Na+/K+-ATPase-Targeted Cytotoxicity of (+)-Digoxin and Several Semisynthetic Derivatives

Abstract

(+)-Digoxin (1) is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides (2-5) and two new analogues, (+)-8(9)-β-anhydrodigoxigenin (6) and (+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7), were synthesized from 1 and evaluated for their cytotoxicity toward a small panel of human cancer cell lines. A preliminary structure-activity relationship investigation conducted indicated that the C-12 and C-14 hydroxy groups and the C-17 unsaturated lactone unit are important for 1 to mediate its cytotoxicity toward human cancer cells, but the C-3 glycosyl residue seems to be less critical for such an effect. Molecular docking profiles showed that the cytotoxic 1 and the noncytotoxic derivative 7 bind differentially to Na⁺/K⁺-ATPase. The HO-12β, HO-14β, and HO-3'aα hydroxy groups of (+)-digoxin (1) may form hydrogen bonds with the side-chains of Asp121 and Asn122, Thr797, and Arg880 of Na⁺/K⁺-ATPase, respectively, but the altered lactone unit of 7 results in a rotation of its steroid core, which depotentiates the binding between this compound and Na⁺/K⁺-ATPase. Thus, 1 was found to inhibit Na⁺/K⁺-ATPase, but 7 did not. In addition, the cytotoxic 1 did not affect glucose uptake in human cancer cells, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na⁺/K⁺-ATPase but not by interacting with glucose transporters.

Figure 1.

ECD spectra of compounds 1 (blue), 2 (red), 3 (green), and 4 (purple) (upper) and those of compounds 1 (blue), 5 (pink), 6 (aqua), and 7 (burgundy) (below). The data were obtained in HPLC grade MeOH as the average of three scans corrected by subtracting a spectrum of the appropriate solution in the absence of the samples recorded under identical conditions. Each scan in the range 200‒450 nm was obtained by taking points every 0.1 nm with a 50 nm/min scanning speed and a 1 nm band width. No Cotton effects were observed in the range 350‒450 nm.

Figure 2.

The docking box and the predicted binding pose of 7 (cyan sticks) from docking aligned with (+)-digoxin (1) (yellow sticks) from the crystal structure) (A), binding poses of 1 (yellow sticks, from the crystal structure) (B) and 7 (cyan sticks, predicted) (C) in Na⁺/K⁺-ATPase pocket, and inhibition of Na⁺/K⁺-ATPase by 1 (D) and 7 (E).

Figure 3.

Cytotoxicity against H1299 cells and glucose transport inhibition of 1. H1299 human lung cancer cells were treated by vehicle, WZB117 (30 μM, positive control), or (+)-digoxin (30 μM) for 15 min. After glucose uptake was initiated by 2-deoxy-D-[³H]glucose, cells were lysed and the radioactivity of the cell lysates was measured. The results showed that (+)-digoxin showed cytotoxicity toward H1299 cells (IC₅₀ 0.46 μM), but it did not inhibit glucose transport (columns, means, n = 3; bars, SE; **p ≤ 0.01 and ***p ≤ 0.001 when compared with the values from mock).

Scheme 1.

Synthesis of compounds 2–4. Reagents and conditions: (a) Ac₂O and pyridine, r.t., 72 h; (b) Ac₂O and pyridine, 80 °C, 2 h.

Scheme 2.

Synthesis of compounds 5 and 6. Reagents and conditions: (a) conc. HCl, r.t., overnight.

Scheme 3.

Plausible mechanism for the generation of (+)-8(9)-β-anhydrodigoxigenin (6) from (+)-digoxin (1).

Scheme 4.

Synthesis of compound 7. Reagents and conditions: (a) MeOH saturated with NaOH, 60 °C, overnight.

Scheme 5.

Plausible mechanism for the formation of (+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7) from (+)-digoxin (1).