DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003

COMPLETE SPECIFICATION
(Section 10 and Rule 13)

A PROCESS FOR THE PREPARATION OF DAPRODUSTAT AND POLYMORPHS THEREOF

APITORIA PHARMA PRIVATE LIMITED HAVING CORPORATE OFFICE AT
GALAXY, FLOORS: 22-24,
PLOT No.1, SURVEY No.83/1,
HYDERABAD KNOWLEDGE CITY,
RAIDURG PANMAKTHA,
RANGA REDDY DISTRICT,
HYDERABAD – 500 032,
TELANGANA, INDIA
AN INDIAN ORGANIZATION

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed.
FIELD OF THE INVENTION
The present invention provides a simple and cost-effective process for the preparation of Daprodustat of Formula (I).

The present invention also relates to novel crystalline forms of Daprodustat and process for the preparation thereof.

BACKGROUND OF THE INVENTION
Daprodustat of Formula (I) is chemically known as N-[(1,3-dicyclohexylhexa hydro-2,4,6-trioxopyrimidin-5-yl)carbonyl]glycine. Daprodustat (GSK1278863) is an oral hypoxia - inducible factor prolyl hydroxylase (HIF PH) inhibitor developed by GlaxoSmithKline for the treatment of anemias, such as chronic kidney disease related anemia.

US 8,324,208 disclosed a process for the preparation of Daprodustat by reacting a compound of Formula (A) with malonyl dichloride to obtain compound of Formula (B), which is then reacted with ethyl isocyanatoacetate followed by hydrolysis to obtain Daprodustat of Formula (I). The process is shown in below scheme:

Scheme – I
The disadvantage of the above-mentioned process is the use of malonyl chloride which is a hazardous halogenating agent.

The US ‘208 also discloses an alternate process for the preparation of Daprodustat by coupling N,N-dicyclohexyl carbodiimide of Formula (C) with malonic acid to obtain compound of Formula (B), which is then reacted with ethyl isocyanatoacetate followed by hydrolysis to obtain Daprodustat of Formula (I). The process is shown in below scheme:

Scheme – II
However, the above processes for the preparation of Daprodustat involves ethyl isocyanatoacetate which is very expensive material and its preparation involves the usage of phosgene which is toxic by acute (short term) inhalation and likely carcinogenic.

The prior art processes described above for the preparation of Daprodustat have major drawbacks such as difficulties with respect to removal of process related impurities, poor commercial viability, use of column chromatography and / or low yields and purity of intermediates and final product.

In addition, US 11117871 discloses crystalline Form CS1 and Form CS9 of Daprodustat.

WO 2020102302 discloses crystalline Form 1, Form 2, Form 3 and Form 4 of Daprodustat.

WO 2021031102 discloses Daprodustat crystalline Form M and Form K.

Considering the importance of Daprodustat and its use in anemia treatment, nevertheless, besides the known forms of Daprodustat, there is a need to develop an alternate process for the preparation of Daprodustat in pure crystalline form which is stable as an Active Pharmaceutical Ingredient (API) and during formulation.

In an attempt, to improve the purity and yield of Daprodustat, the inventors of the present invention found an alternate process to prepare Daprodustat, which is less expensive, industrially feasible, involves simple chemical conversion. The present invention overcomes the above disadvantages and provides Daprodustat, which is recrystallized to obtain pure and stable crystalline form of Daprodustat with high yield.

OBJECTIVE OF THE INVENTION
The objective of the present invention is to provide a simple and cost-effective process for the preparation of Daprodustat of Formula (I).

Another objective of the present invention is to provide novel crystalline forms of Daprodustat and process for the preparation thereof.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a process for the preparation of Daprodustat of Formula (I), which comprises:

(i) reacting the compound of Formula (B) or pharmaceutically acceptable salt thereof with compound of Formula (D) or pharmaceutically acceptable salt thereof to obtain Daprodustat ester compound of Formula (E);
; ;
wherein, R is C1-6 alkyl or aryl.

(ii) converting the Daprodustat ester compound of Formula (E) to obtain Daprodustat of Formula (I);
(iii) optionally, purifying Daprodustat of Formula (I).

