FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
&
The patent Rules, 2003
COMPLETE SPECIFICATION
A Process for Preparation of Citicoline
SeQuent Scientific Limited
A Company Incorporated Under the Companies Act, 1956
Having Registered Office at
116 Vardhman Industrial Complex,L.B.S Marg,
Thane (W), Mumbai - 400 601,India
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of Invention
The present invention relates to a novel, cost-effective process for preparing psychostimulant drug cytidinediphosphate-choline (CDP-Choline) commonly known as citicoline.
BACKGROUND OF THE INVENTION
Citicoline was originally developed in Japan for stroke. Citicoline or its sodium salt was later introduced as a prescription drug in many European countries. In these countries it is now frequently prescribed for thinking problems related to circulation problems in the brain. In the US, citicoline is marketed as a dietary supplement. Citicoline or its sodium salt is used for Alzheimer's disease and other types of dementia, head trauma, cerebrovascular disease such as stroke, age-related memory loss, Parkinson's disease, and glaucoma.
Citicoline sodium is chemically known as 5'-0-[hydroxy({hydroxy[2-(trimethylammonio)ethoxy]phosphoryl}oxy)phosphoryl]cytidine sodium which is represented by formula I,

There are many process described in the art for the preparation of citicoline. Japanese patent 51028636 describes a process for the preparation of citicoline by neutralisation of Calcium salt of phosphorylcholine chloride with 98% H2SO4 to make phosphorylcholine chloride, which is further treated with cytidine-5'-phosphate in presence of DCC and pyridine at 70°C to obtain citicoline hydrate. The drawback of this process is that citicoline is very unstable

in this harsh reaction condition such as formamide, 98% H2SO4 and high temperature of 70°C.
Chinese patent 1944661 describes an enzymatic process for the preparation of citicoline which involves twice pH adjustment to precipitatethe product,filtration of the solids, charcolisation, washing with pure water, eluting through chloride type ion exchange resin with water ethanol/alcohol reagents, desalting the eluate, decoloring and collecting the liquid, vacuum-concentration of the eluate by adding an alcohol solvent to get the solid to obtain the crude product and dissolving the crude product, microfiltering, ultrafiltering, adding an alcoholic solvent, to obtain the wet productand drying to obtain the final product. The primary disadvantage of this process is that the above reaction involves water and ethanol mixture for elution of ion exchange column and also vacuum concentration of water ethanol mixture which requires high energy, more time, leads to decomposition of product and also leads to the formation of more effluent hence it is not suitable for large scale production.
The primary disadvantage of this process is that the above reaction involves water and ethanol mixture for elution of ion exchange column and also vacuum concentration of water ethanol mixture which requires high energy, more time, leads to decomposition of product and also leads to the formation of more effluent hence it is not suitable for large scale production.
US20090286284 describes a microbial process for preparation of citicoline. This patent also discloses a process for purification of citicoline by passing through acidic cation exchange and anion exchange resin. The drawback of this process is that in this process citicoline is passed through cation /anion exchange resin in free form which is unstable and liable to formation of unwanted impurities. Therefore for the purification it needs very high volume of resin (200 times) and high volume (100 times) of solvent. This process further needs reconcentration of huge volume of solvents, which is time taking and energy consuming.
Chemical and Pharmaceutical Bulletin 1971, 19(5), 1011-16 describes a process for the preparation of citicoline by direct condensation of cytidine 5'-

monophosphate and choline phosphate by using p-toluenesulfonyl chloride or methanesulfonyl chloride combined with DMF. After completion of reaction the mass was diluted with water, pH was adjusted with ammonia solution to 9.5 and product was purified by using Dowex-1 ion exchange resin by eluting with formic acid. Another Chemical and Pharmaceutical Bulletin 1971, 19(12), 2499-71 describes a process for the preparation of citicoline by direct condensation cytidine 5'-monophosphate and choline phosphate in presence of thionyl chloride and DMF.The product obtained was further purified by using Dowex-1 ion exchange resin by eluting with formic acid.
Journal of Biological Chemistry, 1956, 185-191 describes a process for the preparation of citicoline by direct condensation5'-cytidylic acid and phosphorylcholine in a mixture of water and pyridine in presence of DCC, stirred for few days by adding DCC in lots, after completion of reaction, reaction mass was diluted with water and filtered. The pH of the filtrate was adjusted 8-9 using 0.5N KOH, diluted further with water and passed through Dowex-1 formate resin by eluting with formic acid and water.
The drawbacks of these processes are that they use hazardous reagents such as p-toluenesulfonyl chloride, methanesulfonyl chloride, thionyl chloride etc. Hence they are not suitable for large scale production. Also, the prior art processes pass citicoline solution, without isolating it, to ion exchange resins for purification. During this process most of the inorganic impurities present along with citicoline or its salt pass through the column, thus making purification difficult.
The present inventors have developed a scalable, eco-friendly process for the preparation of citicoline, which involves the isolation of metal salt of citicoline before passing through the cation /anion exchange resin.
SUMMARY OF THE INVENTION
The principal aspect of the present invention is to provide a process for the preparation of citicoline sodium of formula I, which comprises:

a. Reacting cytidine-5'-monophosphate of formula V with morpholine in
presence of a coupling reagent and an organic solvent to form
morpholidate compound of formula III;
b. condensing morpholidate compound of formula III with calcium salt of
phosphorylcholinechloride of formula IV in presence of an acid to form
citicoline calcium chloride of formula II;
c. contacting citicoline calcium chloride of formula II with acidic cation
exchange resin, and eluting citicoline adsorbed on to the resin with water
or an aqueous solution and further passing through the column containing
anion exchange resin to purify citicoline; and
d. insitu salification of the obtained citicoline with a sodium hydroxide to
obtain citicoline sodium.
The process of the present invention may be illustratedby the below scheme:


Detail Description of the Invention
Accordingly in an embodiment of the invention the reaction of cytidine 5'-monophosphate of formula V with morpholine is carried out in presence of a coupling reagent selected from dicyclohexylcarbodiimide (DCC), N-ethyl-N'-dimethylaminopropyl carbodiimide, N,N'-Dicyclohexylcarbodiimide etc. preferably dicyclohexylcarbodiimide and an organic solvent preferably alcoholic solvent selected from methanol, ethanol, propanol and the like, most preferably methanol.
In another embodiment of the invention, the morpholidate compound of formula III obtained in step (a) is condensed insitu with calcium salt of phosphorylcholine chloride in presence of an acid selected from 30% hydrochloric acid, acetic acid, phosphoric acid, dry hydrochloric acid gas, hydrobromic acid in acetic acid, hydrobromic acid, methanolic hydrochloric acid, preferably hydrochloric acid gas in isopropanol to obtain citicoline calcium chloride of formula II.
In yet another embodiment of the invention, citicoline calcium chloride of formula II is contacted with acidic cation exchange resin, preferably "styrene and divinyl benzene cross-linked Polymer Material functionalised with Sulfonic Acid groups" types of resin. The amount of cation resin varies from 6 to 15 times preferably 10 times of cytidine-5'-monophosphate. Citicoline adsorbed on cation resin is eluted with water or an aqueous solvent. The amount of water for elution varies from 3 to 10 times preferably about 5 times of citicoline calcium chloride. This removes all the cationic impurities including calcium.Citicoline obtained from the above elution step is further passed through column containing anion exchange resin preferably "Cross linked polystyrene Material functionalised with Tertiary Amine groups" to remove majorly anionic impurities like chloride . The amount of an-ion resin varies from 8 to 15 times preferably 10 times of cytidine-5' -monophosphate.
In yet another embodiment of the invention thus obtained citicoline is reacted with sodium hydroxide to obtain citicoline sodium. This, may be further charcoalated, ultra-filtered and micro-filtered. The pure citicoline sodium is

