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

Article abstract of DOI:10.1021/acs.jnatprod.9b01060

(+)-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.

Full text of DOI:10.1021/acs.jnatprod.9b01060

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Article
Na+/K+‑ATPase-Targeted Cytotoxicity of (+)-Digoxin and Several
Semisynthetic Derivatives
Yulin Ren, Hennrique T. Ribas, Kimberly Heath, Sijin Wu, Jinhong Ren, Pratik Shriwas, Xiaozhuo Chen,
Michael E. Johnson, Xiaolin Cheng, Joanna E. Burdette, and A. Douglas Kinghorn*
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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 synthe-
sized 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.
ardiac glycosides have been investigated extensively for
their potential anticancer activities, including cancer cell
from patients with groups 3 and 4 medulloblastoma (3 and 4
MB, the most common malignant brain tumor of childhood),
(+)-digoxin was characterized as a potential cancer driver
signaling inhibitor. Its anti-MB potential has been validated
subsequently in both in vitro cell-based assays and in vivo
orthotopic patient-derived xenograft models.16 Unfortunately,
the cancer chemotherapeutic features of (+)-digoxin seem not
to translate very well in cancer clinical trials,17 indicating that a
more detailed structural investigation might support the design
and discovery of new anticancer agents based on (+)-digoxin
(1).
C
cytotoxicity, antineoplastic effects in in vivo models, and
anticancer activities in clinical trials.1,2 Among these com-
pounds, (+)-digoxin (1), a well-known therapeutic agent
isolated originally from the plant Digitalis lanata Ehrh.
(Plantaginaceae)3,4 and long used to treat congestive heart
failure through inhibition of Na+/K+-ATPase,5 was found to be
cytotoxic toward human cancer cell panels, when a large
number of compounds were tested in a drug repurposing
study.6
The potential anticancer activity of (+)-digoxin has been
reviewed recently,1,2,7 with its potent cytotoxicity (IC50 40−
200 nM) being reported against a small panel of human cancer
cell lines.8 Interestingly, this cardiac glycoside was also found
to synergize the cytotoxicity of cisplatin against human HeLa
cervical cancer cells,9 while cotreatment of paclitaxel with
(+)-digoxin resulted in an antagonistic cytotoxicity against
HeLa cells.10 Mechanistically, (+)-digoxin was reported to
show anticancer potential through inhibition of Na+/K+-
ATPase,11 and it also mediates cancer cell cytotoxicity through
induction of autophagy and immunogenic cell death12,13 and
through inhibition of HIF-1α and NF-κB activation.14,15
In a driver signaling network identification (DSNI)- and
drug functional network (DFN)-based drug repositioning
program that integrated multiple types of genomic profiles
The importance of the saccharide moiety and the steroid
hydroxy groups of several cardiac glycosides in their interaction
with the Na+/K+-ATPase has been addressed previously.18
These structural components, along with the C-17 unsaturated
heterocycle, were determined as being important in cancer cell
cytotoxicity.19 The cytotoxicity of (+)-digitoxin against a small
panel of human cancer cell lines was enhanced by introducing
a C3-MeON-neoglycose unit to the aglycone digitoxigenin,20
Special Issue: Special Issue in Honor of Jon Clardy
Received: October 24, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
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and the C3-O-neoglycosides of digoxigenin were found to be
more cytotoxic than its C3-MeON-neoglycosides and (+)-di-
goxin itself when evaluated against NIH H460 human non-
small-cell lung cancer cells.21 Interestingly, when the lactone
moiety was replaced with a carboxylic acid group, the cytotoxic
activity of the resultant compound was retained,22 but the
inhibitory potencies of (+)-digoxin and 20,22-dihydrodigoxin-
21,23-diol were found to be different against RORγt
transcriptional activity.23 A new derivative of (+)-digoxin,
namely, BD-4, with a γ-benzylidene group substituted at the C-
17-butenolide, exhibited more potent cytotoxicity than the
parent compound against HeLa human cervical cancer cells,
and this compound mediated its cytotoxicity through a
different mechanism of action,24 as confirmed by the docking
profiles, which showed that (+)-digoxin and 21-benzylidene-
digoxin targeted Na+/K+-ATPase differentially.25
the same procedure, (+)-12,3′a,3′b,3′c,4′c-penta-O-acetyldi-
goxin (4)30 was synthesized at 80 °C for 2 h (Scheme 1).
a
Scheme 1. Synthesis of Compounds 2−4
In our continuing search for anticancer agents,26 (+)-digoxin
was found to show potent cytotoxicity against a small panel of
human cancer cell lines,27 and (+)-digitoxin, devoid of a 12-
hydroxy group, exhibited more potent activity than (+)-digoxin
toward human HT-29 colon cancer and MDA-MB-435
melanoma cells.28 However, (+)-digoxin induced lordosis
(spine malformation or curvature disorder) when it was
examined in a zebrafish toxicity assay.29 In the present
investigation, two new and several known (+)-digoxin
derivatives have been synthesized, of which the mechanism
of the generation of the new compounds (+)-8(9)-β-
anhydrodigoxigenin (6) and (+)-17-epi-20,22-dihydro-21α-
hydroxydigoxin (7) are proposed. The structures of the
synthetic compounds have been determined and confirmed
by analysis of their spectroscopic data followed by comparison
a
Reagents and conditions: (a) Ac2O and pyridine, rt, 72 h; (b) Ac2O
and pyridine, 80 °C, 2 h.
Compound 2 showed a sodium adduct ion at m/z 887.4441
for a molecular formula of C45H68O16Na+, indicating that two
hydroxy groups of (+)-digoxin are acetylated. These acetylated
groups were proposed at the C-12 and C-4′c positions after
comparing the NMR spectroscopic data of 2 with those of
(+)-digoxin (Table 1 and Tables S1 and S2, Supporting
Information),31 of which the NMR spectroscopic data have
been assigned completely.32 This assignment for 2 was also
confirmed by the HMBC cross-peaks observed between H-12
and H-4′c and their attached acetyl carbonyl groups,
respectively (Figure S9, Supporting Information). Similarly,
three acetoxy groups were assigned at C-12, C-3′c, and C-4′c
of 3, as indicated by its sodium adduct ion at m/z 929.4492
(C47H70O17Na+, tri-O-acetyldigoxin) and the HMBC correla-
tions between H-12, H-3′c, and H-4′c and their respective
acetyl carbonyl groups. Five acetoxy groups were proposed at
C-12, C-3′a, C-3′b, C-3′c, and C-4′c of 4, as supported by its
mass spectrometric data (positive-ion HRESIMS m/z
1013.4761 for C51H74O19Na+) and the HMBC correlations
between H-12, H-3′a, H-3′b, H-3′c, and H-4′c and their
respective acetyl carbonyl groups (Figure S9, Supporting
Information).