In another aspect, the present invention provides a process for the preparation of Daprodustat of Formula (I), which comprises:
(i) reacting the compound of Formula (B) or pharmaceutically acceptable salt thereof with compound of Formula (F) to obtain the compound of Formula (G) or pharmaceutically acceptable salt thereof;
; ;
wherein, R1 is OMe, OEt, OPh, imidazole.
(ii) optionally, hydrolyzing the compound of Formula (G) or pharmaceutically acceptable salt thereof to obtain compound of Formula (J);

(iii) reacting the compound of Formula (G) or pharmaceutically acceptable salt thereof (or) the compound (J) with glycine ester compound of Formula (H) or pharmaceutically acceptable salt thereof to obtain Daprodustat ester compound of Formula (E);
;
wherein, R is same as defined above;
(iv) converting the Daprodustat ester compound of Formula (E) to Daprodustat of Formula (I);
(v) optionally, purifying Daprodustat of Formula (I).

In one aspect, the present invention provides a compound of Formula (G) or pharmaceutically acceptable salt thereof,

wherein R1 is same as defined above.

In another aspect, the present invention provides a compound of Formula (J) or pharmaceutically acceptable salt thereof,

In one aspect, the present invention provides crystalline forms of Daprodustat, having PXRD pattern as shown in figures 1, 2 & 3 respectively.

In one aspect, the present invention also provides a process for the purification of Daprodustat to produce a crystalline form of Daprodustat (DAD-8), comprising:
a) providing Daprodustat in a solvent or mixture of solvents;
b) adding a base;
c) adjusting pH with an acid;
d) optionally, adding a second solvent;
e) isolating the crystalline form of Daprodustat.

In another aspect, the present invention also provides a process for the purification of Daprodustat to produce a crystalline form of Daprodustat (DAD-10), comprising:
a) providing Daprodustat in a solvent or mixture of solvents;
b) optionally, adding the above reaction mass to an anti-solvent;
c) optionally, isolating the crystalline solid;
d) optionally, dissolving in a second solvent or mixture of solvents at a suitable temperature;
e) cooling and isolating the crystalline form of Daprodustat.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: PXRD pattern of crystalline Form of Daprodustat (DAD-8);
Figure 2: PXRD pattern of crystalline Form of Daprodustat (DAD-10);
Figure 3: PXRD pattern of crystalline Form of Daprodustat (DAD-6).

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for preparation of Daprodustat of Formula (I) comprises, reacting the barbituric acid compound of Formula (B) or pharmaceutically acceptable salt thereof with imidazole substituted glycine ester compound of Formula (D) or pharmaceutically acceptable salt thereof using a base in the presence or absence of a solvent to obtain Daprodustat ester compound of Formula (E) followed by treating Daprodustat ester with a base or an acid in the presence or absence of a solvent to obtain Daprodustat and optionally purifying Daprodustat of Formula (I).

Daprodustat ester compound of Formula (E) is isolated as a solid or can be directly used in the next step without isolation.

The base used in the above process comprises an organic base selected from lithium methoxide, sodium methoxide, potassium methoxide, tetrabutyl ammonium methoxide, lithium isopropoxide, triethylamine, diisopropyl ethyl amine, methyl amine, dimethyl amine or mixtures thereof or an inorganic base selected from alkaline or alkaline earth metal hydroxide such as sodium hydroxide, potassium hydroxide, cesium hydroxide, calcium hydroxide, magnesium hydroxide or mixtures thereof; alkaline or alkaline earth metal carbonate such as sodium carbonate, potassium carbonate, cesium carbonate, calcium carbonate, magnesium carbonate and similar or mixtures thereof.

The acid used in the above reaction comprises an inorganic acid selected from HCl, HBr, phosphoric acid, sulfuric acid or an organic acid selected from methanesulfonic acid, para toluene sulfonic acid, acetic acid, formic acid.

