FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention
"Synthesis of Regadenoson"
2. Applicant(s)
Name Nationality Address
USV Limited Indian company incorporated Arvind Vitthal Gandhi Chowk, D.S.D. Marg, Govandi,
under Companies Act, 1956 Mumbai-400 088, Maharashtra, India,
3. Preamble to the description
The following specification particularly describes the invention and the manner in which it is to be performed.


Field of the invention:
The present invention relates to a process for preparation of Regadenoson (I).
Background of invention:
Regadenoson, chemically known as (l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxy methyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazo]-4-yl)-N-methylcarboxamide is a selective A2A-adenosine receptor agonist. Regadenoson is a pharmacologic stress agent indicated for radionuclide myocardial perfusion imaging in patients unable to undergo adequate exercise stress. Regadenoson is a low affinity agonist for the A2A-adenosine receptor, with at least 10-fold lower affinity for the A1 adenosine receptor and weak, if any, affinity for the A2B and A3 adenosine receptors. Activation of the A2A-adenosine receptor by Regadenoson produces coronary vasodilation and increases coronary blood flow.
Regadenoson is commercially marketed by Astellas Pharma Inc. under the brand name of Lexiscan® in United States and by Rapidscan Pharma/GE Healthcare under the brand name of Rapiscan® in various European countries. The marketed formulation is a sterile, clear, colorless, non-pyrogenic solution for intravenous administration. It is available as a single-use vial and single-use pre-filled syringe containing Regadenoson 0.4mg/5ml (0.08 mg/ml). The recommended dose is 5 ml (0.4mg regadenoson) administered as a rapid injection into a peripheral vein using a 22 gauge or larger catheter or needle followed by immediate flushing with 5ml of saline and administering the radionuclide myocardial perfusion imaging agent 10-20 seconds after the saline flush. The radionuclide may be injected directly into the

same catheter as Lexiscan®.
US6403567 provides different processes for preparation of Regadenoson. One such process is represented in Scheme I as below:

The process disclosed in this patent involves methylamination of pyrazoloadenosine at a higher temperature .i.e., 65°C which is not desirable at an industrial scale. Methyl amine gets evaporated at higher temperature and hence the reaction does not go to completion. The use of higher temperature also affects the quality of the product. Further higher volumes of methylamine is used.
WO2007092372 discloses the reaction of Ethyll-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-carboxylate with methylamine, initially at a temperature of about 0-5°C, followed by warming to about 50-70°C. Alternatively, the reaction is conducted in a sealed pressure reactor.

Use of pressure reactor avoids the escape of methylamine during reaction at higher reaction temperature. It further teaches the reaction of Ethyl 1- {9-[(4S,2R,3R,5R)-3,4-dthydroxy-5-(hydroxymethyl)oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-carboxylate with methylamine at ambient temperature. However, the temperature range is not specified.
There exists a need to develop a simple, cost-effective, commercially viable and industrially feasible process for the preparation of Regadenoson. The present invention provides a simple, economical, commercially viable and industrially feasible process for large scale preparation of Regadenoson having purity of more than 99.8%.
Object of the invention:
An object of the present invention is to provide a simple, cost effective, consistent, commercially viable and industrially feasible process for large scale preparation of substantially pure Regadenoson.
Another object of the present invention provides Regadenoson substantially free of compound selected from hydrolysis product impurity, namely l-{9-[(4S.2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylic acid, decarboxylated product impurity, namely (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl) tetrahydrofuran-3,4-diol, 2-Hydrazinoadenosine, Ethyl 2-formyl-3-oxopropionate or hydrazine hydrate.
Another object of the present invention provides compound selected from hydrolysis product impurity, namely l-{9-[(4S:2R,3R.5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazoI-4-carboxylic acid or decarboxylated product impurity, namely (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl) tetrahydrofuran-3,4-diol and process for preparation thereof.
Yet another object of the present invention provides use of compound selected from

l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylic acid or (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3,4-diol as reference marker/ reference standard for testing the impurity profile of Regadenoson.
Another object of the present invention provides pharmaceutical composition comprising Regadenoson and at least one pharmaceutically acceptable excipient.
Summary of the invention:
One aspect of the present invention provides a process for preparation of Regadenoson comprising the steps of,
a) treating Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (Pyrazoloadenosine) with methyl amine at a temperature of about 0-5°C to obtain a mixture;
b) allowing the mixture to reach a temperature of about 15 to 25°C;
c) isolating Regadenoson from said mixture; and
d) optionally purifying said Regadenoson.
Preferably said treatment in step a) is carried out using about 15-35 volumes of aqueous solution of methyl amine with respect to said Pyrazoloadenosine.
Another aspect of the present invention provides a process for purification of Regadenoson comprising the steps of,
a) treating Regadenoson with a first solvent selected from N,N-dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, acetone tetrahydrofuran or dimethylsulfbxide to obtain a solution;
b) treating said solution with a second solvent selected from isopropanol. methanol, ethanol, n-propanol or butanol to obtain a mixture; and
c) isolating Regadenoson from said mixture.
Another aspect of the present invention provides a process for preparation of Ethyl l-{9-[(4S,2R,3R.5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yI}pyrazoI-4-carboxyIate (Pyrazoloadenosine) comprising the steps of,

