Novel process for the preparation of pure and crystalline form of Regadenoson monohydrate

FIELD OF INVENTION

The present invention relates to process for preparation of highly pure and crystalline form of Regadenoson monohydrate which is acting as an A2A adenosine receptor agonist that is a coronary vasodilator.

BACKGROUND OF THE INVENTION

Regadenoson 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.

US6403567 and US6642210 disclosed a method for the preparation of Regadenoson from 2-Chloroadenosine. The method involving preparation of 2-hydrazinoadenosine from 2-chloroadenosine which further react with ethyl diformyl acetate to yield 2-(4-Methoxycarbonylpyrazol-1-yl) adenosine. Methylamine treated with 2-(4-Methoxycarbonylpyrazol-1-yl) adenosine to obtain Regadenoson.

US8106183 disclosed polymorphic form of Regadenoson, more particularly monohydrate of Regadenoson prepared by treating Regadenoson with mixture of ethanol and water and mixture of dimethylsulfoxide and water.

There is a problem in the art to achieve less than 0.1% level of single maximum unknown impurity in the final product of Regadenoson.

None of the prior art publications provided a method for making pure Regadenoson and therefore there is a need to provide a method for preparing highly pure Regadenoson having a purity of greater than 99.5%.

The invention encompasses a process for preparing Regadenoson having purity of greater than 99.5% of the following formula,

SUMMARY OF THE INVENTION:

In one aspect of the present invention, process for the preparation of Regadenoson having purity greater than 99.5% is disclosed.

Another aspect of the present invention, process for the preparation of pure Regadenoson comprising;

a) dissolving impure Regadenoson in a mixture of at least one solvent selected from the group consisting of amides, sulphoxide and at least one solvent selected from the group consisting of alcohol;

b) treating the solution obtained in step(a) with one anti-solvent selected from the group comprising of nitriles, alcohols or mixture thereof;

c) filtering the solid obtained in step (b) to attain pure Regadenoson.

Further aspect of the invention, it is provided novel process for preparing Regadenoson monohydrate from impure Regadenoson or pure Regadenoson.

Still another aspect of the invention is to provide a stable polymorphic form of Regadenoson monohydrate.

Yet another aspect of the present invention to provide a crystalline form of Regadenoson monohydrate comprising; dissolving Regadenoson in at least one solvent selected from the group consisting of amides, sulphoxide and further treating with a co-solvent to obtain crystalline form of Regadenoson monohydrate.

Further aspect of the invention is to provide industrially viable process to prepare pure Regadenoson with purity is not less than 99.5%.

Brief Description of the Drawings

The accompanying drawings illustrate embodiments in the present invention according to the best modes presently devised for the practical application of the principles.

Figure 1 illustrates 'H NMR Spectrum of Regadenoson obtained from the process of present invention

Figure 2 illustrates ,3C NMR Spectrum of Regadenoson obtained from the process of present invention

Figure 3 illustrates HPLC Chromatogram of Regadenoson

Figure 4 Powder X-Ray diffraction spectrum of Regadenoson

These and other objects of the present invention will become apparent to those familiar in the art when reviewing the following detailed description, combination and elements as herein described, and more particularly defined by the claims, it being understood that changes in the embodiments to the herein disclosed invention are meant to be included as coming within the scope of the claims, except insofar as they may be precluded by the prior art.

DETAILED DESCRIPTION OF PRESENT INVENTION:

It is provided a process for the preparation of Regadenoson having purity greater than 99.5% which can be used for the preparation of medicaments without any further purification.

The process for the preparation of pure Regadenoson comprising;

a) dissolving impure Regadenoson in a mixture of at least one solvent selected from the group consisting of amides, sulphoxide and at least one solvent selected from the group consisting of alcohol;

b) treating the solution obtained in step(a) with one anti-solvent selected from the group comprising of nitriles, alcohols or mixture thereof;

c) filtering the solid obtained in step (b) to attain pure Regadenoson;

d) dissolving Regadenoson in atleast one solvent selected from the group consisting of amides, sulphoxide and further treating with a co-solvent to obtain crystalline form of Regadenoson monohydrate

Further aspect of the invention, it is provided novel process for preparing highly pure Regadenoson monohydrate from impure Regadenoson.

