FIELD OF INVENTION
The present invention relates to an improved process for the preparation of crystalline Regadenoson. Specifically, the process of the present invention provides Regadenoson in a crystalline form having pharmaceulically acceptable level of residual solvent.
BACKGROUND OF THE INVENTION
Regadenoson is chemically known as l-[6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxyl methy!) oxolan-2-yl]purin-2-yl]-N-methylpyrazole-4-carboxamide (Formula-I). It is a selective A2A adenosine receptor agonist belonging to the class of organic compounds called as purine nucleosides, comprising a purine base attached to ribosyl or a deoxy ribosyl moiety and is acting as a coronary vasodilator in cardiac imaging. It is commonly used in the pharmacologic stress testing. The chemical structure of Regadenoson is as given below in the formula (I):
Regadenoson was first approved on April 10, 2008 by the US FDA under the brand name of LEXISCAN and is marketed byAstellas Pharma. Regadenoson is disclosed in US patent 6,403,567. This patent discloses a process for the preparation of Regadenoson which is purified using preparative TLC.
The US patent 8,106,183 discloses polymorphs A, B, C and amorphous form of Regadenoson.

The US patent 8,859,522 discloses crystalline form-D of Regadenoson prepared from a mixture of DMSO and methanol which is further isolated using reverse phase chromatography.
The US patent 9,441,006 discloses polymorph E of Regadenoson and its process for preparation.
The US patent 9,624,258 discloses polymorph of Regadenoson which is a propylene glycol solvate and process of its preparation.
The US patent 10,144,756 discloses polymorphs E, F. G corresponding to the Regadenoson trifluoroethanol solvate, ethanol solvate and anhydrous forms and their process of preparation.
The US patent 10,392,417 discloses process for the preparation of crystalline form-C of Regadenoson. This patent disclosed the purification process of crystalline form-C of Regadenoson from benzyl alcohol resulting in purity of greater than 99%.
The US patent 10.815,265 discloses the process of preparation of Regadenoson, its polymorphs and its purification methods thereof.
The US patent 11,034,714 discloses polymorph H of Regadenoson which is prepared by azeotropic distillation of the Regadenoson in a water immiscible solvent such as n-butano! and also from a solution of ammonia in methanol.
The US patent, application 2018/0208622 discloses polymorph S of Regadenoson prepared by azeotropic distillation in a solvent mixture of ethyl acetate and toluene.
The Indian patent application 3428/MUM/20I5 discloses polymorph S of Regadenoson prepared by the azeotropic distillation in a solvent like toluene and a co-solvent like acetone.

The Indian patent IN 329516 discloses the process for the preparation of form-A or Form-C of Regadenoson and process of purification of the same. The aforementioned Regadenoson is said to be pure and free from conventional impurities.
The Indian patent IN 302980 discloses the process for the preparation of crystalline form-A of Regadenoson from a mixture of organic solvents such as dimethyl sulfoxide and methanol followed by using co-solvents preferably acetonitrile for isolation of the final compound.
The Indian patent application IN 20181031309 discloses stable N-methyl-2-pyrrolidine solvate and Form-C of Regadenoson along with their process of preparation.
The Indian patent application 1470/MUM/2011 discloses various polymorphic forms of Regadenoson such as Form-D, Form-E, Forrn-F, Form-G corresponding to anhydrous, hemihydrate, sesquihydrate, dihydrate and amorphous form along with their process of preparation. Also disclosed are the acid addition salts of Regadenoson along with their process of preparation.
There is a need in the art to prepare Regadenoson with high purity and pharmaceutically
acceptable level of residual solvent.
The present invention provides addresses the drawbacks associated with the prior arts.
SUMMARY OF THE INVENTION
One embodiment of the present invention relates to a process for the preparation of crystalline Form-S of Regadenoson having pharmaceutically acceptable level of residual solvent comprising:
a) Suspending Regadenoson in a solvent mixture comprising hydrocarbon solvent and polar aprotic solvent;
b) heating the suspension obtained in the step a) to 40-110°C and maintaining the reaction mixture at the same elevated temperature;
c) cooling the reaction mixture to 25-30°C and

