Structure-activity relations of amiloride derivatives, acting as antagonists of cation binding on Na⁺/K⁺-ATPase

In a search for an organic analogue of K⁺ or Na⁺ ions that binds to the cation binding sites of Na⁺/K⁺-ATPase with high affinity, the potency of the diuretic amiloride and its derivatives in blocking Rb⁺ occlusion has been tested. Although amiloride itself has a low affinity (> 200 μM), insertion of short alkyl chains in position 5 of the pyrazine ring of the molecule dramatically increased the affinity of the compound. For example, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) competes with a K_i ≈ 10 μM. In derivatives lacking a halogen in position 6 of the ring, a 6-fold decrease in affinity was found. Substitutions in the guanidinium moiety did not produce high affinity inhibitors of Rb⁺ occlusion. Several derivatives at positions 5 and 6 of the pyrazine ring were found to be strictly competitive inhibitors with respect to Rb⁺ ions. The highest affinity was observed around pH 8.0-8.2, and low temperature. EIPA and 5-(N-methyl-N-isobutyl)amiloride (MIBA) stabilized the E₁ form of FITC¹-labelled Na⁺/K⁺- ATPase, behaving as Na⁺ analogues. The present findings are similar to our previous results, showing that alkyl- and arylguanidinium derivatives are competitive Na⁺-like antagonists in cation sites. Conclusions concerning the structural features of amiloride derivatives which are necessary to produce the highest binding affinity, are being exploited in synthesis of competitive cation analogues. Derivatives with sufficiently high affinity (0.1-1 μM) will be converted to affinity and photoaffinity reagents.