may include sodium hydroxide, calcium carbonate and potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, sodium methoxide and mixture of; Preferably by using 30% sodium methoxide solution in methanol.

Another Object of the present invention also provides a method of analyzing the impurity profile of Azacitidine by using chromatography may include Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC), and/or Liquid Chromatography /Mass spectrometry (LC-MS); Preferably by using High Pressure Liquid Chromatography (HPLC).
SUMMARY OF THE INVENTION:
One aspect of the present invention relates to process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine comprising the purification steps as below.
a) adding methanol at 25-30°C;
b) dissolving crude Azacitidine at 25-30°C;
c) stirring the reaction mixture at 25-30°C;
d) adjusting pH to 1-2 by using 10%-15% methanolic hydrochloride solution at 20-30°C;
e) stirring the reaction mixture at 25-30°C;
f) adjusting pH to 6-7 by using 30% sodium methoxide solution in methanol at 20-30°C;
g) stirring the reaction mixture at 25-30°C;
h) cooling the reaction mixture to 10-15°C;
i) filtering the reaction mixture and washing with methanol at 10-15°C;
j) drying at 50-55°C.
Another aspect of the present invention also provides a method of analyzing the impurity profile of Azacitidine by using chromatography; such as liquid or gas chromatography. Methods of liquid chromatography include, for example, Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC), and/or Liquid Chromatography /Mass spectrometry (LC-MS), preferably by using High Pressure Liquid Chromatography (HPLC).
In another aspect of the present invention, the process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine may be depicted as a whole in below scheme-4.

SCHEME-4

DETAILED DESCRIPTION OF THE INVENTION:
The present invention will now be disclosed by describing certain preferred and optional embodiments, to facilitate various aspects thereof.
Azacitidine (CAS Number: 320-67-2) is chemically known as 4-amino-1-ß-D-ribofuranosyl-s-triazin-2(1H)-one, its molecular weight is 244 g/mol and the molecular formula is C8H12N4O5. Azacitidine (U-18496 and CC-486) is indicated for the treatment of myelodysplastic syndrome, myeloid leukemia and juvenile myelomonocytic leukemia.
The present invention relates to an improved process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine.
The present invention is to provide a cost-effective and technically facile process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine.
The present invention is to provide a large-scale industrially advantageous process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine which circumvent the use of water.
The present invention is to provide a highly pure Azacitidine, which contains minimal amounts of degradation impurity, such as N-Formyl Azacitidine.
Another aspect of the present invention is to provide an efficient process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine with yield of product and high purity.
Yet an another object of present invention is to provide process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine using alcohol as a solvent may include methanol, ethanol, propanol, n-butanol, iso-propanol, 2-butanol and mixture of; Preferably by using methanol.
One more aspect of present invention is to provide process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine using first reagents may include sodium hydroxide, calcium carbonate and potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, sodium methoxide and mixture of; Preferably by using 30% sodium methoxide solution in methanol.
