A Direct Comparison of the Anticancer Activities of Digitoxin MeON-Neoglycosides and O-Glycosides: Oligosaccharide Chain Length-Dependent Induction of Caspase-9-Mediated Apoptosis

Abstract

Digitoxin is a cardiac glycoside currently being investigated for potential use in oncology. While a number of structure-activity relationship studies have been conducted, an investigation of anticancer activity as a function of oligosaccharide chain length has not yet been performed. We generated mono-, di-, and tri-O-digitoxoside derivatives of digitoxin and compared their activity to the corresponding MeON-neoglycosides. Both classes of cardenolide derivatives display comparable oligosaccharide chain length-dependent cytotoxicity toward human cancer cell lines. Further investigation revealed that both classes of compounds induce caspase-9-mediated apoptosis in non-small cell lung cancer cells (NCI-H460). Since O-glycosides and MeON-neoglycosides share a similar mode of action, the convenience of MeON-neoglycosylation could be exploited in future SAR work to rapidly survey large numbers of carbohydrates to prioritize selected O-glycoside candidates for traditional synthesis.

Figure 1

Digitoxin mono-, di-, and tri-O-digitoxosides 1−3 and mono-, di-, and tri-MeON-digitoxosides 4−6.

Scheme 1. Synthesis of Digitoxin Mono-, Di-, and Tri-O-digitoxosides 1−3

NBSH, o-nitrobenzenesulfonylhydrazide; DEAD, diethyl azodicarboxylate; and NMO, N-methylmorpholine-N-oxide.

Scheme 2. Synthesis of Digitoxin Mono-, Di-, and Tri-MeON-digitoxosides 1−3

Figure 2

Summary of GI₅₀ data from growth inhibition assays. Reciprocal GI₅₀ values are displayed for clarity.

Figure 3

Summary of IC₅₀ data from cytotoxicity assays. Reciprocal IC₅₀ values are displayed for clarity; standard errors are depicted with error bars.

Figure 4

O-Digitoxosides 1−3 and MeON-digitoxosides 4−6 induce apoptosis in NCI-H460 non-small cell lung cancer cells. Apoptotic cells were quantified by Hoechst dye staining and microscopy. ZV = pan-caspase inhibitor ZVAD-FMK.

Figure 5

O-Digitoxoside 1 and MeON-digitoxoside 4 activate caspase-9, not caspase-8, in NCI-H460 non-small cell lung cancer cells. Cell lysates were incubated in fluorometric substrates for either caspase-8 (IETD-AFC) or caspase-9 (LEHD-AFC); samples were then read using a fluorescence plate reader. Ntx = no treatment. ZV = pan-caspase inhibitor ZVAD-FMK.