REGADENOSON RELATED IMPURITIES AND A PROCESS FOR PREPARATION THEREOF
FIELD OF INVENTION
The present invention relates to impurities of Regadenoson and a process for preparation thereof. Further, the present invention also relates to characterization of impurities of regadenoson.
BACKGROUND OF THE INVENTION
Regadenoson (I) is an A2A adenosine receptor agonist which is used as a coronary vasodilator. It produces maximal hyperemia quickly and maintains the same for an optimal duration which is practical for radionuclide myocardial perfusion imaging.
US patent 6403567 and US patent 6642210 disclose a method for the preparation of Regadenoson from 2-Chloroadenosine. The method involves a preparation of 2-hydrazinoadenosine from 2-chloroadenosine which further react with ethyl diformyl acetate to yield 2-(4-Methoxycarbonylpyrazol-l-yl) adenosine. Methylamine was treated with 2-(4-Methoxycarbonylpyrazol-l-yl) adenosine to obtain Regadenoson.
The regulatory authorities such as the USFDA, European Medicine Agency (EMA) and Canadian Drug and Health Agency (CDHA) are emphasizing on the purity of the drug substance and they are incorporating impurity limits to the allowable levels present in API and finished drug product. This reveals the need and

scope of impurity profiling of drug substance in pharmaceutical research. Thus impurity profiling like identification, isolation and characterization are done and their threshold values comply with the limits set and specified by official bodies. The presence of these unwanted chemical compounds even in trace amount may influence the efficacy and the safety of the pharmaceutical product. The control and identification of impurities has been a critical issue in the pharmaceutical industry. Regadenoson related impurities profile was not studied extensively. It is mandatory for the manufacturer to identify and characterize the unknown impurities that are present in active pharmaceutical ingredient (API) at a level below 0.10%.
The patent US 6403567, Palle et al, Bioorganic & medicinal chemistry letters. 2002, 12(20), 2935-2939 and Gates, Chem Res Toxicol 2009, 22, 1747 describe a process for preparation of regadenoson as given below in scheme-I.

During the process development of a drug substance, it is necessary to identify all the possible process related, degradation and by product impurities in each stages and develop analytical method to monitor all the impurities and control those impurities to be well within the 1CH limit.
There is a need in the prior art to identify, characterize and synthesize the unknown impurities formed during the synthetic process development of regadenoson.

SUMMARY OF THE INVENTION
One embodiment of the present invention relates to 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A) (I).

Another embodiment of the present invention relates to a process for the preparation of 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A) (I) comprising;
a) reacting 2-haloadenine with hydrazine or its salts
b) isolation of 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A). Another embodiment of the present invention relates to methyl l-(6-amino-
9H-purin-2-yl)-lH pyrazole-4-carboxylate (Regadenoson impurity-B) (II).

Compound (II)
Another embodiment of the present invention relates to a process for the preparation of methyl l-(6-amino-9H-purin-2-yl)-lH pyrazole-4-carboxylate (Regadenoson impurity-B) (II) comprising;
a) treating l-[6-amino-9-((2R,3R,4S]5R)-3J4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-9H-purin-2-yl]-lH-pyrazole-4-carboxylic acid methyl ester with HC1 and
b) isolation.
Another embodiment of the present invention relates to 2-(N-Acetyl hydrazino) adenosine (Regadenoson impurity-C) (III).


Another embodiment of the present invention relates to a process for the preparation of 2-(N-Acetyl hydrazino) adenosine (Regadenoson impurity-C) (III) comprising;
a) treating 2-Hydrazino adenosine with acetic acid and
b) isolation.
Another embodiment of the present invention relates to (6-amino-9H-purin-2-yl)-N-methy!-lH- pyrazole-4-carboxamide (Regadenoson impurity-D) (IV).

Another embodiment of the present invention relates to a process for the preparation of (6-amino-9H-purin-2-yl)-N-methyl-IH-pyrazole-4-carboxamide (Regadenoson impurity-D) (IV) comprising;
a) treating regadenoson with hydrochloric acid and
b) isolation.
Another embodiment of the present invention relates to (6-amino-9H-purin-2-yl)-N, N-dimethyl-1 H-pyrazole-4-carboxamide (Regadenoson impurity-E) (V).


