PREPARATION OF ESTERS OF PURINE DERIVATIVES Field of the Invention This invention relates to the preparation of esters of purine derivatives or pharmaceutically acceptable salts thereof. The invention further relates to the preparation of enantiomerically pure esters of purine derivatives or pharmaceutically acceptable salts thereof. hi particular the present invention relates to the preparation of valganciclovir or a pharmaceutically acceptable salt thereof. The present invention further relates to the preparation of enantiomerically pure valganciclovir or a pharmaceutically acceptable salt thereof. Background of the Invention Valganciclovir of Formula I, wherein Z is hydrogen is chemically, L-valyl ester of 2-(2-amino- 1 ,6-dihydro-6-oxo-purin-9-yl)-methoxy-3 -hydroxy- 1 -propane of Formula I. It is commercially available in the form of its hydrochloride salt. Valganciclovir or pharmaceutically acceptable salt thereof is mono-L-valyl ester prodrug of antiviral compound ganciclovir.
FORMULA I Ganciclovir of Formula II, wherein Pi and P
2 are hydrogen is disclosed in US Patent No. 4,355,032. Ganciclovir inhibits replication of human cytomegalovirus in vitro and in vivo. Ganciclovir is highly efficacious against viruses of herpes family, for example, against herpes simplex and cytomegalovirus. Ganciclovir is mostly used as intravenous infusion as it has a very low rate of absorption when administered orally.
FORMULA II British Patent Application GB 2122618 discloses derivatives of 9-(2- hydroexyethoxy methyl) guanine or physiologically acceptable salts thereof having Formula III, wherein X is oxygen or sulphur; Ri is hydroxy or amino; R is hydrogen or a group of Formula CH2OR3a and R3 and R3a are independently amino acid residues; which are useful for the treatment of viral infections and have high water solubility, which renders them of value in the formulation of aqueous pharmaceutical preparations which include oral, rectal, nasal, topical, vaginal or parenteral formulations. However, the application does not describe either the preparation of mono- or bis L-valyl esters of ganciclovir.
FORMULA III Various mono and diacyl esters of ganciclovir of Formula IV, wherein R represents an acyl group are disclosed inJ Pharm. Sci. 76 (2), p. 180-184. The preparation of diacyl esters is detailed in this article. Preparation of monoacyl esters of ganciclovir is also described which involves selectively protecting one of the hydroxy groups of ganciclovir with a conventional protecting group and treating the resulting protected ganciclovir with
acyl residue to get monoacyl esters. However, L-valyl ester of ganciclovir and their process of preparation are not discussed in this article.
FORMULAIVA FORMULAIVB European Patent No 375329 discloses amino acid ester prodrugs of ganciclovir and physiologically acceptable salts thereof having advantageous bioavailability when administered by oral route. The ester disclosed includes Bis-L-valyl ester of ganciclovir of Formula V,
FORMULA V The patent describes a process for preparation of compound of Formula V which involves refluxing ganciclovir in N,N-dimethylformamide with protected L-valine of Formula VI,
FORMULA VI wherein Z is an amino protecting group and deprotecting the resulting compound using palladium on carbon in presence of 10% palladium on carbon and acetic acid to get Bis-L- valyl ester of ganciclovir wherein Z is hydrogen. The patent however, does not disclose the utility as well as process for preparation of mono L-valyl ester of ganciclovir. US Patent Nos. 5,856,481; 5,840,891; 6,083,953; US Patent application no. 2002/0042424; European Patent No. EP 694547 provide a selective process for preparation of mono L-valyl ester of ganciclovir of Formula I, wherein Z is hydrogen or an amino protecting group or pharmaceutically acceptable salt thereof which involves protection of either of the two hydroxy groups of optionally amino protected ganciclovir, and treating the mono-hydroxy protected ganciclovir of Formula II, wherein Pi is an amino protecting group and P2 is hydroxy protecting group with protected L-valine of Formula VI, wherein Z is amino protecting group, to get protected mono L-valyl ester of ganciclovir of Formula VII,
FORMULA VII which is then deprotected to get valganciclovir of Formula I, wherein Z is hydrogen or pharmaceutically acceptable salt thereof in crystalline form. US Patent Nos. US 6,040,446; 6,215,017; 6,218,568; 5,840,890; 6,103,901; 5,700,936 and 5,756,736 describe similar processes for preparation of valganciclovir which involve mono-hydroxy protection of optionally amino-protected ganciclovir
followed by esterification of compound of Formula II, wherein Pi is an amino protecting group and P2 is hydroxy protecting group with protected L-valine and then deprotection of the product to get valganciclovir. Summary of the Invention Present inventors have found that while carrying out esterification of unprotected ganciclovir, the prior-art processes have used organic solvents in which ganciclovir was poorly soluble, thus during reaction with the esterifying residue, the concentration of ganciclovir is low compared to that of the esterifying residue, and therefore invariably bis-esterification resulted. While protection of one of the two hydroxy groups in ganciclovir provided some benefit, the cost of additional steps of protection, deprotection and purification added to the already low yields of esterification. The present inventors while working on the problem have now found that it is possible to prepare mono L-valyl ester of ganciclovir in higher yields and excellent purity by treating totally unprotected ganciclovir with optionally protected L-valine in an organic solvent which is characterized by having a good solubility for ganciclovir. Thus, additional steps of protecting ganciclovir and deprotecting the resulting product are not required. This resulted in significant improvement in process economics. The process is easy and simple even at commercial scale. Bis L-valyl ester of ganciclovir (hereinafter referred to as Bis-VGNC) refers to a compound of Formula V,
FORMULA V wherein Z is hydrogen or a conventional amino protecting group.