a) treating (4S,2R,3R,5R)-2-(6-amino-2-chloropurin-9-yl)-5-(hydroxymethyl) oxolane-3,4-diol (2-chloroadenosine) with about 29-31 fold molar excess of hydrazine hydrate at a temperature of about 20-30°C to obtain (4S,2R,3R,5R)-2-(6-amino-2-hydrazinyl-9H-purin-9-yl)-5-(hydroxymethyl) oxolane-3,4-diol (2-hydrazinoadenosine);
b) treating said 2-hydrazinoadenosine with about 1-1.5 moles of ethyl 2-formyl-3-oxopropionate to obtain Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxoIan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (pyrazoloadenosine); and
c) optionally purifying said pyrazoloadenosine.
Preferably purification of said pyrazoloadenosine is carried out using a solvent selected from N;N-dimethylformamide, acetonitrile, dichioromethane. ethyl acetate, acetone, tetrahydrofuran, dimethylsulfoxide or water. Preferably. Regadenoson obtained is Regadenoson Form A (monohydrate) or Regadenoson Form C.
Preferably, Regadenoson obtained is substantially free of compound selected from the group consisting of l-{9-[(4S,2RJ3R,5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylic acid, (4S,2R,3R,5R)-2-[6-amJno-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(bydroxymethyl) tetrahydrofuran-3.4-diol, 2-Hydrazinoadenosine, Ethyl 2-formyl-3-oxopropionate and hydrazine hydrate.
Another aspect of the present invention provides the use of l-{9-[(4S,2R.3R,5R)-3,4-di hydroxy- 5 -(hydroxymethyl)oxolan-2 -y 1 ] -6 -aminopurin- 2-y 1} pyrazo I -4 -carboxylic acid or (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3,4-diol as a reference marker or reference standard for determining the purity of a sample of Regadenoson or a pharmaceutical dosage form comprising Regadenoson.
Yet another aspect of the present invention provides (4S,2R,3R,5R)-2-[6-amino-2-(1 H-pyrazol-1-yl)-9H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3,4-diol. Preferably, said compound is prepared by treating (4S,2R,3R,5R)-2-(6-amino-2-

hydrazinyl-9H-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol with malonaldehyde bis(dimethylacetal).
Brief description of the Drawings:
Fig. 1: X-ray diffraction pattern of Regadenoson Form A (monohydrate) obtained by the process of the present invention.
Fig. 2: X-ray diffraction pattern of Regadenoson Form C obtained by the process of the present invention.
Detailed description of the invention:
The present invention relates to a simple, cost effective, consistent, commercially viable and industrially feasible process for the preparation of Regadenoson.
According to one embodiment of the present invention, there is provided a process for preparation of Regadenoson comprising the steps of,
a) treating Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV) with methyl amine at a temperature of about 0-5°C to obtain a mixture;
b) allowing the mixture to reach a temperature of about 15 to 25°C;
c) isolating Regadenoson from said mixture; and
d) optionally, purifying said Regadenoson.
In a preferred embodiment, to an aqueous solution of methylamine is added Ethyl 1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxoian-2-yl]-6-aminopurin-2-yI}pyrazoI-4-carboxyIate (IV) or Pyrazoloadenosine at a temperature of about 0-5°C to obtain a mixture. The reaction mixture is stirred at the same temperature for about 3 to 8 hours, preferably for 5 hours to obtain a slurry. The temperature of the slurry is allowed to reach about 15 to 25°C, preferably 18 to 20°C. The slurry is maintained under stirring at the same temperature for about 2 to 6 hours, preferably for 3 hours. The slurry is then treated with water to obtain a mixture. This mixture is stirred for about 30 to 90 minutes, preferably for 60 minutes at the same temperature followed by filtration to obtain a solid. The solid thus obtained is washed with water and/or

