Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization

Abstract

Glycosylated natural products are reliable platforms for the development of many front-line drugs, yet our understanding of the relationship between attached sugars and biological activity is limited by the availability of convenient glycosylation methods. When a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation is used, the glycorandomization of digitoxin leads to analogs that display significantly enhanced potency and tumor specificity and suggests a divergent mechanistic relationship between cardiac glycoside-induced cytotoxicity and Na+/K+-ATPase inhibition. This report highlights the remarkable advantages of glycorandomization as a powerful tool in glycobiology and drug discovery.

Fig. 1.

Glycorandomization is a tool used to convert a single aglycon molecule into a library of analogs with a diverse array of sugar attachments. (A) Neoglycorandomization involves the chemoselective formation of glycosidic bonds between reducing sugars and a secondary alkoxylamine to form a library of neoglycosides (path A). The utility of neoglycorandomization is limited only by the ease of installation of the reactive secondary alkoxylamine group onto a complex natural product aglycon. Chemoenzymatic glycorandomization (path B) exploits nucleotide sugar activation enzymes (E₁ and E₂) and glycosyltransferase enzymes (GlyT) that display natural or engineered promiscuity to glycosylate secondary metabolites. This method is limited to natural products for which promiscuous glycosylation machinery is available. (B) Whereas primary alkoxylamines react with reducing sugars to form open-chain oximes, secondary alkoxylamines react to form closed-ring neoglycosides.

Fig. 2.

The synthesis of cardiac neoglycosides. (A) Aglycon 3β and its C3 epimer 3α were generated in three simple steps from the parent natural product, digitoxin. pyr., pyridine. (B) Aglycons 3β and 3α reacted with d-glucose (2 eq) in 3:1 DMF/acetic acid at 60°C to form neoglycosides 4β and 4α, respectively, in >70% yield by ¹H NMR.

Fig. 3.

The crystal structure of a neoglycoside. (A) Solid-state structure of 4β shown with 50% thermal probability ellipsoids. Hydrogen atoms are omitted for clarity. Crystallographic structure refinement data for 4β and for 3β (structure not shown) can be found, respectively, in Tables 4 and 5, which are published as supporting information on the PNAS web site. (B) The solid-state structure of 4β (red) superimposed on the solid-state structure of a homologous O-glucoside, actodigin (blue). (C) A Newman projection along the C2–C3–N3–C1′ torsion of neoglycoside 4β (red) and the corresponding torsion of 23 known cardiac glycosides (black) reveals that the neoglycoside torsion falls within the range of torsions displayed in the solid state by the known cardiac glycosides. (D) A Newman projection along the C3–N3–C1′–C2′ torsion of 4β (red) and the corresponding torsions of 23 known cardiac glycosides (black) reveals that the neoglycoside torsion falls on the periphery of the narrow range of orientations displayed by natural O-glycosides (citations for the 23 structurally characterized cardiac glycosides can be found in the Supporting Text).

Fig. 4.

Summary of IC₅₀ data from the high-throughput cytotoxicity assay. Reciprocal IC₅₀ values are displayed for clarity, with the current illustration representing an IC₅₀ range of 18 nM (5β, HCT-116) to >25 μM (e.g., 35β, all cell lines). In the assay, live cells were distinguished by the presence of a ubiquitous intracellular enzymatic activity that converts the nonfluorescent cell-permeant molecule calcein AM to the intensely fluorescent molecule calcein, which is retained within live cells. The IC₅₀ value for each library member represents at least six replicates of dose–response experiments conducted over five concentrations at 2-fold dilutions. For the entire panel of 81 compounds in 10 cell lines, the average error was 17%. IC₅₀ values and corresponding error values can be found in Tables 4 and 5, which are published as supporting information on the PNAS web site. The six library member “hits” are depicted in pyranose ⁴C₁ conformations to facilitate structural comparisons. Du145, human colon carcinoma; MCF7, human breast carcinoma; HCT-116, human colon carcinoma; Hep3B, human liver carcinoma; SF-268, human CNS glioblastoma; SK-OV-3, human ovary adenocarcinoma; NCI-H460, human lung carcinoma; A549, human lung adenocarcinoma; NCI/ADR-RES, human breast carcinoma; NmuMG, mouse mammary normal epithelial cells.