N -(2-{3-[3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide (6b): A novel anticancer glycyrrhetinic acid derivative that targets the proteasome and displays anti-kinase activity

Article abstract of DOI:10.1021/jm200285z

18-β-Glycyrrhetinic acid (GA; 1) and many of its derivatives are cytotoxic in cancer cells. The current study aims to characterize the anticancer effects of 17 novel 1 derivatives. On the basis of these studies, N-(2-{3-[3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide (6b) appeared to be the most potent compound, with IC₅₀in vitro growth inhibitory concentrations in single-digit micromolarity in a panel of 8 cancer cell lines. Compound 6b is cytostatic and displays similar efficiency in apoptosis-sensitive versus apoptosis-resistant cancer cell lines through, at least partly, the inhibition of the activity of a cluster of a dozen kinases that are implicated in cancer cell proliferation and in the control of the actin cytoskeleton organization. Compound 6b also inhibits the activity of the 3 proteolytic units of the proteasome. Compound 6b thus represents an interesting hit from which future compounds could be derived to improve chemotherapeutic regimens that aim to combat cancers associated with poor prognoses.

Full text of DOI:10.1021/jm200285z

ARTICLE
pubs.acs.org/jmc
N-(2-{3-[3,5-Bis(trifluoromethyl)phenyl]ureido}ethyl)-
glycyrrhetinamide (6b): A Novel Anticancer Glycyrrhetinic Acid
Derivative that Targets the Proteasome and Displays Anti-Kinase
Activity
Benjamin Lallemand,† Fabien Chaix,§ Marina Bury,‡ Cꢀeline Bruyꢁere,‡ Jean Ghostin,§ Jean-Paul Becker,^
Cꢀedric Delporte,# Michel Gelbcke,# Vꢀeronique Mathieu,‡ Jacques Dubois,† Martine Prꢀevost,^ Ivan Jabin,§
and Robert Kiss*,‡
†Laboratoire de Chimie Bioanalytique, Toxicologie et Chimie Physique Appliquꢀee, ‡Laboratoire de Toxicologie, and #Laboratoire de
Chimie Pharmaceutique Organique, Facultꢀe de Pharmacie, Universitꢀe Libre de Bruxelles (ULB), and §Laboratoire de Chimie Organique
and ^Laboratoire de Structure et Fonction des Membranes Biologiques, Facultꢀe des Sciences, ULB, Brussels, Belgium
S Supporting Information
b
ABSTRACT: 18-β-Glycyrrhetinic acid (GA; 1) and many of its
derivatives are cytotoxic in cancer cells. The current study aims to
characterize the anticancer effects of 17 novel 1 derivatives. On
the basis of these studies, N-(2-{3-[3,5-bis(trifluoromethyl)-
phenyl]ureido}ethyl)-glycyrrhetinamide (6b) appeared to be
the most potent compound, with IC50 in vitro growth inhibitory
concentrations in single-digit micromolarity in a panel of 8
cancer cell lines. Compound 6b is cytostatic and displays similar
efficiency in apoptosis-sensitive versus apoptosis-resistant can-
cer cell lines through, at least partly, the inhibition of the activity
of a cluster of a dozen kinases that are implicated in cancer
cell proliferation and in the control of the actin cytoskeleton
organization. Compound 6b also inhibits the activity of the 3 proteolytic units of the proteasome. Compound 6b thus represents an
interesting hit from which future compounds could be derived to improve chemotherapeutic regimens that aim to combat cancers
associated with poor prognoses.
’ INTRODUCTION
that triggers the pro-apoptotic pathway. Lee et al.9 report that 1
potentiates the apoptotic effects of trichostatin A, a histone deace-
tylase inhibitor, in ovarian cancer cells by increasing the activation of
the caspase-8-dependent pathway as well as the activation of the
mitochondria-mediated cell death.
