Structure-activity relationship analysis of bufadienolide-induced in vitro growth inhibitory effects on mouse and human cancer cells

Article abstract of DOI:10.1021/np400034d

The in vitro growth inhibitory effects of 27 bufadienolides and eight degradation products, with two cardenolides (ouabain and digoxin) chosen as reference compounds, were analyzed by means of an MTT colorimetric assay in six human and two mouse cancer cell lines. A structure-activity analysis was then performed to highlight the most important substituents relating to the in vitro growth inhibitory activity of bufadienolides in cancer cells. Thus, the current study revealed that various bufadienolides, including gamabufotalin rhamnoside (1a), bufotalin (2a), and hellebrin (3a), displayed higher growth inhibitory activities for various human cancer cell lines when compared to ouabain and digoxin. Gamabufotalin rhamnoside (1a) was the only compound that displayed growth inhibitory effects of <1 μM in mouse cancer cells that expressed mutated forms of the Na⁺,K⁺-ATPase α-1 subunit. In addition, all genins and degradation products displayed weaker (if any) in vitro growth inhibitory effects on cancer cells when compared to their respective glycosylated homologue, with the exception of hellebrigenin (3b), which was as active as hellebrin (3a).

Full text of DOI:10.1021/np400034d

Article
pubs.acs.org/jnp
Structure−Activity Relationship Analysis of Bufadienolide-Induced in
Vitro Growth Inhibitory Effects on Mouse and Human Cancer Cells
Laetitia Moreno Y. Banuls,† Ernst Urban,‡ Michel Gelbcke,§ Francois Dufrasne,§ Brigitte Kopp,⊥
̧
Robert Kiss,† and Martin Zehl*,⊥
§
†Laboratoire de Toxicologie and Laboratoire de Chimie Pharmaceutique Organique, Faculte
́
de Pharmacie, Universite
́
Libre de
Bruxelles (ULB), 1050 Brussels, Belgium
⊥Department of Pharmacognosy and Department of Medicinal Chemistry, University of Vienna, 1090 Vienna, Austria
‡
S
* Supporting Information
ABSTRACT: The in vitro growth inhibitory effects of 27
bufadienolides and eight degradation products, with two
cardenolides (ouabain and digoxin) chosen as reference
compounds, were analyzed by means of an MTT colorimetric
assay in six human and two mouse cancer cell lines. A
structure−activity analysis was then performed to highlight the
most important substituents relating to the in vitro growth
inhibitory activity of bufadienolides in cancer cells. Thus, the
current study revealed that various bufadienolides, including
gamabufotalin rhamnoside (1a), bufotalin (2a), and hellebrin
(3a), displayed higher growth inhibitory activities for various
human cancer cell lines when compared to ouabain and digoxin. Gamabufotalin rhamnoside (1a) was the only compound that
displayed growth inhibitory effects of <1 μM in mouse cancer cells that expressed mutated forms of the Na+,K+-ATPase α-1
subunit. In addition, all genins and degradation products displayed weaker (if any) in vitro growth inhibitory effects on cancer
cells when compared to their respective glycosylated homologue, with the exception of hellebrigenin (3b), which was as active as
hellebrin (3a).
ancer is the leading cause of death in economically
Cdeveloped countries and the second leading cause of
death in developing countries.1 As emphasized by Jemal et al.,2
based on the GLOBOCAN project, approximately 12.7 million
cancer cases and 7.6 million cancer deaths are estimated to have
occurred in 2008. Of these, 56% of the cases and 64% of the
deaths occurred in the economically developing world. Various
types of cancers, including gliomas, melanomas, non-small-cell
lung cancers (NSCLC), esophageal cancers, and pancreatic
cancers, among others, remain associated with dismal
prognoses because they resist current radio- and chemo-
therapeutic treatments.3 Indeed, these cancer types display
various levels of resistance to pro-apoptotic stimuli and/or the
multidrug resistance (MDR) phenotype,3 and most of the
cytotoxic agents used in oncology as well as radiotherapy
induce pro-apoptotic stimuli. Novel types of anticancer drugs
are thus needed to overcome apoptosis resistance and/or MDR
phenotypes in those cancer types associated with dismal
prognoses. Certain cardiotonic glycosides could be potential
candidates; for example, 19-hydroxy-2″-oxovoruscharine dis-
plays similar and marked anticancer activity in chemosensitive
versus apoptosis-resistant4,5 and/or MDR6 cancer cell lines. In
addition, various cardiotonic steroids are also able to sensitize
apoptosis-resistant cancer cells to pro-apoptotic stimuli.7−9 At
the clinical level, digoxin has already been suspected to provide
therapeutic benefits to breast cancer,10 leukemia/lymphoma,11
and prostate cancer12 patients. For example, Platz and
colleagues12 have conducted an in vitro prostate cancer cell
cytotoxicity screen of 3187 compounds, and digoxin emerged
as the leading candidate. These authors then evaluated the
association between digoxin treatment (in heart failure
patients) and prostate cancer risk in 47 884 men including
regular digoxin users versus nonusers. Users of ≥10 years
showed a 25% lower prostate cancer risk compared to
nonusers.12
Cardiotonic steroids include two chemical classes, cardeno-
lides (e.g., ouabain and digoxin) and bufadienolides, which both
bind to the α subunits of the sodium/potassium pump (Na+/
K+-ATPase; NaK hereafter) as specific ligands.13,14 Bufadieno-
lides are characterized by the presence of a six-membered
lactone (α-pyrone) ring located at position C-17β.14 While the
PubMed database contained ∼37 000 references concerning
cardenolides in January 2013, it included only ∼800 references
concerning bufadienolides. When the PubMed search is
restricted to the oncology area, i.e., using “cardenolides versus
tumor” and “bufadienolides versus tumor” as keywords, the
PubMed database displayed ∼1400 and ∼130 references,
respectively. Thus, it seems that the potential anticancer effects
Received: February 5, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 1. Determination by Means of the MTT Colorimetric Assay of the Concentration that Reduces in Vitro by 50% (after
Three Days of Culture with the Test Compound) the Growth of Various Mouse and Human Cancer Cell Lines
IC50 in vitro growth inhibitory concentration (nM) after 3 days of culture
mouse cancer cell
a
b
b
compound
name
human cancer cell lines
lines
SKMEL-
28
code Hs683
MCF-7
PC-3
<1
A549
U373
mean SEM CT26.WT B16F10
gamabufotalin rhamnoside
1a
2a
3a
4a
5a
6a
7a
8a
2
9
8
<1
14
3
5
65
<3
877
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
659
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
bufotalin
37
44
34
11
74
36
39 11
29 10
227 86
50 11
533 382
506 224
943 517
hellebrin
7
6
68
5α-bufalin
21
9
428
36
67
22
362
86
463
70
argentinogenin
bovogenin A
scillarenone
43
53
40
73
312
278
341
726
2431
284
426
51
62
336
1607
3506
333
302
399
384
3α-acetylamino-16-acetoxy-14,15-
epoxybufadienolide
arenobufagin diacetate
bufarenogin
9a
2617
2684
3656
7264
9301
>10 000
7417
5910
3843
7135
8809
>10 000
7046
7985
>10 000
>10 000
>7098
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
10a
11a
>7208
>7196
3-O-acetyl-14,15-epoxy-19-
>10 000
5343
oxobufadienolide
c
ouabain
28
40
133
426
43
75
27
50
77
211
266
86 30
167 62
>10 000
>10 000
>10 000
>10 000
c
digoxin
146
a
b
The chemical structure of each compound is depicted in Table 2. The origin and histological type of each cell line analyzed are as follows. Human
glioma model lines included the Hs683 oligodendroglioma (ATCC code HTB-138) and the U373 glioblastoma (ECACC code 89081403) cell lines.
