DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, rule 13]

SOLID STATE FORMS OF REGADENOSON

PIRAMAL ENTERPRISES LIMITED, a company incorporated under the Companies Act, 1956, of Piramal Ananta, Agastya Corporate Park, Opposite Fire Brigade, Kamani Junction, LBS Marg, Kurla West, Mumbai 400070, State of Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The field of the present invention relates to solid state forms of Regadenoson.
In particular, the present invention relates to N-Methyl-2-pyrrolidone (NMP) solvate of Regadenoson and preparation thereof.
Further, the invention also relates to a stable Form C of Regadenoson and preparation thereof.
BACKGROUND OF THE INVENTION
Regadenoson [(l-{9-[(4S, 2R, 3R, 5R)-3,4-dihydroxy-5-(hydroxymethyl)oxalan-2-yl]-6- aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamine] is a selective A2A-adenosine receptor agonist, which is a coronary vasodilator. The selective nature of the drug makes it preferable to other stress agents such as adenosine, which are less selective and therefore cause more side-effects.
Regadenoson was approved by the United States Food and Drug Administration on April 10, 2008 and is marketed by Astellas Pharma under the tradename Lexiscan. It is approved for use in the European Union and under the name of Rapiscan. It is currently being marketed by GE Healthcare and is being sold in both the United Kingdom and Germany.
Regadenoson (CAS Registry Number: 313348-27-5) was described for the first time in U.S. Pat. No. 6,403,567 (henceforth US`567) and has the following structural formula:

From the reported literature on Regadenoson, it is to be understood that Regadenoson exists in various crystalline forms. US 7,732,595 (US`595) discloses many crystalline forms of Regadenoson, including crystal forms A, B, C and an amorphous form.
US`595 illustrate that Regadenoson polymorphic form A is a monohydrate form, this form may be produced by crystallization of Regadenoson from ethanol or ethanol/water mixtures or from a dimethylsulfoxide/water mixture. Regadenoson Form A was identified as more stable than polymorphs B and C at ambient temperature and also under relative humidity stress conditions up to its melting point (identified by DSC as 194.5° C). However, DMSO is not advisable for the crystallisation of active pharmaceutical ingredient (API).
Polymorphic form B was a crystal form obtained by dissolving Regadenoson in a solvent trifluoroethanol at ambient temperatures followed by concentrating under vacuum. The crystal form contains varying amounts of crystal water, and it is very difficult to reproduce the preparation process thereof. Polymorph C was prepared by slurrying Regadenoson in acetonitrile at 60° C. for a long period of time. This polymorph showed a distinct X-ray spectrum, but showed to be a variable hydrate, unstable at high temperatures.
The amorphous form of Regadenoson was formed by heating crystal form A up to 200° C., which, however, is unstable in atmospheric moisture, forming various crystalline hydrates thereof.
WO2012149196A1 describes a polymorph Form-D of Regadenoson. Form D was made by using a mixture of DMSO and methanol, which is further isolated by using reverse phase chromatography. The isolated fractions were concentrated under vacuum at 150° C. The obtained Form D contains 0.8% water. However, use of DMSO and reverse phase chromatography is not advisable at plant level.
Therefore, keeping view of the prior art, the inventors of the present invention have reported a solid form of Regadenoson which is stable and easy to prepare at plant level.
OBJECTS OF THE PRESENT INVENTION
An object of the invention is to provide solid state forms of Regadenoson.
Another object of the present invention is to provide a NMP solvate of Regadenoson.
Another object of the present invention is to provide a process of preparation of NMP solvate of Regadenoson.
Yet another object of the invention is to provide stable Form C of Regadenoson.
Yet another object of the present invention is to provide a process for preparation of Form C of Regadenoson.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1: X-ray powder diffraction (XRD) pattern of NMP solvate of Regadenoson, prepared according to example 4.
Figure 2: Thermal gravimetric analysis (TGA) of NMP solvate of Regadenoson, prepared according to example 4.
Figure 3: DSC thermogram of NMP solvate of Regadenoson, prepared according to example 4.
Figure 4: X-ray powder diffraction (XRD) pattern of stable Form C of Regadenoson, prepared according to example 5.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
According to one of the embodiments, the present invention provides a NMP solvate of Regadenoson, the said process comprising:
a) providing solution of Regadenoson in N-Methyl-2-pyrrolidone (NMP);
b) isolating NMP solvate of Regadenoson.
In a preferred embodiment of the present invention, Regadenoson used in step (a) can be either in crystalline form, or in mixture of crystalline and amorphous form, solvate form or hydrate form thereof.
The obtained solution can be optionally treated with carbon for removal of undesired color or with sodium sulfate for moisture removal.
In an embodiment, the term “isolating” refers to the filtering and drying of the compound.
The drying may be done at atmospheric pressure or reduced pressures, between 100 mmHg and 700 mmHg, at temperatures such as about 30 °C to about 50°C.
The drying can be carried out for about 1 to 15 hours.
The dried material can be optionally milled to get desired particle size, Milling operation can be performed prior to drying or after the drying. Suitable milling techniques includes, air jet milling, or other conventional milling equipment.
According to yet another embodiment, the present invention provides a novel process for preparation of NMP solvate of Regadenoson, the said process comprising:
a) reacting compound of formula (II) with sodium bisulfite in a solvent to provide compound of formula (III);

