Synthesis and evaluation of the α- D -/α-l-rhamnosyl and amicetosyl digitoxigenin oligomers as antitumor agents

Article abstract of DOI:10.1021/ml100290d

A highly regio- and stereoselective asymmetric synthesis of rhamnosyl- and amicetosyl-digitoxigenin analogues has been established via palladium-catalyzed glycosylation followed by bis-/tris-dihydroxylation or bis-/tris-diimide reduction. The α-l-rhamnose and α-l-amicetose digitoxin monosaccharide analogues displayed stronger apoptosis inducing activity and cytotoxicity against nonsmall cell human lung cancer cells (NCI-H460) than its d-diastereomeric isomers in a sugar-chain length dependent manner.

Full text of DOI:10.1021/ml100290d

LETTER
pubs.acs.org/acsmedchemlett
Synthesis and Evaluation of the R-D-/R-L-Rhamnosyl and Amicetosyl
Digitoxigenin Oligomers as Antitumor Agents
Hua-Yu Leo Wang,† Yon Rojanasakul,*,‡ and George A. O’Doherty*,†
†Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
‡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 rhamnosyl- and
amicetosyl-digitoxigenin analogues has been established via palladium-catalyzed glycosyla-
tion followed by bis-/tris-dihydroxylation or bis-/tris-diimide reduction. The R-L-rhamnose
and R-L-amicetose digitoxin monosaccharide analogues displayed stronger apoptosis indu-
cing activity and cytotoxicity against nonsmall cell human lung cancer cells (NCI-H460)
than its D-diastereomeric isomers in a sugar-chain length dependent manner.
KEYWORDS: Digitoxin, digitoxigenin trirhamnoside, digitoxigenin triamicetoside, apoptosis,
cancer, NCI-H460
igitoxin 3 (Figure 1) is a well-known cardiac glycoside found in
Digitalis purpurea that has been used to treat heart congestive
D
failure by the inhibition of plasma membrane Naþ/Kþ ATPase.1 In
this event, the accumulated intracellular Naþ concentration causes
Ca2þ influx to enhance myocardial cell contractility. These findings
triggered a significant amount of research on the evaluation of the
structure activity relationship (SAR) of the carbohydrate moiety in
Naþ/Kþ ATPase binding affinity.2-5 Karlish demonstrated that
inhibition of R2- over R1-Naþ/Kþ ATPase isoform could induce
cardiac contraction with minimal Ca2þ overload and, hence, less
cardiotoxicity.5 Digitoxin has also been recognized for its excellent
antitumor activity against a wide range of human cancer cell lines.6
In fact, a digitalis clinical study on breast tumor patients showed
both reduced tumor size and reduced rate of reoccurrence.7,8
Digitoxin consists of digitoxigenin (pharmacophore) and the
trisaccharide moiety, which is known to be a critical component
for both its cardiotoxic and anticancer activity.9 Although the
detailed mechanism of anticancer activity is not fully understood,
many apoptosis signals have been found to be affected by
digitoxin at subcardiotoxic concentration in plasma.10 Studies
also suggested that an R2-selective cardiac glycoside could result
from sugar modification, because the structural differences in
these isoforms primarily lie in the extracellular carbohydrate
binding loops.5,11 Thus, sugar modification opens the potential
for discovery of new and safer digitoxin analogues with improved
anticancer activity. In addition to the study of Naþ/Kþ ATPase
in myocardial cells, Thorson and co-workers demonstrated the
potential for improving cytotoxicity against a wide range of
human cancer cell lines by screening a library of digitoxin
MeNO-neoglycoside analogues.12 Although few SAR conclu-
sions could be made, the axial C20-alcohol in the D-sugar was
suggested to be critical for anticancer activity.
Figure 1. Digitoxin 3 and Related Carbohydrate Analogues.