suitable solvent and then dried to obtain Regadenoson as an off-white to pale brown coloured solid. The obtained Regadenoson is further purified.
Preferably, said treatment of Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate with methyl amine is carried out at atmospheric pressure. Preferably, about 15-35 volumes of methyl amine is used with respect to Ethyl l-{9-[(4S,2R,3R,5R)-3.4-dihydroxy-5-(hydroxymethyl)oxoIan-2-yl]- 6-aminopurin-2-yl} pyrazol-4-carboxylate. More preferably, about 30 volumes of methylamine is used.
Suitable solvent is selected from C1-C4 alcohol such as isopropyl alcohol(IPA). methanol or ethanol. preferably isopropyl alcohol.
Prior art teaches the reaction with methylamine in a sealed reactor at higher temperature. The present invention avoids the use of sealed reactor for the methylamination reaction. In the process of the present invention, this reaction is carried out initially at low temperature followed by maintaining the reaction mixture under stirring at about 18-20°C. It has been surprisingly found by the inventors of the present invention that the completion of the reaction is achieved at the above mentioned low temperature conditions. Regadenoson obtained by the process of the present invention is substantially free of impurities, especially impurities such as 1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylic acid and/or (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-y])-9H-purin-9-y]]-5-(hydroxymethyl) tetrahydrofuran-3,4-diol.
According to another embodiment of the present invention, there is provided a process for purification of Regadenoson comprising the steps of,
a) treating Regadenoson with a first solvent selected from N,N-dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, acetone, tetrahydrofuran or dimethylsulfoxide to obtain a solution;
b) treating said solution with a second solvent selected from isopropanol, methanol, ethanol, n-propanol or butanol to obtain a mixture; and

c) isolating pure Regadenoson from said mixture.
Preferably, the first solvent is N,N-dimethylformamide and the second solvent is isopropanol (IPA).
In a preferred embodiment, the process for purification of Regadenoson comprises suspending Regadenoson in N.N -dimethylformamide to obtain a suspension. The suspension is wanned to a temperature of about 30 to 50°C, preferably 35 to 40°C and stirred for 10 to 30 min, preferably for 15 to 20 min to obtain a brown solution. The obtained brown solution is filtered to remove any undissolved/suspended particles. The filtrate obtained is treated with isopropanol (IPA) over a period of 5-10 min to obtain a mixture. The mixture is stirred for 2 to 6 hours, preferably for 3 to 4 hours to obtain a slurry. The slurry is filtered to obtain a solid. The solid is washed with IPA and then suspended in water to obtain a slurry. The resultant slurry is maintained for about 1 hour at 25 to 30°C followed by filtration to obtain a solid. The solid is washed with water and/or IPA followed by drying to obtain off-white coloured solid having purity of more than 99.8%.
Prior art teaches purification of Regadenoson using dimethylsulfoxide and water. It has been found by inventors of the present invention that purification using dimethyl sulfoxide and water did not result in ICH quality product. Purification using dimethyl formamide and isopropanol resulted in a product having all impurities less than about 0.15% , preferably less than about 0.1%. more preferably less than about 0.05%.
Another embodiment of the present invention provides a process for preparation of Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-amino purin-2-y]}pyrazol-4-carboxy]ate (IV) comprising the steps of,
a) treating (4S,2R,3R,5R)-2-(6-amino-2-chloropurin-9-yl)-5-(hydroxymethyl) oxolane-3,4-diol (II) or 2-chloroadenosine with about 29-31 fold molar excess of hydrazine hydrate at a temperature of about 20-30°C to obtain (4S,2R,3R,5R)-2-(6-amino-2-hydrazinyl-9H-purin-9-yl)-5-(hydroxymethyl)

oxolane-3.4-diol (III) or 2-hydrazinoadenosine;

b) treating (4S,2R,3R,5R)-2-(6-amino-2-hydra2inyl-9H-purin-9-yl)-5-(hydroxy methyl)oxolane-3,4-diol (III) with about l-l.5moles of ethyl 2-formyl-3-oxopropionate to obtain Ethyl l-{9-[(4S,2R,3R,5R)-3;4-dihydroxy-5-(hydroxymethyi)oxolan-2-yl]-6-aminopurin-2-yl}pyrazoI-4-carboxyIate (IV) or Pyrazoloadenosine; and

c) optionally purifying Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxy methyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV).
Preferably, the process for preparation of Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxoIan-2-yl]-6-aminopurin-2-yI}pyrazoi-4-carboxylate(TV) comprises the steps of, Step 1:
2-chloroadenosine and about 29-31 fold molar excess of hydrazine hydrate are taken in a flask to obtain a mixture. The obtained mixture is stirred at a temperature of about 20-40°C; preferably at 25-30°C for about 3 to 6 hours, preferably for 4 hours. After the completion of the reaction, the mixture is treated with an alcohol selected from isopropanol (IPA), ethanol or methanol, preferably IPA over a period of 20 to

