Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol

Background: rac-Fenoterol is a β₂-adrenoceptor agonist (β₂-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R′- and S,S′-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the β₂-AR. Methods: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. β₂-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human β₂-AR and standard membrane binding studies using the same membranes. The effect of R-F and SF on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. Results: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound β₂-AR as the stationary phase and rac-³H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-³H-fenoterol retention, while similar addition of S-F produced no change in rac-³H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 ± 55 nM (R-F) and 109,000 ± 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 ± 11.6)% to (306 ± 11.8)% of resting cell length (P < 0.05) and reduced EC₅₀ from -7.0 ± 0.270 to -7.1 ± 0.2 log[M] (P < 0.05), while S-F had no significant effect. Discussion: Previous studies have shown that rac-fenoterol acts as an apparent β₂-AR/G_s selective agonist and fully restores diminished β₂-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for β₂-AR/G_s activation and if it can be used in the treatment of congestive heart failure.