FIELD OF THE INVENTION:
The present invention relates to an improved process for the preparation of Nitisinone of formula (1) having purity greater than 99.0% by HPLC, more preferably greater than 99.5%. It further relates to crystalline form I and crystalline form II of Nitisinone of formula (1) and their preparation thereof.
BACKGROUND OF INVENTION
Nitisinone is a 4-hydroxyphenyl-Pyruvate Dioxygenase Inhibitor. Chemically it is known as 2-[2-nitro-4-(trifluoromethyl) benzoyl] cyclohexane-l,3-dione or NTBC being used as a medication used to slow the effects of hereditary tyrosinemia type 1 (HT-1) in adult and pediatric patient. Since its first use for this indication in 1991, it has replaced liver transplantation as the first-line treatment for this rare condition. NTBC is a drug marketed by Swedish Orphan Biovitrum International AB under the brand name Orfadin®. It has been approved by Food and Drug Administration in January 2002.
The synthesis of Nitisinone of formula (1) has been reported in few patents/articles, the contents of which are hereby incorporated as reference in their entirety.
US 4,774,360 patent discloses the conversion of enol ester precursor of 2-(2'-nitrobenzoyl)-l,3-cyclohexanedione to Nitisinone by heating the enol ester precursor of 2-(2'-nitrobenzoyl)-l,3-cyclohexanedione in the presence of triethylamine and a catalytic amount of 2-cyano-2-(trimethylsilyloxy) propane. It does not disclose the purity of final compound Nitisinone.
US 5,728,889 patent discloses the synthesis of Nitisinone by reacting 1,3-cyclohexanediones with 2-chloro-4-(methylsulphonyl) benzoyl chloride in acetonitrile with 82% yield.
US 9,783,485 patent discloses process for the synthesis of Nitisinone by reacting 2-nitro-4-(trifluoromethyl) benzoyl chloride, with 1,3-cyclohexanedione acetonitrile

solution in the presence of potassium carbonate. The process uses 2-nitro-4-(trifiuoromethyl) benzoic acid and thionyl chloride to prepare 2-nitro-4-(trifluoromethyl) benzoyl chloride. Further it reports the crystallization process to obtain crystalline Form A of Nitisinone in 3/1 (w/w) acetonitrile/toluene. The process described herein is time consuming and tedious resulting in low yield which is not applicable at commercial level.
US 5,550,165 patent discloses the synthesis of Nitisinone by reacting 2-nitro-4-trifluromethyl benzoyl chloride with triethylamine, acetone cyanohydrin and hydrochloric acid. The process uses 2-nitro-4-(trifluoromethyl) benzoic acid, oxalyl chloride and cyclohexane-l,3-dione to prepare 2-nitro-4-(trifluoromethyl) benzoyl chloride. The obtained Nitisinone was recrystallized from ethyl acetate. The process does not disclose any yield and purity of the final Nitisinone obtained.
US20160324785 application discloses the synthesis of Nitisinone. It further discloses a crystalline form of Nitisinone obtained by recrystallizing a sample of Nitisinone from a mixture of acetone: water solvent system.
Prior art methods do not disclose the yield and purity of or the final compound. Further the reaction time involved is very long and tedious, hence, the present inventors provide a cost-effective and less tedious process for the preparation of Nitisinone (1).
SUMMARY OF THE INVENTION
Accordingly, in one embodiment of the present invention provides an improved process for the preparation of Nitisinone of formula (1) with purity greater than 99.0% by High-performance liquid chromatography (HPLC), more preferably greater than 99.5%.
In another embodiment, the present invention provides crystalline form I and crystalline form II of Nitisinone of formula (1) and their preparation thereof.

