Stereochemical survey of digitoxin monosaccharides

Article abstract of DOI:10.1021/ml100219d

A stereochemically diverse array of monosaccharide analogues of the trisaccharide-based cardiac glycoside natural product digitoxin has been synthesized using a de novo asymmetric approach. The analogues were tested for cytotoxicity against the NCI panel of 60 human cancer cell lines and in more detail against nonsmall cell human lung cancer cells (NCI-H460). The results were compared with digitoxin and its aglycone digitoxigenin. Three novel digitoxin monosaccharide analogues with β-d-digitoxose, α-l-rhamnose, and α-l-amicetose sugar moieties showed excellent selectivity and activity. Further investigation revealed that digitoxin α-l-rhamnose and α-l-amicetose analogues displayed similar antiproliferation effects but with at least 5-fold greater potency in apoptosis induction than digitoxin against NCI-H460. This study demonstrates the ability to improve the digitoxin anticancer activity by modification of the stereochemistry and substitution of the carbohydrate moiety of this known cardiac drug.

Full text of DOI:10.1021/ml100219d

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Stereochemical Survey of Digitoxin Monosaccharides
Hua-Yu Leo Wang,† Wenjun Xin,‡ Maoquan Zhou,‡ Todd A. Stueckle,§ Yon Rojanasakul,*,§
and George A. O'Doherty*,†
†Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States, and
‡Department of Chemistry and §Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown,
West Virginia 26506, United States
ABSTRACT A stereochemically diverse array of monosaccharide analogues of the
trisaccharide-based cardiac glycoside natural product digitoxin has been synthe-
sized using a de novo asymmetric approach. The analogues were tested for
cytotoxicity against the NCI panel of 60 human cancer cell lines and in more detail
against nonsmall cell human lung cancer cells (NCI-H460). The results were com-
pared with digitoxin and its aglycone digitoxigenin. Three novel digitoxin mono-
saccharide analogues with β-D-digitoxose, R-L-rhamnose, and R-L-amicetose sugar
moieties showed excellent selectivity and activity. Further investigation revealed
that digitoxin R-L-rhamnose and R-L-amicetose analogues displayed similar anti-
proliferation effects but with at least 5-fold greater potency in apoptosis induction
than digitoxin against NCI-H460. This study demonstrates the ability to improve
the digitoxin anticancer activity by modification of the stereochemistry and
substitution of the carbohydrate moiety of this known cardiac drug.
KEYWORDS Digitoxin, digitoxin monosaccharide, rhamnose, amicetose, cancer,
apoptosis, NCI-H460
igitoxin (Figure 1, 1), a cardiac glycoside found in
Digitalis purpurea, has been well studied and used
over centuries and is still used today for treating
many Naþ/Kþ ATPase independent pathways have also been
suggested.8-10
D
Structurally, digitoxin (1) consists of digitoxigenin (aglycone/
pharmacore) and the trisaccharide moiety, which is known
to play a crucial role in cell growth inhibition and tumor cell
death.11 Several research groups have studied the structure-
activity relationship (SAR) of the carbohydrate moiety of
digitoxin, although no systematic study of stereochemistry
(R/β/D/L) has been conducted.12-14 To date, the most sig-
nificant SAR study of carbohydrate portion of digitoxin on
cancer cells is that of Thorson.15 Thorson demonstrated the
potentiality to enhance the anticancer activity of digitoxin by
screening a library of MeON-neoglycoside link monosaccha-
rides to digitoxigenin.16 A limited SAR conclusion could be
made to conclude that a C20-axial alcohol in the D-sugar was
essential for cytotoxicity in many human cancer cell lines.