Mono L-valyl ester of ganciclovir (hereinafter referred to as Mono-VGNC) refers to a compound of Formula I,
FORMULA I or a pharmaceutically acceptable salt thereof in the form of racemic mixture, specific diastereomers, a crystalline form, solvate, hydrate or amorphous form, wherein Z is hydrogen or a conventional amino protecting group. Pure Mono-VGNC refers to a compound of Formula I or a pharmaceutically acceptable salt thereof in the form of racemic mixture, specific diastereomers, a crystalline form, solvate, hydrate or amorphous form, wherein Z is a hydrogen or a conventional amino protecting group, having purity above 98% wherein Bis-VGNC of Fonnula V, is less than 1% by weight as determined by HPLC. Highly pure Mono-VGNC refers to compound of Formula I or a pharmaceutically acceptable salt thereof in the form of racemic mixture, specific diastereomers, a crystalline form, solvate, hydrate or amorphous form, wherein Z is a hydrogen or a conventional amino protecting group, having purity above 98% wherein ganciclovir of Formula II (herein after referred to as GNC) is less than 1% by weight as determined by HPLC. Significantly pure Mono-VGNC refers to compound of Formula I or a pharmaceutically acceptable salt thereof in the form of racemic mixture, specific diastereomers, a crystalline form, solvate, hydrate or amorphous form, wherein Z is a hydrogen or a conventional amino protecting group, having purity above 98% wherein GNC of Formula π, and Bis-VGNC of Formula V, each less than 1% by weight as determined by HPLC. Substantially pure Mono-VGNC refers to compound of Formula I or a pharmaceutically acceptable salt thereof in the form of racemic mixture, specific
diastereomers, a crystalline form, solvate, hydrate or amorphous form, wherein Z is hydrogen or a conventional amino protecting group, having purity above 99% wherein GNC of Formula II, and Bis-VGNC of Formula V, each less than 1% by weight as deten ined by HPLC. Detailed Description of the Invention A first aspect of the present invention provides a process for preparation of Mono- VGNC or a pharmaceutically acceptable salt thereof wherein the said process comprises of a) treating GNC with optionally protected L-valine of Formula VI,
FORMULA VI in presence of a single organic solvent or mixture of two or more organic solvents, characterized by the fact that GNC is soluble in it, optionally containing a catalyst, b) if desired, deprotecting the product obtained in step c), and c) isolating Mono-VGNC or pharmaceutically acceptable salt thereof from the reaction mass. GNC is dissolved in a single organic solvent or in a mixture of two or more organic solvents, wherein the solvent or solvent system is characterized by the fact that ganciclovir is soluble in it. Such solvents are known to a person of ordinary skills in art. Examples of such solvents include dimethylsulphoxide and sulpholane. To this solution is slowly added a solution of N-Carbobenzyloxy-L-valine (hereinafter referred to as CBZ- protected L-valine) in the same solvent containing a catalyst selected from a group comprising of dicyclohexylcarbidumide and 4-dimethylaminopyridine. The reaction mass is stirred at a temperature of 5 to 60°C for 1 to 2 hours and then quenched by addition of water. The separated dicyclohexylurea (DCU) is filtered and washed with aqueous dimethylsulphoxide. To the filtrate is added ethyl acetate and the separated solids were filtered.
