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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.

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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.

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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.

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The aims of the study included: to explore the protein structure basis for the differences in digitalis sensitivity between isoforms of Na/K-ATPase from human and guinea-pig cardiac muscle; to determine the relative significance of the constituents of tripartite digitalis compounds in their inhibitory action on these Na/K-ATPase isoforms; to evaluate the potential significance of the receptor kinetics for pharmacological characteristics. The analytical method has been the recording of the inhibitory interaction of various digitalis derivatives with the Na/K-ATPase isoforms. The protein structure basis for the isoform differences in digitalis susceptibility has been explored by analysing in free-energy plots the kinetics of their inhibitory interaction with 53 digitalis derivatives of grossly different structure. The slope of the regression line and the parameters of the regression equation proved to be similar for the two isoforms in spite of the great difference in their digitalis susceptibilities. This surprising uniformity indicates that a uniform "macroscopic" mechanism underlies the inhibitory effect of the various derivatives on the two isoforms. On the other hand, the differences in the positions of delta G*on and delta G*off values for particular inhibitors relative to the regression line reveal differences in the "microscopic" interaction energy surfaces of the two isoforms. In conclusion, the origin of the isoform distinctions in their susceptibility towards inhibition by various digitalis derivatives is essentially confined to differences in the chemotopology of the digitalis recognition matrix and binding cleft. Specific observations allowed to disentangle the impact of various steroid derivatizations at carbon atoms 3, 17, and diverse other positions on the kinetics of their interaction with the enzyme isoforms. The steroid nucleus of the cardiac glycosides, 5 beta, 14 beta-androstane, proves to be the basal structural element for discrimination of Na/K-ATPase isoforms. This discrimination becomes much enlarged by steroid glycosidation at C3 beta-OH and/or by steroid substitution of C17 beta-H by a lactone ring. The higher inhibitory sensitivity of the human isoform is based either on an increased association rate or a decreased dissociation rate, depending on the nature of derivatization.(ABSTRACT TRUNCATED AT 400 WORDS)

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Twenty-five compounds [digitalis (generic name for cardenolides, bufadienolides and their glycosides) representatives and derivatives, various steroids as well as some customary carcinostatics] have been compared in terms of their potency to suppress the proliferation of Ehrlich mouse ascites carcinoma (EMAC) cells and to inhibit the activity of Na/K-ATPase from EMAC cells and from human cardiac muscle. The inhibitor susceptibilities of the Na/K-ATPase isoforms of EMAC and cardiac muscle are very different, in favour of the cardiac muscle with the digitalis-like acting steroids, whereas they are quite similar with the digitalis-unlike acting compounds. Whereas the K0.5 values for the inhibition of EMAC Na/K-ATPase display the expected dependence on steroid structure, the IC50 values for the suppression of EMAC cell proliferation all lie within a narrow concentration range. With ouabain, the IC50 value for the suppression of proliferation of oestrogen receptor-negative, human mammary carcinoma (MCA) cells is four orders of magnitude higher than the K0.5 value for inhibition of the activity of human cardiac muscle Na/K-ATPase. In contrast to this effectivity order, some synthetic derivatives of digitalis steroids develop primarily antiproliferative potency.

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Since 1985, several research groups have shown that a number of amino acids in the catalytic alpha-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 alpha-subunits in the Na+/K(+)-ATPase protodimer (alpha beta)2 provides the cleft for inhibitory digitalis intercalation.

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The effects of 19 nitrate esters of genins and glycosides on the isolated guinea pig atrium and ileum were studied. The positive inotropic effect of glycoside nitrates correlated with the contracting effect on the ileum. Genin nitrates with weak cardiac activity were found to exert smooth muscle relaxing effects. Compared to genin nitrates, glyceryl trinitrate had a weaker relaxing effect on the ileum. On the coronary strip it had a stronger effect than delta 14-anhydrodigitoxigenin-3 beta-mononitrate, the most effective smooth muscle relaxing genin nitrate.

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Penta-acetyl-gitoxin (PAG) shows species-specific deacylation to 16-acetyl-gitoxin (16 AG; I and III) or gitoxin (II and IV) by homogenates of liver and intestinal mucosa of man (I), rabbit (II), guinea-pig (III) and rat (IV), whereas it is degraded into tri- and tetra-acetates by homogenates of guinea-pig myocardium as well as by human blood and serum. The identity of the principal and chloroform-extractable metabolities in human urine after PAG administration with 16-AG has been demonstrated by mass spectrometry.

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Penta-acetyl-gitoxin is a suitable prodrug of gitoxin since it shows--side-effect latentiation, due to its inactive application form;--bioavailability, due to its improved solubility and thus good enteral absorption and--bioactivation, due to rapid de-acetylation in the body after absorption.

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