Importantly, they also noted that these substrates were
inactive toward Naþ/Kþ ATPase inhibition in human em-
bryonic kidney (HEK-293) and mammalian (CHO-K) cell
lines. In a direct comparison of digitoxin MeON-neoglycoside
to natural O-glycoside against a range of cancer cell lines, our
recent study has found digitoxin O-glycoside to be more
potent than MeON-neoglycoside in both apoptosis and cas-
pases activity.17 This study showed the viability of digitoxin
congestive heart failure by the inhibition of plasma mem-
brane Naþ/Kþ ATPase.1 The accumulated intracellular Naþ
concentration from this inhibition causes an increase in
Ca2þ concentration, which enhances myocardial cell con-
tractility. In addition, digitoxin is known to have antiprolif-
erative effects on cancer cells. Stenkvist found that breast
cancer patients on digitalis (digitoxin 1, digoxin 1a) had
better outcomes than untreated patients.2 They also found a
9.6-fold lower rate of recurrences for digitalis-treated pa-
tients after mastectomy.3 Because of its faster elimination
rate from the body, digoxin (1a) is more widely prescribed as
a cardiac drug than digitoxin (1). Conversely, we believe that
pharmacokinetic properties may make digitoxin a better
candidate for cancer therapy.
While no detailed mechanism of tumor cell death has
been demonstrated, many apoptosis signal pathways have
been found to be affected by digitoxin.4 For instance, digi-
toxin was shown in vitro to induce apoptosis and inhibit
cancer cell growth at a subcardiotoxic concentration in
plasma.5 It is known that the inhibition of Naþ/Kþ ATPase
activates tyrosine kinase Src, phosphoinositide-3 kinase
(PI3K), and phospholipase C signalosome complex, which
can induce many antiproliferative downstream effects re-
lated to cell growth and apoptosis.6,7 Because the anticancer
effects of digitoxin occur below its cardiotoxic concentration,
Received Date: September 18, 2010
Accepted Date: October 25, 2010
Published on Web Date: October 29, 2010
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Scheme 1. De Novo Synthesis of Digitoxin Pyranone Monosac-
charides 16a, 16b, 17a, and 17ba
Figure 1. Digitoxin 1 and related carbohydrate analogues.
with C20-deoxy D-sugar as a cytotoxic agent. In addition, we
demonstrated that the cytotoxicity was dependent upon the
sugar chain length with digitoxin monosaccharide 3 showing
at least 10-fold stronger potency than disaccharide 2 and tri-
saccharide 1 (digitoxin) against a range of cancer cell lines.
Although Thorson's neoglycorandomization study was
groundbreaking for digitoxin analogue drug discovery, it
was limited in its search of stereochemistry (e.g., β-anomer
predominately without C20-deoxysugar).16 Thus, we decided
to test additional digitoxin monosaccharide analogues to
further elucidate the role that carbohydrate chemistry plays
in the anticancer effect. In this present study, we synthesized
a series of digitoxin monosaccharide analogues (Figure 1,
4-10), as both R- and β-anomer, via a highly stereo/chemo-
selective palladium-catalyzed glycosylation.18 This de novo
sugar replacement strategy can be essentially used to sys-
tematically install a broad range of carbohydrates on natural
products in a SAR-amenable fashion. Herein, we report the
synthesis and the study of a range of digitoxin monosaccha-
rides by the use of our de novo glycosylation strategy in a
stereochemistry divergent manner. The analogues were
screened against the National Cancer Institute's (NCI) panel
of 60 human cell lines with further study of the apoptotic
effects of the most potent analogue.
Recently, we have had success using our de novo asym-
metric approach for the introduction of rare/unnatural
sugars in biologically important natural products.19-21 This
approach has great potential for the systematic synthesis of
stereochemically diverse sets of carbohydrates (R/β-D/L). The
route begins with the use of a palladium-catalyzed glycosyla-
tion to install any of the four corresponding digitoxigenin-
pyranones (e.g., R-L-14a to R-L-16a, β-L-14b to β-L-16b, R-D-15a
to R-D-17a, and β-D-15b to β-D-17b) from a given aglycone
(Scheme 1).
a Reagents: NBS, N-bromosuccinimide; DMAP, 4-dimethylaminopyridine.
rearrangement and diastereoselective Boc protection give
the resulting R-/β-diastereomeric mixtures, which were sepa-
rated by flash chromatography to give a moderate yield of
the diastereomeric pure R-/β-D- and R-/β-L-Boc-pyranones
(15a/b and 14a/b).24,25 With a complete set of Boc-pyra-
nones in hand, we employed our Pd-catalyzed glycosylation
[2.5 mol % Pd2(dba)3CHCl3, 10 mol % PPh3] to convert each
sugar building block 15a/b and 14a/b to the desired digitoxin
monosaccharide precursors 16a/b and 17a/b with excellent
yield and stereoretention at the anomeric center. The result-
ing enone functionality was then set for further postglyco-
sylation transformation into various stereochemistries and
functionalities without the reliance on protecting groups.