40 minutes, preferably for 30 minutes at the same temperature to obtain a slurry. The obtained slurry is maintained under stirring for 1 to 3 hours, preferably for 2 hours and then filtered to separate out the solid. The solid obtained is washed with an alcohol selected from isopropanol (IPA), ethanol or methanol, preferably IPA and then treated with water to obtain a slurry. The slurry thus obtained is filtered to separate out a solid, which is washed with water followed by alcohol and dried to obtain (4S.2R,3R,5R)-2-(6-amino-2-hydrazinyi-9H-purin-9-yl)-5-(hydroxymethyl) oxolane-3,4-diol.
Step 2:
(4S,2R,3R.5R)-2-(6-amino-2-hydrazinyl-9H-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol is treated with an alcohol selected from isopropanol (IPA), ethanol or methanol, preferably IPA to obtain a suspension. To this suspension, is added ethyl 2-formyl-3-oxopropionate to obtain a mixture. The obtained mixture is stirred for 10 to 20 minutes, preferably for 15 minutes and then heated to reflux (78 to 82°C). The mixture is maintained at the same temperature for about 2 to 5 hours, preferably for 3 hours. The mixture is then cooled to 10 to 30°C, preferably 15 to 20°C and stirred at the same temperature for 1-5 hours, preferably for 2-3 hours. The mixture is filtered to obtain a solid, which is washed with a solvent selected from alcohol, ether or mixture thereof. The solid .i.e., Ethyl l-{9-[(4S,2R,3R)5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl} pyrazol-4-carboxylate (IV) thus obtained is dried.
Another embodiment of the present invention provides a process for the purification of Ethyl 1 - {9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV) comprising the steps of,
a) dissolving Ethyl l-{9-[(4S,2R,3R;5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV) in an organic solvent and/or water to obtain a mixture;
b) optionally heating the mixture obtained in step a) to obtain a solution; and
c) isolating pure Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)

oxoIan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxyIate(IV).
Preferably, Ethyl 1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5(hydroxymethyl)oxoIan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV) is suspended in a polar aprotic solvent-water mixture to obtain a suspension. The suspension is heated to 80 - 100°C, preferably 85 to 90°C and maintained for 20 to 40 minutes, preferably for 30 min to obtain a clear yellow solution. The solution is cooled to 20 to 40°C. preferably 25 to 30°C and maintained under stirring at the same temperature for 1 to 3 hours, preferably for 2 hours to obtain a yellow coloured slurry. The slurry is filtered and the solid thus obtained is washed with water, organic solvent and dried to obtain pure Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl} pyrazol-4-carboxylate (IV).
Polar aprotic solvent is selected from N,N-dimethylformarnide (DMF), acetonitrile, dichloromethane, ethyl acetate, acetone, tetrahydrofuran or dimethylsulfoxide. preferably, N,N-dimethylformamide. Organic solvent used is selected from alcohol or ether. Alcohol used is selected from G-G alcohol such as isopropyl alcohol. methanol or ethanol, preferably isopropyl alcohol (IPA). Ether is selected from diisopropyl ether, tetrahydrofuran (THF), diethyl ether, methyl tertiary butyl ether (MTBE) or 1,4-dioxane. preferably diisopropyl ether.

Another embodiment of the present invention provides Regadenoson substantially free of compound selected from hydrolysis product impurity, namely l-{9-
The process for preparation of Regadenoson as described above is represented in the below Scheme II,

[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl} pyrazol-4-carboxylic acid, Decarboxylated product impurity, namely (4S,2R,3R,5R)-2-[6-amino-2-(l H-pyrazol-1 -yl)-9H-purin-9-yl]-5-(hydroxymethyl) tetrahydrofuran-3,4-dioi. 2-Hydrazinoadenosine, Ethyl 2-formyl-3-oxopropionate or hydrazine hydrate.
Hydrolysis product impurity and Decarboxylated product impurity are as shown below

Impurity Structure Name
Hydrolysis product impurity l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-
5-(hydroxy methyI)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-
carboxylic acid
Decarboxylated product impurity (4S,2R,3R,5R)-2-[6-amino-2-(l H-pyrazol-1 -yl)-9H-purin-9-yI] -5-

(hydroxymethyl) tetrahydrofuran-3.4-
. diol
Yet another embodiment of the present invention provides compound selected from l-{9-[(4S,2R53Rs5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylic acid or (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3,4-diol.
Another embodiment of the present invention provides the use of these compounds as reference marker and/or reference standard for determining the purity of Regadenoson or pharmaceutical composition containing Regadenoson.
Another embodiment of the present invention provides a method for isolation of the above impurities, the chromatographic conditions are as given below,

Column: Phenomenex prodigy ODS-3,250 mm x 4.6 mm, 5μ.
Eluent:
Mobile phase A: 0.1% orthophosphoric acid in H2O; adjust pH 4.0 with dilute
triethylamine
Mobile phase B: Methanol: Buffer (80:20)
Gradient System:

Time in min Mobile Phase A(%) Mobile Phase B(%)
0 80 20
20 80 20
35 40 60
65 40 60
70 80 20
85 80 20
Flow rate: 1.0 ml/min Injection Vol.: 10.0 μL Wavelength: 246 nm Column temperature: 40°C
Compounds thus separated are atleast 80% pure, preferably at least 90% pure, more preferably at least 95% pure, as judged by GC or HPLC.
Another embodiment of the present invention provides a process for the preparation of hydrolysis product impurity namely, l-{9-[(4S,2R,3R.5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]- 6-aminopurin-2-yl}pyrazol-4-carboxylic acid comprising the steps of.
a) treating Ethyl l-{9-[(4S,2R,3R?5R)-3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV) with a base to obtain a slurry; and
b) isolating the hydrolysis product impurity from said slurry.
In a preferred embodiment, Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxy

methyl)oxolan-2-yIJ-6-aminopurin-2-yl}pyrazol-4-carboxylate (IV) is hydrolyzed using aqueous sodium hydroxide solution at a temperature of about 15 to 30°C. preferably 20 to 25°C to obtain a slurry. The slurry is maintained at the same temperature for 2 to 4 hours, preferably for 3 hours followed by dilution with water. The pH of the slurry is adjusted to 4 by adding conc. HC1 and the slurry is stirred for 30 to 90 min. preferably for 60 min at the same temperature to precipitate out the solid. The solid thus obtained is filtered, washed with water, solvent or mixture thereof and dried to isolate the hydrolysis product impurity.
Solvent is selected from acetonitrile, dichloromethane, N,N-dimethylformamide. ethyl acetate, acetone, tetrahydrofuran, dimethylsulfoxide, diisopropyl ether. tetrahydrofuran (THF), diethyl ether, methyl tertiary butyl ether or 1.4-dioxane preferably acetonitrile or diisopropyl ether.
The process for preparation of hydrolysis product impurity as described above is represented in the below Scheme III,

Another embodiment of the present invention provides a process for preparation of Decarboxylated product impurity, namely (4S.2R,3R.5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl)tetrahydromran-3,4-diol comprising the steps of,
a) treating (4S,2R,3R,5R)-2-(6-amino-2-hydrazinyl-9H-purin-9-yl)-5-(hydroxy methyl)oxolane-3,4-diol with malonaldehyde bis(dimethyl acetal) in

presence of a solvent to obtain a mixture;
b) heating the mixture obtained in step a); and
c) isolating decarboxyiated product impurity from the mixture of step a).
Preferably, (4S,2R,3R,5R)-2-(6-amino-2-hydrazinyl-9H-purin-9-yl)-5-(hydroxy methyl)oxolane-3,4-diol is treated with malonaldehyde bis(dimethyl acetal) in a solvent selected from isopropyl alcohol, methanol or ethanol, preferably isopropyl alcohol to obtain a mixture. The mixture is stirred for 10 to 20 minutes, preferably for 15 minutes. The mixture is then heated to reflux and maintained under stirring at the same temperature for 5 to 10 hours, preferably for 8 hours. The mixture is cooled to 10 to 30°C, preferably 15 to 20°C and stirred for 1 to 5 hours, preferably for 2 to 3 hours. The mixture is filtered to obtain a solid. The solid is washed with a suitable solvent and dried to obtain the Decarboxyiated product impurity.
Suitable solvent used for washing is selected from isopropyl alcohol, methanol, ethanol, diisopropyl ether, tetrahydrofuran (THF). diethyl ether, methyl tertiary butyl ether or 1,4-dioxane, preferably isopropyl alcohol (IPA) or diisopropyl ether.
The process for preparation of the Decarboxyiated product impurity as described above is represented in the below Scheme IV,

Hydrolysis product impurity and Decarboxyiated product impurity are generated at the Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-ammopurin-2-yl}pyrazo!-4-carboxylate (IV) stage. Hydrolysis product impurtity can

also get generated at the Regadenoson stage.
Another embodiment of the present invention provides an analytical method for testing the impurity profile of Regadenoson which comprises the steps of,
a) providing a sample of Regadenoson containing or suspected of containing hydrolysis product impurity, decarboxylated product impurity, 2-hydrazinoadenosine, ethyl 2-formyl-3-oxopropionate, hydrazine hydrate or mixture thereof;
b) subjecting the sample of step a) to chromatographic technique, preferably HPLC; and
c) determining the peak corresponding to one or more of the above impurities.
It has been found that purifying Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazoI-4-carboxylate (IV) prior to methylamination reaction provides Regadenoson substantially free of impurities, in particular hydrolysis product impurity and/or decarboxylated product impurity. Regadenoson obtained by the process of the present invention is substantially free of the above mentioned impurities.
Preferably, Regadenoson obtained according to the present invention is Form A (monohydrate) characterized by X-ray powder diffraction pattern as shown in Fig. 1. It is further characterized by peaks expressed as 2-theta values at about 5.56, 8.93, 11.14, 14.37, 16.77, 18.07, 18.41, 22.82, 23.32, 23.73, 26.00, 28.77 and 34.44 degrees.
Another embodiment of the present invention provides process for the preparation of Regadenoson Form C comprising the steps of, suspending Regadenoson in acetonitrile or methanol to obtain a mixture. The mixture is stirred at 60 to 65°C for 5-6 hours to obtain a slurry. The hot slurry is filtered to obtain a solid. The solid thus obtained is washed and dried to obtain Regadenoson Form C.
Regadenoson Form C obtained according to the present invention is characterized by

