Identification of Differential Protein Binding Affinities in an Atropisomeric Pharmaceutical Compound by Noncovalent Mass Spectrometry, Equilibrium Dialysis, and Nuclear Magnetic Resonance

Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs ( Angew. Chem., Int. Ed. 2009, 48, 6398−6401 ). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer ( Tetrahedron 2004, 60, 4337−4347 ) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers ( Chirality 1996, 8, 364−371 ). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining K D values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and K D values.