The solvent used for the above reaction and purification steps comprises water, alcohols, nitriles, halogenated hydrocarbons, hydrocarbons, amides, sulfoxides, nitriles, esters, ethers, ketones and mixtures thereof. The alcohols comprises C1-6 alcohols comprises methanol, ethanol, butanol, isopropanol; nitriles comprises acetonitrile, propionitrile; halogenated hydrocarbons comprises methylene chloride, ethylene chloride, chloroform; hydrocarbons comprises hexane, cyclohexane, toluene, xylene; amides comprises dimethyl formamide, dimethyl acetamide, N-methyl pyrrolidinone; sulfoxides comprises dimethyl sulfoxide; esters comprises ethyl acetate and butyl acetate; ethers comprises diethyl ether, diisopropyl ether, t-butyl methyl ether, 1,4-dioxane, tetrahydrofuran; ketones comprises acetone, methyl ethyl ketone, methyl isopropyl ketone, methyl tertiary butyl ketone or mixtures thereof.

The pharmaceutically acceptable salts thereof in above process comprises hydrochloride, hydrobromide, sulphate, bisulphate, para toluene sulphonate and methane sulphonate.

In one embodiment, the present invention also provides a process for the preparation of Daprodustat of Formula (I) comprises, reacting the barbituric acid compound of Formula (B) or pharmaceutically acceptable salt thereof with disubstituted carbonyl compound of Formula (F) using a base and in the presence or absence of a solvent to obtain the substituted carbonyl barbituric acid compound of Formula (G) or pharmaceutically acceptable salt thereof. The compound of Formula (G) is optionally hydrolyzed by using an acid or a base in presence or absence of a solvent to obtain an acid compound of formula (J). The compound obtained in the previous step i.e. compound of Formula (G) or Formula (J) is treated with glycine ester compound of Formula (H) or pharmaceutically acceptable salt thereof to obtain Daprodustat ester compound of Formula (E) followed by treating Daprodustat ester with a base or an acid in the presence or absence of a solvent to obtain Daprodustat and optionally purifying Daprodustat of Formula (I).

The solvent(s), base and acid used in the above reaction are the same as described above.

The process of reacting compound (J) with glycine ester compound of Formula (H) in the above process is carried out using coupling agent, base and in the presence or absence of a solvent.

The coupling agent used in the above process is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC. HCl), dicyclohexyl carbodiimide (DCC), carbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI) and like in the presence of additives like hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 4-(dimethylamino)pyridine (DMAP).

The base and solvent used in the above process are the same as described above.

In another embodiment, the present invention provides a substituted carbonyl barbituric acid compound of Formula (G) or pharmaceutically acceptable salt thereof.

wherein, R1 is OMe, OEt, OPh, imidazole.

In yet another embodiment, the present invention specifically encompasses an acid compound of Formula (J) or pharmaceutically acceptable salt thereof.

In another preferred embodiment, the present invention provides a crystalline form of Daprodustat (DAD-8), characterized by its powder X-ray diffraction (PXRD) pattern having peaks at 5.5; 10.2; 11.0 and 20.5 ± 0° 2?.

The crystalline form of Daprodustat (DAD-8) is further characterized by its PXRD pattern having additional peaks at 8.2; 15.5; 22.1 and 24.4 ± 0.2° 2?, wherein PXRD pattern is characterized as shown in Fig-1.

In another embodiment, the present invention also provides a process for the purification of Daprodustat to produce a crystalline form of Daprodustat (DAD-8), which comprises Daprodustat in a solvent or mixture of solvents is treated with a base followed by adjusting pH with an acid and then optionally adding a second solvent and isolating the crystalline form of Daprodustat (DAD-8).

The solvent and second solvent used in the above process comprises alcohols selected from methanol, ethanol, n-propanol, isopropanol and ethers selected from tetrahydrofuran, 2-methyl tetrahydrofuran, water or mixtures thereof, whereas, the base and the acid used in the this reaction are same as described above.

Daprodustat used in the above process is crystalline solid or amorphous.

In another embodiment, the present invention provides a crystalline form of Daprodustat (DAD-10), wherein PXRD pattern is characterized as shown in Fig-2.

In another embodiment, the present invention provides a crystalline form of Daprodustat (DAD-6), wherein PXRD pattern is characterized as shown in Fig-3.