In another embodiment, the present invention provides an improved process for the preparation of Nitisinone, which comprises of the following steps:
a) hydrolyzing 2-nitro-4-(trifluoromethyl) benzonitrile of formula (5)

in presence of a suitable acid to form 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4); and purifying compound of formula (4);

b) chlorinating 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4) using suitable
chlorinating agent to form 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula
(3);
c) reacting 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3) with
cyclohexane-l,3-dione (2)


in presence of a suitable base to form Nitisinone of formula (1); and d) purifying Nitisinone of formula (1).
In yet another embodiment, the present invention involves a process for the purification of Nitisinone of formula (1), comprising:
i. adding a suitable first solvent to Nitisinone of formula (1);
ii. optionally, heating the reaction mixture at a suitable temperature.
iii. optionally, adding a second solvent at a suitable temperature
iv. adding activated carbon;
v. heating the reaction mixture at suitable temperature; and filtering;
vi. optionally, adding suitable third solvent to the filtrate; vii. isolating crystalline form Nitisinone of formula (1).
In another preferred embodiment the Nitisinone obtained by any of the prior-art process can be purified according to the process given in the present invention.
In another preferred embodiment of the invention provides crystalline Nitisinone of formula (1) obtained after purification is having purity greater than 99.0% by HPLC.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the X-Ray powder diffraction pattern of pure Nitisinone of formula (1) prepared by example 4 & 5.
Figure 2 illustrates the X-Ray powder diffraction pattern of pure Nitisinone of formula (1) prepared by example 6.

DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present invention provides an improved process for the preparation of substantially pure Nitisinone of formula (1) having purity greater than 99.0%, more preferably greater than 99.5%.

Accordingly, the steps involved in the preparation of Nitisinone of formula (1) as shown in scheme-1 are as follows:
a) hydrolyzing 2-nitro-4-(trifluoromethyl) benzonitrile of formula (5) in presence
of a suitable acid to form 2-nitro-4-(trifluoromethyl) benzoic acid of formula
(4); and purifying compound of formula (4);
b) chlorinating 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4) using
suitable chlorinating agent to form 2-nitro-4-(trifluoromethyl) benzoyl chloride
of formula (3);

c) reacting 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3) with cyclohexane-l,3-dione (2) in presence of a suitable base to form Nitisinone of formula (1); and
d) purifying Nitisinone of formula (1).
Step a) involves hydrolysis of 2-nitro-4-(trifluoromethyl) benzonitrile of formula (5) in presence of a suitable acid preferably, sulphuric acid in presence of a protic solvent. The said reaction may be carried out at a temperature of 100-140 °C, preferably 120-125 °C. The reaction mass was may be cooled to 0-20 °C, preferably 5-10 °C, filtered and washed with a suitable protic solvent. The solid so obtained, was dissolved in a protic solvent and adjusted pH to 11 to 12 with a suitable base to remove unreacted starting material and impurities. Optionally, the reaction mass may be purified by dissolving in a suitable aprotic solvent and treating with an activated carbon at 25-30 °C .The reaction mass may be then filtered and the pH of the filtrate may be adjusted below 1.0 using a suitable acid at 15-20 °C to isolate pure 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4)of formula (4).
Step b) proceeds with the chlorination of the 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4) using a suitable chlorinating agent to form 2-nitro-4-(trifiuoromethyl) benzoyl chloride of formula (3). A suitable chlorinating agent may be then added at 0-15 °C, preferably 0-5 °C. The temperature of the reaction mass may be then maintained at 25-30°C. The final reaction mixture so obtained may be cooled tol0-40°C, preferably 25-30°C to obtain 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3), which was used directly for the next step without isolation.
The suitable activating agent may be selected from a group comprising of dimethyl formamide (DMF), dimethyl sulfoxide (DMSO) or the like, preferably dimethyl formamide (DMF) was used in the present invention.

