C5′-alkyl substitution effects on digitoxigenin α-l-glycoside cancer cytotoxicity

Article abstract of DOI:10.1021/ml100291n

A highly regio- and stereoselective asymmetric synthesis of various C5′-alkyl side chains of rhamnosyl- and amicetosyl-digitoxigenin analogues has been established via palladium-catalyzed glycosylation with postglycosylated dihydroxylation or diimide reduction. The C5′-methyl group in both α-l-rhamnose and α-l-amicetose digitoxin analogues displayed a steric directed apoptosis induction and tumor growth inhibition against nonsmall cell human lung cancer cells (NCI-H460). The antitumor activity is significantly reduced when the steric hindrance is increased at the C5′-stereocenter.

Full text of DOI:10.1021/ml100291n

LETTER
pubs.acs.org/acsmedchemlett
C50-Alkyl Substitution Effects on Digitoxigenin R-L-Glycoside
Cancer Cytotoxicity
Hua-Yu Leo Wang,† Bulan Wu,‡ Qi Zhang,† Sang-Woo Kang,*,† Yon Rojanasakul,*,§ and
George A. O’Doherty*,†
†Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
‡Department of Chemistry, and §Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown,
West Virginia 26506, United States
S Supporting Information
b
ABSTRACT: A highly regio- and stereoselective asymmetric synthesis of
various C50-alkyl side chains of rhamnosyl- and amicetosyl-digitoxigenin
analogues has been established via palladium-catalyzed glycosylation with
postglycosylated dihydroxylation or diimide reduction. The C50-methyl group
in both R-L-rhamnose and R-L-amicetose digitoxin analogues displayed a steric
directed apoptosis induction and tumor growth inhibition against nonsmall
cell human lung cancer cells (NCI-H460). The antitumor activity is signifi-
cantly reduced when the steric hindrance is increased at the C50-stereocenter.
KEYWORDS: Digitoxin, rhamnose, amicetose, apoptosis, cancer, NCI-H460
or centuries, cardiac glycosides have been used for the treat-
ment of congestive heart failure. The mechanism leading to
F
this cardiotonic activity is the result of plasma membrane Naþ/
Kþ ATPase inhibition.1,2 The accumulated intracellular Naþ
concentration increases Ca2þ sequestration by the sarcoplasmic
reticulum and enhances myocardial cell contractility via excita-
tion-contraction coupling.3 Digitoxin (3; Figure 1), a com-
monly used cardiac glycoside originally isolated from Digitalis
purpurea, has been recognized for its positive inotropic activity,
and more recently for its potential application in oncology.4,5
The cause of digitoxin cancer cell cytotoxicity has inevitably
been associated with Naþ/Kþ ATPase inhibition and the resultant
change in Ca2þ ion concentration. However, many research
groups have found digitoxin induces apoptotic cell death at a
subcardiotoxic concentration in plasma.6-10 These findings
suggest the possible role of digitoxin binding to other biological
targets as an apoptotic inducing event. In fact, direct evidence
correlating Naþ/Kþ ATPase inhibition anticancer effects is still
lacking.
Figure 1. Digitoxin (3) and related carbohydrate analogues.
differences in these isoforms lie primarily in the extracellular
carbohydrate binding loops. Thus, sugar modification could
provide the potential for the discovery of new and safer digitoxin
analogues with improved anticancer activity.
The first anticancer SAR study of digitoxin was conducted by
Thorson in 2005, when they screened a neoglycoside library of
digitoxin monosaccharide. Several β-L-sugar analogues were
found to have improved cytotoxicity over digitoxin.18 More
recently, we have had success in performing a series of systematic
SAR studies on the carbohydrate portion of digitoxin for apoptotic
cytotoxicity in the human lung cancer (NCI-H460) cell line.