Another embodiment of the present invention relates to a process for the preparation of (6-amino-9H-purin-2-yl)-N, N-dimethyl-lH-pyrazole-4-carboxamide (Regadenoson impurity-E) (V) comprising;
a) treating2-{4-[(Dimethylamino) carbonyl]-IH- pyrazol-1-yl} adenosine with hydrochloric acid and
b) isolation.
Another embodiment of the present invention relates to l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-]H-pyrazole-4-carbaldehyde (regadenoson impurity-F) (VI).

Another embodiment of the present invention relates to a process for preparation of l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazole-4-carbaldehyde (Regadenoson impurity-F) (VI) comprising;
a) reacting 2-Hydrazinoadenosine with triformylmethane and
b) isolation .

Another embodiment of the present invention relates to 2-(6-amino-2-(2-((i-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-IH-pyrazol-4-yl)methylene)hydrazineyl)-9H-purin-9-yl)-5-(hydroxymethyl) tetrahydrofuran-3,4-diol (Regadenoson impurity-G) (VII).

Another embodiment of the present invention relates to a process for preparation of 2-(6-amino-2-(2-((l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazo!-4-yl)methylene)hydrazineyl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Regadenoson impurity-G) (VII) comprising;
a) reacting 2-Hydrazinoadenosine with triformylmethane and
b) isolation
Another embodiment of the present invention relates to substantially pure regadenoson comprising less than 0.10% each of impurity-A, Impurity-B, impurity-C, impurity-D, impurity-E, impurity-F and impurity-G as determined by HPLC.
BRIEF DESCRIPTION OF THE DRAWING:
Figure-1: Process related and degradation impurities of Regadenoson.

DETAILED DESCRIPTION OF PRESENT INVENTION:
One embodiment of the present invention relates to 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A) (I).
NH2 [j N NHNH2
Compound (I) Another embodiment of the present invention relates to a process for the preparation of 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A) (I) comprising;
a) reacting 2-haloadenine with hydrazine or its salts
b) isolation of 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A). The halogen in 2-haloadenine is selected from fluorine, chlorine, bromine and
iodine. The reaction is carried out in the temperature range of 30-70°C; preferably 40-60°C. Suitable sajt of hydrazine is used in the reaction. The salt is selected from HCI, HBr and HI salt. The step (a) reaction optionally involves use of organic solvent which is selected from polar and non-polar organic solvent.
Isolation of 2-hydrazinyl-9H-purin-6-amine (Regadenoson impurity -A) is carried out by methods known in the literature such as precipitation, distillation etc; preferably distillation.
Another embodiment of the present invention relates to methyl l-(6-amino-9H-purin-2-yl)-lH pyrazole-4-carboxylate (Regadenoson impurity-B) (III).
NHZ
Compound (II)

Another embodiment of the present invention relates to a process for the preparation of methyl l-(6-amino-9H-purin-2-yl)-lH pyrazole-4-carboxylate (Regadenoson impurity-B) (II) comprising;
a) treating l-[6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-
tetrahydrofuran-2-yl)-9H-purin-2-yl]-IH-pyrazole-4-carboxylic acid methyl
ester with HC1 and
b) isolation. i
The hydrolysis reaction is carried out in the temperature range of 40-120°C;
preferably 70-110°C. The step (a) reaction optionally involves use of organic solvent which is selected from polar and non-polar organic solvent. 0.05N to 0.5N Hydrochloric acid is used in step (a); preferably 0.1 N HCI.
Methyl l-(6-amino-9H-purin-2-yl)-lH pyrazole-4-carboxyIate (Regadenoson impurity-B) is isolated by methods known in the literature such as precipitation, distillation etc; preferably precipitation.
Another embodiment of the present invention relates to 2-(N-Acetyl '
hydrazino) adenosine (Regadenoson impurity-C) (III). !
NH2
«NYS «
HO...>-^ OH
Compound (III)
Another embodiment of the present invention relates to a process for the preparation of 2-(N-Acetyl hydrazino) adenosine (Regadenoson impurity -C) (III) comprising;
a) treating 2-Hydrazino adenosine with acetic acid and
b) isolation.

