Title of Invention

NOVEL METHOD OF MANUFACTURE OF HIGH PURITY AMIFOSTINE TRIHYDRATE

Abstract The present invention relates to an improved process for the preparation of high purity pharmaceuticaly acceptable (greater than 99.9%) Amifostine trihydrate (I) having chemical name S-2- (3-aminopropylamino)ethyl dihydrogen phosphorothioate. Amifostine trihydrate is widely used as a cytoprotective and radio-protective agent.
Full Text

A NOVEL PROCESS FOR MANUFACTURE OF HIGH PURITY AMIFOSTINE TRIHYDRATE
The present invention relates to an improved process for the preparation of high purity Amifostine trihydrate (I) of Pharmaceutically acceptable grade, which is widely used as a cytoprotective and radio-protective agent. The particular hydrate form envisaged by the invention is the Amifostine trihydrate having chemical name S-2- (3-aminopropylamino) ethyl dihydrogen phosphorothioate (amifostine) trihydrate of formula-I.

Amifostine is developed by Southern Research Institute as radio-protective agent. Amifostine trihydrate is available in the market as Ethyol, an injection dosage form containing a lyophilized powder formulation of Amifostine trihydrate for intravenous injection.
Background of the Invention:
The compound S-2- (3-aminopropylamino)ethyl dihydrogen phosphorothioate (also known as Amifostine, ethiofos, Ethyol, NSC 296961, and WR-2721) and other aminoalkylamino dihydrogen phosphorothioates are first reported in J. Med. Chem. Vol 12, 236-243, 1969 later it was patented as US 3892824. This patent also discloses a process for making Amifostine monohydrate (V) (Scheme-I).


In this process, 2-(3-aminopropylamino) ethanol (II) and aqueous hydrobromic acid were reacted and the resulting crystalline 2-(3-aminopropylamino) ethyl bromide dihydrobromide (III) derivative was then reacted with anhydrous trisodium phosphorothioate (IV) in dimethyl formamide and water medium to get the S-2- (3-aminopropylamino) ethyl phosphorothioate monohydrate (V) (Amifostine monohydrate).
In the second process disclosed in DD 289448 and DD 289449, the drug substance is prepared by following the same synthetic methodology but using trisodium phosphorothioate dodecahydrate (VI) instead of anhydrous trisodium phosphorothioate (IV) to get Amifostine trihydrate of formula-1 (Scheme-II).

The process disclosed in US 3892824 for Amifostine monohydrate is not suitable for commercial production and is beset with the following disadvantages.
(a) Anhydrous trisodium phosphorothioate (IV) is not commercially available.
(b) The analytical quality of Amifostine monohydrate (V) obtained from the process is not disclosed.
(c) Process for the preparation of pharmaceutically acceptable trihydrate form is not
mentioned in the patent.
(d) By following the same experimental procedure, we observed that complete elimination of
sodium bromide (by product) is not easy since usage of excess of methanol precipitates
sodium bromide.
(e) The complete elimination of dimethyl formamide (DMF) for the drug substance is not an
easy task since the molecule is unstable above 40° C.

Process disclosed in the second patent (DD 289448 or DD 289449) for Amifostine trihydrate suffers from the following disadvantages.
(a) Here also the commercial availability of trisodium phosphorothioate dodecahydrate is questionable.
(b) The yields and quality of Amifostine trihydrate are not reproducible.
(c) By our observation, in this process, the amount of Amifostine thiol (VIII) formed as an
impurity is above the pharmacopoeial limits and is not acceptable.

(d) The HPLC purity of Amifostine trihydrate obtained from the process is not mentioned.
In the present scenario, a simplified and commercially viable process for the production of high
purity Amifostine trihydrate is imminent.
The difficulties in sourcing raw material like trisodium phosphorothioate commercially, and the
problems encountered in obtaining high purity Amifostine trihydrate, prompted us to prepare
Amifostine trihydrate by adopting a novel industrially feasible methodology.
To meet the market requirement for high purity Amifostine trihydrate of formula-I, we wish to
disclose a simple and commercially viable process comprising of the reaction of 2-(3-
aminopropylamino)ethyl bromide dihydrobromide and thiophosphoryl chloride in aqueous
alkaline medium (Scheme-Ill).

