This application claims priority to Indian patent application No.3177 /CHE/2008 filed on December 1£, 2008, the contents of which are incorporated by reference in their entirety

Fl ELD OF THE INVENTION

The present invention relates to regioselective biocatalytic hydrolysis of bis-valine ester of genciclovir using an enzyme

BACKGROUND OF THE INVENTION

Valcyte (Valgancyclovir HCI tablets) contains Valgancyclovir hydrochloride, a hydrochloride salt of the L-valyl ester of ganciclovir that exist as a mixture of two diastereomers. Ganciclovir is a synthetic guanine derivative active against cytomegalovirus (CMV). Valgancyclovir hydrochloride is a white to off-white crystalline powder with a molecular formula of C14H22N605.HCI and a molecular weight of 390, 83, The chemical name for Valgancyclovir hydrochloride is L-valine, 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)-methoxy-3-hydroxy-1 -propanyl ester monohydrochloride.

European patent No. 375329 discloses ester prodrugs of Ganciclovir i.e. Valgancyclovir and its physiologically acceptable salts thereof having advantageous bioavailability when administered by an oral route. The patent also teaches about the process for the preparation of Valgancyclovir.

Valgancyclovir is prepared in various ways. PCT Patent application WO 94/ 29311 discloses a process for the preparation of amino esters of a nucleoside analogue, including acyclovir and Ganciclovir. This process comprises reacting a nucleoside analogue having a esterifiable hydroxyl group in its linear or cyclic ether moiety, with a 2-oxa-4-aza-cycloalkane-1,3-dione of the formula (a)

Wherein R1 may represent hydrogen, a CM alkyl or alkenyl group or other amino acid side chain, and R2 may represent hydrogen or a group COOR3 where R3 is a benzyl, t-butyl, fluorenylmethyl or an optionally halo substituted linear or branched C alkyl group. Preferred R2 groups include hydrogen, methyl, iso-propyl and isobutyl, yielding respectively the glycine, alanine, valine and isoleucine esters of acyclovir or ganciclovir. Examples 1-3 of the PCT patent application WO 94/029311 discloses only the condensation of acyclovir with the valine-substituted 2-oxa-4-aza-cycloalkane-1,3-dione (Z-valine-N-carboxy anhydride) by conventional

procedures. While the amino acid esters of the PCT application include both the acyclovir and Ganciclovir esters, the application does not disclose how to prepare the Ganciclovir esters, much less the mono-esters of Ganciclovir.

WO 1997/27194 describes process for the preparation of Valgancyclovir. The process includes the reaction of Ganciclovir with N-Boc-Valine-NCA in presence of silylating agent and base for 72 hours at tower temperature to give 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl)-methoxy-3-hydroxy-1-propyl-N-(benzyloxycarbonyl)-L-valinate i.e. protected Valgancyclovir. This process takes more time for completion of reaction.

U.S. Patent Nos. 5,700,936, 5,756,736, 5,840,890, 5,856,481 discloses for the preparation of Valgancyclovir, which involve bis-valinate, N,0-bistrityl, monocarboxylate-monovalinate, bis(L-valinate) intermediates and U.S. Patent. Nos. 6,040,446, 6,215,017 & 6,218,568, involve persilyl guanine or glycerol derivatives as intermediates.

The present invention has an advantage over the prior art processes by way of using the enzyme catalytic conversion instead of chemical conversion. Also another advantage of the present process is that the enzymes used in the reaction process can be reused thus making the process more economical.

OBJECTIVE OF THE INVENTION

The main objective of the present invention relates to regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir, an intermediate of valgancyclovir.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir, an intermediate of valgancyclovir using an enzyme selected from lipases.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses an enzyme engrossed regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir, an intermediate of valgancyclovir.