Further, the present invention is to provide process for removal of degradation impurity N-Formyl Azacitidine from Azacitidine using second reagents may include methanolic sulfuric acid, Ethanolic hydrochloride, Isopropyl hydrochloride, methanolic hydrochloride and mixture of; Preferably by using 10-15% methanolic hydrochloride.
Moreover, the present invention also provides a method of analyzing the impurity profile of Azacitidine by using chromatography may include Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC), and/or Liquid Chromatography /Mass spectrometry (LC-MS); Preferably by using High Pressure Liquid Chromatography (HPLC).
One embodiments of the present invention involve the purification steps as below.
a) adding methanol at 25-30°C;
b) dissolving crude Azacitidine at 25-30°C;
c) stirring the reaction mixture at 25-30°C;
d) adjusting pH to 1-2 by using 10%-15% methanolic hydrochloride solution at 20-30°C;
e) stirring the reaction mixture at 25-30°C;
f) adjusting pH to 6-7 by using 30% sodium methoxide solution in methanol at 20-30°C;
g) stirring the reaction mixture at 25-30°C;
h) cooling the reaction mixture to 10-15°C;
i) filtering the reaction mixture and washing with methanol at 10-15°C;
j) drying at 50-55°C.
The present invention also provides a method of analyzing the impurity profile of Azacitidine, typically using chromatography, such as liquid or gas chromatography. Methods of liquid chromatography include, for example, Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC), and/or Liquid Chromatography /Mass spectrometry (LC-MS), preferably by using High Pressure Liquid Chromatography (HPLC).
The method of analyzing a sample containing Azacitidine comprises: Azacitidine and Azacitidine degradation product N-Formyl Azacitidine in the sample using a High Pressure Liquid Chromatography (HPLC), wherein the High Pressure Liquid Chromatography (HPLC) system is equipped with a mobile phases and is capable of separating the Azacitidine and Azacitidine degradation product N-Formyl Azacitidine; and identifying and/or detecting the presence and/or amount of the Azacitidine degradation product N-Formyl Azacitidine in the sample.
The HPLC measurements were performed using a system equipped with a Zorbax Bonus-RP (2:>0mm x 4.6mmx 5Jlm) Other parameters of the system were as follows:
TABLE 1: PARAMETERS
Column temperature 10°C
Detector wavelength 242 nm
Run time 82 minutes
Injection volume 5µL
Flow rate 1.0 mL
Sample set temperature Ambient Temperature
Sample concentration About 8.0 mg/mL
Retention time of main peak 8.2 minutes
Diluent Dimethyl Sulfoxide
Analyses were performed using the following mobile phase:
TABLE 2: MOBILE PHASES
Mobile Phase (Eluent) A 20mm Ammonium Acetate
Mobile Phase (Eluent) B Acetonitrile
The HPLC gradient is detailed in table below:
TABLE 3: GRADIENT PROGRAMME
Time (Minutes) %Mobile Phase-A %Mobile Phase-B
0.01 100 0
10.00 100 0
26.00 93 7
37.00 85 15
51.00 73 27
61.00 20 80
71.00 20 80
72.00 100 0
82.00 100 0
82.01 STOP
Accordingly, the parameters, mobile phases and gradient were set in High Pressure Liquid Chromatography (HPLC) system. After this, the analysis of crude Azacitidine and pure Azacitidine, which was purified as described in the present invention were performed and the results were found very much significant.
A sample of crude Azacitidine in the diluent Dimethyl sulfoxide (about 8.0 mg/mL) was withdrawn from the flask on every consecutive hour and injected to the HPLC system. Accordingly, the result of the present invention is summarized in below Table 4, which demonstrate the percentage of Azacitidine and percentage of Azacitidine degradation N-Formyl Azacitidine before purification.