The suitable chlorinating agent used herein in step b) may be selected from a group comprising of thionyl chloride, oxalyl chloride, phosphorous trichloride, phosphorous pentachloride, phosphorous oxychloride or the like, preferably oxalyl chloride was used in the present invention.
Step c) involves condensation reaction of cyclohexane-l,3-dione of formula (2) with 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3) in presence of a suitable base to form Nitisinone of formula (1). Cyclohexane-l,3-dione of formula (2) was dissolved in a suitable aprotic solvent and base. This reaction mass was then added to 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3) formed in step b). The said reaction may be carried out at a temperature of 0-25°C, preferably at 5-10°C with the addition of acetone cyanohydrin at 25-30 ° C. The total reaction mass may be heated to 40-60° C, preferably 40-45° C. On completion of reaction, the reaction mass may be cooled to 15-20°C with addition of a suitable protic solvent. The pH of the reaction mass may be adjusted to 1.0 using a suitable acid and extracted with a suitable protic and aprotic solvents to obtain Nitisinone of formula (1).
Suitable bases used in step a) and step c) may be selected from a group comprising of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, piperidine, pyridine, triethylamine, methyl amine, ethyl amine, dimethyl aminopyridine (DMAP), isopropyl amine, diisopropyl amine, diisopropylethylamine, or the like. Preferably the bases employed in the present invention were sodium hydroxide, potassium carbonate and triethylamine.
The suitable acids used in step a) and step c) may be selected from a group of organic or inorganic acids, preferably inorganic acids comprising of but not restricted to sulphuric acid, hydrochloric acid, phosphoric acid, hydrobromic acid or the like; more preferably sulphuric acid and hydrochloric acid were used in the present invention.

In another embodiment the protic solvents used in step a), step b) and step c) were selected from a group comprising of water, methanol, ethanol, isopropyl alcohol (IP A), n-propanol or the like, preferably water was used in the present invention.
In another embodiment the aprotic solvents used in step a), step b) and step c) were selected from a group comprising of acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N,N-dimethylformamide, methyl tertiary butyl ether, hexane, cyclohexane, xylene, toluene, tetrahydrofuran or the like, preferably acetonitrile, dimethylformamide, dichloromethane, toluene were used in the present invention.
In some embodiment, step d) proceeds with the purification of Nitisinone of formula (1) comprising:
i. adding a suitable first solvent to Nitisinone of formula (1); ii. heating the reaction mixture at a suitable temperature iii. optionally, adding activated carbon and filtering; iv. optionally, adding suitable second solvent to the filtrate; v. isolating crystalline form Nitisinone of formula (1).
In another embodiment, the purification of Nitisinone of formula (1) may be carried out by adding Nitisinone of formula (1) to a suitable first solvent at 25-30°C and heating to a suitable temperature of 40-85°C, preferably at 50-55°C. Optionally, the reaction mass may be treated with an activated carbon and filtered through Hyflo. The filtrate may be collected and optionally treated with a suitable second solvent at 50-55°C. The reaction mass may be then cooled to 0-30 °C, preferably 25-30 °C. The solid so formed may be filtered and washed with a mixture of suitable protic and aprotic solvent and dried under vacuum at below 45°C to obtain crystalline Nitisinone of formula (1).

In yet another embodiment, the present invention provides an alternative process for the purification of Nitisinone of formula (1), comprising:
1. adding a suitable solvent to Nitisinone of formula (1);
2. heating the reaction mixture at a suitable temperature
3. cooling the reaction mixture to a suitable temperature
4. isolating crystalline form Nitisinone of formula (1).
The alternative process for the purification of Nitisinone proceeds with adding Nitisinone of formula (1) to a suitable aprotic solvent and stirring for 15-20 minutes at suitable temperature of 60-90°C, preferably at 70-75°C. The reaction mass may be then cooled to 50-55°C and stirred for 24hrs. The reaction mass may be further cooled to 25-30°C and stirred for 3hrs and filtered under vacuum at below 45°C to obtain pure crystalline form of Nitisinone of formula (1).
In some embodiment, the solvents used in purification of Nitisinone may be selected from a group comprising of organic and inorganic solvents. Preferably, organic solvents comprising of protic and aprotic solvents selected from a group comprising but not limited to water, methanol, ethanol, isopropyl alcohol (IPA), n-propanol , acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N,N-dimethylformamide, methyl tertiary butyl ether, hexane, cyclohexane, xylene, toluene, tetrahydrofuran or mixtures thereof.
In some embodiment of the invention provides pure crystalline form of Nitisinone of formula (1) obtained after purification, which may have purity greater than 99.0%, preferably greater than 99.5% and total impurities less than 1.0 (% w/w), particularly less than 0.5% (w/w).