When (+)-digoxin (1) was reacted with concentrated HCl at
room temperature overnight, the compounds (+)-digoxigenin
(5) and (+)-8(9)-β-anhydrodigoxigenin (6) were generated
(Scheme 2).33 These compounds were separated by silica gel
column chromatography followed by purification over
Sephadex LH-20. Compound 5 was assigned as the aglycone
of (+)-digoxin (1) by comparing its NMR spectroscopic data
with those of 1, from which the NMR resonances assigned for
the saccharide moiety of 1 were found to be absent in 5 (Table
1 and Tables S1 and S2, Supporting Information).34 This was
supported by its positive-ion HRESIMS data at m/z 413.2336,
390 Da (C18H30O9) less than 1. A sodium adduct ion at m/z
395.2211 [18 Da (H2O) less than 5] for a molecular formula
of C23H32O4Na+ observed for 6 indicated that this compound
1
of these data with those of (+)-digoxin, with the H and 13C
NMR spectroscopic data of all of these compounds assigned
completely. In addition, the cytotoxicity against a small panel
of human cancer cell lines of all compounds and the inhibition
of Na+/K+-ATPase and glucose transport of (+)-digoxin (1)
have been evaluated, with docking profiles for 1 and its new
and noncytotoxic derivative 7 being carried out. The Na+/K+-
ATPase-related mechanism of action and the relative
importance of the hydroxy groups, the C-3 saccharide moiety,
and the C-17 unsaturated lactone unit of (+)-digoxin in
mediating its cytotoxic activity are also discussed.
RESULTS AND DISCUSSION
■
It has been reported that both the C-3 saccharide and C-17
unsaturated heterocycle units are of importance for cardiac
glycosides to exhibit their cancer cell cytotoxicity,19 while the
sugar moiety and the steroidal hydroxy groups are functional
components required for binding between the cardiac
glycosides and Na+/K+-ATPase.18 To test the importance of
these structural components in effecting the cytotoxicity of
(+)-digoxin (1), several derivatives (2−7) were prepared
synthetically (Figures S1−S8, Supporting Information) and
evaluated biologically.27,28
Of these, three acetylated analogues with different numbers
of acetyl groups (2−4) were produced by acetylation of
(+)-digoxin (1). After 1 was stirred in Ac2O and pyridine at
room temperature for 72 h, (+)-12,4′c-di-O-acetyldigoxin
(2)30 and (+)-12,3′c,4′c-tri-O-acetyldigoxin (3)30 were gen-
erated. Both 2 and 3 were separated from a mixture of reaction
products, using silica gel column chromatography followed by
further purification by passage over Sephadex LH-20. Using
B
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a
1
Table 1. H and 13C NMR Spectroscopic Data of 1 and 7
b
c
b
c
b
c
b
c
position
1
1
1
7
7
position
1
1
7
7
30.16 CH2 α 1.29 m
β 1.36 m
26.00 CH2 α 1.53 m
β 1.53 m
72.09 CH α 3.90 br s
29.58 CH2 α 1.74 m
β 1.33 m
36.31 CH β 1.53 m
26.43 CH2 α 1.18 m
β 1.72 m
21.35 CH2 α 1.08 m
β 1.69 m
40.47 CH β 1.44 m
31.61 CH α 1.59 m
34.67 C
29.70 CH2 α 1.41 m
β 1.06 m
72.96 CH α 3.23 m
55.70 C
30.25 CH2 α 1.30 m
β 1.30 m
25.90 CH2 α 1.49 m
β 1.49 m
72.20 CH
29.74 CH2 α 1.67 m
β 1.27 m
36.38 CH
26.20 CH2 α 1.08 m
β 1.73 m
21.08 CH2 α 1.02 m
β 1.61 m
36.81 CH
31.58 CH
34.82 C
2′a
3′a
4′a
5′a
6′a
1′b
2′b
β 1.56 m
β 1.53 m
66.27 CH β 4.03 m
81.67 CH β 3.14 m
67.49 CH α 3.70 m
18.03 CH3 β 1.10 d (7.0)
99.09 CH β 4.80 m
37.92 CH2 α 1.64 m
β 1.84 m
66.13 CH α 4.05 m
81.92 CH α 3.11 m
67.61 CH β 3.76 m
18.03 CH3 α 1.12 d (7.0)
99.00 CH α 4.83 m
38.31 CH2 α 1.87 m
β 1.59 m
67.01 CH β 3.85 m
72.67 CH β 3.02 m
69.05 CH α 3.66 m
18.37 CH3 β 1.13 d (5.7)
β 4.63 s
66.31 CH
81.69 CH
67.52 CH
18.06 CH3 β 1.09 d (5.8)
99.12 CH β 4.79 m
37.95 CH2 α 1.60 m
β 1.83 m
66.16 CH
81.94 CH
67.64 CH
18.06 CH3 α 1.11 d (6.1)
99.02 CH α 4.81 m
38.34 CH2 α 1.86 m
β 1.58 m
67.03 CH
72.70 CH
69.08 CH
18.40 CH3 β 1.12 d (5.6)
β 4.34 br s
β 4.04 m
β 3.13 m
α 3.69 m
2
3
4
α 3.88 m
5
6
β 1.55 m
3′b
4′b
5′b
6′b
1′c
2′c
α 4.05 m
α 3.11 m
β 3.73 m
7
8
9
10
11
β 1.44 m
α 1.64 m
28.69 CH2 α 1.38 m
β 1.21 m
68.15 CH
49.93 C
85.66 C
3′c
4′c
5′c
6′c
OH-12
OH-14
OH-21
OH-3′a
OH-3′b
OH-3′c
OH-4′c
β 3.84 m
β 2.99 m
α 3.64 m
12
13
14
15
α 3.36 m
84.33 C
32.42 CH2 α 1.88 m
β 1.61 m
28.46 CH2 α 1.87 m
β 1.50 m
β 4.12 s
β 4.07 br s
α 3.67 m
α 4.21 br s
β 4.28 br s
α 4.63 br s
α 4.61 br s
16
26.79 CH2 α 1.98 m β 1.84 m 19.87 CH2 α 1.67 m β
α 4.22 s
β 4.28 s
α 4.65 s
α 4.61 s
1.53 m
β 2.06 m
β 0.89 s
17
18
19
20
21
45.18 CH α 3.26 m
9.44 CH3 β 0.65 s
23.67 CH3 β 0.85 s
176.94 C
73.31 CH2 β 4.85 overlapped
42.88 CH
9.84 CH3
23.63 CH3 β 0.87 s
39.28 CH
94.16 CH
a
Assignments of chemical shifts are based on the analysis of 1D and
β 2.10 m
β 4.40 m
2D NMR spectra. The 13C NMR spectroscopic data CH3, CH2, CH,
and C multiplicities were determined by DEPT 90, DEPT 135, and
HSQC experiments, and the overlapped 1H NMR signals were
(17.9)
α 4.94 br d (17.9)
assigned from H−1H COSY, HSQC, and HMBC spectra without
1
b
designating multiplicity. The 13C NMR spectroscopic data (δ) were
22
115.83 CH 5.82 br s
35.06 CH2 α 2.30 m
β 2.03 m
174.17 C
95.39 CH
38.47 CH2 α 1.76 m
measured in DMSO-d6 at 100.61 MHz and referenced to the solvent
d
residual peak at δ 39.52.31 The 1H NMR spectroscopic data (δ) were
c
23
1′a
2′a
173.96 C
95.32 CH α 4.78 m
38.45 CH2 α 1.77 m
α 4.77 m
measured in DMSO-d6 at 400.13 MHz and referenced to the solvent
residual peak at δ 2.50.31 Shown in the 2D HMBC NMR spectra.
