Abstract

The present invention relates to the bicalutamide active ingredient substantially free of the impurities 4-Cyano-N-methacryloyl-3-trifluoromethylaniline (BIC-O) and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide (BIC-1). The invention also relates to a novel analytical method for estimation of impurities in bicalutamide active ingredient.

Background

Bicalutamide, an acylanilide, is a non-steroidal anti-androgen, is a 'second generation' anti-androgen, which has more advantageous properties than the other known anti-androgens, like Flutamide or Nilutamide. The commercially available product by AstraZeneca is Casodex™ and its chemical name is 4-cyano-3-trifluoromethyl-N-(3- p-fluorophenylsulphony 1- 2- hydroxy-2-methylpropionyl) aniline. It is used as a 50mg tablet once daily in combination with a luteinising hormone-releasing hormone analogue or surgical castration for the treatment of advanced prostate cancer. It is also given at a dosage of 150 mg once daily as monotherapy for the treatment of early (localized or locally advanced) non-metastatic prostate cancer.

State of the art

Bicalutamide is disclosed in patents like United States patent No. 4636505 and the corresponding European Patent No. 100172.
Literature methods are available for the separation of enantiomers of bicalutamide by use of chiral stationary phase and also for the HPLC analysis of the active ingredient several analytical methods are reported for estimating bicalutamide in a given sample.
G.S.Subramanian etal (Journal of planar chromatography,22 (2009),4,273-276) reported a HPTLC method for analysis of bicalutamide from bulk drug and liposomal formulations. The authors claim that the method is suitable for routine industrial quality control of the bulk drug.

R.Nageswararao etal (Journal of Pharmaceutical and Biomedical Analysis, Volume 46, Issue 3, 13 February 2008, Pages 505-519) reported a reversed-phase high-performance liquid chromatographic method for determination of process impurities and degradation products of bicalutamide in bulk drug and pharmaceutical formulations. The separation was accomplished on a Symmetry CO18 (4.6 mm x 250 mm; particle size 5 μ m) column under isocratic mode. The mobile phase was 0.01 M KH2PO4 (pH 3.0):acetonitrile (50:50 v/v) and a PDA detector set at 215 nm was used for detection.

EP1679306 covers a process for producing bicalutamide and method of purifying intermediate crystals therefor. The patent application describes method for reducing the process related impurities like the deoxy sulphide compound. The content of Deoxy-Sulfide Compound was reported as 0.052% in liquid chromatographic area percentage. The conditions used in HPLC analysis are SUMIPAX ODS A-212; acetonitrile/0.1% aqueous acetic acid solution.

R.Nageswara Rao etal (Journal of Pharmaceutical and Biomedical Analysis, Volume 42, Issue 3, 26 September 2006, Pages 347-353) reported an improved HPLC method for determination of enantiomeric purity of bicalutamide in drugs and pharmaceuticals. Baseline separation with resolution ≥6.0 was achieved within 10 min on Chiralpak AD-H (250 mm x 4.6 mm; particle size 5μ m) column using n-hexane:2-propanol (65:35 v/v) as mobile phase at a flow rate of 1.0 ml/min at 25 °C. The detection was made at 270 nm using UV detector while a polarimetric detector connected in series was used for identification of enantiomers. The limits of detection (LOD) and quantification (LOQ) of enantiomers were (2.4, 3.0 and 7.6, 9.3) x 10-8 g/ml for (S)-(+)-BCT and (R)-(-)-BCT enantiomers, respectively.
However, there is no known method which can analyse trace quantities of impurities 4-Cyano-N-methacryloyl-3-trifluoromethylaniline (hereinafter referred to as 'BIC-0') and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide (hereinafter referred to as 'BIC-1') which are known to be genotoxic.

Brief description of invention

It is an object of the present invention to provide bicalutamide active ingredient substantially free of BIC-0 and BIC-1.

It is another object of the invention to provide bicalutamide active ingredient with less than 2% of BIC-0 and BIC-1.

It is another objective of the invention to quantify the potentially genotoxic impurities in bicalutamide.

It is another object of the invention to provide a HPLC method for the estimation of impurities.

It is a further object of the invention to provide a HPLC method that has a high degree of sensitivity to estimate the impurities in bicalutamide.

It is further object of the invention to ensure that the HPLC method can detect BIC-0 and BIC-1 even in trace quantities.

Detailed description of the invention

A general concept of qualification of impurities is described in the guidelines for active substances (Q3A, Impurities in New Active Substances) or medicinal products (Q3B, Impurities in New Medicinal Products), whereby qualification is defined as the process of acquiring and evaluating data that establishes the biological safety of an individual impurity or a given impurity profile at the level(s) specified. In the case of impurities with a genotoxic potential, determination of acceptable dose levels is generally considered as a particularly critical issue.