b) reacting compound of formula (IV) with compound of formula (III) in a solvent to provide compound of formula (V);

c) reacting compound of formula (V) with alcoholic solution of methyl amine in NMP solvent to provide a NMP solvate of Regadenoson;

d) isolating NMP solvate of Regadenoson.

Wherein: R is selected from the group consisting of alkyl, aryl, substituted aryl, imidazole.
In an embodiment, solvent used in step (a) and step (b) is selected from the group consisting of water, alcohols, ethers, amides, esters, nitriles, sulfoxides, ketones, hydrocarbons and halogenated hydrocarbons, wherein alcohol is selected from methanol, ethanol, iso-propanol. n-butanol or iso-butanol; ester is selected from ethyl acetate or isopropyl acetate; ketone is selected from the group consisting of acetone, methyl isobutyl ketone and methyl ethyl ketone; ether is selected from the group consisting of methyl tert-butyl ether, diisopropyl ether, diethyl ether tetrahydrofuran, 2-methyl tetrahydrofuran, cyclopentyl methyl ether and dioxane; halogenated solvent is selected from the group consisting of dichloromethane, chloroform, chlorobenzene and bromobenzene; hydrocarbons is selected from the group consisting of toluene, xylene and cyclohexane; nitrile is selected from acetonitrile or propionitrile; amide is selected from N,N-dimethylformamide or N,N-dimethyl acetamide; sulfoxide is dimethyl sulfoxide; sulfone; or mixtures thereof.
The alcohol used in step (c) to prepare alcoholic solution of methyl amine is selected from methanol, ethanol and isopropyl alcohol.
The novel process for the preparation of Regadenoson is schematically represented ad follows:

According to the present invention, isolation of NMP solvate of Regadenoson in step (d) comprises the steps of:
I. heating the reaction mass obtained in step (a) and removing the alcohol to obtain the concentrate;
II. dilute the concentrate by adding NMP solvent;
III. isolating NMP solvate of Regadenoson.
In an embodiment, the term “isolating” refers to the filtering and drying of the compound.
Further, in a preferred embodiment of the present invention, the removal of solvent in step (I) is carried out by distillation, evaporation, atmospheric distillation, distillation under vacuum such as rotary evaporator.
The NMP solvate of Regadenoson has an X-Ray Powder Diffractogram (XRPD) substantially as depicted in Figure 1 of the accompanied drawing.
In one embodiment, the present invention provides a NMP solvate of Regadenoson characterized by a X-ray powder diffraction pattern having peaks at 12.89, 13.28, 13.50, 14.98, 17.13 & 25.48 ±0.2 degrees 2?.
The NMP solvate of Regadenoson has a DSC thermogram substantially as depicted in Figure 2 of the accompanied drawings. The DSC thermogram shows characteristic endothermic peak at 187.19.
The crystalline NMP solvate of Regadenoson has a TGA substantially as depicted in Figure 3 of the accompanied drawings. The TGA of the NMP solvate of Regadenoson shows a weight loss of about 20%.
In another embodiment, the present invention describes Form C of Regadenoson in a stable form. The stable Form C of the present invention essentially does not convert to any other form of Regadenoson. The stable Form C of Regadenoson has an X-Ray Powder Diffractogram (XRPD) substantially as depicted in Figure 4 of the accompanied drawing, having peaks at 10.25, 10.68, 14.12, 16.27, 17.76, 18.25, 19.01, 21.47, 22.54, 24.66, 25.37, 26.15 & 27.62 ±0.2 degrees 2?.
The stable Form C of Regadenoson can be prepared by a process comprising:
a) combining NMP solvate of Regadenoson with acetonitrile to form a mixture;
b) heating the mixture;
c) cooling and stirring the heated mixture of step (b) at room temperature;
d) isolating stable Form C of Regadenoson.

