FORM 2
THE PATENT ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
Title of the invention
"NOVEL POLYMORPHIC FORMS OF REGADENOSON."
Enaltec Labs Pvt. Ltd. an Indian Company, having its Registered Office at 17thFloor, Kesar Solitaire, Plot No.5 Sector-19, Sanpada, Navi Mumbai Maharashtra, India. Pin Code: 400705
1. The following specification particularly describes the invention and the manner in which it is to be performed.

NOVEL POLYMORPHIC FORMS OF REGADENOSON
FIELD OF THE INVENTION:
The present invention relates to novel polymorphic forms of regadenoson. The present invention further relates to processes of preparing novel polymorphic forms of regadenoson and pharmaceutical composition thereof.
BACKGROUND OF THE INVENTION:
Regadenoson is chemically adenosine, 2-[4-[(methylamino) carbonyl]-lH-pyrazol-l-yl] -or l-(6-amino-9-((3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-9H-purin-2-yl)-N-methyl-lH-pyrazole-4-carboxamide and is known from U.S. Patent No. 6,403,567 and is represented by compound of structural formula I.

Regadenoson has been approved in USA as regadenoson monohydrate and sold in market under the proprietary name LEX1SCAN. It is a pharmaceutical stress agent indicated for radionuclide myocardial perfusion imaging (MP1) in patients unable to undergo adequate exercise stress.

U.S. Patent No. 6,403,567 does not describe crystallization of regadenoson compound of structural formula I.
U.S. Patent No. 7,671,192 discloses three different crystalline forms, referred to herein as monohydrate Form A, Form B, Form C, and an amorphous form of regadenoson compound of structural formula I.
Regadenoson Form A is monohydrate form and can be produced by crystallizing regadenoson from protic solvents, for example ethanol or ethanol/water mixtures, or from a polar solvent, for example dimethylsulfoxide/water.
Regadenoson Form B can be produced by evaporating under vacuum a solution of regadenoson in trifluoroethanol at ambient temperatures. The X-ray analysis of the crystals was distinctly different from any other polymorph, but it was difficult to determine its constitution, as the X-ray analysis gave disordered broad peaks, and the polymorph contained varying amounts of water. It was found to be difficult to reliably reproduce the preparation of this polymorph.
Regadenoson Form C is an acetonitrile solvate and can be produced by slurrying regadenoson in acetonitrile for a long period of time at 60° C. The X-ray analysis of the crystals was distinctly different from any other polymorph. Regadenoson Polymorph C was shown to be a variable hydrate, which, at elevated temperatures, desolvates to an unstable form.
Regadenoson amorphous form can be produced by heating Regadenoson Form A at a temperature of up to 200° C. This polymorph is unstable in the presence of atmospheric moisture, forming variable hydrates.
The present applicant of the patent has surprisingly found that regadenoson can exist in anhydrous form and different hydrated forms. Thus the invention relates to novel

polymorphic forms of regadenoson i.e. anhydrous regadenoson, regadenoson hemihydrate, regadenoson sesquihydrate, and regadenoson dihydrate.
SUMMARY OF THE INVENTION:
A first aspect of the invention is to provide anhydrous form of regadenoson herein after referred as crystalline form D.
A second aspect of the present invention is to provide hemihydrate form of regadenoson herein after referred as crystalline form E.
A third aspect of the present invention is to provide sesquihydrate form of regadenoson herein after referred as crystalline form F.
A fourth aspect of the present invention is to provide dihydrate form of regadenoson herein after referred as crystalline form G.
Another aspect of the present invention is to provide acid addition salts of regadenoson.
Another aspect of the present invention is to provide acid addition salts of regadenoson with an organic acid.
Another aspect of the present invention is to provide acid addition salts of regadenoson with an inorganic acid.
The acid addition salts of regadenoson may be selected from the group comprising of regadenoson hydrochloride, regadenoson hydrobromide, regadenoson maleate, regadenoson fumarate, regadenoson succinate, regadenoson sulfate, regadenoson acetate, regadenoson mesylate, regadenoson tartrate or regadenoson lactate.

Another aspect of the present invention is use of acid addition salts of regadenoson in purification of regadenoson.
Another aspect of the present invention is use of acid addition salts of regadenoson in the preparation of amorphous form of regadenoson.
Another aspect of the present invention present invention is to provide processes for the preparation of crystalline form D of regadenoson.
The regadenoson form D may be prepared by azeotropic distillation of organic solvent from the solution of regadenoson in an organic solvent.
Another aspect of the present invention present invention is to provide processes for the preparation of crystalline form E of regadenoson.
Another aspect of the present invention present invention is to provide processes for the preparation of crystalline form F of regadenoson.
Another aspect of the present invention present invention is to provide processes for the preparation of crystalline form G of regadenoson.
Another aspect of the present invention present invention is to provide processes for the preparation of amorphous form of regadenoson.
Another aspect of the present invention present invention is to provide pharmaceutical composition comprising crystalline form D, crystalline form E, crystalline form F, crystalline form G or amorphous form of regadenoson.
The amorphous form of regadenoson may be prepared by spray-drying or freeze-drying the solution of regadenoson in an organic solvent.