Melanoma models included the human SKMEL-28 (ATCC code HTB-72) and the mouse B16F10 (ATCC code CRL-6475) cell lines. Carcinoma
models included the A549 NSCLC (DSMZ code ACC107), the MCF-7 breast (DSMZ code ACC115), and the PC-3 prostate (DSMZ code
c
ACC465) human cancer cell lines, as well as the mouse CT26.WT colon (ATCC code CRL-2638) cancer cell line. Positive control substance.
of bufadienolides have been investigated far less than those
relating to cardenolides.
and digoxin. For example, gamabufotalin rhamnoside (1a) was
approximately 100 times more potent in terms of cancer cell
growth inhibition than ouabain or digoxin (Table 1). Bufotalin
(2a) and hellebrin (3a), while more active than ouabain and
digoxin, were about 10 times less active than gamabufotalin
rhamnoside (Table 1). Cardenolides and bufadienolides target
the NaK α subunits. NaK is an integral membrane protein
found in the cells of all higher eukaryotes and is responsible for
translocating sodium and potassium ions across the cell
membrane, utilizing ATP as the driving force.3,18 It is also a
versatile signal transducer and a key player in cell adhesion, and
its aberrant expression and activity are implicated in the
development and progression of different types of cancers.3
NaK is composed of two subunits. The catalytic α subunit (of
which four different isoforms have been characterized to date)
is a multipass transmembrane protein containing the binding
sites for Na+, K+, ATP, and cardiotonic steroids. The β
regulatory subunit (of which three different isoforms have been
characterized) is a transmembrane protein with several
glycosylation sites that is required for the biogenesis and
activity of the enzyme complex.3,17
The aim of the present study, therefore, is the character-
ization of the in vitro growth inhibitory effects of 27
bufadienolides and eight degradation products thereof, with
two cardenolides (ouabain and digoxin) used as reference
compounds. The current study then attempted a structure−
activity (SAR) analysis to determine the most important
substituents for the growth inhibitory activity of bufadienolides
and derivatives thereof in six human and two mouse cancer cell
lines.
RESULTS AND DISCUSSION
■
One of the current coauthors recently contributed to an in-
depth review concerning bufadienolides and their antitumor
activity,14 and it appears that several studies have already
highlighted the in vivo antitumor efficacy of various
bufadienolides. Bufalin exhibits significant antitumor effects in
human xenografts of hepatocellular carcinoma,15 and resibufo-
genin showed similar effects on HeLa human tumor growth in
nude mice.16
Gamabufotalin rhamnoside (1a) was the only compound
that displayed growth inhibitory activity of <1 μM in the mouse
cancer cell lines studied, while the observed effects were
markedly less pronounced than in human cancer cells (Table
1). In fact, rodent NaK shows a roughly 1000-fold lower affinity
for ouabain compared to human NaK.19 This feature is caused
by two mutations (human-rat Q117R and N128D) in the
extracellular loop between TM1 and TM2 in murine α1
compared to human α1. These mutations also account for the
approximately 1000-fold weaker sensitivity of murine cancer
cells to the growth inhibitory effects of cardiotonic steroids.17
Yang et al.20 demonstrated that the relative lack of the NaK α3
subunits in rodent cancer cells may also account for the
unresponsiveness to cardiotonic steroids, but Lin et al.21
In the present study, the growth inhibitory activity of
selected bufadienolides and bufadienolide degradation products
on six human and two mouse cancer cell lines was determined
by an MTT colorimetric assay, and the results were compared
to the two cardenolides ouabain and digoxin (Table 1). These
two reference compounds are very well studied with respect to
their in vitro activities on various cancer cell lines. Furthermore,
digoxin is a marketed drug for treating congestive heart failure
and atrial fibrillation (Lanoxin), so that plenty of in vivo and
pharmacokinetic data are available, and ouabain is of particular
interest since it is assumed to be an endogenous hormone in
humans.10−13,17
The data in Table 1 indicate that several bufadienolides
display higher in vitro growth inhibitory activities than ouabain
B
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C
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recently emphasized the importance of the NaK α1 subunit in
tumor growth and cancer cell survival. According to the data
illustrated in the present study, gamabufotalin rhamnoside (1a)
would mainly target the NaK α1 subunit because it is 100 to
1000 times less active in rodent than in human cancer cells.
While the α1 subunit is upregulated in non-small-cell lung
cancers, gliomas, melanomas, and kidney cancers, the α3
subunit is upregulated in colon cancers.3 Impairing α1 NaK
subunit activity is much more detrimental to cancer than to
normal cells.3,5,17 We are currently accumulating the necessary
amount of gamabufotalin rhamnoside (1a) to characterize its in
vivo antitumor activity in various cancer models.
relative configuration of rings A, B, C, and D, only the cis
configuration between rings C and D is crucial for the growth
inhibition of cancer cells; rings C and D cis-configured
compounds are the most potent growth inhibitors.
In conclusion, the current study reveals that various
bufadienolides, including gamabufotalin rhamnoside (1a),
bufotalin (2a), and hellebrin (3a), display higher growth
inhibitory activities in various human cancer cell lines when
compared to two cardenolides chosen as references, ouabain
and digoxin. While genins of cardiac glycosides typically display
weaker growth inhibitory effects on cancer cells when
compared to their glycosylated homologues, the exception
was hellebrigenin (3b), which was as active as hellebrin (3a).
None of the tested bufadienolide degradation products lacking
the α-pyrone showed activity at 10 μM or below. In agreement
with previous studies, our SAR analysis showed that the 14β-
OH group is very important for the activity and that a 10β-
CHO moiety rather increases while a 16β-O-acetyl or a 15α-
OH group rather decreases growth inhibitory activities in
various human cancer cell lines.