These efforts began with the synthesis of R-D-/L-rhamnose
and R-D-/L-amicetose digitoxin monosaccharide analogues.
The desired C40-sugar stereochemistry was diastereoselec-
tively installed via Luche reduction (NaBH4/CeCl3, 78 °C) to
give R-D-/L-allylic alcohol 5 and 8 as a single isomer (Scheme 2).
The resulting C20-C30 olefin was readily hydrolyzed to give
exclusively rhamnose 18 and 6 through Upjohn dihydrox-
ylation (OsO4/NMO) in excellent yield.26 In addition, this
functional ene moiety was reduced under a facile diimide
condition to generate R-D-/L-amicetose 4 and 7. The addition
of NMM as a solvent seemed to improve the overall yield of
this reaction.27,28
The desired glycosyl donor pyranones of any given ste-
reochemistry were easily prepared by a three-step sequence
from acylfuran 11 (Scheme 1). The absolute stereocenters
were securely installed via a highly enantioselective Noyori
asymmetric reduction to obtain the corresponding optically
pure furanyl alcohols 12 and (ent)-12 with high enantios-
electivity (ee > 98%).22,23 An Achmatowicz oxidative
Our effort toward the β-D/L-sugars began from our pre-
viously reported synthesis of 3.29,30 Briefly, a reduction of
digitoxin pyranones 17b and 16b under Luche reduction,
followed by Myers reductive rearrangement (NBSH, PPh3/
DIAD, NMM, -30 °C to room temperature) and Upjohn
dihydroxylation gave exclusively diol 3 and 9 with excel-
lent yield in three steps (Scheme 3).31 The resulting axial
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Scheme 2. Synthesis of R-D-/L-Rhamnose 18 and 6 and R-D-/
L-Amicetose 4 and 7 Digitoxin Analoguesa
appeared to be a better inhibitor of tumor cell growth by a
factor of 10 than its D-sugar diastereomer R-D-amicetose 4
(GI50 ∼ 113 nM across the 60 cell lines). Similarly, we also
found β-D-digitoxose 3 (GI50 ∼ 25 nM across the 60 cell lines)
showing greater potency than its L-sugar diastereomer β-L-
digitoxose 9 (GI50>50 nM across the 60 cell lines). Because
of the instability of the glycosidic bond upon long-term
storage, allylic alcohol 5 was not tested. We were, however,
able to test the activity of its L-sugar diastereomer 8, although
its decreased cytotoxicity could also be due to the same issue
of instability. The GI50 value for R-L-rhamnose 6 was below
the minimum concentration that could be accurately deter-
mined. On the basis of these findings, we decided to further
test the most active digitoxin analogues β-D-digitoxose 3, R-L-
amicetose 7 and R-L-rhamnose 6 against the nonsmall cell
human lung cancer cell (NCI-H460) to investigate its mode of
action. The choice of NCI-H460 was based on its intermedi-
ary activity (i.e., not the most or least sensitive cell line) to be
the representative in vitro model for our SAR study.
a Reagents: NMO, N-methylmorpholine-N-oxide; NBSH, o-nitrobenze-
nesulfonylhydrazide; and NMM, N-methylmorpholine.
We have previously shown that β-D-digitoxose 3 induces
apoptosis in SK-OV-3, NCI-H460, NCI/ADR-RES, and HT-29
cell lines, which is consistent with other findings for
digitoxin.5,11,17 Digitoxin initiates apoptosis via both intrinsic
and extrinsic pathways recognized to associate with differ-
ent downstream effectors. To evaluate the pro-apoptotic
properties of our novel digitoxin monosaccharide analogues 3,
R-L-rhamnose 6, and R-L-amicetose 7, we conducted a
cytotoxicity assay with both digitoxin 1 and the aglycone as a
control. This assay was performed with a 12 h exposure of
drugs in increasing concentration (10 nM to 10 μM). The
apoptosis activity was differentiated by Hoechst 33342
nuclear stain and propidium iodide to score the cells having
intensely condensed chromatin and/or fragmented nuclei
from the cells that undergo necrosis.