18-β-Glycyrrhetinic acid (GA; 1) is the main constituent of
Glycyrrhiza glabra, which has long been used as an antitussive,
anti-inflammatory, antiulcer, antiallergic, immunotropic, and
hypolipidemic agent.1 The in vitro growth inhibiting activity of
1 was reported three decades ago in mouse2 and human3 cancer
cell lines, and the 1-induced in vivo activity has also been reported
in various experimental cancer models.4ꢀ6 1 displays both
cytostatic5,7,8 and cytotoxic7,9 effects in cancer cells, depending
on the concentrations, the cell line, and durations of treatment.
Cytostatic effects are mediated through the arrest of cancer cells
into the G1 phase of the cell cycle as it was shown for 1 and
related compounds including ursolic acid and 18 β-erythrotriol at
20ꢀ25 μM range concentrations.7
Schwarz and Csuk12 have claimed that improving the anti-
tumor activity of 1 without affecting its pro-apoptotic effects is
expected to be a major challenge of the derivatization of 1.
However, the aim of the current study was to obtain cytostatic
and noncytotoxic (pro-apoptotic) 1 derivatives because many
cancers develop acquired chemoresistance during chronic treat-
ment with cytotoxic drugs in the form of the multidrug resistance
(MDR) phenotype.13,14 In addition, various cancer types display
intrinsic resistance to pro-apoptotic stimuli, and thus to conven-
tional chemotherapy and radiotherapy. One solution to apopto-
sis resistance and to the MDR phenotype entails supplementing
cytotoxic therapeutic regimens with cytostatic agents.
The 1-induced cytotoxic effects in cancer cells occur at higher
doses and relate to pro-apoptotic stimuli.4,7,9ꢀ11 Indeed, 1 at
micromolar concentrations, when added to rat liver mitochon-
dria, induces swelling, loss of membrane potential, pyridine nu-
cleotide oxidation, and release of cytochrome c and apoptosis
inducing factor.10 Salvi et al.10 argue that altogether these data
indicate that 1 is an inducer of mitochondrial permeability transition
Received: March 11, 2011
Published: September 02, 2011
r
2011 American Chemical Society
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We have based our 1-related derivatization strategy on the fact
that 1 and several 1 derivatives are already known to target
various kinases15ꢀ19 or to act as proteasome inhibitors.20 1 and
its derivatives can also target peroxysome proliferator-activated
receptors (PPARs),15,21ꢀ24 which control the expression of
networks of genes that encode proteins involved in all aspects
of lipid metabolism.25 In addition, PPARs are tumor growth
modifiers that act via the regulation of cancer cell apoptosis,
proliferation, and differentiation and also through their action on
the tumor cell environment, angiogenesis, inflammation, and
immune cell functions.25,26
Figure 1. Chemical structure of GA (1) illustrating the R1 and R2 sites
on which modifications were carried out (see Table 1).
The current study characterizes the in vitro anticancer activity
of 17 novel 1 derivatives and 3 1 derivatives that have already
been described in the literature, namely, compounds 2,27 8,28 and
lines, the human U37333,34 and T98G34 GBM, the A549
NSCLC,35,36 and the SKMEL-2837 cell lines were previously
demonstrated to show various levels of resistance to distinct pro-
apoptotic stimuli. Similarly, the sensitivity to apoptosis was
reported for the human Hs683 oligodendroglioma,33,34 the
MCF-7 breast,38 the PC-3 prostate38 cell lines, and for the mouse
B16F1037 melanoma cell line. Table 1 shows that all compounds
under study display similar growth inhibitory activity whether the
cancer cell lines are sensitive or rather resistant to pro-apoptotic
stimuli. It must nevertheless be emphasized that most 1-related
monoamides, i.e., 3a, 3b, 3d, 3e, and 3f, seem to be less efficient
in apoptosis-resistant (U373, T98G, A549, SKMEL-28) than in
apoptosis-sensitive cell lines (Hs683, MCF-7, PC-3, B16F10)
(Table 1). However, these differences did not reach statistical
levels of significance, because n = 4 in each group only, and
because heterogeneous responses were observed between the
cancer cell lines with respect to a given compound (Table 1).