d
a
Scheme 2. Synthesis of Compounds 5 and 6
This assignment has been supported by the HMBC
correlations observed between H3-18 and C-12, C-13, C-14,
and C-17 and between H3-19 and C-1, C-5, C-9, and C-10, as
well between H-11 and C-8 and between H-16 and C-14 of 6
(Figures S8−S10 and Tables S1 and S2, Supporting
Information). A plausible mechanism for the generation of 6
from 1 could involve an acid-mediated hydrolysis of 1 to form
5 and a 1,2-hydride shift concurrent with hydration of 5
(Scheme 3).
Another new analogue, (+)-17-epi-20,22-dihydro-21α-hy-
droxydigoxin (7), was produced when (+)-digoxin (1) was
stirred in NaOH-saturated MeOH at 60 °C overnight (Scheme
4). Comparison of the NMR spectroscopic data of 7 with
those of 1 indicated these compounds to contain the same
saccharide moiety and A/B/C/D-ring system, but a different
lactone unit. Compound 7 gave a sodium adduct ion at m/z
821.4299 for a molecular formula of C41H66O15Na+, 18 Da
(H2O) more than 1, indicating that this derivative is a
hydration product of 1. In turn, the Δ20(22) double bond in 1
was saturated in 7, with a hydroxy group being substituted at
C-21, as supported by its 13C NMR spectroscopic resonances
at δC 39.28 (C-20), 94.16 (C-21), and 35.06 (C-22), as
compared to those at δC 176.94 (C-20), 73.31 (C-21), and
a
Reagents and conditions: (a) concentrated HCl, rt, overnight.
is a dehydrated analogue of 5, as inferred from its NMR
resonances at δC 128.13 for C-8 and at δC 131.57 for C-9 but
not at δC 40.51 and δC 31.50 for the respective carbons of 5.
C
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1i and deconjugation of the unsaturated lactone 1i followed by
opening of the resulting β,γ-unsaturated lactone unit and a
subsequent hydration of the resulting formyl group followed by
cyclization of the intermediate hydrate (or derivatives)
(Scheme 5).
Scheme 3. Plausible Mechanism for the Generation of
(+)-8(9)-β-Anhydrodigoxigenin (6) from (+)-Digoxin (1)
Scheme 5. Plausible Mechanism for the Formation of
(+)-17-epi-20,22-Dihydro-21α-hydroxydigoxin (7) from
(+)-Digoxin (1)
a
Scheme 4. Synthesis of Compound 7
The structure of (+)-digoxin (1) was established previously
by analysis of its single-crystal X-ray diffraction data,4 with the
(3′cS,4′cS) absolute configuration determined from its
[Mo2(OAc)4]-induced ECD.27 In turn, its (3S, 5R, 8R, 9S,
10S, 12R, 13S, 14S, 17R, 1′aR, 3′aS, 4′aS, 5′aR, 1′bS, 3′bS,
4′bS, 5′bR, 1′cS, 3′cS, 4′cS, 5′cR) absolute configuration was
determined and supported by the 2D NOESY NMR spectrum
(Figure S10, Supporting Information). Following this deter-
mination, the (3S, 5R, 8R, 9S, 10S, 12R, 13S, 14S, 17R)
absolute configuration for the steroid core was assigned for
compounds 2−5, with those for their glycosyl unit being
defined as (1′aR, 3′aS, 4′aS, 5′aR, 1′bS, 3′bS, 4′bS, 5′bR, 1′cS,
3′cS, 4′cS, 5′cR) for 2, (1′aR, 3′aS, 4′aS, 5′aR, 1′bS, 3′bS, 4′bS,
5′bR, 1′cS, 3′cS, 4′cR, 5′cR) for 3, and (1′aR, 3′aS, 4′aR, 5′aR,
1′bS, 3′bS, 4′bR, 5′bR, 1′cS, 3′cS, 4′cR, 5′cR) for 4. In
addition, the (3S, 5R, 10S, 12R, 13S, 14R, 17R) and (3S, 5R,
8R, 9S, 10S, 12R, 13S, 14S, 17S, 20S, 21S, 1′aR, 3′aS, 4′aS,
5′aR, 1′bS, 3′bS, 4′bS, 5′bR, 1′cS, 3′cS, 4′cS, 5′cR) absolute
configurations were assigned for compounds 6 and 7, from
analysis of their specific rotation values and ECD (Figure 1)
and 2D NOESY NMR spectroscopic data and from
comparison of all of these data with those of (+)-digoxin (1)
(Figures S8−S10, Supporting Information).
a
Reagents and conditions: (a) MeOH saturated with NaOH, 60 °C,
overnight.
