FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
[See section 10; rule 13]
"PROCESS FOR PREPARATION OF N2-ACETYL-9-(l, 3-DIACETOXY-2-PROPOXY METHYL) GUANINE"


(a) M/S. BAKUL FINECHEM RESEARCH CENTRE
(b) Sterling Centre, 4th Floor, 16/2, Dr. Annie Besant Road, Worli, Mumbai 400018. India
(c) Indian company incorporated under the Companies Act 1956
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

Technical field of invention:
This invention relates to a process for preparation of N -acetyl-9-(l, 3-diacetoxy -2-propoxy methyl) guanine, which is useful intermediate for the preparation of ganciclovir, an anti-viral compound.
Background of the invention:
Viral infections are wide spread and result in a wide variety of symptoms. Some viral infections are easily overcome by body's defense mechanism, while others are more serious in nature leading to permanent damage such as blindness and some times even to death. One such family of viruses which may cause serious infection and weakened the immune system is the herpes virus group.
Ganciclovir, is chemically known as 9-(l, 3, dihydroxy-2-propoxy methyl) guanine an important nucleoside having significant anti-viral properties which is used for treating cytomegalo virus infections. The structure of the ganciclovir is shown below.

A number of methods are reported in the literature for preparation of ganciclovir. US patent 4146715 describes substituted purine compounds of 9-(2-hydroxy ethoxy methyl) guanine such as adenine, guanine, thioguanine and 2, 6- diamino purine and pharmaceutically acceptable salts of these compounds and the process for preparation of these compounds.

Preparation of 9-(l, 3-dihydroxy-2-propoxymethyl) guanine, its pharmaceutically acceptable salts and pharmaceutical compositions are also described in the US patent 4355032.
US patent 4199574 discusses anti-viral compounds and its pharmaceutically acceptable salts thereof.
US 5821367 discloses a region specific process for the synthesis of acyclic nucleosides such as ganciclovir and acyclovir and intermediates thereof starting from diacyl guanine and an alkylating agent, selected from 2-oxa-butane, 1, 4-dibenzyloxy-3-acetoxy methyl-2-oxabutane, 2-oxa-l,4-butane diol diacetate (OBDDA). Method of recycling N-7 isomer to its thermodynamically more stable N-9 isomer by heating the said N-7 isomer in presence of OBDDA for a period of 13-15 hrs in the absence of acid catalyst and solvent, thereby produce a mix of N-7 and N-9 isomers was also disclosed.
Synthesis of acyclic purine nucleosides, particularly acyclovir and 9-[(l, 3-dihydroxy-2-propoxy) methyl]guanine (DHPG) was disclosed in US 5583225, wherein the N2, N9-di protected guanine was reacted with di acetoxy propane or 2-acetoxy methoxy-1, 3, acetoxy-propane in the presence of a mixture of an acid and acetic anhydride, or in the presence of an acid, wherein, the said acid can be phosphoric acid or poly phosphoric acid at 120°C for 3 hours, but, results in mixture of N-7 and N-9 isomers ultimately leads to lower yield of desired N-9 isomer in case of ganciclovir.
Synthesis of 9-(l, 3-dihydroxy-2-propoxymethyl) N2-acetyl guanine was reported by Boryski et.al [Synthesis, 1999, 4. (625-628)], wherein 9-(2,'3,'5'-Tri-0-acetyl-β-D-ribofuranosyl-N -acetylguanine is reacted with 1,3 Diacetoxy-2-(acetoxymethoxy)propane in presence of chlorobenzene and p-toluene sulphonic acid.

WO 03033498 discusses an improved process for the preparation of N2 acetyl-9-(l,3-diacetoxy-2-propoxy methyl)guanine , which is an intermediate of ganciclovir, wherein, the process includes reacting di-acetyl guanine in presence of mono-acetyl guanine with acetic acid or with 2-acetoxymethoxy-l, 3-diacetoxy propane for 3-60 hours at a temperature range of 50-150°C.
The other major disadvantage involves with the high reaction temperatures and reaction times. If the reaction is carried out at the high temperature, the product results in a mixture, this is difficult to separate the mixture and obviously result in low yields.
Consequently, it is very desirable to provide better process for the preparation of the N -acetyl-9-(l, 3-diacetoxy-2-propoxy methyl) guanine, with improved yields and good purity.
The present invention discloses new synthetic route started with guanine instead of starting with monoacetyl guanine and di acetyl guanine as in prior art. The acetic anhydride will act as solvent as well as reactant in some of the embodiments.
In this present invention, crystallizing the desired N-9 isomer in different solvents such as polar to non-polar solvents enhances the yields and purity which is commercially feasible and industrially applicable in the larger scale. Solvents are used for crystallizing the N-9 isomer selected from water, aceto nitrile, ethyl acetate, methanol, hexane and toluene.
Accordingly, the present invention provides a process for preparation of N -acetyl-9-(l, 3-diacetoxy-2-propoxy methyl) guanine, started with a mixture of guanine, acetic anhydride, and 2-acetoxy methoxy-1, 3-diacetoxy propane in presence of an acid catalyst such as p-toluene sulphonic acid with DMF and without solvent at a temperature of 130°C to refluxing temperature of the solvent for 3-4 hrs.

