FORM-2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
[See section 10, rule 13]
A novel process for the preparation of Cytidine 5'-diphosphate choline and its monosodium salt
APPLICANT:
HERBERT BROWN PHARMACEUTICAL & RESEARCH LABORATORIES
W-256/257/258A, M.I.D.C. Phase II, Shivaji UdyogNagar, Dombivli (E)-421203, District- Thane, Maharashtra, India.
Indian Company incorporated under the Companies Act 1956
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention relates to a novel process for the preparation of Cytidine 5'-diphosphate choline and its monosodium salt, avoiding use of ion exchange resin for purification. More particularly the present invention relates to a novel process for the preparation of monosodium salt of Cytidine 5'-diphosphate choline having HPLC purity of more than 99.5%, avoiding use of ion exchange resin for purification.
BACKGROUND OF THE INVENTION
Cytidine 5'-diphosphate choline of Formula I, hereinafter abbreviated as CDP-choline, is a psychostimulant or nootropic.

CDP-choline is used for treatment of Alzheimer's disease and other types of dementia, head trauma, cerebrovascular disease such as stroke, age-related memory loss, Parkinson's disease, and glaucoma. CDP-choline is administered as free compound or as a pharmaceutically acceptable salt thereof.
CDP-choline has conventionally been prepared by extracting it from living tissues and purifying the extract. The known processes for preparation of CDP-choline can broadly be classified into two types: chemical process and enzymatic process.

Journal of Biological Chemistry, 222, 185 (1956) discloses a process for preparation of CDP-choline by condensation of cytidine 5'-monophosphate with choline phosphate in hydrous pyridine in presence of dicyclohexylcarbodiimide. After completion of the reaction the semisolid obtained was washed with water and the washings were collected and extracted with ether. The pH of aqueous layer was adjusted to 8-9 followed by elution of the CDP-choline from ion exchange resin column (Dowex 1 x 2 in formate form) with 0.04N formic acid.
Chemical and Pharmaceutical Bulletin 19 (12), 2466-2471 (1971) discloses a process for preparation of CDP-choline by condensation of cytidine 5' -monophosphate with choline phosphate in dimethylformamide in presence of p-toluenesulfonyl chloride. After completion of the reaction, water was added to the reaction mixture. The pH of this aqueous mixture was adjusted followed by introduction to the ion exchange resin column (Dowex 1 x 4 in formate form). The CDP-choline was then eluted from this ion exchange resin column using 0.01M formic acid.
Chemical and Pharmaceutical Bulletin 19(5), 1011-1016, (1971) discloses a process for preparation of CDP-choline by condensation of cytidine 5'-monophosphate with choline phosphate in dimethylformamide in presence of thionyl chloride. After completion of the reaction, water was added to the reaction mixture. The pH of the aqueous mixture was adjusted and then the mixture was introduced to the ion exchange resin column (Dowex 1 x 4 in formate form). The CDP-choline was then eluted from the ion exchange resin column using 0.01M formic acid.
US3787392 describes an improved process for preparation of CDP-choline by reacting cytidine 5'-monophosphate with calcium salt of phosphorylcholine chloride in methanol in presence of morpholine and dicyclohexylcarbodiimide, wherein the

improvement lies in slowly bringing together a methanolic solution of cytidine 5'-monophosphate, morpholine and dicyclohexylcarbodiimide with methanolic solution of calcium salt of phosphorylcholine chloride and hydrochloric acid. After completion of the reaction, the product was precipitated using isopropanol. The crude obtained was purified using anion exchange resin (Dowex 1 x 2 in formate form).
Indian patent application No. 192/MUM/2012 discloses a process for preparation of CDP-choline sodium wherein cytidine 5'-monophosphate is condensed with morpholine in presence of coupling agent and organic solvent to obtain morpholidate. The morpholidate thus obtained was further condensed with calcium salt of phosphorylcholine chloride which leads to formation of calcium chloride salt of CDP-choline. The calcium chloride salt thereby obtained was subjected to purification first by using cation exchange resin and then by anion exchange resin to obtain pure CDP-choline. The CDP-choline thus obtained was then treated with sodium hydroxide to form sodium salt of CDP-choline.
US2009286284 provides a process for purification of CDP-choline comprising steps of contacting a CDP-choline solution having pH 0.5 to 5 containing nucleic acid analogue with H-type strongly acidic cation exchange resin and eluting CDP-choline adsorbed onto the resin with water or aqueous solution having ion concentration less than 0.1 mol/liters.
Almost in all the processes of prior art after completion of reaction, CDP-choline is precipitated by addition of anti-solvent like isopropanol to the reaction mixture, thereby making recovery and reuse of solvents difficult. Also, due to precipitation of crude CDP-choline number of operations like isolation and drying increases thereby requiring large amount of time and energy.

