Modifying the glycosidic linkage in digitoxin analogs provides selective cytotoxins

Article abstract of DOI:10.1016/j.bmcl.2007.11.058

A chemoselective reaction between oxyamines and unprotected, unactivated reducing sugars was used to construct for the first time a panel of linkage-diversified neoglycosides. This panel of digitoxin analogs included potent and selective tumor cytotoxins; cytotoxicity was dependent on the structure of the glycosidic linkage. These results validate linkage diversification through neoglycosylation as a unique and simple strategy to powerfully complement existing methods for the optimization of glycoconjugates.

Full text of DOI:10.1016/j.bmcl.2007.11.058

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 670–673
Modifying the glycosidic linkage in digitoxin analogs
provides selective cytotoxins
Joseph M. Langenhan,* Jeffery M. Engle, Lauren K. Slevin, Lindsay R. Fay,
Ryan W. Lucker, Kyle R. Smith and Matthew M. Endo
Department of Chemistry, Seattle University, 901 12th Avenue, Seattle, WA 98122, USA
Received 31 October 2007; revised 14 November 2007; accepted 15 November 2007
Available online 21 November 2007
Abstract—A chemoselective reaction between oxyamines and unprotected, unactivated reducing sugars was used to construct for the
first time a panel of linkage-diversified neoglycosides. This panel of digitoxin analogs included potent and selective tumor cytotox-
ins; cytotoxicity was dependent on the structure of the glycosidic linkage. These results validate linkage diversification through neo-
glycosylation as a unique and simple strategy to powerfully complement existing methods for the optimization of glycoconjugates.
Ó 2007 Elsevier Ltd. All rights reserved.
Glycoconjugates are vital probes for the exploration of
glycobiology1 and have long been used as powerful ther-
apeutics.2 Optimization of these molecules has typically
involved modification of aglycons and attached sugars
since both entities influence target recognition, selectiv-
ity, and pharmacology. Tailoring the atomic structure
of the glycosidic linkage between aglycons and attached
sugars represents a third distinct optimization strategy
that has received little attention. We hypothesized that
such structural variants would also provide compounds
with enhanced activities, and employed the well-known
model glycoside digitoxin (1) as a test platform. Here
we validate our hypothesis by developing chemoselective
glycosylation chemistry that readily supplies different
glycosidic linkages and showing that some of the result-
ing digitoxin analogs are more selective tumor cytotox-
ins than the parent natural product.
growth6 and proliferation,7 and the inducement of apop-
tosis.7a The sugar and aglycon region that encompasses
the glycosidic linkage of cardiac glycosides such as 1 ap-
pears to strongly influence the rich spectrum of biologi-
cal properties exhibited by these molecules.3,8 Thus,
digitoxin is an excellent system to test the influence of
the intervening glycosidic linkage. However, creating
structural variation at this position requires non-tradi-
tional glycosylation chemistry.
While most glycoconjugates are generated by labor
intensive chemoenzymatic or synthetic methods,9 many
chemoselective ligation strategies have been employed
to form glycosides quickly and conveniently.10 One of
the most successful methods is oxyamine neoglycosyla-
tion, the chemoselective reaction between secondary
oxyamines and unprotected, unactivated reducing sug-
ars to form stable ‘neoglycosides’ (Scheme 1).11 This
method has provided glycopeptide mimics,12 oligosac-
charide mimics,13 and large libraries of natural product
MeON-neoglycosides14 including potent digitoxin-based
cytotoxins (e.g., 3a, IC50 = 18–59 nM).14a Since digi-
toxin neoglycosides containing a non-natural MeON-
linkage displayed strong cytotoxicity, we suspected tun-
ing the structure of such glycosidic linkages would mod-
ulate glycoconjugate activities. Testing this hypothesis
required the development of oxyamine neoglycosylation
to provide other RON-linkages.
Digitoxin (1) is a cardiac glycoside drug used to treat
congestive heart failure and supraventricular arrhyth-
mias through inhibition of plasma membrane Na+/K+-
ATPase.3 In addition to this well-known activity, several
studies have suggested an enhancement in the survival
rate of cancer patients taking cardiac glycosides.4 Digi-
toxin has also been shown to display anticancer proper-
ties in vitro,5 including the inhibition of cancer cell
Keywords: Chemoselective ligation; Glycosylation; Cardiac glycoside;
Glycorandomization; Cytotoxicity.
Literature precedents suggest a variety of secondary oxy-
amines can be chemoselectively glycosylated to provide
*
Corresponding author. Tel.: +1 206 296 2368; fax: +1 206 296
5786; e-mail: langenha@seattleu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.058

