Supercritical fluid chromatography approach for a sustainable manufacture of new stereoisomeric anticancer agent.

Two routes aimed at the manufacture of unprecedented stereoisomeric combretastatin A-4 analogue were described: flash chromatography vs supercritical fluid chromatography. The latter has many advantages over liquid chromatography and was therefore chosen for the small scale separation of methyl 1-[(3-hydroxy-4-methoxyphenyl) (3,4,5-trimethoxyphenyl)methyl]-5-oxo-l-prolinate 5, with potential antitumoral activity. After a screening of six different polysaccharide based chiral stationary phases and four co-solvents, the percentage of co-solvent, the flow-rate and the outlet pressure were optimized through a design of experiments (DoE). The preparation of 50mg of each stereoisomer was achieved successfully on a Chiralpak AD-H with isopropanol as a co-solvent. Productivity (kkd), solvent usage and environmental factor (E Factor) were calculated. Flash chromatography and supercritical fluid chromatography approaches were compared in terms of yield and purity of each stereoisomer manufactured.

Small scale separation of isoxazole structurally related analogues by chiral supercritical fluid chromatography.

Chromatographic preparative enantioseparation is now the preferred method to obtain milligram amounts of pure enantiomers in the first step of the development of a therapeutic molecule. Supercritical fluid chromatography has many advantages over liquid chromatography and was therefore chosen for the small scale enantioseparation of four original 3-carboxamido-5-aryl isoxazole molecules, ligands of the CB2 cannabinoid receptors. The preparation of about 10mg of each of the eight enantiomers was achieved successfully on a Chiralpak® AD-H (tris-3,5-dimethylphenylcarbamate of amylose) polysaccharide based stationary phase with various percentages of ethanol as a co-solvent, through mixed-stream injections and touching-band approach. For the all compounds, no peak distortion is observed during the volume overloading, in spite of the injection mode. Production rate (mgmin-1), productivity (kilogram of racemate separated per kilogram of CSP per day (kkd)) and solvent usage were found higher and environmental factors (E Factor) were found lower for compounds 1 and 3. The yields of each purified enantiomer were comprised between 60 and 94%. In order to improve the limit of detection calculated with the diode array detector, the hyphenation with an evaporating light scattering detector was explored and a factor of ten was won. Lastely, the enantiomeric excess and achiral purity of each of the eight individual enantiomer generated was determined and found higher than 98%.

Exploring chiral separation of 3-carboxamido-5-aryl isoxazole derivatives by supercritical fluid chromatography on amylose and cellulose tris dimethyl- and chloromethyl phenylcarbamate polysaccharide based stationary phases.

Four polysaccharide based chiral stationary phases were chosen, two chlorinated: Lux™ Amylose-2 (tris-5-chloro-2-methylphenylcarbamate of amylose) and Lux™ Cellulose-2 (tris-3-chloro-4-methylphenylcarbamate of cellulose) and two methylated: Chiralpak® AD-H (tris-3,5-dimethylphenylcarbamate of amylose) and Chiralcel® OD-H (tris-3,5-dimethylphenylcarbamate of cellulose) to separate four 3-carboxamido-5-aryl isoxazole derivatives by supercritical fluid chromatography. The effect of chiral stationary phase, co-solvent nature (MeOH, EtOH, 2-PrOH and ACN) and percentage (10-20%), temperature (20-45°C) and chemical structure of the compounds on retention, resolution and elution order were thoroughly studied. In addition, thermodynamic parameters were determined from the linear portion of the Van't Hoff plots. For all the derivatives, the Lux™ Cellulose-2 and Chiralpak® AD-H provided excellent resolutions (Rs=9.78) in short run time (under 6min). The preparation of about 10mg of each of the eight enantiomers was achieved successfully on a Chiralpak® AD-H with various percentages of ethanol as a co-solvent. Lastly, the enantiomeric purity of each of the eight individual enantiomer generated was determined and found higher than 98%.