Novel Compounds

FORM2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10; rule 13) 1. Title of the invention. - "Novel Compounds" 2. Applicant(s) (a) NAME : ALEMBIC LIMITED (b) NATIONALITY : An Indian Company (c) ADDRESS : Alembic Campus, Alembic Road, Vadodara-390 003, Gujarat, India 3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed :

Field of invention: The present invention relates to novel compounds useful as reference markers for the analysis of Ropinirole hydrochloride of formula (la) and pharmaceutical formulations thereof. It also discloses method of testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride.

Background of the invention: The chemical name of Ropinirole is 4-[2-(Dipropylamino)ethyl]-l,3-dihydro-2//-indol-2-one, formula C16H24N20 and molecular weight is 260.37. Ropinirole is marketed in the form of its hydrochloride salt. The current pharmaceutical product containing this drug is being sold by Glaxosmithkline using the tradename Requip®, in the form of tablets. The structural formula of Ropinirole is represented by formula (I)

Ropinirole is useful in the treatment of Parkinson's disease. Ropinirole is a dopamine agonist and having selective affinity for dopamine D2-like receptors and little or no affinity for non-dopaminergic brain receptors. Ropinirole is indicated as adjunct therapy to levodopa in patients with advanced Parkinson's disease. Also, recent clinical trials have focused on its use, as monotherapy in patients with early Parkinson's disease. To obtain marketing approval for a new drug product, it is required to submit data to the regulatory authorities proving the safety and efficacy of the drug. This data include 2

analytical data which shows that (i) drug is free from impurities or impurities are present only in negligible amount and (ii) shelf-life or storage stability of drug is acceptable. In order to generate this data the drug is tested against an external standard or reference marker. Impurities generally found in pharmaceutically active agents and formulations containing them include residual amounts of synthetic precursors to the active agent, by-products which arise during synthesis of the active agent, residual solvent, isomers of the active agent, contaminants which were present in materials used in the synthesis of the active agent or in the preparation of the pharmaceutical formulation, and unidentified adventitious substances. Other impurities which may appear on storage include substances resulting from degradation of the active agent, for instance by oxidation or hydrolysis, the ICH Q7A guidance for API manufacturers requires that process impurities be maintained below set limits by specifying the quality of raw materials, controlling process parameters, such as temperature, pressure, time, and stoichiometric ratios, and including purification steps, such as crystallization, distillation, and liquid-liquid extraction, in the manufacturing process. At certain stages during processing of an API, such as Ropinirole hydrochloride, it must be analyzed for purity, typically, by HPLC or GC analysis, to determine if it is suitable for continued processing and, ultimately, for use in a pharmaceutical product. The API need not be absolutely pure, as absolute purity is a theoretical ideal that is typically unattainable. Rather, purity standards are set with the intention of ensuring that an API is as free of impurities as possible, and, thus, is as safe as possible for clinical use. As discussed above, in the United States, the Food and Drug Administration guidelines recommend that the amounts of some impurities be limited to less than 0.1 percent. Generally, side products, by-products, and adjunct reagents (collectively "impurities") are identified spectroscopically and/or with another physical method, and then associated with a peak position, such as that in a chromatogram, or a spot on a TLC plate. (Strobel p. 953, Strobel, H. A.; Heineman, W. R., Chemical Instrumentation: A Systematic 3

Approach, 3rd dd. (Wiley & Sons: New York 1989)). Thereafter, the impurity can be identified, e.g., by its relative position in the chromatogram, where the position in a chromatogram is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector. The relative position in the chromatogram is known as the "retention time." The retention time varies daily, or even over the course of a day, based upon the condition of the instrumentation, as well as many other factors. To mitigate the effects such variations have upon accurate identification of an impurity, practitioners use the "relative retention time" ("RRT") to identify impurities. (Strobel p. 922). The RRT of an impurity is its retention time divided by the retention time of a reference marker. In theory, Ropinirole Hydrochloride itself could be used as the reference marker, but as a practical matter it is present in such a large proportion in the mixture that it can saturate the column, leading to irreproducible retention times, as the maximum of the peak can wander (Strobel, FIG. 24.8(b), p. 879, illustrates an asymmetric peak observed when a column is overloaded). Thus, it may be advantageous to select a compound other than the API that is added to, or present in, the mixture in an amount sufficiently large to be detectable and sufficiently low as not to saturate the column, and to use that compound as the reference marker. Those skilled in the art of drug manufacturing research and development understand that a compound in a relatively pure state can be used as a "reference standard." A reference standard is similar to a reference marker, which is used for qualitative analysis only, but is used to quantify the amount of the compound of the reference standard in an unknown mixture, as well. A reference standard is an "external standard," when a solution of a known concentration of the reference standard and an unknown mixture are analyzed using the same technique. (Strobel p. 924, Snyder p. 549, Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed. (John Wiley & Sons: New York 1979)). The amount of the compound in the mixture can be determined by comparing the magnitude of the detector response. See also U.S. Pat. No. 6,333,198, incorporated herein by reference. The reference standard can also be used to quantify the amount of another compound in the mixture if a "response factor," which compensates for differences in the sensitivity of 4