Objective and Statement of Invention.
Amifostine trihydrate is a well-known radio-protectant that has now been in the market and has shown great promise as a valuable radio-protective agent with less toxic effects. Keeping in view, the above-mentioned difficulties in commercialization of the known processes for the preparation of Amifostine monohydrate or Amifostine trihydrate, we aimed to develop a simple and economical process for commercial production of Amifostine trihydrate.

We observed that a promising approach for such a process is to
(a) avoid the usage of trisodium phosphorothioate anhydrous or trisodium phosphorothioate dodecahydrate
(b) Carryout the reaction using commercially available reagents like thiophosphoryl chloride and sodium hydroxide
(c) remove the unwanted by products like sodium chloride or sodium bromide from the reaction mixture by simple crystallization in aqueous alcohol
(d) avoid the usage of dimethyl formamide (DMF) from the reaction since the complete removal of dimethyl formamide from the drug substance at low temperatures is very difficult.
Accordingly, the main objective of the present invention is to provide a new and novel process
for the preparation of S-2- (3-aminopropylamino)ethyl phosphorothioate trihydrate (I), which
obviates the drawbacks of the prior art processes and use cheaper and easily available reagents.
Accordingly, another main objective of the present invention provides a new process for the
preparation of S-2- (3-aminopropylamino) ethyl phosphorothioate trihydrate (I), which
comprises the usage of 2-(3-aminopropylamino)ethyl bromide dihydrobromide (III),
thiophosphoryl chloride (VII) and sodium hydroxide as principle raw materials.
According to still another objective of the present invention is to obviate the usage of anhydrous
and hydrate form of trisodium phosphorothioate (sodium thiophosphate) (IV & VI) since these
are not commercially available.
According to yet another objective of the present invention is to provide an improved process for
the preparation of Amifostine trihydrate of formula-I by avoiding the usage of dimethyl
formamide.
According to yet another objective of the present invention is to provide an improved process for
the preparation of Amifostine trihydrate of formula-I by novel crystallization method using
aqueous alcohol.
According to still another objective of the present invention is to provide an improved
commercial method of preparation of amifostine trihydrate (I) of high purity (99.9-99.95% by
HPLC).

According to still another objective of the present invention is to provide a new method of elimination of amifostine thiol impurity of formula-VIII (Limit:0. 2%) listed in United States Pharmacopoeia 2005 (USP 28) monograph for amifostine trihydrate using a novel crystallization and isolation technique.
According to still another objective of the present invention is to provide a method of minimization of total impurity content in amifostine trihydrate using a novel crystallization and isolation technique.
Detailed description of the invention:
According to the present process of the invention as outlined in Scheme-Ill, thiophosphoryl
chloride (VII) is treated with aqueous alkali solution at room temperature and after getting
uniform solution, 2-(3-aminopropylamino)ethyl bromide dihydrobromide (III) (prepared
according to the procedure reported in US 3892824) is added to get S-2- (3-
aminopropylamino)ethyl phosphorothioate trihydrate.
After crystallization, the purity of Amifostine trihydrate according to the present process has
>99.9% purity with all the impurities listed in United States Pharmacopoeia 2005 monograph
well below the specified limits.
Accordingly, the present invention provides an improved process for the preparation of
Amifostine trihydrate of formula-I,


Which comprises of the following steps:
(i) Dissolution of thiophosphoryl chloride (VII) in aqueous alkali

(ii) Addition of 2-(3-aminopropylamino)ethyl bromide dihydrobromide (III) to the above aqueous solution
(iii) Isolation of Amifostine trihydrate by the addition of lower alcohols such as methanol, ethanol, isopropyl alcohol, ketonic solvents like acetone or ester solvents like ethyl acetate or dipolar aprotic solvents like acetonitrile or tetrahydrofuran.
(iv) Crystallization of Amifostine trihydrate obtained above by dissolving in aqueous alcohol, the alcohol being methanol or ethanol or isopropanol or in aqueous acetone or in aqueous dipolar aprotic solvents like acetonitrile or tetrahydrofuran to get Amifostine trihydrate of USP grade.