115.83 (C-22) in 1 (Table 1). The stereostructure of 7 was
assigned by analysis of its specific rotation value and 1D and
2D NMR spectra, which indicated that 7 and 1 have the same
configuration for their glycosidic moiety and A-, B-, and C-ring
system but differ in the D-ring and lactone unit. This was
supported by the 2D NOESY correlations between H-17 and
H-7β, H-16β, and H3-18, between H-20 and H-7β, H-16β, and
H3-18, and between H-21 and H-7β, H-16β, and H-20 in 7
(Figure S10, Supporting Information). Thus, 7 was determined
as having a β-oriented proton at C-17, C-20, and C-21, as
implied by its 13C NMR spectroscopic resonances at δC 28.69,
68.15, 49.93, 85.66, 28.46, 19.87, 42.88, and 9.84, rather than
at δC 29.70, 72.96, 55.70, 84.33, 32.42, 26.79, 45.18, and 9.44
in 1 for their respective C-11−C-18 carbons (Table 1), due to
the effects from the different orientation of the C-17 lactone
unit.35,36
As shown in Figure 1, the positive Cotton effect (CE)
around 241 nm shown in the ECD spectrum of (+)-digoxin
(1) was absent in the ECD spectrum of 7, due to the loss of
the α,β-unsaturated lactone functional unit in 7. The ECD
spectrum of 5 was found to be closely comparable with that of
A plausible mechanism for the generation of 7 from 1 could
involve a base-mediated isomerization of C-17 to form isomer
D
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a
Table 2. Cytotoxicity of 1−7
b
c
d
e
compound
HT-29
MDA-MB-231 OVCAR3
MDA-MB-435
1
2
3
4
5
6
7
0.28
5.1
>10
>10
3.6
>10
>10
0.068
0.0008
0.31
8.2
>10
>10
3.2
>10
>10
0.48
0.0027
0.10
2.5
6.2
8.2
2.4
>10
>10
0.12
0.0033
0.17
5.4
>10
>10
0.9
>10
>10
0.043
0.0002
f
(+)-digitoxin
paclitaxel
g
a
IC50 values are the concentration (μM) required for 50% inhibition
of cell viability for a given test compound with a 72 h treatment and
were calculated using nonlinear regression analysis with measure-
ments performed in triplicate and representative of three independent
b
experiments, where the values generally agreed within 10%. IC50
c
value toward the HT-29 human colon cancer cell line. IC50 value
d
toward the MDA-MB-231 human breast cancer cell line. IC50 value
e
toward the OVCAR3 human ovarian cancer cell line. IC50 value
f
toward the MDA-MB-435 human melanoma cell line. Data reported
g
previously.28 Positive control.
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 bandwidth. No Cotton effects were
observed in the range 350−450 nm.
found to exhibit activity toward these cells, with IC50 values in
the range 0.9−3.6 μM (Table 2), indicating that the glycosyl
unit is not critical for 1 to mediate cellular cytotoxicity.
However, the cytotoxicity against all of these cell lines
decreased in the sequence 2, 3, and 4, and no discernible
activity was observed for compounds 6 and 7 (Table S3,
Supporting Information). This indicates that O-acetylation
reduces the cytotoxicity of (+)-digoxin (1), and either removal
of its C-14 hydroxy group followed by unsaturation of the C-8
and C-9 bond or inversion of its C-17 configuration followed
by saturation of the lactone unit and introducing simulta-
neously a hydroxy group at C-21 results in activity being
abolished. Thus, the C-12 and C-14 hydroxy groups and C-17
lactone unit seem to be important for 1 to mediate its
cytotoxicity against human cancer cells and to interact with its
molecular targets.
(+)-digoxin (1), indicating that the glycosyl unit present does
not affect the ECD curve of 1 (Figure 1). A (17R)
configuration was assigned for compounds 2−4 from their
2D NOESY NMR spectra and specific rotation values, which
are consistent with those of 1. However, around a 20 nm shift
was found in the positive CEs (in the range 213−221 nm) for
2−4 when compared with that around 241 nm for (+)-digoxin
(1), indicating that an acetoxy group at C-12 results in the
ECD spectrum of (+)-digoxin being modified. In addition, a
(17R) configuration was indicated for 6 from its consistent 2D
NOESY correlations with those of 5 and from its sequential
negative and positive CEs at 241.8 and 216.1 nm in the ECD
spectrum. These reversed CEs of 6 may result from the B-/C-/
D-ring conformational change upon formation of the C-8/C-9
double bond.27 Thus, the presence of a double bond in the B-/
C-/D-ring system could reverse the CEs observed for this type
of cardiac glycosides.
Based on the established NMR spectroscopic assignments of
(+)-digoxin (1),32 the 1H and 13C NMR spectroscopic data of
the synthetic cardiac glycosides 2−7 presented herein were
assigned completely, by analysis of their 1D and 2D NMR
spectra (Table 1 and Tables S1 and S2, Supporting
Information).
Compounds 1−7 were evaluated for their cytotoxicity
against human HT-29 colon, MDA-MB-231 breast, and
OVCAR3 ovarian cancer and MDA-MB-435 melanoma cells,
using a protocol reported previously, with paclitaxel as the
positive control.28 This appears to be the first report of the
cytotoxicity of compounds 2−7 toward these cancer cell lines.
The parent compound, (+)-digoxin (1), exhibited potent
cytotoxicity toward all the cell lines tested, with IC50 values in
the range 0.1−0.3 μM (Table 2). The aglycone 5 was also
It is well known that Na+/K+-ATPase is a potential target for
cardiac glycosides to mediate their antitumor properties,11 and
the U-shaped cis−trans−cis fused steroid core (steroid core),
the β-oriented C-14 hydroxy group (HO-14β), the C-17 five-
membered unsaturated lactone unit (17β-lactone), and the C-3
carbohydrate moiety (3β-glycosyl) are all critically important
for these compounds to bind to Na+/K+-ATPase.18,24,25 The
crystal structure of Na+/K+-ATPase and bound ouabain [a
hydroxylated analogue of (+)-digoxin] at a low affinity showed
that this cardenolide binds to a cavity formed by the
transmembrane helices M1, M2, M4, M5, and M6 that are
close to the K+-binding sites of Na+/K+-ATPase.37 Ouabain
inserts deeply into the transmembrane domain, with the
steroid core being held tightly in a cavity lined by the M4−M6
helices. Hydrogen bonds were found to be formed between the
HO-14β and 3β-glycosyl and Thr-804 and Arg-887 (in the L7/
8 loop and Glu-319 on M4), respectively, and the 17β-lactone
docked closely to Val-329 and Ala-330 in the M4E helix,
indicating that the position of the conjugated carbonyl group
in the 17β-lactone is important.37
In the crystal structure for a complex of Na+/K+-ATPase and
ouabain at a high affinity state, an extensive hydrogen-bonding
network formed between HO-5β, HO-14β, and HO-19β of
ouabain and Glu117 (αM2), Thr797 (αM6), and Gln111
(αM1) and Asn122 (αM2) of Na+/K+-ATPase, respectively,
E
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Figure 2. Docking box and the predicted binding pose of 7 (cyan sticks) from docking aligned with (+)-digoxin (1) (yellow sticks, from the crystal
structure) (A), and binding poses of 1 (yellow sticks, from the crystal structure) (B) and 7 (cyan sticks, predicted) (C) in the Na+/K+-ATPase
pocket, and inhibition of Na+/K+-ATPase by 1 (D) and 7 (E).