The reaction of 2-Hydrazino adenosine with acetic is carried in the temperature range of 40-IOO°C; preferably 60-80°C. The reaction is carried out in the presence of organic solvent such as alcohol, amide and the like or a water mixture thereof. Preferably, the solvent is alcohol which is selected from methanol, ethanol, isopropanol and the like.
The impurity-C is isolated from the reaction mixture by methods known in the literature such as precipitation, distillation etc; preferably distillation under vacuum.
Another embodiment of the present invention relates to (6-amino-9H-purin-2-yl)-N-methyl-lH- pyrazoIe-4-carboxamide (Regadenoson impurity-D) (IV).
NHZ
H N N^3 0 H
Compound (IV)
Another embodiment of the present invention relates to a process for the preparation of (6-amino-9H-purin-2-yl)-N-methyl-lH-pyrazole-4-carboxamide (Regadenoson impurity-D) comprising;
c) treating regadenoson with hydrochloric acid and
d) isolation.
Regadenoson is treated with hydrochloric acid in the temperature range of 60-120°C; preferably 90-110°C. The impurity-D is isolated from the reaction mixture by methods known in the literature such as precipitation, distillation etc.
Another embodiment of the present invention relates to (6-amino-9H-purin-2-yl)-N, N-dimethyl-lH-pyrazole-4-carboxamide (Regadenoson impurity-E) (V).

NH2
Compound (V)
Another embodiment of the present invention relates to a process for the preparation of (6-amino-9H-purin-2-yl)-N, N-dimethyl-]H-pyrazole-4-carboxamide (Regadenoson impurity-E) (V) comprising;
c) treating 2-{4-[(Dimethylamino) carbonyl]-]H- pyrazol-1-yl} adenosine with hydrochloric acid and
d) isolation.
The reaction of 2-{4-[(Dimethylamino) carbonyl]-lH- pyrazol-1-yl} adenosine with hydrochloric acid is carried out in the temperature range of 60-120°C; preferably 90-110°C. Isolation of(6*amino-9H-purin-2-yl)-N,N-dimethyl-lH-pyrazole-4-carboxamide (Regadenoson impurity-E) is carried out by methods known in the literature such as precipitation, distillation etc.
Another embodiment of the present invention relates to l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazole-4-carbaldehyde (regadenoson impurity-F) (VI).
OH
Compound (VI) Another embodiment of the present invention relates to a process for preparation of I-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-

yl)-9H-purin-2-yl)-lH-pyrazole-4-carbaldehyde (Regadenoson impurity-F) (VI) comprising;
c) reacting 2-Hydrazinoadenosine with triformylmethane and
d) isolation.
The reaction of 2-Hydrazinoadenosine with trimethylformate is carried in a mixture of alcohol and acetic acid. The alcohol is selected from methanol, ethanol, propanol, isopropanol and the like; preferably methanol. The ratio between alcohol and acetic acid is 1:1. The mole ratio of triformylmethane to 2-Hydrazinoadenosine is between 4 and 8 mole; preferably 6 mole.
Isolation of I-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazole-4-carbaldehyde (Regadenoson impurity-F) is carried out by methods known in the literature such as precipitation, distillation etc.
Impurity-F is optionally purified by using ether solvent such as diethyl ether, tertiary butyl methyl ether, and the like. Further, the mixture of haloalkane and alcohol or aqueous mixture thereof used for purification. The haloalkane solvent is selected from dichloromethane, dichloroethane and the like and the alcohol solvent is selected from methanol, ethanol, propanol and the like. Further embodiment of the present invention provides a process for purification of Impurity-F by chromatography using silica gel and 5% ethyl acetate /hexane as an eluent. The pure form of impurity-F is isolated methods known in the literature such as precipitation, distillation etc; preferably distillation.
Another embodiment of the present invention relates to 2-(6-amino-2-(2-((l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazol-4-yl)methyIene)hydrazineyl)-9H-purin-9-yl)-5-(hydroxymethyI) tetrahydrofuran-3,4-diol (Regadenoson impurity-G) (VII).

