Field of the invention:

The present invention relates to an improved process for the preparation of Docetaxel and further relates to process for the purification of protected Docetaxel. The invention is also related to the process for the enrichment of phenyl isoserine intermediate, one of the key intermediates used during the preparation of Docetaxel.

Back ground of the invention

Docetaxel is an esterified product of 10-deacetyl baccatin III, which is extracted from the
renewable and readily available European yew tree. It has been found to be an effective anticancer agent and is presently sold under the name of Taxotere®. Docetaxel is chemically known as (2R, 3S)-Ar-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5,20-epoxy-l,2,4,7,10,13-hexahydroxytax-ll-en-9-one 4-acetate 2-benzoate, having the following structural formula I.

US 4,814,470 first discloses Docetaxel, its preparation and purification, wherein pure Docetaxel is obtained from protected 10-deacetyl baccatin III cinnamate derivative and sodium salt of t-butyl N-chlorocarbamate followed by removal of protecting groups by using acetic acid in presence of zinc to obtain Docetaxel as residue, which is purified by thin layer chromatography to get the same in pure form.

The foregoing process renders the formation of mixture of isomers which has to be separated thus the complete conversion of starting materials into final desired compound
is not attainable.

According to US 4,924,012 Docetaxel is obtained by the condensation of the threo-2-(l- Ethoxyethoxy)-3-tert-butyloxycarbonylamino-3-phenylpropionic acid and protected 10- deacetyl baccatin III (10-DAB) in aromatic solvent in presence of condensing agent such as dicyclohexyl carbodiimide and an activating agent such as 4-dimethylaminopyridine at a temperature of between 60 to 90°C, followed by column chromatography purification and subsequent deprotection. This process is depicted in the following scheme-1

Taxotere, which is obtained by the key starting material 10-deacetyl baccatin III, contains potential impurities that are derived from esterification process and other impurities originating, for example, from the epimerization of the carbon at the position 2" of the side chain. More especially the removal of 2"S, 3"S epimer impurity is much more difficult and it requires numerous successive purification steps involving various solvent systems thus finally leads to yield loss in the final product.

Protected Docetaxel obtained by the previously known processes must be purified and it is generally carried out by column chromatography in order to separate the 2"R,3"S and 2"S, 3"S epimers. Although, the process of US 4,924,012 is viable in its entirety, but the
process entails the complexity in removing the impurities particularly epimer impurity if the crude protected Docetaxel is solely purified by column chromatography. Therefore further crystallization step has to be implemented and suitable solvents have to be identified in order to remove this impurity.

Moreover, in US 4,924,012 there is no disclosure regarding the nature of threo-2-(l- Ethoxyethoxy)-3-tert-butyloxycarbonylamino-3-phenylpropionic acid side chain which is the factor to be considered in order to obtain the taxotere in high optical purity and high yields. There is no disclosure in the art in favor of diasteriomerically enriched (2R, 3S)-
N-Boc- O-(ethoxyethyl)-3-phenylisoserine side chain usage while condensing with protected 10-DAB.

It has been known that the methoxy methyl and substituted methoxy methyl groups were found to be well suited among the diverse protecting groups of hydroxyl groups particularly when the acidic carboxylic group is present in the adjacent position. During the preparation of (2R, 3S)-N-Boc-0-(ethoxyethyl)-3-phenylisoserine, there are two diastereomers formed due to the presence of ethoxyethyl protecting group.

US 5,578,739 discloses process for the preparation of Docetaxel comprising the steps of: treating a taxane derivative compound-VI in an acid medium by means of an alcoholic solution of an inorganic acid to remove the protecting group R2 in order to provide compound-I, purifying the compound-I by chromatography or crystallization and treating the purified compound-I under non-epimerizing conditions to remove the R1 and G1 protecting groups there from and provides Docetaxel. This process involves many deprotection steps and purification steps. The described process is depicted under Scheme-2.

US 6,838,569 discloses the preparation of pure Docetaxel trihydrate by repeated purifications using three sets of solvents such as mixture of alkane/chlorinated alkane, alkyl ketone/alkane and aliphatic nitrile/purified water respectively.

