FORM-2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
[See section 10, rule 13]
A process for preparation of N2-acetyl-9-(l,3-diacetoxy-2-propoxymethyl)guanine, an intermediate of Ganciclovir
APPLICANT:
CALYX CHEMICALS AND PHARMACEUTICALS LTD.
2, Marwah's Complex, Sakivihar Road, Sakinaka, Andheri (E), Mumbai-400 072, 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 process for preparation of substantially pure N -acetyl-9-(l,3-diacetoxy-2-propoxymethyl)guanine referred to herein as N-9 alkylated isomer of structural Formula I, an intermediate of antiviral compound, Ganciclovir.
O

The present invention further relates to a process for separation of N -acetyl-7-(l,3-diacetoxy-2-propoxymethyl)guanine referred to herein as N-7 alkylated isomer of structural Formula II,

Formula II
from N-9 alkylated isomer without involving use of tedious and cumbersome processes such as column chromatography.
BACKGROUND OF THE INVENTION
Ganciclovir is chemically, 9-(l,3-dihydroxy-2-propoxymethyl) guanine and has the structural Formula III. It is a nucleoside analogue of guanosine and the first antiviral

drag to be effective in the treatment of cytomegalovirus (CMV) disease in humans. It is one of the most important acyclic nucleosides which inhibit not only all the herpes viruses but also the transformation of normal cord-blood lymphocytes by Epstein-Barr virus.

Formula III
A number of methods for preparation of Ganciclovir are reported in literature but the simplest synthetic route is through formation of N-9 alkylated isomer, an intermediate. The synthesis of N-9 alkylated isomer involves the direct alkylation of appropriately substituted 2-aminopurines e.g. guanine derivative which on deprotection of functional group results in formation of Ganciclovir.
EP74306 discloses a process whgrein a guanine derivative is alkylated using acetoxymethyl-l,3-diacetoxy-2-propyl ether which is carried out at elevated temperature under vacuum in presence of acidic catalyst. This process results in mixture of N-9 alkylated isomer and N-7 alkylated isomer. The N-9 alkylated isomer is separated from the mixture and is further hydrolyzed to form Ganciclovir.
EP532878 describes a process in which alkylation of guanine derivative with 1,4-Diacetoxy-3-acetoxymethyl-2-oxa-butane in presence of acidic catalyst and an acid anhydride is carried out to yield mixture of N-9 alkylated isomer and N-7 alkylated isomer. The N-9 alkylated isomer is further allowed to undergo saponification which results in formation of Ganciclovir.

US6043364 describes a process for conversion of N-7 isomer to N-9 isomer by heating the N-7 isomer in presence of alkylating agent at high temperature.
It can be observed that the major drawback in all the processes described above is that the N-9 alkylated isomer is always formed along with corresponding N-7 alkylated isomer. Hence, in the preparation of Ganciclovir separation of the desired N-9 alkylated isomer from the N-7 alkylated isomer becomes important.
However, the separation of N-9 alkylated isomer from the mixture of N-9 alkylated isomer and N-7 alkylated isomer is difficult. All the above mentioned processes for preparation of Ganciclovir utilize costlier and tedious methods like column chromatography for separation of N-7 and N-9 alkylated isomer.
Hence, there is a need to develop a novel, efficient and industrially viable process for preparation of substantially pure N-9 alkylated isomer.
The inventors of present invention have developed a process for preparation of substantially pure N-9 alkylated isomer without involving use of tedious and cumbersome processes such as column chromatography. The inventors of present invention have rationally designed use of combination of solvents for separation and purification of N-9 alkylated isomer. With the purification to the extent of more than 99% in formation of intermediate, no further purification is required for preparation of Ganciclovir. The process is cost effective, operationally simple and yields the final product with desired purity.
4
1 1 JAN 2011

OBJECT OF THE INVENTION
i) An object of the present invention is to provide a process for the preparation of substantially pure N-9 alkylated isomers.
ii) Another object of the present invention is to separate N-9 alkylated isomer from N-7 isomer without involving use of tedious and cumbersome processes such as column chromatography.
iii) Another object of the present invention is to obtain N-9 alkylated isomer with
a purity of greater than 99%.
« iv) Yet another object of the present invention is to provide simple, economic and
industrially viable process for the preparation of N-9 alkylated isomer.
v) Yet another object of the present invention is to provide simple, economic and industrially viable process for the preparation of Ganciclovir.
SUMMARY OF THE INVENTION
According to an aspect of the present invention, there is provided a process for the preparation of substantially pure N-9 alkylated isomer of structural Formula I


comprising,
a) alkylating diacetyl guanine of structural Formula IV

with 1,3-diacetoxy 2-(acetoxy methoxy)-propane of structural Formula V

in presence of an acid catalyst in a suitable polar organic solvent such as N, N-dimethylacetamide
b) isolating the crude product by usual workup
c) separating N-9 isomer from N-7 isomer in crude product obtained in step b) by crystallizing in a combination of two or more organic solvents.
According to another aspect of the present invention, the process for preparation of substantially pure N-9 alkylated isomer of Formula I is carried out in a suitable polar organic solvent such as N, N- dimethylacetamide.
According to yet another aspect of the present invention, an improved process for separation of N-9 alkylated isomer from N-7 alkylated isomer comprising crystallization of isomers in a combination of two or more of organic solvents.

