Enantioseparation of Orciprenaline, Betaxolol, and Propranolol using HPLC and New Chiral Reagents Based on 1,5-Difluoro-2,4-dinitrobenzene

Abstract

Diastereomers of three β-blockers (orciprenaline, betaxolol, and propranolol) were synthesized using three new chiral derivatizing reagents that were prepared by substituting one fluorine atom in 1,5-difluoro-2,4-dinitrobenzene with two chirally pure amines and one L-amino acid. The reagents were characterized using ultraviolet-visible spectroscopy, infrared spectroscopy, elemental analysis, and proton nuclear magnetic resonance. Diastereomers were synthesized under microwave irradiation at 50 s at 80% of 800 W and also by stirring for 50 min at 45°C. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C₁₈ column with detection at 340 nm using acetonitrile and aqueous trifluoroacetic acid as the mobile phase components. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection, and limit of quantification.

Keywords:

• 1,5-Difluoro-2,4-dinitrobenzene
• β-blocker
• Enantioseparation
• Liquid chromatography

Acknowledgments

The authors are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for the award of a senior research fellowship to H. Nagar. Thanks are due to the Alexander von Humboldt Foundation, Bonn, Germany, for donating Knauer HPLC equipment to R. Bhushan.

Notes

Chromatographic conditions: LiChrospher C₁₈ (250 × 4.6 mm i.d., 5 µm particle size); mobile phase 2, linear gradient (35 to 65%) of acetonitrile with 0.10% trifluoroacetic acid for diastereomers of propranolol and betaxolol, and linear gradient (30 to 75%) of acetonitrile with 0.10% trifluoroacetic acid for diastereomers of orciprenaline in 45 min; flow rate, 1.0 mL/min; detection, 340 nm. t _R1 is retention time, k ₁ is retention factor of [(R)-L]-diastereomers; α, separation factor; R _s, resolution.

1 and 2 are representing experimental yield of [(R)-L]- and [(S)-L]-diastereomer, respectively in mg and % yield of diastereomer is shown below in parentheses.

Mol. wt. of propranolol, orciprenaline and betaxolol are 259.34, 211.26, and 307.43, respectively. The molecular weights of the diastereomers of three title compounds prepared with chiral derivatizing reagent 1, 2, and 3 respectively are, 558.60, 550.65, and 544.60 for diastereomers of propranolol; 510.52, 502.56, and 496.51 for diastereomers of orciprenaline; and 606.69, 598.73, and 592.68 for diastereomers of betaxolol.

^A and ^Brepresent the R _s values for the diastereomers of (R,S)-propranolol and (R,S)-betaxolol.