US 6,881,852 discloses a process for the purification of Docetaxel, starting with purity 40-55%, in six sequential steps comprises:

a) purifying crude Docetaxel with purity 40-55% in a mixture of alkane and chlorinated alkane solvents

b) again repeating the step(a) one or more times to increase the Docetaxel purity up to 65-75%

c) purifying the step (b) solid in a mixture of alkyl ketone and alkane to increase the purity up to 85-90%

d) yet again repeating the step (c) one or more times to increase Docetaxel purity up to 90-95%

e) purifying the Docetaxel obtained from step (d) in alkanol and water to obtain Docetaxel with purity 97-98%

f) Finally the step (e) Docetaxel is purified by using alkyl ketone and alkane in order to attain pure Docetaxel.

According to US 6,881,852 each step further requires one or more times repeated purifications but still there is no remarkable increase in purity. More especially this purification process makes the method laborious and involves more number of solvent systems thereby the process turns out to be ineffective in view of cost.

WO 2008/123751 discloses anhydrous stable crystalline Docetaxel and its preparation by dissolving Docetaxel in an organic solvent selected from ether, ester, ketone, dichloromethane- methanol mixture, dichloromethane-Acetonitrile mixture, and a mixture thereof, inducing crystallization by adding an anti-solvent such as pentane, hexane or heptane, recovering the resulting crystals and drying the crystalline anhydrous crystals under reduced pressure.

WO 2007/044950 provides novel anhydrous crystalline Docetaxel characterized by XRD pattern and further provides process for preparing the said crystalline Docetaxel from a mixture of methyl isobutyl ketone and organic anti-solvent selected from C5-C8 linear and branched alkane. This application further provides process for preparation of known anhydrous crystalline from a variety of organic solvents and mixtures thereof.

According to WO 2007/078050, anhydrous crystalline Docetaxel is prepared by colloidizing Docetaxel in colloid-formable solvent such as acetone.

Docetaxel may normally be purified by using column chromatographic techniques and by high performance liquid chromatographic technique on a silica column. However, these chromatographic techniques may be convenient for small scale production as it requires large quantities of solvent system.

None of the methods described in the prior art are not efficient in removing the unwanted 2"- epimer impurity from Docetaxel.

Surprisingly the present inventors have found a process which is capable of removing the unwanted 2"- epimer impurity from Docetaxel by selective purification methods.

Summary of the invention

The object of the present invention is to provide a process for the preparation of Docetaxel in high purity and yield.

Another object of the present invention is to provide process for the preparation of diastereomerically enriched (2R,3S)-N-Boc-0-(ethoxyethyl)-3-phenylisoserine and its use in the preparation of protected Docetaxel.

Further object of the present invention is to provide the purification of protected Docetaxel in a solvent selected from alcohols, chlorinated alkanes, aliphatic hydrocarbons, aliphatic esters and mixtures thereof.

Yet another object of the present invention is to provide a process for the purification of Docetaxel using the solvents selected from chlorinated alkanes, Acetonitrile and mixtures thereof and anti-solvent is selected from saturated acyclic aliphatic hydrocarbons.

Detailed description of the invention

In an embodiment the present invention relates to process for preparing Docetaxel in high purity and yield comprising the steps of:

a) Preparing diastereomerically enriched (2R,3S)-N-Boc-0-(ethoxyethyl)-3-phenyl isoserine, herein after called as enriched protected Boc-phenyl isoserine

b) Condensing the protected Boc-phenyl isoserine obtained from step (a), with protected 10-DAB to obtain crude protected Docetaxel

c) Subjecting thus obtained crude protected Docetaxel to column chromatography to remove protected 10-DAB

d) Purifying the protected Docetaxel obtained from step (c) from the solvent selected from the group alcohols, chlorinated alkanes, aliphatic hydrocarbons, esters and mixtures thereof to get the protected Docetaxel with less than 1% of 2"-epimer impurity.
e) Deprotecting the compound obtained from step (d) by using zinc and acetic acid gives crude Docetaxel

f) Purification of the crude Docetaxel to give pure Docetaxel.
The above described method of this present invention is depicted in the scheme-3.

In another embodiment of the invention, 80% - 90% diastereomerically enriched 2R, 3S)- N-Boc-0-(ethoxyethyl)-3-phenylisoserine is used for the condensation of step (b). The column chromatography of step (c), wherein the eluent used is the mixture of ethyl acetate and hexane in increased order of polarity.

