DESC:FIELD OF THE INVENTION
The present invention relates to isolation and identification of novel oxidative degradation products of Pitavastatin and a process for the preparation thereof. The invention also relates to use of these oxidation products as a reference marker and/or reference standard.

The invention also deals with analytical standards and analytical methods used for the control of the production process and final quality of Pitavastatin.

BACKGROUND AND THE PRIOR ART
Pitavastatin Calcium is a novel, synthetic 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitor. The HMG-CoA reductases are known as therapeutically effective active substances for the treatment of dyslipidemias and cardiovascular diseases, selected from the group consisting of dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, arteriosclerosis, coronary artery diseases, coronary heart diseases and the like, associated with the metabolism of lipids and cholesterol.

Pitavastatin calcium is chemically known as monocalcium salt of bis {(3R, 5S, 6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]-3,5-dihydroxy-6-heptenoate} represented by structural formula (1).

Pitavastatin calcium is sold under the trade name LIVALO® in the US.

Pitavastatin was first described in US 5,753,675 patent and the calcium salt was disclosed in US 5,856,336 patent.

In view of the importance of obtaining a drug substance with high level of purity, as per regulatory norms, it is necessary to identify and characterize each impurity or degradation product present in the drug substance. Moreover impurity profile of drug substances is critical for its safety assessment and manufacturing process. It is therefore important to identify and characterize the process and degradation impurities in a pharmaceutical product, if present above the acceptable limit.

Moreover, the stability test guideline issued by ICH suggests that stress studies should be carried out on active pharmaceutical ingredients to establish its inherent stability characteristics, leading to separation of degradation products and hence supporting the suitability of the proposed analytical procedures.

A research review reveals that there are several reports on the degradation products of Pitavastatin.

Grobelny et al (Journal of Pharmaceutical and Biomedical Analysis 50,597–601, 2009) have investigated the photochemical processes of Pitavastatin in solution. The main photochemical reaction observed was photo cyclisation leading to formation of four photoproducts, namely PP-1, PP-2, PP-3 and PP-4.

Gomas et al (American Journal of Analytical Chemistry, 2, 83-90, 2010) discusses the degradation pathway for Pitavastatin Calcium as per ICH guidelines by validated stability indicating UPLC method. The stress conditions employed for degradation study were acid, base, oxidation, photolytic and thermal. The prominent degradation impurity identified under peroxide stress condition i.e. 10% H2O2 upon heating for 2 hours is 3-hydroxymethyl-2-cyclopropyl-4-(4-fluorophenyl)-quinoline.

The Indian patent application 3872/CHE/2010 discusses process for the preparation of novel Pitavastatin calcium impurities i.e. hydrogenated impurity of formula-A, two degradation products of formula B1 and B2 two dehydroxyl impurities of formulae C1 and C2 and amide impurity of formula D.

Neelima et al (Int. J Pharma Sci., 3(4), 309-315, 2013) discusses degradation studies done under oxidative conditions by heating the drug sample with 30% H2O2 at 60 oC, however, degraded product peaks observed were not identified.

However, till date no report on epoxide and the corresponding N-oxide oxidative degradation products of Pitavastatin calcium has been located. So there exists a scope to identify and prepare the oxidative degradation products of Pitavastatin Calcium and avoid the presence of these oxidative degradation products in the drug substance to meet regulatory norms.

DESCRIPTION OF THE INVENTION
As describe herein, the term pitavastatin includes a compound (3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl]-3,5-dihydroxyhept-6-enoic acid (CAS No.: 147511-69-1) and pharmaceutically acceptable salts thereof. In particular, its includes pitavastatin acid (CAS No.: 147511-69-1), pitavastatin calcium salt (CAS No.: 147526-32-7), pitavastatin sodium salt (CAS No.: 574705-92-3), pitavastatin magnesium salt (CAS No.: 956116-90-8), pitavastatin ammonium salt (CAS No.: 1035622-90-2) and pitavastatin tert-butyl amine salt (CAS No.: 956034-96-1).

Present invention relates to isolated oxidative degradation products of Pitavastatin.

Accordingly, the present invention meets a need in the art for obtaining Pitavastatin Calcium product in a highly pure form having a low content of oxidative degradation products, by a technologically simple manner, by isolation and characterization of the oxidative degradation products of Pitavastatin Calcium.

One object of the present invention relates to novel compounds of formula (2) and formula (3), which are oxidative degradation products of Pitavastatin. The formation of these oxidative degradation products is observed when Pitavastatin calcium is subjected to oxidative degradation studies and/or when it is exposed to an oxidizing environment e.g. air/oxygen.

In another object of the present invention epoxide, (3R, 5S)-5-(3-(2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)oxiran-2-yl)-3,5-dihydroxypentanoic, compound of formula (2) and the corresponding N-oxide, 3-(3-((1S,3R)-4-carboxy-1,3-dihydroxybutyl)oxiran-2-yl)-2-cyclopropyl-4-(4-fluorophenyl)quinoline-1-oxide, compound of formula (3) are the major oxidative degradation products of Pitavastatin observed during peroxide degradation studies done at 80oC in the presence of 10% hydrogen peroxide.

In yet another object of the present invention compound of formula (2) and compound of formula (3) are isolated by preparative HPLC and characterized by NMR, MS and IR spectroscopy.