We later showed that digitoxin O-glycosides consistently
displayed stronger potency in apoptosis activation than MeNO-
neoglycosides regardless of sugar-chain length (i.e., mono-1 is
better than di-2 and trisaccharide 3; Figure 1).13 We further
investigated the O-linked monosaccharides for the optimal
stereochemistry and functionality required for initiating apoptosis.14
Surprisingly, this survey found two L-sugar digitoxin monosac-
charides (4 and 7) that displayed equal or better growth inhibition
(against the NCI panel of 60-cancer cell lines) than the previous
Received: December 6, 2010
Accepted: January 10, 2011
Published: January 24, 2011
r
2011 American Chemical Society
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Scheme 1. De Novo Approach to the Key Pyranone Building
Blocks 17a/b and 18a/b
Scheme 2. Stereodivergent Approach to D- and L-Digitoxin
Monosaccharides
best, digitoxin monosaccharide 1. For example, both R-L-rham-
noside (4) (IC50 46.7 nM) and R-L-amicetoside (7) (IC50 55.7 nM)
demonstrated a similar potency to β-D-digitoxoside (1) (IC50
74.8 nM) in apoptosis induction against nonsmall cell human lung
cancer cell (NCI-H460), which are all ∼10 fold more potent than
digitoxin (3) (IC50 357 nM).14,15 The effects of C50 alkyl substituents
on cytotoxicity for both the R-L-rhamno- and R-L-amiceto- mono-
saccharides were also studied (see DOI 10.1021/ml100291n).
The fact that the structurally different monosaccharide ana-
logues 1, 4, and 7 (e.g., sharing only one stereocenter and having
different numbers of hydroxyl groups) possess similar activity
suggests that the carbohydrate binding site of the target must
accommodate several distinct orientations upon binding to its
target.15 To further probe this hypothesis, we decided to test
whether di- and trisaccharide analogues of 4 and 7 would have
a similarly weakened anticancer activity. Thus, we decided to
synthesize and test di- and trisaccharide R-L-rhamnoside (5 and 6)
and R-L-amicetoside (8 and 9) analogues. In addition, all
D-rhamnoside (10-12) and all D-amicetoside (13-15) analogues
could be made and tested as a control group, to account for
nonspecific effects such as solubility and membrane transport
properties.
Scheme 3. Stereodivergent Approach to D- and L-Digitoxin
Bis- and Tris-pyran Oligomers
To rapidly assess this structurally diverse set of analogues, we
employed our de novo asymmetric synthesis of carbohydrates to
systematically prepare both stereo- and oligo-isomers of rham-
nosyl and amicetosyl digitoxin derivatives. Herein, we report the
synthesis and biological activity of digitoxin R-D-/R-L-rhamnose
and amicetose mono-, di-, and trisaccharide analogues (4-15,
Figure 1) against human lung cancer NCI-H460 cell line.
Retrosynthetically, we imagined that the desired target mole-
cules could be obtained by coupling with the digitoxigenin
(DigOH) and the R-D/L-Boc pyranones (D-17a and L-18a,
Scheme 1). Previously, we have described a de novo approach
where all the R/β-D/L-Boc pyranones could be prepared in three
steps from acetylfuran 16.16,17 Briefly, the absolute D- and L-sugar
stereochemistry was installed by Noyori asymmetric reduction.18
The pyranyl ring was prepared by Achmatowicz oxidative
rearrangement (NBS/H2O), with subsequent Boc-protection
under various conditions to give 17a/b and 18a/b in good
overall yield (60-70%).19
With the desired R-D/R-L-sugar building blocks 17a and 18a
in hand, we next employed our palladium-catalyzed glycosylation
to couple them with digitoxigenin to afford the corresponding
digitoxin pyranones 19 and 20, with complete stereocontrol at the
anomeric center (Scheme 2). The desired C40-hydroxyl group was
stereoselectively installed via Luche reduction to give allylic
alcohols 21 and 22, respectively. The resulting C20-C30 olefin
was readily oxidized or reduced in order to provide rhamnosyl 10
and 4 or amicetosyl 13 and 7 digitoxin analogues under Upjohn
dihydroxylation or diimide reduction conditions.20,21
To rapidly construct the R-linked 1,4-oligosaccharide digitox-
in (Scheme 3), we utilized the versatile digitoxin C40-allylic
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Scheme 4. Post-glycosylation Installation of Bis-rhamno-/
amiceto-functionality
Scheme 5. Post-glycosylation Installation of Tris-rhamno-/
amiceto-functionality
alcohol intermediates 21 and 22 as the desired glycosyl receptors
to couple with R-D/L-Boc-pyranones 17a and 18a via palladium
mediated glycosylation.22,23 The resulting digitoxin bis-enones
23 and 24 were reduced by NaBH4 to give exclusively C40-
equatorial alcohols 25 and 26. By simply repeating these two
steps of glycosylation and reduction, the corresponding digitoxin
tris-pyranyl alcohols 29 and 30 were prepared in both excellent
yield and diastereoselectivity.
the syntheses of the rhamnosyl 12 and 6 and amicetosyl 15 and 9
derivatives of digitoxin trisaccharide were completed by repeat-
ing these two versatile dihydroxylation and diimide reduction
transformations (Scheme 5). The key to this success relied on
the highly stereoselective bis-/tris-dihydroxylation and diimide
reduction to install four/six stereocenters in one transformation
without any reliance on protecting groups. And these complex
digitoxin trirhamnoside/amicetoside analogues were concisely
prepared in a total of seven linear steps, with 41%-46% overall
yield from the sugar building blocks.