Precise SAR analysis appeared difficult to perform due to the
large variety of chemical groups present in bufadienolides and
their degradation products. For instance, no conclusion can be
drawn regarding the effects of substituents on positions 10, 11,
and 12. The only elements worth mentioning are the potent
activities of compounds possessing an aldehyde moiety and an
α-OH on C-11, as already observed for numerous other
bufadienolides.22,23 Other positions show trends only because
too few compounds have been analyzed. This is the case for the
sugar moiety at position 3, the double bond at position 9, and
the −OH group at position 5 (only two or three compounds
were available for comparison in each case; for the double bond
between C-9 and C-11, only one compound was available). In
contrast, more relevant observations can be made about the
importance of some chemical functions, as detailed below. The
double bond between C-4 and C-5 has no effect on the
biological activity of the compounds (Table 2, compare
compound 2c vs 7a). The β-configured alcohol on position
14 is of critical importance, as it is the case for structurally
related steroids.22,23 None of the 14 most active bufadienolides
and only three of the 22 most active compounds lack this
substituent. This hydroxy group is responsible for donating a
hydrogen bond to an asparagine in the binding pocket.3,22
When this donating effect is prevented by inclusion of the
oxygen atom in an epoxide, the activity is lower (see
compounds 6b and 11a).24 As already observed by Kamano
et al.,22,23 polar or sterically hindered substituents at position 15
are rather unfavorable in terms of the growth inhibition of
cancer cells (see compound 4b or 4d). Substituents on position
16 also have a deleterious effect on the growth inhibition of
cancer cells (see compounds 2a vs 1b and 2b vs 4b), although
some discrepancies were observed (e.g., compare compounds
8b and 11a). However, when substitution does take place, an
acetoxy group is preferred to a hydroxy group, a feature
previously observed in cinobufagin derivatives.24 The reason for
these effects is that this face of the steroidal backbone is
directed toward a rather lipophilic area of the binding pocket.3
The α-pyrone is essential for the growth inhibition of cancer
cells, because the 23 active compounds (i.e., with a mean IC50
value of <10 μM on the six human cancer cell lines) possess
this heterocyclic moiety at position 17. Only chemical groups
efficiently mimicking the unsaturated lactone group in terms of
interactive chemical group and size can be used in this position,
as already pointed out by Cerri et al.25 and Gupta.26 Within this
framework, alkylcarboxylate moieties (the alternative chemical
groups for the α-pyrone in the test compound group) are not
relevant for this purpose. Indeed, these groups fail in having the
optimal size requested to fill the pocket (in being either too
small or too large), while the α-pyrone matches it very well.
Additionally, they are unable to make the right interactions,
while unsaturated esters are more suitable.22,26 Regarding the
EXPERIMENTAL SECTION
■
General Experimental Procedures. CD spectra in the range
230−650 nm were obtained from solutions in CHCl3 (1 mg/mL) with
a cell length of 0.1 cm on a JASCO J-810 spectropolarimeter. NMR
spectra were recorded on a Bruker Avance 500 NMR spectrometer
(UltraShield) using a 5 mm switchable probe (PA BBO 500SB BBF-
1
H-D-05-Z, H, BB = 19F and 31P−15N) with z axis gradients and an
automatic tuning and matching accessory (BrukerBioSpin, Rheinstet-
1
ten, Germany). The resonance frequencies were 500.13 MHz for H
NMR and 125.75 MHz for 13C NMR. All measurements were
performed in solutions of fully deuterated chloroform or methanol at
298 K. Standard 1D and gradient-enhanced (ge) 2D experiments, such
as double-quantum filtered (DQF) COSY, NOESY, HSQC, and
HMBC, were performed according to the manufacturer’s instructions.
The chemical shifts are referenced internally to the residual,
nondeuterated solvent signal for chloroform 1H (δ 7.26 ppm) or
methanol 1H (δ 3.31 ppm) and to the carbon signal of the solvent for
chloroform 13C (δ 77.00 ppm) or methanol 13C (δ 49.00 ppm).
ESIMSn spectra were obtained on a 3D-ion trap mass spectrometer
(HCT, Bruker Daltonics, Bremen, Germany), and HRESIMS spectra
were recorded on an ESI-Qq-TOF mass spectrometer (micrOTOF-Q
II, Bruker Daltonics) in the positive-ion mode by direct infusion. The
HPLC analyses were performed as previously described14,27 using a
Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with an
LC-10AD VP binary pump, an SPD-M10A VP diode array detector, an
SCL-10A VP system controller, an autosampler, and a DGU-14A
degasser. The samples were separated on a Nucleosil C18 column (4.0
× 250 mm, 5 μm, Macherey-Nagel, Duren, Germany). The mobile
̈
phase consisted of MeCN and water (adjusted to pH 3.3 with acetic
acid). A multistep gradient program was used as follows: 8% MeCN (0
min), 54% MeCN (45 min), 54% MeCN (55 min), and 95% MeCN
(70 min). The flow rate was 0.8 mL/min. Online UV spectra were
recorded in the range 190−400 nm.
Isolation and Derivatization of the Bufadienolides. All
investigated compounds (1a−11b) were obtained from the
“bufadienolide collection” of the Department of Pharmacognosy,
University of Vienna, and were in part gifts from Prof. Dr. Tadeus
Reichstein (University of Basel, Switzerland). The purities of the above
listed compounds were >98%, as determined by HPLC analysis. The
acetylation of bufadienolides28 and the degradation of bufadieno-
lides28−33 were described previously. Gamabufotalin 3-O-α-L-rhamno-
side (1a) was isolated from red-colored bulbs of plants of the Urginea
maritima aggregate34 and bulbs of tetraploid Urginea aphylla,35 Urginea
maritima sensu strictu,36 and Urginea hesperia.37 Hellebrigenin (3b)
was in part isolated from the venom of Bufo melanosticus38 and in part
obtained by deacetylation of 3-acetylhellebrigenin. For the deacetyla-
D
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tion, 36 mg of 3-acetylhellebrigenin was dissolved in 20 mL of dry
methanol. Then, 5 mL of sodium methanolate solution (500 mg of
sodium was dissolved in 50 mL of dry methanol) was added, and the
mixture was stirred for 1 h at 20 °C. Next, 50 mL of NH4Cl solution
(5% in water) was added, and the mixture was extracted with
dichloromethane (50 mL). The organic layer was separated, dried with
sodium sulfate, and evaporated to dryness to give 17 mg of
hellebrigenin (3b). The structures of all compounds were verified or
determined by extensive NMR and mass spectrometry experiments.
The compounds gamabufotalin (1b),39 (3β,5β,11α,14β,17β)-3,11-
bis(acetyloxy)-14-hydroxyandrostane-17-carboxylic acid methyl ester
(1c),40,41 bufotalin (2a),39 3-O-acetylbufotalin (2b),42 bufotalone
(2c),43 (5β,14β,17β)-5,14-dihydroxy-3-diphenylmethoxyandrostane-
17β-carboxylic acid diphenylmethyl ester (2d),31 hellebrin (3a),44
hellebrigenin (3b),45 5α-bufalin (4a),46 3-O-acetylbufalin (4b),45 3-
epi-bufalin (4c),47 15α-hydroxybufalin 3-acetate (4d),22
(3β,5β,14α,15α)-14,15-epoxy-3-hydroxybufa-20,22-dienolide (4g),22
argentinogenin (5a),28 bovogenin A (6a),42 bovogenin A 3-acetate
(6b),42 scillarenone (7a),42,48 desacetylcinobufagin 3-acetate (8b),22
(3β,5β,14β,15β,16β,17β)-3-(acetyloxy)-14,15-epoxy-16-hydroxyan-
drostane-17-carboxylic acid methyl ester (8c),32 arenobufagin 3,11-
diacetate (9a),32,33,42 (3β,5β,11α,14β,17β)-3,11-bis(acetyloxy)-14-hy-
droxy-12-oxoandrostane-17-carboxylic acid methyl ester (9b),32,33
bufarenogin (10a),33,49 11α,12β-dihydroxybufalin (10b),28,50 bufar-
enogin 3,12-diacetate (10c),28,33,42 (3β,5β,12α)-3,12-bis(acetyloxy)-
14-hydroxy-11-oxobufa-20,22-dienolide (10d),33,42 (3β,5β,12β,17β)-
3,12-bis(acetyloxy)-11-oxoandrostane-17-hydroxymethyl-α-acetate
(10e),33 resibufagin acetate (11a),51 and 3β-azidoresibufogenin
(11b)41 were previously described.
