*Article* **Steroid Glycosides Hyrcanoside and Deglucohyrcanoside: On Isolation, Structural Identification, and Anticancer Activity**

**Silvie Rimpelová 1,†, Tomáš Zimmermann 2,†, Pavel B. Drašar 2, Bohumil Dolenský 3, Jiˇrí Bejˇcek 1, Eva Kmoníckov ˇ á 4,5, Petra Cihlárov ˇ á 2, So ˇna Gurská 6, Lucie Kuklíková 1, Marián Hajd ˚uch 6, Tomáš Ruml 1, Lubomír Opletal 7, Petr Džubák 6,\* and Michal Jurášek 2,\***


**Abstract:** Cardiac glycosides (CGs) represent a group of sundry compounds of natural origin. Most CGs are potent inhibitors of Na+/K+-ATPase, and some are routinely utilized in the treatment of various cardiac conditions. Biological activities of other lesser known CGs have not been fully explored yet. Interestingly, the anticancer potential of some CGs was revealed and thereby, some of these compounds are now being evaluated for drug repositioning. However, high systemic toxicity and low cancer cell selectivity of the clinically used CGs have severely limited their utilization in cancer treatment so far. Therefore, in this study, we have focused on two poorly described CGs: hyrcanoside and deglucohyrcanoside. We elaborated on their isolation, structural identification, and cytotoxicity evaluation in a panel of cancerous and noncancerous cell lines, and on their potential to induce cell cycle arrest in the G2/M phase. The activity of hyrcanoside and deglucohyrcanoside was compared to three other CGs: ouabain, digitoxin, and cymarin. Furthermore, by in silico modeling, interaction of these CGs with Na+/K+-ATPase was also studied. Hopefully, these compounds could serve not only as a research tool for Na+/K+-ATPase inhibition, but also as novel cancer therapeutics.

**Keywords:** cardiac glycosides; secondary plant metabolites; natural product isolation; hyrcanoside; deglucohyrcanoside; ouabain; cymarin; digitoxin; anticancer activity; Na+/K+-ATPase inhibitors