According to yet another aspect of the present invention, N-9 alkylated isomer with a purity of greater than 99% is obtained.
According to yet another aspect of the present invention, N-9 alkylated isomer is further hydrolyzed to obtain Ganciclovir.
DETAILED DESCRIPTION OF INVENTION
The present invention relates to a process for the preparation of substantially pure N2-acetyl-9-(l,3-diacetoxy-2-propoxymethyl)guanine referred to herein as N-9 alkylated isomer of structural Formula I

Formula IV
with 1,3-diacetoxy 2-(acetoxy methoxy)-propane of structural Formula V


Formula V
in presence of an acid catalyst in a suitable polar organic solvent such as N, N-dimethylacetamide
b) isolating the crude product by usual workup
c) separating N-9 isomer from N-7 isomer in crude product obtained in step b) by crystallizing in a combination of two or more organic solvents.
According to an embodiment of the present invention, the acid catalyst used in step a) is selected from methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, phosphoric acid, sulphuric acid, polyphosphoric acid, preferably methane sulfonic acid is used.
The solvent used in step a) is polar organic solvent such as N, N- dimethylacetamide.
According to an embodiment of the present invention, step a) is carried out at temperature of about 115 °C to 130°C, preferably 120 °C to 125 °C for about 1 to 5 hours, preferably 3 hours.
According to another embodiment of the present invention, after completion of step a) the crude product is isolated by concentrating the reaction mixture followed by addition of an organic solvent having lower solubility of N-7 and N-9 alkylated isomers, heating the reaction mixture to reflux temperature, cooling and filtering the solid crude product obtained.

The organic solvent having lower solubility of N-7 and N-9 alkylated isomer is selected from low molecular weight ketone like acetone, methyl isobutyl ketone, esters like ethyl acetate, methyl acetate, ethers like diisopropyl ether, diphenyl ether, tetrahydrofuran, alcohols like isopropanol, butanol or hydrocarbons like toluene, xylene, hexane, heptane, preferably acetone is used.
The solid crude product isolated in step b) comprises of about 40-45% N-7 alkylated isomer and 55-60% N-9 alkylated isomer.
According to yet another embodiment of the present invention, the N-9 alkylated isomer is separated from N-7 alkylated isomer by method of crystallization wherein combination of two or more organic solvents is used.
The N-7 alkylated isomer is crystallized by utilizing combination of polar aprotic solvent like dimethylformamide, JV-methylpyrrolidone, dimethylacetamide, dimethylsulfoxide with ketones like acetone, methyl isobutyl ketone, esters like ethyl acetate, methyl acetate, alcohols like methanol, ethanol, isopropanol, butanol, hydrocarbons like toluene, xylene, hexane, heptane or ethers like diisopropyl ether, diphenyl ether, tetrahydrofuran. The N-7 alkylated isomer is preferably crystallized using a combination of dimethylformamide and ethyl acetate. The volume ratio of the dimethylformamide to the ethyl acetate preferably is 2:8
After separation of N-7 isomer, the concentration of filtrate containing N-9 isomer is adjusted by evaporation or by dilution.
The N-9 alkylated isomer is crystallized by utilizing polar aprotic solvent like dimethyl formamide -/V-methylpyrrolidone, dimethylacetamide, dimethylsulfoxide with ketones like acetone, methyl isobutyl ketone, esters like ethyl acetate, methyl

