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 EFFICIENT PROCESS FOR PREPARATION OF PHENYLALANINE MUSTARD HYDROCHLORIDE"
Emcure Pharmaceuticals Limited.,
an Indian Company, registered under the Indian Company's Act 1957
and having its Registered Office at
Emcure House, T-184, 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 a process for preparation of phenylalanine mustard hydrochloride salt of desired purity. More specifically, the invention relates to a method for preparation of 4-bis(2-choloroethyl)ammo]-L-phenylalariine hydrochloride (I) of desired purity, by reaction of melphalan free base with concentrated hydrochloric acid in water and isolating the compound of formula (I) having desired purity.
BACKGROUND OF THE INVENTION
Melphalan, chemically known as 4-[bis(2-chloroethyl) amino]-L-phenylalanine and also by other names like L-phenylalanine mustard, L-sarcolysine or L-PAM is a bifunctional alkylating agent that acts against selected human neoplastic diseases and is the L-isomer of 4-[bis(2-chloroethyl) amino]-phenylalanine, which exhibits superior antitumor activity than the D-isomer (medphalan) and racemic (DL)-form (sarcolysine). Melphalan is marketed as its hydrochloride salt, under the brand name 'Alkeran' by The Wellcome Foundation.

Various researchers have attempted to synthesize the active hydrochloride salt of the L-form of 4-[bis(2-chloroethyl) amino]-phenylalanine.
The synthesis of melphalan was first disclosed in US 3,032,584 and US 3,032,585 wherein melphalan free base is prepared by a synthetic route which employs a phthalimide functional group for protecting the glycine amino functional group. However, these and the subsequent patents GB 1,377,336 and EP 233 733, however do

not disclose a method for isolating the acid addition salts of the L-form of 4-[bis(2-chIoroethyl)-amino]-phenylalanine, specifically the hydrochloride salt.
US 4,997,651 discloses a method for preparing melphalan hydrochloride comprising addition of hydrochloric acid to a slurry of melphalan free base in an alcohol, preferably ethanol and refluxing the mixture for minimum duration to reduce the level of impurities. It should be noted that complete conversion of the free base to the hydrochloride salt in a short time on a commercial scale is not practically feasible since it results in large amounts of unreacted material. Further, an extended duration of heating in alcohols was found to result in associated impurities, which required successive purifications for obtaining a product of the desired purity. Also, an additional purification step reduces the yield considerably, thereby limiting the method only for laboratory scale preparation.
Similarly, GB 1,064,972, FR 1 360 836 and DE 1 292660 also describes the use of an alcohol for preparing the hydrochloride salt with the same disadvantages.
RO 57195 describes a method for purification of melphalan free base through insitu formation of the hydrochloride salt followed by treatment with a suitable base such as sodium bicarbonate or sodium acetate. However, the isolation of melphalan hydrochloride salt using alcohol as solvent results in associated impurities, which eventually requires extensive purification.
Similarly, US 4,997,651 and DE 1 292660 also utilize an alcoholic solution of hydrochloric acid with the same result. A replication of these prior art methods by the present inventors also revealed that associated impurities were indeed formed thus requiring subsequent purifications. Further, the inventors tried out several experiments to replace alcoholic solvent with other solvents like toluene, wherein the salt formation was incomplete due to the limited solubility of hydrochloric acid while in ethyl acetate or tetrahydrofuran the results were very unsatisfactory due to the high level of

impurities. Regulatory guidelines necessitated the removal of associated impurities by purification, which eventually reduced the yield.
It is clearly evident from the above prior art that
i) none of the prior art methods provides melphalan hydrochloride having the desired
purity; ii) the product obtained by prior art methods requires extensive purification resulting
in high production cost and thereby making such process commercially unviable.
Therefore, to overcome the problems associated with prior art, there was a need to
develop
i) a simple, efficient, high yielding process which does not result in unreacted starting
material remaining after treatment with hydrochloric acid and ii) a process which yields a product of desired purity with minimal impurities, thereby
obviating the need for extensive purification. Moreover, health authorities all over
the world have very stringent norms for permissible limits of these associated
impurities in the final formulation.
The present inventors after carrying out several experiments with different solvents have unexpectedly found mat the hydrochloride salt could be obtained using water during treatment with hydrochloric acid and in absence of an organic solvent. Further, the isolation of the highly water miscible compound was achieved with the help of a water-immiscible solvent and finally by the addition of a water-miscible solvent preferably an alcohol to obtain melphalan hydrochloride of desired purity and with level of impurities conforming to regulatory specifications. It should be noted that such a process using water as medium was not attempted in prior art as the isolation of the highly water soluble melphalan hydrochloride is quite tedious and cumbersome.
The addition of an alcohol after concentration of the reaction mixture makes the product free flowing and constitutes an inbuilt purification step as it helps in removal of associated impurities during filtration.