The remaining stereocenters of C20/C30-hydroxyl groups
were rapidly installed via bis-dihydroxylation to give the desired
rhamno-stereochemistry in 11 and 5 (Scheme 4). Alternatively,
R-D-/L-amicetose digitoxin disaccharides were prepared by bis-
diimide reduction to give 14 and 8 in relatively high yield. Finally,
We next evaluated the anticancer activity of the diastereo-/
oligo-isomers of the rhamnosyl and amicetosyl digitoxigenin
Figure 2. Apoptotic cell death as effect of stereochemistry. (A and B) Apoptotic cell death (%) was compared for each R-L-/D-pair of digitoxin
rhamnoside and amicetoside at 50 nM concentration (Student t test; ***, P < 0.001). (C) Hoechst stained apoptotic cell appears blue and propidium
iodide stained necrotic cell appears red at 50 nM drug concentration.
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Figure 3. Cytotoxicity as a function of drug concentration in the comparison of sugar stereochemistry and chain length. The dose response curve of total
cell death (apoptosis/necrosis) mediated by digitoxin analogues in 12 h treatment at increasing concentrations (10 nM to 100 μM). All data were
analyzed by two-way ANOVA (N = 6; *, P < 0.05; **, P < 0.01; ***, P < 0.001).
Table 1. Cytotoxicity of Digitoxin Analogues on NCI-H460
Epithelial Human Lung Cancer Cells
(48.6%) and L-amicetose (7) (45.7%, Figure 2A and B). Thus,
the change in the D-/L-stereochemistry of the sugar has a greater
effect on the degree of apoptosis activation than substitution at
the C-2/C-3 position of the sugar (i.e., L-rhamnose (4) is
significantly more active than D-rhamnose (10) (∼20 fold),
whereas L-rhamnose (4) and L-amicetose (7) are equally active
(P > 0.05)). Regardless of sugar substitution and stereochemis-
try, the major mode of cell death is apoptosis (>85%). As shown
in Figure 2C, NCI-H460 cells underwent apoptosis, with con-
densed and fragmented nuclei seen in blue Hoechst nuclear stain,
whereas cells appeared completely ruptured in red propidium
iodide stain, indicative of necrosis. It is worth noting that the ratio
of apoptosis and necrosis become difficult to estimate at high
dose concentration (>500 nM), due to the high cell mortality
rate.
To further study the anticancer activity as the effect of sugar
chain length, the cytotoxicity assay was conducted in a 12 h expo-
sure of drugs at increasing concentrations (10 nM to 100 μM).
Our result clearly demonstrated that both L-rhamnose (4) and
L-amicetose (7) induced cell death (apoptosis and necrosis) in
both concentration dependent and sugar-chain length depen-
dent manners (Figure 3A and B). Significantly, both L-rhamnose
(4) (IC50 47 nM) and L-amicetose (7) (IC50 48 nM) showed
at least ∼10-fold stronger potency than the corresponding di- (5
and 8) and trisaccharide analogues (6 and 9, Table 1). We found
IC50
(nM) ( S.E.a
IC50
(nM) ( S.E.a
compd
compd
R-L-rhamnoside
mono-4
di-5
R-D-rhamnoside
mono-10
di-11
47 ( 1
365 ( 1
706 ( 1
4271 ( 1
18032 ( 1
tri-6
1347 ( 1
tri-12
R-L-amicetoside
mono-7
di-8
R-D-amicetoside
mono-13
di-14
48 ( 1
510 ( 1
387 ( 1
5992 ( 1
33098 ( 2
tri-9
3963 ( 1
tri-15
a All values represent the standard error of the mean value of three
independent experiments with duplicate determinations.