The alkali solution used in step (i) to dissolve thiophosphoryl chloride is selected from alkali
metal hydroxides like sodium hydroxide, potassium hydroxide and lithium hydroxide preferably
sodium hydroxide.
The molar ratio between thiophosphoryl chloride and alkali metal hydroxide used in step (i) is
1: 6.0 or 1: 6.25 preferably 1: 6.0.
The temperature at which dissolution of thiophosphoryl chloride in aqueous alkali metal
hydroxide in step (i) is 25-40 °C preferably 25-35° C.
The time of addition of thiophosphoryl chloride to the alkali metal hydroxide solution in step (i)
is 3-6 hours preferably 4-5 hours.

In step (ii), the temperature at which the addition of 2-(3-aminopropylamino)ethyl bromide
dihydrobromide to the above aqueous solution is 10-25° C preferably 15-20° C.
In step (iii), solvent employed for isolation Amifostine trihydrate is selected from alcohols such
as methanol, ethanol, isopropyl alcohol or ketonic solvents like acetone or ester solvents like
ethyl acetate or dipolar aprotic solvents like acetonitrile or tetrahydrofuran. The preferred solvent
is methanol.
In step (iii), the temperature at which the addition of the above said solvent to the reaction
mixture for isolation Amifostine trihydrate is 0-20° C preferably 5-10° C.
In step (iii), the temperature at which isolation of Amifostine trihydrate is 0-10° C preferably 5-
10° C.
In step (iv), the solvent employed for crystallization of Amifostine trihydrate is selected from
Water-methanol, water-ethanol, water-isopropyl alcohol, water-acetone, water-ethyl acetate,
water-acetonitrile or water-tetrahydrofuran preferably water-methanol.
In step (iv), the percentage of aqueous alcohol used for crystallization of amifostine trihydrate
may be in the range of 5-40% preferably 10-20%.
In step (iv), the volume of aqueous alcohol used to crystallize amifostine trihydrate may be in the
range of 2-10 times to its weight, preferably 5-6 times and the temperature at which Amifostine
trihydrate is dissolved may be in the range of 25-40° C preferably 35-40° C.
In step (iv), the temperature at which isolation of crystalline Amifostine trihydrate is 0-10° C
preferably 5-10° C.
In step (iv), the percentage of aqueous alcohol used to wash the wet cake of crystalline
Amifostine trihydrate may be in the range of 5-40%, preferably 10-20%.
In step (iv), the temperature at which drying of crystalline Amifostine trihydrate of USP grade
may be in the range of 15-30° C preferably 25° C.

Advantages of the Invention:
1. The present invention provides a process for the preparation of crystalline high purity (>99.9% by HPLC) Amifostine trihydrate.
2. The present process provides a novel method of manufacture of pharmaceutical grade Amifostine trihydrate containing substantially low levels of the thiol impurity ( 3. The present process avoids the usage of trisodium phosphorothioate (sodium thiophosphate), which is not commercially available.
4. The present invention provides a novel process for the manufacture of Amifostine trihydrate using commercially available raw material like thiophosphoryl chloride.
The details of the process of the invention are provided in the examples given below which are provided by way of illustration only and therefore should not be construed to limit of the scope of the invention.

Example-1: Preparation of Amifostine Trihydrate (I):
To a stirred solution of sodium hydroxide (124g; 3.1 mole) and water (336 ml), thiophophoryl chloride (84g; 0.49 mole) is added drop wise over a period of 4-5 hours at 30-40° C. After addition, the solution is stirred till uniform solution is observed (approximately 2-6 hours). After getting uniform solution, 2-(3-aminopropylamino)ethyl bromide dihydrobromide (III) (lOOg; 0.29 mole) is added lot wise at 15-20° C. After addition the reaction mixture is stirred for another 4 hours and further cooled to 5° C. To this solution methanol (1.4 Lt) is added drop-wise to precipitate Amifostine trihydrate (I). Yield: 78 g
Purification of Amifostine trihydrate(I):
The above wet product of Amifostine trihydrate (I) (78g) is dissolved in water (195 ml) at 35-38°C under nitrogen atmosphere. After complete dissolution, the solution is cooled to room temperature (25°C) and the solution is treated with activated charcoal (2g) for 10-15 minutes and to the filtered solution is added methanol (39 ml) at 25-30° C. The clear solution is further cooled to 0-5° C and stirred for 2 hours and filtered. The wet cake is then washed with aqueous methanol and dried in a vacuum oven until the moisture content limit (19-22%) is reached. The purity of Amifostine trihydrate is 99.92% and the thiol impurity is 0.04%. Yield: 45 g (57.7%)

