FORM 2
THE PATENTS ACT 1970
(Act 39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
(SECTION 10 and Rule 13)
TITLE OF THE INVENTION "AN IMPROVED PROCESS FOR BENDAMUSTINE HYDROCHLORIDE''
Emcure Pharmaceuticals Limited.
an Indian Company, registered under the Indian Company's Act
1957 and having its Registered Office at
Emcure House, T-l 84, M.I.D.C, Bhosari, Pune-411026, India.
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 an improved process for preparation of bendamustine hydrochloride. Specifically, the invention relates to a process for controlling the level of associated impurities formed during the chlorination of hydroxy ester intermediate.
BACKGROUND OF THE INVENTION
Bendamustine hydrochloride, chemically known as 4-{5-[Bis(2-chloroethyl)-amino]-l-methyl-benzimidazolyl} butyric acid hydrochloride of formula (I), is an alkylating agent having therapeutic utility in treating Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and breast cancer.

Bendamustine hydrochloride was approved by USFDA on March 20, 2008 and is marketed under the brand name 'Treanda' in the form of a sterile non-pyrogenic white to off-white lyophilized powder in a single use vial, with strength of 100 mg /vial and 25 mg / vial, for the treatment of chronic lymphocytic leukemia (CLL).
Various researchers have attempted to synthesize the active pharmaceutical ingredient bendamustine hydrochloride of formula (I).
DD 34727 and DD 159877 describe a process for preparing bendamustine and pharmaceutically acceptable salts, by a method involving chlorination of hydroxyl ester with thionyl chloride in chloroform as solvent at 0°C to 5°C, followed by

removal of excess thionyl chloride and saponification of the resulting ester with aqueous hydrochloric acid. However, the specification does not mention the purity and yield of bendamustine or its hydrochloride salt. Further, the replication of these experiments at our laboratory resulted in formation of an associated N-chloroalkyl impurity of formula (III) in the range of 18-25%; which necessitated additional purification steps for obtaining a product conforming to regulatory specification.

CN 101691359 describes a process for preparation of bendamustine by utilizing phosphorous oxychloride as chlorinating agent in an organic solvent like chloroform, toluene, dichloromethane or xylol. The method employs abnormally high quantity of phosphorous oxychloride (10-30 moles) with respect to the hydroxy ester substrate viz. 4-(6-bis(2-hydroxyethylamino)-3-methylbenzimidazoylbutyrate for chlorination reaction. Although the specification, indicates a purity of 99.5% and yield of 65.5%; however replication of experiments at our laboratory showed that the formation of N-chloroalkyl impurity of formula (III) was quite appreciable. Further, since this impurity is very difficult to remove, therefore additional purification steps like crystallization or column chromatography were required to obtain the product conforming to regulatory specifications, which further increased the number of steps, lowers the yield and thereby increasing the cost.

Monatshefte fur Chemie (1997), 128, 291-299 discloses a process for preparation of bendamustine hydrochloride by employing a eleven step sequence starting from 2,4-dinitrochlorobenzene and the crucial step, involves chlorination of ethyl 4-(6-bis(2-hydroxyemylammo)-3-methylbenzimidazoylbutyrate (dihydroxy ester) with thionyl chloride, to give ethyl 4-(6-bis(2-cUoroethyl)arnmo-3-memylbenzimidazol-2-ylbutyrate (dichloro ester) followed by the subsequent ester hydrolysis with hydrochloric acid to obtain bendamustine hydrochloride. Under the disclosed reaction conditions, bendamustine was found to hydrolyze further to N-chloroalkyl impurity of formula (HI), upto 20%.
WO 2011079193 provides a process for the preparation of bendamustine hydrochloride, wherein reaction of 4-(5-Amino-l -methyl- lH-benzoirnidazol-2-yl)-butyric acid isopropyl ester with 2-haloethanol in the presence of an organic base gave 4-{5-[bis-(2-hyo^oxyethyl)arnmo]-l-methyl-l-H-benzoimidazol-2-yl}-butyric acid isopropyl ester, which on treatment with a chlorinating agent provides 4-{5-[bis-(2-chloroemyl)arnino] -1 -methyl-1 H-benzoimidazol-2-yl} -butyric acid isopropyl ester. Further, hydrolysis gives bendamustine hydrochloride with a yield of 61%. However, it was found that during quenching of reaction mixture with water in acidic environment, the N-chloroalkyl impurity of formula (III) was significantly formed.
Additionally, the PCT application does not disclose any specific process with phosphorous oxychloride as chlorinating agent; however replication of experiments with phosphorous oxychloride as per the method disclosed in the application, resulted in the formation of unidentified impurity along with N-chloroalkyl impurity of formula (III) to the extent of 0.22% which resulted in a low yield of 45%. Regulatory authorities all over the world have very stringent norms for permissible limits of such impurities in either the active ingredient or the final formulation.

