Crystalline inhibitor of 4-hydroxyphenylpyruvate dioxygenase, and a process of synthesis and crystallization thereof

Abstract

The present invention relates to an improved synthesis and crystallization process of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, also known as nitisinone or NTBC.

Claims

1. A process of synthesis of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione crystalline Form A, comprising: a) reacting 2-nitro-4-(trifluoromethyl) benzoyl chloride and 1,3-cyclohexanedione in a CH.sub.3CN solution in the presence of potassium carbonate; b) acidifying by means of HCl 37% to about pH=1 and warming to about 55° C.; c) washing the organic phase and concentrating it to about half the initial volume of CH.sub.3CN used in a); d) adding the crude nitisinone from c) to about an 3/1 (w/w) binary acetonitrile/toluene mixture, wherein the ratio nitisinone/binary mixture is around ¼ (w/v) and heating the resultant combination at a temperature of about 55° C. until complete dissolution; e) concentrating the solution from d) to a final volume of about twice the initial volume of toluene added in d) at a temperature below 50° C. to obtain a solution of nitisinone in toluene containing about 0.5-0.6 g of nitisinone per ml of solvent; f) adding toluene to the mixture obtained in e) in order to double the final volume obtained from e); g) repeating e); h) heating the product of g) to about 55° C. for about 1 hour; i) cooling the product resulting from h) to about 10° C. in about 10 to about 12 h; j) filtering off the solid thus obtained in i) and rinsing it with toluene pre-cooled to 0° C.; and k) drying the resulting crystals under vacuum at a temperature of about 60° C. for about 4 hours, wherein 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione crystalline Form A has a X-ray powder diffraction pattern with at least five specific peaks at about 2-theta=7.4, 14.7, 15.7, 22.9, and 29.7, wherein said values may be plus or minus 0.2° 2-theta and have an intensity of at least 30%.

2. The process according to claim 1, wherein the purity of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione crystalline Form A obtained in j) is greater than 98%.

3. The process according to claim 1, wherein the amount of each single impurity is less than 0.05%.

4. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione crystalline Form A which has a X-ray powder diffraction pattern with at least five specific peaks at about 2-theta=7.4, 14.7, 15.7, 22.9, and 29.7, wherein said values may be plus or minus 0.2° 2-theta and have an intensity of at least 30%.

5. The crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione of claim 4, which has a X-ray powder diffraction pattern with at least ten specific peaks at about 2-theta=7.4, 14.7, 15.7, 22.9, 23.5, 23.8, 25.7, 29.7, 30.3, and 31.9, wherein said values may be plus or minus 0.2° 2-theta.

6. The crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione of claim 5, with a purity of at least 99.94%.

7. The crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione of claim 6, containing less than 10 ppm of 2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.

8. The crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione of claim 7, containing less than 5 ppm of 2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.

9. The crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione of claim 8, containing less than 1 ppm of 2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.

10. The crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione of claim 7, containing less than 0.05% of any single impurity after storage for six months at a temperature of 40° C. and 75% relative humidity, wherein said impurity is selected from the group consisting of 2-nitro-4-(trifluoromethyl) benzoic acid, 1,3-cyclohexanedione, 4-(trifluoromethyl)salicylic acid, and 6-trifluoromethyl-3,4-dihydro-2H-xanthene-1,9-dione.

11. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione crystalline Form A, obtainable by the process of claim 1.

12. A pharmaceutical composition comprising the crystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione according to claim 4.

13. The pharmaceutical composition according to claim 12 comprising less than 1 ppm of 2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.

Description

DESCRIPTION OF THE DRAWING

(1) FIG. 1: represents the X-ray spectra of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione Form A polymorph.

EXAMPLES

(2) Abbreviations:

(3) CH.sub.3CN: acetonitrile

(4) HCl: hydrochloric acid

(5) HPLC: high-performance liquid chromatography

(6) HPLC/MS: high-performance liquid chromatography-mass spectrometry

(7) General Remarks:

(8) Crystalline form has been characterised by Bruker D8 Advance X-ray powder diffraction (XRPD), Bragg-Brentano geometry, CuKα radiation with wavelength λ=1.54; scanning with 2θ angle range of 3° to 40°, step size of 0.02° for 0.5 seconds per step. Linear solid-state detector (Lynx Eye). Micronization has been performed with a laboratory scale micronizer FLUID JET MILL J-20 (Tecnologia Meccanica Srl) using the following milling conditions: Grind air: dry nitrogen gas Ring pressure: 3.9 bar Venturi pressure: 4.1 bar Feed rate: 0.30 g/min

(9) Particle size and d(90) has been determined using laser light scattering technique using a Malvern Mastersizer 3000 and water as dispersant.