J. M. Langenhan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 670–673
671
O
1. CrO3, H2SO4,
acetone, H2O
2. RONH2*HCl,
pyr, MeOH
O
OH
O
3. tBuNH2*BH3,
10 % aq. HCl,
EtOH
HO
O
O
O
O
O
HO
HO
HO
digitoxin (1)
O
"oxyamine
structural
variation
O
neoglycosylation"
O
O
L
-ribose or
-xylose
OH
OH
L
MeOH/CHCl3
AcOH (1 eq.)
40 oC
R
O
N
H
O
HO
R
N
O
secondary oxyamine
neoglycoside
L
-ribose
L
-xylose
2a: R = Me
2b: R = Et
2c: R = i Pr
2d: R = tBu
2e: R = allyl
2f: R = Bn
3a: R = Me
3b: R = Et
3c: R = i Pr
3d: R = tBu
3e: R = allyl
3f: R = Bn
4a: R = Me
4b: R = Et
4c: R = i Pr
4d: R = tBu
4e: R = allyl
4f: R = Bn
Scheme 1. Synthesis of oxyamines followed by oxyamine neoglycosylation.
hydrolytically stable glycosides without sugar activation
or protection,11,12 but in most published examples few
oxyamines other than methoxyamines such as 2a have
been utilized, leading only to MeON-glycosidic linkages
(e.g., 3a).13–15 To determine if neoglycosides with alter-
native linkages can be generated easily, we synthesized
aglycons 2 from digitoxin in three simple steps16 and
incubated them in parallel with L-ribose and L-xylose,
sugars that previously produced cytotoxic digitoxin neo-
glycosides.14a We were gratified to learn that under the
optimized reaction conditions indicated in Scheme 1,
all digitoxin aglycons containing non-methyl O-alkyl
groups except for 2d (R = tert-Bu) were compatible with
oxyamine neoglycosylation, leading to a panel of 10 digi-
toxin analogs (3a–c,e,f and 4a–c,e,f) containing different
RON-glycosidic linkages.17 The panel was purified in
parallel via solid phase extraction on silica gel cartridges
to remove unreacted aglycon and sugar, and LC–MS was
used to confirm product identity and assess product pur-
ity. Similar to previous results with MeON-glycosides,14
the average purity of the panel of neoglycosides as esti-
mated by LC–MS was 87%, and all but one compound
(3b) contained greater than 90% of a single product iso-
mer. The isolated yields for these compounds ranged
from 7% to 61% (average 30%).18 To our knowledge, this
represents the first panel of natural products that contain
diversified neoglycosidic linkages.
25.000
SKOV-3: ovarian carcinoma
NCI/ADR-RES: multi-drug resistant ovarian carcinoma
NCI-H460: lung adenocarcinoma
20.000
15.000
10.000
5.000
HT-29: colorectal adenocarcinoma
0.000
Compound
Figure 1. Summary of IC50 data from cytotoxicity assays. Reciprocal
IC50 values are displayed for clarity; standard errors are depicted with
error bars. In the assay, live cells are quantified by measuring the
luminescent signal resulting from the reaction between cellular ATP
and luciferin to form oxyluciferin. The IC50 value for each compound
represents at least four replicates of dose–response experiments
conducted over six concentrations at twofold dilutions. The IC50 of
4f against HT-29 cells was >1 lM.
cytotoxicity profile similar to digitoxin, in accord with
previous results.14a However, EtON-, AllylON-, and
BnON-glycosides (3b/4b, 3e/4e, and 3f/4f, respectively)
were markedly less potent cytotoxins than digitoxin or
MeON-glycosides 3a and 4a; like digitoxin, 3a, and 4a,
they were also non-specific.
The anticancer activities of the panel and digitoxin (1)
were assessed using a high-throughput cytotoxicity assay
on four human cancer cell lines, representing lung, colo-
rectal, and ovarian carcinomas, as well as NCI/ADR-
RES, a drug-resistant ovarian carcinoma line (Fig. 1).19
Our data clearly show that the structure of the glycosidic
linkage affects both the potency and selectivity of neogly-
cosides. Digitoxin displayed strong, non-specific cyto-
toxicity toward three of the four cell lines tested, and
MeON-glycosides 3a and 4a displayed a non-specific
Most significantly, i-PrON-glycosides 3c and 4c dis-
played enhanced selectivity relative to digitoxin and
MeON-glycosides 3a and 4a. Specifically, while 3c and
4c were only modestly cytotoxic toward lung, colorectal,
and ovarian carcinomas, these molecules were 5–6 times
more potent cytotoxins against NCI/ADR-RES cells
(IC50 = 110 20 nM and 120 10 nM, respectively)
than any other cell line tested. To the best of our knowl-