ExampIe-2:
Preparation of Amifostine Trihydrate (I):
To a stirred solution of sodium hydroxide (124g; 3.1 mole) and water (336 ml), thiophophoryl chloride (84g; 0.49 mole) is added drop wise over a period of 4-5 hours at 30-40° C. After addition, the solution is stirred till uniform solution is observed (approximately 2-6 hours). After getting uniform solution, 2-(3-aminopropylamino) ethyl bromide dihydrobromide (III) (lOOg; 0.29 mole) is added lot wise at 15-20° C. After addition, the reaction mixture is stirred for another 4 hours and further cooled to 5° C. To this solution ethanol (1.4 Lt) is added drop-wise to precipitate Amifostine trihydrate (I). Yield: 76.5 g
Purification of Amifostine trihydrate (I):
The above wet product of Amifostine trihydrate (I) (76g) is dissolved in water (195 ml) at 35-38°C under nitrogen atmosphere. After complete dissolution, the solution is cooled to room temperature (25°C) and the solution is treated with activated charcoal (2g) for 10-15 minutes and to the filtered solution is added ethanol (38 ml) at 25-30° C. The clear solution is further cooled to 0-5° C and stirred for 2 hours and filtered. The wet cake is then washed with aqueous ethanol and dried in a vacuum oven until the moisture content limit (19-22%) is reached. The purity of Amifostine trihydrate is 99.95% and the thiol impurity is 0.03%. Yield: 42.3 g (54.2%)

Example-3:
Preparation of Amifostine Trihydrate (I):
To a stirred solution of sodium hydroxide (3.1 Kg; 77.5 mole) and water (8.4 Lt), thiophophoryl chloride (2.IK; 12.38 mole) is added drop wise over a period of 4-5 hours at 30-40° C. After addition, the solution is stirred till uniform solution is observed (approximately 2-6 hours). After getting uniform solution, 2-(3-aminopropylamino) ethyl bromide dihydrobromide (III) (2.5 Kg; 7.28 mole) is added lot wise at 15-20° C. After addition, the reaction mixture is stirred for another 4 hours and further cooled to 5° C. To this solution methanol (35 Lt) is added drop-wise to precipitate Amifostine trihydrate (I). Yield: 2.1 Kg
Purification of Amifostine trihydrate (I):
The above wet product of Amifostine trihydrate (I) (2.1 Kg) is dissolved in water (5.25 Lt) at 35-38°C under nitrogen atmosphere. After complete dissolution, the solution is cooled to room temperature (25°C) and the solution is treated with activated charcoal (50g) for 10-15 minutes and to the filtered solution is added methanol (1.05 Lt) at 25-30° C. The clear solution is further cooled to 0-5° C and stirred for 2 hours and filtered. The wet cake is then washed with aqueous methanol and dried in a vacuum oven until the moisture content limit (19-22%) is reached. The purity of Amifostine trihydrate is 99.93% and the thiol impurity is 0.04%. Yield: 1.2 Kg (61.4%)





We claim:
1. Amifostine trihydrate of formula (I) in a purity equal to or greater than 99.9%
2. The Amifostine trihydrate of formula (I) as claimed in claims 1, where in the thiol
impurity of formula (VIII) does not exceed 0.05% (according to HPLC).
3. A novel process for the manufacture of high purity Amifostine trihydrate of the formula-I as claimed in claim 1 which comprises:

(i) reacting 2-(3-aminopropylamino)ethylbromide dihydrobromide of formula-Ill

with thiophosphoryl chloride of formula VII dissolved in alkali metal hydroxide solution