The amorphous form of regadenoson may be prepared by concentrating the solution of regadenoson in an organic solvent under reduced pressure.
DETAIL DESCRIPTION OF THE INVENTION:
Regadenoson used for present invention may be formed by methods disclosed in the art such as those described in U.S. Patent Nos. 6,403,567; 7,671,192 which are incorporated herein by reference only.
An anhydrous crystalline form D of regadenoson may be characterized by having moisture content less than 0.1% wt/wt.
A crystalline form E of regadenoson hemihydrate may be characterized by having moisture content in the range of 2% wt/wt to 2.5% wt/wt.
A crystalline form F of regadenoson sesquihydrate may be characterized by having moisture content in the range of 6% wt/wt to 7.5% wt/wt
A crystalline form G of regadenoson dihydrate may be characterized by having moisture content in the range of 8.0% wt/wt to 9.0% wt/wt..
The crystalline form D of regadenoson may be formed by azeotropic distillation of organic solvent from the solution of regadenoson in an organic solvent.
The solution of regadenoson in an organic solvent having moisture content in the range of 0.5% weight / weight to 8% weight / weight may be formed by dissolving regadenoson in an organic solvent at a temperature in the range of-10° to 60°C.
The examples of organic solvent may include but not limited to alcohol solvents.

The examples of alcohol solvents may include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, pentanol or mixture(s) thereof.
The crystalline form D of regadenoson may be isolated by the steps filtration, centrifugation, washing, drying or the combinations thereof.
The crystalline form E of regadenoson may be isolated from the solution of regadenoson in an organic solvent having moisture content in the range of 2% wt/wt to 2.5% wt/wt at a temperature in the range of-5°C to 75°C.
The examples of organic solvent may include but not limited to polar protic solvents.
The example of polar protic solvents is selected from the group comprising of methanol, ethanol, n-propanol, isopropanol, n-butanol or mixture(s) thereof.
The crystalline form E of regadenoson may be isolated by the steps filtration, centrifugation, washing, drying or the combinations thereof.
The crystalline form F of regadenoson may be formed by treating a solution of regadenoson in an organic solvent with water at a temperature in the range of 0°C to 40°C.
The solution of regadenoson in an organic solvent may be formed by dissolving regadenoson in an organic solvent at a temperature in the range of 20° to 60°C.
The water used for the preparation of crystalline form F of regadenoson may be in the range 1.5 moles to 1.8 moles of regadenoson.
The examples of organic solvent may include but not limited to alcohol solvents.

The examples of alcohol solvents may include methanol, ethanol, n-pfopanol, isopropanol, n-butanol, isobutanol, pentanol or mixture(s) thereof.
The crystalline form F of regadenoson may be isolated by the steps filtration, centrifugation, washing, drying or the combinations thereof.
The crystalline form G of regadenoson may be formed by treating a solution of regadenoson in an organic solvent with water at a temperature in the range of 0°C to 80°C.
The solution of regadenoson in an organic solvent may be formed by dissolving regadenoson in an organic solvent at a temperature in the range of 0° to 80°C.
The water used for the preparation of crystalline form F of regadenoson may be in the range 2.0 moles to 2.5 moles of regadenoson.
The examples of organic solvent may include but not limited to alcohol solvents.
The examples of alcohol solvents may include methanol, ethanol, n-pfopanol, isopropanol, n-butanol, isobutanol, pentanol or mixture(s) thereof.
The crystalline form G of regadenoson may be isolated by the steps filtration, centrifugation, washing, drying or the combinations thereof.
An amorphous form of regadenoson may be prepared by spray-drying or freeze-drying the solution of regadenoson in an organic solvent.
A solution of regadenoson in organic solvents may be prepared by dissolving crystalline regadenoson in an organic solvent at a temperature in the range of 0°C to 75°C.