d) isolation.
Other embodiment of the present invention relates to a process for the preparation of crystalline Form-S of Regadenoson having pharmaceutically acceptable level of residual solvent comprising:
a) Suspending Regadenoson in a solvent mixture comprising hydrocarbon solvent and s polar prolic solvent;
b) heating the suspension obtained in the step a) to 40-110°C and maintaining the reaction mixture at the same elevated temperature;
c) cooling the reaction mixture 25-30°C and
d) isolation.
Another embodiment of the present invention relates to a process for the preparation of crystalline Form-S of Regadenoson with high purity greater than 99.5% and good yield.
Crystalline Form-S of Regadenoson which is obtained according to the present invention is characterized by a Powder X-ray diffraction pattern comprising one or more peaks which is selected from 6.20°, 10.27°, 10.72°, 14.18°, 16.30°, 18.29°, 19.02°, 21.51°, 22.59°, 24.71°, 25.44° and 27.66° ± 0.2 theta. The PXRD pattern of crystalline Form-S of Regadenoson obtained from the present invention is shown in figure-1.
Crystalline Form-S of Regadenoson which is obtained according to the present invention is characterized by endotherm peak at range of about 271°C to 279°C.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure.l illustrates a powder X-ray diffraction pattern of crystalline Form-S of Regadenoson
obtained in example-2.
Figure.2 illustrates a powder X-ray diffraction pattern of crystalline Form-S of Regadenoson
obtained in example-3.
Figurc.3 illustrates a powder X-ray diffraction pattern of crystalline Form-S of Regadenoson
obtained in example-4.

DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.
The term as used herein "polymorphism" means the capacity to exist in different crystalline forms and is known to occur in many organic molecules. These different crystalline forms are known as "polymorphic modifications" or "polymorphs". In these forms the chemical composition remains the same but the difference lies in the packing, geometrical distribution and other solid-state physical properties which affect the solubility, dissolution rate, bioavailability, chemical and physical stability, flowability, fracturability and compressibility of the compound. These indirectly affect the safety and efficacy of the drug products on the basis of the drug substance.
These crystalline forms may be prepared by a variety of methods, including methods other than the methods disclosed in the present invention, for example, crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, and jet spraying. Techniques for crystallization or recrystallization of crystalline forms from a solvent mixture includes, for example, evaporation of the solvent, decreasing the temperature of the solvent mixture, crystal seeding a supersaturated solvent mixture of the molecule and/or salt, freeze drying the solvent mixture, and addition of anti-solvents (counter solvents) to the solvent mixture.
The term as used herein "purity" is based on the "organic" purity of the compound. Purity does not include a measure of any amount of water, solvent, metal, inorganic salt, etc. In one aspect, the purity of desired compound is compared to the purity of the reference standard by comparing the area under the peak.

The term used as herein "improved yield" means a sufficiently good overall weight of the chemical compound or the drug molecule. In another aspect of the present invention it essentially means the overall weight of the chemical compound or the drug molecule is preferably increased by using the said process disclosed in the present invention.
In another embodiment the non-limiting examples of the organic solvents used in the present invention for purification of Regadenoson includes but are not limited to polar aprotic solvents such as dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidinone; ethereal solvents such tetrahydrofuran, 2-methyl tetrahydrofuran, methyl t-butyl ether, dimethyl ether, diisopopyl ether, 1,4-dioxane and diethoxymethane;; halogenated solvents such as dichloromethane, ethylene bromide, ethylene chloride; acetates such as ethyl acetate, isopropyl acetate, and butyl acetate, and other solvents such as acetonitrile, methyl vinyl ketone, N,N-dimethylacetamide and or mixtures thereof; non-polar solvents such as toluene, hexanes, xylene and heptane and mixtures thereof; polar protic solvents such as water, alcohols and glycols such as methanol, ethanol, 1-propanol, isopropanol (IPA), l-butanol , 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-nitroethanol5 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 2-methoxyethanol, 2-ethoxyethanol, diethylene glycol, propylene glycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol, glycerol and mixtures thereof. The solvent used is preferably a mixture of dimethyl sulfoxide and methanol.
In another embodiment the ratio of solvent used for the purification of Regadenoson is in between 1:1 to 10:1 or vice-versa.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, comprises isolating Regadenoson from the reaction medium by crystallization or precipitation by the addition of anti-solvents selected from the group consisting of hexane, toluene, 1,4-dioxane, chloroform, diethyl ether, methyl teri. butyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, isopropyl acetate, acetone, dimethyl formamide, acetonitrile, dimethyl sulfoxide, n-butanol, isopropanol, n-propanol, ethanol,