m, H-5), 1.65 (1H, m, H-2a), 1.65 (1H, m, H-2b), 1.65 (1H, m, H-
12a), 1.60 (1H, m, H-12b), 1.61 (1H, m, H-1a), 1.58 (1H, m, H-7a),
1.44 (1H, d, J = 14.2 Hz, H-4b), 1.38 (1H, m, H-1b), 1.37 (1H, m, H-
11a), 1.34 (1H, m, H-7b), 1.19 (1H, m, H-6b), 1.10 (1H, m, H-11b),
0.99 (3H, s, H-19), 0.67 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz)
δ 161.71 (C, C-24), 160.72 (HCOO), 148.64 (CH, C-21), 145.22
(CH, C-22), 117.19 (C, C-20), 115.77 (CH, C-23), 72.75 (C, C-14),
70.72 (CH, C-3), 57.66 (CH, C-15), 43.39 (CH, C-17), 42.11 (C, C-
13), 36.95 (CH, C-5), 36.53 (CH, C-9), 34.93 (C, C-10), 34.16 (CH2,
C-12), 32.31 (CH, C-8), 30.37 (CH2, C-1), 30.23 (CH2, C-4), 29.26
(CH2, C-16), 25.30 (CH2, C-6), 24.99 (CH2, C-2), 23.44 (CH3, C-
19), 20.71 (CH2, C-7), 19.52 (CH2, C-11), 16.04 (CH3, C-18);
ESIMS m/z 413.2 [M + H]+; ESIMS2 (413.2 →) m/z 413.1 (11),
367.1 (100), 349.1 (13); ESIMS3 (413.2 → 367.1 →) m/z 367.1 (40),
349.1 (11), 225.0 (10), 223.0 (14), 221.0 (14), 215.1 (40), 213.0 (11),
211.0 (14), 209.0 (20), 207.0 (17), 205.0 (12), 199.0 (17), 197.0 (22),
195.0 (35), 193.0 (18), 187.0 (42), 185.0 (23), 183.0 (36), 181.0 (35),
180.0 (11), 179.0 (22), 175.0 (12), 173.0 (23), 171.0 (31), 169.0 (65),
167.0 (28), 166.0 (11), 165.0 (29), 161.0 (22), 159.0 (73), 157.0 (53),
155.0 (41), 154.0 (11), 153.0 (33), 149.0 (12), 147.0 (33), 145.0 (73),
144.0 (12), 143.0 (62), 142.0 (25), 141.0 (62), 135.0 (21), 133.1 (59),
131.1 (100), 129.0 (52), 128.1 (61), 121.1 (23), 119.1 (53), 117.1
(51), 115.1 (36), 109.1 (25), 107.1 (36), 105.1 (71); HRESIMS m/z
+
413.2355 [M + H]+ (calcd for C25H33O5 , 413.2323, Δ = 7.9 ppm).
(3α,5β,15β,16β)-3-(Acetylamino)-16-(acetyloxy)-14,15-epox-
ybufa-20,22-dienolide (8a): colorless powder (acetone); mp 294−
306 °C; CD (c 2.1 × 10−3 M, CHCl3) λmax (Δε) 230 (−1.54) nm; 1H
NMR (CDCl3, 500 MHz) δ 7.92 (1H, brd, H-22), 7.15 (1H, brd, H-
21), 6.21 (1H, d, J = 9.8 Hz, H-23), 5.46 (1H, d, J = 9.5 Hz, H-16),
3.79 (1H, m, H-3), 3.64 (1H, s, H-15), 2.77 (1H, d, J = 9.5 Hz, H-17),
2.02 (1H, td, J = 12.0, 3.8 Hz, H-8), 1.96 (3H, s, NCOCH3), 1.90 (3H,
s, OCOCH3), 1.81 (1H, m, H-1a), 1.79 (1H, m, H-6a), 1.76 (1H, m,
H-12a), 1.72 (1H, m, H-2a), 1.60 (1H, td, J = 12.0, 3.2 Hz, H-9), 1.55
(1H, m, H-4a), 1.51 (1H, m, H-7a), 1.51 (1H, m, H-11a), 1.50 (1H,
m, H-4b), 1.49 (1H, m, H-5), 1.38 (1H, m, H-12b), 1.29 (1H, m, H-
11b), 1.28 (1H, m, H-6b), 1.16 (1H, m, H-1b), 1.16 (1H, m, H-2b),
0.95 (3H, s, H-19), 0.90 (1H, m, H-7b), 0.80 (3H, s, H-18); 13C NMR
(CDCl3, 125 MHz) δ 170.29 (OCOCH3), 169.35 (NCOCH3), 161.70
(C, C-24), 151.50 (CH, C-21), 148.23 (CH, C-22), 116.19 (C, C-20),
113.87 (CH, C-23), 74.60 (CH2, C-16), 72.44 (C, C-14), 59.35 (CH,
C-15), 50.25 (CH, C-17), 49.28 (CH, C-3), 45.11 (C, C-13), 41.62
(CH, C-5), 39.97 (CH, C-9), 39.95 (CH2, C-12), 35.28 (CH2, C-1),
34.84 (C, C-10), 33.27 (CH2, C-4), 33.14 (CH, C-8), 27.88 (CH2, C-
2), 25.87 (CH2, C-6), 23.46 (NCOCH3), 23.33 (CH3, C-19), 20.71
(CH2, C-11), 20.63 (CH2, C-7), 20.56 (OCOCH3), 17.19 (CH3, C-
18); ESIMS m/z 484.2 [M + H]+; ESIMS2 (484.2 →) m/z 424.2
(100); ESIMS3 (484.2 → 424.2 →) m/z 424.2 (11), 382.1 (56), 365.1
(100), 347.1 (19), 215.1 (11); ESIMS4 (484.2 → 424.2 → 365.1 →)
m/z 365.1 (14), 347.1 (100), 319.1 (12), 305.1 (16), 283.0 (18),
265.0 (11), 251.0 (11), 241.1 (47), 237.0 (12), 225.1 (14), 223.0 (11),