These studies include a comparison of digitoxin MeNO-neo- and
Digitoxin consists of digitoxigenin (pharmacophore) and the
trisaccharide moiety, which are known to play a crucial role in
both its cardiotoxicity and anticancer activity.11 Because digitoxin’s
cardiotonic activity has been known for a much longer time,
most structure activity relationship (SAR) studies have focused
on the Naþ/Kþ ATPase inhibition and examined the effect of
modifying the carbohydrate portion of the cardiac glycosides.12-15
More recently, Karlish demonstrated that inhibition of the R2-
isoform over the R1-Naþ/Kþ ATPase isoform could induce
cardiac contraction with minimal Ca2þ overload and less cardio-
toxicity.16,17 These studies also suggest that an R2-selective cardiac
glycoside could result from sugar modification, as the structural
Received: December 6, 2010
Accepted: January 10, 2011
Published: January 24, 2011
r
2011 American Chemical Society
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Scheme 1. Retrosynthetic Analysis of Digitoxin Analogues
Scheme 2. De Novo Approach to R-L-Boc-pyranone 16a
O-glycosides (with O- better than MeNO-neo-glycosides),8 a
study of sugar-chain length (with mono- better than di- or tris-
accharides),10 and a stereochemical survey of digitoxin mono-
saccharides (with β-D-digitoxo-, R-L-amiceto-, and R-L-rhamno-
glycosides being the most active).9 From these studies, we
concluded that O-glycosides were better than MeNO-neoglyco-
sides8 and that sugars with β-D-digitoxo-, R-L-amiceto-, and R-L-
rhamno-stereochemistry were the most active.9 Regardless of the
glycosidic linkage (O-/neo- and/or R-/β-), the sugar stereo-
chemistry of the monosaccharides was more effective than those
of di- and trisaccharides.10
Scheme 3. De Novo Approach to the Pyranone Building
Blocks 16b-e
It is worth noting that these observations were stereospecific
(i.e., R-D-rhamno- and R-D-amiceto-glycosides were significantly
less active than their R-L-sugar diastereomers) and, thus, not
attributable to nonspecific solubility and/or membrane transport
properties. To further probe the specificity of the sugar portion of
the R-L-digitoxin glycosides, we decided to test the SAR-impor-
tance of the C50-alkyl position. Herein, we report our successful
synthesis and cytotoxicity study of four digitoxigenin R-L-rhamno-
glycosides (5-8) and three digitoxigenin R-L-amiceto-glycosides
(10-12) using our de novo asymmetric approach to glycoside
assembly (Figure 1).
Outlined in Scheme 1 is our retrosynthetic analysis for the
preparation of various C50-alkyl substituted digitoxin analogues.
We envisioned that the desired rhamno- and amiceto-stereoche-
mistries and functionalities could be installed by a diastereose-
lective ketone reduction and a dihydroxylation, or alternatively a
diimide reduction, of the digitoxin pyranone precursors 17a-e.
These structurally diverse pyranones, in turn, could be prepared
by a stereospecific palladium-catalyzed glycosylation to couple
digitoxigenin (DigOH) with the corresponding R-L-Boc pyra-
nones 16a-e. The preparation of these desired glycosyl donors
could be simply amended by the use of our de novo asymmetric
carbohydrate synthesis from an achiral furan starting material
with different R-substituted alkyl carboxylic acids.
Boc-pyranone sugar building blocks (16b-e, Scheme 3). The
required acetyl furans (14b-e) with various R-alkyl substitutions
(i.e., ethyl, n-propyl, isopropyl, and isobutyl) could be prepared
by a one-pot, two-step procedure.22-24 Thus, the addition of
furan (13) to a hexane solution of n-BuLi generated a solution of
2-lithiofuran, to which was added a THF solution of the appro-
priate substituted carboxylic acid (<0.5 equiv) to provide 14b-e
in good yields. Exposure of the acylfurans to our preferred Noyori
asymmetric reduction phase transfer conditions (Noyori (S, S),
HCO2Na (aq), 10 mol % CTAB) provided good yields of the
furan alcohols 15b-ewith excellent enantiomeric purity (>98% ee).
The furan alcohols 15b-e were then oxidized under the
Achmatowicz conditions (NBS/H2O), and the resulting pyra-
none intermediates were protected with Boc2O to give the desired
Boc-pyranones 16b-e in good overall yields with modest
R-selectivity.