was reported.38 The 3β-attached glycosyl moiety of ouabain
was found to occupy a wide cavity lined with polar residues,
including Glu116 (loop αM1−2), Glu312 (αM4), Arg880, and
Asp884 (loop αM7−8), with the 17β-affixed lactone unit being
located in a hydrophobic funnel composed of Leu125 (αM2),
Ala323 (αM4), and Ile800 (αM6).38 Following this, the crystal
structure of the E2P form of the pig kidney Na+/K+-ATPase
(α1β1γ) in complex with (+)-digoxin (1) was adopted, which
showed that the HO-14β of 1 and the Thr797 (αM6) side-
chain of Na+/K+-ATPase could be within hydrogen bond
distance, and HO-12β forms a hydrogen bond with Asn122
(αM2). The first proximal digitoxose unit of (+)-digoxin (1) is
located in a wide cavity lined with polar residues Gln111,
Thr114, Glu116, Gln117 (loop αM1−2), Glu312 (αM4),
Arg880 (loop αM7−8), and Gln84 (β-ectodomain), and
similarly to ouabain, its 17β-lactone unit occupies a hydro-
phobic funnel formed by Leu125 (αM2), Ala323 (αM4), and
Ile800 (αM6).39 This indicated that the HO-12β, HO-14β, 3β-
glycosyl, and the 17β-attached lactone unit are all important
for (+)-digoxin (1) to bind to Na+/K+-ATPase, consistent
with the previous conclusions from the crystal structures of the
complex of Na+/K+-ATPase and ouabain.37,38
way that the lactone unit penetrates deep inside the
hydrophobic funnel formed by α-M4−M6, where the 17β-
lactone unit of 1 binds to the protein (Figure 2A). The relative
orientation between the lactone and the steroid moieties of 7 is
opposite to that of 1, and thus, if 7 adopts a similar pose to 1, it
would clash with the αM4 helix (Figure 2B and C). In
addition, the lactone unit of 7 rotates away from the cation-
binding site of Na+/K+-ATPase to assume a different pose,
with the carbonyl group being directed to a small groove on
the side of the pocket made by Ile320, Ala323 (αM4) and
Ile780, Phe783 (αM5) (Figure 2C). This rotation makes
enough room for its HO-21α substituent to occupy this site,
but the presence of this group impairs the binding of 7 to the
hydrophobic groove.
The nonpolar concave α-surface of the steroid core of 7
interacts with bulky hydrophobic side chains of αM4−M6
(Figure 2C), which constitute a generally conserved docking
platform for most cardiac glycosides, including (+)-digoxin
(4RET), bufalin (4RES),39 and ouabain (4HYT).38 The
hydroxy groups on the β-surface of (+)-digoxin (1) could
form hydrogen bonds with the side-chains of Asp121 (αM2)
and Asn122 (αM2) (HO-12β) and Thr797 (αM6) (HO-14β)
of Na+/K+-ATPase (Figure 2B). However, upon rotation of the
lactone unit of 7, its steroid core is also rotated by nearly 30°
and is oriented toward the entrance of the hydrophobic cavity.
This results in the hydrogen bonds between HO-12β of 7 and
Na+/K+-ATPase being lost (Figure 2C), which, in turn,
weakens the binding between 7 and the protein, and thus, it
In the present work, computer-simulated docking profiles for
the cytotoxic (+)-digoxin (1) and its noncytotoxic analogue,
(+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7), were inves-
tigated. In contrast to 1, for which the 17β-lactone and steroid
core overlaid the crystal structure very well, 7 seemed not to fit
into the cation-binding site of Na+/K+-APTase. Only 28 out of
320 (8.75%) final predicted binding poses of 7 oriented in a
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does not show any positive activities in the cytotoxicity assays
(Table 2).
This was supported by ouabain and thevetin, two cardiac
glycosides showing inhibitory effects on the active transport of
3-methylglucose across the intestine of frogs in vitro.48
Similarly, (+)-digoxin (1) has a sterically unfavorable HO-
12β substituent, which could have an impact on its binding to
Na+/K+-ATPase,37 and thus, it showed a decreased binding
affinity and cancer cell cytotoxicity when compared with
(+)-digitoxin.27,28,40 Introducing a 3β-glycosyl unit to the
steroid core of a cardenolide could fill the large vacant space
around the glycosyl residue to enhance the binding affinity,
and thus 1 was found to show a more potent inhibitory activity
against Na+/K+-ATPase than its aglycone40 and exhibited more
potent cytotoxicity toward human cancer cells than (+)-digox-
igenin (5) (Table 2).
Cancer cells require an increase in glucose uptake and
metabolism to meet the energy and biomass synthesis for their
fast proliferation rates,49 and these cells take in the majority of
glucose via glucose transporters, which have become important
targets for the development of anticancer agents.50,51 Using a
procedure reported previously,52 (+)-digoxin (1) was tested
for its effects on glucose uptake in H1299 human lung cancer
cells, against which 1 showed potent cytotoxicity, with an IC50
value of 0.46 μM (Figure 3). However, the results showed that
To test the effect of the C-17 configuration on the binding
between 7 and Na+/K+-ATPase, a docking profile was
produced for its 17,20,21-triepimer (7a), which showed that
the binding between 7 or 7a and Na+/K+-ATPase is different
(Figure 2 and Figure S11, Supporting Information). Unlike 7,
compound 7a binds to Na+/K+-ATPase in a similar way to
(+)-digoxin (1). However, the HO-21β of 7a was found to be
surrounded by several hydrophobic residues within a 5 Å
distance, including L125 and I800 of Na+/K+-ATPase (Figure
S11, Supporting Information), which could negatively impact
the binding between 7a and Na+/K+-ATPase.41 Also, a
negative effect may occur for HO-21β to dock a hydrophobic
pocket, due to the effect of desolvation.41 Thus, 7a could bind
to Na+/K+-ATPase, but the binding is not as strong as 1,
indicating that the substituent, configuration, and conforma-
tion of the C-17 lactone unit all affect the binding between
cardiac glycosides and Na+/K+-ATPase.
It has been reported previously that (+)-digoxin (1) inhibits
potently Na+/K+-ATPase activity,42 and the substituents at C-
10, C-12, and C-17 of the steroid core of 1 or other cardiac
glycosides could affect critically this enzyme inhibitory effect.43
Thus, the cytotoxic (+)-digoxin (1) and its noncytotoxic
analogue, (+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7),
were tested for their ability to inhibit Na+/K+-ATPase activity
herein. The cellular enzyme adenosine 5′-triphosphatase from
the porcine cerebral cortex was treated with various
concentrations of both cardiac glycosides, and, as expected, 1
inhibited Na+/K+-ATPase activity in a concentration-depend-
ent manner, with an IC50 value of 0.23 μM (Figure 2C), but 7
did not at concentrations less than 100 μM (IC50 160.2 μM)
(Figure 2D), indicating that the cytotoxicity of 1 could result
from its Na+/K+-ATPase inhibitory activity.
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).