Still another aspect of the invention is to provide a stable polymorphic form of Regadenoson monohydrate.

Yet another aspect of the present invention to provide a crystalline form of Regadenoson monohydrate comprising; dissolving Regadenoson in at least one solvent selected from the group consisting of amides, sulphoxide and further treating with a co-solvent to obtain crystalline form of Regadenoson monohydrate.

Amide solvents of step (a) is selected from N,N-Dimethylacetamide, N,N-Dimethyl formamide, Formamide, N-Methyl formamide, preferably formamide, N-methyl formamide, N,N-Dimethyl acetamide, more preferably N,N-Dimethyl formamide.

Sulphoxide solvent used in step (a) is Dimethyl sulphoxide and alcohol is methanol, ethanol, propanol and the like.

Solvent used in step (a) is a mixture of Dimethyl sulphoxide and methanol or N, N-Dimethyl formamide and methanol, most preferably Dimethyl sulphoxide and methanol.

The ratio of solvent mixture used in step (a) is between 1:2 and 2:1, preferably 1:1, more preferably 1:2 and most preferably 2:1.

The anti-solvent used in step (b), nitriles is acetonitrile; alcohol is methanol or ethanol. Anti-solvent used in step (b) is preferably methanol, and most preferably acetonitrile.

The ratio of anti-solvent used in step (b) with solution obtained from step (a) is 2:1, preferably 4:1 and most preferably 3:1.

The solvent used in step (d) is Dimethyl sulphoxide N, N-Dimethyl formamide or mixture thereof, most preferably N, N-Dimethyl formamide.
The co-solvent used to isolate the pure compound in step (d) is methanol, ethanol, acetonitrile and water or mixture thereof; preferably aqueous methanol, aqueous ethanol, and aqueous acetonitrile; most preferably water.

The ratio of co-solvent used in step (d) with solution is 2:1; preferably 3:1 most preferably 4:1.

Reverse-phase High Performance Liquid Chromatography (HPLC) method for determining purity of Regadenoson:

The purity of the Regadenoson prepared by the process of the present invention is estimated by HPLC and the purity is measured by area value of the peak. An exemplary HPLC method comprises:

a) Combining Regadenoson sample with as solution of Triethyl ammonium phosphate buffer and a mixture of solvent.

b) Injecting the solution into a 250X4.6 mmX5um Symmetry C-8 reverse phase column.

c) Eluting the sample from the column using a mixture of Eluent A (Buffer) and Eluent B (Mixture of solvent)

d) Meseauring the Regadenoson content in the relevant sample with UV detector.

The purity of Regadenoson has been estimated by HPLC. Regadenoson obtained by the process of the present invention is dissolved in a mixture of Triethyl ammonium phosphate buffer (solution A) and 1:1 mixture of acetonitrile and methanoI.(Solution B). The buffer (solution A) and solvent mixture .(Solution B) required to dissolve Regadenoson is 90:10, preferably 85:15 and most preferably 95:5. The solution is injected into a C-8 reverse phase column, most preferably 250X4.6 mm X 5\im Symmetry reverse phase column. The eluent used may be a mixture of Eluent A (Solution A) and Eluent B (Solution B) wherein the ratio of components in the mixture varies over time, in other words, a gradient eluent. The gradient programme most preferably at time 0 minutes, the eluent contains 95% of Eluent A and 5% of Eluent B; at 20 minutes the eluent contains 83% of Eluent A and 17% of Eluent B; at 35 minutes the eluent contains 55% of Eluent A and 45% of Eluent B; at 40 minutes the eluent contains 50% of Eluent A and 50% of Eluent B; at 42 minutes the eluent contains 95% of Eluent A and 5% of Eluent B; at 55 minutes the eluent contains 95% of Eluent A and 5% of Eluent B. UV detector is set in at a

Table -1

wavelength of about 246nm to measure the area value of the peak separated by the HPLC method to estimate the purity of Regadenoson. It is evident from the Table - 1, Area % obtained in the last column shows 99.96% purity of Regadenoson and level of all other impurities are less than 0.01%.