Compound 1 is Cytotoxic, while Compound 6b Is Cyto-
static. Flow cytometry analyses revealed that 1 at its IC50 in vitro
growth inhibitory concentrations (see Table 1) induced slight
pro-apoptotic effects in U373 GBM cells, but marked ones in
A549 ones (Figure 2A) as expected from data published in the
literature.7,9 1 did not induce cytostatic effects in U373 GBM and
A549 NSCLC cells as revealed by the absence of accumulation of
cancer cells in the G1 phase of the cell cycle (Figure 2B), a feature
expected to occur when 1 displays cytostatic effects.7 Compound
6b appeared cytostatic according to the fact that it did not induce
apoptosis in U373 GBM and A549 NSCLC cells (Figure 2A).
However, this cytostatic effects did not relate to the accumulation
of cancer cells in the G1 phase (Figure 2B), a feature that
prompted us to analyze 6b-induced effects on the kinome and on
the proteasome as detailed below.
9
24 (see Figure 1 and Table 1 for the chemical structures). The
IC50 in vitro growth inhibitory concentration of each compound
has been determined in 8 cancer cell lines, leading to a structureꢀ
activity relationship. The influences of the most interesting
compounds have then been analyzed at the levels of proteasome
activity inhibition, supported by a molecular docking analysis.
The PPAR-γ activation and the inhibition of several kinases have
also been evaluated. Lastly, the pro-apoptotic effects of 1 and 6b
have been characterized in two cancer cell lines.
’ CHEMISTRY
To increase the anticancer activity of 1 and to obtain potent
cytostatic compounds, various families of derivatives have been
synthesized through chemical modifications at the carboxylic
acid group (see R1 in Figure 1 and Table 1).
First, monoamides (3aꢀf) were synthesized through peptidic
coupling reactions between 1 and various primary amines
(Scheme 1). These reactions were conducted in the presence
of N,N0-dicyclohexylcarbodiimide (DCC) and hydroxybenzo-
triazole (HOBt) and produced the desired derivatives in approxi-
mately 60ꢀ80% yields after flash chromatography (FC)
purification (see the Supporting Information). The keto deriva-
tive 3e was prepared by the oxidation of 3d. In addition to these
monoamides, we were also interested in the grafting of a primary
amino group to have ready access to different families of com-
pounds. Thus, the peptidic coupling reaction was also applied to
the monoprotected ethylenediamine,29 leading to a 64% yield of
the amido derivative 5. A subsequent deprotection of the amino
group under acidic conditions produced the versatile intermedi-
ate 227 in 97% yield. Reaction of 2 either with acyl chlorides,
isocyanates, or thioisocyanates led to diamido (4aꢀe), ureido
(6aꢀc), and thioureido (7a,b) derivatives, respectively, in good
yields after FC purification.
Characterizing 6b-Induced Effects on a Panel of 333 Kinases.
Compound 6b was assayed at 1 μM in a panel of 333 kinases (see
the Supporting Information). 6b displayed in vitro growth inhibitory
concentrations that range between 4 and 12 μM among the 8 cancer
cell lines analyzed in the current study (Table 1). Therefore, 1 μM
represents between 8% and 25% of these in vitro growth inhibitory
concentrations. In this very low concentration range, 6b inhibited
the activity of the 12 kinases listed in Table 2 by g50%. Among
these kinases, 4 already are inhibitors that have been studied in
clinical trials, whereas no inhibitors are being examined in clinical
trials for the remaining 8 kinases.
Finally, the reference compounds, 828 and 9,24 were synthe-
sized according to procedures described in the literature (see the
Supporting Information).