Example 1

(10) Thionyl chloride (162 g, 1.36 mol) was added dropwise into a suspension of 2-nitro-4-trifluoromethylbenzoic acid (228 g, 0.97 mol) in toluene (630 g) at 80° C. The thus obtained solution was kept under stirring at 80° C. for 20 hours, and then cooled to 50° C. The volatiles were removed under reduced pressure in order to get the expected 2-nitro-4-trifluoromethylbenzoyl chloride as an oil. The latter, cooled to 25° C. was added dropwise to a suspension of 1,3-cyclohexanedione (109 g, 0.97 mol) and potassium carbonate (323 g, 2.33 mol) in CH.sub.3CN (607 g). After 18 h the mixture was diluted with water (500 ml) and slowly acidified to about pH=1 with HCl 37%. The mixture was then warmed to about 55° C. and the phases were separated. The organic layer was washed with a 10% aqueous solution of sodium chloride and then, concentrated under reduced pressure at a temperature below 55° C. to reach a volume of 380 ml. The thus obtained mixture was stirred at 55° C. for 1 h and then cooled to 0° C. in 16 to 18 h. The resulting solid was filtered and rinsed several times with pre-cooled (0° C.) toluene. The wet solid was dried at 60° C. under vacuum for 6 h to provide nitisinone (164 g) as a white to yellowish solid with a purity of 98.4% as measured by HPLC and a content of potentially genotoxic impurity no 4 of 6.1 ppm measured by HPLC/MS.

Example 2

(11) Nitisinone as obtained from example 1 (164 g) was added to a 3/1 (w/w) mixture of CH.sub.3CN/toluene (volume of solvent: 638 ml). The mixture was warmed gently to about 55° C. under stirring until solids were completely dissolved. The solution was then concentrated under reduced pressure maintaining the internal temperature below 50° C. to reach a volume of 290 ml. Then, more toluene (255 g) was added and the solution was concentrated again under reduced pressure until the residual volume reached 290 ml. The solution was heated to about 55° C. for 1 h and successively cooled slowly in 10 to 12 h to 10° C. The resulting solid was filtered and rinsed several times with pre-cooled (0° C.) toluene. The wet solid was dried at about 60° C. under vacuum for 4 h to provide nitisinone (136 g) as a white to yellowish solid, with a purity of 99.94% and a 99.8% assay measured by HPLC and a d(90) particle size between 310 and 350 μm. The content of potential genotoxic impurity no 4 resulted below 1 ppm.

(12) Stability Studies

(13) As evidenced in Table 1, nitisinone obtained through the process of the invention resulted extremely stable even in accelerated conditions for a period of at least six months. Importantly, the potentially genotoxic impurity-4 resulted below the limit of quantification, independently from the storage conditions. The presence of impurity-4 was checked by reverse HPLC/MS using the method described in the table underneath.

(14) TABLE-US-00001 Column Ascentis Express C18 5 μm, 250 × 4.6 mm Flow 1 ml/minute Injection volume 10 μl Wavelength 235 nm Column temperature 30° C. Detector MS Polarity: positive; SIM Mode; m/z = 330 Gas temp: 300° C. Gas flow: 13.1 l/min Nebulizer: 20 psi Capillary: 4500 nA Step 0 to SIM > to MS Step 1 to SIM > to waste Mobile phase A CH.sub.3CN Mobile phase B H.sub.2O/0.1% TFA HPLC Gradient Time % A % B  0 50 50 10 70 30 12 70 30 13 50 50 23 50 50 Retention time 5.8 minutes

(15) TABLE-US-00002 TABLE 1 Tests Specifications 6 months 9 months 6 months 9 months 6 months Appearance While to C C C C C (Visual) yellowish crystalline powder Water content (KF) NMT 0.5% w/w    0.0%    0.0%    0.2%    0.0%    0.2% Assay-on 98.0-102.0%   98.8%   98.8%   99.4%   101.4%   100.4% anhydrous basis (HPLC) Impurity-1 NMT 0.15% <0.01% <0.01% <0.01%  <0.01%  <0.01% Impurity-2 NMT 0.15% <0.01% <0.01% <0.01%  <0.01%  <0.01% Impurity-3 NMT 0.15% <0.01% <0.01% <0.01%  <0.01%  <0.01% Impurity 4 NMT 10 ppm <5 ppm <5 ppm <5 ppm <5 ppm  <5 ppm Impurity 5 NMT 0.15%   0.01% <0.01%   0.01%  <0.01%    0.01% Any unspecified NMT 0.10% <0.01% <0.01% <0.01%  <0.01%  <0.01% impurities Total impurities NMT 0.50%   0.01% <0.01%   0.01%  <0.01%    0.01% C = Conform; NMT = No more than