In another embodiment, of the invention provides pure Nitisinone of formula (1) obtained after purification may be having heavy metals less than 10 ppm.
In another embodiment, the pure crystalline Nitisinone of formula (1) obtained after purification may have X-Ray powder diffraction (XRPD) pattern as shown in figure 1 and the 2 theta values provided in Table 1

In another embodiment, the pure crystalline Nitisinone of formula (1) obtained after purification may have X-Ray powder diffraction (XRPD) pattern as shown in figure 2 and the 2 theta values provided in Table 2


The following examples further illustrate the present invention but should not be construed in any way as to limit its scope.

EXAMPLES EXAMPLE-1 Preparation of 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4)
lOOg of 2-nitro-4-(trifluoromethyl) benzonitrile (100 g) of formula (5) was added to an aqueous solution of Sulphuric acid dissolved in 1400mL water and heated at 120-125°C. On completion of reaction, the reaction mixture was cooled to 25-30°C and then poured onto chilled water, at 0-5°C and stirred for lhr. The solid formed was filtered under vacuum and washed with chilled water. Purification process:
The solid was dissolved in 300mL of water and cooled to 10-15°C. The pH of the reaction mass was adjusted to 11-12 with aqueous sodium hydroxide. The temperature of the reaction mass was raised to 25-30°C and lOOmL of methyl tertiary butyl ether was added. The aqueous layer was separated and extracted with 200 mL of toluene. The aqueous layer was then separated, lOg of activated carbon was added to it and the reaction mixture was filtered through Hyflo. The filtrate was cooled to 15-20 °C and pH was adjusted to below 1.0 with concentrated hydrochloric acid. The temperature of the filtrate was then raised to 25-30 °C and filtered under vacuum. The solid so formed was washed with water and dried to obtain 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4). Yield %: 78; Purity: 99.8% (HPLC).
EXAMPLE-2 Preparation of 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3)
lOOg of 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4) was dissolved in 500mL dichloromethane , with addition of 0.2mL of dimethyl-formamide and the reaction mass cooled to 0-5°C. 70g of oxalyl chloride was added 0-5 °C to the reaction mass and temperature was raised to 25-30°C. On completion of reaction, the solvent was distilled off under vacuum and co-distilled with dichloromethane below 45°C to

yield 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3), which was directly used in the next step.
EXAMPLES Preparation of Nitisinone of formula (1)
48 g of 1,3-cyclohexadione was dissolved in 500 mL of dichloromethane and cooled to 5-10 °C. 172g of triethylamine added to the reaction mass at 5-10 °C. This reaction mass was then added to the reaction mass containing 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3) obtained in step b) and the temperature of the total reaction mass raised to 25-30 °C. 3.62 g of acetone cyanohydrin was further added, and the reaction mass heated to 40-45 °C. On completion of reaction, the reaction mass was cooled to 15-20 °C and the pH was adjusted to 1.0 using hydrochloric acid. The resulting reaction mass was then extracted with different volumes of water, dichloromethane, acetonitrile and finally the biphasic mixture was separated. The organic layer was washed with water and dried over sodium sulphate and filtered. The filtrate was distilled off under vacuum below 40°C to obtain Nitisinone of formula (1). Yield %: 60.
EXAMPLE-4
Purification of Nitisinone of formula (1)
400mL of acetonitrile was added to the Nitisinone of formula (1) and heated to 50-55°C. lOg of activated carbon was added to the reaction mass and filtered through Hyfio at 50-55°C. 500 mL of water was slowly added to the filtrate , stirred and then cooled to 25-30°C. The solid formed was then filtered and washed with 100 mL of acetonitrile and water mixture (1:1) and dried under vacuum below 45 °C to yield pure Nitisinone of formula (1). Yield%: 65%; Purity (HPLC): 99.75%. XRD: Figure-1.

EXAMPLES
Process for the purification of Nitisinone of formula (1)
5g of Nitisinone of formula (1) was mixed with 30mL of toluene at 25-30°C and the reaction mass was heated to 60-65°C and stirred for 15-20 minutes. The reaction mass was then cooled to 50-55°C and stirred for 24hrs and further cooled to 25-30°C and stirred for 3hrs. The solid formed was filtered under vacuum to obtain Nitisinone of formula (1). Yield: 75.3%; Purity (HPLC): 99.8%. XRD: Figure-1.
EXAMPLE-6
Process for the purification of Nitisinone of formula (1)
5g of Nitisinone of formula (1) was mixed with 30mL of xylene and stirred for 15-20 minutes at 70-75 °C. The reaction mass was then cooled to 50-55°C and stirred for 24hrs. The reaction mass was then further cooled to 25-30 °C and stirred for 3hrs and filtered under vacuum to obtain Nitisinone of formula (1). Yield: 75.5%; Purity (HPLC): 99.82%. XRD: Figure-2.