methanol, water and or combinations thereof. In a specific embodiment the anti-solvent used is preferably acetonitrile.
In another embodiment the non-limiting examples of the organic solvents used in the present invention for the washings of Regadenoson is performed by organic solvent selected from alcohols and glycols, methanol, ethanol, 1-propanol, isopropanol. 1-butanol , 2-butanol, i-butyl alcohol, /-butyl alcohol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 2-methoxyethanol, 2-elhoxyethanol, dielhylene glycol, propylene glycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, dichloromethane, dichlorobenzene, ethyl acetate, n-butyl acetate, isopropyl acetate, n-propyl acetate, propenyl acetate, pentyl acetate, diethyl ether, tetrahydrofuran, dioxane, methyl tetrahydrofuran, methyl ethyl ether, methyl ethyl ketone, acetonitrile, xylene, toluene, or mixture thereof. In a specific embodiment the organic solvent used for washing Regadenoson is methyl tertiary butyl ether.
In still another embodiment the purification reaction may be performed at temperatures -5° C to about 120° C, preferably in the range of from 0° C to about 110° C, preferably in the range of from 20° C to about 110° C and more preferably i n the range of from 40° C to about 110° C.
In another embodiment the non-limiting examples of the organic solvents used in the present invention for crystallization includes but are not limited to, polar aprotic solvents such as dimethyl formamide, dimethyl sulfoxide, and N-methyl pyrrolidinone; ethereal solvents such tetrahydrofuran, 2-methyl tetrahydrofuran, methyl t-butyl ether, dimethyl ether, diisopopyl ether, 1,4-dioxane and diethoxymethane; halogenated solvents such as dichloromethane, ethylene bromide, ethylene chloride; acetates such as ethyl acetate, isopropyl acetate, and butyl acetate, and other solvents such as acetonitrile, methyl vinyl ketone, N,N-dimethylacetamide and mixtures thereof; non-polar solvents such as toluene, hexanes, xylene and heptane and mixtures thereof ; polar protic solvents such as alcohols and glycols, methanol, ethanol, l-propanol, isopropanol, 1-butanol , 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 2-methoxyethanol, 2-ethoxyethanol, diethylene glycol, propylene glycol, 1-, 2-, or 3-pentanol,

neo-pentyl alcohol, t-pentyl alcohol, dielhylene glycol monomethyl elher, diethylene glycol monoethyl elher, cyclohexanol, benzyl alcohol, phenol, glycerol and mixtures thereof. The solvent used for recrystallization is preferably a mixture of toluene and isopropyl alcohol or a mixture of toluene and acetonitrile.
In another embodiment the ratio of solvent used for the crystallization of Regadenoson is in between 1:1 to 10:1 or vice-versa.
In another embodiment the non-limiting examples of the organic solvents used in the present invention for the washings of crystalline Regadenoson is performed by organic solvent selected from alcohols and glycols, methanol, ethanol, 1-propanol, isopropanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroelhanol, ethylene glycol, 2-methoxyethanol, 2-ethoxyethanol, diethylene glycol, propylene glycol, 1 -, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, dichloromethane, dichlorobenzene, ethyl acetate, n-butyl acetate, isopropyl acetate, n-propyl acetate, propenyl acetate, pentyl acetate, diethyl ether, tetrahydrofuran, dioxane, methyl tetrahydrofuran, methyl ethyl ether, methyl ethyl ketone, acetonitrile, xylene, toluene, or mixture thereof. In a specific embodiment the organic solvent used for washing the pure Regadenoson is isopropyl alcohol.
In another embodiment the purification reaction may be performed at temperatures -5° C to about 120°C, preferably in the range of from 0°C to about 110°C, preferably in the range of from 20° C to about 110° C and more preferably in the range of from 80° C to about 110° C. In another embodiment the reaction may be carried out for any desired time periods to achieve the desired product yield and purity, with time periods from about 1 to 20 hours, or longer, as required for the reaction completion.
Isolation of crystalline Form-S of Regadenoson is carried out by conventional methods such as solvent evaporation, precipitation etc.
In another embodiment the term used herein "residual solvents" refers to the organic volatile chemicals used in the preparation of the drug substances or excipients, or in the manufacture