1 did not inhibit glucose uptake in H1299 cells (Figure 3),
indicating that this cardiac glycoside mediates its cytotoxicity
toward H1299 cells through a molecular signaling process
exclusive of glucose transport. Similar evidence has been
obtained previously, which showed that excretion of Ca2+,
3−
Mg2+, and PO4 by the ipsilateral kidney was enhanced by
(+)-digoxin (1), but no change in the maximal transport of
glucose was observed at the dose levels studied when 12−27 kg
female dogs were renal-artery infused by (+)-digoxin (1)
(0.05−0.08 mg/kg). This suggested that cardiac glycosides
may affect renal tubular excretion of inorganic phosphate
differently when compared with that of glucose.53
Na+/K+-ATPase is an electrogenic pump located in the cell
membrane to transport potassium ions in and sodium ions out.
Inhibition of this pump could result in a higher intracellular
Na+/K+ ratio followed by a prevented calcium ion exit and a
resultant higher concentration of cytoplasmic Ca2+. This, in
turn, increases calcium uptake into the sarcoplasmic reticulum
by the sarco/endoplasmic reticulum (ER) calcium ATPase2
(SERCA2) transporter and leads to tumor cell apoptosis
through cell cycle arrest and caspase activation.44 In addition,
Na+ was found to be required in the extracellular solutions to
drive uphill glucose transport, for which the energy is provided
by the sodium gradient maintained by the Na+/K+-ATPase
across the brush-border membrane, and Na+/glucose cotrans-
porters (SGLTs) depend critically on the stoichiometry of the
Na+ and sugar fluxes to accumulate sugar.45 Both the Na+/K+-
ATPase membrane protein and SERCA pump were found to
play an important role in the uptake of glucose,46,47 indicating
that the sugar pump could be closely related to the Na+ pump.
Therefore, (+)-digoxin (1) targets Na+/K+-ATPase to
mediate its cytotoxicity, as evidenced by the inhibition of
Na+/K+-ATPase observed for 1 but not for its noncytotoxic
derivative 7 and by the presence of the important HO-14β and
17β-lactone functionalities in mediating its cytotoxicity and in
its binding to Na+/K+-ATPase.
There are serious challenges for the development of
(+)-digoxin (1) as a new anticancer drug, including its narrow
clinic therapeutic index54 and poor clinical outcomes,17 of
which both could result from the inhibition of Na+/K+-ATPase
of 1.1 In the present study, both HO-14β and the 17β-lactone
moiety were characterized as important structural components
for mediation of the cytotoxicity of 1 and in the binding
between this cardiac glycoside and Na+/K+-ATPase. In
addition, the Na+/K+-ATPase inhibitory activity of 1 was
found to be blocked by synthetic derivatization of its 17β-
lactone unit to produce 7. These indicate that modifications of
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hexane−acetone (5:1 → 1:1), followed by purification using a
Sephadex LH-20 column, eluted with CH2Cl2−MeOH (1:1), and 5.0
mg (0.013 mmol) of 5 (7.9%) and 2.0 mg (0.0054 mmol) of 6 (3.3%)
were yielded.
either the HO-14β or the 17β-lactone moiety of (+)-digoxin
(1) could generate new compounds that target Na+/K+-
ATPase differentially or orient different targets to improve the
therapeutic index and clinical trial outcomes of 1.
(+)-Digoxigenin (5): amorphous, colorless powder; [α]20 +22 (c
D
0.1, MeOH); UV (MeOH) λmax (log ε) 216 (3.92) nm; ECD
(MeOH, nm) λmax (Δε) 235.5 (+4.54); IR (dried film) νmax 3390,
1732, 1622, 1446, 1382, 1028, 958, 865 cm−1; 1H and 13C NMR data,
see Tables S1 and S2 (Supporting Information); positive-ion
HRESIMS m/z 413.2336, calcd for C23H34O5Na+, 413.2299.
(+)-8(9)-β-Anhydrodigoxigenin (6): amorphous, colorless powder;
[α]20D +30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.97) nm;
ECD (MeOH, nm) λmax (Δε) 216.1 (+5.44), 241.8 (−2.58); IR
(dried film) νmax 3418, 1739, 1621, 1444, 1368, 1022, 901, 846 cm−1;
1H and 13C NMR data, see Tables S1 and S2 (Supporting
Information); positive-ion HRESIMS m/z 395.2211, calcd for
C23H32O4Na+, 395.2193.
Alkaline Hydrolysis of (+)-Digoxin (1). To a 25 mL glass vial
equipped with a magnetic stirrer, containing 10.8 mg (0.014 mmol) of
(+)-digoxin (1), was added 8 mL of MeOH saturated with NaOH,
and the vial was sealed. After the mixture was stirred at 60 °C
overnight, the mixture was acidified with 1 N HCl solution to a pH
value of 7.0. The mixture was extracted with CH2Cl2, and the CH2Cl2
partition was washed with H2O. The CH2Cl2 solution was evaporated
at reduced pressure, and the residue was separated by silica gel
column chromatography using n-hexane−acetone (5:1 → 1:1),
followed by purification over a Sephadex LH-20 column, eluted
with a mixture of CH2Cl2 and MeOH (1:1) to afford 7.0 mg (0.0088
mmol) of 7 (63.3%).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured at room temperature on an Anton Paar polarimeter
(Anton Paar, Graz, Austria). UV spectra were recorded on a Hitachi
U2910 ultraviolet spectrophotometer. ECD measurements were
performed using a JASCO J-810 spectropolarimeter. IR spectra
were recorded on a Nicolet 6700 FT-IR spectrometer. H and 13C,
1
DEPT 90, DEPT 135, HSQC, HMBC, NOESY, and COSY NMR
spectra were recorded at room temperature on a Bruker Avance II
400, Bruker Avance III HD 700, or Bruker Avance III HD 800 MHz
NMR spectrometer. ESIMS or HRESIMS data were collected on a
Bruker Maxis 4G Q-TOF mass spectrometer in the positive-ion mode.
Column chromatography was conducted using silica gel (65 × 250 or
230 × 400 mesh, Sorbent Technologies, Atlanta, GA, USA).
Analytical TLC was performed on precoated silica gel 60 F254 plates
(Sorbent Technologies). Sephadex LH-20 was purchased from
Amersham Biosciences, Uppsala, Sweden. For visualization of TLC
plates, H2SO4 was used as a spray reagent. All procedures were carried
out using solvents purchased from commercial sources and employed
without further purification. (+)-Digoxin, paclitaxel, and other
reagents for chemical synthesis were purchased from Sigma-Aldrich
(St. Louis, MO, USA) (purity ≥98%).