’ RESULTS
Determination of the IC50 in Vitro Growth Inhibitory
Concentration. The IC50 in vitro growth inhibitory concentra-
tions of all of the synthesized compounds were determined with a
MTT colorimetric assay30ꢀ33 in a panel of seven human and one
mouse cancer cell lines after three days of culturing the cancer
cells with the respective drugs (Table 1).
Most, if not all, of these kinases (Table 2), including ALK,39
BMX,40 EPHB1 and EPHB4,41,42 Fes and Fyn,43,44 FGF-R2,45
IGFR1,46 RET,47 ZAP70,48 and PAK2,49 play somewhatimportant
roles in cancer cell proliferation, adhesion, motility and invasion,
and thus in cancer cell migration and cancer metastatic processes.
The histological origin of each cancer cell line in this study is
detailed in the legend of Table 1, and of these eight cancer cell
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Table 1. Characterization of the in Vitro Growth Inhibitory Activity of 17 Novel and 3 (2, 8, 9) Already Known GA (1) Derivatives
versus 1 in a Panel of 8 Cancer Cell Lines
a P indicates purity. b The stability of the products was measured with HPLC analysis following incubation in MEM cell culture medium at 37 ꢀC over
7 days. Results are expressed as the % of the incubated compound recovered. c The IC50 in vitro growth inhibitory concentrations were determined using
the MTT colorimetric assay. The cell lines include the following: human U373 (ECACC code 89081403), T98G (ATCC code CRL1690), and Hs683
(ATCC code HTB-138) glioblastoma, the A549 (DSMZ code ACC107) NSCLC, the MCF-7 (DSMZ code ACC115) breast cancer, and the PC-3
(DSMZ code ACC465) prostate cancer and SKMEL-28 (ATCC code HTB-72) melanoma. One mouse cell line, i.e., the B16F10 melanoma model
(ATCC code CRL-6475) was also analyzed. d Six data points were available for each concentration tested and nine concentrations (from 0.01 to 100 μM,
with semilog increases) were available for each cell line.
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Scheme 1a
a Reagents and conditions: (a) 1ꢀ. DCC, HOBt, DIPEA, DMF, RT, 30 min; 2ꢀ. R1NH2, RT, overnight; (b) 1ꢀ. DCC, HOBt, DIPEA, DMF, RT, 30 min;
2ꢀ. H2N(CH2)2NHBoc, RT, overnight, 64%; (c) TFA, DCM, 0 ꢀC, 3 h, 97%; (d) DMAP, RCOCl, DCM; (e) THF, RNCO, RT, 20 h; (f) THF, RNCS,
RT, 20 h; (g) Jones reagent, acetone, 0 ꢀC, 45 min, 98%.
Several Compounds under Study Target the Proteasome.
On the basis of the fact that 1 derivatives can target the
proteasome, we biochemically analyzed the effects of six 1
derivatives in addition to compounds 1, 2, 8, and 9 on the three
proteasome catalytic units, including the trypsin-like, the cas-
pase-like, and the chymotrypsin-like units. Each compound was
tested at its mean MTT colorimetric assay-related IC50 concen-
trations calculated on the eight cancer cell lines (see Table 1).
The proteasome inhibitor MG-13250 was analyzed at a concen-
tration of 0.5 μM.
Figure 3A shows that 60 μM 2 inhibited the three proteasome
catalytic units with an efficiency that was similar to that of the
0.5 μM MG-132. Compound 6b (7 μM) also inhibited the three
catalytic units of the proteasome, but at a much lower concen-
tration than 2 (Figure 3A). While the remaining compounds, 1
(90 μM), 3a (60 μM), 3b (70 μM), 3e (80 μM), 3f (50 μM), 7a
(25 μM), 8 (3 μM), and 9 (30 μM), inhibited the activity of the
trypsin-like catalytic unit, they also activated the caspase-like and
the chymotrypsin-like catalytic units (Figure 3A).
crystal structure complexed to fellutamide B51 (PDB code 3D29).