In an embodiment, the term “isolating” refers to the filtering and drying of the compound.
BEST MODE OR EXAMPLES FOR WORKING OF THE INVENTION
The present invention is described in the examples given below; further these are provided only to illustrate the invention and therefore should not be construed to limit the scope of the invention.
EXAMPLE-1: Synthesis of Sodium 2-(ethoxycarbonyl)-1, 3-dihydroxypropane-1,3-disulfonate ( Sodium bisulphite adduct of Ethyl 2-formyl 3-oxo propanoate):

A dry 500 ml jacketed flask was equipped with a mechanical stirrer, thermometer pocket and condenser was charged with Ethyl-2-formyl-3-oxo-propanoate (20 g), Sodium Bisulphite (1.3 g), methanol (160 ml) and 10 ml demineralised water (DM water). The mixture was stirred at 25-30°C for 10-12 h. The solids were filtered, washed with methanol and dried in a vacuum oven at 50 °C to obtain 37.2 g of Sodium 2-(ethoxycarbonyl)-1, 3-dihydroxypropane-1, 3-disulfonate.
% Yield: 75.7%

EXAMPLE-2: Synthesis of ethyl 1-(6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5 (hydroxymeth- yl) tetrahydrofuran-2-yl)-9H-purin-2-yl)-1H-pyrazole-4-carboxylate:

A dry 500 ml jacketed flask was equipped with a mechanical stirrer, thermometer pocket and condenser was charged with (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxy methyl)tetrahydrofuran-3,4-diol (5 g), Sodium 2-(ethoxycarbonyl)-1,3-dihydroxypropane-1,3-disulfonate (8.2 g), Isopropanol (50 ml). The mixture was heated at 80-85 °C for 3-4 hours, cooled to 10-15 °C and stirred at 25-30 °C for 1-2 hours. The solids were filtered, washed with methanol. Again the flask were charged with obtained wet solid and washed with DM water.
The solids were dried in a vacuum oven at 50°C to obtain 6.1g of ethyl 1-(6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-1H-pyrazole-4-carboxylate.
% Yield: 99.1% HPLC purity: 99.1%

EXAMPLE-3: Synthesis of 1-(6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetra-hydrofuran-2-yl)-9H-purin-2-yl)-N-methyl-1H-pyrazole-4 carboxamide:

A dry 500 ml jacketed flask was equipped with a mechanical stirrer, thermometer pocket and condenser was charged with 1-(6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-N-methyl-1H-pyrazole-4-carboxa-mide (RGS-II) (10 g), Oxalic acid (3.7 g), DMSO (40 ml). The mixture was stirred at 25-35 °C for 1-2 hours. Ethanol was added to the mixture and stirred for 5 to 10 minutes. The solids were filtered, washed with ethanol. Again the flask was charged with obtained wet solid and acetone was added into that flask. The mixture was stirred at 25-35 °C for 15 to 20 minutes. The solids were filtered and washed with acetone. Again the flask was charged with obtained wet solid and 10% sodium carbonate solution (200 ml) was added into that flask. The mixture was stirred at 25-35 °C for 1 to 2 hours. The solids were filtered, washed with DM water followed by ethyl acetate and acetone. The solids were dried in a vacuum oven at 50 °C to obtain 6.6 g of 1-(6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-N-methyl-1H-pyrazole-4-carboxamide.
HPLC purity: 99.84%, Any individual unknown impurity: 0.06%.

