Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

Na,K-ATPase α4 is a testis-specific
plasma membrane Na+ and K+ transporter expressed
in sperm flagellum.
Deletion of Na,K-ATPase α4 in male mice results in complete
infertility, making it an attractive target for male contraception.
Na,K-ATPase α4 is characterized by a high affinity for the cardiac
glycoside ouabain. With the goal of discovering selective inhibitors
of the Na,K-ATPase α4 and of sperm function, ouabain derivatives
were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin
analogue 25, carrying a benzyltriazole moiety at C17,
is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding
α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm
membrane potential, intracellular Ca2+, pH, and hypermotility.
These results proved that the new ouabagenin triazole analogue is
an effective and selective inhibitor of Na,K-ATPase α4 and sperm
function.