FIELD OF THE INVENTION
The present invention relates to an improved process for the preparation of Chlorambucil of formula (1), having purity greater than 99% as determined by HPLC.
BACKGROUND OF THE INVENTION
Chlorambucil is an orally active aromatic nitrogen mustard alkylating agent used as antineoplastic agent for the treatment of various malignant and nonmalignant diseases. Chemically it is known as 4-[4-[bis(2-chloroethyl) amino] phenyl] butanoic acid. Chlorambucil is sold under the brand name LEUKERAN and used to treat chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, and non-Hodgkin lymphoma.
The synthesis of Chlorambucil of formula (1) has been reported in few patents and non¬patent literature, the contents of which are hereby incorporated as reference in their entirety.
US2944079 discloses the synthesis of Chlorambucil of formula (1) by treating methyl p-amino phenyl butyrate with ethylene oxide, benzene, phosphoryl chloride under reflux conditions to form intermediate methyl para-NN-di-(2-hydroxyethyl)-amino phenylbutyrate. This intermediate on treating with phosphoryl chloride to form intermediate methyl para-NN-di-(2-chloroethyl)-amino phenylbutyrate which on hydrolysis by hydrochloric acid formed Chlorambucil of formula (1) which was recrystallized from petrol-ether.
US3046301 discloses the synthesis of intermediate methyl-[p-amino-phenyl] butyrate in high yield which is used for the further synthesis of Chlorambucil of formula (1).

Organic and Biomolecular Chemistry, 2012, vol. 10, p. 5238 - 5244 reports the preparation of Chlorambucil by photolysis of series of styryl conjugated 2-nitrobenzyl-caged (SNB) chlorambucil prodrugs.
Prior art reports found difficulty in isolation of intermediates and are silent about the purity of the final Chlorambucil of formula (1) so obtained. Hence, the present inventors hereby reported an improved synthetic process to obtain Chlorambucil of formula (1) with purity greater than 99.0%.
OBJECTIVE OF THE INVENTION
Accordingly, one objective of the present invention is to provide an improved process for the preparation of Chlorambucil of formula (1).
Another objective of the present invention is to provide a process for the purification of Chlorambucil of formula (1).
Yet, another objective of the present invention is to provide Chlorambucil of formula (1) with purity greater than 99.0% by High-performance liquid chromatography (HPLC).
SUMMARY OF THE INVENTION
Accordingly, in one embodiment the present invention provides an improved process for the preparation of substantially pure Chlorambucil of formula (1) and its pharmaceutical acceptable salts with purity greater than 99.0%.
In another embodiment, the steps involved in the preparation of Chlorambucil of formula (1) as shown in scheme-1 are as follows:
a) Addition reaction of methyl 4-(4-aminophenyl) butanoate of formula (4)
3

with ethylene oxide to form methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3)
[3)
) chlorination of methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3) with a suitable chlorinating agent to form methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula (2)
which was directly used for the next step without purification. c) acidic hydrolysis of methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula (2) to form Chlorambucil of formula (1)

d) purification of Chlorambucil of formula (1)
According to another embodiment, the present invention discloses an improved process for the preparation of methyl 4-(4-aminophenyl) butanoate of formula (4), as shown in scheme-2 comprises of the following steps: i. Esterification of 4-(4-nitrophenyl) butanoic acid of formula (6)
reduction of the nitro group of methyl-4-(4-nitrophenyl) butanoate of formula (5) to amino group to form methyl 4-(4-aminophenyl) butanoate (4)

In another embodiment the present invention provides a process for the purification of Chlorambucil of formula (1) comprises:
a) providing a solution of Chlorambucil of formula (1) in an aprotic solvent;
b) removing the solvent at suitable temperature;
c) adding a non-polar solvent and
d) isolating pure Chlorambucil of formula (1).
In another part of the embodiment the pure Chlorambucil of formula (1) prepared has purity greater than 99.0 % and having the impurities B, E and G are less than 0.15% (w/w).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Illustrates the X-Ray powder diffraction pattern (XRPD) of Chlorambucil of formula (1).
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, this invention provides an improved process for the preparation of substantially pure Chlorambucil of formula (1) with purity greater than 99.0% as illustrated in scheme 1.