analogues with both R-L-rhamnose (4) and R-L-amicetose (7) as
a control against NCI-H460 human lung cancer cells. To identify
the occurrence of apoptosis, we used Hoechst 33342 nuclear
stain and propidium iodide to differentiate cells that undergo
apoptosis or necrosis.24 Comparing the effects of D- vs L-
stereochemistry on apoptosis induction, both D-rhamnose (10)
(13.6%) and D-amicetose (13) (15.3%) exhibited a significantly
reduced apoptosis activity as compared to L-rhamnose (4)
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LETTER
for these rhamnosyl and amicetosyl glycosides that the R-L-sugar
stereochemistry is essential for the potency (cf., D-analogues 10
and 13, Table 1). It is worth noting that R-L-amicetose (7) has
previously shown greater growth inhibitory effect against an
NCI-panel of 60 human cancer cell lines than R-D-amicetose
(13) (see Supporting Information). Consistent with our pre-
vious hypothesis, all the D-rhamnose (10-12) and D-amicetose
(13-15) showed a dramatically reduced cytotoxic activity with
increasing sugar chain length (Table 1).
In summary, we have prepared and evaluated the anticancer
activity of the diastereo- and oligo-isomers of rhamnosyl and
amicetosyl digitoxigenin. The syntheses of digitoxin di-/trir-
hamnoside and di-/triamicetoside analogues were successfully
achieved in a linear and highly stereoselective fashion from a
commercially available acetylfuran. Based on the SAR study in
the comparison of our previously found potent digitoxin R-L-
rhamnose (4) and R-L-amicetose (7), we identified a significant
change of cytotoxicity by altering the diastereomeric relation-
ship; L-sugar stereochemistry is suggested to exhibit a distinct
binding orientation to its target. In addition, we demonstrated
that the important structural motif for inducing anticancer
activity can be greatly optimized by shortening the carbohy-
drate chain of digitoxin analogues. Most importantly, this work
illustrates the use of palladium-catalyzed glycosylation and de
novo asymmetric synthesis to install an array of rare sugars,
which are not readily accessible via enzymatic synthesis, in
digitoxin for the development of a potential cardiac glycoside
anticancer drug.
(3) Brown, L.; Erdmann, E.; Thomas, R. Digitalis structure-activity
relationship analyses: Conclusions from indirect binding studies with
cardiac (Naþ þ Kþ)-ATPase. Biochem. Pharmacol. 1983, 32, 2767–2774.
(4) Rathore, H.; From, A. H. L.; Ahmed, K.; Fullerton, D. S. Cardiac
Glycosides. 7. Sugar Stereochemistry and Cardiac Glycoside Activity.
J. Med. Chem. 1986, 29, 1945–1952.
(5) Katz, A.; Lifshitz, Y.; Bab-Dinitz, E.; Kapri-Pardes, E.; Goldshleger,
R.; Tal, D. M.; Karlish, S. J. D. Selectivity of Digitalis Glycosides for Isoforms
of Human Na,K-ATPase. J. Biol. Chem. 2010, 285, 19582–19592.
(6) Newman, R. A.; Yang, P.; Pawlus, A. D.; Block, K. I. Cardiac
Glycosides as Novel Cancer Therapeutic Agents. Mol. Interventions
2008, 8, 36–49.
(7) Stenkvist, B.; Bengtsson, E.; Eriksson, O.; Holmquist, J.; Nordin,
B.; Westman-Naeser, S. Cardiac glycosides and breast cancer. Lancet
1979, 1, 563.
(8) Stenkvist, B.; Bengtsson, E.; Dahlqvist, B.; Eriksson, O.; Jarkrans,
T.; Nordin, B. N. Cardiac glycosides and breast cancer, revisited. N. Engl.
J. Med. 1982, 306, 484.
(9) 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.
(10) Haux, J.; Solheim, O.; Isaksen, T.; Angelsen, A. Digitoxin, in
non-toxic concentrations, inhibits proliferation and induces cell death in
prostate cancer cell lines. Z. Onkol. 2000, 32, 11–16.
(11) Wansapura, A. N.; Lasko, V.; Xie, Z.; Fedorova, O. V.; Bagrov,
A. Y.; Lingrel, J. B.; Lorenz, J. N. Marinobufagenin enhances cardiac
contractility in mice with ouabain-sensitive R1 Naþ-Kþ-ATPase. Am. J.
Physiol. Heart Circ. Physiol. 2009, 296, 1833–1839.
(12) Langenhan, L. M.; Peters, N. R.; Guzei, I. A.; Hoffmann, M.;
Thorson, J. S. Enhancing the anticancer properties of cardiac glycosides
by neoglycorandomization. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
12305–12310.
’ ASSOCIATED CONTENT
(13) Iyer, A. K. V.; Zhou, M.; Azad, N.; Elbaz, H.; Wang, L.;
Rogalsky, D. K.; Rojanasakul, Y.; O’Doherty, G. A.; Langenhan, J. M.