EXAMPLE-4: Synthesis of Regadenoson NMP Solvate
A dry 100 ml capacity jacketed flask was equipped with a mechanical stirrer, thermometer pocket, and condenser was charged with N-Methyl-2-pyrrolidone (NMP) (10 ml), (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxy methyl)tetrahydrofuran-3,4-diol (2 g) and 25% solution of methyl amine in methanol (10 ml). This mixture was stirred overnight at room temperature and then heated at 110 °C. The solvent was removed by simple distillation to obtain a residue. To this residue another 5 ml N-methyl pyrrolidone was added and the obtained mixture was cooled at room temperature. The mixture was filtered and dried under vacuum for 12 hours at 50 °C to provide 1.2 g of NMP Solvate of Regadenoson.

EXAMPLE-5: Synthesis of Stable Form-C of Regadenoson
A dry 100 ml jacketed flask was equipped with a mechanical stirrer, thermometer pocket, and condenser was charged with Regadenoson NMP solvate (1.5 g) and Acetonitrile (100 ml). The mixture was heated at 80-85 °C for 1 to 2 hours and the solvent was distilled to half its volume after which the reaction mixture was cooled to room temperature. The obatined mixture was stirred for 1 to 2 hours at room temperaure. The mixture was filtered and dried under vacuum at 50 °C to provide 1.3 g of Stable Form-C of Regadenoson.
,CLAIMS:We Claim:
1. A Regadenoson NMP solvate.

2. The compound according to claim 1, wherein the Regadenoson NMP solvate is characterized by a X-ray powder diffraction pattern having peaks at 12.89, 13.28, 13.50, 14.98, 17.13 & 25.48 ±0.2 degrees 2?, which is depicted in Figure 1.

3. The compound according to claim 1, wherein the Regadenoson NMP solvate has a DSC endothermic peak at 187.19oC, which is depicted in Figure 3.

4. The compound according to claim 1, wherein the Regadenoson NMP solvate is prepared by the process comprising:
a) providing solution of Regadenoson in N-Methyl-2-pyrrolidone (NMP); and
b) isolating Regadenoson NMP solvate.

5. The compound according to claim 1, wherein the Regadenoson NMP solvate is prepared by the process comprising:
a) reacting compound of formula (II) with sodium bisulfite in a solvent to provide compound of formula (III);

wherein: R is selected from the group consisting of alkyl, aryl, substituted aryl, imidazole.

b) reacting compound of formula (IV) with compound of formula (III) in a solvent to provide compound of formula (V);

c) reacting compound of formula (V) with alcoholic solution of methyl amine in NMP solvent to provide a NMP solvate of Regadenoson;

d) isolating NMP solvate of Regadenoson.

6. The process as clamed in claim 5, wherein the solvent used in step (a) and step (b) is an alcohol selected from methanol, ethanol, n-propanol, isopropanol.

7. The process as claimed in claim 5, wherein the alcohol in the alcoholic solution of methyl amine in the step (c) is selected from methanol, ethanol, isopropanol.

8. Stable Form C of Regadenoson.

9. The compound according to claim 8, wherein the stable Form C of Regadenoson is prepared by a process comprising:
a) combining NMP solvate of Regadenoson with acetonitrile to form a mixture;
b) heating the mixture;
c) cooling and stirring the heated mixture of step (b) at room temperature;
d) isolating stable Form C of Regadenoson.

10. The compound according to claim 8, wherein the stable Form C of Regadenoson is characterized by a X-ray powder diffraction pattern having peaks at 10.25, 10.68, 14.12, 16.27, 17.76, 18.25, 19.01, 21.47, 22.54, 24.66, 25.37, 26.15 & 27.62 ±0.2 degrees 2?, which is depicted in Figure 4.

Dated this 19th day of August 2019
_________________________
SRIDEVI KRISHNAN
GENERAL MANAGER – CORPORATE PATENTS
PIRAMAL ENTERPRISES LIMITED
(APPLICANT)
To,
The Controller of Patents
The Patent Office
At Mumbai.