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.

Na,K-ATPase α4 为一种睾丸特异性血浆膜钠钾转运蛋白，主要表达于精子鞭毛中。雄性小鼠Na,K-ATPase α4基因的敲除导致完全不育，使其成为男性避孕的潜在靶点。Na,K-ATPase α4以其对心脏糖苷类药物ouabain的高亲和力而著称。旨在发现对Na,K-ATPase α4及精子功能具有选择性的抑制剂，对ouabain衍生物在糖基（C3）和内酯（C17）区域进行了修饰。携带C17位苯基三唑基团的Ouabagenin类似物25，是Na,K-ATPase α4的皮摩尔级抑制剂，展现出卓越的α4同种型选择性。此外，化合物25在体外和体内均降低了精子活力，并影响了精子膜电位、细胞内钙离子浓度、pH值以及过度运动。这些结果表明，新型的ouabagenin三唑类似物是Na,K-ATPase α4及精子功能的有效且选择性抑制剂。