The process is convenient and cost effective for commercial scale since the process obviates the need for an additional purification step.
OBJECT OF THE INVENTION
An object of the present invention is to provide a cost effective process for synthesis of 4-[bis(2-chloroethyl)-amino]-L-phenylalanine hydrochloride of desired purity and conforming to regulatory specifications.
Another object of the present invention is to provide a simple cost effective process for 4-[bis(2-chloroethyl)-amino]-L-phenylalanine hydrochloride which eliminates the use of subsequent purifications.
Yet another object of the present invention is to provide a simple cost effective process for 4-[bis(2-chloroethyl)-amino]-L-phenylalanine hydrochloride which avoids utilization of an organic solvent during treatment of 4-[bis(2-chloroethyl)-amino]-L-phenylalanine free base with hydrochloric acid.
SUMMARY OF THE INVENTION
An aspect of the invention is to provide a simple method for preparation of 4-[bis(2-chloroethyl)-amino]-L-phenylalanine hydrochloride having desired purity.
Another aspect of the present invention relates to providing a cost effective method for synthesis of melphalan hydrochloride formula (I) comprising treatment of 4-[bis(2-chloroethyl)-amino]-L-phenylalanine with hydrochloric acid in water followed by concentration in presence of a solvent, stirring with an alcohol and filtering to yield 4-[bis(2-chloroethyl)-amino]-L-phenylalanine hydrochloride (I) of desired purity.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a simple, economical and commercial viable process for the preparation of melphalan hydrochloride (I).

Melphalan free base is conventionally prepared in the prior art from N-t-
butyloxycarbonyl-bis-[(2-hydroxyethyl)-amino]-L-phenylalanine methyl ester (II), as
represented in Scheme-I, and which in turn is prepared by the process disclosed in CN
101100440. N-t-butyloxycarbonyl-bis-[(2-hydroxyethyl)-amino]-L-phenylaIanine
methyl ester (II) is subjected to chlorination with phosphorus oxychloride or thionyl chloride followed by acid hydrolysis for the removal of the ester protection followed by neutralization with aqueous ammonia to produce 4-[bis(2-chloroethyl)-amino]-L-phenylalanine i.e. melphalan (IV) free base as represented in scheme-I.

The present invention relates to a process for preparation of 4-[bis(2-chloroethyl)-amino]-L-phenylalanine hydrochloride (I) from its free base.
In an embodiment, aqueous hydrochloric acid is added to melphalan free base (IV) suspended in water at ambient temperature 15 to 20°C. The temperature is stirred at 15°C to 45°C in 2 to 3 hours to give a clear solution containing melphalan hydrochloride (I).
The acid addition salt formation reaction is preferably carried out between 25°C and 30°C.

The isolation of the compound of formula (I) comprises addition of an organic solvent preferably a hydrocarbon solvent to the reaction mixture and concentrating the mixture under reduced pressure.
The addition of the organic solvent followed by concentration of the reaction mixture may be carried out more than once if desired to achieve moisture content less than 5%.
The aromatic hydrocarbon is selected from the group comprising of toluene, cumene, xylene etc, but preferably toluene.
The reaction mixture is stirred with an alcohol at ambient temperature, filtered, washed with the alcohol and dried to give melphalan hydrochloride of desired purity.
It should be noted that the prior to the addition of the alcohol, the concentrated residue is sticky and not filterable due to the presence of impurities which hinder the formation of an easily filterable free flowing solid.
The present inventors had tried out several solvents like esters, ethers, hydrocarbons etc for obtaining a filterable solid; however, it was found that the desired objective could be achieved by addition of an alcohol. Therefore, the addition of an alcohol before filtration constitutes an inbuilt purification step, as it helps in dissolving the impurities without affecting the yield and helps in obtaining the compound of formula (I) having the desired purity.
The alcoholic solvents used for isolation are selected from the group comprising of ethanol, isopropyl alcohol, n-propanol, isobutanol and t-butanol or mixture thereof, but preferably isopropyl alcohol.
Thus, the present invention is a simple, cost effective method for preparation of melphalan hydrochloride having the desired purity.

