Rudolf Megges

LOCATION AND PROPERTIES OF THE DIGITALIS RECEPTOR SITE IN NA+/K+-ATPASE

Since 1985, several research groups have shown that a number of amino acids in the catalytic α‐subunit of Na+/K+‐ATPase more or less strongly modulate the affinity of a digitalis compound like ouabain to the enzyme. However, scrutiny of these findings by means of chimeric Na+/K+‐ATPase constructs and monoclonal antibodies has recently revealed that the modulatory effect of most of these amino acids does not at all result from direct interaction with ouabain, but rather originates from long‐range effects on the properties of the digitalis binding matrix. Starting from this knowledge, the present review brings together the various pieces of evidence pointing to the conclusion that the interface between two interacting α‐subunits in the Na+/K+‐ATPase protodimer (αβ)2 provides the cleft for inhibitory digitalis intercalation.

The nitration of canrenone with acetic anhydride/nitric acid

3-Oxo-17 alpha-pregna-4,6-diene-21,17-carbolactone (canrenone, II) is produced from the potassium salt of 17-hydroxy-3-oxo-17 alpha-pregna-4,6-diene-21-carboxylic acid (I) by acid catalyzed lactonization. II reacts with acetic anhydride/nitric acid to give one main product (III) and some minor products. The structure of III was determined by chemical and spectral analysis to the 4-nitro derivative of canrenone. This result is in contrast to the known reactions of II with most other reagents that were found to add at delta(6), and also in contrast to the reactions of acetic anhydride/nitric acid with alkenes. Electrophilic substitution at the ambident C4 is discussed as the reaction path. The 4-nitro group enhances the inhibitory activity of II against Na+/K(+)-ATPase, the target enzyme of the cardioactive digitalis glycosides, which appears to indicate increased cardioactivity.

Modeling of the three-dimensional structure of the digitalis intercalating matrix in Na+/K(+)-ATPase protodimer.

Based on the knowledge that the digitalis receptor site in Na+/K(+)-ATPase is the interface between two interacting alpha-subunits of the protodimer (alpha beta)2, the present review makes an approach towards modeling the three-dimensional structure of the digitalis intercalating matrix by exploiting the information on: the primary structure and predicted membrane topology of the catalytic alpha-subunit; the determinants of the secondary, tertiary and quaternary structure of the membrane-spanning protein domains; the impact of mutational amino acid substitutions on the affinity of digitalis compounds, and the structural characteristics in potent representatives. The designed model proves its validity by allowing quantitative interpretations of the contributions of distinct amino acid side chains to the special bondings of the three structural elements of digitalis compounds.

Synthesis of acetates of gomphogenin and gomphoside and evaluation of structure-activity relationships

Summary Acetates of gomphogenin and gomphoside have been synthesized and their structures established by NMR measurements, optical rotation, mass and infrared spectrometry. The kinetic and equilibrium parameters of the inhibitory interaction of the compounds with guinea-pig heart muscle Na + /K + -ATPase are presented and discussed. Acetylation of the 2α-OH group in gomphogenin or its 3β-acetate increases the binding affinity by 15- and 24-fold, respectively, whereas 3β- O -acetylation of gomphogenin and its 2α-acetate increases the affinity only two- and threefold. Acetylation of 4′-OH or 3′,4′-OH of gomphoside, instead, reduces the high affinity of gomphoside towards Na + /K + -ATPase.

Structure-activity relationship at the glycosidic moiety of digitalis compounds as found in tests with NA/K-ATPase isoforms from cardiac muscle of guinea-pig and man.

AbstractThe glycosidic moiety plays an important role in the pharmacokinetic and pharmacodynamic behaviour of cardiac glycosides like digitoxin and digoxin. Their tridigitoxoside side chain becomes slowly removed in the animal body and hence delays the inactivation of the drugs by epimerization and conjugation of the C3β-hydroxy group to which the glycosidic side chain is attached.1,2 In addition, the glycosidic component affects the kinetics of the glycoside interaction with the receptor enzyme, i.e., the Na/K-ATPase, by influencing both the lag time for the onset of action and the half-life time for the length of action. 3The determination of the association and dissociation rate constants has shown that these parameters of receptor kinetics are modulated by the structure of the monoside bound proximately to the steroid moiety.4 Although repeatedly analysed, the attempts of modelling the structure-activity relationships (SAR) have not reached general significance and predictive capacity5 as required for...

Chromatographische differenzierung von cardenoliden durch anwendung von boraten und anderen diolreagenzien

Abstract The paper probes 13 diol reagents known to interact with the cis -1,2,- and/or -1,3-diol groups of carbohydrates and other compounds, as to their influence on the relative mobilities of 16-acetylgitoxin and acetylgitoxin-α as representative for cis -1,2- and cis -1,3-diols in the cardenolide series. The influence on mobility has been studied using paper partition chromatography with formamide as stationary phase. Metaboric aced, boric acid, arsenic pentoxide, phenyl arsonic acid, ammonium metavanadate, sodium molybdate, and sodium tungstate are without influence on the relative mobilities of both diols. Sodium metaborate, disodium tetraborate, and potassium borooxalate decrease and arsenic trioxide increases specially the mobility of the 1,2-diol. Phenylboronic acid and diphenylborinic acid increase the relative mobility of both diols. In the case of phenylboronic acid a differentiation between both diols is possible: at low concentration the relative mobility of the 1,3-diol only and at high concentration that of the 1,2-diol, too, increases. The effect of the diol reagents is completely lost by acetylation of one OH group of the reacting diol group. Apparently, the reagents effects on chromatographic mobility are due to the formation of cyclic complexes or cyclic esters with the cis -1,2- or cis -1,3-diol group.

Über den einfluss von phenyl- und diphenylborsäure auf die mobilität von cardenoliden un dbufadienoliden bei der papierchromatographie

Abstract On the influence of phenylboronic and diphenylborinic acid on the paper chromatographic mobilities of cardenolides and bufadienolides Phenylboronic acid and diphenylborinic acid were studied as to their influence on the paper chromatographic mobilities of cardenolides and bufadienolides with and without a cis -1,2- or cis -1,3-diol group. At low concentrations, phenylboronic acid and at higher concentrations, diphenylborinic acid increases the mobility of nearly all investigated cis -1,3-diols with a tertiary OH-group. At higher concentrations both acids enhance the mobilities of most of the cis -1,3- and cis -1,2-diols without a tertiary OH-group, too. It is concluded that there are two basic prerequisites for the derivatization of cis -1,2- or cis -1,3-diols: (1) the capabilities of the diol to reach an O-O distance like that in phenylboronic acid esters or in diphenylborinic acid complexes; (2) the absence of a considerable steric hindrance by a substituent near the reactive diol group. Among the four studied rhamnosides being 1,2-diols two do not react from hitherto unknown reasons.

Partial synthetic derivatization of canrenone and characterization of its impact on the inhibitory effect on Na+/K+-ATPase activity in human heart muscle

To improve the weak inhibitory effect of 3-oxo-17 alpha-pregna-4,6-diene-21,17-carbolactone (canrenone, II) on Na+/K(+)-ATPase activity in human heart muscle, we have investigated the impact of hydrogenation, reduction, glycosidation, and the introduction of a 3-sulfonamido residue on the inhibitory potency of canrenone. The greatest increase in potency (> 20 times) was found for 3 beta-(alpha-L-rhamnopyranosyloxy)-5 beta, 17 alpha-pregnane-21, 17-carbolactone (IX). The 3-O-glycosides IX-XI are the first representatives of C/D-trans steroids with effector-receptor complex decay half-times longer than those of therapeutically used cardenolides.