Furthermore, in all the above described processes, the aqueous solution of crude CDP-choline is purified by ion exchange resin. Use of ion exchange resin suffers from several drawbacks listed below
i) the large volumes of water is required for loading, washing and eluting the
desired product ii) the large volumes of eluent comprising desired product is to be
concentrated iii) the huge amount of energy is required to concentrate the eluent
comprising desired product iv) the need to regenerate the resin column after every use in order to
maintain the ion exchange capacity v) the time cycle for ion exchange resin chromatography is long
All these above discussed drawbacks increases the over all time cycle and cost of the process and also indicates that the process is not environment friendly as it generates huge amount of effluent and is less feasible on industrial scale.
There are also enzymatic processes for preparation of CDP-choline discussed in literatures, some of the references are Bioresource Technology 101 (22), 8807-8813 (2010), Bioscience, Biotechnology, and Biochemistry 61(6), 956-959 (1997), USRE28886, US6387667, GB1294769, US20050164359. However these enzymatic processes are difficult to handle for large scale production.
None of the processes known in prior art gives an alternate process for preparation CDP-choline, avoiding use of ion exchange resin for purification. There is, therefore, an unfulfilled need for developing alternative process for preparation of CDP-choline and its monosodium salt avoiding the use of tedious ion exchange resin for

purification and allowing industrial preparation of this product in simplified and economic manner and in shorter period of time.
OBJECT OF THE INVENTION
1. An object of the present invention is to provide a novel process for the preparation of CDP-choline and its monosodium salt with substantial purity, avoiding use of ion exchange resin for purification
2. Another object of the present invention is to provide a monosodium salt of CDP-choline having HPLC purity of more than 99.5%.
3. Yet another object of the present invention is to provide a novel process for preparation of CDP-choline and its monosodium salt using single organic solvent for the reaction and the isolation of crude CDP-choline.
4. Yet another object of the present invention is to provide a process for preparation of CDP-choline and its monosodium salt wherein the solvent used in the process can be recovered and reused.
5. Yet another object of the present invention is to provide simple, economic and industrially feasible process for preparation of CDP-choline and its monosodium salt, avoiding use of ion exchange resin for purification.
SUMMARY OF THE INVENTION
According to an aspect of the present invention, there is provided a novel process for the preparation of CDP-choline of Formula I and its monosodium salt of Formula II,


avoiding use of ion exchange resin for purification.
comprising,
a) reacting cytidine 5'-monophosphate with morpholine in presence of dicyclohexylcarbodiimide in methanol at 20-60°C for 3 to 6 hours to form cytidine 5'-monophosphate morpholidate of Formula III


b) insitu condensing cytidine 5'-monophosphate morpholidate of Formula III with phosphorylcholine chloride calcium salt in methanol in presence of hydrochloric acid at 20-60°C for 3 to 6 hours
c) recovering the methanol used in steps a) and b) to obtain a thick semisolid mass of crude CDP-choline
d) dissolving or suspending the thick semisolid mass in water and optionally, removing insolubles by filtration
e) adding a calcium precipitating agent to precipitate water insoluble calcium salt
f) removing the water insolubles formed in step d) or e) by filtration to obtain a clear solution
g) subjecting the clear solution to nanofiltration and subsequently to charcoalization by adjusting the pH to 2 to 5 to obtain a solution comprising CDP-choline of Formula I
h) concentrating the solution comprising CDP-choline of Formula I to obtain a
concentrated solution i) optionally, converting the CDP-choline in the concentrated solution into its
monosodium salt and isolating thus formed monosodium salt of CDP-choline
of Formula II by adding organic solvent selected from a group consisting of
alcohol, ketone or mixture thereof.
According to another aspect of the present invention, there is provided an alternate novel process for the preparation of CDP-choline of Formula I and its monosodium salt of Formula II, avoiding use of ion exchange resin for purification


comprising,
a) reacting cytidine 5'-monophosphate with morpholine in presence of dicyclohexylcarbodiimide in methanol at 20-60°C for 3 to 6 hours to form cytidine 5'-monophosphate morpholidate of Formula III