X-ray powder diffraction pattern as shown in Fig. 2. It is further characterized by peaks expressed as 2-theta values at about 6.17, 10.24, 10.68, 12.39, 14.16, 17.68, 17.84,18.26, 19.08, 21.49, 22.59, 24.67, 25.43, 27.83 and 43.12 degrees.
Another embodiment of the present invention provides process for the preparation of Regadenoson monohydrate from Regadenoson Form C comprising the steps of, suspending Regadenoson in water to obtain a mixture. The mixture is stirred at 15 to 30°C, preferably at 20 to 25°C for 1 to 3 hours, preferably for 2 hours to obtain a slurry. The slurry is filtered to obtain a solid. The solid is washed with water and dried to obtain Regadenoson monohydrate.
Apart from potential process related impurities, there are some genotoxic impurities. The control of these impurities is more difficult while scaling-up the processes according to the prior art, resulting in extensive isolation/purification procedures so as to obtain pure Regadenoson. The process of the present invention provides Regadenoson wherein these impurities are controlled well below 1500 ppm thus complying with the ICH guidelines.
Genotoxic impurities which may be present in the final API are as described below,
a) Ethyl 2-formyl-3-oxopropionate: starting material for preparation of Regadenoson; and/or
b) (4S,2R,3R,5R)-2-(6-amino-2-hydra2inyl-9H-purin-9-yl)-5-(hydroxy methyl) oxolane-3,4-diol or 2-Hydrazinoadenosine: an intermediate in synthesis of Regadenoson.
Starting materials, (4S,2R.3R,5R)-2-(6-amino-2-chloropurin-9-yl)-5-(hydroxy methyI)oxolane-3,4-dioI and Ethyl 2-formyI-3-oxopropionate are commercial products.
(4S,2R.3R,5R)-2- (6-amino-2-chloropurin-9-yl)-5-(hydroxymethyl) oxolane-3,4-diol and Ethyl 2-formyl-3-oxopropionate can be prepared by any method known in the art. Ethyl 2-formyl-3-oxopropionate is prepared by a process comprising the steps of

treating ethyl 3,3-diethoxypropionate with ethyl formate in the presence of sodium hydride. The reaction is carried out at a temperature of about 0-5°C for about 24 hours followed by the addition of water to the reaction mixture. This reaction mixture is then subjected to extraction with an organic solvent such as methyl tert-butyl ether (MTBE). The aqueous layer is acidified with hydrochloric acid followed by extraction with dichloromethane. The organic layer is separated and dried under vacuum. Ethyl 2-formyl-3-oxopropionate thus obtained can be further purified by conventional techniques, if required.
Regadenoson obtained by the process of the present invention has particle size distribution such that 90% particles have particle size less than about 500 microns. The particle size may be reduced using conventional techniques so as to obtain particles having particle size less than about 300 microns, preferably less than about 200 microns, more preferably less than about 150 microns.
Another embodiment of the present invention provides pharmaceutical composition comprising Regadenoson, prepared by the process of the present invention. Regadenoson obtained by the process of the present invention may be combined with pharmaceutically acceptable exctpients to obtain suitable pharmaceutical compositions.
Unless otherwise indicated, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
The term "substantially pure" or "substantially free" means Regadenoson or Regadenoson monohydrate having less than about 1%, preferably less than about 0.5%, more preferably less than about 0.3%, most preferably less than about 0.15% of undesired compounds or other polymorphic forms.
The term "reflux temperature" means the temperature at which the solvent or solvent system refluxes or boils at atmospheric pressure.

X-ray powder diffraction pattern was obtained on PANalytical XperfPRO diffractometer equipped with accelerator detector using Copper Ka (n = 1.5406 A0) radiation with scanning range between 2-theta 4-50° at a scanning speed of 2o/min,
The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.
Examples
Example 1: Preparation of (4S,2R,3R,5R)-2-(6-amino-2-hydrazinyl-9H-purin-
9-yI)-5-(hydroxymethyl)oxolane-3,4-diol (2-hydrazinoadenosine)
To a 1 L RB flask, hydrazine hydrate (500 ml, 10.24 mol) and 2-chloroadenosine (100 g. 0.33 mol) were charged to obtain a mixture. The mixture was stirred at 25-30°C for 4 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the mixture was added slowly to IPA (3000 ml) at the same temperature over a period of 30 min under stirring to obtain a slurry. The obtained slurry was stirred for 2 hours and filtered to obtain a solid. The solid was washed with IPA (100 ml) and the wet cake was charged in water (1000 ml) followed by stirring for 12 -14 hours at 25-30°C to obtain a slurry. The slurry was filtered and the solid thus obtained was washed with water (100 ml), IPA (2 x 100 ml) followed by drying at 55-60°C to obtain off-white to pale brown coloured solid. Yield: 72 g (73%); Purity: more than 90%.
Example 2: Preparation of Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy -5-(hydroxymethyl)oxolan-2-yl] -6-aminopurin-2-yl} pyrazol-4-carboxylate (pyrazoloadenosine)
To a 2 L RB flask, IPA (750 ml, 15 vol) and 2-hydrazinoadenosine (50 g, 0.168 mol) were charged to obtain a suspension. To this suspension, was added ethyl-2-formyl-3-oxopropionate (36.3 g, 0.252 mol) to obtain a mixture. The mixture was stirred for 15 min and then heated to reflux (78-82°C). The mixture was maintained at the same temperature for 3 hours. The mixture thus obtained was cooled to 15-20OC and maintained under stirring at the same temperature for 2-3 hours. The mixture was fdtered to obtain a solid. The obtained solid was washed with IPA