The carbon atoms indicated by the asterisks in formula (2) and formula (3) are chiral, thus the scope of the present invention includes all the possible stereoisomers individually and mixture thereof in any ratio.

Another object of the present invention is the process of preparation of compound of formula (2) and compound of formula (3). According to the present invention, the oxidative degradation products of Pitavastatin Calcium are prepared by reacting Pitavastatin free acid with m-chloro perbenzoic acid in ethyl acetate at room temperature.

The degradation impurities were purified using preparative HPLC and completely characterized on the basis of NMR, MS and IR spectroscopy.

The oxidative degradation products may form during the synthesis of Pitavastatin Calcium or upon storage.

In a further aspect, the object of present invention relates to the use of isolated oxidative degradation products formula (2) and formula (3), as a reference marker in a qualitative analysis of Pitavastatin Calcium.

In another aspect, the invention is directed to a method of using oxidative degradation products formula (2) and formula (3) as reference standard to analytically quantify the purity of Pitavastatin Calcium.

In yet a further aspect, the invention is directed to a method for the quantification of the purity of Pitavastatin Calcium, comprising the use of oxidative degradation impurities formula (2) and formula (3) as reference standard, where the reference standard may be either external standard or internal standard.

Present invention is further illustrated with the following non-limiting examples.

A. Synthesis of oxidative degradation impurities:

1. Preparation of Pitavastatin free acid:

10 gm of Pitavastatin calcium was charged in a RBF followed by 100 ml of ethyl acetate and 100 ml of DM water. The reaction mass was cooled to 5-10°C and stirred for 30 minutes. The pH of reaction mass was adjusted between 3 and 4 using dilute hydrochloric acid and was stirred for 15 minutes. Layers were separated and organic layer was washed with 100 ml of DM water, dried over sodium sulphate and concentrated under reduced pressure to get 9 gm of Pitavastatin acid as oil.

2. Preparation of Epoxides and corresponding N-oxide of Pitavastatin:

9 gm of Pitavastatin free acid was dissolved in 60 ml of ethyl acetate and charged in a RBF. This solution was cooled to 5-10°C and 6.7 grams (1 mole equivalent) of meta-chloro per benzoic acid in 30 ml of ethyl acetate was added slowly using a dropping funnel. The reaction mass was brought to room temperature and stirred for 18-20 hours after which the solution turned into a white suspension. The solids were filtered and washed with ethyl acetate. The filtrate containing desired products was washed with water dried over sodium sulphate and concentrated under reduced pressure to get a mixture of both epoxide (2) and the corresponding N-oxide (3) oxidative degradation impurity. These degradation impurities were purified using preparative HPLC and completely characterized on the basis of NMR, MS and IR spectroscopy.

B. Pitavastatin Calcium Degradation Study:

Sample preparation: - 15 mg of Pitavastatin calcium sample taken in 50 ml volumetric flask was dissolved and made up the volume with diluent. Oxidative degradation (1.0 hrs. heating @80 oC):- 15 mg of Pitavastatin calcium taken in 50 ml volumetric flask was dissolved in 5ml of diluent. Thereafter 5.0ml 10% hydrogen peroxide solution was added to the dissolved sample and heated at 80 o C for 15 min. Cooled to room temperature and made up the volume with diluent for analysis.

C. Analytical Apparatus and conditions:

1. LCMS Method/conditions:

LCMS was performed on a Waters Xevo G2 QToF using Ascentis express C18 (4.6x100 mm) 2.7µm column, with UV detector at 245nm. 2 Mobile phases were used in a gradient programme. First mobile phases consisted of an 80:20 mixture of buffer prepared by dissolving 1.27 grams of Ammonium fomate in 1000 ml of milliQ water and the pH adjusted to 4.9 with formic acid and an 80:20 solution of Acetonitrile and THF. Second mobile phase consisted of a 20: 80 mixture of buffer prepared by dissolving 1.27 grams of Ammonium fomate in 1000 ml of milliQ water and the pH adjusted to 4.9 with formic acid and an 80:20 mixture of Acetonitrile and THF. Flow rate was set at 1.2ml per minute and the column oven temperature was set at 25°C.

2. Preparative HPLC method/conditions:

Impurities were isolated using a ShimadzuLC-8A preparative HPLC system consisting of UV/Vis detector and data was processed using Class VP, version 6.14SP-1 software. An YMC-Actus triart PREP C-18 (200x20) mm, 10 micron column was used for separation and the mobile phase consisted of a mixture of Acetonitrile and water. The flow rate was maintained at 30ml/min and run time was 25 minutes. Sample solution of 75 mg/ml was prepared in acetonitrile and a drop of formic acid. 2 ml sample was injected and monitored at 254 nm.

Synthetic Scheme:-


,CLAIMS:1. A compound of formula (2):
.
2. A compound of formula (3):
.
3. A process for the preparation of the compound of formula (2)

comprising reacting pitavastatin with an oxidizing reagent.
4. A process for the preparation of the compound of formula (3)

comprising reacting pitavastatin with an oxidizing reagent.
5. A process according to claim-4 or claim-5 wherein the oxidizing reagent is meta-chloroperbenzoic acid.