b) insitu condensing cytidine 5'-monophosphate morpholidate of Formula III with phosphorylcholine chloride calcium salt in methanol in presence of hydrochloric acid at 20-60°C for 3 to 6 hours
c) recovering the methanol used in steps a) and b) to obtain thick semisolid mass of crude CDP-choline
d) dissolving or suspending the thick semisolid mass in water and optionally, removing insolubles by filtration
e) adding a calcium precipitating agent to precipitate water insoluble calcium salt
f) removing the water insolubles formed in step d) or e) by filtration to obtain a clear solution
g) subjecting the clear solution to charcoalization and subsequently to nanofiltration by adjusting the pH to 2 to 5 to obtain a solution comprising CDP-choline of Formula I
h) concentrating the solution comprising CDP-choline of Formula I to obtain a
concentrated solution i) optionally, converting the CDP-choline in the concentrated solution into its
monosodium salt and isolating thus formed monosodium salt of CDP-choline
of Formula II by adding organic solvent selected from a group consisting of
alcohol, ketone or mixture thereof.
According to yet another aspect of the present invention, the monosodium salt of CDP-choline obtained in step i) can be further purified with a mixture of water and an organic solvent selected from group consisting of alcohol, ketone or mixture thereof.
DETAILED DESCRIPTION OF INVENTION
The present invention relates to a novel process for the preparation of CDP-choline of

Formula I and its monosodium salt of Formula II, avoiding use of ion exchange resin for purification

comprising,
a) reacting cytidine 5'-monophosphate with morpholine in presence of dicyclohexylcarbodiimide in methanol at 20-60°C for 3 to 6 hours to form cytidine 5' -monophosphate morpholidate of Formula III

b) insitu condensing cytidine 5'-monophosphate morpholidate of Formula III
with phosphorylcholine chloride calcium salt in methanol in presence of
. hydrochloric acid at 20-60°C for 3 to 6 hours
c) recovering the methanol used in steps a) and b) to obtain a thick semisolid mass of crude CDP-choline
d) dissolving or suspending the thick semisolid mass in water and optionally, removing insolubles by filtration
e) adding a calcium precipitating agent to precipitate water insoluble calcium salt
f) removing the water insolubles formed in step d) or e) by filtration to obtain a clear solution
g) subjecting the clear solution to nanofiltration and subsequently to charcoalization by adjusting the pH to 2 to 5 to obtain a solution comprising CDP-choline of Formula I
h) concentrating the solution comprising CDP-choline of Formula I to obtain a
concentrated solution i) optionally, converting the CDP-choline in the concentrated solution into its
monosodium salt and isolating thus formed monosodium salt of CDP-choline
of Formula II by adding organic solvent selected from a group consisting of
alcohol, ketone or mixture thereof.
The present invention further relates to an alternate novel process for the preparation of CDP-choline of Formula I and its monosodium salt of Formula II, avoiding use of ion exchange resin for purification


comprising,
a) reacting cytidine 5' -monophosphate with morpholine in presence of dicyclohexylcarbodiimide in methanol at 20-60°C for 3 to 6 hours to form cytidine 5'-monophosphate morpholidate of Formula III


b) insitu condensing cytidine 5'-monophosphate morpholidate of Formula III with phosphorylcholine chloride calcium salt in methanol in presence of hydrochloric acid at 20-60°C for 3 to 6 hours
c) recovering the methanol used in steps a) and b) to obtain thick semisolid mass of crude CDP-choline
d) dissolving or suspending the thick semisolid mass in water and optionally, removing insolubles by filtration
e) adding a calcium precipitating agent to precipitate water insoluble calcium salt
f) removing the water insolubles formed in step d) or e) by filtration to obtain a clear solution
g) subjecting the clear solution to charcoalization and subsequently to nanofiltration by adjusting the pH to 2 to 5 to obtain a solution comprising CDP-choline of Formula I
h) concentrating the solution comprising CDP-choline of Formula I to obtain a
concentrated solution i) optionally, converting the CDP-choline in the concentrated solution into its
monosodium salt and isolating thus formed monosodium salt of CDP-choline
of Formula II by adding organic solvent selected from a group consisting of
alcohol, ketone or mixture thereof.
The process for the preparation of monosodium salt of CDP-choline can be depicted in scheme I