(50 ml) followed by diisopropylether (2 x 50 ml) and dried at 60°C. Yield: 50 g (73.5%); Purity: more than 90 %.
Example 3: Purification of Pyrazoloadenosine:
Pyrazoloadenosine (50 g) was suspended in DMF (250 ml) and water (1000 ml) to obtain a mixture. The mixture was heated to 85-90°C and maintained for 30 min at the same temperature to obtain a clear yellow solution. The solution was cooled to 25-30°C followed by stirring for 2 hours to obtain a yellow coloured slurry. The slurry was filtered and the obtained solid was washed with water (100 ml), IPA (2 x 100 ml), diisopropylether (2 x 100 ml) and then dried at 50-60°C to obtain yellow to pale orange coloured solid. Yield: 42 g (85%); Purity: more than 95 %
Example 4: Preparation of Regadenoson Form A (monohydrate)
40 % aqueous solution of methylamine (1500 ml, 17.37 mol) was charged in a flask and cooled to 0-5°C. To this cooled solution, Pyrazoloadenosine (50 g, 0.123 mol) was added to obtain a mixture. The mixture was stirred at the same temperature for 5 hours to obtain a slurry. The temperature of the slurry was allowed to reach to 18-20°C and the slurry was maintained under stirring at the same temperature for 3 hours. Water (500 ml) was added to the slurry to obtain a mixture. The mixture was stirred for 1 hour at 18-20°C and filtered to obtain a solid. The obtained solid was washed with water (3x 100 ml), IPA (3 x 100ml) and dried at 25-30°C to obtain off-white to pale brown coloured solid. Yield: 40 g (80%); Purity: more than 98%
Example 5: Purification of Regadenoson Form A (monohydrate)
Regadenoson (10 g) was suspended in DMF (100 ml) to obtain a suspension. The suspension was warmed to 35-40°C and stirred for 15-20 min to obtain a brown solution. The brown solution was filtered to remove undissolved/suspended particles. The filtrate obtained was added in IPA (300 ml) over a period of 5-10 min to obtain a mixture. The mixture was stirred for 3 - 4 hours to obtain a slurry. The slurry was filtered to obtain a solid. The solid was washed with IPA (3 x 40 ml) and

was suspended in water (100 ml). The resultant slurry was maintained for 1 hour at 25-30°C followed by filtration to obtain a solid. The solid was washed with water (20 ml) followed by IPA (2 x 20 ml) and dried at 25-30°C to obtain off-white coloured solid. Yield: 6.5 g (65%); Purity: more than 99.8%
Example 6: Preparation of Regadenoson Form C
Regadenoson (10 g) was suspended in acetonitrile (50 ml) to obtain a mixture. The mixture was stirred at 60 to 65°C for 5-6 hours. The hot mixture was filtered to obtain a solid. The solid thus obtained was washed with acetonitrile and dried at 25-30°C for 12 hours to obtain Regadenoson Form C.
Example 7: Preparation of Regadenoson Form A (monohydrate) from Regadenoson Form C
Regadenoson Form C (2 g) was suspended in water (10 ml) to obtain a mixture. The mixture was stirred at 20-25°C for 2 hours to obtain a slurry. The obtained slurry was filtered to obtain a solid. The solid was washed with water and dried at 25-30°C for 12 hours to obtain Regadenoson Form A (monohydrate).
Example 8:
Preparation of l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-
yl]-6-aminopurin-2-yl}pyrazol-4-carboxylic acid (Hydrolysis product impurity)
In a 500 ml RB flask, water (100 ml) and sodium hydroxide (1.1 g, 0.027 mol) were charged followed by addition of 2-pyrazoloadenosine (10 g, 0.0246 mol) at 20-25°C to obtain a slurry. The slurry was maintained under stirring at the same temperature for 3 hours. The slurry was diluted with water (50 ml) and the pH of the mixture was adjusted to 4 using cone. HC1. The mixture was stirred for 1 hour at the same temperature and filtered to obtain a solid. The solid was washed with water (3 x 40 ml), acetonitrile (4 x 40 ml), diisopropyl ether (3 x 40 ml) and dried at 60-70°C to obtain yellowish brown powder, Yield: 8 g (75%); Purity: more than 90%