215.1 (16), 213.1 (32), 211.0 (16), 195.0 (12), 187.0 (15), 185.0 (11),
181.0 (12), 175.0 (13), 173.0 (14), 171.0 (18), 159.1 (54), 157.1 (59),
155.0 (14), 147.1 (15), 145.1 (36), 143.1 (23), 142.0 (12), 133.1 (11),
131.1 (91), 129.1 (15), 128.1 (11), 119.1 (13), 105.1 (13); HRESIMS
(3β,5β,14α)-3-(Acetyloxy)-14-hydroxy-15-oxo-bufa-20,22-di-
enolide (4e): colorless solid (acetone/diethyl ether); mp 256−262
°C; CD (c 2.3 × 10−3 M, CHCl3) λmax (Δε) 231 (+0.53), 299 (+1.21)
1
nm; H NMR (CDCl3, 500 MHz) δ 7.28 (1H, d, J = 2.6 Hz, H-21),
7.22 (1H, dd, J = 9.3, 2.6 Hz, H-22), 6.33 (1H, d, J = 9.3 Hz, H-23),
5.09 (1H, s, H-3), 3.46 (1H, t, J = 9.5 Hz, H-17), 2.72 (1H, dd, J =
18.9, 9.5 Hz, H-16a), 2.23 (1H, m, H-7a), 2.19 (1H, dd, J = 18.9, 9.5
Hz, H-16b), 2.05 (3H, s, OCOCH3), 2.01 (1H, m, H-8), 1.98 (1H, m,
H-6a), 1.95 (1H, m, H-9), 1.93 (1H, m, H-4a), 1.91 (1H, m, H-12a),
1.69 (1H, m, H-5), 1.62 (1H, m, H-2a), 1.55 (1H, m, H-2b), 1.53 (1H,
m, H-1a), 1.46 (1H, m, H-11a), 1.46 (1H, m, H-4b), 1.38 (1H, m, H-
12b), 1.35 (1H, m, H-1b), 1.27 (1H, m, H-6b), 1.23 (1H, m, H-11b),
1.20 (1H, m, H-7b), 0.99 (3H, s, H-19), 0.65 (3H, s, H-18); 13C NMR
(CDCl3, 125 MHz) δ 211.53 (C, C-15), 170.73 (OCOCH3), 161.54
(C, C-24), 149.20 (CH, C-21), 144.75 (CH, C-22), 116.34 (C, C-20),
115.88 (CH, C-23), 81.30 (C, C-14), 70.31 (CH, C-3), 45.73 (C, C-
13), 40.97 (CH, C-17), 36.86 (CH, C-5), 36.52 (CH2, C-16), 35.34
(C, C-10), 35.17 (CH, C-8), 32.62 (CH, C-9), 30.84 (CH2, C-1),
30.46 (CH2, C-4), 28.14 (CH2, C-12), 25.92 (CH2, C-6), 24.86 (CH2,
C-2), 23.38 (CH3, C-19), 21.49 (OCOCH3), 19.73 (CH2, C-7), 19.56
(CH2, C-11), 14.98 (CH3, C-18); ESIMS m/z 443.2 [M + H]+;
ESIMS2 (443.2 →) m/z 443.2 (15), 383.1 (100); ESIMS3 (443.2 →
383.1 →) m/z 329.1 (12), 231.0 (11), 221.0 (11), 213.1 (61), 211.0
(17), 209.0 (13), 203.1 (20), 199.0 (11), 197.0 (15), 195.0 (16), 193.0
(12), 187.0 (20), 185.0 (32), 183.0 (19), 181.0 (14), 175.0 (12), 173.0
(19), 171.0 (58), 169.0 (20), 167.0 (11), 165.0 (11), 161.0 (20), 159.0
(44), 157.0 (100), 155.0 (20), 153.0 (10), 151.0 (11), 149.0 (13),
147.1 (39), 145.0 (61), 143.0 (70), 142.0 (29), 141.0 (22), 135.1 (18),
133.1 (49), 131.1 (84), 129.1 (54), 128.1 (51), 123.1 (17), 121.1 (25),
119.1 (22), 117.1 (35), 115.1 (23), 109.1 (23), 107.1 (18); HRESIMS
+
m/z 484.2682 [M + H]+ (calcd for C28H38NO6 , 484.2694, Δ = −2.4
ppm).
(3β,5β,11α,14β,17β)-3,11-Bis(acetyloxy)-14-(formyloxy)-12-
oxo-androstane-17-carboxylic acid methyl ester (9c): colorless
solid (diethyl ether); mp 131−134 °C; [α]25D +66 (CHCl3); 1H NMR
(CDCl3, 500 MHz) δ 8.08 (1H, s, OCHO), 5.46 (1H, d, J = 11.7 Hz,
H-11), 5.08 (1H, s, H-3), 4.00 (1H, dd, J = 9.5, 6.0 Hz, H-17), 3.66
(3H, s, COOCH3), 2.84 (1H, dd, J = 14.8, 7.6 Hz, H-15a), 2.76 (1H,
td, J = 12.1, 3.8 Hz, H-8), 2.21 (1H, m, H-16a), 2.16 (1H, m, H-9),
2.12 (3H, s, OCOCH3 at 11), 2.05 (3H, s, OCOCH3 at 3), 1.95 (1H,
m, H-6a), 1.89 (1H, m, H-16b), 1.81 (1H, m, H-4a), 1.77 (1H, m, H-
5), 1.71 (1H, m, H-1a), 1.70 (1H, m, H-7a), 1.69 (2H, m, H-2),1.51
(1H, m, H-4b), 1.48 (1H, m, H-7b), 1.46 (1H, m, H-1b), 1.39 (1H, m,
H-15b), 1.34 (1H, m, H-6b), 1.33 (3H, s, H-18), 1.16 (3H, s, H-19);
13C NMR (CDCl3, 125 MHz) δ 202.73 (C, C-12), 174.68 (C, C-20),
+
m/z 443.2440 [M + H]+ (calcd for C26H35O6 , 443.2428, Δ = 2.7
ppm).