According to an embodiment of the present invention, the amount of dicyclohexylcarbodiimide used with respect to cytidine-5'-monophosphate is in the range of 1.5 to 2.5 moles equivalent.
The amount of morpholine used with respect to cytidine 5'-monophosphate is in the range of 1.5 to 3.5 moles equivalent, preferably 2.5 to 3 moles equivalent.
The amount of phosphorylcholine chloride calcium salt used with respect to cytidine 5'-monophosphate is in the range of 1.5 to 3 moles equivalent, preferably 2 to 2.5 moles equivalent.
The total amount of methanol used in the step a) and b) with respect to cytidine 5'-monophosphate is in the range of 10 to 50 volumes, preferably 20 to 40 volumes.
According to an embodiment of the present invention, in step c) more than 80% of the total amount of the methanol used in step a) and step b) is recovered.
The inventors of present invention have skillfully developed a process for preparing CDP-choline using single solvent system, wherein the low boiling solvent like methanol is used. Unlike in prior art processes, after completion of reaction the crude CDP-choline is not isolated using anti-solvent instead the methanol in the reaction mixture is recovered quantitatively by known processes like distillation under vacuum. The recovered solvent is used in the next batch, thus making the process highly economical at production scale.
During the course of reaction dicyclohexylurea is formed as a by-product. The water insoluble dicyclohexylurea is separated from water soluble CDP-choline by dissolving or suspending the thick semisolid mass of crude CDP-choline in water.

According to another embodiment of the present invention, in step d) the water insoluble dicyclohexylurea is removed by filtration and is quantitatively recovered with respect to dicyclohexylcarbodiimide used. The dicyclohexylurea, thus recovered can be converted to dicyclohexylcarbodiimide by the known methods and can be reused in the reaction, thereby making the process economical and environment friendly.
According to another embodiment of the present invention, in step e) the calcium precipitating agent is added to remove by precipitation calcium present in the crude CDP-choline in form of water insoluble calcium salt.
The calcium precipitating agent like oxalic acid, sodium phosphate, sodium hydrogen phosphate, potassium phosphate, potassium hydrogen phosphate is used, preferably oxalic acid is used.
The inventors of present invention studied use of different precipitating agent to facilitate maximum removal of calcium. An appropriate precipitating agent for the present invention is the one that efficiently precipitates calcium in form of water insoluble salt without affecting the quality of final product i.e. CDP-choline. It was observed by inventors of present invention that use of oxalic acid as precipitating agent ensures removal of calcium to the extent of 95% with respect to input calcium content.
The amount of the calcium precipitating agent used with respect to phosphorylcholine chloride calcium salt is in the range of 0.8 to 1.2 moles equivalent.
According to another embodiment of the present invention, in step f) the water

insolubles like dicyclohexylurea and calcium salt formed in step d) and step e) respectively, are removed by filtration.
According to another embodiment of the present invention, the clear solution obtained in step f) is extracted with a water immiscible organic solvent selected from esters like ethyl acetate, halogenated solvents like dichloromethane, chloroform, ethers like diethyl ether or hydrocarbons like hexane, toluene.
According to another embodiment of the present invention in step g), nanofiltration herein refers to passage of solution through nanofiltration membrane followed by washing and flushing the membrane with water at temperature ranging from 20-35°C. to obtain retentate. The nanofiltration membrane used is selected from organic or inorganic membrane. The pore size of the nanofiltration membranes allows a molecular weight cut off of 100-300 Daltons.
According to another embodiment of the present invention, in step g) the nanofilteration ensures removal of low molecular weight compounds.
According to another embodiment of the present invention, in step i) the organic solvent is selected from group consisting of alcohols like methanol, ethanol, propanol, butanol, pentanol, ketones like acetone, methyl ethyl ketone, methyl isobutyl ketone, diethyl ketone or mixture thereof.
According to yet another embodiment of the present invention, the monosodium salt of CDP-choline obtained in step i) can be further purified with a mixture of water and an organic solvent selected from group consisting of alcohol like methanol, ethanol, propanol, butanol, pentanol, ketone like acetone methyl ethyl ketone, methyl isobutyl ketone, diethyl ketone or mixture thereof.

