Jürgen Weiland

The lead structure in cardiac glycosides is 5 β,14 β-androstane-3 β,14-diol

SummaryThe purpose of the present study was to determine the lead structure in cardiac glycosides at the receptor level, i.e. the minimal structural requirement for specific and powerful receptor recognition. Accordingly 73 digitalis-like acting steroids were characterized as to the concentration effecting half-maximum inhibition of Na,K-ATPase from human cardiac muscle under standardized turnover conditions. Since the Ki value equaled the apparent KD value, K′D was expressed in terms of the apparent standard Gibbs energy change ΔGo′ of steroid interaction with Na,K-ATPase. This allowed the use of the extrathermodynamic approach as a rational way of correlating in a quantitative manner, the potency and structure of the various steroidal compounds.The results of the present analysis taken in conjunction with relevant findings reported in the literature, favour the following conclusions.1.Cassaine, canrenone, prednisolone- and progesterone-3,20-bisguanylhydrazone, and chlormadinol acetate are compounds that are not congeneric with digitalis.2.The butenolide ring of cardenolides or the analogous side-chains at C17β of 5β,14β-androstane-3β,14-diol are not pharmacophoric substructures, but merely amplifiers of the interaction energy of the steroid lead.3.All modifications of the structure, geometry and spatial relationship between the steroid nucleus and butenolide side chain of digitoxigenin all at once weaken the close fit interaction with the steroid and butenolide binding subsites of the enzyme in such way that the cardenolide derivatives interact with the receptor binding site area in whatever orientation that will minimize the Gibbs energy of the steroid-receptor-solvent system.4.The “butenolide carbonyl oxygen distance model” (Ahmed et al. 1983) for the interpretation of the differences in potency of the cardenolide derivatives describes the change in interaction energy through structural modification as a function of the entire molecule.5.5β,14β-androstane-3β,14-diol, the steroid nucleus of cardiac glycosides of the digitalis type, is the minimum structure for specific receptor recognition and the key structure for inducing protein conformational change and thus Na,K-ATPase inhibition. It is also the structural requirement for maximum contributions of the butenolide substituent at C17β and the sugar substituent at C3β-OH to the overall interaction energy, i.e. this steroid nucleus is the lead structure.6.The tridigitoxose side-chain at C3β-OH of digitalis glycosides can be more than isoenergetically replaced by glucose, 2′,3′-O-isopropylidene-rhamnose, digitoxose, rhamnose and 4′-deoxy-4′-amino-rhamnose (increasing order of interaction energy increments) indicating a remarkable degree of conformational adaptability of the sugar binding subsite.

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.

Glycosidation of Chlormadinol Acetate Alters its Actions on Na+/K+-Transporting ATPase and Cardiac Contractility: A Contribution to the Endogenous Digitalis Problem

Compared to the progesterone derivative chlormadinol acetate 1, the arabinofuranoside 2, rhamnoside 3 and glucoside 4 of 1 are less potent in the Na/K-ATPase assay, but evoke, contrary to 1, positive inotropy in vivo. In anaesthetized cats the circulation effects of 2 and 3 appear to be more favourable than those of the digitalis glycoside digitoxin. Hence, the progestin 1 is transformed through glycosidation into an interesting cardioactive steroid.

Chemical Models for the Chemical Nature of Endogenous Digitalis

The inability or the capacity to promote the phosphorylation of Na+/K(+)-transporting ATPase (Na/K-ATPase) from [32P]Pi is shown to differentiate between mechanistically digitalis-unlike and digitalis-like inhibitors of this enzyme known to be the receptor for all digitalis actions. A negative or positive response in the phosphorylation promotion assay introduced here appears thus to be suitable to diagnose the chemical species in the isolates of animal origin related to the putative endogenous digitalis. Various digitalis-congeneric C/D-cis steroids, progesterone-congeneric C/D-trans steroids and the Erythrophleum alkaloid cassaine promote the enzyme phosphorylation and show a similar pattern of discrimination between three Na/K-ATPase variants. Thus, their cyclopentanoperhydrophenanthrene or perhydrophenanthrene nuclei appear to serve as the minimal pharmacophoric lead structures for bimolecular recognition and to represent chemical models for the chemical nature of endogenous digitalis. Specifically, the hormonal C/D-trans steroids could provide the basic skeleton in endogenous digitalis biosynthesis.

Interaction of progesterone derivatives with the digitalis target enzyme: Impact of glycosidation on inhibitory potency

As a function of the structural modification of the steroid nucleus, the inhibitory interaction of 11 progesterone derivatives with human Na/K-ATPase (Na+/K(+)-transporting ATPase, EC 3.6.1.37), through C3-O-rhamnosylation, is either much decreased or weakly up to strongly increased, so that the rhamnosyl residue contributes to the complementary Gibbs energy of interaction, at the most, the same Gibbs energy increment as realized in ouabain. After C3 beta-O-rhamnosylation, the activity of some progesterone derivatives considerably surpasses that of 3 beta-O-rhamnosyl-chlormadinolacetate, which has been known to elicit positive inotropy in cats. The progesterone derivatives (aglycons and glycosides), that have been analysed more closely, produce their effects by the same molecular mechanism of interaction with Na/K-ATPase as characteristic for digitalis aglycons and glycosides. The results promise to pave the way for the identification of the chemical nature of endogenous digitalis and for the design of novel inotropic drugs.

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...

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.