We Claim:
1. A process for the preparation of Nitisinone of formula (1), comprising the steps of:
a) hydrolyzing 2-nitro-4-(trifluoromethyl) benzonitrile of formula (5);

in presence of a suitable acid to form 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4) and purifying compound of formula (4);

b) chlorinating 2-nitro-4-(trifluoromethyl) benzoic acid of formula (4) using
suitable chlorinating agent to form 2-nitro-4-(trifluoromethyl) benzoyl chloride
of formula (3);

c) reacting 2-nitro-4-(trifluoromethyl) benzoyl chloride of formula (3) with
cyclohexane-l,3-dione (2)

in presence of a suitable base to form Nitisinone of formula (1); and
d) purifying Nitisinone of formula (1).
2. The process as claimed in claim 1, wherein the suitable acids used in step a) is selected from a group comprising of sulphuric acid, hydrochloric acid, phosphoric acid, hydrobromic acid or any combination thereof.
3. The process as claimed in claim 1, wherein chlorinating agent used in step b) is selected from a group comprising of thionyl chloride, oxalyl chloride, phosphorous trichloride, phosphorous pentachloride, phosphorous oxychloride or mixtures thereof.
4. The process as claimed in claim 1, wherein suitable bases used in step c) is selected from a group comprising of piperidine, pyridine, triethylamine, methyl amine, ethyl amine, dimethyl aminopyridine (DMAP), isopropyl amine, diisopropyl amine, diisopropylethylamine, or mixtures thereof.
5. A process for the purification of Nitisinone of formula (1), which comprises:
i. adding a suitable first solvent to Nitisinone of formula (1);
ii. heating the reaction mixture at a suitable temperature;
iii. optionally, adding activated carbon and filtering;
iv. optionally, adding suitable second solvent to the filtrate; and

v. isolating Nitisinone of formula (1).
6. The process as claimed in claim 5, wherein Nitisinone is characterized by X-ray powder diffraction pattern comprising the following characteristic peaks expressed by 26 degree, when measured using Cu-Ka radiation: 7.28, 9.15, 13.27, 13.66, 14.67, 15.07, 15.67, 16.72, 17.22, 18.14, 19.28, 20.55, 20.87, 21.94, 22.26, 22.79, 23.01, 23.46, 23.81, 24.07, 24.75, 25.16, 25.61, 27.08, 27.67, 28.30, 28.66, 29.64, 30.01±0.2°.
7. A process for the purification of Nitisinone of formula (1), which comprises:

1. adding a suitable solvent to Nitisinone of formula (1);
2. heating the reaction mixture at a suitable temperature;
3. cooling the reaction mixture to a suitable temperature; and
4. isolating crystalline Nitisinone of formula (1).

8. The process as claimed in claim 7, wherein Nitisinone is characterized by X-ray powder diffraction patterns comprising the following characteristic peaks expressed by 29 degree, when measured using Cu-Ka radiation: 7.44, 9.30, 12.46, 13.41, 13.81, 14.82, 15.22, 15.80, 16.85, 17.37, 18.27, 18.60, 19.41, 20.71, 20.99, 22.06, 22.44, 22.98, 23.15, 23.61, 23.95, 24.20, 24.90, 25.28, 25.77, 27.23, 27.82, 28.41,28.79,29.80, 31.18 ±0.2°.
9. The process as claimed in claim 5 and claim 7, wherein the purity of Nitisinone of formula (1) obtained is greater than 99.5%.

10. The process as claimed in claim 1, claim 5 and claim 7, wherein, the solvents used is selected from a group comprising of water, methanol, ethanol, isopropyl alcohol (IP A), n-propanol , acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N,N-dimethylformarnide, methyl tertiary butyl ether, hexane, cyclohexane, xylene, toluene, tetrahydrofuran or mixtures thereof.