According to yet another embodiment of the present invention, the monosodium salt of CDP-choline obtained has HPLC purity of more than 99.5%.
The detail of the invention provided in the following examples is given by the way of illustration only and should not be construed to limit the scope of the present invention.
EXAMPLES Example 1:
To lOOg of cytidine 5'-monophosphate in 1500ml of methanol was added 74.12g of morpholine. The mixture was heated to 40-45°C and 114.9g of dicyclohexycarbodiimide was added. The mixture thus obtained was heated to 50-55°C for 3-6hrs. To this mixture was then added methanolic solution of phophorylcholine chloride (200g of phosphorylcholine calcium salt tetrahydrate and 88ml of concentrated hydrochloric acid dissolved in 1500ml of methanol) at 50-55°C and the reaction mixture was stirred at same temperature for 3-6hrs. After completion of reaction, the methanol was recovered under vacuum to obtain thick semisolid mass. The thick semisolid mass was dissolved /suspended in water and the insolubles formed were removed by filtration. To the filterate was added 76.47g of oxalic acid dihydrate at 25-30°C and the mixture was stirred for 1 hour. The precipitated calcium oxalate was filtered off and the pH of filterate was adjusted to 2 to 5 using aqueous sodium hydroxide. The clear solution thus obtained was extracted with dichloromethane. The aqueous solution was then subjected to nanofilteration to obtain retentate. The retentate was charcoalized and filtered to obtain a solution comprising CDP-choline. The solution comprising CDP-choline was concentrated under vacuum. The CDP-choline in the concentrated solution was converted into its monosodium salt by using aqueous sodium hydroxide thereby forming solution-A.

The monosodium salt of CDP-choline in the solution-A was precipitated using 2400ml of ethanol. The crude monosodium salt of CDP-choline thus obtained was treated with methanol, filtered and dried to obtain 75g of monosodium salt of CDP-choline having HPLC purity of 99.56% and UV assay of 98.71%. The monosodium salt of CDP-choline obtained is further purified by dissolving in water and once again precipitating it with ethanol. The precipitate obtained was filtered and dried to obtain 72g of monosodium salt of CDP-choline having HPLC purity of 99.77% and UV assay of 99.65%.
Example 2:
The procedure is carried out as example 1 by starting with l00g of cytidine 5'-monophosphate except for following
The monosodium salt of CDP-choline in the solution-A was precipitated using 2400ml of ethanol. The crude monosodium salt of CDP-choline thus obtained was treated with methanol, filtered and dried to obtain 79g of monosodium salt of CDP-choline having HPLC purity of 99.56% and UV assay of 98.92%. The monosodium salt of CDP-choline obtained is further purified by dissolving in water and precipitating it with acetone. The precipitate obtained was filtered and dried to obtain 76g of monosodium salt of CDP-choline having HPLC purity of 99.58% and UV assay of 98.60%.
Example 3:
The procedure is carried out as in example 1 by starting with 50g of cytidine 5'-monophosphate except for following
The monosodium salt of CDP-choline in the solution-A was precipitated using 900ml of (50:50) acetone: methanol mixture. The precipitate was filtered and dried to obtain 43g of monosodium salt of CDP-choline having HPLC purity of 99.80% and UV assay of 96.67%. The monosodium salt of CDP-choline obtained was further purified

by dissolving the monosodium salt of CDP-choline in water and once again precipitating with (50:50) acetone: methanol mixture. The precipitate obtained was filtered and dried to obtain 40g of monosodium salt of CDP-choline having HPLC purity of 99.83% and UV assay of 99.70%. Example 4:
To 50g of cytidine 5'-monophosphate in 750ml of methanol was added 37.06g of morpholine. The mixture was heated to 40-45°C and 57.46g of dicyclohexycarbodiimide was added. The mixture thus obtained was heated to 50-55°C for 3-6hrs. To this mixture was then added methanolic solution of phophorylcholine chloride (l00g of phosphorylcholine calcium salt tetrahydrate and 42.5ml of concentrated hydrochloric acid dissolved in 750ml of methanol) at 50-55°C and the reaction mixture was stirred at the same temperature for 3-6hrs. After completion of reaction, the methanol was recovered under vacuum to obtain thick semisolid mass. The thick semisolid mass was dissolved /suspended in water and the insolubles formed were removed by filtration. To the filterate thus collected was added 38.3g of oxalic acid dihydrate at 25-30°C and the mixture was stirred for 1 hour. The precipitated calcium oxalate was filtered off and the pH of filterate was adjusted to 2 to 5 using aqueous sodium hydroxide. The clear solution thus obtained was extracted with dichloromethane. The aqueous layer was separated and was charcoalized and filtered to obtain clear solution. The clear solution was subjected to nanofilteration to obtain retentate comprising CDP-choline. The retentate comprising CDP-choline was concentrated under vacuum. The CDP-choline in the concentrated solution was converted into its monosodium salt by using aqueous sodium hydroxide thereby forming solution-B.
The monosodium salt of CDP-choline in the solution-B was precipitated using 1200ml of ethanol. The crude monosodium salt of CDP-choline thus obtained was treated with methanol, filtered and dried to obtain 39g of monosodium salt of CDP-choline having HPLC purity 99.62% and UV assay of 98.39%. The monosodium salt