672
J. M. Langenhan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 670–673
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edge, this is the first observation of cell line selectivity
resulting from a simple alteration of glycosidic linkage.
This outcome is particularly important since NCI/
ADR-RES is a multi-drug resistant line that has high
levels of P-glycoprotein expression.19 While cardiac gly-
cosides are generally substrates for P-glycoprotein,21
such tumor specificity suggests that 3c and 3d may no
longer serve as P-glycoprotein substrates. Alternatively,
the i-PrON-glycosides may be interacting with a unique
cellular target. Supporting this notion, neoglycosides
have previously been shown to be significantly less po-
tent Na+/K+-ATPase inhibitors in HEK-239 human
embryonic kidney cells than digitoxin.14a Also, digitoxin
itself can target cellular components in addition to Na+/
K+-ATPase21 such as the TNF-a/NF-jB signaling path-
way, a regulator of inflammation responses relevant to
cancer therapy.22 Neoglycosides 3a–c and 4a–c were
more potent cytotoxins than the corresponding aglycons
(2a–c), confirming the importance of sugar attachments
to the cytotoxicity of cardiac neoglycosides (data not
shown).
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In summary, we harnessed a mild, chemoselective reac-
tion between oxyamines and unprotected, unactivated
reducing sugars to construct for the first time a panel
of linkage-diversified neoglycosides. This modestly-sized
panel of digitoxin analogs included selective tumor cyto-
toxins, validating linkage diversification through neogly-
cosylation as a unique and simple strategy to powerfully
complement existing methods for the optimization of
glycoconjugates.
13. (a) Peri, F.; Nicotra, F. Chem. Comm. 2004, 623; (b) Peri,
F.; Jimenez-Barbero, J.; Garcia-Aparicio, V.; Tvaroska, I.;
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Acknowledgments
We thank Noe¨l Peters (Keck-UWCCC Small Molecule
Screening Facility) for performing cytotoxicity assays,
Gary Girdaukus (UW-Madison School of Pharmacy
Analytical Facility) for performing LC–MS, and P.J.
Alaimo for helpful discussions. The research was sup-
ported by the Camille and Henry Dreyfus Foundation,
the Research Corporation, and Seattle University.
14. (a) Langenhan, J. M.; Peters, N. R.; Guzei, I. A.;
Hoffman, F. M.; Thorson, J. S. Proc. Natl. Acad. Sci.
2005, 102, 12305; (b) Ahmed, A.; Peters, N. R.; Fitzgerald,
M. K.; Watson, J. A., Jr.; Hoffmann, F. M.; Thorson, J. S.
J. Am. Chem. Soc. 2006, 128, 14224; (c) Griffith, B. R.;
Krepel, C.; Fu, X.; Blanchard, S.; Ahmed, A.; Edmiston,
C. E.; Thorson, J. S. J. Am. Chem. Soc. 2007, 129, 8150.
15. The distribution of pyranose, and sometimes furanose,
anomers in these glycosides is dependent on sugar identity,
but usually a single isomer predominates (see Ref. 14a).
16. Example procedure: Digitoxigenone (see Ref. 14a) (1.82 g,
4.89 mmol) was dissolved in methanol (11 mL, 2.2 mL/
mmol) and pyridine (0.87 mL, 10.8 mmol). Methoxyamine
hydrochloride (0.653 g, 7.82 mmol) was added, and the
solution was stirred for 2.5 h and then concentrated. The
resulting residue was dissolved in CH2Cl2 and washed with
1 M HCl, brine, dried over MgSO4, filtered, and then
concentrated. The mixture of oxime diastereomers was
suspended in ethanol (7 mL, 0.7 mL/mmol) and cooled to
0 °C. Borane tert-butylamine complex (1.40 g, 16.2 mmol)
was added, followed by the dropwise addition of 10% aq
HCl (13.2 mL, 2.7 mL/mmol). The reaction mixture was
stirred at 0 °C for 2.5 h. After this time, Na2CO3 was
added until gas evolution ceased, and the mixture was
partitioned between water and CHCl3. The organic layer
was washed with brine, dried over MgSO4, filtered, and
concentrated. The resulting diastereomeric mixture was
purified by SiO2 column chromatography eluting with 3:2
EtOAc/hexane to elute 2a (TLC Rf = 0.33 in 3:2 EtOAc/
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.11.058.
References and notes
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1
hexane). H NMR (CDCl3, 400 MHz) d 5.88 (s, 1H), 4.99