Example 9:
Preparation of (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol(Decarboxylated product impurity)
In a 500 ml RB flask, IPA (150 ml) and 2-hydrazinoadenosine (10 g, 0.0336 mol) were charged to obtain a suspension. To this, malonaldehyde bis (dimethyl acetal) [8.3 g, 0.052 mol] was added to obtain a mixture. The mixture was stirred for 15 min and then heated to reflux (78-82°C). The mixture was maintained under stirring for 8 hours at the same temperature. The mixture was cooled to 15-20°C and stirred for 2-3 hours followed by filtration to obtain a solid. The solid was washed with IPA (10 ml) followed by diisopropylether (3x10 ml) and dried at 60°C to obtain yellowish brown powder. Yield: 6 g (60%); Purity: more than 90%.

We claim,
1. A process for preparation of Regadenoson comprising the steps of.
a) treating Ethyl l-{9-[(4S,2R,3R,5R)- 3,4-dihydroxy-5-(hydroxymefhyl) oxoIan-2-yl] -6-aminopurin-2-yl} pyrazol -4-carboxylate (Pyrazoloadenosine) with methyl amine at a temperature of about 0-5°C to obtain a mixture;
b) allowing the mixture to reach a temperature of about 15 to 25°C;
c) isolating Regadenoson from said mixture; and
d) optionally purifying said Regadenoson.

2. The process as claimed in claim 1, wherein said treatment in step a) is carried out using about 15-35 volumes of aqueous solution of methyl amine with respect to said Pyrazoloadenosine.
3. The process as claimed in claim 1. wherein said Regadenoson is purified by a process comprising the steps of,

a) treating Regadenoson with a first solvent selected from N.N-dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, acetone, tetrahydrofuran or dimethylsulfoxide to obtain a solution:
b) treating said solution with a second solvent selected from isopropanol, methanol, ethanol, n-propanol or butanol to obtain a mixture; and
c) isolating pure Regadenoson from said mixture.
4. The process as claimed in claim 1, wherein said Ethyl l-{9-[(4S,2R,3R,5R)-
3,4-dihydroxy-5-(hydroxymethyl) oxolan-2-yI]-6-aminopurin-2-yl}pyrazol-
4-carboxylate (Pyrazoloadenosine) is prepared by a process comprising the
steps of,
a) treating (4S,2R,3R,5R)-2-(6-amino-2-chloropurin-9-yI) -5- (hydroxy methyl)oxolane-3,4-diol (2-chloroadenosine) with about 29-31 fold molar excess of hydrazine hydrate at a temperature of about 20-30°C to obtain (4S,2R;3R,5R)-2- (6-amino-2-hydrazinyl-9H-purin-9-yl) -5-

(hydroxymethyl)oxolane-3.4-dio] (2-hydrazinoadenosine);
b) treating said 2-hydrazinoadenosine with about 1-1.5 moles of ethyl 2-formyl-3-oxopropionate to obtain Ethyl l-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-carboxylate (pyrazoloadenosine); and
c) optionally purifying said pyrazoloadenosine.

5. The process as claimed in claim 4. wherein purification of said pyrazoloadenosine is carried out using a solvent selected from N,N-dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, acetone, terrahydrofuran, dimethylsulfoxide or water.
6. A process for preparation of Regadenoson, wherein Regadenoson is purified using solvent selected from DMF, water or isopropanol.
7. The process as claimed in claim 3, wherein said Regadenoson is Form A (monohydrate) or Form C and wherein said Regadenoson is substantially free of compound selected from the group consisting of l-{9-[(4S,2R,3R,5R)-3;4-dihydroxy- 5- (hydroxymethyl)oxolan-2-yl]- 6 -amino purin-2-yl}pyrazol-4-carboxylic acid, (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]- 5- (hydroxymethyl) tetrahydrofuran-3,4-diol, 2-Hydrazinoadenosine, Ethyl 2-formyl-3-oxopropionate and hydrazine hydrate.
8. The process as claimed in claim 3, wherein l-{9-[(4S,2R.3R,5R)-3,4-dihydroxy- 5 -(hydroxymethyl)oxolan-2-yl]- 6- aminopurin-2-yl} pyrazol-4-carboxylic acid or (4S!2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3,4-diol is used as a reference marker or reference standard for determining the purity of a sample of Regadenoson or a pharmaceutical dosage form comprising Regadenoson.

9. (4S,2R,3R,5R)-2-[6-amino-2-(lH-pyrazol-l-yl)-9H-purin-9-yl]-5-(hydroxy methyl) tetrahydrofuran-3.4-diol.
10. A process for preparation of compound of claim 9, comprising treating (4SJ2R,3R,5R)-2-(6-amino-2-hydrazinyl-9H-purin-9-yl)-5-(hydroxymethyl) oxolane-3,4-diol with malonaldehyde bis(dimethylacetal).