of CDP-choline obtained is further purified by dissolving in water and once again precipitating with ethanol. The precipitate obtained was filtered and dried to obtain 37g of monosodium salt of CDP-choline having HPLC purity of 99.66% and UV assay of 98.54%. Example 5:
The procedure is carried out as in example 4 by starting with 50g of cytidine 5'-monophosphate except for following
The monosodium salt of CDP-choline in the solution-B was precipitated using 1200ml of (50:50) acetone: methanol mixture. The precipitate of monosodium salt of CDP-choline was filtered and dried to obtain 44g of monosodium salt of CDP-choline having HPLC purity 99.39%) and UV assay of 97.63%. The monosodium salt of CDP-choline obtained is further purified by dissolving the monosodium salt of CDP-choline in water and once again precipitating with (50:50) acetone: methanol mixture. The precipitate obtained was filtered and dried to obtain 42g of monosodium salt of CDP-choline having HPLC purity of 99.50%and UV assay of 98.10%.
Example 6:
To 50g of cytidine 5'-monophosphate in 750ml of methanol was added 37.06g of morpholine. The mixture was heated to 40-45°C and at this temperature 57.46g of dicyclohexycarbodiimide was added. The mixture thus obtained was heated to 50-55°C for 3-6hrs. To this mixture was then added methanolic solution of phophorylcholine chloride (l00g of phosphorylcholine calcium salt tetrahydrate and 43ml of concentrated hydrochloric acid dissolved in 750ml of methanol) at 50-55°C and the reaction mixture was stirred at the same temperature for 3-6hrs. After completion of reaction, the methanol was recovered under vacuum to obtain thick semisolid mass. The thick semisolid mass was dissolved /suspended in water. To this slurry was added 38.3g of oxalic acid dihydrate at 25-30°C and the mixture was stirred for 1 hour. The insolubles (comprising dicyclohexylurea and calcium oxalate)

formed were filtered off and the pH of filtrate was adjusted to 2 to 5 using aqueous sodium hydroxide. The clear solution thus obtained was extracted with dichloromethane. The aqueous layer was separated and then subjected to nanofilteration to obtain retentate. The retentate was charcoalized and filtered to obtain a solution comprising CDP-choline. The solution comprising CDP-choline was concentrated under vacuum. The CDP-choline in the concentrated solution was converted into its monosodium salt by using aqueous sodium hydroxide thereby forming solution-C.
The monosodium salt of CDP-choline in the solution-C was precipitated using 1200ml of ethanol. The crude monosodium salt of CDP-choline thus obtained was treated with methanol, filtered and dried to obtain 38g of monosodium salt of CDP-choline having HPLC purity of 99.88% and UV assay 98.13%. The monosodium salt of CDP-choline obtained is further purified by dissolving in water and once again precipitating it with ethanol. The precipitate obtained was filtered and dried to obtain 37g of monosodium salt of CDP-choline having HPLC purity of 99.88% and UV assay of 99.71%
Example 7:
The procedure is carried out as in example 6 by starting with 50g of cytidine 5'-monophosphate except for following
The monosodium salt of CDP-choline in the solution-C was precipitated using 1200ml of (50:50) acetone: methanol mixture. The precipitate was filtered and dried to obtain 43g of monosodium salt of CDP-choline having HPLC purity of 99.79% and UV assay of 98.42%. The monosodium salt of CDP-choline thus obtained was further purified by dissolving the monosodium salt of CDP-choline in water and once again precipitating it with (50:50) acetone: methanol mixture. The precipitate obtained was filtered and dried to obtain 41 g of monosodium salt of CDP-choline having HPLC purity of 99.83% and UV assay of 99.32%.