The wet solids are then dissolved in trifluoroacetic acid and water and the solution is extracted with dichloromethane. After concentration of the dichloromethane layer, the residue is recrystallized from denatured spirit to get N-Carbobenzyloxy-Mono-VGNC. The protected ester is subjected to hydrogenolysis at 0.5 to 1.5 Kg pressure of hydrogen in presence of 10% palladium on carbon as catalyst and an acid. After completion of reaction, the catalyst is filtered and the solution is concentrated to get a residue, which is recrystallized, from isopropanol and water to get Mono-VGNC or a pharmaceutically acceptable salt thereof. A second aspect of the present invention provides a process for depletion of Bis- VGNC from a mixture comprising of Mono-VGNC and Bis-VGNC optionally also containing GNC wherein the process comprises a) dissolving a mixture of Mono-VGNC and Bis-VGNC wherein Bis-VGNC is more than 1% by weight optionally containing ganciclovir in water containing an organic solvent, b) adding second organic solvent to the solution obtained in step a) wherein the second organic solvent is characterized by the fact that it has selective solubility for Bis-VGNC and optionally Mono-VGNC precipitates out c) if desired, deprotecting the product and converting it to a pharmaceutically acceptable salt, and d) isolating pure Mono-VGNC or a pharmaceutically acceptable salt thereof containing less than 1% of Bis-VGNC of Formula V from the reaction mass. To a mixture of Mono-VGNC and Bis-VGNC wherein Bis-VGNC is more than P/o by weight optionally containing GNC obtained after reacting ganciclovir and CBZ- protected L-valine and present in the filtrate is added a second organic solvent in such a manner that Mono-VGNC and GNC precipitate out from the reaction mass. Optionally the biphasic solution can be heated to about 35-45°C by which Mono-GNC and GNC remains in aqueous layer and Bis-VGNC gets completely extracted in ethyl acetate. The layers can be separated while still hot and the aqueous layer after repeated washing with ethyl acetate in a similar manner can be cooled, diluted further with water to precipitate out Mono-VGNC and GNC.
The second organic solvent is characterized by the fact that Bis-VGNC is soluble in it whereas Mono-VGNC and GNC are not soluble in it. Examples of second organic solvents are ethyl acetate, dichloromethane and the like. The filtrate is stored separately for the recovery of Bis-VGNC. The precipitated product is isolated by conventional means and subjected to hydrogenolysis as described in first aspect of the invention followed by purification of the Mono-VGNC or pharmaceutically acceptable salt thereof to get pure product having less than 1% of Bis-VGNC. A third aspect of the present invention provides a process for recovering Bis-VGNC from a mixture of Mono-VGNC and Bis-VGNC, optionally also containing GNC, wherein the process comprises a) dissolving a mixture of Mono-VGNC and Bis-VGNC, optionally containing GNC, in water containing an organic solvent, b) adding second organic solvent to the solution obtained in step a) in such a manner that Bis-VGNC selectively dissolves in it, c) separating the resulting layers, d) concentrating the second organic solvent layer, e) if desired, deprotecting the product and converting it to a pharmaceutically acceptable salt, and e) isolating Bis-VGNC from the reaction mass. The filtrate containing Bis-VGNC as obtained in second aspect of the invention is concentrated to dryness and the residue obtained is washed with water and purified optionally from denatured spirit to get Bis-VGNC. A fourth aspect of the present invention relates to a process of converting Bis-VGNC of Formula V to GNC of Formula II wherein the process comprises a) hydro lysing Bis-VGNC with a base, and b) isolating GNC from the reaction mass. The Bis-VGNC obtained in the third aspect of the invention is treated with an aqueous solution of sodium hydroxide at a temperature of about 25 to 100°C for
hydrolysis of the ester to get GNC. After completion of the reaction pH of the resultant mixture is adjusted to 6.5 to 7 with an acid and the separated solids were filtered and washed with water. After recrystallization from water pure GNC is obtained which can be recycled in the process for preparation of Mono-VGNC. A fifth aspect of the invention provides depletion of ganciclovir from a mixture comprising of Mono-VGNC and GNC optionally containing Bis-VGNC wherein the process comprises a) dissolving the mixture comprising Mono-VGNC and GNC wherein GNC is more than 1% by weight optionally containing Bis-VGNC in aqueous first organic solvent using trifluoroacetic acid, b) selectively extracting Mono-VGNC from the aqueous solution obtained in step a) with a non-polar water immiscible or partially miscible second organic solvent c) separating layers and concentrating the organic layer, d) if desired, deprotecting the product and converting it to a pharmaceutically acceptable salt, and e) isolating highly pure Mono-VGNC or a pharmaceutically acceptable salt thereof, containing less than 1% of GNC from the reaction mass. The mixture comprising Mono-VGNC and GNC wherein GNC is more than 1% by weight optionally containing Bis-VGNC obtained as wet solid in second aspect of the invention is dissolved in aqueous first organic solvent using trifluoroacetic acid. The first organic solvent can be a water miscible organic solvent, for example, lower alkanol, polar aprotic solvents, ketones, acetonitrile or mixtures thereof. The lower alkanols can be, for example, methanol, ethanol, n-propanol and isopropanol. Polar aprotic solvent can be, for example, tetrahydrofuran, 1,4-dioxane, dimethylsulphoxide, N,N-dimethylformamide, N,N-dimethylacetamide and sulpholane. The ketone can be, for example, acetone. The solution is extracted with non-polar water immiscible or partially miscible second organic solvent to selectively remove the Mono-VGNC. The aqueous layer retains GNC, which is stored separately for recovery.