the detector to the two compounds, has been predetermined. (Strobel p. 894). For this purpose, the reference standard is added directly to the mixture, and is known as an "internal standard." (Strobel p. 925, Snyder p. 552). The reference standard can even be used as an internal standard when, without the addition of the reference standard, an unknown mixture contains a detectable amount of the reference standard compound using a technique known as "standard addition." In a "standard addition," at least two samples are prepared by adding known and differing amounts of the internal standard. (Strobel pp. 391-393, Snyder pp. 571, 572). The proportion of the detector response due to the reference standard present in the mixture without the addition can be determined by plotting the detector response against the amount of the reference standard added to each of the samples, and extrapolating the plot to zero. (See, e.g., Strobel, FIG. 11.4 p. 392). As is known by those skilled in the art, the management of process impurities is greatly enhanced by understanding their chemical structures and synthetic pathways, and by identifying the parameters that influence the amount of impurities in the final product. It has been observed by inventors of the present invention that Ropinirole hydrochloride contains five known impurities. These impurities are: (i) 4-[2-(N-Propylamino)ethyl]-1,2-dihydro-2H-indol-2-one hydrochloride (Compound A) (ii) 4-[2-(N,N-Di-n-propylamino)ethyl]-l,4-dihydro-3H-2,l-benzoxazin-3-one hydrochloride (Compound B) (iii) 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid hydrochloride (Compound C) (iv) 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid-N-oxide hydrochloride (Compound D) (v) 4-(2-di-n-propylaminoethyl)-7-hydroxy-2(3H)-indolone (Compound E) 5

Out of these, compounds A and E are impurities arising from side reactions during the synthesis of Ropinirole hydrochloride and compound C is synthetic precursor of the active agent. The inventors of present invention have found that out of these five impurities, compound B and D can be used as reference markers for the analysis of Ropinirole hydrochloride or of pharmaceutical dosage forms comprising Ropinirole hydrochloride. Object of the invention: It is an object of the present invention to provide novel compounds, Compound B and Compound D which are useful as reference markers for the analysis of Ropinirole hydrochloride and pharmaceutical formulations thereof. Another object of the present invention is to provide a method of testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride. A further object of the present invention is to provide a method of testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride comprising assaying the said sample for the presence of a compound selected from Compounds B and Compound D. Yet another object of the present invention is to provide highly pure Ropinirole hydrochloride. Summary of the invention: In the first embodiment, this invention is directed to an isolated Ropinirole hydrochloride impurity; the isolated 4-[2-(N,N-Di-n-propylamino)ethyl]-l,4-dihydro-3H-2,l-benzoxazin-3-one hydrochloride (Compound B) 6

In another embodiment, this invention is directed to an isolated Ropinirole hydrochloride impurity; the isolated 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid-N-oxide hydrochloride (Compound D) In another embodiment, the invention is directed to a method of using Compound B and Compound D as reference markers to analytically quantify the purity of Ropinirole hydrochloride. In yet another embodiment, the invention is directed to a method for the quantification of the purity of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride, comprising the use of Compound B and Compound D as reference markers. In a further embodiment of the present invention provides highly pure Ropinirole hydrochloride. Detailed description of the invention: Ropinirole was first reported in US patent no. 4,452,808. It discloses the process for the preparation of Ropinirole hydrochloride as shown in following Scheme.