The present invention provides the preparation of stable polymorphic Form a of Regadenoson monohydrate. It is provided a novel process for preparing Form A which involves;

a) dissolving Regadenoson in a solvent selected from the group consisting of amides or sulphoxide and precipitate with water; or

b) dissolving Regadenoson with one solvent selected form the group consisting of amides or sulphoxide and precipitate by water containing alcohol; or

c) dissolving Regadenoson with one solvent selected form the group consisting of amides or sulphoxide and precipitate with water containing alkyl nitriles.

The solvent used in step (a) is Dimethyl sulphoxide, N, N-Dimethyl acetamide, orN, N-Dimethyl formamide and preferably N, N-Dimethyl acetamide and most preferably N, N-Dimethyl formamide.

The solvent used in step (a) is Dimethyl sulphoxide, N, N-Dimethyl acetamide, or N, N-Dimethyl formamide and preferably N, N-Dimethyl acetamide and most preferably N, N-Dimethyl formamide.

The ratio of water used step (a) with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein amide is N, N-Dimethyl formamide and sulphoxide is Dimethyl sulphoxide.

The solvent used in step (b) is with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein solvent is aqueous ethanol; amide is N, N-Dimethyl formamide and sulphoxide is Dimethyl sulphoxide.

The solvent used in step (b) is with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein solvent is aqueous methanol; amide is N, N-Dimethyl formamide and sulphoxide is Dimethyl sulphoxide.

The solvent used in step (c) is with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein solvent is aqueous acetonitrile amide is N, N-Dimethyl formamide and sulphoxide is Dimethyl sulphoxide.
1H NMR and 13C NMR Analysis in DMSO :

Table -2 shows 'H NMR chemical shift values obtained for the Regadenoson monohydrate of the present invention. Table - 3 shows ,3C NMR chemical shift values obtained for the Regadenoson monohydrate of the present invention.

Example I: Preparation of pure Regadenoson monohydrate (Form A)

Regadenoson (lOOgm) having purity about 96% was dissolved in a 2:1 mixture of DMSO and methanol (1500ml) at room temperature. To that, acetonitrile 3 volume (4.5 lit) was added with stirring. After addition, continue stirring for 1 hr. The crystallized materia! was filtered and the wet material was dissolved in N, N-Dimethyl formamide (500ml). The solution was then added into distilled water (2Iit) slowly with stirring. The solid formed was filtered and dried to yield pure Regadenoson having purity greater than 99.5%

Yield: 85 gm.

Example II: Preparation of pure Regadenoson monohydrate (Form A)

Regadenoson (lOOgm) having purity about 96% was dissolved in a 2:1 mixture of DMSO and methanol (1500ml) at room temperature. To that, acetonitrile 3 volume (4.5 lit) was added with stirring. After addition, continue stirring for 1 hr. The crystallized material was filtered and the wet material was dissolved in N,N-Dimethyl formamide (500ml). The solution was then added into 10% aqueous methanol (21it) slowly with stirring. The solid formed was filtered and dried to yield Regadenoson monohydrate Form A.

Yield: 85 gm.

Example III: Preparation of Regadenoson monohydrate (Form A)

Regadenoson (lOOgm) having purity about 96% was dissolved in a 2:1 mixture of DMSO and methanol (1500ml) at room temperature. To that, acetonitrile 3 volume (4.5 lit) was added with stirring. After addition, continue stirring for 1 hr. The crystallized material was filtered and the wet material was dissolved in N,N-Dimethyl formamide (500ml). The solution was then added into 10% aqueous acetonitrile (21it) slowly with stirring. The solid formed was filtered and dried to yield Regadenoson monohydrate Form A.

Yield: 85 gm.

Example IV: Preparation of Regadenoson monohydrate (Form A)

Regadenoson (lOOgm) having purity about 96% was dissolved in a 2:1 mixture of DMSO and methanol (1500ml) at room temperature. To that, acetonitrile 3 volume (4.5 lit) was added with stirring. After addition, continue stirring for 1 hr. The crystallized material was filtered and the wet material was dissolved in N,N-Dimethyl formamide (500ml). The solution was then added into 10% aqueous ethanol (21it) slowly with stirring. The solid formed was filtered and dried to yield Regadenoson monohydrate Form A.