Recrystallization i.e. purification of protected Docetaxel involves the solvent selected from the group alkanols, chlorinated alkanes, aliphatic hydrocarbons, esters and mixtures thereof, wherein the alkanols is selected from but not limited to methanol, ethanol,
2- propanol and mixtures thereof. The chlorinated alkanes such as chloroform and dichloromethane are used and the aliphatic hydrocarbons, esters are selected from heptane, hexane, ethyl acetate, isopropyl acetate are used, preferably alcohols are used and more preferably methanol is used.

It has been observed that the recrystallization of crude protected Docetaxel in most of the solvents is not useful in getting rid of unwanted epimer impurity and the crude material was remained as residue without being purified. If the epimer impurity is not removed substantially in the penultimate stage then it is very difficult to remove the unwanted 2"- epimer of Docetaxel in the final stage.

In one specific embodiment, the present invention relates to process for the purification of protected Docetaxel from the solvent selected from the group alcohols, chlorinated alkanes, aliphatic hydrocarbons, esters and mixtures thereof to get the protected Docetaxel with less than 1 % of 2"-epimer impurity.

In a preferred embodiment Purification of the crude Docetaxel comprising the steps of;

a) Subjecting the crude Docetaxel to first crystallization by dissolving crude Docetaxel in a solvent selected from chlorinated alkanes and adding the anti solvent which is selected from saturated acyclic aliphatic hydrocarbons

b) Subjecting the Docetaxel obtained from step (a) to second crystallization by dissolving the step (a) product in a solvent mixture selected from the group chlorinated alkanes and Acetonitrile and adding the anti solvent selected from saturated acyclic aliphatic hydrocarbons to obtain Docetaxel with high pure and high yield.

c) Optionally, repeating the first purification step as mentioned in the step (a)
Wherein chlorinated alkanes such as chloroform and dichloromethane are used and the aliphatic hydrocarbon is selected from but not limited to heptane, hexane and mixtures thereof.

In a preferred embodiment diastereomerically enriched (2R,3S)-N-Boc-0-(ethoxyethyl)-
3- phenyl isoserine is prepared by;

• Reacting (2R,3S) N-tert-butoxy carbonyl-3-phenylisoserine methyl ester with Ethyl vinyl ether at 25-30°C in presence of pyridinium p-toluene sulphonate

• Evaporating the reaction mass and isolating the solid

• Treating the obtained solid with LiOH monohydrate

• Distilling the solvent and extracting the product into dichloromethane at a pH of about 5-6

• Evaporating dichloromethane and isolating the diastereomerically enriched product in an aromatic hydrocarbon

Wherein the preferred organic solvent is methanol and the aromatic hydrocarbon is selected from hexane, heptane, cyclohexane and mixtures threof.
The invention is now illustrated with a few non-limiting examples.

Example: Preparation of pure Docetaxel
Stage-1: Preparing diastereomerically enriched (2R,3S)-N-Boc-0-(ethoxyethyl)-3- phenyl isoserine (2R,3S) N-tert-butoxy carbonyl-3-phenylisoserine methyl ester (50gm) was dissolved in dichloromethane (230ml). PPTS (2.1g) was added to the above solution. Ethyl vinyl ether (32.5 ml) was added to the reaction mixture and stirred at 25-30°C for 4 hrs. After reaction completion, reaction mixture was washed with, 3% NaHCC3 solution (150ml), DM water (150ml) followed by brine (150ml). Dichloromethane layer was evaporated to dryness. Hexane (300ml) was added to the above thick paste and stir for VA hr at 25- 30°C. The precipitated solid was filtered and washed with hexane (50 ml) and dried under vacuum at 40-45°C.

The above solid was dissolved in Methanol (550ml) and Lithium hydroxide (7.12 gm) was added slowly in 60-90 minutes to the above solution. Reaction mixture was stirred at 25-30°C for 4 hrs. After reaction completion methanol was distilled under vacuum completely. The volume of the reaction mass was making up to 300ml with DM water and washed with dichloromethane (200ml). The reaction mass was cooled to 5-10°C. To the reaction mass dichloromethane (500ml) was added and adjusted pH to 4.5 with acetic acid (50ml) at 5-10°C. Stirring was continued for 10 minutes at 5-10°C and the temperature was raised to 25-30°C and allowed for separation of layers. Organic layer was separated and washed with DM water (300ml) and brine (100ml). Organic layer was dried over anhydrous sodium sulphate and evaporated upto 30-35 ml of reaction mass remains inside and hexane (200ml) was added at 25-30°C. The precipitated solid was filtered, washed with hexane (50ml) and dried under vacuum at 40-45°C.

