Synthesis and Characterization of Potential Dimers of Gatifloxacin – an Antibacterial Drug

Gatifloxacin is an antibacterial agent belonging to the fourth-generation fluoroquinolone family. Four piperazine-linked fluoroquinolone dimers of Gatifloxacin were observed during the laboratory process for Gatifloxacin and they were identified. The present work describes the origin, synthesis, characterization, and control of these dimers along with the synthesis of Despropylene Gatifloxacin (metabolite).


Introduction
Gatifloxacin 1 is chemically known as 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, one of the most important broad-spectrum antibacterial agents and a member of the fourth-generation fluoroquinolone family. Gatifloxacin 1 is marketed by Bristol-Myers Squibb in the brand name of Tequin ® for the treatment of respiratory tract infections. It is available as aqueous solutions for intravenous therapy. The ophthalmic solution is marketed by Allergan in the brand name of Zymer ® .
The presence of impurities in an Active Pharmaceutical Ingredient (API) will influence the quality and safety of the drug product. In the regulatory guidelines of the International Conference on Harmonization (ICH), it is recommended that impurities more than 0.1% [1] should be identified and characterized. Impurities are required in pure form to check the analytical performance characteristics such as specificity, linearity, range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), robustness, system suitability testing, and relative retention factor [2].
During the process development of Gatifloxacin 1 in the laboratory, we observed four unknown impurities along with the known impurities [3,4]. These unknown impurities were detected, monitored, and their structures were tentatively assigned on the basis of their fragmentation patterns in LC-MS. In the present work, the identified impurities of Gatifloxacin were synthesized and characterized by various spectroscopic techniques and further confirmed by co-injection studies in the qualitative HPLC analysis.

Gatifloxacin dimer-1:
Gatifloxacin dimer-1 6 originated from the substitution reaction of the piperazine moiety of Gatifloxacin 1 with the fluoro group present at the C-7 position of 8-methoxyquinoline carboxylic acid 7 during the hydrolysis of Gatifloxacin borondifluoride chelate 4.
Compound 6 was independently prepared by the condensation of compound 1 with compound 3 in DMSO to produce compound 5, which was hydrolyzed with triethylamine in methanol to produce compound 6 (Scheme 2). The mass spectrum showed a molecular ion at m/z 651.1 amu [(M+H) + ] and a sodium adduct at m/z 673.1 amu [(M+Na) + ]. In comparison with Gatifloxacin 1, twice the number of quinoline moiety protons in 1 H NMR were observed.

Gatifloxacin dimer-2:
Gatifloxacin dimer-2 9 originated from the condensation of the piperazine moiety of Gatifloxacin 1 with the carboxylic acid of 8-methoxyquinolinecarboxylic acid 7 during the hydrolysis of Gatifloxacin borondifluoride chelate 4.
Compound 8 was independently prepared from compound 7. Reaction of 7 with thionyl chloride in dichloromethane to produce compound 8, which was further reacted with Gatifloxacin 1 in dichloromethane in presence of diisopropylethylamine, produced compound 9 (Scheme 3). The mass spectrum showed a molecular ion at m/z 653.2 amu [(M+H) + ]. The amide group was confirmed by observing C=O stretching at 1599 cm −1 in the IR spectrum. In comparison with Gatifloxacin 1, twice the number of quinoline moiety protons in 1 H NMR and twice the number of carbon signals in 13 C NMR were observed.

Gatifloxacin dimer-4:
Gatifloxacin dimer-4 16 originated by the condensation of ethylenediamine, present as an impurity in the raw material, and 2-methylpiperazine with two molecules of compound 3.  Gatifloxacin dimers 1-4, 6, 9, 11, 16 are novel and process-related compounds. These impurities were eliminated during the purification of Gatifloxacin 1. Compounds 14 & 16 originated due to the presence of the ethylenediamine impurity found in the raw material 2methylpiperazine, and thus ethylenediamine is controlled within a specified limit (0.1%) in the specifications of the 2-methylpiperazine raw material.

Experimental
Solvents and reagents were obtained from the commercial sources and used without purification. 1 H NMR and 13 C NMR spectral data were performed on the Bruker-Avance 300-MHz spectrometer in DMSO-d 6 . The chemical shift values were reported on the δ scale in parts per million (ppm), downfield from tetramethylsilane (TMS) as an internal standard. IR spectra were recorded in the solid state as KBr pellets using a Perkin-Elmer FT-IR spectrophotometer. The mass spectrum was recorded using a Perkin-Elmer PE SCIEX-API 2000, equipped with ESI source used online with a HPLC system after the ultraviolet (UV) detector.
The reaction mass was concentrated under reduced pressure to remove excess thionyl chloride and dissolved the residue in dichloromethane (100mL). In a separate flask, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxoquinoline-3carboxylic acid 1 (10 g, 2.66 mmol) was dissolved in dichloromethane (750 mL), N,Ndiisopropylethylamine (13.4 g, 10.66 mmol) was added and cooled the reaction mass to 0-5 °C. Thereafter, the above prepared acid chloride 10 solution was added at 0-5 °C. After 1 h stirring at 20-25 °C, the reaction mass was washed with DM water (250 mL). DM water (250 mL) was added to the organic layer and adjusted pH to 7.0 with 10% aqueous hydrochloric acid. The organic layer was separated, washed with DM water (250 mL), and concentrated at 35-40 °C under reduced pressure to obtain a residue, which was triturated with hexanes and further purified by using column chromatography to obtain a white solid 11. (