Acetylation of (+)-Digoxin (1). To a 25 mL glass vial equipped
with a magnetic stirrer, containing 14.1 mg (0.018 mmol) of
(+)-digoxin (1), were added 50 μL of Ac2O and 1 mL of pyridine, and
the vial was sealed. After the mixture was stirred at room temperature
for 72 h, it was cooled to room temperature. A 5 mL amount of
CH2Cl2 was added, and the solution was washed with distilled H2O
and evaporated at reduced pressure. The residue was separated by
silica gel column chromatography, using n-hexane−acetone (5:1 →
1:1), followed by purification by passage over a Sephadex LH-20
column, eluted with CH2Cl2−MeOH (1:1), and 5.0 mg (0.0058
mmol) of 2 (32.1%) and 2.0 mg (0.0022 mmol) of 3 (12.1%) were
afforded. Using the same procedure, with 26.8 mg (0.034 mmol) of
(+)-digoxin (1) being reacted with 1 mL of Ac2O and 200 μL of
pyridine at 70 °C for 2 h, 24.6 mg (0.025 mmol) of compound 4
(71.9%) was produced.
(+)-17-epi-20,22-Dihydro-21α-hydroxydigoxin (7): amorphous,
colorless powder; [α]20 +16 (c 0.1, MeOH); IR (dried film) νmax
D
1
3445, 1725, 1458, 1375, 1161, 1067, 939, 867 cm−1; H and 13C
NMR data, see Table 1; positive-ion HRESIMS m/z 821.4299, calcd
for C41H66O15Na+, 821.4294.
Cell Lines. All cell lines were purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37
°C in 5% CO2. The human HT-29 colon, MDA-MB-231 breast, and
OVCAR3 ovarian cancer and MDA-MB-435 melanoma cell lines were
cultured in RPMI 1640 medium, supplemented with FBS (10%),
penicillin (100 units/mL), and streptomycin (100 μg/mL). The
H1299 human non-small-cell lung cancer cell line was maintained in
Dulbecco’s modified Eagle medium (DMEM containing 25 mM
glucose) supplemented with FBS (10%), penicillin (100 units/mL),
and streptomycin (100 μg/mL).
(+)-12,4′c-Di-O-acetyldigoxin (2): amorphous, colorless powder;
[α]20 +33 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 210 (4.17)
Cytotoxicity Assays. The cytotoxicity of the cardiac glycosides
was screened against the human cancer HT-29, MDA-MB-231, MDA-
MB-435, or OVCAR3 cell lines, with a procedure reported
previously,27,28 with the vehicle and paclitaxel used as the negative
and positive control, respectively. Briefly, after log-phase-growth, cells
were seeded in 96-well clear flat-bottomed plates (Microtest 96,
Falcon) and treated with a test sample or paclitaxel (both dissolved in
DMSO and diluted to different concentrations required) or the
vehicle (DMSO) for 72 h. Viability of cells was evaluated using a
commercial absorbance assay (CellTiter 96 AQueous One Solution
Cell Proliferation Assay, Promega Corp, Madison, WI, USA), with the
IC50 values calculated from the vehicle control. For cytotoxicity
testing against the H1299 human non-small-cell lung cancer cell line,
cell proliferation was assessed using the MTT proliferation assay kit
(Cayman Chemical, Ann Arbor, MI, USA). Cells were seeded in each
well of a 96-well plate and treated with the samples for 24 h followed
by a treatment with MTT for 4 h. The medium was removed, and 100
μL of Crystal Dissolving Solution was added to each well. The
absorbance of the solution was measured at 570 nm, with IC50 values
calculated from the vehicle control.
D
nm; ECD (MeOH, nm) λmax (Δε) 221.4 (+5.53); IR (dried film) νmax
1
3479, 1738, 1620, 1371, 1242, 1165, 1070, 860 cm−1; H and 13C
NMR data, see Tables S1 and S2 (Supporting Information); positive-
ion HRESIMS m/z 887.4441, calcd for C45H68O16Na+, 887.4400.
(+)-12,3′c,4′c-Tri-O-acetyldigoxin (3): amorphous, colorless pow-
der; [α]20 +40 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 211
(4.29) nmD; ECD (MeOH, nm) λmax (Δε) 213.5 (+4.88); IR (dried
film) νmax 3495, 1747, 1628, 1557, 1372, 1246, 1165, 1076, 856 cm−1;
1H and 13C NMR data, see Tables S1 and S2 (Supporting
Information); positive-ion HRESIMS m/z 929.4492, calcd for
C47H70O17Na+, 929.4505.
(+)-12,3′a,3′b,3′c,4′c-Penta-O-acetyldigoxin (4): amorphous, col-
orless powder; [α]20D +54 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
215 (4.30) nm; ECD (MeOH, nm) λmax (Δε) 219.0 (+10.26); IR
(dried film) νmax 3501, 1747, 1634, 1371, 1246, 1156, 1058, 950
1
cm−1; H and 13C NMR data, see Tables S1 and S2 (Supporting
Information); positive-ion HRESIMS m/z 1013.4761, calcd for
C51H74O19Na+, 1013.4717.
Acid Hydrolysis of (+)-Digoxin (1). To a 25 mL glass vial
equipped with a magnetic stirrer, containing 126.4 mg (0.16 mmol) of
(+)-digoxin (1), was added 8 mL of concentrated HCl, and the vial
was sealed. After the mixture was stirred at room temperature
overnight, 5 mL of CH2Cl2 was added. The solution was washed with
H2O, and the organic layer was evaporated at reduced pressure. The
residue was separated by silica gel column chromatography using n-
Molecular Modeling. For the docking profiles shown in Figure 2,
the chain A of the crystal structure 4RET [(+)-digoxin] was used as
the receptor, which was prepared by MGLTools55 to add nonpolar
hydrogens and charges. The 3D structure of (+)-17-epi-20,22-
dihydro-21α-hydroxydigoxin (7) was built in Maestro and prepared
̈
by LigPrep from Schrodinger Suite 2018-2 [Schrodinger Suite 2018-2
H
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̈
Protein Preparation Wizard (Schrodinger, LLC, New York, NY,
2018)]. The geometric optimization was performed using the OPLS3
(optimized potentials for liquid simulation 3) force field with all
possible ionization states at pH 7.4 0.1 created by Epik [a software
program for pK(a) prediction and protonation state generation].56
Sixteen conformations of 7 generated by LigPrep were used for the
Mapleton, OR, USA). Differences among samples were assessed by
one-way ANOVA followed by Tukey−Kramerʼs test, and the
significance level was set at p < 0.05.
ASSOCIATED CONTENT
■
molecular docking against the receptor by Autodock Vina.57,58
A
sı
* Supporting Information
rectangular box (25 × 25 × 30 Å3) centered around the center of
Leu793, Ile787, Glu117, and Gln111 defines the region that the
ligands can explore, which could cover nearly all of the α-M1−M6 of
Na+/K+-ATPase. The top ranked 20 binding poses for each ligand
conformation, totally 320 poses, were selected for the further analysis.