Yield: 25g
Stage-2: Preparation of 7,10-Bis Troc 10-DeacetyIbaccatin-III
To the 10-DAB-III (50 gm) solution in pyridine (100ml) at 25-30°C under stirring was added acetone (100ml) and cooled the reaction mass to 0-3°C. 2,2,2-Trichloroethyl chloroformate (l00gm) was added to the reaction mass slowly at 0-3°C. The reaction mixture was stirred for 5-10 minutes at 0-3°C. After completion of reaction DM water (1500ml) was added and stirred the reaction mass at 25-30°C for 2 hrs. The precipitated material was filtered and washed with DM water (200ml). The obtained product was suspended in Methanol (250ml) and stirred for V2 hr. The suspension was filtered and washed with Methanol (50ml) and allowed for suck drying for 1 hr. Again the obtained material was added to a solvent mixture of Hexane (200ml) and Ethyl acetate (75ml). The suspension was stirred for V2 hr and filtered the product and washed with a mixture of Hexane (36ml) and Ethyl acetate (14ml). The obtained wet material was dried under vacuum at 50-55°C.

Yield: 80gm. Purity by HPLC 98%
Stage-3: Preparation of 7,10-Bis Troc 13-{(2R,3S)-3-f-butoxycarbonyl amino - 2-(l-ethoxyethoxy) -3-phenyl propanoate} 10-Deacetylbaccatin-III
(2R,3S)-3-tert-butoxycarbonyl amino 2-(l-ethoxyethoxy)3-phenyl propanoic acid (30gm) was added to toluene (475ml) and stirred for lOmin at 25-30°C. To this 7, 10-Bis Troc 10-Deacetylbaccatin-III (19gm) was added and continued stirring for lOmin. Toluene (190ml) was distilled under vacuum at 50-53°C to remove moisture. DCC (17.5gm dissolved inl90ml toluene) was added to the reaction mixture at 60-65°C under N2. DMAP (2.6gm) was added to the reaction mixture. The reaction mixture was stirred at 60-65°C under N2 atm. The reaction mixture was cooled to 25-30°C and the solid (by product) was filtered and washed with toluene. The filtrate was washed with DM water (1000ml), NaHC03 solution (1000ml), and DM water (500ml). The organic layer was evaporated at 40-45°C under vacuum to remove toluene completely and dichloromethane (50ml) was added to the above crude. The obtained crude was purified by column chromatography using silica gel. The column was run using Ethyl acetate and hexane in increasing order of polarity.

i) 15% ethyl acetate in Hexane
ii) 25% ethyl acetate in Hexane
iii) Ethyl acetate

Fractions containing product were distilled under vacuum below 40°C. To the crude product Methanol (105ml) was added and stirred for 1 hr. The suspension was filtered and washed with methanol (20ml). The wet product was dried under vacuum at 40-45°C.

Yield: l0gm. Purity by HPLC 99%
Stage-4: Preparation of Docetaxel (crude)
Stage-3 product (lO.Ogm) was dissolved in Ethyl acetate (100ml) and stirred for 15-30 minutes at 25-30°C. To the obtained clear solution acetic acid (100ml) was added slowly over a period of 30 minutes. The reaction mixture was heated to 40-43°C under N2 and Zinc powder (lO.Ogm) was added slowly over a period of 30 minutes at 40-43°C. Stirring was continued for 2 hrs and the reaction mass was cooled to 25-30°C. The reaction mass was filtered and the solids were washed with ethyl acetate (100ml). The filtrate was washed with DM water (600ml) followed by Sodium bicarbonate solution (12.0gm dissolved in 240ml water). Sodium bicarbonate layer was extracted with ethyl acetate (120ml). All the organic layers were combined and wash with DM water (80ml), Brine solution (80ml) and finally dried over anhydrous sodium sulphate.

The organic layer was evaporated below 40°C and n-Heptane (120ml) was added slowly over a period of 3-4 hrs and stirred for 1-2 hrs. The precipitated material was filtered and washed with n-Heptane (20ml). The wet product was dried under vacuum at 35- 40°C.
Yield: 6.0gm. Purity by HPLC 93%.

