Preparative Scale Resolution of Enantiomers Enables Accelerated Drug Discovery and Development

Abstract

The provision of pure enantiomers is of increasing importance not only for the pharmaceutical industry but also for agro-chemistry and biotechnology. In drug discovery and development, the enantiomers of a chiral drug depict unique chemical and pharmacological behaviors in a chiral environment, such as the human body, in which the stereochemistry of the chiral drugs determines their pharmacokinetic, pharmacodynamic and toxicological properties. We present a number of challenging case studies of up-to-kilogram separations of racemic or enriched isomer mixtures using preparative liquid chromatography and super critical fluid chromatography to generate individual enantiomers that have enabled the development of new candidate drugs within AstraZeneca. The combination of chromatography and racemization as well as strategies on when to apply preparative chiral chromatography of enantiomers in a multi-step synthesis of a drug compound can further facilitate accelerated drug discovery and the early clinical evaluation of the drug candidates.

Keywords: active pharmaceutical ingredient; chiral resolution; chiral stationary phases; drug development; drug discovery; preparative chromatography; racemization; supercritical fluidchromatography.

Figure 1

Chiral separation of a basic compound A. (a) Analytical SFC chromatogram using a Chiralpak IA 150 mm × 4.6 mm, 3 µm column. Mobile phase was 15% methanol/dichloromethane/diethyl amine 50/50/0.5 (v/v/v %) in CO₂ at 40 °C, 120 bar and a flow rate of 4 mL/min; (b) Analytical NPLC chromatogram using a Chiralpak IA 250 mm × 4.6 mm, 20 µm column. Mobile phase was heptane/ethyl acetate/diethyl amine 50/50/0.5 (v/v/v %) at a flow rate of 1 mL/min; (c) Preparative chromatogram on Chiralpak IA 250 mm × 110 mm, 20 µm. 9.45 g (270 mg/mL dichloromethane) was injected every 13 min using heptane/ethyl acetate/diethyl amine 50/50/0.5 (v/v/v %) at a flow rate of 600 mL/min. The blue area corresponds to the product fraction, 1.8 L/injection each cycle.

Figure 2

Chiral separation of compound B, a racemic carboxylic acid. (a) Analytical SFC chromatograms using 150 mm × 4.6 mm, 3 µm columns. Mobile phases were 20% methanol/formic acid 100/0.5 (v/v %) on Lux Cellulose 4 and 30% ethanol/formic acid 100/0.5 (v/v %) in CO₂ at 40 °C, 120 bar and a flow rate of 4 mL/min; (b) Loadability study on Chiralpak IC. Racemate (600 mg, 200 mg/mL ethanol) was injected on a 250 mm × 30 mm, 5 µm column using 20% ethanol/formic acid 100/0.5 (v/v %) in CO₂ at 40 °C, 120 bar and a flow rate of 150 g/min. The blue area corresponds to the product fraction, on this column the first eluting enantiomer; (c) Loadability study on Lux Cellulose 4. Racemate (600 mg, 200 mg/mL ethanol) was injected on a 250 mm × 30 mm, 5 µm column using 15% methanol/formic acid 100/0.5 (v/v %) in CO₂ at 40 °C, 120 bar and a flow rate of 150 g/min. The blue area corresponds to the product fraction, on this column the second eluting enantiomer; (d) Preparative chromatogram on Chiralpak IC 250 mm × 50 mm, 5 µm. 3.4 g (200 mg/mL ethanol) was injected every 155 s using 20% ethanol/formic acid 100/0.5 (v/v %) in CO₂ at 40 °C, 120 bar and a flow rate of 450 g/min. The blue area corresponds to the product fraction, 70 mL/injection each injection.

Figure 3

Chiral separation of compound C, a racemic amide. (a) Analytical SFC chromatogram using a Chiralart SA 150 mm × 4.6 mm, 3 µm column. Mobile phase was 40% 2-propanol (v/v %) in CO₂ at 40 °C, 120 bar and a flow rate of 4 mL/min; (b) Preparative chromatogram on Chiralart SA 250 mm × 20 mm, 5 µm. Then 0.5 g (100 mg/mL acetonitrile) was injected every 190 s using 40% 2-propanol (v/v %) in CO₂ at 40 °C, 130 bar and a flow rate of 80 g/min. The blue area corresponds to the product fraction and the dashed blue area corresponds to the unwanted enantiomer; (c) The feed solution (100 mg/mL acetonitrile) was injected and after chiral chromatography the enantiomers were collected and evaporated. The unwanted enantiomer was re-dissolved in methanol and trimethylamine (30 mg/mL and 3:1 molar equivalents to compound) and left overnight on rotation. The next day, full racemization had occurred and the racemate was re-used as feed solution.