Example 8:
The procedure is carried out as in example 6 by starting with 50g of cytidine 5'-monophosphate except for following
The monosodium salt of CDP-choline in the solution-C was precipitated using 1200ml of (50:50) acetone: methanol mixture. The crude monosodium salt of CDP-choline precipitated was treated with methanol, filtered and dried to obtain 39g of monosodium salt of CDP-choline having HPLC purity of 99.82% and UV assay of 99.50%. The monosodium salt of CDP-choline thus obtained was further purified by dissolving in water and once again precipitating it with (50:50) acetone: methanol mixture. The precipitate obtained was filtered and dried to obtain 37g of monosodium salt of CDP-choline having HPLC purity of 99.86% and UV assay of 99.89%.
Example 9:
To 50g of cytidine 5'-monophosphate in 750ml of methanol was added 37.06g of morpholine. The mixture was heated to 40-45°C and at this temperature 57.46g of dicyclohexycarbodiimide was added. The mixture thus obtained was heated to 50-55°C for 3-6hrs. To this mixture was then added methanolic solution of phophorylcholine chloride (l00g of phosphorylcholine calcium salt tetrahydrate and 42.5ml of concentrated hydrochloric acid dissolved in 750ml of methanol) at 50-55°C and the reaction mixture was stirred at the same temperature for 3-6hrs. After completion of reaction, the methanol was recovered under vacuum to obtain thick semisolid mass. The thick semisolid mass was dissolved /suspended in water. To this slurry was added 38.3g of oxalic acid dihydrate at 25-30°C and the mixture was stirred for 1 hour. The insolubles (comprising dicyclohexylurea and calcium oxalate) formed were filtered off and the pH of filtrate was adjusted to 2 to 5 using aqueous sodium hydroxide The clear solution thus obtained was extracted with dichloromethane. The aqueous layer was separated and was charcoalized and filtered

to obtain a solution. The solution thus formed was subjected to nanofilteration to obtain retentate. The retentate was concentrated under vacuum. The CDP-choline in the concentrated solution was converted into its monosodium salt by using aqueous sodium hydroxide thereby forming solution-D.
The monosodium salt of CDP-choline in the solution-D was precipitated using 1200ml of ethanol. The crude monosodium salt of CDP-choline thus obtained was treated with methanol, filtered and dried to obtain 39g of monosodium salt of CDP-choline having HPLC purity of 99.86% and UV assay of 98.92%. The monosodium salt of CDP-choline obtained is. further purified by dissolving the monosodium salt of CDP-choline in water and once again precipitating with ethanol. The precipitate obtained was filtered and dried to obtain 37g of monosodium salt of CDP-choline having HPLC purity of 99.84% and UV assay of 98.99%.
Example 10:
The procedure is carried out as in example 9 by starting with 100g-of cytidine 5'-monophosphate except for following
The monosodium salt of CDP-choline in the solution-D was precipitated using 2400ml of (50:50) acetone: methanol mixture. The precipitate of monosodium salt of CDP-choline was filtered and dried to obtain 95g of monosodium salt of CDP-choline with HPLC purity of 99.27% and UV assay of 97.96%. The monosodium salt of CDP-choline was further purified by dissolving it in water and once again precipitating with (50:50) acetone: methanol mixture. The precipitate obtained was filtered and dried to obtain 86g of monosodium salt of CDP-choline having HPLC purity of 99.50% and UV Assay of 99.66%.
Example 11:
The procedure is carried out as in example 9 by starting with 50g of cytidine 5'-monophosphate except for following

The monosodium salt of CDP-choline in the solution-D was precipitated using 960ml of (50:50) acetone: methanol mixture. The crude monosodium salt of CDP-choline thus obtained was treated with methanol, filtered and dried to obtain 41g of monosodium salt of CDP-choline having HPLC purity of 99.84% and UV assay of 98.61%. The monosodium salt of CDP-choline obtained is further purified by dissolving in water and once again precipitating with (50:50) acetone: methanol mixture. The precipitate obtained was filtered and dried to obtain 39g of monosodium salt of CDP-choline having HPLC purity of 99.88% and UV Assay of-99.94%.