J. M. Langenhan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 670–673
673
(A of ABX, 1H, J = 18.0, 1.6), 4.81 (B of ABX, 1H,
J = 18.0, 1.5), 3.55 (s, 3H), 3.26 (br s, 1H), 2.79 (m, 1H),
2.15 (m, 2H), 1.85 (m, 3H), 1.74–1.22 (m, 17H), 0.94 (s,
3H), 0.87 (s, 3H); 13C NMR (CDCl3, 100 MHz): d 174.7,
174.6, 117.8, 85.7, 73.6, 62.6, 55.1, 51.1, 49.7, 42.0, 40.1,
36.7, 35.8, 35.7, 33.3, 30.5, 28.8, 27.0, 26.7, 23.9, 22.9, 21.3,
21.2, 15.9; Electrospray ionization–MS m/z (M+H) calcu-
lated for C24H37NO4 404.3, observed 404.4. The undesired
a isomer can be obtained by further elution with 100%
EtOAc.
0.89 (s, 3H), 0.77 (s, 3H); 13C NMR (DMSO-d6,
100 MHz) d 176.4, 173.9, 116.2, 90.3, 83.8, 78.5, 73.1,
70.0, 69.6, 67.3, 62.9, 56.2, 50.2, 49.5, 41.1, 36.3, 35.4,
35.3, 32.3, 30.2, 28.9, 26.6, 26.3, 23.7, 23.0, 21.1, 20.8,
15.7; Electrospray ionization–MS m/z (M+H) calculated
for C30H47NO8 550.3, obsd 550.5.
18. The isomeric mixtures were not resolved during product
purification since equilibration between product isomers
occurs (see Ref. 14a).
19. For years, NCI/ADR-RES cells have been misidentified as
drug-resistant MCF-7 breast adenocarcinoma cells, but
recently this line has been shown definitively to be a drug-
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17. Example procedure: Aglycon 2a (8.1 mg, 20 lmol) and
L-xylose (6.6 mg, 33 lmol) were added to a glass vial
equipped with a stirring flea and then were dissolved in
9:1 MeOH/CHCl3 (200 lL). AcOH was added (1.1 lL,
33 lmol) and the reaction mixture was stirred at 40 °C
for 4 days. The crude reaction mixture was concen-
trated, then suspended in 2% MeOH in CHCl3. The
crude suspension was purified on a disposable SiO2
solid-phase extraction column. Three treatments with
2 mL of 3% MeOH/CH2Cl2 eluted unreacted aglycon
and five treatments with 2 mL of 5% MeOH/CH2Cl2
eluted product 4b (100% pyranose form, b-anomer).
(TLC Rf = 0.40 in 10% MeOH/CH2Cl2). 1H NMR
(DMSO-d6, 400 MHz) d 5.91 (s, 1H), 4.97–4.90 (m,
4H), 4.52 (d, 1H, J = 5.2), 4.08 (s, 1H), 3.88 (d, 1H,
J = 8.7), 3.68 (dd, 1H, J = 10.9, 5.1), 3.51 (s, 3H), 3.40–
3.08 (m, 4H), 2.93 (dd, 1H, J = 10.6, 10.6), 2.73 (m,
1H), 2.05 (m, 2H), 1.93 (m, 1H), 1.87–1.05 (m, 18H),
22. Yang, Q.; Huang, W.; Jozwik, C.; Lin, Y.; Glasman, M.;
Caohuy, H.; Srivastava, M.; Esposito, D.; Gillette, W.;
Hartley, J.; Pollard, H. B. Proc. Natl. Acad. Sci. 2005, 102,
9631.