Objective of the invention:
The objective of the present invention is to develop cost effective process for preparation of N2-acetyl-9-(l, 3-diacetoxy-2-propoxy methyl) guanine with increased yields and purity from Guanine.
Another objective of the invention is to remove the undesired N-7 isomer without any difficult procedures such as column chromatography.
Further objective of the invention is to achieve the desired isomer in good purity by crystallizing the same in polar to non- polar solvents.
Summary of the invention:
The present invention discloses a process for preparation of N2-acetyl-9-(l, 3-diacetoxy -2-propoxy methyl) guanine, which is useful intermediate for the preparation of ganciclovir, an anti-viral compound wherein, the said process comprises mixing guanine, acetic anhydride, 2-acetoxy methoxy-1, 3,- diacetoxy propane; adding p-toluene sulphonic acid with DMF as a solvent and/or with out solvent ; (or) mixing guanine, acetic anhydride; adding p-toluene sulphonic acid; refluxing for 1 hr; cooling to 40°C; adding 2- acetoxy-methoxy-1, 3-diacetoxy propane; refluxing the mixture at 130-150°C for 3-4 hrs; removing the solvent by distillation; adding ethyl acetate; filtering the mixture of isomers; refluxing the crude in methanol; separating the N-7 isomer by crystallization; removing the solvent from the filtrate by distillation; crystallizing the desired N-9 isomer from polar to non-polar solvents.
The polar to non-polar solvents are selected from water, aceto nitrile, ethyl acetate, methanol, hexane and toluene.

Detailed description of the invention:
The present invention discloses a process for preparation of N -acetyl-9-(l,3-diacetoxy-2-propoxy methyl) guanine in high purity and in good yield.

N -atetyl-9-(1, 3-diacetoxy-2-propoxy N -acetyl-7-(1,3-diacetoxy-2-propoxy
methyl) guanine methyl) guanine
In one embodiment of the invention, the reaction between guanine, acetic anhydride and 2-acetoxy-l, 3-diacetoxy propane in presence of acid catalyst at a temperature of 130°C to refluxing temperature of the solvent for 3-4 hours results in a mixture of N-7 and N-9 isomers. After completion of the reaction, the solvent which is acetic anhydride removed by distillation to get the thick mass. Ethyl acetate is added to this mass, and filtered to get the N-7 and N-9 isomers in crude from. This crude product is refluxed in methanol and the undesired N-7 isomer is isolated as a crystalline form by cooling the solvent. The solvent from the filtrate is removed by distillation and the desired N-9 isomer is obtained in a pure form by crystallizing the product in water.
In another embodiment of the invention, the reaction between the mixture of guanine, acetic anhydride, 2-acetoxy methoxy-1, 3-diacetoxy propane, p- toluene sulphonic acid in DMF solvent takes place at a temperature range of 140-150°C for 3-4 hrs. After completion of the reaction, the undesired isomer is removed by refluxing the crude in methanol and separated out in crystalline form by cooling the solvent. The solvent from

the filtrate is distilled out completely and crystallized the desired N-9 isomer from hexane as a solvent.
In a further embodiment of the invention, the reaction between the mixture of guanine, acetic anhydride and p-toluene sulphonic acid takes place at refluxing temperature for 1 hour, and the reaction mass is cooled 40°C. 2- acetoxy methoxy- 1, 3- diacetoxy propane is added and refluxed at 130-140°C for 3 hours. After completion of the reaction, the solvent which is acetic anhydride removed by distillation. The undesired isomer is removed by refluxing the crude in methanol and separated out in crystalline form by cooling the solvent. The solvent from the filtrate is distilled out completely and crystallized the desired N-9 isomer from toluene as a solvent.
The process for preparation of 2-acetoxy methoxy-1, 3-diacetoxy propane, which is also one of the starting material of N -acetyl-9-(l, 3-diacetoxy-2-propoxy methyl) guanine, was already disclosed in our earlier Patent application No. 288/MUM/2003 is incorporated as a reference herein.