With the desired C50-alkyl substituted R-L-Boc-pyranone
sugar building blocks 16a-e in hand, we next employed them
in a palladium-catalyzed glycosylation with digitoxigenin (DigOH)
to provide R-L-digitoxin pyranones 17a-e (82-98%) as a single
diastereomer with complete stereocontrol at the anomeric center
(Scheme 4). The desired C40-hydroxyl group was stereoselec-
tively installed via Luche reduction to give equatorial allylic
alcohols 18a-e (77-99%). The resulting C20-C30 double bonds
in 18a-e were readily dihydroxylated under Upjohn conditions
to provide C50-alkyl substituted rhamnosyl digitoxin analogues
4-8 (Condition A).25 Alternatively, the double bonds were
reduced to provide C50-alkyl substituted amicetosyl digitoxin
analogues 9-12 via a diimide reduction (Condition B).26
With the desired rhamno- and amiceto-glycosides in hand,
we decided to first evaluate their apoptotic cytotoxicity against
As we have previously described, the synthesis of C60-deoxy-
R-L-Boc-pyranone 16a was successfully achieved by a practical
three-step sequence from acetylfuran 14a (Scheme 2).19 The
absolute L-sugar stereochemistry was installed via an enantiose-
lective Noyori asymmetric reduction.20 The resulting furfural
alcohol 15a (>99% ee) was converted to the corresponding
pyranone via an Achmatowicz oxidative rearrangement and a
stereodivergent Boc-protection to yield an easily separable 4 to 1
ratio of R-/β-mixtures of Boc-pyranones in good overall yield
(60-70%).21
The flexibility of this de novo Achmatowicz approach al-
lowed us to further expand the diversity of C50-alkyl substituted
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LETTER
NCI-H460 human lung cancer cells, with R-L-rhamnose (4) and
R-L-amicetose (9) serving as controls. Using our standard
conditions, the nine compounds were tested at a single concentra-
tion (50 nM, 12 h), with the degree and the mechanism of cell death
(apoptosis or necrosis) quantified using the Hoechst 33342
nuclear stain/propidium iodide method (Figure 2).27 The blue
Hoechst nuclear stained cells with condensed and fragmented
nuclei are indicative of apoptosis, whereas the completely ruptured
cells (indicative of necrosis) were stained red with propidium
iodide (Figure 2C/D). While all the digitoxin analogues 4-12
displayed varying degrees of cytotoxicity, the C50-Me analogues
rhamno-4 and amiceto-9 were the most cytotoxic (Figure 2A
and B). Regardless of potency, apoptosis was the predominant
mechanism of cell death for all the analogues. Interestingly, the
activity trend of apoptosis significantly decreased when the size
of the C50-alkyl substituent increased.
The cytotoxicity (apoptosis and necrosis) was further evalu-
ated in a 12 h treatment of increasing concentration (10 nM to
10 μM) of drugs. These results were plotted in Figure 3, which
showed that all the analogues induced apoptotic cell death in a
dose-dependent manner. The degree of cytotoxicity resulting in
R-L-rhamno-C50-Me (4) and R-L-amiceto-C50-Me (9) concur
with the literature data from the previous study, where both
analogues 4 and 9 showed at least 4-fold stronger potency than
natural digitoxin.9 The IC50 values were determined by a non-
linear regression analysis and recorded in Table 1. As in the single
dose experiments, the IC50 values increased commensurately
with the size of the C50-substituent. This trend could be seen in
both the rhamnosides (4-8, Figure 3A) and amicetosides
(9-12, Figure 3B). Although demonstrably different, the po-
tency of the C50-Et-rhamno-analogue (5; IC50 57 nM) was only
slightly weaker than that of the C50-Me-rhamno-analogue (4;
IC50 52 nM). The sensitivity to this negative steric effect greatly
increased as subsequent methylene groups were added (e.g., C50-
n-Pr (6; IC50 116 nM), C50-i-Pr (7; IC50 130 nM), and C50-i-Bu
Scheme 4. Systematic Approach to R-L-Digitoxin C50-Alkyl
Analogues (4-12)
Figure 2. Apoptotic cell death and modification of C50 bulk. (A and B) Apoptotic cell death (%) was compared for each C50-alkyl substituted digitoxin
rhamnoside and amicetoside at 50 nM concentration (one-way ANOVA; N = 6; ***, P < 0.001). (C and D) Hoechst 33342 stained apoptotic cells appear
in blue and propidium iodide stained necrotic cells in red at 50 nM drug concentration.
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Figure 3. Cytotoxicity as a function of drug concentration in the comparison of C50-alkyl substitution. Dose response curve of total cell death
(apoptosis/necrosis) mediated by digitoxin analogues in a 12 h treatment at increasing concentrations (10 nM to 10 μM). All the data were analyzed by
two-way ANOVA (N = 6; *, P < 0.05; **, P < 0.01; ***, P < 0.001).
Figure 4. Cell viability as a function of drug concentration in the comparison of C50-alkyl substitution. The dose response curve of cell viability in a 48 h
treatment at increasing concentrations (1 nM to 10 μM). All the data were analyzed by two-way ANOVA (N = 9; *, P < 0.05; **, P < 0.01; ***, P < 0.001).
(8; IC50 180 nM)). Interestingly, this effect of the C50-alkyl
substitution appeared to be even more significant on the amiceto-
series (9-12), where there was a more significant increase in the
IC50 values with the addition of methylene groups (cf., C50-Me (9;
IC50 52 nM), C50-Et (10; IC50 87 nM), C50-i-Pr (11; IC50 533
nM), and C50-i-Bu (12; IC50 458 nM)). It is worth noting that R-L-
amiceto-C50-Me (9) has shown consistently stronger potency in
growth inhibition against the NCI-panel of 60 human cancer cell
lines than amiceto-C50-i-Pr (11) and C50-i-Bu (12) (see Supporting
Information).