Alternatively, such a solution may be obtained directly from a reaction in which regadenoson is formed.
Examples of organic solvent may include ketones, alcohols, esters, nitriles, halogenated aliphatic hydrocarbon solvents, ethers or mixtures thereof.
The ketone solvents may be selected from the group comprising of acetone, methyl ethyl ketone, methyl isobutyl ketone, dibutyl ketone, diethyl ketone, dipropyl ketone, diisopropyl ketone, methyl butyl ketone, methyl propyl ketone, methyl isopropyl ketone, &k?j\ ".s^y-TOp^ ketone- os mvxtaire^s) \h«.rectf.
The alcohol solvents may be selected from the group comprising of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butajiol, pentanol or mixture(s) thereof.
The ester solvents may be selected from the group comprising of ethyl acetate, propyl acetate, isopropyl acetate; butyl acetate, tertiary butyl acetate, pentyl acetate or mixture(s) thereof.
The nitrile solvents may be selected from the group comprising of acetonitrile, propionitrile or mixture(s) thereof.
The halogenated aliphatic hydrocarbon solvents may be selected from the group comprising of dichloromethane, dichloroethane, chloroform, carbon tetrachloride or mixture(s) thereof.
The ether solvents may be selected from the group comprising of tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, dibutyl ether, methyl tertiary butyl ether, methyl ethyl ether, methyl isobutyl ether or mixture(s) thereof.
The solvent may be removed from the solution by a technique which includes, for example, spray drying and freeze drying.

In one aspect, regadenoson amorphous form may be recovered from the solution using a spray drying technique. A Mini-Spray Dryer (Model: Buchi 190, Switzerland) can be used. The Buchi 190 Mini-Spray Dryer operates on the principle of nozzle spraying in a parallel flow, i.e., the sprayed product and the drying gas flow in the same direction. The drying gas can be air or inert, gases such as nitrogen, argon and carbon dioxide.
In another aspect, regadenoson amorphous form may be recovered from the solution using a freeze drying technique. A freeze dryer (Model; Virtis Genesis SQ Freeze Dryer) can be used in this technique. The Virtis Genesis SQ Freeze Dryer operates on the principle of lyophilization, i.e., a process of stabilizing initially wet materials (aqueous solution or suspensions) by freezing them, then subliming the ice while simultaneously desorbing some of the bound moisture (primary drying). Following removal of the ice, desorption may be continued (secondary drying). This process may be carried out under vacuum.
The spray drying may be accomplished using a spray dryer which operates on the principle of nozzle spraying in a parallel flow, i.e., the sprayed product and the drying gas flow in the same direction. The drying gas can be air or one or more inert gases such as nitrogen, argon, and carbon dioxide. Moreover, the product obtained may be further or additionally dried to achieve the desired moisture values. For example, the product may be further or additionally dried in a tray drier, dried under vacuum and/or in a Fluid Bed Dryer.
The acid addition salts of regadenoson may be selected from the group comprising of regadenoson hydrochloride, regadenoson hydrobromide, regadenoson maleate, regadenoson fumarate, regadenoson succinate, regadenoson sulfate, regadenoson acetate, regadenoson mesylate, regadenoson tartrate or regadenoson lactate.
The salts of regadenoson compound of structural formula I with hydrochloric acid, hydrobromic acid, maleic acid, fumaric acid, succinic acid, sulphuric acid, acetic acid,

methanesulfonic acid, tartaric acid or lactic acid may be formed by reacting regadenoson compound of structural formula I with hydrochloric acid, hydrobromic acid, maleic acid, fumaric acid, succinic acid, sulphuric acid, acetic acid, methanesulfonic acid, tartaric acid or lactic acid in an organic solvent at a temperature in the range of 0°C to 30°C for a period of 30 minutes to 8 hours.
The examples of organic solvent may include but not limited to alcohol solvents.
The examples of alcohol solvents may include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, pentanol or mixture(s) thereof.
The salts of regadenoson compound of structural formula I with hydrochloric acid, hydrobromic acid, maleic acid, fumaric acid, succinic acid, sulphuric acid, acetic acid, methanesulfonic acid, tartaric acid or lactic acid may be isolated by the steps of filtration, centrifugation, washing drying and the combinations thereof.
The salts of regadenoson compound of structural formula I with hydrochloric acid, hydrobromic acid, maleic acid, fumaric acid, succinic acid, sulphuric acid, acetic acid, methanesulfonic acid, tartaric acid or lactic acid may be further converted into substantially pure regadenoson compound of structural formula I.
The salts of regadenoson compound of structural formula 1 with hydrochloric acid, hydrobromic acid, maleic acid, fumaric acid, succinic acid, sulphuric acid, acetic acid, methanesulfonic acid, tartaric acid or lactic acid may be neutralized with inorganic base to get substantially pure regadenoson compound of structural formula I.
The examples of inorganic base may include alkali metal hydroxide such as lithium hydroxide, sodium hydroxide or potassium hydroxide, alkali earth metal hydroxide such as calcium hydroxide, magnesium hydroxide, alkali metal carbonate such as lithium carbonate, sodium carbonate, potassium carbonate, alkali earth metal carbonate such as