(ii) Isolation of Amifostine trihydrate from aqueous solution by addition of solvents such as a lower alcohol or a ketonic solvent or an ester of lower alcohol or a dipolar aprotic solvent.
(iii) Purification of the resulting Amifostine trihydrate by dissolving it in water at 35-40° C, decolorising with activated carbon and adding a lower alcohol or a ketonic

solvent or an ester or lower alcohol or a dipolar aprotic solvent at 25-30° C and allowing the resulting clear solution to cool to 0-5° C following by filtration to get high purity (>99.9% by HPLC) Amifostine trihydrate of formula-I meeting the pharmacopoeial requirements.
4. An improved process for Amifostine trihydrate (I) as claimed in claim (3) where in the
alkali metal hydroxide in step (i) is selected from sodium hydroxide or potassium
hydroxide or lithium hydroxide.
5. An improved process for Amifostine trihydrate (I) as claimed in claims 3 & 4 where in isolation of Amifostine trihydrate from aqueous solution of step (ii) is carried out by addition of solvents like methanol, ethanol, isopropyl alcohol or a ketonic solvent like acetone or an aliphatic ester like ethyl acetate or dipolar aprotic solvents like acetonitrile or tetrahydrofuran.
6. An improved process for Amifostine trihydrate of formula (I) as claimed in claims 3-5, where in purification of Amifostine trihydrate in step (iii) is carried out by dissolving the product in water at 35-40° C, decolorising the resulting solution with activated carbon at 25-30° C followed by filtration and crystallization of Amifostine trihydrate by the addition of lower alcohols like methanol, ethanol, isopropanol or ketonic solvent like acetone or an aliphatic ester like ethyl acetate or a dipolar aprotic solvents like acetonitrile or tetrahydrofuran.
7. An improved process for Amifostine trihydrate of formula (I) as claimed in claims 3-6 where in the purity of Amifostine trihydrate (I) is 99.9-99.95 by HPLC.
8. An improved process for Amifostine trihydrate of formula (I) as claimed in claims 3-7, where in the thiol impurity of formula (VIII) does not exceed 0.05% by HPLC.

9. An improved process for Amifostine trihydrate of formula (I) as claimed in claims 3-8
where in the purity of Amifostine trihydrate (I) complies the pharmacopoeial
requirements specified in USP-28.
10. An improved process for the manufacture of high purity Amifostine trihydrate of formula
(I) substantially as herein described with reference to examples 1-3.


Documents:

1592-CHE-2005 AMENDED PAGES OF SPECIFICATION 18-04-2011.pdf

1592-CHE-2005 AMENDED CLAIMS 18-04-2011.pdf

1592-CHE-2005 CORRESPONDENCE OTHERS.pdf

1592-CHE-2005 CORRESPONDENCE PO.pdf

1592-che-2005 form-1 18-04-2011.pdf

1592-CHE-2005 FORM-18.pdf

1592-CHE-2005 EXAMINATION REPORT REPLY RECIEVED 18-04-2011.pdf

1592-che-2005-absimage.jpg

1592-che-2005-abstract.pdf

1592-che-2005-claims.pdf

1592-che-2005-correpondense othes.pdf

1592-che-2005-discription complete.pdf

1592-che-2005-discription provisional.pdf

1592-che-2005-form 1.pdf

1592-che-2005-form 5.pdf

abs-1592-che-2005.jpg


Patent Number 248804
Indian Patent Application Number 1592/CHE/2005
PG Journal Number 36/2011
Publication Date 09-Sep-2011
Grant Date 25-Aug-2011
Date of Filing 02-Nov-2005
Name of Patentee NATCO PHARAMA LIMITED
Applicant Address NATCO PAHRMA LIMITED NATCO HOUSE, ROAD NO.2, BANJARA HILLS, HYDERABAD
Inventors:
# Inventor's Name Inventor's Address
1 ADIBHATLA KALI SATYA BHUJANGA RAO NATCO PAHRMA LIMITED NATCO HOUSE, ROAD NO.2, BANJARA HILLS, HYDERABAD
2 KONDURI SRINIVASA KRISHNA MURTHY NATCO PAHRMA LIMITED NATCO HOUSE, ROAD NO.2, BANJARA HILLS, HYDERABAD 500 033, ANDRA PRADESH INDIA
3 GUMMALAMPATI PEDA ANKAIAH NATCO PAHRMA LIMITED NATCO HOUSE, ROAD NO.2, BANJARA HILLS, HYDERABAD 500 033, ANDRA PRADESH INDIA
4 VENKAIAH CHOWDARY NANNAPANENI NATCO PAHRMA LIMITED NATCO HOUSE, ROAD NO.2, BANJARA HILLS, HYDERABAD 500 033, ANDRA PRADESH INDIA
PCT International Classification Number A61K 31/66
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA