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Type 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) transforms 4-androstene-3,17-dione (Delta(4)-dione) into the androgen testosterone. To produce potent inhibitors of this key steroidogenic enzyme, we performed parallel liquid-phase synthesis of 3beta-substituted androsterone (ADT) libraries (A-D) in good yields and average high-performance liquid chromatography (HPLC) purities of 92-94%. The first library (A) of 3 beta-amidomethyl-ADT derivatives (168 members), including two levels of molecular diversity on the amide (R(1) and R(2)), was synthesized with a parallel liquid-phase method (method I) in less time than with the classic chemistry method. The screening of library A revealed that relatively small hydrophobic chains at R(1) (5-8 carbons) and small hydrophobic substituents at R(2) (1-4 carbons) provided the most potent inhibitors. In accordance with these inhibition results, a second library (B) of 3 beta-amidomethyl-ADT derivatives (56 members) was generated in a very short time using an improved method based on scavenger resins and liquid-phase parallel chemistry. Library B produced more potent inhibitors than library A and provided useful structure-activity relationships that directed the design of a third library (C) of 49 members. Once again, very potent inhibitors were identified from library C and 3 beta-[(N-adamantylmethyl-N-butanoyl)aminomethyl]-3 alpha-hydroxy-5 alpha-androstan-17-one (C-7-3) was identified as the most potent inhibitor of the three libraries with an inhibitory activity (IC(50) = 35 nM) 18-fold higher than that of the natural substrate of the enzyme, Delta(4)-dione, (IC(50) = 650 nM) used itself as inhibitor. Finally, we designed a library (D) of 3-carbamate-ADT derivatives (25 members) using the efficient parallel liquid-phase method III, which allowed the synthesis of more rigid molecules with two levels of molecular diversity (R(1)/R(2) and R(3)) in the local area occupied by the adamantane group of C-7-3. Interestingly, one of the most potent inhibitors of library D, the 3R-spiro-[3'-[3' '-N-morpholino-2' '-(3' "-cyclopentyl-propionyloxy)propyl]-2'-oxo-oxazolidin-5'-yl]-5 alpha-androstan-17-one (D-5-4), showed an inhibitory activity on type 3 17 beta-HSD similar to that of compound C-7-3, while exhibiting a nonandrogenic profile.

The synthesis and binding affinities to the digitalis Na(+),K(+)-ATPase receptor of a series of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane derivatives bearing a 17 alpha-(aminoalkoxy)imino chain are reported; some derivatives were also studied for their inotropic activity. Our recently proposed model of interaction of molecules with the digitalis receptor was used to design these compounds. On that basis, the possibility to design novel potent inhibitors of Na(+),K(+)-ATPase without being constrained by the stereochemistry of the classical digitalis skeleton in the D-ring region was predicted. The binding affinities of the most potent compounds in the two series, (EZ)-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-5 beta-androstane-3 beta,14 beta-diol (6f) and (EZ)-3 beta-hydroxy-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-14,15-seco-5 beta-androstan-14-one (24c) are higher than that of the potent natural compound digitoxigenin, despite the unusual alpha-exit of the substituent in position 17 of 6f or the disruption of the D-ring in 24c. These results further support the validity of our recently proposed model of binding at the digitalis receptor. Results of the inotropic tests on guinea pig atrium deserve further investigation on the pharmacological profile of these derivatives.

A series of digitalis-like compounds with a 17-aminoalkoxyiminoalkyl or -alkenyl substituent was synthesized and evaluated for inhibition of Na(+),K(+)-ATPase and for inotropic activity. The highest inhibition was found with compounds having the substituent in configuration 17beta and the amino group at a distance of 6 or 7 bonds from C(17) of the digitoxigenin skeleton. The presence of the oxime function strengthens the interaction with the receptor, more if alpha,beta-unsaturated, thus mimicking the electronic situation of the unsaturated lactone in natural digitalis compounds. The most active compounds showed Na(+),K(+)-ATPase inhibitory potencies (IC(50)) 17-25 times higher than the standards digitoxigenin and digoxin and 3-11 times higher inotropic potencies (EC(50)) in isolated guinea pig left atria. These features are supported by a molecular model suggesting the possible interactions of the groups described above with particular amino acid residues in the H1-H2 domains of Na(+),K(+)-ATPase. Some interactions are the classical ones already described in the literature; a new, very strong interaction of the basic group with the Cys138 was found and adds new possibilities to design compounds interacting with this region of the receptor. The most interesting compounds were also studied in vivo in the anesthetized guinea pig for evaluating their inotropic effect versus the lethal dose. Compounds 9 and 12 showed a slightly higher safety ratio than digoxin and deserve further evaluation.