,CH3 H3C_ Anhy. THF, borane o Reflux 2hrs CH3 HCI;NaOH (IV) NO, H3C 10°C, NaOH, H202; HCI 5% Pd/C, ethanol, hydrogenation, 5.5 h COOH

(ID H,C

COOH

SOCI, 75°C, reflux 1 hrs. CH,

N02 Na metal, abs. ethanol, rt, Diethyl oxalate; reflux, N2 atmosphere

7

The process of preparation of Ropinirole hydrochloride is also disclosed in pending PCT application no. PCT/IN06/00052. 4-[2-(N,N-Di-n-propylamino)ethyl]-1,4-dihydro-3H-2,1 -benzoxazin-3-one hydrochloride (Compound B) is formed due to side reactions occurring during the synthesis of Ropinirole hydrochloride. 3 \ /^\ ^-^"x. /^''3

H Compound B Compound (VII) is hydrogenated to get the Ropinirole hydrochloride of formula (la). During the hydrogenation, the nitro group is reduced to -NHOH group which reacts with the hydroxy group of-COOH group to give cyclic compound B as impurity.

(VII) (Vllb) Compound B Compound B is substantially pure and having purity at least 97.6%. Compound B is isolated by carrying of column chromatography of the crude Ropinirole hydrochloride. 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid-N-oxide hydrochloride (Compound D) is also formed due to side reactions occurring during the synthesis of Ropinirole hydrochloride. 8

Compound D Compound (VI) is hydrolysed with 2% sodium hydroxide in the presence of 30% hydrogen peroxide, followed by treatment with concentrated HC1 to give compound (VII) as hydrochloride salt. During the reaction, nitrogen of compound (VI) is oxidized to give N-oxide compound of formula (VI). The compound of formula (VI) is also hydrochloride in a similar fashion. The hydrolysis of compound of formula (VI) leads to the compound of formula D as impurity.

H3C

OEt I Jv N02 Compound D Compound D is isolated by carrying of column chromatography of the crude Ropinirole hydrochloride. Compound D obtained by this method is substantially pure and has purity at least 97.24%. When compounds B and D are used as reference markers they must be in a suitably pure form. The test sample of drug substance (Ropinirole hydrochloride) or drug product (pharmaceutical dosage form of Ropinirole hydrochloride) to be analyzed may be analysed by one or more conventional analytical techniques. The analytical technique includes high performance liquid chromatography (HPLC). The results obtained are compared with the results obtained from testing a substantially pure reference sample of 9

compound B or D. The content or each compound in the test sample can then be determined. In one aspect of the present invention, it provides novel compounds B and D. In another aspect of the present invention, the method for testing the purity of a sample of Ropinirole hydrochloride which comprises steps of: (i) dissolving a reference standard of impurities and ropinirole hydrochloride in a solvent (diluent) to produce a reference solution, (ii) dissolving a sample of Ropinirole hydrochloride in a solvent (diluent) to produce a sample solution (iii) injecting the diluent, reference solution and sample solution on to an HPLC column, and determining the area of each peak and calculating the purity of Ropinirole hydrochloride. In this further aspect it is necessary to run a mobile phase as blank through the HPLC column prior to step (i). The following examples illustrate the invention further. It should be understood, however, that the invention is not confined to the specific limitations set forth in the individual examples but rather to the scope of the appended claims. EXAMPLE-1 Preparation of 4-[2-(N,N-Di-n-propylamino)ethyl]-l,4-dihydro-3H-2,l-benzoxazin- 3-one hydrochloride (Compound B). Compound-B was isolated from crude Ropinirole hydrochloride by column chromatography. Purity of the product was determined by HPLC as 97.6%. The HPLC conditions were as follows. Column : Symmetry C8 (250x4.6mm,5u) or equivalent Mobile Phase-(A): Mix Buffer and acetonitrile in the ratio of 90:10 and filter through 10

0.45u filter paper and degas. Mobile Phase (B): Mix 800 ml of Acetonitrile with 200ml of Milli-Q water;add 2.0ml of Triethylamine; mix and Adjust pH 6.0with 10% ortho phosphoric acid and filter through 0.45 u filter paper and degas. Gradient Profile:

Step Time Mobile Phase A Mobile Phase B 0 00.00 95.0 05.0 1 20.00 95.0 05.0 2 37.00 40.0 60.0 3 47.00 40.0 60.0 4 50.00 95.0 05.0 5 60.00 95.0 05.0 Temperature: 45°C. Flow rate: l.OmL/min. Wavelength: 250nm. Injection Volume: 20uL. Diluent: Mix M.P.A and M.P.B in the ratio of 95:05v/v respectively. Phosphate buffer: Dissolve 7.80 g of Sodium dihydrogen Orthophophate dihydrate in 1000 ml of water; mix to dissolve. Add 5.0 ml of Triethylamine;mix and adjust pH 6.0with 10% orthophosphoric acid,filter through 0.45 u filter paper and degas. Pre-equilibrate the column for about 60min with mobile phase or until a stable base line is obtained. When a stable baseline was obtained, the diluent was injected. Compound B solution was injected and found the purity by area normalization method. The product had the following characteristics: Molecular formula: C16H25N2O2CI Molecular mass: 312.5 IR (in KBr): 3432; 2962-2819; 1727; 931-638 cm"1 11

lH NMR (in DMSO / 300 MHz): 10.91 (lH,b); 7.26-7.21 (lH,t); 6.95-6.92 (lH,d); 6.82-6.8 (lH,d); 3.63 (2H,s); 3.2-3.18 (2H,m); 3.04 (6H,m); 1.78-1.70 (4H,m); 0.93-0.88 (6H,t) 8 ppm. Mass spectra: m/z=277.1 (M+H)+ EXAMPLE-2 Preparation of 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid-N-oxide hydrochloride (Compound D) Compound-D was isolated from crude Ropinirole hydrochloride by column chromatography. Purity of the product was determined by HPLC as 97.24%. The HPLC conditions were as follows. Column : Symmetry C8 (250x4.6mm,5u) or equivalent Mobile Phase-(A): Mix Buffer and acetonitrile in the ratio of 90:10 and filter through 0.45 u filter paper and degas. Mobile Phase (B): Mix 800 ml of Acetonitrile with 200ml of Milli-Q water;add 2.0ml of Triethylamine; mix and Adjust pH 6.0with 10% ortho phosphoric acid and filter through 0.45 u filter paper and degas. Gradient Profile:

Step Time Mobile Phase A Mobile Phase B 0 00.00 95.0 05.0 1 20.00 95.0 05.0 2 37.00 40.0 60.0 3 47.00 40.0 60.0 4 50.00 95.0 05.0 5 60.00 95.0 05.0 Temperature: 45°C. Flow rate: l.OmL/min. Wavelength: 250nm. 12

Injection Volume: 20uL. Diluent: Mix M.P.A and M.P.B in the ratio, of 95:05v/v respectively. Phosphate buffer: Dissolve 7.80 g of Sodium dihydrogen Orthophophate dihydrate in 1000 ml of water; mix to dissolve. Add 5.0 ml of Triethylamine;mix and adjust pH 6.0with 10% orthophosphoric acid,filter through 0.45u filter paper and degas. Pre-equilibrate the column for about 60min with mobile phase or until a stable base line is obtained. When a stable baseline was obtained, the diluent was injected. Compound D solution was injected and found the purity by area normalization method. The product had the following characteristics: Molecular formula: C16H25N2O5CI Molecular mass: 360.5 IR(in KBr): 3424; 2973-2882; 1725; 1527.6; 882-640 cm"1 'H NMR (in DMSO / 300 MHz): 12.76 (lH,b); 7.9-7.88 (lH,d); 7.81-7.78 (lH,d); 7.56-7.5 (lH,t); 3.94 (2H,s); 3.83-3.78 (2H,m); 3.67-3.62 (4H,m); 3.33-3.28- (2H,m); 1.81-1.73 (4H,m); 0.95-0.9 (6H,t) 5 ppm. Mass spectra: m/z= 325.6 (M+H)+ EXAMPLE-3 Determination of Related Compounds Related substances for Ropinirole in drug by HPLC comprises following steps: Preparation of Reference Solution Impurity stock solution: Accurately weigh and transfer about 5.0mg each of Impurities A, B, C, into a 5 ml volumetric flask, add about 2 ml of diluent and sonicate to dissolve. Make up the volume with diluent. 13