isolated by water miscible solvents selected from the group methanol, ethanol, isopvopanol, propanol, butanol, acetone, methylethylketone or the mixtures thereof, preferably isopropanol and Methanol mixture.
Thus the in present invention by isolating the citicoline calcium chloride, most of the inorganic impurities get removed only anionic and cationic impurities related to citicoline, are further purified through the column. In the present process no hazardous reagent is used hence there is no probability of degradation of citicoline. The process is very industry friendly and the product is highly pure.
The present invention can be illustrated by the following non-limiting examples.
Example:
(a) Preparation of citicoline calcium chloride:
S'-Cytidine mono phosphate (1.25 kg)and morpholine (1.12 kg) were added into methanol {6.25 L) and DCC (1.50 kg) at 25 to 35°C.The reaction mixture was heated to 50 to 55° C and stirred for 7 hours. After completion of the reaction, the reaction mass was cooled to 25 to 35°C and the obtained reaction mass was added slowly to phosphoryl choline chloride calcium salt (1.9 kg) in methanol (8.75 L) solution. The pH was maintained to 3.8 to 4.2 using HC1 gas in IPA and stirred for 6 hours at 25 to 35°. The reaction mass was further heated to 45 to 5Q°C. After completion of reaction the yeactkm mass was cooled and stirced for 1 hour. The product was filtered, washed with chilled methanol at 0 to 5°Cand suck dried to obtain citicoline calcium chloride.
Yield; 3.70-4.0 kg (b) Preparation of citicoline sodium:
The above obtained crude citicolinecalcium chloride was dissolved in water (6.25 L), filtered, washed with water and suck dried. Filtrate containing the product was re-filtered through Hyflo bed. The clear filtrate was eluted through column containing acidic cation exchange resins (12.5 L). The material was washed with water. The eluent was further passed through anion exchange resin (12.5 L). column and washed with water.

Complete aqueous solution after the passing through an-ion exchange resin was collected, pH of the solution was adjusted to 6.5 to 7.0 using 30 % sodium hydroxide solution (0.3Kg in 0.45L) and solution was concentrated using reverse osmosis. The solution was cooled to 25 to 35°C and charcoalated. The solution was filtered through hyflo bed at 25 to 35°C, washed with water. The solution was further filtered through ultra-filter at 25 to 35°C.
Clear filtrate and mixture of isopropanol and Methanol (1:1) (25 L) were stirred, the reaction mass was cooled to 0 to 5°C, and stirred for 2 hours. The product was filtered under nitrogen atmosphere, solid was washed with the mixture of IPA and methanol (1:1) (1.25 L) at 0 to 5°C and dried under vacuum below 95°C until moisture/LOD is less the 2.0%.
Yield: 1 to 1.2 kg

We claim:
1. A process for the preparation of 5'-0-[hydroxy({hydroxy[2-(trimethylammonio)ethoxy]phosphoryl}oxy)phosphoryl]cytidine sodium of formula I,

which comprises:
a. reacting cytidine 5'-monophosphate of formula V with morpholine in presence of a coupling reagent and an organic solvent to form morpholidate compound of formula III;

b. condensing morpholidate compound of formula III with calcium salt of phosphorylcholinechloride of formula TV in presence of an acid to form citicoline calcium chloride of formula II;



c. contacting citicoline calcium chloride of formula II with acidic
cation exchange resin, and eluting citicoline adsorbed on to the resin
with water or an aqueous solution and further passing through the
column containing anion exchange resin to purify citicoline; and
d. insitu salification of the obtained citicoline with a sodium hydroxide
to obtain citicoline sodium.
2. A process according to claim 1, wherein coupling reagent in step (a) is selected from dicyclohexylcarbodiimide (DCC), N-ethyl-N'-dimethylaminopropyl carbodiimide, N, N'-Dicyclohexylcarbodiimide etc. preferably dicyclohexylcarbodiimide.
3. A process according to claim 1, wherein coupling reagent in step (a) is dicyclohexylcarbodiimide (DCC).
4. A process according to claim 1, wherein the organic solvent in step (a) is an alcoholic solvent selected from methanol, ethanol, propanol, etc. preferably methanol.
5. A process according to claim 1, wherein the organic solvent in step (a) is methanol.
6. A process according to claim 1, wherein the acid in step (b) is selected from 30% hydrochloric acid, acetic acid, phosphoric acid, dry hydrochloric acid gas, hydrobromic acid in acetic acid, hydrobromic acid,

mehanolic hydrochloric acid, preferably hydrochloric acid gas in isopropanol
7. A process according to claim 1, wherein the acid in step (b) is hydrochloric acid in isopropanol.
8. A process according to claim 1, wherein the pure citicoline sodium is isolated by water miscible solvents selected from the group methanol, ethanol, isopropanol, propanol, butanol, acetone, methylethylketone or the mixtures thereof, preferably isopropanol and Methanol mixture.
9. A process according to claim 1, wherein the pure citicoline sodium is isolated by isopropanol and Methanol mixture.