36 August / September 2016

Resurgence in Fluorinated Chiral Polysaccharide Phases for

Supercritical Fluid Chromatography Christine M. Aurigemma1 , William P. Farrell1 , Jeff R. Elleraas1 and Matthew Przybyciel2

1. Pfizer Worldwide Research and Development, Oncology Medicinal Chemistry, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121 USA 2. ES Industries, Inc, West Berlin, NJ 08091 USA

The incorporation of fluorine within biologically active molecules in pharmaceutical drug discovery has resulted in chiral recognition challenges using existing chiral stationary phases (CSPs). Building upon decades-old research, several new phases were created to explore fluorophilic retention mechanisms towards enantioseparation of fluorinated compounds. While the concept of aromatic halogenated substituents within polysaccharide CSPs is not new, the availability of these types of phases with fluorine substituents is limited. The initial set of fluorinated phases, incorporating either 4-fluoro-3-methyl phenylcarbamate or 2-fluoro-5-methyl phenylcarbamate, were prepared on cellulose and evaluated by SFC using drug- like compounds with fluorinated functional groups. During the evaluation, it was discovered that these prototype CSPs also separated other halogenated compounds, but with less overall retention and solvent consumption. Based on these results, modifications were made, and two new iterations of fluorinated phases were created, and 3 of the 4 prototypes were later commercialised. The separation of racemic compounds achieved using these fluorinated phases using SFC will be presented, as well as comparative separations using other halogenated stationary phases.


Chiral SFC is the primary tool for the separation of enantiomers in support of drug discovery efforts. SFC uses a simple mobile phase system consisting of non- toxic CO2

and an organic modifier, typically an alcohol such as methanol [1]. The lower toxicity of solvents and reduced overall waste generation of SFC make this a greener alternative to normal phase HPLC, and is thus ideal for preparative scale applications including chiral separations [2-6]. Not only is SFC considered a green technique, but high diffusivity and low viscosity of supercritical CO2

enable higher flow rates

and shorter run times, which leads to faster and more efficient separations relative to HPLC [7]. The most frequently used chiral selectors for SFC are the polysaccharides phases, which are substituted phenyl carbamates and benzoates bonded to either amylose or cellulose. These coated cellulose and amylose phases do have some solvent restrictions, and are often limited to the use of alcohol modifiers, while some of the newer immobilised phases such as the Chiralcel IA, IB and IC (Chiral Technologies, Inc, West Chester, PA USA) have expanded solvent compatibility [8]. Other examples of CSPs include the Pirkle-type, such as the Regis Whelk-O1 (Regis Technologies, Inc., Chicago, IL USA) and macrocyclic glycoprotein phases exemplified by Astec® ChirobioticTM

V, T,

and R-series (Sigma-Aldrich, St. Louis, MO, USA). While the Pirkle-type and peptide macrocyclic glycoprotein phases have a broader solvent compatibility range than coated polysaccharide derivatives and are usually applied in reverse phase (aqueous/ organic) or in polar organic mode (e.g. 20mM ammonium acetate in methanol), the diversity of the polysaccharide-based phases and their variable chiral recognition abilities make them more versatile across a broad range of compounds. This is especially true for the substituted phenyl carbamates and benzoates of these polysaccharides, whose chiral recognition abilities have been recognised in a number of publications over the years [9-14].

The recognition of the importance of 3D geometry of lead molecules is leading to the synthesis of novel molecular entities [15] which are creating enantioseparation challenges for which an increasing number of chiral compounds are not separating on any traditional polysaccharide CSPs. In addition, compounds with low molecular weight (e.g. MW < 200 Da), and those with limited complexity/functionality near the chiral centre contribute to diminished chiral recognition ability with these phases. While gas chromatography is probably best suited for separating these compounds, it is inconsistent with the goals of isolating pure enantiomers at a satisfactory scale for medicinal chemistry support. In Figure

Figure 1. Traditional polysaccharide CSP usage over the last 10 years compiled from purification methods of internal compounds.

1, a review of over 3000 internal chiral compounds over several years and the methods used to resolve them indicates an exponential shift from traditional phases towards non-traditional CSPs. Interestingly, this data also coincides with the continued trend in the increased incorporation of halogens (in particular, fluorine) in medicinal chemical designs.

While the 3,5-dimethylphenylcarbamates of cellulose and amylose (Chiralcel OD and Chiralpak AD, respectively) stationary phases (Chiral Technologies, Inc) remain an integral component of our chiral SFC screening protocol, reduced success with the other amylose and cellulose-based CSPs has led to an expansion of our screening set to incorporate newer phases to potentially provide alternative mechanisms for chiral

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