Scheme 3. Synthesis of β-D-/L-Digitoxose 3 and 9 and β-L-Olivose
10 Digitoxin Analogues
Our result clearly showed that digitoxin monosaccharide
analogues 3, 6, and 7 have significantly stronger potency
than digitoxin 1 within a range of 10-500 nM (P < 0.001) in
a dose-dependent manner (Figure 3A). A nonlinear regres-
sion analysis showed that R-L-rhamnose 6 was at least 7-fold
more active (IC50∼46.7 nM) than digitoxin 1 (IC50 ∼ 357 nM).
Interestingly, R-L-rhamnose 6 also showed better cytotox-
icity than R-L-amicetose 7 (IC50 ∼ 55.7 nM) and β-D-digitoxose
3 (IC50 ∼ 74.8 nM) in a range of 50-250 nM (P < 0.001). In
addition, R-L-amicetose 7 is comparatively more potent than
β-D-digitoxose 3at 50 nM (P<0.01). The aglycone digitoxigenin
was nearly ineffective (IC50 > 323 nM) against NCI-H460 cells
in vitro, which was consistent to the results reported by Lopez-
Lazaro.11
To further differentiate the cytotoxicity between apoptosis
and necrosis, the NCI-H460 cells were treated with Hoechst
and propidium iodide. Cellular degradation with condensed
and fragmented nuclei as shown by Hoechst stain in blue is
indicative of apoptosis, while necrotic cells appear complete-
ly ruptured in red when stained with propidium iodide. We
found for the most potent digitoxin analogue 6 that the ratio
of apoptotic and necrotic cell death was 80:20 at 50 nM
concentration (Figure 3C). It is worth noting that the ratio of
apoptosis and necrosis becomes difficult to estimate at a
high dose concentration (>500 nM) due to the high cell
C30-alcohol 9 was inverted via Mitsunobu reaction (DIAD/
PPh3, p-NO2PhCO2H) and subsequently hydrolyzed with
K2CO3 to generate β-L-olivose analogue 10. We then sub-
mitted our eight novel digitoxin monosaccharides (3-10),
along with digitoxin disaccharide 2 and trisaccharide 1 to
NCI for the evaluation of growth inhibitory effect toward
panel of 60 human cancer cell lines.
Previously, we have shown that 1, 2, and 3 possess potent
antitumor activity against 60 members of NCI cancer cell
lines.17 Expectedly, we found that nearly all of the cell lines
significantly reduced growth ability when treated with digi-
toxin monosaccharide analogues (3-10, GI50 ∼ 1-10 nM),
which showed at least 5-fold greater potency than digitoxin 1
and digitoxin disaccharide 2 (GI50 ∼ 50-100 nM, Figure 2).
In particular, digitoxin monosaccharide 3 and R-L-amicetose 7
showed excellent selectivity and growth inhibition against
several cancer cell lines, including leukemia (MOLT-4), hu-
man lung carcinoma (A459/ATCC and NCI-H460), human
colon carcinoma (HCT-116), human ovary adenocarcinoma
(OVACR-3/8), human prostate carcinoma (DU-145), and all
six human renal adenocarcinoma cell lines. Interestingly,
R-L-amicetose 7 (GI50 ∼ 12 nM across the 60 cell lines)
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Figure 2. Carbohydrate survey of digitoxin monosaccharide analogues (1 to 5 and 7 to 10) against NCI panel cell lines, where the values for
6 are represented by the dotted line. Reciprocal GI50 value is displayed for clarity.
Figure 3. (A) Concentration-response curve of the apoptosis-mediated total cell death by digitoxin analogues in 12 h treatment. (B)
Apoptotic cell death percentage was compared for each compound at 50 nM concentration (one-way ANOVA; ***, P < 0.001). (C) Hoechst-
stained apoptotic cells appear in blue, and propidium iodide-stained necrotic cells appear in red at 50 nM for compounds 1, 3, 6, 7, DIGOH.