Thus, it would be evident from the foregoing specification that prior art methods for chlorination of 4-(6-bis(2-hydroxyethylamino)-3-methylbenzimidazoyIbutyrate ester (hydroxy ester) are less efficient, yield unidentified impurities along with compound of formula (III), and do not provide the product of desired quality even after repeated purification. Since, the impurity formed during chlprination reaction is not easily removed during purification by recrystallization or column chromatography; hence these methods are not practical on a commercial scale.
Thus, there is a need to develop a process for the preparation of bendamustine hydrochloride (I), which obviates the formation of impurities and does not require column chromatography purification for getting the desired purity.
Further, considering the commercial importance of bendamustine hydrochloride, the present inventors have developed a method which provides the desired product and conforms to regulatory specifications by overcoming prior art drawbacks.
OBJECTS OF THE INVENTION
An object of the present invention is to provide an improved process for preparation of bendamustine hydrochloride, which is free from N-chloroalkyl impurities and does not utilize column chromatography purification.
Another object of the present invention is to provide a process for controlling the formation of undesired impurities during the chlorination of hydroxy ester and isolating the desired product by a method which removes unidentified impurities and provides a product confirming to regulatory specification.
SUMMARY OF THE INVENTION
An aspect of the present invention relates to a process for preparation of bendamustine hydrochloride comprising chlorination of hydroxy ester and isolating

the intermediate ester in a definite pH range, hydrolyzing with hydrochloric acid at 25 to 30°C and providing bendamustine hydrochloride free from the corresponding N-chloroalkyl and other unidentified impurities.
Another aspect of the present invention relates to a process for preparation of bendamustine hydrochloride comprising reaction of 4-{5-[Bis-(2-hydroxyethyl)-amino]-l-methyl-l-H-ben2irnidazol-2-yl}-butyric acid ester (II) with a phosphorous oxychloride in absence of a solvent at 80-90°C, concentrating the mixture and quenching with aqueous alcohol mixture, adjusting to neutral pH with a base, treating the filtered solid with hydrochloric acid at 25-30°C, concentrating the mixture and filtering to give bendamustine hydrochloride of desired purity with associated impurities below regulatory limits.
Yet another aspect of the invention relates to preparation of bendamustine hydrochloride having N-chloroalkyl impurity of formula (III) and other associated impurities below regulatory limits.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an improved process for isolation of bendamustine comprising chlorination of 4-{5-[Bis-(2-hydroxyethyl)-amino]-l-methyl-l-H-benzimidazol-2-yl}-butyric acid ester (II) in absence of an organic solvent, wherein R is C1-C4 alkyl group.
The present inventors have found that the chlorination of hydroxy ester with chlorinating agent in presence of organic solvent and aqueous work up results in the formation of an impurity of formula (III) ranging from 0.25 to 0.5%. The present inventors have separated the impurity by preparative HPLC and identified with the help of NMR & mass spectroscopy.

It was unexpectedly found after several experiments that formation of the compound of formula (III) could be avoided during the chlorination reaction. Further, the inventors were amazed to note that, not only the reaction was facile, but the absence of associated impurities provided bendamustine which not only had the desired purity, even the associated impurities were found to be below permissible regulatory limits. The inventors found that the present invention provide a solution to the problem associated with the prior art methods.
One embodiment of the present invention relates to a process for chlorination of hydroxy ester of formula (II) with chlorinating agent in absence of an organic solvent. The synthetic sequence for bendamustine is represented in the following scheme I:

4-{5-{bis-(2-hydroxyethyl)-amirjol- Bendamustine
1-methy1-1-H-benzimidazol-2-yl}- hydrochloride (I)
butyric acid ester (II)
(R -C1-C4AIkyl group)
The starting 4- {5-[Bis-(2-hydroxyemyl)-amino]-l-methyl-1 -H-benzimidazol-2-yl}-butyric acid ester (II) may be obtained by the process disclosed in the prior art.
The detailed reaction process for bendamustine is described in the following steps; i) chlorination of compound of formula (II) with phosphorous oxychloride in absence of an organic solvent,

ii) concentration of the reaction mixture, optionally in presence of an organic
solvent, iii) quenching of the residue with an aqueous mixture of water and alcohol, iv) adjusting the pH of the mixture to 7.0-7.5 with a base, v) ester hydrolysis with hydrochloric acid at 25-30°C, and vi) partially concentrating the mixture and filtering to give bendamustine
hydrochloride (I) of desired purity with impurity level according to
regulatory limits.
Another embodiment of the present invention relates to an improved process for preparation of bendamustine hydrochloride comprising, chlorination of compound of formula (II) with chlorinating agent in absence of an organic solvent. Prior art methods disclose aqueous work-up after chlorination reaction, which generates the N-chloroalkyl impurity by degradation of the product.
In view of the above drawback, the present inventors have developed a process for preparation of bendamustine wherein chlorination reaction is carried out in absence of an organic solvent, wherein the chlorination reaction utilizes optimum quantity of the chlorinating agent in absence of an organic solvent, followed by removal of excess phosphorous oxychloride and quenching with an aqueous alcohol mixture, which results in minimal formation of N-chloroalkyl impurity of formula (III).
The chlorinating agent for chlorination reaction is selected from the group comprising of phosphorous oxychloride, phosphorous trichloride, but preferably phosphorous oxychloride. The amount of chlorinating agent utilized for chlorination reaction is in the range of 2 to 8 moles per mole of compound of the formula (II), but preferably in the range of 4 to 7 moles.

The chlorination reaction may be carried out at temperature ranging from 60°C to 1 lO°C, but preferably temperature in the range from 80°C to 90°C.
After complete of the chlorination reaction, excess of chlorinating agent is distilled under reduce pressure at temperature below 60°C. The distillation of chlorinating agent is optionally carried out in presence of an organic solvent. The solvent for distillation is selected from aromatic or aliphatic hydrocarbon and more preferably selected from toluene and cyclohexane.
In another embodiment of the invention, after removal of chlorinating agent, the residue containing the product is quenched with mixture of water and alcohol at 25°C to 35°C. The mixture of water and alcohol employed is in the ratio of 3:2, preferably in the ratio of 4:1. The alcohol used is selected from the group methanol, ethanol, n-propyl alcohol, isopropyl alcohol or t-butyl alcohol but preferably isopropyl alcohol.
The pH of the reaction mixture is adjusted between 7 and 7.5 with a base. The base for pH adjustment is selected from organic and inorganic base, preferably inorganic base. The inorganic base is selected from alkali or alkaline earth metal carbonates or bicarbonates, preferably alkali metal carbonates like sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate.
The pH adjustment is a vital part of the invention during isolation of "bendamustine hydrochloride. From the prior art experiments it was observed that during isolation of bendamustine hydrochloride, if the pH is in acidic region, the N-chloroalkyl impurity formula (III) was formed during work up and further if pH is in the basic region, degradation of the product generates an associated impurities.

Therefore, the present inventors have established a process wherein bendamustine is isolated at neutral pH. Thus, the present invention provides no formation of impurity, by degradation of product at neutral pH.
At neutral pH, reaction mixture is cooled to 0°C to 5°C and the precipitated solid is filtered and washed with water. Finally, hydrolysis of ester in presence of hydrochloric acid to give bendamustine hydrochloride of desired purity and yield were in the range of 75% to 80%.
A further advantage of the invention is that the chlorination reaction is carried out in solvent free condition which reduces burden on environment and in turn has a positive effect on production cost. Furthermore, the invention provides a process for preparation of bendamustine hydrochloride, which eliminates the formation of N-chloroalkyl impurity with good yield in comparison to the conventional processes reported in the prior art.
The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The present invention is described herein below with reference to examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner.
EXAMPLE:
Example 1: Preparation of bendamustine hydrochloride (I) (with 2.0 mole
phosphorous oxychloride):
4- {5-[Bis-(2-hydroxyethyl)-amino]-1 -methyl-1 -H-benzoimidazol-2-yl} -butyric acid isopropyl ester (lOOgms, 0.2754 moles) and phosphorous oxychloride (84.43gms, 0.550 moles) were suspended in flask at 0°C to 5°C. The reaction mixture was heated to 80-90°C for 2 hours under stirring. After reaction completion as monitored by HPLC, the mixture was cooled to 45°C to 50°C and excess phosphorous oxychloride was removed under reduced pressure below 60°C. Toluene (200ml) was added to the

residue and concentrated to obtain a residue. Water (400 ml) and isopropyl alcohol (100ml) mixture were added to the residue and the pH was adjusted to 7.0-7.5 with aqueous solution of potassium carbonate (115gms). The reaction mass was cooled to 0°C to 5°C for 1 hour and the solid filtered and washed with water (100 ml). The wet cake was stirred with hydrochloric acid (900 ml) at 25°C to 30°C for 14. to 16 hours, after complete hydrolysis as monitored by HPLC, the mixture was concentrated until bendamustine hydrochloride separated out from the mixture, which was then filtered and optionally purified from acetone/water mixture. Yield: 73gms, Purity > 99.5%.
Example 2: Preparation of bendamustine hydrochloride (I) (with 3 mole phosphorous oxychloride):
4- { 5 -[Bis-(2-hydroxyethyl)-amino] -1 -methyl-1 -H-benzoimidazol-2-yI} -butyric acid isopropyl ester (lOOgms, 0.2754 moles) and phosphorous oxychloride (126.64gms, 0.826 moles) were suspended in flask at 0°C to 5°C. The reaction mixture was heated to 80- 90°C for 2 hours under stirring. After reaction completion as monitored by HPLC, the mixture was cooled to 45°C to 50°C and excess phosphorous oxychloride was removed under reduced pressure below 60°C. Toluene (200ml) was added to the residue and continues the distillation till trace of phosphorous oxychloride removed from the residue. Water (400ml) and isopropyl alcohol (100ml) mixture were added to the residue and pH adjusted to 7.0-7.5 with aqueous solution of potassium carbonate (115gms). The reaction mass was cooled to 0°C to 5°C for 1 hour, solid filtered and washed with water (100ml). The wet cake was stirred with hydrochloric acid (900ml) at 25°C to 30°C for 14 to 16 hours, after complete hydrolysis as monitored by HPLC, the mixture was concentrated until bendamustine hydrochloride separates out from the mixture, which was then filtered and optionally purified from acetone- water mixture. Yield 79gms, Purity > 99.5%.

Example 3: preparation of bendamustine hydrochloride (I) (with 6.4 mole phosphorous oxychloride):
4-{5-[Bis-(2-hydroxyethyl)-amino]-l-methyl-l-H-ben2oimidazol-2-yl}-butyric acid isopropyl ester (l00gms, 0.2754 moles) and phosphorous oxychloride (211.15gms, 1.77 moles) were suspended in flask at 0°C to 5°C. The reaction mixture was heated to 80- 90°C for 2 hours under stirring. After reaction completion as monitored by HPLC, the mixture was cooled to 45CC to 50°C and excess phosphorous oxychloride was removed under reduced pressure below 60°C. Toluene (200ml) was added to the residue and continues the distillation till trace of phosphorous oxychloride removed from the residue. Water (400ml) and isopropyl alcohol (100ml) mixture were added to the residue and pH adjusted to 7.0-7.5 with aqueous solution of potassium carbonate (115gms). The reaction mass was cooled to 0°C to 5°C for 1 hour, solid filtered and washed with water (100ml). The wet cake was stirred with hydrochloric acid (900ml) at 25°C to 30°C for 14 to 16 hours, after complete hydrolysis as monitored by HPLC, the mixture was concentrated until bendamustine hydrochloride separates out from the mixture, which was then filtered and optionally purified from acetone- water mixture. Yield 74gms, Purity > 99.5%.
Example 4: Preparation of bendamustine hydrochloride (I) (with 8.0 mole phosphorous oxychloride):
4- {5-[Bis-(2-hydroxyemyl)-amino]-1 -methyl-1 -H-benzoimidazol-2-yl} -butyric acid isopropyl ester (lOOgms, 0.2754 moles) and phosphorous oxychloride (337.72gms, 2.203 moles) were suspended in flask at 0°C to 5°C. The reaction mixture was heated to 80- 90°C for 2 hours under stirring. After reaction completion as monitored by HPLC, the mixture was cooled to 45°C to 50°C and excess phosphorous oxychloride was removed under reduced pressure below 60°C. Toluene (200ml) was added to the residue and continues the distillation till trace of phosphorous oxychloride removed from the residue. Water (400ml) and isopropyl alcohol (100ml) mixture were added