The Q3A guideline states that actual and potential impurities most likely arise during synthesis, purification and storage of the new drug substance and should be identified, based on a sound scientific appraisal of the chemical reactions involved in the synthesis, impurities associated with raw materials that could contribute to the impurity profile of the new drug substance, and possible degradation products. The guideline also recommends that the identification of genotoxic impurities should be guided by existing genotoxicity data or the presence of structural alerts.

The European guideline on the limits of genotoxic impurities opines that genotoxic compounds have the potential to damage DNA at any level of exposure and that such damage may lead/contribute to tumour development. Thus for genotoxic carcinogens it is prudent to assume that there is no discernible threshold and that any level of exposure carries a risk. The impossibility of defining a safe exposure level (zero risk concept) for genotoxic carcinogens without a threshold and the realization that complete elimination of genotoxic impurities from drug substances is often unachievable, requires implementation of a concept of an acceptable risk level.

The inventors have surprisingly found a bicalutamide active ingredient substantially free of the impurities, BIC-0 and BIC-l. The active ingredient has excellent storage stability when stored at the ambient and accelerated conditions.

Bicalutamide of this invention can be made by the following process:
N-[4-Cyano-3-(trifluoromethy)phenyl]-3-[(4-fluorophenyl)sulphonyl]-2-hydroxy-2- methyl propanamide is prepared by oxidation of N-[4-Cyano-3- (trifluoromethyl)phenyl]-3-[(4-fluorophenyl)thio]-2-hydroxy-2-methyl propanamide with Hydrogen peroxide in a . stoichiometric ratio of the substrate to Hydrogenperoxide of 1:1.5. The reaction is preferably carried out at temperature between 20°C to 50°C. The reaction mixture is washed with sodiumthiosulphate solution or sodium metabisulphite solution to nullify unreacted Hydrogen peroxide, water and treated with charcoal. The resulting reaction mixture is evaporated followed by addition of Isopropyl ether or t-Butylmethyl ether or n-Hexane or Toluene, and cooling to 25 to 30°C and isolation of the product by filtration.

Inlight of the recommendations, apart from the identification of genotoxic impurities, it becomes equally important to have a suitable analytical method that can determine the presence of these impurities even in trace amounts.

The following impurities are possible in bicalutamide API.

Imp-I: 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide (BIC-I)

lmp-II: N-[4-Cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)thio]-2-hydroxy-2-methyl propanamide (BIC-II) BIC-0: 4-Cyano-N-methacryloyl-3-trifluoromethylaniline (BIC-0) Imp-III N-[4-Cyano-3-(Trifluoromethyl)phenyl]-3-[(4-flurophenyl)sulfinyl]-2-

Hydroxy-2-methyl-propanamide
BIC-Amine 4-Cyano-3-(trifluoromethyl)Aniline
Imp-IV N-[4-Cyano-3-(Trifluoromethyl)phenyl]-3-[(2-Fluorophenyl)sulfonyl]-2-
Hydroxy-2-methyl-propanamide (BIC 2-Fluoro isomer)

Des hydroxy analogue: N-[4-Cyano-3-(Trifluoromethyl)phenyl]-3-[(4-Fluorophenyl)sulfonyl] -2-methyl-propanamide

Desfluoroanalogue: N-[4-Cyano-3-(Trifluoromethyl)phenyl]-3-(phenylsulfonyl)-2-Hydroxy-2-methyl-propanamide -

The USP monograph for bicalutamide requires the estimation of the following impurities by HPLC, apart from individual unspecified impurity, total unspecified impurities and total impurities:
Desfluoro analog
2-fluoro isomer
Deshydroxy analog

The method recommends the use of High performance liquid chromatography equipped with UV or PDA detector and auto sampler using water, methanol, and tetrahydrofuran (55:30: 15) (v/v/v) as mobile phase. This method does not require reporting of 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide hereinafter referred to as BIC-1 and 4-Cyano-N-methacryloyl-3-trifluoromethylaniline hereinafter referred to as BIC-0. Further the method does not have sufficient sensitivity to detect these impurities in the sample.

The inventors have developed a method with a high degree of sensitivity and which is designed to detect the presence of the genotoxic impurities BIC-0 and BIC-1. Method of analysis for BIC-1 and BIC-0 Equipment: High performance liquid chromatography equipped with PDA detector and auto
Sampler

Chromatographic conditions

Column . Octadecyl silane (ODS) C18,150 x 4.6mm, 5 microns
Flow rate . l.0mL/min (Gradient)
Detector. UV-283nm
Runtime 40 minutes
Oven Temp. 40°C

Procedure

Buffer solution preparation:

Take 1000ml of MilliQ. water and adjust pH to 2.8 with dilute acetic acid.