In one embodiment the present invention emcompasses the process for the regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir using an enzyme. The selective hydrolysis of the bis-valine ester of ganciclovir is performed using an enzyme such as lipases and suitable solvent such as dioxane in the presence of a buffer such as phosphate at a pH of around 7,

In another embodiment, the present invention encompasses the process for the regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir that may be illustrated by the following Scheme Scheme

In yet another embodiment the present invention encompasses the process for the regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir comprising the steps of treating bi-valine ester of ganciclovir with suitable solvent in presence of enzyme and suitable buffer such as phosphate buffer of pH 7 isolating N-BOC protected valgancyclovir which can be hydroiyzed to obtain valgancyclovir as a free base
In yet another embodiment the enzyme is added at room temperature In yet another embodiment the enzyme is added at elevated temperature In yet another embodiment the enzyme is added at about 30 to 70°C In yet another embodiment the protected form of valgancyclovir is precipitated at acidic pH conditions

In yet another embodiment the protected form of valgancyclovir is precipitated at acidic pH conditions wherein the pH is about 3 to 7

The enzyme used in the present invention is selected from Lipozymes are Porcine Pancreatic Lipase, CAL-A, lyophilized Candida lipolytica Lipase, Geotrichum candidum Lipase, Pseudomonas aroginosa Lipase, Aspergillus niger Lipase, Pseudomonas cepacia Lipase, Pseudomonas fluorescens Lipase, Candida rugosa Lipase, Rhizopus delemar Lipase, Rhizopus oryzae Lipase, Penicillium camembertii Lipase, Penicillium camembertii Lipase, Mucor javanicus Lipase, Penicillium roqueforti Lipase, Pseudomonas cepacia Lipase, CAL-B, lyophilized microbial, lyophilized Lipase, Thermomyces sp, Lipase.Alcaligines sp., Chromobacterium viscosum Lipase.Candida utis Lipase, Rhizopus niveus Lipase, Pseudomonas sp. Lipoprotein Lipase, Thermomuces lanijginosus Lipase, Rhizomucor miehei Lipase, Pseudomonas species Lipase, Wheat Germ Lipase, Rhizopus arrhizus Lipase, Pancreatic Lipase 250, Novozyme-435 and the like.

The solvent used in the selective hydrolysis step of ganciclovir is selected from but not limited to Dioxane water, ketones such as acetone, methyl isobuty! ketone, methyl ethyl ketone and cyclohexanone; alcohols such as methanol, ethanol, isopropanol, n-propanol, n- butanol, tertiary-butyl alcohol, cyclohexanol or polar aprotic solvents like dimethyl sulfoxide and dimethyl formamide or a mixture thereof.

An advantage of the present invention is that the enzyme used in the preparation process can be recovered and reused thus making the process more economical

The following non-limiting examples illustrate specific embodiments of the present invention. They should not construe it as limiting the scope of present invention in any way.

Examples
Example 1
Bis-valine ester of ganciclovir (5 gram, 6.93 mM) was dissolved in 70 mL solvent (35 ml_ Dioxane +35 mL phosphate buffer pH 7.0, 0.1 M) by stirring at room temperature, thereafter Novozyme-435 (2.5 g) was added and the resulting mixture was stirred at 50 °C and the reaction was periodically monitored by HPLC. Reaction was stopped after 5 days. HPLC analysis showed (N-Benzyloxy carbonyl valgancyclovir 2 (78 %), 1 (15.5 %) and 3 (6.5%). The enzyme was removed by filtration, and the solvent was evaporated using rotary evaporator at 40 ° C. The residue was treated with methylene dichloride (MDC) (100 mL) and dil HCI (70 mL) and the organic and aqueous layer was separated.

The organic layer containing compound 1 are combined, dried and concentrated. The W-Benzyloxy carbonyl valgancyclovir was precipitated out at pH 5 from aqueous layer. The solid was filtered and dried.

Example 2
Bis-valine ester of ganciclovir (5 gram, 6.93 mM) was dissolved in 70 mL solvent (35 mL Dioxane +35 mL phosphate buffer pH 7.0, 0.1 M) by stirring at room temperature, thereafter Pseudomonas cepacia lipase {2.5 g) was added and the resulting mixture was stirred at 50 °C and the reaction was periodically monitored by HPLC. Reaction was stopped after 5 days. HPLC analysis showed W-Benzyloxy carbonyl valgancyclovir 2 (75.5 %), 1 (19.0 %) and 3 (5.5%). The enzyme was removed by filtration, and the solvent was evaporated using rotary evaporator at 40 °C. The residue was treated with methylene dichloride (MDC) (100 mL) and dil HCI (70 mL) and the organic and aqueous layer was separated. The organic layer containing compound 1 are combined, dried and concentrated. The W-Benzyloxy carbonyl valgancyclovir was precipitated out at pH 5 from aqueous layer. The solid was filtered and dried.