Accordingly, the steps involved for the preparation of Chlorambucil free base of formula (1) as shown in scheme-1 are as follows:
Step a) involves the addition of methyl 4-(4-aminophenyl) butanoate of formula (4) to ethylene oxide to form methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3). Methyl 4-(4-aminophenyl) butanoate of formula (4) was dissolved in a suitable protic solvent at 25-30 °C, heated to 45-50 °C and then cooled to 25-30 °C. Ethylene oxide gas was then passed into the solution for 12-15hrs. Finally, the reaction mass was extracted with a suitable aprotic solvent at 25-30 °C and the organic layer

was dried, filtered and the filtrate distilled off under vacuum to obtain intermediate of formula (3).
Step b) proceeds with the chlorination of methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3) with a suitable chlorinating agent to form methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula (2). Intermediate of formula (3) was treated with a suitable chlorinating agent at 25-30 °C, heated and stirred for 3-4hrs at 80-85 °C. On completion of reaction, the solvent was distilled off and the residue was washed with a suitable aprotic solvent to form intermediate methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula (2). Washing with a suitable aprotic solvent is important to remove the excess of chlorinating agent which otherwise can cause the reaction to become exothermic and it also help to reduce the formation of impurity E.
The suitable chlorinating agent used herein could be selected from a group comprising of oxalyl chloride, thionyl chloride, phosphorus pentachloride, phosphorus trichloride, phosphorous oxychloride or the like, preferably phosphorous oxychloride was used in the present invention.
Step c) involves the acidic hydrolysis of intermediate (2). The solid intermediate (2) was treated with a suitable inorganic acid at 25-30 °C, heated to 90-95 °C and cooled to 25-30 °C. The reaction mass was then treated with a suitable protic solvent and a decolorizing agent and filtered through Hyflo at 25-30 °C. The filtrate was extracted with a suitable aprotic solvent and the organic layer collected, dried and filtered. The filtrate was distilled off to obtain a crude solid. The crude was dissolved in a suitable protic solvent and stirred to form a clear solution. To the clear solution was extracted with different volumes of suitable aprotic solvent and the total organic layer was collected. The organic layer was dried over sodium sulphate and filtered to obtain solid crude. The crude was dissolved in a protic solvent and stirred to obtain a clear solution.

A suitable protic solvent was then added to the clear solution, stirred and filtered to obtain crude. The crude was then washed with an aprotic solvent and dried under vacuum to obtain Chlorambucil of formula (1). The suitable inorganic acid used herein was selected from a group comprising of hydrochloric acid, sulfuric acid, phosphoric acid or the like, preferably hydrochloric acid was used in the present invention.
Step d) proceeds with the purification of crude Chlorambucil of formula (1). The crude Chlorambucil of formula (1) was dissolved in a suitable aprotic solvent at 20-25 °C and stirred for 10-20 min to obtain clear solution. The clear solution was dried over sodium sulphate and filtered under vacuum. The filtrate again filtered through 0.2 u filter and distilled off the solvent under vacuum. The residue so obtained was cooled to 20-25 °C, treated with an aprotic solvent with stirring and filtered. The solid so obtained was washed with a suitable chilled aprotic solvent and dried under vacuum to obtain crude Chlorambucil of formula (1).
The aprotic solvents used in step a), step b), step c) and step d) of scheme 1 were selected from a group comprising of acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N, N-dimethylformamide, methyl tertiary butyl ether, n-hexane, cyclohexane, toluene, n-heptane, tetrahydrofuran or the like, preferably ethyl acetate, acetonitrile, n-heptane and methyl tertiary butyl ether were used in the present invention.
The protic solvents used in step a) and step c) of scheme 1 were selected from a group comprising of methanol, ethanol, isopropyl alcohol (IPA), n-propanol, n-butanol, water or the like, preferably water and methanol were used in the present invention.

In another embodiment, the present invention provides an improved process the preparation of intermediate methyl 4-(4-aminophenyl) butanoate of formula (4) as depicted in scheme-2.
Step i) proceeds with the esterification of 4-(4-nitrophenyl) butanoic acid of formula (6) in suitable protic solvents to form methyl 4-(4-nitrophenyl) butanoate of formula (5).
Step ii) proceeds with the reduction of the nitro group of methyl-4-(4-nitrophenyl) butanoate of formula (5) dissolved in a suitable protic solvent in presence of a suitable metal catalyst under pressure. The reaction mixture was then filtered, the filtrate was distilled off and cooled. The residue was then treated with an aprotic solvent, filtered and dried to form methyl 4-(4-aminophenyl) butanoate (4). The metal catalyst was selected from a group comprising of platinum, palladium, and nickel catalyst, preferably Palladium/Carbon, Platinum/Carbon or Raney nickel, more preferably Raney nickel was used in the present invention.
The protic solvents used in step i) and step ii) of scheme 2 were selected from a group comprising of methanol, ethanol, isopropyl alcohol (IP A), n-propanol, n-butanol, water or the like, preferably methanol and water were used in the present invention.
The aprotic solvents used in step i) and step ii) of scheme 2 were selected from a group comprising of acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N, N-dimethylformamide, methyl tertiary butyl ether, n-hexane, cyclohexane, toulene, n-heptane, tetrahydrofuran or the like, preferably n-heptane was used in the present invention.
In another embodiment the present invention provides a process for the purification of Chlorambucil of formula (1) comprises:

a) providing a solution of Chlorambucil of formula (1) in an aprotic solvent;
b) removing the solvent;
c) adding a non-polar solvent; and
d) isolating pure Chlorambucil of formula (1).
The aprotic solvent used in the said purification can be selected from acetonitrile, methyl tertiary butyl ether, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N, N-dimethylformamide, preferably methyl tertiary butyl ether was used. The non-polar solvent used in the said purification can be selected from n-hexane, n-heptane preferably n-heptane was used.
In another embodiment, Chlorambucil of formula (1) obtained after purification by HPLC was reported to have water content by Karl Fischer method was less than 0.5 (% w/w).
In another embodiment, Chlorambucil of formula (1) obtained after purification by HPLC was having unspecified single maximum impurity less than 0.10 (% w/w) and total impurities less than 1.0(% w/w), more specifically 0.5(% w/w).
In another embodiment of the invention the pure Chlorambucil of formula (1) obtained after purification was having purity greater than 99.0% and was having impurity E less than 0.6 (%, w/w) and having impurities B and G are less than 0.4 (% w/w).

EXAMPLE-1
Preparation of methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate (3)
lOOg of methyl 4-(4-aminophenyl) butanoate of formula (4) was dissolved in 2000 mL of water at 25-30 °C. The reaction mass was heated to 45-50 °C, stirred for 30minutes and cooled to 25-30 °C. 130g of ethylene oxide gas was then passed through the reaction mixture for l-2hrs at 25-30 °C. On completion of reaction, the ethylene oxide gas was degassed, and the reaction mas was extracted with 1200mL of ethyl acetate at 25-30 °C and the layers separated. The total organic layer was collected, dried over 40g of sodium sulphate at 25-30 °C and filtered. The fdtrate was distilled under vacuum to obtain methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3). Yield: 90%

EXAMPLE-2
Preparation of methyl 4-(4-(bis(2-chIoroethyl) amino) phenyl) butanoate of formula (2)
100 g of intermediate of formula (3) was treated with 250mL of phosphorous oxychloride over a period of 60-90 minutes at 25-30 °C and heated to 80-85 °C with stirring for 3-4 hrs. On completion of reaction, the solvent was removed by distillation under vacuum below 75 °C. The solid residue was stripped off with 300mL of acetonitrile to remove excess phosphorous oxychloride to obtain crude methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula (2), which was directly used for the next step without purification.
EXAMPLE-3
Preparation of crude Chlorambucil of formula (1)
The crude intermediate (2) was hydrolyzed by adding 1000 mL of hydrochloric acid over a period of 20-30 min at 25-30 °C. The reaction mass was heated to 90-95 °C and stirred for 8-10hrs.On completion of reaction, the reaction mass was cooled to 25-30 °C and treated with 3000mL of water and 20g of Neutral charcoal (Norit CN1). The reaction mixture was stirred for 10-15 minutes and filtered under vacuum through Hyfio. The filtrate was then extracted with 500 mL of ethyl acetate and the layers separated. The organic layer was further extracted with lOOOmL of ethyl acetate. The total organic layers were collected, dried over sodium sulphate at 25-30 °C and filtered under vacuum. The filtrate was then treated with 150mL of methanol, stirred for 10-15 min at 20-25 °C to obtain clear solution. 250 mL of n-heptane was added to the obtained clear solution and stirred fori 0-20 min at 20-25 °C. 100ml of water was then added to the reaction mixture, stirred for 10-15 min at 20-25 °C and filtered under vacuum. The solid so obtained was washed with 50 mL of n-heptane and suck dried to obtain crude Chlorambucil of formula (1).