Preparation of Mobile phase A:

Filtered and degassed mixture of buffer, methanol, in the ratio of 76:24
Mobile phase B: 1,4-Dioxane
Diluent: Mixture of buffer, Acetonitrile in the ratio of 50:50(v/v)
Test solution preparation:

Prepare a known concentration of Bicalutamide 2.0mg/ml in diluent.

Standard solution preparation:

Accurately weigh about 50.0mg of BIC-1 and BIC-0 working standard, into a 25mL volumetric flask, add about 10 mL of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Dilute lmL of this solution to 100 mL with diluent and, again dilute 1 mL to 100 mL with diluent and further dilute 1 mL to l0mL with diluent (0.00002mg/mL)

A comparison of analytical results by the USP method and the present method are tabulated
below:

USP method

LOD LOQ

BIC-0 106.5ppm 353.8ppm BIC-1 168.4ppm 560.6ppm

In house method

LOD LOQ

BIC-0 1.3ppm 4.33ppm BIC-1 2.14ppm 7.13ppm

The inventors have also found that the bicalutamide of their invention has very low level of impuritiesand is substantially free of BIC-0 and BIC-1. The samples of the active ingredient were analysed by the inventor's method and official USP method.
The results are tabulated below:

Examples
Ethyl Acetate 720 It is charged into 1.6 kl Glasslined reactor, Methanesulfonic acid 1.2 kg,
Sodiumtungstate 1.2 kg and N-[4-Cyano-(3-trifluoroinethyl)phenyl]-3-f(4-fluorophenyl)thio]-2-hydroxy-2-methyl propanamide 60 kg were added at 30+5°C. Hydrogen peroxide(50%) is added at 25- 32°C and maintained for 9 hours at 25-32°C. Bicalutamide purity in reaction mass is minimum up to 99.5%.lf HPLC did not comply this condition the mixture is maintained further at 25-32°C. After completion of the reaction Sodium thio sulphate solution was charged and stirred to 30+5min, settled for 30+5min and the aqueous later was separated. The organic layer was washed with Sodium Bicarbonate Solution, Sodium Chloride solution and DM Water. Ethyl Acetate was distilled out at below 80°C, vacuum is applied for distilling out Ethyl Acetate completely. The mass is filtered and washed with t-Butyl methylether and the material is dried at 65+-5°C to get Bicalutamide 64 kg. Yield -9" %. HPLC Purity 99.7%.

We claim:

1. A reverse phase HPLC method to determine all the impurities of bicalutamide preferably the genotoxic impurities 4-Cyano-N-methacryloyl-3-trifluoromethylaniline and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide.

2. A reverse phase HPLC method using phosphate, acetic acid buffer of pH 2.5-3.5 and a polar organic solvents, Acetonitrile, methanol, tetra hydrofuran, dioxan and an octadecylsilane(cl8) column of 4.6 x 100mm - 4.6 x 250mm, 3.0-5.0 micron particle size columns.

3. A reverse phase gradient HPLC method using a buffer pH 2.5-3.5 preferably pH 2.8 acetic acid buffer in combination with Acetonitrile, methanol preferably methanol as mobile phase-A and Tetra hydrofuran, acetonitrile, Dioxan preferably Dioxan as mobile phase-B.

4. A reverse phase gradient HPLC method using a acetic acid buffer pH 2.8 in combination with methanol in the ratio 50:50 to 90:10 preferably 76:24 as mobile phase-A and Dioxan as mobile phase-B. and a octadecyl silane(cl8) 4.6 x 250mm -4.6 x 150mm 5 micron column preferably 4.6 x 150mm 5 micron C18 column. With mobile phase flow rates of 1.2 -0.8ml/min preferably 1.0 ml/min .

5. Bicalutamide substantially free of 4-Cyano-N-methacryloyl-3-trifluoromethylaniline and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide.

6. Bicalutamide of claim 5 wherein the content of 4-Cyano-N-methacryloyl-3-trifluoromethylaniline and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide is less than 2%.

7. Bicalutamide of claim 5 wherein the content of 4-Cyano-N-methacryloyl-3-trifluoromethylaniline is less than 10ppm.

8. Bicalutamide of claim5 wherein the content of 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide is less than 10ppm.

9. A method for the estimation of 4-Cyano-N-methacryloyl-3-trifluoromethylaniline and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide which can detect and quantify 2% or less.

10. A method for the estimation of 4-Cyano-N-methacryloyl-3-trifluoromethylaniline and 4-cyano-3-(trifluoromethyl) benzamido-2-methyl-l,2-epoxide which can detect and quantify 0.1% or less.