The structure of the proteasome has also been determined with
bortezomib, the sole antiproteasome marketed compound to
date, bound at all six sites. We did not use bortezomib for our
docking analyses because it contains a boron atom that is not
supported in the default force field used and optimized for the
Glide software that was employed in this study.
To assess the reliability of our docking procedure, the crystal-
lographic ligand, fellutamide B, was docked in the three different
pairs of binding sites of the yeast proteasome after removal of all
water molecules and ligands. The docked poses were compared
with the positions of the crystal ligand by computing the root-
mean-square deviation (rmsd), which measures the distance
between the poses. A rmsd value for the backbone atoms was
found to range between 0.6 and 2.0 Å for all sites, indicating a
high similarity between the docked and the experimental posi-
tions of the crystallographic ligand.
Compound 6b, the molecule exhibiting the best affinity, was
docked in the three different catalytic sites. A detailed analysis
identified in all three sites two types of binding modes. In one of
these two modes, the 3,5-bis(trifluoromethyl)phenyl moiety
accommodates the S1 specificity pocket52 and the diamide group
forms several hydrogen bonds. This mode appears to present
similarities to the mode of binding of several known peptide
inhibitors,53 which mimic the proteasome susbstrate backbone.
In order to get a deeper insight into the molecular basis of the
proteasome recognition by the different compounds, we per-
formed molecular docking experiments at each of the caspase-
like (C-L; Figure 3B), trypsin-like (T-L; Figure 3B), or chymo-
trypsin-like (CT-L; Figure 3B) sites of proteolytic functions.
Figure 3B illustrates these three doublets of proteolytic sites in a
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A unique feature of 6b is to inhibit with a similar potency the
three catalytic units, whereas our other GA derivatives exhibit a
large discrepancy in their activity toward the different sites. We
thus carried out the docking of 3e in the chymotrypsin-like and
the trypsin-like binding sites which feature the largest potency
difference. In the chymotrypsin-like site, 3e occupies none of the
specificity pockets in contrast to 6b (see Figure 3C and D). In the
trypsin-like site, however, the best binding poses of 3e show that
its aromatic moiety visits the S1 specificity pocket, as for
compound 6b. Interestingly, a significantly higher potency was
measured at the chymotrypsine-like site for a noncovalent
inhibitor featuring a 3,4,5-trimethoxybenzylamine at the S3
position relative to the derivative containing only one methoxy.55
Several Compounds in this Study Were Found to Inhibit
PPAR-γ. On the basis of the fact that 1 derivatives can target
PPAR-γ, we also biochemically analyzed PPAR-γ inhibition
versus the activation induced by the 10 compounds described
above for the proteasome inhibition analyses. The compounds
were studied at the same concentrations as those reported above
for the proteasome inhibition analyses. Figure 4 shows that 1, 2,
and 6b did not significantly (p > 0.05; KruskalꢀWallis with
Dunn’s procedure) modify PPAR-γ activity as compared to the
control, whereas the remaining seven 1 derivatives inhibited
PPAR-γ activity (p > 0.05; KruskalꢀWallis with Dunn’s proce-
dure, except for 3f p < 0.05) (Figure 4B).
’ DISCUSSION
Cancer remains a devastating disease, and the number of
cancer-related deaths is still increasing. More than 90% of cancer
patients die from tumor metastases, which are intrinsically
resistant to apoptosis;56 therefore, these tumors are unresponsive
to a large majority of currently available anticancer drugs, which
generally work through apoptosis induction.57,58 Before metas-
tasizing, many cancer types also display intrinsic resistance to
proapoptotic stimuli; these include head and neck cancers,59
non-small-cell lung cancer (NSCLC),35,60 melanoma,61 pancrea-
tic cancer,62 esophageal cancer,63,64 and gliomas.65 As empha-
sized by Landis-Piwowar et al.,66 drug resistance limits the
effectiveness of treatment options and is a major challenge faced
by current cancer research. As detailed below, an additive effect
has been observed in cancer cells treated with bortezomib in
combination with traditional chemotherapeutics, and in some
cases, drug resistance has been overcome.66 In the same manner,
one of the kinases targeted by 6b, i.e., BMX,67 is implicated in
cancer drug resistance. Thus, 6b, apart its intrinsic anticancer
activity that makes it able to overcome resistance to pro-apoptotic
stimuli, could also overcome cancer drug resistance if adminis-
tered with conventional chemotherapeutics.