Yield: 85 gm.

We claim:

1. A process for the preparation of pure Regadenoson comprising;

a. dissolving impure Regadenoson in a mixture of at least one solvent selected form the group consisting of amides, sulphoxide and at least one solvent selected from the group consisting of alcohol;

b. treating the solution obtained in step(a) with one anti-solvent selected from the group
comprising of nitriles, alcohols or mixture thereof;

c. filtering the solid obtained in step (b) to attain pure Regadenoson;

d. dissolving Regadenoson in at least one solvent selected from the group consisting of amides, sulphoxide or mixture thereof;

e. treating the solution obtained in step (d) with co-solvent to get crystalline form of Regadenoson monohydrate.

2. The process as claimed in claim 1, wherein amide solvents of step (a) is selected from N,N-Dimethylacetamide, N,N-Dimethyl formamide, N-Methyl formamide, preferably formamide, N-Methyl formamide, N,N-Dimethyl formamide, more preferably N,N-Dimethyl formamide; Sulfoxide solvent used in step (a) is Dimethyl sulphoxide and alcohol is methanol, ethanol, propanol and the like.

3. The process as claimed in claim 1, wherein solvents used in step (a) is a mixture of Dimethyl sulphoxide and methanol orN, N-Dimethyl formamide and methanol, most preferably Dimethyl sulphoxide and methanol; the ratio of solvent mixture used in step (a) is between 1:2 and 2:1, preferably 1:1, and most preferably 2:1.

4. The process as claimed in claim 1, wherein anti-solvent used in step (b), nitrile is acetonitrile; alcohol is methanol or ethanol.

5. The process as claimed in claim 4, wherein anti-solvent used in step (b) is preferably methanol, acetonitrile or mixture thereof, most preferably acetonitrile.

6. The process as claimed in claim 1, wherein ratio of anti-solvent used in step (b) with solution obtained from step (a) is 2:1, preferably 4:1 and most preferably 3:1.

7. The process as claimed in claim 1, wherein solvent used in step (d) is Dimethyl sulphoxide N, N-Dimethyl acetamide or mixture thereof, most preferably N, N-Dimethyl formamide;

8. The process as claimed in claim 1, wherein co-solvent used to isolate the pure compound in step (e) is methanol, ethanol, acetonitrile and water or mixture thereof; preferably aqueous methanol, aqueous ethanol, aqueous acetonitrile; most preferably water.

9. The ratio of co-sol vent used in step (e) with solution of step (d) is 2:1; preferably 3:1 most preferably 4:1.

10. A process for the preparation of crystalline Form A of Regadenoson monohydrate comprising;

a. dissolving Regadenoson in a solvent selected form the group consisting of amides or sulphoxide and precipitate with water; or

b. dissolving Regadenoson with one solvent selected form the group consisting of amides or sulphoxide and precipitate by water containing alcohol; or

c. dissolving Regadenoson with one solvent selected form the group consisting of amides or sulphoxide and precipitate with water containing alkyl nitriles.

11. The process as claimed in claim 10, wherein ratio of water used step (a) with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein amide is N, N-Dimethyl acetamide and sulphoxide is Dimethyl sulphoxide.

12. The process as claimed in claim 10, wherein solvent used in step (b) is with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein solvent is aqueous ethanol; amide is N, N-Dimethyl acetamide and sulphoxide is Dimethyl sulphoxide.

13. The process as claimed in claim 10, wherein the ratio of solvent used in step (b) is with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1, wherein solvent is aqueous methanol; amide is N, N-Dimethyl acetamide and sulphoxide is Dimethyl sulphoxide.

14. The process as claimed in claim 10, wherein alkyl nitrile used in step (c) is with amides or sulphoxide is between 4:1 and 8:1, preferably 6:1 and most preferably 4:1; alkyl nitrile is aqueous acetonitrile.

15. Process for the preparation of pure Regadenoson and crystalline Form A of Regadenoson monohydrate is such as herein described and exemplified.