calcium carbonate, magnesium carbonate, alkali metal bicarbonate such as sodium bicarbonate, potassium bicarbonate.
The term "substantially pure regadenoson" means regadenoson having HPLC purity greater than 99.0%.
EXAMPLES:
In the following example, the preferred embodiment of the present invention is described only by way of illustrating the processes of the invention. However, these are not intended to limit the scope of the present invention in any way.
Example no. 1 Preparation of Regadenoson anhydrous crystalline form D.
Regadenoson (l0gm) having moisture content 2% was dissolved in an ethanol solvent
(100ml) at 50°C and the resulting solution was distilled and azeotrope was isolated for 2
hours. The resulting solution was cooled to -5°C and resulting solids were filtered,
washed with anhydrous ethanol (moisture content 0.001% wt/wt) and dried at 50°C under
reduced pressure.
Yield: 9 gm
Moisture content: 0.002% wt/wt
Example no. 2 Preparation of Regadenoson hemihydrate crystalline form E.
The anhydrous regadenoson (lOgm) was dissolved in butanol (75ml) at 60°C and the
resulting solution was stirred for 12 hours and then it was cooled to 20°C and the
resulting solids were filtered, washed with butanol (15ml) and dried at 40°C under
reduced pressure.
Yield: 7.7gm
Moisture Content: 2.30% wt/wt

Example no. 3 Preparation of Regadenoson sesquihydrate crystalline form F.
The anhydrous regadenoson (l00gm) was dissolved in isopropanol (1250ml) at 40°C and
the resulting solution was added water (5ml) and it was stirred for 6 hours at 0°C. The
resulting solids were filtered, washed with isopropanol (120ml) and dried at 40°C under
reduced pressure.
Yield: 86gm
Moisture content: 6.5% wt/wt
Example no. 4 Preparation of Regadenoson dihydrate crystalline form G.
The anhydrous regadenoson (lOOgm) was dissolved in isobutanol (1000ml) at 60°C and
the resulting solution was added water (7.5ml) and it was stirred for 9 hours at 25°C. The
resulting solids were filtered, washed with isobutanol (50ml) and dried at 40°C under
reduced pressure.
Yield: 92gm
Moisture content: 8.5% wt/wt
Example no. 5 Preparation of Regadenoson amorphous form
Crystalline regadenoson (15 gm) was dissolved in ethanol (100 ml) at 40°C. The clear
solution was subjected to spray drying in a mini spray dryer at an inlet temperature of
60°C and an outlet temperature of 40°C with a feed rate of 15 ml/minute. Regadenoson in
an amorphous form was thus isolated.
Yield: 7 gm
Moisture content: 0.025% wt/wt

Example no. 6 Preparation of Regadenoson hydrochloride
The solution of regadenoson (50gm) in ethanol (100ml) was treated with an ethanolic
hydrochloride solution (25ml, 10% HC1 wt/wt) at 10°C for 2 hours and resulting solids
were filtered, washed with ethanol (20ml) and dried at 40°C to obtain regadenoson
hydrochloride salt.
Yield: 48gm
Purity: 99.89 % (HPLC)

WE CLAIM:
1. Regadenoson crystalline form D characterized by having moisture content less than 0.1% wt/wt.
2. Regadenoson crystalline form E characterized by having moisture content in the range of 2% wt/wt to 2.5% wt/wt.
3. Regadenoson crystalline form F characterized by having moisture content in the range of 6% wt/wt to 7.5% wt/wt.
4. Regadenoson crystalline form G characterized by having moisture content in the range of 8.0% wt/wt to 9.0% wt/wt.
5. Acid addition salts of regadenoson selected from the group comprising of regadenoson hydrochloride, regadenoson hydrobromide, regadenoson maleate, regadenoson fumarate, regadenoson succinate, regadenoson sulfate, regadenoson acetate, regadenoson mesylate, regadenoson tartrate or regadenoson lactate.
6. A pharmaceutical composition comprising crystalline form D, crystalline form E, crystalline form F, crystalline form G or amorphous form of regadenoson.
7. A process of preparing amorphous form of regadenoson by spray-drying or freeze-drying the solution of regadenoson in an organic solvent.
8. The process according to claim no. 7 wherein organic solvent is selected from the group comprising of ketones, alcohols, esters, nitriles, halogenated aliphatic hydrocarbon solvents, ethers or mixtures thereof.
9. Substantially pure regadenoson having HPLC purity greater than 99.0%.

10. A process of preparing substantially pure regadenoson comprising treating Acid addition salts of regadenoson selected from the group comprising of regadenoson hydrochloride, regadenoson hydrobromide, regadenoson maleate, regadenoson fumarate, regadenoson succinate, regadenoson sulfate, regadenoson acetate, regadenoson mesylate, regadenoson tartrate or regadenoson lactate with an inorganic base.