The IC50 in vitro growth inhibitory concentrations of all newly
synthesized compounds are lower than that of 1 (Table 1),
clearly showing the importance of the amido subunit to increase
anticancer activity, at least in vitro. According to the fact that the
oxidation of the hydroxy group on the C-3 position decreases
the in vitro anticancer activity (3d vs 3e), this position was not
further modified in the frame of the current study. The results
reveal that most 1-related monoamides could display weaker
growth inhibitory activity in apoptosis-resistant cancer cell lines
(i.e., >100 μM) compared to apoptosis-sensitive cancer cell lines
(Table 1). Therefore, monoamide 1 derivatives (3aꢀf) do not
seem to be appropriate drugs to combat those cancer types
associated with poor prognoses because they are able to resist
Figure 2. (A) Determination of the percentages of A549 NSCLC and
U373 GBM cells undergoing apoptosis during 48 and 72 h of treatment
with 80 (U373 cells) and 100 (A549 cells) μM for compound 1 and
10 μM (for both cell lines) for compound 6b. Negative (Ctꢀ) and
positive (Ct+) controls available in the kit were used along with 1 μM
narciclasine-treated human PC-3 prostate cancer cells as controls for this
experiment. (B) Growth rate determination (i.e., the determination of
the % of cells in the G1 phase of the cell cycle) for the U373 GBM and A549
NSCLC cells treated as in (C). Each experimental condition was performed
in triplicate, and the data are presented as the mean ( SD values.
The second mode which is more largely represented in the
chymotrypsin and caspase-like sites depicts the 3,5-bis(tri-
fluoromethyl)phenyl moiety bound in the S3 specificity pocket.
This mode agrees with the inhibition potency, at the chymo-
trypsin-like site and to a lesser extent at the caspase-like site, of
proteasome inhibitors containing 3,5-bis(trifluoromethyl)-
phenyl at the S3 position.54 To attempt to discriminate between
these two binding modes, we performed docking of 6b in a series
of proteasome crystal structures in complex with different
inhibitors, most of them bound to the chymotrypsin-like site
(PDB codes 3NZJ, 3MG8, 3MG4, and 3GPJ). Overall, the
most common docked pose portrayed the 3,5-bis(trifluoro-
methyl)phenyl moiety of 6b in the S3 specificity pocket. Docking
experiments including protein flexibility (induced-fit protocol
from Schrodinger Inc.) in the form of flexible side chain residues
selected in the vicinity of the S1 specificity pocket corroborate
this binding mode.