2-acetoxy methoxy-1, 3-diacetoxy propane
The invention is further illustrated with the following examples which do not limit the scope of the invention described in the claims.
Example 1
To a mixture of guanine (lOgm), acetic anhydride (27gm), 2-acetoxy methoxy-1, 3-diacetoxy propane (28gm), p-toluene sulphonic acid (0.5gm) is added and refluxed for 3-4 hrs at 130-140°C. After completion of the reaction, the solvent is removed by

distillation to obtain a thick mass. To this mass, ethyl acetate (50ml) is added, and filtered to get crude mixture of N-7 and N-9 isomers.
This mixture is refluxed in methanol (150ml), N-7 isomer (4gm) separated out by
crystallization. The solvent from the filtrate is removed by distillation and the desired N2
acetyl-9-(l,3-diacetoxy-2-propoxy methyl)guanine (lOgm) obtained by crystallizing in
water.
Yield = 39% Purity = 95 %
Example 2
To a mixture of guanine (lOgm), acetic anhydride (30gm), and 2-acetoxy methoxy-1, 3-diacetoxy propane (25gm), p-toluene sulphonic acid (0.5gm) is added in DMF as solvent and refluxed for 3-4 hrs at a temperature range of 140-150°C. The solvent is removed completely after completion of the reaction by distillation to get the thick mass. To this mass ethyl acetate is added and filtered to get the mixture of N-7 and N-9 isomers.
The above isomers are refluxed in methanol (100ml) and the un desired isomer is separated out by crystallization. The solvent from the filtrate is distilled out completely and crystallized by hexane to get the desired N2 acetyl-9-(l,3-diacetoxy-2-propoxy methyl)guanine (8.0gm).
Yield = 30% Purity = 92 %
Example 3
To a mixture of guanine (lOgm) and acetic anhydride (35gm), p-toluene sulphonic acid (0.25gm) is added and refluxed for one hr. The reaction mass is cooled to 40°C, 2-acetoxy methoxy-1, 3-diacetoxy propane (25gm) added and refluxed at 130-140°C for 3 hrs.

After the complete removal of the solvent, methanol (100ml) is added, refluxed and the undesired N-7 isomer (5mg) crystallized from the solvent. The filtrate is distilled completely and the desired N2 acetyl-9-(l, 3-diacetoxy-2-propoxy methyl) guanine (7gm) crystallized from toluene (30ml).
Yield = 27 % Purity = 90%
While the present invention is described above in connection with preferred or illustrative embodiments, these embodiments are not intended to be exhaustive or limiting of the invention. Rather, the invention is intended to cover all alternatives, modifications and equivalents included within its scope, as defined by the appended claims.

claim
1. A process for preparation of N2 acetyl-9-(l,3-diacetoxy-2-propoxy
methyl)guanine, comprising the steps of;
reacting guanine with acetic anhydride and 2-acetoxy methoxy-1, 3,- diacetoxy
propane in the presence of an acid catalyst and optionally in presence of a single
solvent; at a temperature of 130-150°C for 3-4 hrs;
adding ethylacetate to the residue of the reaction mass to precipitate the mixture
of N7 and N9 isomers;
isolating N7 isomer by precipitating from methanol; and
crystallizing the N9-isomer from crude using polar to non-polar solvents.
2. The process as claimed in claim 1, wherein said acid catalyst is p-toluene
sulphonic acid.
3. The process as claimed in claim 1, wherein said acetic anhydride used as a solvent as well as reactant in the reaction.
4. The process as claimed in claim 1, wherein the said reaction is carried out at the reflux temperature of the solvent used.
5. The process a claimed in claim 1, wherein ethyl acetate is added after distilling
the solvent.
6. The process as claimed in claim 1, wherein said optional solvent is
Dimethylformamide.
7. The process as claimed in claim 1, wherein the said polar to non-polar solvents
are selected from water, aceto nitrile, ethyl acetate, methanol, hexane or toluene.
8. A process for preparation of N acetyl-9-(l,3-diacetoxy-2-propoxy
methyl)guanine as substantially described herein with reference to the foregoing
examples 1 to 3.
Dated this the 19th day of April 2004

Dr. Gopakumar G. Nair
Agent for the Applicant