Example 3
8is-valine ester of ganciclovir (5 gram, 6.93 mM) was dissolved in 70 ml_ solvent (35 ml_ Dioxane +35 ml phosphate buffer pH 7.0, 0.1 M) by stirring at room temperature, thereafter porcine pancreatic lipase PPL {5 g) was added and the resulting mixture was stirred at 50 °C and the reaction was periodically monitored by HPLC. Reaction was stopped after 5 days. HPLC analysis showed N-Benzyloxy carbonyl valgancyclovir 2 (70 %), 1 (27.5 %) and 3 (2.5%). The enzyme was removed by filtration, and the solvent was evaporated using rotary evaporator at 40 ° C. The residue was treated with methylene dichloride (MDC) {100 ml_) and dil HCI (70 ml_) and the organic and aqueous layer was separated. The organic layer containing compound 1 are combined, dried and concentrated. The W-Benzyloxy carbonyl valgancyclovir was precipitated out at pH 5 from aqueous layer. The solid was filtered and dried.

We claim

1. A process for the regioselective biocatalytic hydrolysis of bis-valine ester of ganciclovir comprising the steps of

a) treating bis-valine ester of ganciclovir with suitable solvent in presence of enzyme and suitable buffer such as phosphate buffer of pH 7

b) isolating N-BOC protected valgancyclovir which can be hydrolyzed to obtain valgancyclovir as a free base

2. The process according to claim 1, wherein the solvent is selected from dioxane, water, ketones such as acetone, methyl isobutyl ketone, methyl ethyl ketone and cyclohexanone; a\coho\s such as methanol, ethanol, isopropanol, n-propanol, n- butanol, tertiary-butyl alcohol, cyclohexanol or polar aprotic solvents such as dimethyl sulfoxide and dimethyl formamide or their mixtures thereof.

3. The process according to claim 1, wherein the enzyme is selected from Upozymes

4. The process according to claim 3, wherein the enzyme is selected from Porcine Pancreatic Lipase, CAL-A, lyophilized Candida lipolytics Lipase, Geotrichum candidum Lipase, Pseudomonas aroginosa Lipase, Aspergillus niger Lipase, Pseudomonas cepacia Lipase, Pseudomonas fluorescens Lipase, Candida rugosa Lipase, Rhizopus delemar Lipase, Rhizopus oryzae Lipase, Penicillium carrtembertii Lipase, Penicillium camembertii Lipase, Mucor javanicus Lipase, Penicillium roqueforti Lipase, Pseudomonas cepacia Lipase, CAL-B, lyophilized microbial, lyophilized Lipase, Thermomyces sp. Lipase.Alcaligines sp„ Chromobacterium viscosum Lipase,Candida utilis Lipase, Rhizopus niveus Lipase, Pseudomonas sp. Lipoprotein Lipase, Thermomuces lanuginosus Lipase, Rhizomucor miehei Lipase, Pseudomonas species Lipase, Wheat Germ Lipase, Rhizopus

5. The enzyme according to claim 4 is selected from arrhizus Lipase, Pancreatic Lipase 250, Novozyme-435,

6. The process according to claim 1, wherein the enzyme can be added at room temperature or elevated temperature.

7. The process according to claim 1, wherein the enzyme is added at about 30 to 70°C

8. The process according to claim 1, wherein the protected valgancyclovir is precipitated at acidic pH conditions.

9. The process according to claim 8, wherein the pH is about 3 to 7.

10. The N-BOC protected valgancyclovir is hydrolysed to obtain valgancyclovir free base which can be treated with HCI to obtain valgancyclovir hydrochloride.