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Table 2. Identification of the Kinases Targeted by Compound 6b at a Concentration of 1 μMa
% of 6b-induced in
hibition at 1 μM
major cancer types in which the
compounds currently in clinical trials
kinases
name or family
kinase displays significant roles in cell biology
targeting this kinase versus cancer types
ALK
Anaplastic lymphoma
kinases
69
Anaplastic large cell lymphoma,
Crizotinib, an oral ALK
inhibitor, was demonstrated to
provide dramatic clinical benefit
with little toxicity in patients with
advanced NSCLC
inflammatory myofibroblastic tumors,
nonsmall cell lung cancer, and neuroblastoma
BMX
B-tyrosine
52
Androgen-independent prostate
cancers. Also implicated in the
None
kinases family
chemoresistance of small-cell-lung cancers,
play a role in cell growth and differentiation
Ovarian cancers, regule the actin cytoskeleton
during cell motility and invasion
Ovarian cancers; colon cancers, play a
role in the cell adhesion and migration
Ovarian cancers; colon cancers; gastric cancers;
prostate cancers, module integrin-mediated adhesion
Thyroid cancers; Multiple endocrine
neoplasia type 2 (MEN 2), play a role in the
cell adhesion and migration
PAK2 p-21 associated
kinases family
51
64
64
48
None
None
None
None
EPHB1 Eph receptor
tyrosine kinases
EPHB4 Eph receptor
tyrosine kinases
RET
R tyrosine kinases family
ZAP70 Zeta-chain (TCR)
67
67
Chronic lymphocytic leukemia (CLL),
play a role in cell migration
None
associated protein kinases
FGF-R2 Fibroblast growth
factor receptor type 2
Breast cancers; gastric cancers; endometrial
cancers; prostate cancers, play a role in the
cell proliferation
AZD2171 - Ki23057 -
PD173074 - SU5402
FES
Fes tyrosine kinases
Src kinase family
59
72
Not yet clearly defined
None
FYN
Prostate cancers, play a role in the
cell proliferation, morphogenesis
and motility
Dasatinib, an orally available small-molecule
multikinase inhibitor
IGF-R1 Insulin-like growth factor type 1
64
50
Breast cancers; colon cancers; head
and neck cancers
Dalotuzumab
None
MATK Megakaryocyte-associated
Not yet clearly defined
tyrosine kinases
a The panel of 333 kinases that have been investigated are provided in the Supporting Information.
pro-apoptotic stimuli. In comparison with monoamido (3) and
diamido (4) groups, the ureido (6) and thioureido (7) ones lead
to a better in vitro anticancer activity. Finally, the addition of
electron-withdrawing groups on the aromatic part of the ureido
moieties (6a vs 6b) seems to reinforce this in vitro anticancer
activity as revealed by the MTT colorimetric assay.
As discussed below, 6b emerges as a hit from the current 1
derivatives. At first glance, one could estimate that 6b would be
difficult to be further developed due to its high molecular weight
and poor hydrosolubility. However, preliminary data we ob-
tained already indicate that 6b can be formulated as specific
microemulsions for oral and i.v. administrations, a project that is
currently ongoing in our laboratory.
Together, these data led us to focus less on the monoamide
(3aꢀf) and diamido-1 (4aꢀe) derivatives and more on the
remaining compounds in this study. We thus decided to pay
particular attention to 6b, which appeared to be the most potent
compound from the current series in terms of in vitro growth
inhibitory activity in a panel of four apoptosis-resistant and four
apoptosis-sensitive cancer cells lines (Table 1). In addition, 6b
also appeared to be an actual cytostatic compound as revealed by
flow cytometry analyses (Figure 2).
We then analyzed 6b’s ability to inhibit proteasome activity
because 1 and certain of its derivatives have already been shown
to act as proteasome inhibitors.20 Proteasomes are large, multi-
catalytic proteinase complexes located in the cytosol and in the
nucleus of eukaryotic cells.68,69 The ubiquitinꢀproteasome
system is responsible for the degradation of most intracellular
proteins and therefore plays an essential regulatory role in critical
cellular processes, including cell cycle progression, proliferation,
differentiation, angiogenesis, and apoptosis.68ꢀ70 Human cancer
cells possess elevated levels of proteasome activity and are more
sensitive to proteasome inhibitors than normal cells.68 Protea-
some inhibition bypasses, at least partly, the resistance of cancer
cells to apoptosis because it activates macroautophagy, a com-
pensatory protein degradation system,71 and other pro-survival
signaling pathways.72 Inhibition of these autoprotective re-
sponses sensitizes cancer cells to the antiproliferative effects of
proteasome inhibitors.72 The proteasome inhibitor bortezomib
(PS-341) was the first clinically available proteasome inhibi-
tor and was approved by the FDA in 2003 for the treatment of
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Figure 3. (A) Determination of the effects of the 10 compounds in this study on the proteolytic activity of the proteasome in human U373 GBM
cells treated with each compound at their IC50 in vitro growth inhibitory concentrations, which are detailed in Table 1. MG-132 (0.5 μM) was
used as a reference compound. The data are presented as mean ( SD values calculated in triplicate for each experimental condition. The Y-axis is
on a logarithmic scale. (B) Ribbon representation of the 20S proteasome crystallographic structure bound to the fellutamide B ligand in the three
pairs of binding sites (PDB code: 3D29): chymotrypsin-like sites (CT-L), trypsin-like sites (T-L), caspase-like sites (C-L). Fellutamide B is
depicted as balls. (C,D) Comparison of the predicted best affinity binding modes of compounds 6b (C) and 3e (D) in the chymotrypsin-like site
of the 3D29 crystal structure. The ligands are depicted as sticks and the protein is represented by its molecular surface. The S3 specificity pocket
is indicated.