Another embodiment of the present invention relates to a process for preparation
of 2-(6-amino-2-(2-(( 1 -(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl)
tetrahydrofuran-2-yl)-9H-purin-2-yl)-IH-pyrazol-4-yl)methylene)hydrazineyl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Regadenoson impurity-G) (VII) comprising;
c) reacting 2-Hydrazinoadenosine with triformylmethane and
d) isolation
The reaction of 2-Hydrazinoadenosine with trimethylformate is carried in a mixture of alcohol and acetic acid. The alcohol is selected from methanol, ethanol, propanol, isopropanol and the like; preferably methanol. The ratio between alcohol and acetic acid is 1:1. The mole ratio of triformylmethane to 2-Hydrazinoadenosine is between 1 and 3.5 mole; preferably 1.85 mole.
Isolation of l2-(6-amino-2-(2-((l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazol-4-yl)methylene)hydrazineyl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Regadenoson impurity-G) is carried out by methods known in the literature such as precipitation, distillation etc.
Impurity-G is optionally purified by using ether solvent such as diethyl ether, tertiary butyl methyl ether, and the like. The pure form of impurity-G is isolated by methods known in the literature such as precipitation, distillation etc.
The present invention is useful to develop analytical method in order to monitor all the impurities formed during the process for preparation of regadenoson and during the degradation of regadenoson and control those impurities to be well within thelCH limit.

Another embodiment of the present invention relates to substantially pure regadenoson comprising less than 0.10% each of lmpurity-A, Impurity-B, impurity-C, impurity-D, impurity-E, impurity-F and impurity-G as determined by HPLC.
Another embodiment of the present invention relates to substantially pure regadenoson comprising less than 0.05% of impurity-G (VII).
Another embodiment of the present invention relates to a pharmaceutical composition comprising regadenoson having less than 0.10% each of Impurity-A, Impurity-B, impurity-C, impurity-D, impurity-E, impurity-F and impurity-G as determined by HPLC. The pharmaceutical composition is preferable injectable composition such as lyophilized powder for reconstitution and a solution for injection.
The following example further describes certain specific embodiments of the invention and demonstrates the practice and advantages thereof. It is to be understood that the examples are given by way of illustration only and are not intended to limit the scope of the invention in any manner.
EXPERIMENTS Example-1: Synthesis of 2-hydrazinyl-9H-purin-6-amine (Impurity-A)
2-Chloroadenine (2.0 g, 12.0 mmoles) was added to 30 ml of 50% hydrazine hydrate. Then the reaction mass was heated to 50-55°C. After completion of the starting material monitored by TLC, the reaction mass was evaporated and kept for drying under high vacuum at 50-55°C to yield a white color solid of 2-hydrazinyl-9H-purin-6-amine. Yield: 95%
1H-NMR (DMSO-d6, 400 MHz,): SH 8.14 (1H, s, C6-H), 8.40 (1H, brs, imidazole NH), 7.69 (2H, s, -NH2).13C-NMR (DMSO-d6, 100 MHz): 8C 162.07 (C-2), 152.74 (C-8 & C-9), 155.71 (C-4), 140.47 (C-6). MS (ESI) (C5H7N7) 166.0 ([M+H]+). IR (KBr, cm-1): 3278.99(NH2), 3I16.97(NH), 3008.95(=CH), 2808.36(C-H), 1680.00 (ON), 1612.49 (C=C) and 1384.89 (C=N). ExampIe-2: Synthesis of methyl l-(6-amino-9H-purin-2-yl)-lH pyrazole-4-carboxylate (Impurity-B)

