Isolation and Characterization of an Impurity Obtained During the Synthesis of the Antibiotic Drug Sparfloxacin

During the synthesis of Sparfloxacin, a fluoroquinolone antibiotic drug, an unknown impurity ( SF5-IMP ) was identified in the fifth stage of the synthetic process. The impurity has been isolated from the mother liquor of intermediate SF5 . The mother liquor was concentrated to dryness added dichloromethane and stirred for 1 h and filtered to generate SF5-IMP and the molecular structure was elucidated as 7-amino-1-cyclopropyl-5,6,8-trifluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid by 19 F NMR and single crystal X-ray diffraction studies. The structural features of SF5-IMP and SF5 have been discussed here.


Introduction
Sparfloxacin, a fluoroquinolone antibiotic sold under the trade name, Zagam and other drugs belonging to the same class are Ofloxacin and Ciprofloxacin. Sparfloxacin has demonstrated better activity in the clinical trials than other quinolones [1] against a broad range of gram-positive, gram-negative [2] and atypical pathogens. These organisms cause Sci Pharm. 2009; 77; 67-77. common lower respiratory tract infections and are responsible for severe mortality and morbidity around the world. Sparfloxacin was granted final FDA approval for the treatment of community-acquired pneumonia (CAP) [3] and acute bacterial exacerbations of chronic bronchitis [4] in 1996. In addition, Sparfloxacin showed in vitro activity against many penicillin-resistant strains of the gram-positive pathogen, streptococcus pneumoniae, as well as multi-drug resistant strains of the gram-negative pathogens haemophilus influenzae and Moraxella catarrhalis, although clinical efficacy has not yet been established.
In general, the synthesis of Active Pharmaceutical Ingredient (API) constitutes multiple steps. In an effort to synthesize Sparfloxacin, at fifth stage an impurity was isolated in very small amounts (0.15 % by HPLC). The purity of the drug substance is of paramount importance and any impurity should be controlled at 0.1 %. The impurities have to be adequately identified and characterized as per the International Conference on Harmonization (ICH) guidelines [5] and the United States Food and Drugs Administration (US-FDA) guidelines [6]. The structural characterization of this impurity is presented here.
About 0.15% (by HPLC) of unknown impurity was isolated at the fifth stage (Scheme 1). The impurity is labeled as SF5-IMP. Isolation and structural characterization of this impurity has been carried out by 19 F NMR and single crystal X-ray diffraction. The spectroscopic data ( 1 H NMR, 2D-NMR, 19 F NMR and HR-MS) of all the intermediates and impurity were recorded. The structure elucidation of key intermediates SF3, SF4, SF5 and SF5-IMP are described below.

Structure elucidation of SF3
The HR-MS of SF3 has shown molecular ion at m/z 330.0749 (calculated mass = 330.0753), corresponding to the chemical composition C 15 H 12 NO 3 Figure 1; CAS No. 110872-04-3). LC-MS reveals minor quantities of impurity (SF4-IMP) with same molecular formula (not isolated). The data indicates that the impurity could be an isomer of SF4.

Structure elucidation of SF5 and SF5-IMP
To obtain structural insight of SF5 and SF5-IMP, the spectroscopic data was generated. The HR-MS data of SF5 and SF5-IMP displayed the protonated molecular ions at m/z 297.0490 (calculated mass = 297.0487), corresponding to the molecular formula C 13 H 9 N 2 F 3 O 3 and at m/z 297.0481 (calculated mass = 297.0487), corresponding to the molecular formula C 13 H 9 N 2 F 3 O 3 , respectively. Since both SF5 and SF5-IMP have the same mass, the impurity could be an isomer of SF5. The 1 H and 2D NMR data revealed that the basic quinolone ring is intact in both SF5 and SF5-IMP. Hence, it is speculated that the position of amino group could be different on the aromatic ring.