(+)-Digoxin (1) was also docked to the receptor as the reference.
For molecular modeling shown in Figure S11 (Supporting
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01060.
Mass and NMR spectra of compounds 1−7; diagrams of
COSY, key HMBC, and selective NOESY correlations of
1
compounds 1−7; assignments of the H and 13C NMR
data of the known compounds 2−5; cytotoxicity toward
human cancer cells of 1−7, and analytical data of the
known compound 1 (PDF)
̈
Information), proteins and ligands were prepared from Schrodinger
Release 2016-1 [Schrodinger Release 2016-1 Protein Preparation
Wizard, Epik version 3.5, Impact version 7.0, Prime version 4.3
̈
̈
(Schrodinger, 2016)]. The Protein Preparation Wizard was used to
optimize the crystal structure of the Na+/K+-ATPase in complex with
(+)-digoxin (PDB code 4RET). Restrained minimization was
performed on the hydrogens, with the root-mean-square deviation
(RMSD) of heavy atoms being converged to less than 0.30 Å, using
the OPLS3 force field. The 3D structure of 7a with defined
stereogenic atoms was built in Maestro by modifying the 3D crystal
AUTHOR INFORMATION
■
Corresponding Author
A. Douglas Kinghorn − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States;
orcid.org/0000-0002-6647-8707; Phone: +1 (614) 247-
8094; Email: kinghorn.4@osu.edu; Fax: +1 (614) 247-8642
̈
structure of (+)-digoxin (1) (Schrodinger Release 2016-1: Ligprep,
version 3.7, Schrodinger, 2016). OPLS3 was used for ligand geometric
optimization, with all possible ionization states created at pH 7.4 by
̈
̈
̈
Epik (Schrodinger Release 2016-1: Epik, version 3.5, Schrodinger,
2016). All the stereogenic atoms were kept during LigPrep, and
default values were utilized for other parameters for protein and
ligand preparations. Molecular docking was performed using GOLD
v5.2.259 with the above prepared protein and ligands. The active site
for Na+/K+-ATPase was defined as being within 10 Å around the
catalytic site in the prepared protein. The best scoring pose for each
compound was selected for further analysis, and illustrations were
made using Chimera.60
Authors
Yulin Ren − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Hennrique T. Ribas − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Kimberly Heath − Department of Pharmaceutical Sciences,
College of Pharmacy, University of Illinois at Chicago, Chicago,
Illinois 60612, United States
Sijin Wu − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States
Jinhong Ren − Center for Biomolecular Sciences, College of
Pharmacy, University of Illinois at Chicago, Chicago, Illinois
60612, United States
Pratik Shriwas − Department of Biological Sciences, Edison
Biotechnology Institute, and Molecular and Cellular Biology
Program, Ohio University, Athens, Ohio 45701, United States
Xiaozhuo Chen − Department of Biological Sciences, Edison
Biotechnology Institute, Molecular and Cellular Biology
Program, and Department of Biomedical Sciences, Ohio
University, Athens, Ohio 45701, United States
Michael E. Johnson − Department of Pharmaceutical Sciences,
College of Pharmacy and Center for Biomolecular Sciences,
College of Pharmacy, University of Illinois at Chicago, Chicago,
Illinois 60612, United States
Na+/K+-ATPase Activity Assay. Na+/K+-ATPase activity was
assessed using a luminescent ADP detection assay (ADP-Glo Max
Assay; Promega) that measures enzymatic activity by quantitating the
ADP produced during the enzymatic first half-reaction.28 Specifically,
10 μL of assay buffer containing adenosine 5′-triphosphatase (ATP)
from porcine cerebral cortex (Sigma) was added to the wells of a 96-
well plate followed by 10 μL of DMSO or the test compounds
dissolved in DMSO. After 5.0 μL of ATP was added to each well
followed by a 15 min incubation, 25 μL of ADP-Glo Reagent was
added. After a 40 min incubation, 50 μL of kinase detection reagent
was added to each well followed by a 60 min incubation. ATP was
measured via a luciferin/luciferase reaction using a Synergy Mx
(BioTek, Winooski, VT, USA) to assess luminescence.
Glucose Uptake Assay. Using a reported protocol,52,61 after
H1299 cells grown in 24-well plates were washed and treated with
FBS-free DMEM for 1.5 h, the cells were subsequently washed and
incubated for 30 min in glucose-free KRP buffer. WZB117 (used as
the positive control) or (+)-digoxin was added to cells at a final
concentration of 30 μM. The cells were incubated for 15 min, and
glucose uptake was initiated by adding 37 MBq/L 2-deoxy-D-
[3H]glucose and 1 mM regular glucose as the final concentrations
to cells. After 40 min of incubation, the glucose uptake was terminated
by phosphate-buffered saline. Cells were lysed, and the radioactivity
retained in the cell lysates was measured by an LS 6000 series liquid
scintillation counter (Beckman Coulter, Inc., Fullerton, CA, USA).
The data were analyzed statistically using the Student’s t test, by
comparison of the data from the experimental samples with those
from the vehicle control, and p < 0.05 was set as the level of significant
difference.
Xiaolin Cheng − Division of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio 43210, United States;
orcid.org/0000-0002-7396-3225
Joanna E. Burdette − Department of Pharmaceutical Sciences,
College of Pharmacy, University of Illinois at Chicago, Chicago,
Illinois 60612, United States; orcid.org/0000-0002-7271-
6847
Statistical Analysis. The in vitro measurements were performed
in triplicate and are representative of three independent experiments,
where the values generally agreed within 10%. The dose−response
curve was calculated for IC50 determinations using nonlinear
regression analysis (Table Curve2DV4; AISN Software Inc.,
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b01060
I
https://dx.doi.org/10.1021/acs.jnatprod.9b01060
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This investigation was supported by grant P01 CA125066
funded by the National Cancer Institute, NIH, Bethesda, MD.
We thank Dr. David J. Hart, Emeritus Professor, Department
of Chemistry and Biochemistry, The Ohio State University, for
many helpful comments and suggestions about the mechanism
of the formation of compound 7. Dr. Chunhua Yuan, Nuclear
Magnetic Resonance Laboratory, and Drs. Arpad Somogy and
Nanette M. Kleinholz, Mass Spectrometry and Proteomics,
Campus Chemical Instrument Center, The Ohio State
University, are acknowledged for their assistance with NMR
and MS data collection. Drs. Craig A. McElroy and Deepa
Krishnan, College of Pharmacy, The Ohio State University, are
thanked for providing access to some of the NMR
instrumentation used in this investigation.
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