The Powder X-ray diffraction (PXRD) pattern measured on an X-ray diffractometer (instrument name Bruker D8 advance-Eco with lynex detector equipped with Cu source ?=1.58Å) measured using CuKa radiation. Methodology of X-ray diffraction is as follows:

Scanning Type: Continuous scan; Scan range: at least 3-40° 2theta; Step size: 0.8°; Time/Step: 0.05 sec; Divergence slit: V20; Rotation: 30 rpm.

In yet another embodiment, the present invention provides a process for the preparation of a crystalline form of Daprodustat (DAD-6), which comprises dissolving Daprodustat in a solvent at a temperature ~ 75? C to 85? C, followed by adding the reaction mass to anti-solvent at a suitable temperature , then filtering and drying the reaction mass to obtain a crystalline form of Daprodustat (DAD-6).

In yet another embodiment, the present invention provides a process for the preparation of a crystalline form of Daprodustat (DAD-10), which comprises dissolving the crystalline form of Daprodustat (DAD-6) in a second solvent followed by cooling to a temperature of -45? C to -55? C and dried to obtain the crystalline form of Daprodustat (DAD-10).
In yet another embodiment, the present invention provides a process for the preparation of a crystalline form of Daprodustat (DAD-10), which comprises dissolving Daprodustat in a solvent or mixture of solvents at temperature ~ 55? C to 80? C followed by cooling to a temperature of -45? C to -55? C and then dried to obtain crystalline form of Daprodustat (DAD-10).

Daprodustat used in the above processes is crystalline solid or amorphous.

The solvent, second solvent and anti-solvent used in the above processes comprising water, alcohol selected from methanol, ethanol, isopropanol and alkanes selected from n-pentane, n-heptane, n-hexane and mixture thereof.

The following example(s) illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention.

EXAMPLE - 1:
Preparation of 1,3-dicyclohexyl-2,4,6(1H, 3H,5H)-pyrimidinetrione
Process – 1:
Dicyclohexyl urea (25 grams, 1.0 m.eq), malonic acid (14.6 grams, 1.25 m.eq) were added to a mixture of acetic acid (125ml) and acetic anhydride (150ml) at 20-30°C. The reaction mass was heated to 50-60°C and stirred for 16-18 hours at 50-60°C to complete the reaction. The reaction mass was cooled to 0-10°C and the solid mass was stirred for 1hour. The solid was filtered and washed with pre-cooled acetic acid and then dried under vacuum at 50-60°C to obtain title product.
Yield: ~ 77%

Process – 2:
Dicyclohexyl carbodimide (250 grams, 1.0 m.eq) was suspended in THF (2500 ml). A solution of malonic acid (63 grams, 0.5 m.eq malonic acid dissolved in 250 ml THF) was added drop wise slowly at 0-10°C to the above reaction mass. The reaction mass temperature was raised to 20-30°C and stirred for 3-5 hours. After completion of the reaction, the solid was filtered and washed with THF (250 ml). The filtrate was concentrated under vacuum at below 50°C. Methanol (1250 ml) was added to the concentrated mass at 20-30°C and stirred for 2 hours. The solid was filtered and washed with methanol (250 ml) and then dried under vacuum at 50-60°C to obtain 1,3-dicyclohexyl-2,4,6(1H, 3H,5H)-pyrimidinetrione.
Yield:~ 74%

EXAMPLE - 2:
Preparation of Glycine methyl ester HCl
Glycine (200 grams, 1.0 m.eq) was suspended in methanol (2000 ml), then thionyl chloride (1268 grams, 4.0 eq) was slowly added drop wise at 0-10°C. The reaction mass was heated to 60-70°C and stirred at 60-70°C for 7 hours to complete the reaction. Then, the reaction mass was cooled to 0-10°C and the solid mass was stirred for 1 hour. The solid was filtered and washed with pre-cooled methanol and then dried under vacuum at 45-55°C to obtain Glycine methyl ester HCl.
Yield: ~82%

EXAMPLE - 3:
Preparation of methyl (1H-imidazole-1-carbonyl)glycinate
Triethyl amine (20.14 grams, 1.0 m.eq) was slowly added dropwise to the reaction mass of glycine methyl ester HCl (25 grams, 1.0 m.eq), 1,1'-Carbonyldiimidazole (48.5 grams, 1.5 m.eq) in methylene chloride (250 ml) at 20-30°C. The reaction mass was stirred at 20-30°C for 5 hours and reaction was monitored by TLC. After completion of the reaction, the reaction mass was quenched with water (250 ml) at 20-30°C and extracted with methylene chloride (2 x 50ml). Then, the organic layer was concentrated at a temperature below 45°C to obtain crude product. The crude product was then crystallized in ethyl acetate (100 ml) to obtain methyl-(1H-imidazole-1-carbonyl)glycinate.