of drug products. Appropriate selection of solvents determines the characteristics such as crystal nature, solubility, purity and improves the yield. Therefore, residual solvents are critical parameters in the synthetic process. Allowable limits or permitted daily exposure (PDE) for residual solvents is given in ICH Q3C. The PDE is based on the toxicity of the solvents. The residual solvents as defined above are classified into class-i, II and III solvents on the basis of their toxicity. Class-1 solvents need to be avoided as considered to be producing potential carcinogenic effect, whereas class-ll solvents should be within a permissible limits and class-Ill are with least toxic potential and no limit is needed.
In another embodiment of the present invention makes use of class-ll solvents within the permissible limits as described by the ICH.
In one embodiment of the present invention relates to a process for the preparation of crystalline Form-S of Regadenoson having pharmaceutically acceptable level of residual solvent comprising:
a) Suspending Regadenoson in a solvent mixture comprising hydrocarbon solvent and polar aprotic solvent;
b) heating the suspension obtained in the step a) to 40-110°C and maintaining the reaction mixture at the same elevated temperature;
c) cooling the reaction mixture and
d) isolation.
Hydrocarbon solvent used in step a) is toluene, xylene, cyclohexane or heptane and preferably using toluene.
Polar aprotic solvent used in step-a) is acetone, acetonitrile, dichloromethane, tetrahydrofuran, dimethyl formamide, dimethyl sulfoxide, ethyl acetate and preferably using acetonitrile.
The ratio of hydrocarbon solvent and polar aprotic solvent used in step-a) is between 1:1 to 10:1 and or vice versa; preferably using the ratio of 1:1, where in hydrocarbon solvent is toluene.

The reaction may be performed al temperatures in the range from 5° C to about 120° C, preferably in the range from 10° C to about 110° C, preferably in the range from 20° C to about 110° C and more preferably in the range from 40° C to about 110° C.
In other embodiment of the present invention relates to a process for preparation of crystalline Form-S of Regadenoson having pharmaceutical^ acceptable level of residual solvent comprising:
a) Suspending Regadenoson in a solvent mixture comprising hydrocarbon solvent and polar protic solvent;
b) heating the suspension obtained in the step a) to 40-110°C and maintaining the reaction mixture at the same elevated temperature;
c) cooling the reaction mixture and
d) isolation.
The ratio of hydrocarbon solvent and polar protic solvent used in step-a) is between 1:1 to 10:1 and or vice versa; preferably using the ratio of 1:1.
Hydrocarbon solvent used in step a) is toluene, xylene, cyclohexane or heptane and preferably using toluene.
Polar protic solvent used in step-a) is methanol, ethanol, propanol, butanol or isopropanol and preferably using isopropanol.
The reaction may be performed at temperatures in the range from 5° C to about 120° C, preferably in the range of from 10° C to about 110° C3 preferably in the range of from 20° C to about 110° C and more preferably in the range of from 40° C to about 110° C.
Crude Regadenoson used in the present invention is also prepared from the any known methods disclosed in the prior art.
In another embodiment of the present invention provides a process for the preparation of crystalline Form-S of Regadenoson is carried out by suspending the crude Regadenoson in a mixture of organic solvents as described herein specifically in a mixture of toluene and isopropyl alcohol (1:1 to 10:1 and or vice versa) and or toluene and acetonitrile (1:1 to 10:1

and or vice versa). The suspension is heated at an elevated temperature for a time period and cooled to room temperature. It is then filtered and the crystalline Form-S of Regadenoson is isolated.
Crystalline Form-S of Regadenoson which is obtained from the present invention is further characterized by a PXRD pattern with characteristic 20 peaks at 6.20°, 10.27°, 10.72°, 14.18°, 16.30°, 18.29°, 19.02°, 21.51°, 22.59°, 24.71°, 25.44°and 27.66° ± 0.26 shown in figure-1.
Crystalline Form-S of Regadenoson which is obtained from the present invention is having an endotherm at range of about 271°C to 279°C.
In one embodiment, crystalline Form-S of Regadenoson which is obtained according to the present invention has a residual solvent within the ICH limit. The below Table. 1 provides a comparison of residual solvents content on the basis of type of solvents used alone or in combination of other solvents. Crystalline Form-S of Regadenoson which is obtained according to the present invention having residual solvent content well within the ICH limit. Compared to the prior art procedures for preparing the crystalline Form-S of Regadenoson, the present invention resulted in low level of residual solvent and is simple, cost effective and industrially viable.
i i i
In one embodiment, the purity of crystalline Regadenoson is greater than about 98.5%. In one embodiment, the purity is greater than about 99.0%. In one embodiment, the purity is greater than about 99.5%. In one embodiment, the purity is greater than 99.8%. In one embodiment, the purity is determined by chiral HPLC technique.