The non-polar water immiscible or partially miscible second organic solvent is characterized by the fact that the said solvent dissolves Mono-VGNC and has poor solubility for GNC. Examples of second organic solvent include, for example, dichloromethane, ethyl acetate or mixtures thereof. The organic layer after concentration gives a residue which can be recrystallized from denatured spirit to obtain highly pure Mono-VGNC which, if desired, can be deprotected as described in first aspect of the invention to get highly pure Mono-VGNC. A sixth aspect of the invention provides a process for recovering GNC from a mixture comprising of Mono-VGNC and GNC optionally containing Bis-VGNC wherein the process comprises a) dissolving the mixture comprising Mono-VGNC and GNC optionally containing Bis-VGNC in aqueous organic solvent using trifluoroacetic acid, b) selectively extracting Mono-VGNC from the aqueous solution obtained in step a) with a non-polar water immiscible or partially miscible organic solvent, c) separating layers and diluting the aqueous layer with water so that GNC precipitates out from the reaction mass, and d) isolating GNC from the reaction mass. The aqueous layer obtained in fifth aspect of the invention containing GNC is further neutralized to a pH of about 6.5 to 7 and resultant mass is cooled to 15 to -20°C. The separated solids are filtered and washed with water and dried under vacuum to get GNC, which can be recycled in the process for preparation of Mono-VGNC. A seventh aspect of the invention provides a process for preparing Mono-VGNC from recovered GNC wherein the process comprises
a) treating recovered GNC with optionally protected L-valine of Formula VI,
FORMULA VI in the presence of an organic solvent characterized by the fact that GNC is soluble in it, optionally containing a catalyst, b) depleting GNC and Bis-VGNC from the reaction mass, c) if desired, deprotecting the product obtained in step b), and d) isolating Mono-VGNC or pharmaceutically acceptable salt thereof from the reaction mass. An eighth aspect of the invention provides a process for depleting GNC and Bis- VGNC from a mixture comprising Mono-VGNC, Bis-VGNC and GNC wherein the process comprises a) dissolving a mixture of Mono-VGNC, Bis-VGNC and GNC (wherein Bis- VGNC and GNC each are more than 1% by weight) in water containing a first organic solvent, b) adding a second organic solvent to the solution obtained in step a) in such a manner that Bis-VGNC dissolves in the second organic solvent and Mono- VGNC along with GNC optionally precipitate out from the mixture, c) isolating the mixture of Mono-VGNC and GNC from the mixture, d) dissolving the mixture obtained in step c) in aqueous third organic solvent using trifluoroacetic acid, e) selectively extracting Mono-VGNC from the aqueous solution obtained in step d) with a non-polar water immiscible or partially miscible fourth organic solvent, f) separating layers and concentrating the organic layer,
g) if desired, deprotecting the product and converting it to a pharmaceutically acceptable salt, and h) isolating significantly pure Mono-VGNC or a pharmaceutically acceptable salt thereof, containing less than 1% each of Bis-VGNC and GNC from the reaction mass. A ninth aspect of the invention provides a process for preparation of substantially pure Mono-VGNC or a pharmaceutically acceptable salt thereof wherein the process comprises a) treating GNC with optionally protected L-valine of Formula VI,
FORMULA VI
in presence of an organic solvent characterized by the fact that GNC is soluble in it, optionally containing a catalyst, b) depleting GNC and Bis-VGNC from the reaction mass, c) if desired, deprotecting the product and converting it to a pharmaceutically acceptable salt, and d) isolating substantially pure Mono-VGNC or a pharmaceutically acceptable salt thereof, having a purity above 99% and containing less than 1% by weight of each of Bis-VGNC and GNC as determined by HPLC from the reaction mass. A tenth aspect of the present invention provides a process for preparation of enantiomerically pure Mono-VGNC of Formula I,
FORMULA I wherein Z is hydrogen or a conventional amino protecting group, having more than 99% of L-Mono-VGNC and less than 1% of D-Mono-VGNC, wherein the process comprises a) preparing a solution of ganciclovir in a single organic solvent or mixture of two or more organic solvents, characterized by the fact that GNC is soluble in it, b) treating the solution obtained in a) with optionally protected L-valine of Formula VI,
FORMULA VI optionally in presence of a catalyst, c) depleting GNC and Bis-VGNC from the reaction mass, d) if desired, deprotecting the product obtained in step c), and e) isolating Mono-VGNC or pharmaceutically acceptable salt thereof from the reaction mass. Ganciclovir is added to a single organic solvent or mixture of two or more organic solvents, characterized by the fact that GNC is soluble in it and the resultant mixture is warmed to about 40 to 80°C. To the resultant mixture was added in single lot optionally
