The effect of 16β-substitution on the structure and activity of digitoxigenin: Is there an additional binding interaction with Na⁺, K⁺-ATPase?

We have studied the basis of the effect of 16β-substitution on the structure and activity of digitoxigenin derivatives by examining the crystal structures of these compounds and their inhibitory activity towards the receptor for these drugs, Na⁺, K⁺-ATPase. To understand the increase in inhibitory activity of the 16β-ester compounds and the decrease in activity of gitoxigenin (16β-hydroxydigitoxigenin), both with respect to digitoxigenin, we have compared the observed conformations of gitoxigenin, gitoxigenin 16β-formate, and other 16β-esters to that of digitoxigenin. Our data do not support the possibility of hydrogen bonding between the 16β-hydroxyl of gitoxigenin and the lactone ring, previously suggested to account for the decreased activity of gitoxigenin vis a vis digitoxigenin, but, rather, suggest that the decreased activity may be due to an intramolecular hydrogen bond between the hydroxyls on C-14 and C-16 and an unusual D-ring conformation which combine to alter the carbonyl oxygen of the lactone ring away from the putative active position. In contrast, the 16β-ester moiety has a preferred conformation which may serve to fix the lactone ring in the active conformation. Thus, the increased activity of the 16β-esters cannot be explained by altered carbonyl oxygen position and may be related to an additional receptor binding site for the ester moiety.