Instrument Details:
X-ray powder diffraction analysis of Regadenoson which is obtained from the present invention was carried out using BRUKER D8-ADVANCE X-ray powder diffractometer using Cu K alpha radiation. The instrument was equipped with a line focus X-ray tube, and the voltage and current were set to 40 kV and 40 mA respectively. The scanning rate was set as 0.2 second per step and step size is set as 0.02°. The diffractometer was equipped with LYNXEYE_2 (ID mode) detector and rotating sample stage. X-ray diffractometer was used to record diffractogram from 2° to 45° (2-theta).
Certain specific aspects and embodiments of the invention will be explained in more detail with reference to the following examples, which are provided for purposes of illustration only and should not be construed as limiting the scope of the invention in any manner.
The invention can be illustrated with the few examples shown below which are no way limit the scope of the present invention.
EXPERIMENTAL SECTION
Example 1: Purification of crude Regadenoson:
Crude Regadenoson (1 gram) was dissolved in a mixture of dimethyl sulfoxide (lOml/gm) and methanol (5ml/gm) at 25-30°C and stirred for 10 minutes at the same temperature. Acetonitrile (45ml/gm) was added to the resulting reaction mixture at 25-30°C and stirred at the same temperature. The precipitated material is filtered and washed with acetonitrile (5ml/gm) followed by methyl tert. butyl ether (lOml/gm). The obtained wet cake was suspended in methyl tert. butyl ether (30ml/gm) at 25-30°C and stirred at the same temperature, filtered the precipitated material and dried under high vacuum. Yield: 0.75-0.85gm.; purity by HPLC: 99.80%
Example 2: Preparation of Crystalline Form-S of Regadenoson:
The pure Regadenoson (1 gram) was suspended in toluene (15ml/gm) under stirring and argon atmosphere. The suspension was heated to 100-105°C and stirred for 8 hours at the same

temperature. The suspension was cooled to room temperature and the precipitated solid was filtered, washed with methyl tertiary butyl ether and dried under reduced pressure at 85 - 90° C for 10 hours, yield: 85-90%: purity by HPLC >99.80%
Example 3: Preparation of Crystalline Form-S of Regadenoson:
The pure Regadenoson (1 gram) was suspended in a mixture of toluene: isopropyl alcohol (1:1) under stirring and argon atmosphere. The suspension was heated to 75-85° C and stirred for 10 hours at the same temperature. The suspension was cooled to room temperature and the precipitated solid was filtered, washed with isopropyl alcohol (5 ml/gm) and dried under reduced pressure at 65 - 70° C for 10 hours. (Yield: 85-90%; purity by HPLC >99.80%)
Example 4: Preparation of Crystalline Form-S of Regadenoson:
The pure Regadenoson (1 gram) was suspended in a mixture of toluene: acetonitrile (1:1) under stirring and argon atmosphere. The suspension was heated to 80-85° C and heating maintained for 8 hours under stirring at same temperature. The suspension was cooled to room temperature and the solid was filtered, washed with acetonitrile and dried under reduced pressure at 65 -70° C for 10 hours. Yield: 85-90%; purity by HPLC >99.80%.

We Claim:
1. A process for the preparation of crystalline Form-S of Regadenoson having
pharmaceutical!)' acceptable level of residual solvent comprising:
a) Suspending Regadenoson in a solvent mixture comprising hydrocarbon solvent and polar aprotic solvent;
b) heating the suspension obtained in the step a) to 40-110°C;
c) cooling the reaction mixture to 25-30°C and
d) isolation.
2. A process for the preparation of crystalline Form-S of Regadenoson having
pharmaceutically acceptable level of residual solvent comprising:
comprising:
a) Suspending Regadenoson in a solvent mixture comprising hydrocarbon solvent and polar protic solvent;
b) heating the suspension obtained in the step a) to 40-110°C;
c) cooling the reaction mixture to25-30°C and
d) isolation.

3. The process as claimed in claim 1, wherein the polar aprotic solvent used in step a) is selected from nitrile solvents such as acetonitrile or propionitrile.
4. The process as claimed in claim 2, wherein the polar protic solvent used in step a) is selected from alcoholic solvents such as methanol, ethanol, propanol, butanol or isopropanol.
5. The process as claimed in claim 1 and 2, wherein the hydrocarbon solvent used in step a) is selected from toluene, xylene, cyclohexane and heptane.
6. A process for the preparation of crystalline Form-S of Regadenoson as claimed in claim 1-5, wherein Regadenoson is characterized by a powder-X-ray diffraction

spectrum having peaks at 2-theta values of about 6.20°, 10.27°, 10.72°, 14.18°, 16.30°; 18.29°, 19.02°, 21.51°, 22.59°, 24.71°, 25.44° and 27.66° ± 0.2° thela and PXRD pattern as mentioned in figure-1.
7. A process for the preparation of crystalline Form-S of Regadenoson as claimed in claim 1-6, characterized by differential scanning calorimetry having an endotherm at range of about 271 °C to 279°C.
8. A method for preparing pharmaceutical composition comprising pure Regadenoson of claim 1-7 and at least one pharmaceutical^ acceptable excipient.