protected L-valine along with suitable catalyst, if required, such as dicyclohexylcarbodiimide and 4-dimethylaminopyridine and the mass is stirred at ambient temperature for 30 minutes to 5 hours. After quenching the reaction mass with water the reaction mass is stirred at ambient temperature and filtered to remove catalyst. From the filtrate obtained, Bis-VGNC and ganciclovir is depleted as per the process provided in our co-pending provisional application 614/DEL/2004. After deprotecting the resultant product, crude Mono-VGNC is obtained which can be purified to get enantiomerically pure Mono-VGNC of Formula I. An eleventh aspect of the present invention provides substantially pure Mono- VGNC or a pharmaceutically acceptable salt thereof, having a purity above 99% and containing less than 1% by weight of each of Bis-VGNC and GNC as determined by HPLC. A twelfth aspect of the present invention provides substantially pure amorphous form of Mono-VGNC or a pharmaceutically acceptable salt thereof, having a purity above 99% and containing less than 1% by weight of each of Bis-VGNC and GNC as determined by HPLC. A thirteenth aspect of the present invention provides Mono-VGNC or a pharmaceutically acceptable salt thereof wherein the particle size of the powder is 90% less than 50 micron and 50% of the particles have size less than 15 microns. A fourteenth aspect of the present invention provides enantiomerically pure Mono- VGNC of Formula I or a pharmaceutically acceptable salt thereof in crystalline, hydrate, solvate or amorphous form, wherein Z is hydrogen or a conventional amino protecting group, having more than 99% of L-Mono-VGNC and less than 1% of D-Mono-VGNC. A fifteenth aspect of the present invention provides enantiomerically pure Mono- VGNC of Formula I or a pharmaceutically acceptable salt thereof in crystalline, hydrate, solvate or amorphous form, having more than 99.5% of L-Mono-VGNC and less than 0.5% of D-Mono-VGNC. A sixteenth aspect of the present invention provides a pharmaceutical composition for treating viral infections caused by herpes simplex and cytomegalovirus, comprising substantially pure Mono-VGNC or a pharmaceutically acceptable salt thereof, as active ingredient along with pharmaceutically acceptable excipients, diluents or carriers.
A seventeenth aspect of the invention provides a pharmaceutical composition for treating viral infections caused by herpes simplex and cytomegalovirus, comprising Mono- VGNC or a pharmaceutically acceptable salt thereof, wherein the particle size of the powder is 90% less than 50 micron and 50% of the particles have size less than 15 microns, as active ingredient along with pharmaceutically acceptable excipients, diluents or carriers. An eighteenth aspect of the present invention provides a pharmaceutical composition for treating viral infections caused by herpes simplex and cytomegalovirus, comprising enantiomerically pure Mono-VGNC or a pharmaceutically acceptable salt thereof, as active ingredient along with pharmaceutically acceptable excipients, diluents or carriers. A nineteenth aspect of the invention provides a method of treating viral infections caused by herpes simplex and cytomegalovirus, comprising administering to a mammal in need thereof, a therapeutically effective amount of enantiomerically pure Mono-VGNC or a pharmaceutically acceptable salt thereof. A twentieth aspect of the invention provides a method of treating viral infections caused by herpes simplex and cytomegalovirus, comprising administering to a mammal in need thereof, a therapeutically effective amount of substantially pure Mono-VGNC or a pharmaceutically acceptable salt thereof. A twenty- first aspect of the invention provides a method of treating viral infections caused by herpes simplex and cytomegalovirus, comprising administering to a mammal in need thereof, a therapeutically effective amount of Mono-VGNC or a pharmaceutically acceptable salt thereof, wherein the particle size of the powder is 90% less than 50 micron and 50% of the particles have size less than 15 microns. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
EXAMPLE 1: REACTION OF CBZ-L-VALINE WITH GANCICLOVIR To a solution of CBZ-L-valine (88.4 g) in dimethylsulphoxide (350 ml) at 18-20°C was added dicyclohexylcarbidumide (105 g), followed by the addition of 4-dimethylamino pyridine (2.8 g). The reaction mixer was stirred for 10 minutes and was added to a
solution of anhydrous ganciclovir (100 g) in dimethylsulphoxide (650 ml) in one lot at 20- 25°C. The reaction mass was stirred at 20-25°C for 60 minutes and then quenched by adding water (400 ml) slowly at 20-30°C. The reaction mixture was stirred at 20-25°C for 30 minutes. The separated solids were filtered and washed with a solution of dimethylsulphoxide (60 ml) in water (40 ml).