The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention, which is intended to be protected herein, however, is not to be understood as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art, without departing from the spirit of the invention.
The invention is further explained with the help of following illustrative examples, however, in no way these examples should be construed as limiting the scope of the invention.
EXAMPLES:
Example 1:
Preparation of compound of 4-{bis (2-chloroethyl)-ainino]-L-phenylalanine methyl
ester hydrochloride (III):
N-t-butyloxycarbonyl-bis-[(2-hydroxyethyl)-amino]-L-phenylalanine methyl ester (II) (85 grams, 0.222 mole) was stirred in acetonitrile (425 ml) to get a clear solution at 25°C to 30°C. Phosphorous oxychloride (124 ml, 1.33 moles) was charged drop wise to the above clear solution at 25°C to 30°C and the mixture was stirred at 75°C to 80°C. After completion of the reaction, mixture was concentrated under reduced pressure to give crude 4-[bis (2-chloroethyl)-amino]-L-phenylalanine methyl ester hydrochloride (III) which was used as such for the next step.
Example 2:
Preparation of compound of 4-[Bis(2-chloroethyl)-amino]-L-phenylalanine
(Melphalan) (IV):
Concentrated hydrochloric acid (425 ml, 5 volume) was added to the crude 4-[bis(2-chloroethyl)-amino]-L-phenylalanine methyl ester hydrochloride (HI) obtained in Example 1 and the mixture was heated at 80°C to 850C. After completion of the reaction as monitored by HPLC, the mixture was treated with activated carbon and filtered through hyflobed. The filtrate was cooled to 0°C and the pH of the filtrate was adjusted to pH 6.0 by addition of aqueous ammonia solution (450 ml). The product was

filtered, washed with water and the wet 4-[bis(2-chloroethyl)-amino]-L-phenylalanine free base (IV) was used as such for preparation of the hydrochloride salt.
Example 3:
Preparation of 4-[bis (2-chloroethyl)-amino]-L-phenylalanine hydrochloride (I):
4-[Bis(2-chloroethyl)-amino]-L-phenylalanine (V) obtained in Example 2 was treated with concentrated hydrochloric acid (80 ml) at 25°C to 30°C and stirred at same temperature for 15-20 hours. Toluene (1060ml) was added and the resulting mixture was concentrated under reduced pressure till the moisture content of the residue was about 5%. Isopropyl alcohol (765ml) was added to the resulting residue and stirred at 0-5°C for 2-4 hours and filtered. 4-[Bis(2-chloroethyl)-amino]-L-phenylaIanine hydrochloride was filtered, washed with isopropyl alcohol (340ml) and dried. Yield: 38 grams HPLC Purity: > 99%.

CLAIMS:
1. A process for the preparation of 4-bis(2-chloroethyl)-amino]-L-phenylalanine
hydrochloride having the desired purity, comprising,
i. treatment of melphalan free base in water with hydrochloric acid, ii. adding an organic solvent and concentrating the mixture, iii. stirring an alcohol with the residue and filtering.
2. A process as claimed in claim 1, wherein hydrochloric acid is an aqueous solution.
3. A process as claimed in claim l(i), wherein the reaction temperature is between 15°C and 45°C but preferably between 25°C and 30°C.
4. The process as claimed in claim l(ii), wherein the organic solvent is a hydrocarbon and is preferably toluene.
5. The process as claimed in claim 1 (iii), wherein the alcohol is selected from the group comprising of C2 to C6 alcohols.
6. The process as claimed in claim 6, wherein the alcohol is selected from the group comprising of ethanol, isopropyl alcohol, n-propanol, isobutanol and t-butanol or mixtures thereof.
7. The process as claimed in claim 6, wherein the alcohol is preferably isopropyl alcohol.