Table 1. Cytotoxicity Evaluation of Digitoxin Analogues on
NCI-H460 Human Lung Cancer Epithelial Cells
a
b
a
b
compd
IC50
GI50
compd
IC50
GI50
R-L-rhamnoside
C50-Me-4
C50-Et-5
R-L-amicetoside
C50-Me-9
C50-Et-10
C50-i-Pr-11
C50-i-Bu-12
52 ( 1
57 ( 1
2 ( 1
2 ( 1
52 ( 1
87 ( 1
2 ( 1
2 ( 1
8 ( 1
8 ( 1
C50-n-Pr-6
C50-i-Pr-7
C50-i-Bu-8
116 ( 1
130 ( 1
180 ( 1
5 ( 1
533 ( 1
458 ( 1
6 ( 1
13 ( 1
To demonstrate that this steric-demand trend was not specific
to apoptosis, we decided to measure the cytotoxicity of these
digitoxin analogues with a different assay. We chose to use the
MTT assay for this comparison, which evaluated the cell viability
based on the measurement of mitochondrial enzymatic activity
after 48 h of exposure.28,27 These results (Table 1 and Figure 4)
showed a dose-dependent cytotoxic activity for each analogue
that was similar to that of the apoptosis assay. As seen earlier,
a commensurate drop in cytotoxicity was observed with the increas-
ing size of the C50-substituent. This trend was consistent across
bothrhamnoside(Figure4A) andamicetoside(Figure4B) series of
digitoxin analogues. It should be noted, however, that the greater
a The IC50 value was measured by a 12 h treatment in Hoechst and
propidium iodide stain assays. b The GI50 value was measured by a 48 h
treatment in an MTT assay. All values represent the standard error of the
mean value of three independent experiments with duplicate determinations.
steric sensitivity of the amiceto-series in the apoptosis assay was
not observed in the MTT assay.
In summary, we have demonstrated the SAR-effects of C50-alkyl
substitution on digitoxin R-L-rhamnoside (4) and R-L-amicetoside
(9). Our results suggest that both the rhamno- and amiceto-ana-
logues occupy a similar binding site and orientation in its target
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ACS Medicinal Chemistry Letters
LETTER
with a small hydrophobic pocket for the C50-Me. While these
cytotoxicity trends are not inconsistent with the models for
Naþ/Kþ ATPase inhibition, further study is needed. The effi-
ciency with which this systematic SAR-study was conducted was
enabled by the flexibility of this unique de novo approach to
carbohydrate synthesis. Further efforts aimed at optimizing cyto-
toxicity and elucidation of mechanism are ongoing and will be
reported in due course.
(9) Wang, H.-Y. L.; Xin, W.; Zhou, M.; Stueckle, T. A.; Rojanasakul,
Y.; O’Doherty, G. A. Stereochemical Survey of Digitoxin Monosacchar-
ides. ACS Med. Chem. Lett. 2011, 2 (1), pp 73–78.
(10) Wang, H.-Y. L.; Rojanasakul, Y.; O’Doherty, G. A. Synthesis
and Evaluation of the R-D-/R-L-Rhamnosyl and Amicetosyl Digitox-
igenin Oligomers as Anti-tumor Agents. ACS Med. Chem. Lett. 2011,
(see DOI 10.1021/ml100290d).
(11) Lopez-Lazaro, M.; Pastor, N.; Azrak, S. S.; Ayuso, M. J.; Austin,
C. A.; Cortes, F. Digitoxin Inhibits the Growth of Cancer Cell Lines at
Concentrations Commonly Found in Cardiac Patients. J. Nat. Prod.
2005, 68, 1642–1645.
’ ASSOCIATED CONTENT
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2774.
S
Supporting Information. Assay protocols, statistical anal-
b
ysis data, synthetic procedures, characterization data, and NMR
spectra. This information is available free of charge via the
Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: S.-W.K., s.kang@neu.edu; Y.R., yrojan@
hsc.wvu.edu; G.A.O., g.odoherty@neu.edu.
Author Contributions
All the biological experimental work was performed by H.-Y.L.W.
Analogues synthesis was primarily carried out by H.-Y.L.W., with
significant contributions being made by B.W., Q.Z., and S.-W.K.
The experimental design, data analysis and manuscript prepara-
tion was performed by all the authors.
(18) Langenhan, L. M.; Peters, N. R.; Guzei, I. A.; Hoffmann, M.;
Thorson, J. S. Enhancing the anticancer properties of cardiac glycosides
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’ ACKNOWLEDGMENT
We thank Anand Krishnan Iyer (Hampton University), Todd
A. Stueckle, and Yongju Lu (West Virginia University) for their
advice on cell culturing and cytotoxicity assays. We also thank
Jonathan Boyd (West Virginia University) for his help with data
analysis. We are grateful to the NIH (GM088839) and NSF
(CHE-0749451) for the support of our research.
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