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Figure 4. (A) Time-dependent experiment (50 nM). (B) Dose-dependent experiment (48 h). Both time- and dose-dependent data were
analyzed by two-way ANOVA (N = 9, *, P < 0.05; ***, P < 0.001).
mortality rate. However, in the comparison of digitoxin and
its analogues in apoptosis activity at 50 nM, we found that
R-L-rhamnose 6 (45.8%) showed the highest apoptosis activity
than the others (P < 0.001, Figure 3B). The β-D-digitoxose
3 (28.8%) and R-L-amicetose 7 (33%) displayed less activity,
but both showed better apoptosis induction than digitoxin
1 (8.4%) and digitoxigenin (8.5%).
inconsistent with the SAR conclusion by Fullerton for the
cardioglycoside's cardiotonic activity.32
This study highlights our de novo asymmetric method for
the unique introduction of different R/β-D/L stereoisomers,
which can be easily established via palladium-catalyzed gly-
cosylation coupled with a series of postglycosylation trans-
formations. This approach not only shows an efficient pre-
paration of novel digitoxin analogues but also demonstrates
its use for systematic SAR studies. Moreover, this comparison
of digitoxin monosaccharide stereoisomers provides better
insight into the effect of the carbohydrate moiety toward
anticancer activity for designing potential cardiac glycoside
anticancer agents. Further investigations along this line are
ongoing and will be reported in due course.
We next quantified NCI-H460 cell viability by measuring
mitochondrial activity via MTT colorimetric assay to com-
pare cell growth inhibition of our digitoxin analogues. Con-
sistent with our apoptosis results, digitoxin monosaccha-
ride analogues 3, 6, and 7 all showed significantly stronger
cytotoxicity than digitoxin 1 in both time-and dose-depen-
dent manner (P < 0.001, Figure 4A,B). In particular, we
found that digitoxin monosaccharide analogues 3, 6, and 7
showed better growth inhibition than digitoxin 1 in a low
concentration range (10-25 nM) over 48 h of treatment (P<
0.001, Figure 4B). Specifically, both R-L-rhamnose 6 (GI50 ∼ 2
nM) and R-L-amicetose 7 (GI50∼2.2 nM) showed a greater
growth inhibitoryeffect than β-D-digitoxose 3(GI50∼3.8 nM) as
well as digitoxin 1 (GI50 ∼ 10.7 nM). These results also agree to
the GI50 value from our NCI data, where R-L-rhamnose 6 (GI50
is undetermined), R-L-amicetose 7 (GI50 ∼ 3.8 nM), and
β-D-digitoxose 3 (GI50 ∼ 4.5 nM) displayed at least 3-fold
greater potency than digitoxin 1 (GI50 ∼ 12.8 nM) in NCI-
H460 cells.
In summary, we investigated our newly synthesized digi-
toxin monosaccharide analogues, in which we note a distinct
structural activity shift between the D- and the L-sugars in
their NCI growth inhibition data. For instance, this data showed
that R-L-amicetose 7 and β-D-digitoxose 3 with greater potency
than their sugar enantiomers (i.e., diastereomer), R-
D-amicetose 4 and β-L-digitoxose 9. We also found that the β-
D-digitoxose 3, R-L-rhamnose 6, and R-L-amicetose 7 sugar
moieties showed the most promising antineoplastic activity.
Both of our apoptosis and cell viability assays showed en-
hanced activity for the digitoxin monosaccharide analogues
3, 6, and 7 as compared to natural digitoxin 1. Our results
suggested an overall activity trend with R-L-rhamnose 6 g
R-L-amicetose 7 > β-D-digitoxose 3 > digitoxin 1 > digitoxi-
genin in both apoptosis induction and antiproliferation
effects in NCI-H460 cells. It is interesting to note that the
cancer cell cytotoxicity for structures 6, 7, and 3 is not
SUPPORTING INFORMATION AVAILABLE Assay protocols,
statistical analysis data, synthetic procedures, characterization
data, and NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *E-mail: tstueckle@hsc.wvu.edu
(T.A.S.). yrojan@hsc.wvu.edu (Y.R.) or g.odoherty@neu.edu
(G.A.O.).
Funding Sources: We thank the NIH (GM088839) and the NSF
(CHE-0749451) for their support of our research program.
ACKNOWLEDGMENT We thank Anand Krishnan Iyer (Hampton
University) and Yongju Lu (West Virginia University) for advice on
cell culturing and apoptosis assay.
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