l-^-Amino^-^R^R^S^R^^-dihydroxy-S-hydroxymethyl-tetrahydro-furan-2-yl)-9H-purin-2-yl]-lH-pyrazole-4-carboxylic acid methyl ester (350 mg, 0.9 mmoles) dissolved in 30ml of 0.1N HC1 solution and heated to 100-105°C. The reaction mixture become clear and after some time white color solid was thrown out from the reaction mass. The stirring was continued for 4 hours at I00-105°C. The reaction mass was cooled to room temperature and a thick white solid formed was filtered off. The filtered solid was washed with water. The solid was dried under high vacuum to yield methyl 1 -(6-amino-9H-purin-2-yl)-1H pyrazole-4-carboxylate. Yield: 95%.
1H-NMR (DMSO-d6, 400 MHz,): 5H 8.93 (IH, s, C6-H), 8.73 (1H, s, C12-H), 8.16 (1H, s, C14-H), 8.09 (1H5 brs, imidazole NH), 5.61(2H, brs, NH2), 3.81 (3H, s, 0CH3).I3C-NMR (DMSO-d6, 100 MHz): SC 172.24 (C-15), 162.32 (C-8), 154.39 (C-2), 151.08 (C-4), 150.85 (C-9), 142.36 (C-6), 139.88 (C-12), 132.11 (C-14), 115.66 (C-13), 51.59 (C-16). MS (ESI) (CI0H10C1N7O2) 260.! [M-HC1+H]+.1R (KBr, cm-l): 3361.93 & 3197.98 (NH2), 1722.43 (C=0), 1670.3 5 (C=N), 1568.13 (C=C)& 1267.23 (C-N). Example-3: Synthesis of 2-(N-Acetyl hydrazino) adenosine (Impurity-C)
2-Hydrazino adenosine (100 mg, 3.36 mmoles) is heated in a mixture of acetic acid (7.5 ml) and methanol (7.5 ml) at 80-85°C for 8 hrs. The reaction mixture becomes clear. After completion of reaction, distill out the solvent mixture completely under vacuum. The obtained semi solid was dried under high vacuum yield the title compound. Yield: 95%
lH-NMR(DMSO-d6, 300 MHz,): 5H 8.05 (H, s, C6-H), 7.98 (IH, brs, -NH-CO), 6.96 (2H, brs, -NH2), 5.74 (IH, d, C-10H), 4.54-4.58(^^,0-11^, 4.14-4.16 (IH, m, C-I2H), 3.89-3.90 (IH, m, C-13H), 3.49-3.66 (2H, m, C-14H), 1.90 (3H, s, C-16H).
13C-NMR (DMSO-d6, 75 MHz): SC 169.50 (C-15), 160.29 (C-8), 156.49 (C-4), 151.20 (C-2) , 137.82 (C-6) , 115.13 (C-9), 87.78 (C-10), 85.86 (C-13), 73.66 (C-11), 71.10 (C-12), 62.28 (C-14), 20.64 (C-16). MS (ESI) (C12H17N705) 340.2
_ _ — m . . ■— > ■ . . ft -T- * t— I U it s^. * ^ r\ A n -i*"1!. A (~
([M+H]+).1R (KBr, cm-1): 3271.27 (NH2), 2935.66(C-H), 1645.28(C=0), I598.99(C=C)& I267.23(C-N).
Example-4: Synthesis of (6-amino-9H-purin-2-yl)-N-methyl-lH- pyrazole-4-carboxamide (Impurity-D)
Regadenoson (500 mg, 1.28 mmoles) was dissolved in 50ml of O.IN HCI solution and heated to 100-105°C. The reaction mixture become clear and after some time white color solid was thrown out from the reaction mass. The stirring was continued for 4 hours at the same temperature, then the reaction mass was cooled to room temperature and white solid formed was filtered off and washed with water followed by dried under vacuum gave the title compound. Yield: 90%
1H-NMR (DMSO-d6, 400 MHz,): SH 8.96 (IH, s, C-6H), 8.64 (IH, s, C-12H), 8.39 (IH, q, 16 NH), 8.10 (IH, s, C-14H), 7.96 (2H, s,-18NH2) 2.75 (3H, d, C-17H). I3C-NMR (DMSO-d6, 100 MHz): 8C 161.64 (C-15), 154.72 (C-2), 151.22 (C-4), 151.02 (C-9), 141.11 (C-6), 139.92(C-12), 129.51 (C-14), 120.21 (C-8), 113.36 (C-13), 25.54 (C-17). MS (ESI) (C10H11CIN80) 259.1 ([M+H-HC1]+).IR (KBr, cm-1): 3325.28 (N-H), 3151.69 (N-H), 1680.00 (C=0), 1647.2I(C=N), 1583.56(C=C), 1288.45 (C-N).
Example-5: Synthesis of (6-amino-9H-purin-2-yl)-N, N-dimethyl-lH-pyrazole-4-carboxamide (Impurity-E)
2-{4-[(Dimethylamino) carbonyl]-lH- pyrazol-1-yl} adenosine (500 mg, 1.28 mmoles) was dissolved in 50 ml of 0.1N HCI solution and heated to I00-I05°C. The reaction mixture become clear and after some time white color solid was thrown out from the reaction mass. The stirring was continued for 4 hours at 100-105°C. The reaction mass was cooled to room temperature and a thick white solid formed was filtered off. The filtered solid was washed with water and dried under high vacuum resulted the impurity-E. Yield: 90%.
IH-NMR (DMSO-d6, 400 MHz,): SH 8.74 (IH, s, C-6H), 8.33 (IH, s, C-I2H), 8.01 (IH, s, C-14H), 7.73 (IH, brs, NH), 4.24 (3H, brs, -NH &-NH2), 2.99 (3H, s, CH3),3.20(3H,s,CH3).