Yield: ~41%

EXAMPLE – 4:
Preparation of Daprodustat methyl ester
Methyl-(1H-imidazole-1-carbonyl)glycinate (28.2 grams, 1.50 m.eq) was added drop wise to the reaction mass of dicyclohexyl pyrimidinetrione (30 grams, 1.0 m.eq) and diisopropyl ethyl amine( 26.5g, 2.0 m.eq) in methylene chloride (300 ml) at 20-30°C. The reaction mass was stirred at 20-30°C for 16-18 hours to complete the reaction. Then, the reaction mass was quenched with water (300 ml) at 20-30°C and stirred for 30 minutes. Organic layer was separated and concentrated under vacuum at temperature below 40°C to obtain Daprodustat methyl ester as thick oily mass. Weight: ~80g
Yield: ~80 grams

EXAMPLE – 5:
Preparation of Daprodustat API
Aqueous sodium hydroxide solution (16.5 grams, 4.0 m.eq) (dissolved 16.5g sodium hydroxide in 150 ml of purified water) was added drop wise to Daprodustat methyl ester (~80 grams) in methanol (300 ml) at 20-30°C. Then, the reaction mass was stirred tat 20-30°C for 3-5 hour to complete the reaction. After completion of the reaction, pH of the reaction mass adjusted to 1.5-2.0 with Conc. HCl (~40 ml) at 10-30°C. The solid product was then stirred at 20-30°C for 2 hours and then filtered and washed with water (100 ml). Wet solid was charged in purified water (600 ml) at 20-30°C and stirred the slurry mass at 20-30°C for 1 hour. Then, the solid product was filtered and washed with purified water (100 ml). Further, wet solid was charged in acetic acid (600 ml) at 20-30°C and heated the reaction mass to 110-120°C and then stirred for 1 hour. The clear solution was filtered at 110-120°C through hyflo bed and then cooled to 20-30°C. The solid product was stirred at 20-30°C for 3-5 hours. The product was filtered and washed with acetic acid (60 ml) and then dried under vacuum at 50-60°C to obtain Daprodustat API.
Yield: ~50%

EXAMPLE – 6:
Preparation of Daprodustat Form DAD - 8
Daprodustat (200 mg) was dissolved in mixture of methanol (6 ml) and THF (4 ml) at 65°C and then KOH (20 mg) dissolved in water (1 ml) was added. Then the solvent was distilled under vacuum at 65°C and methanol (3 ml) was added to dissolve the obtained solid, the reaction mass was stirred for 30-40 minutes. Then, the pH was adjusted to 4-5 using acetic acid and water and stirred at 25-35°C for 45-50 minutes. Then, the suspension was filtered and re-slurried in water for 7-8 hours at 25-35°C. The obtained solid was filtered and suck dried for 10-15 minutes, followed by drying at 50°C for 4-5 hours and analyzed by PXRD [Ref: Figure – 1].

EXAMPLE – 7:
Preparation of Daprodustat Form DAD - 10
Daprodustat (250 mg) was dissolved in methanol (27 ml) at reflux temperature, then suddenly cooled to -50°C to -60°C. Obtained solid was filtered and suck dried for 10-15mins at room temperature, followed by drying at 40°C under vacuum for 2-3 hours and then analyzed by PXRD [Ref: Figure – 2].

EXAMPLE – 8:
Preparation of Daprodustat Form DAD - 10
Daprodustat (2 grams) was dissolved in IPA (60 ml) at ~80°C and then the reaction mass was added to pre-cooled n-pentane (2°C). The reaction mass was maintained under stirring for 15-20 hours at 2°C. The obtained solid was then filtered and suck dried for 10-15 minutes under vacuum to obtain crystalline solid of Daprodustat (Form DAD-6) and was analyzed by PXRD [Ref: Figure – 3].