(3α,5β,14α,15α)-14,15-Epoxy-3-(formyloxy)-bufa-20,22-di-
enolide (4f): colorless solid (CHCl3); mp 240−243 °C; CD (c 2.4 ×
1
10−3 M, CHCl3) λmax (Δε) 230 (+1.75), 293 (+0.88) nm; H NMR
(CDCl3, 500 MHz) δ 8.06 (1H, s, OCHO), 7.17 (1H, m, H-21), 7.16
(1H, m, H-22), 6.29 (1H, d, J = 11.1 Hz, H-23), 5.21 (1H, s, H-3),
3.53 (1H, s, H-15), 2.46 (1H, dd, J = 11.1, 6.6 Hz, H-17), 2.17 (1H, td,
J = 11.8, 4.4 Hz, H-8), 2.09 (1H, dd, J = 13.6, 6.6 Hz, H-16a), 1.99
(1H, td, J = 14.2, 2.6 Hz, H-4a), 1.90 (1H, tt, J = 14.0, 4.6 Hz, H-6a),
1.78 (1H, dd, J = 11.8, 3.5 Hz, H-9), 1.72 (1H, m, H-16b), 1.68 (1H,
170.55 (C, OCOCH3 at 3), 169.66 (C, OCOCH3 at 11), 97.31 (C, C-
E
dx.doi.org/10.1021/np400034d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
14), 74.24 (CH, C-11), 69.92 (CH, C-3), 64.39 (C, C-13), 51.63
(CH3, OCH3), 45.58 (CH, C-17), 38.09 (CH, C-5), 37.34 (CH, C-8),
37.02 (CH, C-9), 36.95 (C, C-10), 33.22 (CH2, C-1), 30.72 (CH2, C-
4), 29.86 (CH2, C-15), 25.97 (CH2, C-6), 25.30 (CH2, C-2), 23.40
(CH3, C-19), 22.57 (CH2, C-7), 22.53 (CH2, C-16), 21.44 (CH3,
OCOCH3 at 3), 20.76 (CH3, OCOCH3 at 11), 16.33 (CH3,C-18);
ESIMS m/z 515.1 [M + Na]+, 447.1 [M − HCOOH + H]+; ESIMS2
(447.1 →) m/z 447.1 (15), 415.1 (100); ESIMS3 (447.1 → 415.1 →)
m/z 345.0 (23), 327.0 (12), 285.0 (100), 267.0 (42), 239.0 (16),
185.0 (14), 159.0 (34); HRESIMS m/z 515.2210 [M + Na]+ (calcd
for C26H36O9Na+, 515.2252, Δ = −8.1 ppm).
Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany).
The code number and histological type of each of the cell lines used in
the current study are detailed in Table 1.
The U373 cell line was cultured in MEM culture medium (Lonza;
code 12-136F), while the Hs683, SKMEL-28, B16F10, A549, PC-3,
MCF-7, and CT26 cell lines were cultured in RPMI culture medium
(Lonza; code 12-115F) supplemented with 10% heat-inactivated fetal
bovine serum (Lonza; code DE14-801F). Cell culture media were
supplemented with 4 mM glutamine (Lonza; code BE17-605E), 100
μg/mL gentamicin (Lonza; code 17-5182), and penicillin−streptomy-
cin (200 units/mL and 200 μg/mL) (Lonza; code 17-602E).
Determination of the IC50 Growth Inhibitory Concentrations
in Vitro. The MTT colorimetric assay was used as detailed
previously.52,53 Briefly, this test measures the number of metabolically
active (living) cells that are able to transform the yellow substrate 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
into the blue formazan dye via a mitochondrial reduction involving
succinate dehydrogenase. The amount of formazan obtained at the end
of the experiment (measured by spectrophotometry) is directly
proportional to the number of living cells. The determination of the
optical density in the control compared to the treated cells therefore
enables quantitative measurements of the effects of compounds on the
growth of normal as well as cancer cells in vitro. Each experimental
condition was assessed in six replicates.
(3β,5β,11α,14β,17β)-3,11-Bis(acetyloxy)-14-hydroxy-12-oxo-
androstane-17-carboxylic acid γ-lactone (10f): colorless solid
(diethyl ether/petroleum ether); mp 240−258 °C; CD (c 2.3 × 10−3
M, CHCl3) λmax (Δε) 230 (+0.86), 291 (+1.81) nm; 1H NMR
(CDCl3, 500 MHz) δ 5.46 (1H, d, J = 10.1 Hz, H-11), 5.09 (1H, s, H-
3), 3.01 (1H, d, J = 4.1 Hz, H-17), 2.53 (1H, m, H-8), 2.32 (1H, t, J =
10.9 Hz, H-9), 2.16 (1H, m, H-16a), 2.11 (3H, s, OCOCH3 at 11),
2.06 (3H, s, OCOCH3 at 3), 2.01 (1H, m, H-6a), 1.96 (1H, m, H-
15a), 1.88 (1H, m, H-7a), 1.86 (1H, m, H-4a), 1.86 (1H, m, H-5),
1.78 (1H, m, H-2a), 1.68 (1H, m, H-2b), 1.75 (1H, m, H-15b), 1.62
(1H, m, H-16b), 1.55 (1H, m, H-4b), 1.49 (1H, m, H-1a), 1.43 (1H,
m, H-7b), 1.37 (1H, m, H-6b), 1.33 (1H, m, H-1b), 1.29 (3H, s, H-
18), 1.23 (3H, s, H-19); 13C NMR (CDCl3, 125 MHz) δ 200.44 (C,
C-12), 175.98 (C, C-20), 170.57 (OCOCH3 at 3), 169.55 (OCOCH3
at 11), 91.47 (C, C-14), 72.81 (CH, C-11), 69.84 (CH, C-3), 61.26
(C, C-13), 50.53 (CH, C-17), 40.58 (CH, C-9), 37.78 (CH, C-5),
37.19 (C, C-10), 33.98 (CH, C-8), 32.93 (CH2, C-1), 31.00 (CH2, C-
4), 27.45 (CH2, C-15), 26.19 (CH2, C-6), 25.30 (CH2, C-2), 23.76
(CH3, C-19), 22.45 (CH2, C-7), 21.44 (OCOCH3 at 3), 20.54
(OCOCH3 at 11), 18.73 (CH2, C-16), 14.86 (CH3, C-18); ESIMS m/
z 433.1 [M + H]+; ESIMS2 (433.1 →) m/z 433.1 (15), 415.1 (24),
387.1 (13), 373.0 (100), 355.0 (60), 313.0 (31), 295.0 (97), 267.0
(18), 249.0 (11), 213.0 (33), 187.0 (21); ESIMS3 (433.1 → 295.0 →)
m/z 295.0 (12), 267.0 (21), 249.0 (23), 239.0 (38), 213.0 (100),
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR data of compounds 1a−11b, as well as H
1
and 13C NMR spectra of compounds 4e, 4f, 8a, 9c, 10f, and
10g. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
+
187.0 (54); HRESIMS m/z 433.2214 [M + H]+ (calcd for C24H33O7 ,
Corresponding Author
433.2221, Δ = −1.5 ppm).
*Tel: +43 1 4277 55219. Fax: +43 1 4277 9552. E-mail: martin.
zehl@univie.ac.at.
(3β,5β,11α,12β,14β,17β)-3,11,12-Tris(acetyloxy)-14-hydroxy-
androstane-17-carboxylic acid methyl ester (10g): colorless
solid (acetone/diethyl ether); mp 186−190 °C; CD (c 2.0 × 10−3 M,
Notes
1
The authors declare no competing financial interest.