I3C-NMR(DMSO-d6, 100 MHz): 5C 162.85 (C-l5), 155.32 (C-2), 150.56 (C-4), 141.98 (C-9), 140.05 (C-6), 131.73 (C-12), 130.08 (C-14), 118.66 (C-8), 40.67(C-17), 40.46(C-18). MS (ESI) (C11HI2N80) 271.0 [M-H]+.IR (KBr, cm-1): 3377.36 & 3120.82 (NH2), 2893.22 (CH), 1662.64 (CO-N) 1608.63 (OC), 1556.55 (C=N), 1166.93 (C-N).
Example-6: Synthesis of l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-lH-pyrazole-4-carbaldehyde (Impurity-F)
To a suspension of 2-Hydrazinoadenosine (0.53 g, 1.8mmol) in a mixture of MeOH-acetic acid (8.0 ml, 1:1 ratio), triformylmethane (0.6g, 6.0mmol) was added under nitrogen at room temperature. The resultant reaction mixture was stirred at 25-30°C for 2h. After completion of the reaction was monitored by HPLC, the reaction mixture was filtered and the solid washed with 5 ml of ethanol and 2.5ml of MTBE. The obtained solid was stirred with 5.3ml of MTBE at room temperature for 15 minutes, filtered and dried the material under high vacuum for lh at 40°C to gave pale yellow colour solid (Yield: 0.74g). The obtained crude compound was stirred with 5% methanol/MDC (15ml) at 25-30°C for 25minutes filtered the solid (yield: 0.46g). The crude compound obtained was purified by column chromatography using silica gel and 5% ethyl acetate/hexane used an eluent. Collect the pure fractions and solvent was evaporated and dried under high vacuum resulted pale yellow colour solid compound. Yield: 45%
1H-NMR (DMSO-d6, 400 MHz,): 5H 9.96 (IH, s, C-16H), 9.18 (IH, s, C-5H), 5.94 (IH, d, C-8H), 8.43 (IH, s, C-13H), 8.21 (IH, s, C-15H), 7.87 (2H, brs, NH2), 5.50 (IH, d, b-OH), 5.24 (IH, d, c-OH), 5.02 (IH, t, a-OH), 4.60-4.65 (IH, m, C11H), 4.16-4.19 (IH, m, C10H), 3.94-3.97 (IH, m, C9H), 3.55-3.71 (2H, m, CI2H),.
13C-NMR (DMSO-d6, 100 MHz): SC 185.5 (C-l6), 156.43 (C-3), 150.15 (C-2), 150.05 (C-6), 140.76 (C-5), 140.30 (C-l3), 134.13 (C-l5), 124.82 (C-14), 118.18(C-7), 87.12(C-8), 85.66(C-ll),73.58(C-9), 70.42(C-10), 61.41(C-12). MS (ESI) (C14H15N705) 362.12 [M-H]+.