acetate, alcohols like methanol, ethanol, isopropanol, butanol, hydrocarbons like toluene, xylene, hexane, heptane or ethers like diisopropyl ether, diphenyl ether, tetrahydrofuran. The N-9 alkylated isomer is preferably crystallized using a combination of dimethylacetamide and ethyl acetate. The volume ratio of the dimethylacetamide to the ethyl acetate preferably is 1.5:8.
According to an embodiment of the present invention, N-9 alkylated isomer with a purity of greater than 99% is obtained.
According to another embodiment of the present invention, N-9 alkylated isomer can be hydrolyzed further by methods known in the art to obtain Ganciclovir.
The detail of the invention provided in the following example is given by the way of illustration only and should not be construed to limit the scope of the present invention.
EXAMPLES Example 1
Preparation of crude N2-Acetyl-9-(l ,3-diacetoxy-2-prpoxymethyl)guanine
To N, N-dimethylacetamide (220 ml) was added Diacetyl guanine (50g, 0.212 mol), 1,3-diacetoxy 2-(acetoxy methoxy)-propane (84.4g, 0.34 mol) and methane sulfonic acid (1.4 ml). The mixture was then heated at 120-125°C for 3 hours. The reaction mixture was concentrated under 10mm vacuum at 60°C. To the concentrated mass was added acetone (350 ml) and the reaction mixture was refluxed for 1 hour. The reaction mixture was cooled and was stirred for 3 hours at room temperature. The mixture was further cooled to 0° C and was stirred for another 2 hours. The crude product obtained was filtered, washed with precooled acetone (100 ml) and dried in

vacuum oven at 60°C for 1 hour.
Example 2
Separation of N2-Acetvl-9-(l,3-diacetoxy-2-prpoxvmethvl)guanine from N2-Acetyl-7-(l,3-diacetoxy-2-prpoxymethyl)guanine
The dried crude product (70g) from Example 1 was suspended in N,N-Dimethylformamide (140 ml) and the suspension was heated at 60° C for 30 minutes. To this suspension was added ethyl acetate (630 ml). The solution was stirred for one hour at 80°C, then cooled to 25-27° C within 3 hours and was stirred for 1 hour. The suspension was then filtered and the residue obtained (unwanted N-7 alkylated isomer) was washed with ethyl acetate (50 ml).
To the filtrate containing N-9 alkylated isomer was added charcoal (5g) and the mixture was stirred for 1 hour at 60° C. The solution was cooled to 10° C and filtered through high flow bed. The filtrate was concentrated under reduced pressure up to dryness to give crude solid (wt. 55g). To the crude solid was added N,N-Dimethylacetamide (82.5 ml) and the mixture was stirred at 60° C for 1 hour. To this solution was added acetone (440 ml) and the mixture was further stirred for 1 hour at 60° C. The solution was cooled to room temperature within three hours, the solution was further cooled to 5° C and stirred for one hour. The N-9 alkylated isomer obtained was filtered and washed with acetone (50 ml), dried under vacuum at 60° C for two hours.
Yield of N-7 alkylated isomer: - 15 gms
Yield of N-9 alkylated isomer: - 20 gms
Purity of N-9 alkylated isomer: - 99.5% (by HPLC analysis)

Example 3
Preparation of Ganciclovir
To N-9 alkylated isomer (20 g, 0.026 mol) was added 27% liq. ammonia (120 ml). The reaction mixture was stirred at 50°C for one and half hours. After completion of reaction (checked by TLC), the reaction mixture was concentrated to dryness under vacuum at 50° C. To the residue was added water (200 ml) and the suspension was stirred at room temperature for 30 minutes. The aqueous solution was neutralized with glacial acetic acid and then heated at 90° C for one hour to obtain clear solution. The reaction mass was then cooled to room temperature, stirred for one hour, filtered, washed with water and acetone and was dried at 70°C in vacuum oven to obtain Ganciclovir (12.7 gm).
Yield: 12.7 gm
Purity: 99% (by HPLC analysis)

We claim
1. A process for the preparation of substantially pure N-9 alkylated isomer of formula I

comprising, a) alkylating diacetyl guanine of structural Formula IV

Formula IV
with 1,3-diacetoxy 2-(acetoxy methoxy)-propane of structural Formula V

Formula V

in presence of an acid catalyst in a suitable polar organic solvent such as N, N- dimethylacetamide
b) isolating the crude product by usual workup
c) separating N-9 isomer from N-7 isomer in crude product obtained in step b) by crystallizing in a combination of two or more organic solvents.

2. The process as claimed in claim 1, wherein the N-9 alkylated isomer obtained has purity of not less than 99%.
3. The process as claimed in claim 1, wherein the acid catalyst used is selected from methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, phosphoric acid, sulphuric acid, polyphosphoric acid, preferably methane sulfonic acid is used
4. The process as claimed in claim 1, wherein the N-7 isomer in crude product obtained in step b) is crystallized utilizing combination of dimethylformamide and ethyl acetate.
5. The process as claimed in claim 4, wherein the volume ratio of the dimethylformamide to the ethyl acetate preferably is 2:8
6. The process as claimed in claim 1, wherein the N-9 isomer in crude product obtained in step b) is crystallized utilizing combination of dimethylacetamide and ethyl acetate
7. The process as claimed in claim 4, wherein the volume ratio of the dimethylacetamide to the ethyl acetate preferably is 1.5:8

8. The process as claimed in claim 1, wherein the N-9 alkylated isomer is hydrolyzed further to obtain Ganciclovir