CHCl3) λmax (Δε) 231 (+0.62) nm; H NMR (CDCl3, 500 MHz) δ
5.11 (1H, m, H-12), 5.10 (1H, m, H-3), 5.10 (1H, m, H-11), 3.70
(3H, s, COOCH3), 2.65 (1H, m, H-17), 2.28 (1H, m, H-9), 2.26 (1H,
m, H-15a), 2.12 (3H, s, OCOCH3 at 12), 2.06 (1H, m, H-16a), 2.05
(3H, s, OCOCH3 at 3), 1.97 (1H, m, H-7a), 1.94 (1H, m, H-4a), 1.92
(1H, m, H-16b), 1.92 (1H, m, H-6a), 1.90 (3H, s, OCOCH3 at 11),
1.89 (1H, m, H-15b), 1.78 (1H, td, J = 12.1, 3.8 Hz, H-8), 1.68 (1H,
m, H-1a), 1.66 (2H, m, H-2), 1.65 (1H, m, H-5), 1.47 (1H, m, H-4b),
1.35 (1H, m, H-1b), 1.30 (1H, m, H-7b), 1.25 (1H, m, H-6b), 1.13
(3H, s, H-18), 1.01 (3H, s, H-19); 13C NMR (CDCl3, 125 MHz) δ
179.36 (C, C-20), 170.73 (OCOCH3 at 3), 170.13 (OCOCH3 at 12),
169.86 (OCOCH3 at 11), 84.07 (C, C-14), 76.78 (C, C-12), 70.97
(CH, C-11), 70.32 (CH, C-3), 52.28 (CH3, OCH3), 52.03 (CH, C-
17), 51.69 (C, C-13), 39.45 (CH, C-8), 38.59 (CH, C-5), 35.64 (CH2,
C-15), 35.59 (C, C-10), 33.40 (CH2, C-1), 32.46 (CH, C-9), 30.87
(CH2, C-4), 26.57 (CH2, C-16), 26.15 (CH2, C-6), 25.44 (CH2, C-2),
23.36 (CH3, C-19), 21.49 (OCOCH3 at 3), 21.20 (OCOCH3 at 12),
21.17 (OCOCH3 at 11), 21.04 (CH2, C-7), 15.73 (CH3, C-18);
ESIMS m/z 531.2 [M + Na]+, 491.1 [M − H2O + H]+; ESIMS2
(491.1 →) m/z 459.1 (15), 431.1 (100), 389.1 (29), 371.1 (36), 311.1
(25); ESIMS3 (491.1 → 431.1 →) m/z 371.1 (62), 329.1 (12), 311.1
(100), 279.0 (14), 251.0 (83), 229.0 (15); ESIMS3 (491.1 → 251.0
→) m/z 251.0 (16), 223.0 (17), 209.0 (25), 195.0 (60), 183.0 (25),
181.0 (29), 171.0 (17), 169.0 (87), 159.0 (13), 157.0 (45), 155.0 (13),
143.0 (100), 119.0 (20); HRESIMS m/z 531.2566 [M + Na]+ (calcd
for C27H40O9Na+, 531.2565, Δ = 0.2 ppm).
ACKNOWLEDGMENTS
■
We thank J. Konig and S. Haselmaier (Department of
̈
Nutritional Sciences, University of Vienna) for facilitating the
HRESI mass spectrometric measurements and J. Winkler
(Department of Medicinal Chemistry, University of Vienna) for
technical assistance with the CD spectroscopy. L.M.Y.B. is a
Ph.D. researcher and R.K. a director of research with the Fonds
National de la Recherche Scientifique (FRS-FNRS, Belgium).
REFERENCES
■
(1) World Health Organization. The Global Burden of Disease: 2004
Update; World Health Organization: Geneva, 2008.
(2) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman,
D. CA-Cancer J. Clin. 2011, 61, 69−90.
(3) Mijatovic, T.; Dufrasne, F.; Kiss, R. Curr. Med. Chem. 2012, 19,
627−646.
(4) Mijatovic, T.; Roland, I.; Van Quaquebeke, E.; Nilsson, B.;
Mathieu, A.; Van Vynckt, F.; Darro, F.; Blanco, G.; Facchini, V.; Kiss,
R. J. Pathol. 2007, 212, 170−179.
(5) Lefranc, F.; Mijatovic, T.; Kondo, Y.; Sauvage, S.; Roland, I.;
Debeir, O.; Krstic, D.; Vasic, V.; Gailly, P.; Kondo, S.; Blanco, G.; Kiss,
R. Neurosurgery 2008, 62, 211−221.
Cancer Cell Lines. Human and murine normal and cancer cell
lines were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA), the European Collection of Cell
Culture (ECACC, Salisbury, UK), and the Deutsche Sammlung von
(6) Mijatovic, T.; Jungwirth, U.; Heffeter, P.; Hoda, M. A. R.;
Dornetshuber, R.; Kiss, R.; Berger, W. Cancer Lett. 2009, 282, 30−34.
(7) Frese, S.; Frese-Schaper, M.; Andres, A. C.; Miescher, D.;
Zumkehr, B.; Schmid, R. A. Cancer Res. 2006, 66, 5867−5874.
F
dx.doi.org/10.1021/np400034d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(8) Simpson, C. D.; Mawji, I. A.; Anyiwe, K.; Williams, M. A.; Wang,
X.; Venugopal, A. L.; Gronda, M.; Hurren, R.; Cheng, S.; Serra, S.;
Behesthi Zavarreh, R.; Datti, A.; Wrana, J. L.; Ezzat, S.; Schimmer, A.
D. Cancer Res. 2009, 69, 2739−2747.
(37) Krenn, L.; Jambrits, M.; Kopp, B. Planta Med. 1988, 54, 227−
232.
(38) Gao, H.; Zehl, M.; Kaehlig, H.; Schneider, P.; Stuppner, H.;
Moreno Y. Banuls, L.; Kiss, R.; Kopp, B. J. Nat. Prod. 2010, 73, 603−
608.
(9) Dong, Y.; Yin, S.; Li, J.; Jiang, C.; Ye, M.; Hu, H. Apoptosis 2011,
16, 394−403.
(39) Ye, M.; Guo, H.; Guo, H.; Han, J.; Guo, D. J. Chromatogr. B
2006, 838, 86−95.
(10) Stenkvist, B.; Pengtsson, E.; Dahlquist, B.; Eriksson, O.;
Jarkrans, T.; Nordin, B. N. Engl. J. Med. 1982, 306, 484−484.
(11) Goldin, A. G.; Safa, A. R. Lancet 1984, 323, 1134−1134.
(12) Platz, E. A.; Yegnasubramanian, S.; Liu, J. O.; Chong, C. R.;
Shim, J. S.; Kenfield, S. A.; Stampfer, M. J.; Willet, W. C.; Giovannucci,
E.; Nelson, W. G. Cancer Discovery 2011, 1, 68−77.
(13) Mijatovic, T.; Ingrassia, L.; Facchini, V.; Kiss, R. Expert Opin.
Ther. Targets 2008, 12, 1403−1417.
(40) Lardon, A.; Reichstein, T. Pharm. Acta Helv. 1952, 27, 287−302.
(41) Terness, P.; Navolan, D.; Dufter, C.; Kopp, B.; Opelz, G. Int.
Immunopharmacol. 2001, 1, 119−134.
(42) Gsell, L.; Tamm, C. Helv. Chim. Acta 1969, 52, 551−568.
(43) Hoeriger, N.; Zivanov, D.; Linde, H. H. A.; Meyer, K. Helv.