Analysis of the filtrate showed:
Desired product- CBZ-protected mono-L-valyl ester of ganciclovir about 50%
Unreacted ganciclovir about 35%; and
CBZ-protected Bis L-valyl ester of ganciclovir about 15%.
EXAMPLE 2: REMOVAL OF CBZ-protected BIS L-VALYL ESTER OF GANCICLOVIR IMPURITY To the filtrate obtained in Example 1 was added ethyl acetate (1.0 L) and the resultant mixture was stirred at 20-30°C for 10 minutes. Water (1.5 L) was added at 30-35°C and stirred for 30 minutes, the separated solids were filtered and washed with ethyl acetate (200 ml). Analysis of the wet splids showed: Desired product- CBZ- protected mono-L-valyl ester of ganciclovir about 65%; Ganciclovir about 30%; and CBZ-protected Bis L-valyl ester of ganciclovir about 5%. The wet solids obtained above were suspended in ethyl acetate (1.0 Lit) and heated to 50 C, cooled to 20-25°C and separated solids were filtered to get mono L-valyl ester of ganciclovir contaminated with ganciclovir. The ethyl acetate layers were combined and used for recovery of CBZ-protected bis L-valyl ester of ganciclovir.
A alysis of the solid obtained showed:
Desired product- CBZ-protected mono-L-valyl ester of ganciclovir about 70%;
Ganciclovir about 30%; and
CBZ-protected Bis L-valyl ester of ganciclovir less than 1%.
EXAMPLE 3: REMOVAL OF CBZ-protected BIS L-VALYL ESTE NCICLOVIR IMPURITY To the filtrate obtained in Example 1 was added ethyl acetate (1.0 L) and the resultant mixture was stirred at 20-30°C for 10 minutes. Water (300 ml) was added at 36-
38 °C. The contents were stirred for 10 minutes and allowed to settle. The upper organic layer containing CBZ-protected Bis-L-valyl ester of ganciclovir is separated. The aqueous layer was extracted with ethyl acetate (3 x 1 L) at 36-38°C to completely remove CBZ- protected Bis-L-valyl ester of ganciclovir. The aqueous layer was treated with water (1.2 L) and cooled to 0°C and further stirred at 0-5°C for 3 hours. The separated product was filtered to obtain about 180 g of wet material.
Analysis of the solid obtained showed:
Desired product- CBZ-protected mono-L-valyl ester of ganciclovir about 70%;
Ganciclovir about 30%; and
CBZ-protected Bis L-valyl ester of ganciclovir less than 1%.
EXAMPLE 4: REMOVAL OF GANCICLOVIR IMPURITY Trifluoroacetic acid (400 ml) was cooled to 5°C and to it was added wet solids obtained in Example 3. The clear solution was obtained after stirring for 10-20 minutes. To this solution was added water (1.0 L) slowly at 5-10°C and the resultant mixture was extracted with dichloromethane (3 x 500 ml). The aqueous layer was stored separately for recovery of ganciclovir. The combined organic layers were washed with water (500 ml) and concentrated at 35-40°C under vacuum and the residue was recrystallized using denatured spirit.
The product is dried to get pure CBZ-protected mono L-valyl ester of ganciclovir.
Yield: 35-40 g
Purity: more than 98% (by HPLC)
Melting point: 176-178°C.