TABLE 4: BEFORE PURIFICATION
Peak No. Peak Name Retention Time (min) Relative Retention Time Area µAU* Second Height
µAU Area %
1 3.72 0.47 47543 0.000 0.17
2 NFA 4.89 0.61 3161127 127676.455 11 .09
3 6.42 0.80 19979 1165.804 0.07
4 Azacitidine 7.97 1.00 24773969 1242615.432 86.88
5 10.14 1.27 150151 3283.455 0.53
6 12.48 1.57 29112 997.086 0.10
7 17.25 2.16 229865 8839.401 0.81
8 18.87 2.37 39586 1606.661 0.14
9 23.51 2.95 10680 602.322 0.04
10 26.37 3.31 47147 2758.020 0.17
11 48.36 6.07 6561 482.968 0.02
Total 28515719 1390027.60 100
A sample of pure Azacitidine in the diluent Dimethyl sulfoxide (about 8.0 mg/mL), which was purified as described in the present invention was withdrawn from the flask on every consecutive hour and injected to the HPLC system. Accordingly, the result of the present invention is summarized in below Table 5, which demonstrate the percentage of Azacitidine and percentage of Azacitidine degradation N-Formyl Azacitidine after purification.

TABLE 5: AFTER PURIFICATION
Peak No. Peak Name Retention Time (min) Relative Retention Time Area µAU* Second Height
µAU Area %
1 NFA 4.80 0.61 46919 2510.795 0.09
2 5.26 0.67 8776 263.803 0.02
3 Azacitidine 7.91 1.00 54906454 1869339.603 99.79
4 12.40 1.57 22514 585.374 0.04
5 17.24 2.18 8366 280.374 0.02
6 26.39 3.33 28959 977.167 0.05
Total 55021987 1873957.12 100
Above both Table 4 and Table 5 shows that Azacitidine, which was purified by the present invention is highly pure and having minimal or negligible amount of N-Formyl Azacitidine impurity. Hence, it is proven that the present invention is found more efficacious.
Having described the invention with reference to certain preferred embodiment, other aspects will become apparent to one skilled in the art from consideration of the specification. The invention is further defined by reference to the following example describing in detail by the purification of the compounds of the invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.
The following example is provided for illustrative purpose only and this example is in no way limitative on the present invention
EXAMPLE: PROCESS FOR REMOVAL OF DEGRADATION IMPURITY N-FORMYL AZACITIDINE FROM AZACITIDINE

PROCESS:
1. Arrange a 500 mL clean & dry four neck round bottom flask on water bath with thermos-pocket, thermometer, overhead stirrer and condenser under nitrogen atmosphere.

2. Charge 500 mL methanol into the round bottom flask at 25-30°C.

3. Charge 50 g crude Azacitidine into the round bottom flask at 25-30°C.

4. Stir the reaction mixture for 10 minutes under nitrogen atmosphere at 25-30°C.

5. Adjust pH of reaction mixture to 1-2 by using 10%-15% methanolic hydrochloride solution at 20-30°C.

6. Stir the reaction mixture for 10 minutes under nitrogen atmosphere at 25-30°C.

7. Adjust pH of reaction mixture to 6-7 by using 30% sodium methoxide solution in methanol at 20-30°C.

8. Stir the reaction mixture for 2-3 hours at 25-30°C.

9. Cool the reaction mixture to 10-15°C.

10. Stir the reaction mixture for 1-2 hours at 10-15°C.

11. Filter the reaction mixture and wash with 25 mL methanol at 10-15°C.

12. Suck dry wet material for 30 minutes at 20-30°C.

13. Unload wet material into tray and take a wet weight.

14. Dry material under vacuum at 50-55°C for 8 hours.

15. Unload dry material into tray and take a dry weight.
RESULTS:
Physical Results:
TABLE 6: WEIGHT
Wet Weight 20 g
Dry Weight 13 g
HPLC Analysis Results:
TABLE 7: BEFORE & AFTER PURIFICATION
Sr. No. Analysis % of Azacitidine % of NFA
1 Before Purification 86.88% 11.09%
2 After Purification 99.79% 0.09%

The invention described herein comprises in various objects and their description as mentioned above, with respect to characteristics and processes adopted. While these aspects are emphasized in the invention, any variations of the invention described above are not to be regarded as departure from the spirit and scope of the invention as described.
,CLAIMS:1. An improved process for the purification of Azacitidine comprises:
(a) adding methanol at 25-30°C;
(b) dissolving crude azacitidine at 25-30°C;
(c) stirring the reaction mixture at 25-30°C;
(d) adjusting pH to 1-2 by using second reagent at 20-30°C;
(e) stirring the reaction mixture at 25-30°C;
(f) adjusting pH to 6-7 by using first reagent in methanol at 20-30°C;
(g) stirring the reaction mixture at 25-30°C;
(h) cooling the reaction mixture to 10-15°C;
(i) filtering the reaction mixture and washing with methanol at 10-15°C;
(j) drying at 50-55°C and isolating purified azacitidine.

2. The process as claimed in claim 1, wherein the process is carried out in absence of water.

3. The process as claimed in claim 1, wherein the first reagents is selected from sodium hydroxide, calcium carbonate and potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, sodium methoxide and mixture thereof, preferably 30% sodium methoxide solution in methanol.

4. The process as claimed in claim 1, wherein the second reagents is selected from methanolic sulfuric acid and methanolic hydrochloride and mixture thereof, preferably 10-15% methanolic hydrochloride.

5. The process as claimed in Claim 1 wherein the content of N-Formyl Azacitidine degradation impurity in Azacitidine is 0.09% or less.

6. The process as claimed in claim 1, the isolated azacitidine impurity profile is analysed by using high pressure liquid chromatography method.