Chim. Acta 1972, 55, 2549−2562.
(44) Muhr, P.; Kerek, F.; Dreveny, D.; Likussar, W.; Schubert-
Zsilavecz, M. Liebigs Ann. Recl. 1995, 1995, 443−444.
(45) Cunha-Filho, G. A.; Resck, I. S.; Cavalcanti, B. C.; Pessoa, C. O.;
Moraes, M. O.; Ferreira, J. R. O.; Rodrigues, F. A. R.; dos Santos, M. L.
Toxicon 2010, 56, 339−348.
(14) Gao, H.; Popescu, R.; Kopp, B.; Wang, Z. Nat. Prod. Rep. 2011,
28, 953−969.
(15) Han, K. Q.; Huang, G.; Gu, W.; Su, Y. H.; Huang, X. Q.; Ling,
C. Q. World J. Gastroenterol. 2007, 13, 3374−3379.
(16) Wang, C. S.; Li, Y. Y.; Chen, N.; Li, Y. Z.; Xiao, J. J.; Meng, S.
C.; Dong, X. M.; Guo, D. A.; Huang, Q. F. J. Beijing Univ. Tradit. Chin.
Med. 2006, 29, 184−187.
(46) Hauser, E.; Linde, H. H. A.; Zivanov, D. Helv. Chim. Acta 1972,
55, 2625−2628.
(47) Ma, X.; Cui, J.; Zheng, J.; Guo, D. J. Mol. Catal. B Enzym. 2007,
48, 42−50.
(17) Mijatovic, T.; Van Quaquebeke, E.; Delest, B.; Debeir, O.;
Darro, F.; Kiss, R. Biochim. Biophys. Acta Cancer Rev. 2007, 1776, 32−
57.
(48) Zhang, X.; Ye, M.; Dong, Y.; Hu, H.; Tao, S.; Yin, J.; Guo, D.
Phytochemistry 2011, 72, 1779−1785.
(49) Kamano, Y.; Kotake, A.; Takano, R.; Morita, H.; Takeya, K.;
Itokawa, H. Heterocycles 1998, 49, 275−280.
(18) Liang, M.; Tian, J.; Liu, L.; Pierre, S.; Liu, J.; Shapiro, J.; Xie, Z.
J. J. Biol. Chem. 2007, 282, 10585−10593.
(50) Qiao, L.; Huang, Y.; Cao, J.; Zhou, Y.; Qi, X.; Pei, Y. J. Asian
Nat. Prod. Res. 2008, 10, 224−227.
(19) Katz, A.; Lifshitz, Y.; Bab-Dinitz, E.; Kapri-Pardes, E.;
Goldshleger, R.; Tal, D. M.; Karlish, S. J. J. Biol. Chem. 2010, 285,
19582−19592.
(51) Kamano, Y.; Yamamoto, H.; Hatayama, K.; Tanaka, Y.;
Shinohara, M.; Komatsu, M. Tetrahedron Lett. 1968, 54, 5669−5672.
(52) Janssen, T.; Darro, F.; Petein, M.; Raviv, G.; Pasteels, J. L.; Kiss,
R.; Schulman, C. C. Cancer 1996, 77, 144−149.
(20) Yang, P.; Menter, D. G.; Cartwright, C.; Chan, D.; Dixon, S.;
Suraokar, M.; Mendoza, G.; Liansa, N.; Newman, R. A. Mol. Cancer
Ther. 2009, 8, 2319−2328.
(21) Lin, Y.; Ho, D. H.; Newman, R. A. J. Exp. Ther. Oncol. 2010, 8,
271−286.
(22) Kamano, Y.; Kotake, A.; Hashima, H.; Inoue, M.; Morita, H.;
Takeya, K.; Itokawa, H.; Nandachi, N.; Segawa, T.; Yukita, A.; Saitou,
K.; Katsuyama, M.; Pettit, G. R. Bioorg. Med. Chem. 1998, 6, 1103−
1115.
(23) Kamano, Y.; Yamashita, A.; Nogawa, T.; Morita, H.; Takeya, K.;
Itokawa, H.; Segawa, T.; Yukita, A.; Saito, K.; Katsuyama, M.; Pettit, G.
R. J. Med. Chem. 2002, 45, 5440−5447.
(24) Ye, M.; Qu, G.; Guo, H.; Guo, D. J. Steroid Biochem. Mol. Biol.
2004, 91, 87−98.
(53) Mijatovic, T.; Mathieu, V.; Gaussin, J. F.; De Neve, N.;
Ribaucour, F.; Van Quaquebeke, E.; Dumont, P.; Darro, F.; Kiss, R.
Neoplasia 2006, 8, 402−412.
(54) Van Goietsenoven, G.; Hutton, J.; Becker, J. P.; Lallemand, B.;
Robert, F.; Lefranc, F.; Pirker, C.; Vandenbussche, G.; Van Antwerpen,
P.; Evidente, A.; Berger, W.; Prevost, M.; Pelletier, J.; Kiss, R.; Kinzy,
́
T. G.; Kornienko, A.; Mathieu, V. FASEB J. 2010, 24, 4575−4584.
(25) Cerri, A.; Almirante, N.; Barassi, P.; Benicchio, A.; Fedrizzi, G.;
Ferrari, P.; Micheletti, R.; Quadri, L.; Ragg, E.; Rossi, R.; Santagostino,
M.; Schiavone, A. O.; Serra, F.; Zappavigna, M. P.; Melloni, P. J. Med.
Chem. 2000, 43, 2332−2349.
(26) Gupta, S. P. Chem. Rev. 2012, 112, 3171−3192.
(27) Gao, H.; Zehl, M.; Leitner, A.; Wu, X.; Wang, Z.; Kopp, B. J.
Ethnopharmacol. 2010, 131, 368−376.
(28) Spengel, S.; Linde, H. H. A.; Meyer, K. Helv. Chim. Acta 1973,
56, 2827−2834.
(29) Meyer, K.; Linde, H. In Venomous Animals and Their Venoms;
Bucherl, W.; Buckley, E. E., Eds.; Academic Press: New York, 1971;
Vol. 2, Chapter 40, pp 521−556.
(30) Linde, H.; Meyer, K. Helv. Chim. Acta 1959, 42, 807−826.
(31) Bernoulli, F.; Linde, H.; Meyer, K. Helv. Chim. Acta 1962, 45,
240−251.
(32) Hofer, P.; Linde, H.; Meyer, K. Helv. Chim. Acta 1960, 43,
1950−1955.
(33) Huber, K.; Linde, H.; Meyer, K. Helv. Chim. Acta 1967, 50,
1994−2008.
(34) Krenn, L.; Kopp, B.; Deim, A.; Robien, W.; Kubelka, W. Planta
Med. 1994, 60, 63−69.
(35) Krenn, L.; Kopp, B.; Griesmayer-Camus, E.; Kubelka, W. Sci.
Pharm. 1992, 60, 65−72.
(36) Krenn, L.; Ferth, R.; Robien, W.; Kopp, B. Planta Med. 1991, 57,
560−565.
G
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