]R (KBr, cm-1): 3392.79 (NH), 3184.48 (OH), 2926.01(CH),1666.50 (C=0) 1610.56 (C=C),
Example-7: Synthesis of 2-(6-amino-2-(2-((l-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-y!)-9H-purin-2-yl)-lH-pyrazol-4-yl)methylene) hydrazineyl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Impurity-G)
To a suspension of 2-Hydrazinoadenosine (0.5 g, 1.68mmol) in a mixture of MeOH-acetic acid (7.5 ml, 1:1 ratio), triformylmethane (0.18g, 1.85mmol) was added under nitrogen at room temperature. The resultant reaction mixture was stirred at 80-85oC for 4h. After completion of the reaction, the reaction mixture was cooled to room temperature and stirred for 30minutes. The solid formed was filtered and washed with 5 ml of ethanol and 2.5ml of MTBE. The obtained solid compound was stirred with 5.0ml of MTBE at room temperature and the resultant solid obtained was filtered and dried under high gave pale yellow colour solid compound (Yield: 90%)
1H-NMR (DMSO-d6, 400 MHz,): 611 10.58 (IH, bs, 17-NH), 8.39 (IH, s, C-5H, 8.21 (IH, s, C-21H),), 8.82 (IH, s, C-16H),8.0 & 8.06(2H, s, C13 & 15H), ), 7.72 (2H, brs, 4-NH2),), 7.06 (2H, brs, 20-NH2),5.94 (IH, d, C-8H),5.77(lH,d,C-24H), 5.55 (IH, d, a-OH), 5.42-5.43 (2H, m, b-OH & a'-OH), 5.03-5.06 (IH, t, c'-OH),4.61-4.75 (2H, m, C11& 27H), 4.19-4.23 (2H, m, C10 & 26H), 3.97-4.01(2H, m, C9 &25H), 3.55-3.71 (2H, m, CI2H)
13C-NMR (DMSO-d6, 100 MHz): 6C 156.38 (C-3), 156.20 (C-19), 150.72 (C-2 & C-18), 150.33 (C-22), 140.31 (C-16), 139.83 (C-5), 138.17 (C-21), 132.56 (C-15), 127.48(C-13), 120.68(C-14), 117.66(C-7),115.45(C-23), 88.18(C-8), 87.01(C-24), 86.32(C-ll),85.63(C-27), 73.61(C-9), 72.81(C-25), 71.18(C-10), 70.56(C-26), 62.12(C-12),61.47(C-28). MS (ESI) (C24H28N1408) 641.23 [M-H]+.
IR(KBr,cm-l):3332.99(NH), 3265.49(OH),2929.87(CH), 1595.13(C=N),1485.19 (C=C) 1396.46(C-N),

We Claim:
1. Substantially pure regadenoson comprising less than 0.10% each of Impurity-A, Impurity-B, impurity-C, impurity-D, impurity-E, impurity-F and impurity-G as determined by HPLC.
2. A compound of formula (II) (Regadenoson impurity-B):

3. A compound of formula (III) (Regadenoson impurity-C):

4. A process for the preparation of compound of formula (III) (Regadenoson
impurity-C) comprising;
a) treating 2-Hydrazino adenosine with acetic acid and
b) isolation.

5. A compound of formula (V) (Regadenoson impurity-E):

Compound (V)
6. A process for the preparation of compound of formula (V) (Regadenoson
impurity-E) comprising;
a) treating 2-{4-[(Dimethylamino) carbonyl]-] H- pyrazol-1-yl} adenosine with hydrochloric acid and
b) isolation.
7. A compound of formula (VI) (Regadenoson impurity-F)
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8. A process for preparation of compound of formula (VI) (Regadenoson
impurity-F) comprising
a) reacting 2-Hydrazinoadenosine with triformylmethane and
b) isolation
wherein mole ratio of triformylmethane to 2-Hydrazinoadenosine is between 4 and 8 mole.

9. A compound of formula (VII) (Regadenoson impurity-G):

10. A process for the preparation of compound of formula (VII) (Regadenoson
impurity-G) comprising;
a) reacting 2-Hydrazinoadenosine with triformylmethane and
b) isolation
wherein mole ratio of triformylmethane to 2-Hydrazinoadenosine is between 1 |
and 3.5 mole.
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