We claim
1. A novel process for the preparation of CDP-choline of Formula I and its monosodium salt of Formula II, avoiding use of ion exchange resin for purification

comprising,
a) reacting cytidine 5' -monophosphate with morpholine in presence of dicyclohexylcarbodiimide in methanol at 20-60°C for 3 to 6 hours to form cytidine 5' -monophosphate morpholidate of Formula III


b) insitu condensing cytidine 5'-monophosphate morpholidate of Formula III with phosphorylcholine chloride calcium salt in methanol in presence of hydrochloric acid at 20-60°C for 3 to 6 hours
c) recovering the methanol used in steps a) and b) to obtain a thick semisolid mass of crude CDP-choline
d) dissolving or suspending the thick semisolid mass in water and optionally, removing insolubles by filtration
e) adding a calcium precipitating agent to precipitate water insoluble calcium salt
f) removing the water insolubles formed in step d) or e) by filtration to obtain a clear solution
g) subjecting the clear solution to nanofiltration and subsequently to charcoalization by adjusting the pH to 2 to 5 to obtain a solution comprising CDP-choline of Formula I
h) concentrating the solution comprising CDP-choline of Formula I to obtain a
concentrated solution i) optionally, converting the CDP-choline in the concentrated solution into its
monosodium salt and isolating thus formed monosodium salt of CDP-choline
of Formula II by adding organic solvent selected from a group consisting of
alcohol, ketone or mixture thereof.

2. A novel process for the preparation of CDP-choline of Formula I and its monosodium salt of Formula II, without using ion exchange resin for purification

comprising,
a) reacting cytidine 5' -monophosphate with morpholine in presence of dicyclohexylcarbodiimide in methanol at 20-60°C for 3 to 6 hours to form cytidine 5'-monophosphate morpholidate of Formula III


b) insitu condensing cytidine 5'-monophosphate morpholidate of Formula III with phosphorylcholine chloride calcium salt in methanol in presence of hydrochloric acid at 20-60°C for 3 to 6 hours
c) recovering the methanol used in steps a) and b) to obtain thick semisolid mass of crude CDP-choline
d) dissolving or suspending the thick semisolid mass in water and optionally, removing insolubles by filtration
e) adding a calcium precipitating agent to precipitate water insoluble calcium salt
f) removing the water insolubles formed in step d) or e) by filtration to obtain a clear solution
g) subjecting the clear solution to charcoalization and subsequently to nanofiltration by adjusting the pH to 2 to 5 to obtain a solution comprising CDP-choline of Formula I
h) concentrating the solution comprising CDP-choline of Formula I to obtain a . concentrated solution
i) optionally, converting the CDP-choline in the concentrated solution into its monosodium salt and isolating thus formed monosodium salt of CDP-choline of Formula II by adding organic solvent selected from a group consisting of alcohol, ketone or mixture thereof.

3. The process as claimed in claim 1 or 2, wherein the monosodium salt of CDP-choline obtained in step i) is further purified with a mixture of water and an organic solvent selected from group consisting of alcohol like methanol, ethanol, propanol, butanol, pentanol, ketone like acetone methyl ethyl ketone, methyl isobutyl ketone, diethyl ketone or mixture thereof.
4. The process as claimed in claim 1, 2 or 3, wherein the monosodium salt of CDP-choline obtained has HPLC purity of more than 99.5%.
5. The process as claimed in 1 or 2, wherein the amount of phosphorylcholine chloride calcium salt used with respect to cytidine 5'-monophosphate is in the range of 1.5 to 3 moles equivalent, preferably 2 to 2.5 moles equivalent.
6. The process as claimed in 1 or 2, wherein in the step c) more than 80% of the total amount of the methanol used is recovered.
7. The process as claimed in claim 1 or 2, wherein the calcium precipitating agent like oxalic acid, sodium phosphate, sodium hydrogen phosphate, potassium phosphate, potassium hydrogen phosphate is used, preferably oxalic acid is used.
8. The process as claimed in claim 1 or 2, wherein the amount of calcium
precipitating agent used with respect to phosphorylcholine chloride calcium salt is in
the range of 0.8 to 1.2 moles equivalent.
9. The process as claimed in claim 1 or 2, wherein the clear solution obtained in step
f) is optionally, extracted with a water immiscible organic solvent selected from
esters like ethyl acetate, halogenated solvents like dichloromethane, chloroform,
ethers like diethyl ether or hydrocarbons like hexane, toluene.

10. The process as claimed in claim 1 or 2, wherein in step g) the solution is subjected to nanofilteration for removal of low molecular weight compounds and for concentrating the solution.
11. The process as claimed in claim 1 or 2, wherein in step i) the organic solvent is selected from group consisting of alcohols like methanol, ethanol, propanol, butanol, pentanol, ketones like acetone, methyl ethyl ketone, methyl isobutyl ketone, diethyl ketone and mixture thereof.