EXAMPLE 5: REMOVAL OF GANCICLOVIR IMPURITY The wet solids obtained in Example 3 were suspended in methanol (540 ml) and the suspension is cooled to about 2°C. Trifluoroacetic acid (100 ml) was added to the suspension and the resultant mass is stirred to get clear solution. The solution was further diluted with water (1.5 L) and the product is extracted using dichloromethane (3 x 100 ml). The organic layer was separated and concentrated to dryness under vacuum. The
residue was crystallized from isopropanol or mixture of isopropanol and hexane and dried under vacuum to get 28-30 g product.
Purity: more than 98% (by HPLC)
Melting point: 176-178°C.
EXAMPLE 6: PREPARATION OF SUBSTANTIALLY PURE HYDROCHLORIDE SALT OF MONO L-VALYL ESTER OF GANCICLOVIR The pure CBZ-protected mono L-valyl ester of ganciclovir (100 g) was suspended in 20% aqueous ethanol (1200 ml) and to this suspension was added concentrated hydrochloric acid (21 ml, 35% w/w) and palladium on carbon (10%, 50%o wet, llg). The suspension was stirred at 0.5-1.0 Kg hydrogen pressure at 20-40°C. After completion of reaction, catalyst was filtered through celite bed and the bed was washed with 10% aqueous ethanol. The filtrate obtained was concentrated completely at 35-38°C under vacuum and the residue was crystallized using water (0.8 times) and isopropyl alcohol (6.5 times). The crystalline product was washed with isopropyl alcohol and dried under vacuum at 55-60°C to get substantially pure mono L-valyl ester of ganciclovir as its hydrochloride salt.
Yield: 60-65 g.
Purity (by HPLC): greater than 99%; Bis L-valyl ester of ganciclovir less than 1%; Ganciclovir less than 1%.
EXAMPLE 7: PPREPARATION OF SUBSTANTIALLY PURE AMORPHOUS FORM OF HYDROCHLORIDE SALT OF MONO L-VALYL ESTER OF GANCICLOVIR Substantially pure crystalline hydrochloride salt of mono L-valyl ester of ganciclovir (100 g) was dissolved in water (350 ml) at 20-25°C and filtered through celite bed, the bed was washed with water (50 ml). The clear filtrate was spray dried under following conditions. Inlet temperature 160-170 °C. Outlet temperature 75-85 °C Flow rate 1.8-2.0 Lt./hr.
B lower pressure and exhaust adjust to get smooth spray drying of the material having moisture content between 1.5 -3.5%. The amorphous product obtained was further dried in vacuum oven to get substantially pure amorphous form of mono L-valyl ester of ganciclovir.
Yield: 85 g
Purity (by HPLC): greater than 99%; Bis L-valyl ester of ganciclovir less than 1%; Ganciclovir less than 1%.
EXAMPLE 8: RECOVERY OF GANCICLOVIR FROM BIS L- VALYL ESTER
OF GANCICLOVIR The ethyl acetate filtrates obtained in Example 3 were combined and evaporated to dryness. To the residue was added methanol (200 ml) and aqueous sodium hydroxide solution (100 ml, 40% w/v). The resultant mixture was stirred till TLC showed absence of bis L-valyl ester of ganciclovir. The pH of the solution was adjusted to 6.5-7.0 using aqueous hydrochloric acid. The solids started to separate which were filtered and washed with water. The wet solid was suspended in water and heated to 70-80°C to get a clear solution. The solution was cooled to 0-5°C, filtered and dried to get ganciclovir. This recovered ganciclovir was recycled in the process as exemplified in Example 1.
Yield: 8 g.
EXAMPLE 9: RECOVERY OF GANCICLOVIR To the aqueous layer obtained in Example 4 was further diluted with water (500 ml) and the resultant mixture neutralized to pH of about 6.7 using aqueous sodium hydroxide solution (20% w/v). The reaction mass was cooled to 2-5°C and further maintained at the same temperature for 3 hours. The separated solids were filtered and washed with water (2 x 250 ml), the wet material (100 gm) was dissolved in water (600 ml) at 75-80°C. The clear solution obtained was cooled to 5-10°C and further maintained at 5-10°C for 3 hours. The separated solids were filtered and washed with water (100 ml). The recovered ganciclovir was then dried at 50-55°C. The recovered ganciclovir was recycled in the process as exemplified in Example 1.
Yield: 25g
EXAMPLE 10: REACTION OF CBZ-L-VALINE WITH GANCICLOVIR To a solution of anhydrous ganciclovir (100 g) in dimethylsulphoxide (900 ml) (obtained by heating to 60-65°C) was added CBZ-L-valine (88.4 g), dicyclohexylcarbbdiimide (98 g), followed by the addition of 4-dimethylaminopyridine (1.0 g) at 24-25°C. The reaction was stirred at 25-30°C for 60 minutes and then quenched by adding water (400 ml) slowly at 25-30°C. The reaction mixture was stirred at 25-30 °C for 1 hr. The separated solid dicyclohexylurea is filtered and washed with a solution of dimethylsulphoxide (100 ml) in water (40 ml).