Materials and methods:
The investigated samples were synthesized at Custom Pharmaceutical Synthesis (CPS)-II of Dr. Reddy's Laboratories Ltd. The solvents used for synthesis were LR grade purchased from the SD fine chemicals and Ranbaxy Laboratories, India.

b) Synthesis of Ethyl 3-oxo-3-(pentafluorophenyl)propanoate (SF2):
A Grignard reagent of diethylmalonate was prepared by adding 1,2-dibromoethane (0.305 g, 0.16 mmol), magnesium (Mg) flakes (0.843 g, 34.70 mmol) in 5.70 mL ethanol and diethylmalonate (4.342 g, 16.30 mmol) and refluxed for 1 h and cooled to 25-30 °C. SF1 (3.0 g, 13.04 mmol) was added to this mixture and refluxed for ca. 2 hrs. The reaction mass was quenched with 2N H 2 SO 4 at less than 15 °C, and the solvent removed under reduced pressure to afford the crude product which was directly used in the next step.

c) Synthesis of 1-Cyclopropyl-5,6,7,8-tetrafluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (SF3):
To a solution of triethylorthoformate (2.836 g, 19.13 mmol), SF2 (5.0 g, 17.72 mmol) and acetic anhydride (2.116 g, 20.73 mmol) were added at 25-30 °C and the contents were warmed to 100-105 °C, stirred under nitrogen atmosphere for one hour the distillate was collected (without vacuum) using a Dean-stork apparatus. This reaction mass was cooled to 70 °C and the acetic anhydride was distilled under vacuum and allowed to cool to about 30 °C. Toluene (25 mL) was added to this reaction mass, cooled to 5 °C. A solution of cyclopropyl amine (0.830 g, 14.5 mmol) in toluene (10 mL) was added and the contents stirred at 5 °C for about 2 hrs. The contents were subsequently warmed to about 25 °C and the mixture was washed with 5 mL of water, 3 mL of 2 % sodium carbonate (15 mL) and 5 mL of water. The organic layer was collected and potassium carbonate (3.778 g, 27.33 mmol) was added and the contents were heated to about 45 °C for 12 hrs. Toluene was distilled under reduced pressure and the solid obtained was washed with about 5 mL of water followed by 2.5 mL of methanol. The yellow colored solid obtained was dried under vacuum.

f) Isolation
The mother liquor obtained from the above step was concentrated to dryness. Added 2.5 mL of dichloromethane (DCM) and stirred for 1 hr and filtered to obtain SF5-IMP. This method was repeated thrice to obtain pure SF5-IMP in large quantities.

UPLC-TOF
The UPLC-TOF-MS system consisted of an ACQUITY™ Ultra Performance Liquid Chromatography system and a Micromass LCT Premier XE Mass Spectrometer (High sensitivity orthogonal time-of-flight instrument; Waters, Milford, USA) equipped with a lock mass sprayer, operating in either the positive or negative ion mode. The source temperature was set at 120 °C with a cone gas flow of 10 l/h, a desolvation gas temperature of 250 °C, and a desolvation gas flow of 450 l/h. The capillary voltage was set at 2300V and the cone voltage to 30 V. All analyses were acquired using the lock spray to ensure accuracy and reproducibility; leucine-enkephalin was used as the lock mass. Sample of concentration 0.02 mg/mL in methanol was infused in TOF-MS at a flow rate of 10µL/min. High resolution (W mode, FWHM 10500) positive polarity scan responses were collected from m/z 100 to 1000 at a rate of 1.0 s/scan. The dynamic range enhancement (DRE) function was used. The lock spray frequency was set at 5s and data were averaged over 10 scans.

Single crystal X-ray Diffraction
The X-ray data for the single crystal has been collected on Rigaku AFC-7S diffractometer equipped with Mercury CCD detector using graphite monochromated Mo-K α radiation (λ = 0.7107 Å). The structure was solved with direct methods (SIR-2004) and refined using least squares procedure (CRYSTALS) using the crystal structure 3.8.1 software.
CCDC 715190 (for SF5-IMP) and CCDC 715191 (for SF5) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.