to the residue and pH adjusted to 7.0-7.5 with aqueous solution of potassium carbonate (115gms). The reaction mass was cooled to 0CC to 5°C for 1 hour, solid filtered and washed with water (100ml). The wet cake was stirred with hydrochloric acid (900ml) at 25°C to 30°C for 14 to 16 hours, after complete hydrolysis as monitored by HPLC, the mixture was concentrated until bendamustine hydrochloride separates out from the mixture, which was then filtered and optionally purified from acetone- water mixture. Yield 75gms, Purity > 99.5%.
Example 5: Preparation of bendamustine hydrochloride (I) (with 6.4 mole phosphorous oxychloride):
4-{5-[Bis-(2-hydroxyemyl)-arrimo]-l-methyl-l-H-berizoimida2ol-2-yl}-butyric acid isopropyl ester (l0gms, 0.02754 moles) and phosphorous oxychloride (21.1 lgms, 0.177 moles) were suspended in flask at 0°C to 5°C. The reaction mixture was heated to 80- 90°C for 2 hours under stirring. After reaction completion as monitored by HPLC, the mixture was cooled to 45°C to 50°C and excess phosphorous oxychloride was removed under reduced pressure below 60°C and continues the distillation till trace of phosphorous oxychloride removed from the residue. Water (400ml) and isopropyl alcohol (100ml) mixture were added to the residue and pH adjusted to 7.0-7.5 with aqueous solution of potassium carbonate (115gms). The reaction mass was cooled to 0°C to 5°C for 1 hour, solid filtered and washed with water (100ml). The wet cake was stirred with hydrochloric acid (900ml) at 25°C to 30°C for 14 to 16 hours, after complete hydrolysis as monitored by HPLC, the mixture was concentrated until bendamustine hydrochloride separates out from the mixture, which was then filtered and optionally purified from acetone- water mixture. Yield 8gms, Purity > 99.5%.

WE CLAIM,
1. A process for preparation of bendamustine hydrochloride of desired purity comprising reaction of 4-{5-[bis-(2-hydroxyethyl)-amino]-l-methyl-l-H-benzunidazol-2-yl} -butyric acid ester (II) with phosphorous oxychloride in absence of a solvent at 80-90°C, concentrating the mixture and quenching with aqueous alcohol mixture, adjusting to neutral pH with a base, treating the filtered solid with hydrochloric acid at 25-30°C, concentrating the mixture and filtering to give bendamustine hydrochloride of desired purity with associated impurities below regulatory limits.
2. The process as claimed in claim 1 wherein the molar quantity of phosphorous oxychloride is in the range of 2 to 8 moles per mole of compound of formula (II), preferably in the range of 4 to 7 moles.
3. The process as claimed in claim 1 wherein the excess phosphorous oxychloride is removed by distillation; optionally in presence of an organic solvent selected from aromatic or aliphatic hydrocarbons, more preferably toluene or cyclohexane.

4. The process as claimed in claim 1 wherein, pH is adjusted between 7.0 and 7.5.
5. The process as claimed in claim 1 wherein the base used for neutralization is selected from an organic or inorganic base but preferably an inorganic base.
6. The process as claimed in claim 5, wherein the inorganic base is selected from
alkali metal carbonates and bicarbonates but preferably sodium carbonate,
potassium carbonate, sodium bicarbonate or potassium bicarbonate.

7. The process as claimed in claim 1 wherein the isolated bendamustine hydrochloride has N-chloroalkyl impurity less than 0.1%.