Direct Comparison of the Anticancer Activities of Digitoxin MeON-
Neoglycosides and O-Glycosides: Oligosaccharide Chain Length-
Dependent Induction of Caspase-9-Mediated Apoptosis. ACS Med.
Chem. Lett. 2010, 1, 326–330.
S
Supporting Information. Assay protocols, statistical
b
analysis data, synthetic procedures, characterization data, and
NMR spectra. This information is available free of charge via the
Internet at http://pubs.acs.org.
(14) 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.
(15) It is worth noting that other D- and L-sugar monosaccharides
were significantly less active than monosaccharides 1, 4, and 7; see ref 13.
(16) For the synthesis of R-/β-D-Boc pyranones, see: Zhou, M.;
O’Doherty, G. A. De Novo Approach to 2-Deoxy-β-glycosides: Asym-
metric Syntheses of Digoxose and Digitoxin. J. Org. Chem. 2007, 72,
2485–2493.
(17) For the synthesis of R-/β-L-Boc pyranones, see: Guo, H.;
O’Doherty, G. A. De Novo Asymmetric Synthesis of Anthrax Tetra-
saccharide and Related Tetrasaccharide. J. Org. Chem. 2008, 73, 5211–
5220.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: yrojan@hsc.wvu.edu; g.odoherty@neu.edu.
Author Contributions
All the experimental work was performed by H.-Y. L.W. The
experimental design, data analysis and manuscript preparation
was performed by all the authors.
’ 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 (GM090259 and
GM088839) and NSF (CHE-0749451) for the support of our
research.
(18) Fujii, A.; Hashiguchi, S.; Uematsu, N.; Ikariya, T.; Noyori, R.
Ruthenium (II)-Catalyzed Asymmetric Transfer Hydrogenation of
Ketones Using a Formic Acid-Triethylamine Mixture. J. Am. Chem.
Soc. 1996, 118, 2521–2522.
(19) Achmatowicz, O.; Bukowski, P.; Szechner, B.; Zwierzchowska,
Z.; Zamojski, A. Synthesis of methyl 2,3-dideoxy-DL-alk-2-enopyrano-
sides from furan compounds: A general approach to the total synthesis of
monosaccharides. Tetrahedron 1971, 27, 1973–1996.
(20) VanRheenen, V.; Kelly, R. C.; Cha, D. Y. An improved catalytic
OsO4 oxidation of olefins to cis-1,2-glycols using tertiary amine oxides
as the oxidant. Tetrahedron Lett. 1976, 17, 1973–1976.
(21) Haukaas, M. H.; O’Doherty, G. A. Enantioselective Synthesis of
2-Deoxy- and 2,3-Dideoxyhexoses. Org. Lett. 2002, 4, 1771–1774.
(22) Babu, R. S.; Zhou, M.; O’Doherty, G. A. De Novo Synthesis of
Oligosaccharides Using a Palladium-Catalyzed Glycosylation Reaction.
J. Am. Chem. Soc. 2004, 126, 3428–3429.
’ REFERENCES
(1) Greeff, K. Cardiac Glycosides, Part 1: Experimental Pharmacology.
Handbook of Experimental Pharmacology; Springer-Verlag: Berlin, New
York, 1981; Vol. 56.
(2) Yoda, A. Structure-Activity Relationships of Cardiotonic
Steroids for the Inhibition of Sodium- and Potassium-Dependent
Adenosine Triphosphatase. Mol. Pharmacol. 1973, 9, 51–60.
268
dx.doi.org/10.1021/ml100290d |ACS Med. Chem. Lett. 2011, 2, 264–269

ACS Medicinal Chemistry Letters
LETTER
(23) Zhou, M.; O’Doherty, G. A. The De Novo Synthesis of
Oligosaccharides: Application to the Medicinal Chemistry SAR-Study
of Digitoxin. Curr. Top. Med. Chem. 2008, 8, 114–125.
(24) Chanvorachote, P.; Nimmannit, U.; Stehlik, C.; Wang, L.; Jiang,
B.-H.; Ongpipatanakul, B.; Rojanasakul, Y. Nitric Oxide Regulates Cell
Sensitivity to Cisplatin-Induced Apoptosis through S-Nitrosylation and
Inhibition of Bcl-2 Ubiquitination. Cancer Res. 2006, 66, 6353–6360.
269
dx.doi.org/10.1021/ml100290d |ACS Med. Chem. Lett. 2011, 2, 264–269