Stage-5: Purification of Docetaxel
Crude Docetaxel was purified in successive crystallizations.
Crystallization 1:

Crude Docetaxel (l0gm) was dissolved dichloromethane (200ml) and stirred for l0min. To the obtained solution n-Heptane (125ml) was added slowly drop wise over a period of 1-116 hrs at 20-25°C. The reaction mass was stirred for 5 hrs under nitrogen at 20-25°C and filtered. The filtered product was washed with a mixture of Dichloromethane (12.5ml) & Heptane (7.5ml) and dried under vacuum at 45-50°C.

Yield: 7.5gm. Purity by HPLC 99.5%
Crystallization 2.

Docetaxel (7.5gm) obtained in 1st crystallization was dissolved in a mixture of dichloromethane (30ml) and Acetonitrile (30ml). The reaction mixture was stirred for l0min and to the obtained solution n-Heptane (37.5ml) was added slowly drop wise over a period of 1 V% hrs. The reaction mass was stirred for 5 hrs under nitrogen at 20-25°C and filtered. The filtered product was washed with a mixture of Dichloromethane (18ml) Acetonitrile (18ml) & Heptane (23ml) and dried under vacuum at 45-50°C.

Yield: 6.1gm. Purity by HPLC 99.6% Crystallization 3.
Docetaxel (6.0gm) obtained in 2nd crystallization was dissolved dichloromethane (120ml) and filtered through 0.45 micron filter. To the filtered solution n-Heptane (75ml) was added slowly drop wise over a period of l-1/2 hrs at 20-25°C. The reaction mass was stirred for 5 hrs under nitrogen at 20-25°C and filtered. The filtered product was washed with a mixture of Dichloromethane (7.5ml) & Heptane (4.5ml) and dried under vacuum at 45-50°C. Yield: 5.5gm. Purity by HPLC 99.8%

We claim;
1. A process for preparing Docetaxel in high purity and yield comprising the steps of:

a. Preparing diastereomerically enriched (2R,3S)-N-Boc-0-(ethoxyethyl)-3- phenyl isoserine

b. Condensing the protected Boc-phenyl isoserine obtained from step (a), with protected 10-DAB to obtain crude protected Docetaxel

c. Subjecting thus obtained crude protected Docetaxel to column chromatography to remove protected 10-DAB

d. Purifying the protected Docetaxel obtained from step (c) from the solvent selected from the group alcohols, chlorinated alkanes, aliphatic hydrocarbons, esters and mixtures thereof to get the protected Docetaxel with less than 1% of 2"-epimer impurity.
e. Deprotecting the compound obtained from step (d) by using zinc and acetic acid gives crude Docetaxel

f. Purification of the crude Docetaxel to give pure Docetaxel.

2. A process for the purification of crude Docetaxel comprising the steps of;

a. Subjecting the crude Docetaxel to first crystallization by dissolving crude Docetaxel in a solvent selected from chlorinated alkanes and adding the anti solvent which is selected from saturated acyclic aliphatic hydrocarbons

b. Subjecting the Docetaxel obtained from step (a) to second crystallization by dissolving the step (a) product in a solvent mixture selected from the group chlorinated alkanes and Acetonitrile and adding the anti solvent selected from saturated acyclic aliphatic hydrocarbons to obtain Docetaxel with high pure and high yield.

c. Optionally, repeating the first purification step as mentioned in the step (a)

3. The process as claimed in claim 2, wherein the chlorinated alkane is selected from chloroform, dichloromethane and mixture thereof.

4. The process as claimed in claim 2, wherein the aliphatic hydrocarbon is selected from heptane, hexane and mixture thereof.

5. A process for preparing diastereomerically enriched (2R,3S)-N-Boc-0- (ethoxyethyl)-3-phenyl isoserine comprising the steps of;

a. Reacting (2R,3S) N-tert-butoxy carbonyl-3-phenylisoserine methyl ester with Ethyl vinyl ether in presence of pyridinium p-toluene sulphonate

b. Evaporating the reaction mass and isolating the solid

c. Treating the obtained solid with LiOH monohydrate in an organic solvent

d. Distilling the solvent and extracting the product into dichloromethane at a pH of about 5 to 6

e. Evaporating dichloromethane and isolating the diastereomerically enriched product in an aromatic hydrocarbon

6. The process as claimed in claim 5, wherein the preferred organic solvent is methanol.

7. The process as claimed in claim 5, wherein the aromatic hydrocarbon is selected from hexane, heptane, cyclohexane and mixtures thereof.