Crystalline solid of Daprodustat (500 mg) was dissolved in methanol (30 ml) at 65°C and then suddenly cooled to -50 °C. The reaction mass was stirred for 30 minutes at -50°C, the obtained solid was filtered and washed with pre-cooled hexane (15ml). The solid product was suck dried for 10-15 minutes under vacuum, followed by drying at 40°C for 2-3 hours and was analyzed by PXRD [Ref: Figure – 2]. ,CLAIMS:CLAIMS
We claim,
1. A process for the preparation of Daprodustat of Formula (I), which comprises:

(i) reacting the compound of Formula (B) or pharmaceutically acceptable salt thereof with a compound of Formula (D) or pharmaceutically acceptable salt thereof to obtain Daprodustat ester compound of Formula (E);
; ;
wherein, R is C1-6 alkyl or aryl,
(ii) converting the Daprodustat ester compound of Formula (E) to obtain Daprodustat of Formula (I);
(iii) optionally, purifying Daprodustat of Formula (I).

2. A process for the preparation of Daprodustat of Formula (I), which comprises:
(i) reacting the compound of Formula (B) or pharmaceutically acceptable salt thereof with a compound of Formula (F) to obtain a compound of Formula (G) or pharmaceutically acceptable salt thereof:
; ;
wherein, R1 is OMe, OEt, OPh, imidazole,
(ii) optionally, hydrolyzing the compound of Formula (G) or pharmaceutically acceptable salt thereof to obtain compound of Formula (J);

(iii) reacting the compound of Formula (G) or pharmaceutically acceptable salt thereof (or) the compound (J) with glycine ester compound of Formula (H) or pharmaceutically acceptable salt thereof to obtain Daprodustat ester compound of Formula (E);
;
wherein, R is same as defined above;
(iv) converting the Daprodustat ester compound of Formula (E) to Daprodustat of Formula (I);
(v) optionally, purifying Daprodustat of Formula (I).
3. The process as claimed in claim 2, wherein hydrolysis at step (ii) is carried out in the presence of acid or a base.

4. The process as claimed in claims 2 and 3, wherein the acid comprises inorganic acid selected from HCl, HBr, phosphoric acid, sulfuric acid or an organic acid selected from methanesulfonic acid, para toluene sulfonic acid, acetic acid and formic acid.

5. The process as claimed in claims 2 and 3, wherein the base comprises an organic base selected from triethylamine, diisopropyl methyl amine, methyl amine, dimethyl amine or mixtures thereof or an inorganic base selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, calcium carbonate, magnesium carbonate or mixtures thereof.

6. An intermediate compound of Daprodustat comprises;
a) a compound of Formula (G) or pharmaceutically acceptable salt thereof;

wherein R1 is OMe, OEt, OPh, imidazole;
b) a compound of Formula (J) or pharmaceutically acceptable salt thereof,

7. A process for the purification of Daprodustat to produce a crystalline form of Daprodustat (DAD-8), comprising:
a) providing Daprodustat in a solvent or mixture of solvents;
b) adding a base;
c) adjusting pH with an acid;
d) optionally, adding a second solvent;
e) isolating the crystalline form of Daprodustat.

8. A process for the purification of Daprodustat to produce a crystalline form of Daprodustat (DAD-10), comprising:
a) providing Daprodustat in a solvent or mixture of solvents;
b) optionally, adding the above reaction mass to an anti-solvent;
c) optionally, isolating the crystalline solid;
d) optionally, dissolving in a second solvent or mixture of solvents at a suitable temperature;
e) cooling and isolating the crystalline form of Daprodustat.

9. The process as claimed in claims 7 and 8, the crystalline form of Daprodustat characterized by XRPD pattern as depicted in Figure 1, Figure 2 and Figure 3.

10. The process as claimed in claims 8 and 9, wherein the solvent, second solvent and anti-solvent comprise water, C1-6 alcohols selected from methanol, ethanol, butanol, isopropanol; ethers comprise diethyl ether, diisopropyl ether, tetrahydrofuran; the anti-solvent comprises alkanes selected from n-pentane, n-heptane, n-hexane and mixture thereof.