Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: I. Pyrazine-ring modifications.

The overall on- and off-rate constants for blocking epithelial Na channels by amiloride analogs were estimated by noise analysis of frog skin epithelium. The substituents at position-5 and -6 of the pyrazine ring of amiloride were varied in order to obtain the structure/rate constant relationship. (1) The off-rate constant increases with halo-substitutions at position-6 in the order Cl less than Br less than l less than F less than H. Substitution of Cl by H lowers the standard free energy of activation of the off-step by 2.3 kcal mol-1. The on-rate constant is not affected. Apparently the substituent at ring position-6 controls the duration of attachment in the blocking position. pKa considerations show that the duration is longer when the 6-substituent is more negatively polarized. We suggest that this substituent binds to the receptor by virtue of its electronegativity. (2) In contrast, replacement of the adjacent 5-amino group (electron donor) by H or Cl affects both the on-rate and the off-rate. The dual effect may be explained by a decrease of the electronic charge at more remote parts of the molecule (on-rate decrease), as well as at the 6-position (off-rate increase). Apparently the 5-amino group stabilizes the blocking position by increasing the electron density on the 6-ligand.