Reference solution: Accurately weigh about l0.Omg of Ropinirole hydrochloride reference standard and transfer into a 10 ml volumetric flask, add about 4ml of diluent and sonicate to dissolve. Then add 15.0 ul impurity stock solution. Mix and make up the volume with diluent. Preparation of Sample Solution: Accurately weigh about 25.0mg of test sample and transfer into a 25 ml volumetric flask, add about 10ml of diluent and sonicate to dissolve, mix and make up the volume with diluent. Chromatographic procedure: The following conditions were used: Column : Symmetry C8 (250x4.6mm,5|j,) or equivalent Mobile Phase-(A) : Mix Buffer and acetonitrile in the ratio of 90:10 and filter through 0.45 u filter paper and degas. Mobile Phase (B): Mix 800 ml of Acetonitrile with 200ml of Milli-Q water;add 2.0ml of Triethylamine; mix and Adjust pH 6.0with 10% ortho phosphoric acid,filter through 0.45 u filter paper and degas. Gradient Profile:

Step Time Mobile Phase A Mobile Phase B 0 00.00 95.0 05.0 1 20.00 95.0 05.0 2 37.00 40.0 60.0 3 47.00 40.0 60.0 4 50.00 95.0 05.0 5 60.00 95.0 05.0 Temperature: 45°C. Flow rate: l.OmL/min. Wavelength: 250nm. 14

Injection Volume: 20uL. Diluent: Mix M.P.A and M.P.B in the ratio of 95:05v/v respectively. Phosphate buffer:Dissolve 7.80 g of Sodium dihydrogen Orthophophate dihydrate in 1000 ml of water; mix to dissolve. Add 5.0 ml of Triethylamine;mix and adjust pH 6.0with 10% orthophosphoric acid,filter through 0.45n filter paper and degas. Injection Procedure Pre-equilibrate the column for about 60min with mobile phase or until a stable baseline is obtained. When a stable base line was otained the reference solution was injected and the resolution factor between compound B and compound E was calculated and it should be more than 2.0. The sample solution was the injected into the column. Calculations The content of any component was calculated by following formula, % of individual compound(w/w) = %Area of impurity by Area Normalisation RRF TABLE-1 Component Retention Relative Relative Response Time(min.) Retention time (RRT) Factor(RRF) Compound A 5M 032 0/74 Compound B 15.00 0.88 0.64 Compound C 12.75 0.75 0.34 Compound D 11.00 0.65 0.22 Compound E 15.80 0.93 1.08 15

What is claimed is: 1. A method of testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride, which method comprises determination of reference maker B and D in the said sample by HPLC method. 2. A method according to claim 1 for testing the purity of a sample of Ropinirole hydrochloride, which comprises steps of: (i) dissolving a reference standard of impurities (A, B, C, D, E) and ropinirole hydrochloride in a solvent (diluent) to produce a reference solution, (ii) dissolving a sample of Ropinirole hydrochloride in a solvent (diluent) to produce a sample solution (iii) injecting the diluent, reference solution and sample solution on to an HPLC column, and determining the area of each peak and calculating the purity of Ropinirole hydrochloride; wherein compound A is 4-[2-(N-Propylamino)ethyl]-l,2-dihydro-2H-indol-2-one hydrochloride; compound B is 4-[2-(N,N-Di-n-propylamino)ethyl]-l,4-dihydro-3H-2,l-benzoxazin-3-one hydrochloride compound C is 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid hydrochloride; compound D is 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid-N-oxide hydrochloride compound E is 4-(2-di-n-propylaminoethyl)-7-hydroxy-2(3H)-indolone. 3. A compound which is 4-[2-(N,N-Di-n-propylamino)ethyl]-l,4-dihydro-3H-2,l-benzoxazin-3-one hydrochloride having formula 16

H Compound B and is known as compound B. A sample of compound as claimed in claim 3 which is in substantially pure form. A sample according to claim 4 which has a purity level of 97.6% or above. A compound which is 2-Nitro-6-[2-(N,N-Di-n-propylamino)ethyl]phenyl acetic acid-N-oxide hydrochloride of formula

Compound D and is known as compound D. A sample of compound as claimed in claim 6 which is in substantially pure form. A sample according to claim 7 which has a purity level of 97.24% or above. Use of compound B as reference marker for testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride. 17

10. Use of compound D as reference marker for testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropimrole hydrochloride. Dated this 31st day of March 2006. . Ashwini Sandu OfS. Majumdar&Co. Applicant's Agent 18

Abstract: Novel compounds useful as reference markers for the analysis of Ropinirole hydrochloride of formula (la), pharmaceutical formulations therefor and method of testing the purity of a sample of Ropinirole hydrochloride or a pharmaceutical dosage form comprising Ropinirole hydrochloride. 19