EXAMPLE-4
Purification of crude Chlorambucil of formula (1)
The crude Chlorambucil of formula (1) was dissolved in 5 volumes of methyl tertiary butyl ether at 20-25 °C under nitrogen atmosphere and stirred for 10-20 minutes at 20-25 °C to obtain clear solution. The clear solution was dried over sodium sulphate and filtered. The filtrate was passed through 0.2 p filter at 20-25 °C and distilled off under vacuum below 45 °C. The residue was then cooled to 20-25 °C, stirred for lhr with addition of 3.5 volumes of n-heptane at 20-25 °C. The solid so formed was then filtered and washed with 0.5 volume of chilled n-heptane at 20-25 °C, followed by drying under vacuum for 5-6hrs at 25-30 °C under nitrogen atmosphere to obtain pure Chlorambucil of formula (1). Yield: 33 %; Purity: 99.8%
EXAMPLES
Preparation of methyl-4-(4-nitrophenyl) butanoate (5)
lOOg of 4-(4-nitrophenyl) butanoic acid of formula (6) was dissolved in 500mL of methanol at 25-30 °C, stirred for 10-15 minutes and cooled to 0-5 °C. 500mL of methanolic hydrochloric acid was then added to the reaction mixture for 30 minutes at 5-10 °C. The reaction mixture was kept aside till temperature reached to 25-30 °C and stirred 3-4hrs at 25-30 °C. On completion of reaction, the reaction mixture was concentrated to 200 mL volume by distilling under vacuum below 50 °C. The concentrated reaction mass was cooled, treated with water and stirred for lhr at 0-5 °C. The solid so formed was filtered, washed with cold water at 10-15 °C and dried under vacuum to obtain methyl-4-(4-nitrophenyl) butanoate of formula (5). Yield: 90%

EXAMPLE-6
Preparation of methyl 4-(4-aminophenyl) butanoate (4)
lOOg of methyl-4-(4-nitrophenyl) butanoate of formula (5) was dissolved in lOOOmL of methanol at 25-30 °C in a clean and dry autoclave which was set up with a pressure of 4kg of hydrogen gas at 25-30 °C. 20g of Raney nickel was added to the reaction mass and stirred for 10-12hrs. On completion of reaction, the reaction mass was unloaded from the autoclave and filtered under vacuum through Hyflo at 25-30 °C. The filtrate was distilled off under vacuum below 50 °C and the reaction mass cooled to 25-30 °C. 200 mL of n-heptane was then added to the reaction mass at 0-5 °C and stirred for lhr.The solid so formed was filtered and washed with pre-cooled n-heptane and dried under vacuum to obtain methyl 4-(4-aminophenyl) butanoate (4). Yield: 85%.

We claim:
1. A process for the preparation of Chlorambucil of formula (1),
^ ^ ^ OH
which comprises:
a) reacting methyl 4-(4-aminophenyl) butanoate of formula (4) with ethylene oxide in a protic solvent to form methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3);
b) chlorinating methyl 4-(4-(bis(2-hydroxyethyl) amino) phenyl) butanoate of formula (3) with a suitable chlorinating agent to form methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula
(2);
c) hydrolysing methyl 4-(4-(bis(2-chloroethyl) amino) phenyl) butanoate of formula (2) in the presence of an inorganic acid to form Chlorambucil of formula (1); and
d) purifying Chlorambucil of formula (1) to pure Chlorambucil of formula
(1)-
2. The process as claimed in claim 1, wherein the protic solvent used in step a) is selected from methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol, water or mixture thereof.
3. The process as claimed in claim 1, wherein the chlorinating agent used in step b) is selected from oxalyl chloride, phosphorous oxychloride, thionyl chloride, phosphorus pentachloride and phosphorus trichloride.

4. The process as claimed in claim 1, wherein the inorganic acid used in step c) is selected from a group comprising of hydrochloric acid, sulfuric acid and phosphoric acid.
5. A process for the preparation of methyl 4-(4-aminophenyl) butanoate of formula (4) comprises:
A. esterifying 4-(4-nitrophenyl) butanoic acid of formula (6) with
methanolic hydrochloride in a suitable protic solvent to form methyl 4-
(4-nitrophenyl) butanoate of formula (5); and
B. reducing methyl-4-(4-nitrophenyl) butanoate of formula (5) in a protic
solvent and in the presence of metal catalyst under hydrogen pressure
to form 4-(4-aminophenyl) butanoate of formula (4).
6. The process as claimed in claim 5, wherein the metal catalyst used in step B) is selected from Palladium on Carbon, Platinum on Carbon and Raney nickel.
7. A process for the purification of Chlorambucil of formula (1) comprises:
i. providing a solution of Chlorambucil of formula (1) in an aprotic
solvent; ii. removing the solvent at suitable temperature; iii. adding a non-polar solvent; and iv. isolating pure Chlorambucil of formula (1).
8. The process as claimed in claim 7, wherein the aprotic solvent used in step i) is selected from acetonitrile, methyl tertiary butyl ether, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N, N-dimethylformamide or mixtures thereof.
9. The process as claimed in claim 7, wherein the non-polar solvent used in step ii) is selected from n-heptane and n-hexane.

10. The process claimed in claim 7, wherein the purified Chlorambucil of formula (1) obtained by the process has more than 99.0% purity and one or more of the following:
i. less than 0.4% of impurity B; ii. less than 0.6% of impurity E; and hi. less than 0.4% of impurity G.