Analysis of the filtrate showed
Desired product- CBZ-protected mono-L-valyl ester of ganciclovir about 30%;
Unreacted ganciclovir about 55%; and
CBZ-protected Bis L-valyl ester of ganciclovir about 12%.
EXAMPLE 11 : REMOVAL OF CBZ-protected BIS L-VALYL ESTER OF GANCICLOVIR IMPURITY The filtrate obtained in Example 10 was extracted with ethyl acetate (5 x 1.0 Lit) and the resultant aqueous layer was isolated. Water (1.5 Lit) was added at 30-35°C and stirred for 30 minutes, the separated solids were filtered and washed with ethyl acetate (200 ml). The ethyl acetate layers were combined and used for recovery of CBZ-protected bis L-valyl ester of ganciclovir.
Analysis of the solid obtained showed:
Desired product- CBZ-protected mono-L-valyl ester of ganciclovir about 70%;
Ganciclovir about 30%; and
CBZ-protected Bis L-valyl ester of ganciclovir less than 1%.
EXAMPLE 12: REMOVAL OF GANCICLOVIR IMPURITY To a solution of methanol (200 ml) and water (500 ml) was added the wet solid obtained in Example 11 and the resultant mass was cooled to 0°C. Trifluoroacetic acid (105 ml) was then slowly added at 0-5°C. The resultant solution was extracted with dichloromethane (3 x 500 ml). The combined organic layer was washed with water (400
ml) containing trifluoroacetic acid (35 ml) at 0-10°C. The organic layer is concentrated at 35-40°C under vacuum and the concentrated mass was crystallized using IP A and hexane.
The combined aqueous layer was kept separately for ganciclovir recovery.
The product was dried to get pure CBZ-protected mono L-valyl ester of ganciclovir.
Yield: 35 g
Purity: 99.3% (by HPLC)
Enantiomeric Purity: L-enantiomer 99.8%, D-enantiomer 0.2%
Melting point: 122-124°C.
EXAMPLE 13: PREPARATION OF ENATIOMERICALLY PURE HYDROCHLORIDE SALT OF MONO L-VALYL ESTER OF GANCICLOVIR The pure CBZ-protected mono L-valyl ester of ganciclovir (100 g) was suspended in 2O% aqueous ethanol (1200 ml) and to this suspension was added concentrated hydrochloric acid (21 ml, 35% w/w) and palladium on carbon (10%>, 50% wet, 1 lg). The suspension was stirred at 0.5-1.0 Kg hydrogen pressure at 20-40°C. After completion of reaction, catalyst was filtered through celite bed and the bed was washed with 10% aqueous ethanol. The filtrate obtained was concentrated completely at 35-38°C under vacuum and the residue was crystallized using water (0.8 times) and isopropyl alcohol (6.5 times). The crystalline product was washed with isopropyl alcohol and dried under vacuum at 55-60 C to get enantiomerically pure mono L-valyl ester of ganciclovir as its hydrochloride salt.
Yield: 60 g.
Purity (by HPLC): greater than 99%>; Bis L-valyl ester of ganciclovir less than 1%; Ganciclovir less than 1%.
Enantiomeric Purity:
L-Mono-VGNC: 99.64%
D-Mono-VGNC: 0.36%
EXAMPLE 14: PPREPARATION OF SUBSTANTIALLY PURE AMORPHOUS FORM OF HYDROCHLORIDE SALT OF MONO L-VALYL ESTER OF GANCICLOVIR Substantially pure crystalline hydrochloride salt of mono L-valyl ester of ganciclovir (100 g) was dissolved in water (350 ml) at 20-25°C and filtered through celite bed, the bed was washed with water (50 ml). The clear filtrate was spray dried under following conditions. Inlet temperature 160-170 °C. Outlet temperature 75-85 °C Flow rate 1.8-2.0 Lt./hr. Blower pressure and exhaust adjust to get smooth spray drying of the material having moisture content between 1.5 -3.5%. The amorphous product obtained was further dried in vacuum oven to get substantially pure amorphous form of mono L-valyl ester of ganciclovir.
Yield: 85 g
Purity (by HPLC): greater than 99%>; Bis L-valyl ester of ganciclovir less than 1%>; Ganciclovir less than 1%.