multiple myeloma.73 However, despite clinical benefits, bortezo-
mib causes various types of toxicities including peripheral
neuropathy74 and thrombocytopenia.75
The current study reveals that 1 inhibits the trypsine-like
catalytic activity of the proteasome by ∼50% while it activates
the chymotrypsin-like catalytic activity. It also appears to be
without any apparent effects on the caspase-like catalytic activity
when tested at its IC50 in vitro growth inhibitory concentration
(Figure 3A). The already known compound 2,27 i.e., N-(2-
aminoethyl)-glycyrrhetinamide, behaved as a proteasome inhibi-
tor, probably because of its amine function, which is well-known to
have potent activity on cell viability (Table 1). In addition, its IC50
growth inhibitory concentrations in the eight cancer cell lines
under study was similar to the one displayed by 1 (Table 1). The
novel GA derivative 6b (N-(2-{3-[3,5-bis(trifluoromethyl)phenyl]
ureido}ethyl)-glycyrrhetinamide) was also able to inhibit the
three catalytic units of the proteasome (Figure 3A). At first
glance, 6b appears to be less potent than 2 in its inhibitory activity
toward the proteasome (Figure 3A). However, it should be noted
that compound 6b has been tested at a concentration of 7 μM
(i.e., its IC50 in vitro growth inhibitory concentration; Table 1),
whereas compound 2 has been tested at 60 μM (also its IC50 in
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S6 kinase (S6K),18 and the PI3K/Akt/GSK3β pathway.19 Kinase
inhibitors are the largest class of new anticancer drugs,81 and
approximately 150 kinase inhibitors have been subjected to recent
clinical testing, with ∼45 kinases listed as primary targets.82
However, it is already apparent that most cancers can avoid the
inhibition of any single kinase.81 It becomes, in fact, increasingly
obvious that it is mandatory to inhibit multiple kinases,81 and
partial inhibition of a small number of kinases appears to be a more
promising strategy.83 The data from the present study reveal that
6b inhibits the activity of a dozen kinases, and most if not all of
them are implicated in cancer cell proliferation and migration (see
Table 2). It thus seems that the cytostatic activity of 6b relates,
at least partly, to the targeting of this cluster of kinases, a feature
that was observed at 1 μM, which is a concentration ranging
between 8% and 25% of the IC50 in vitro growth inhibitory
concentrations determined in a panel of eight distinct cancer cell
lines. These 6b-mediated effects on kinases that are implicated in
cancer cell proliferation and migration (with the implication of the
actin cytoskeleton that also exerts major roles in cytokinesis, and
thus in cellproliferation) canexplain, atleast partly, the 6b-induced
cytostatic effects.
In conclusion, the present study provides a novel 1 derivative,
6b, which displays cytostatic and not cytotoxic effects in cancer
cells. These cytostatic effects occur, at least partly, through the
targeting of a limited number of 12 kinases that are implicated in
cancer cell proliferation and migration and through antiprotea-
some activity, including the three catalytic units of the protea-
some, while 6b does not inhibit PPAR-γ activity in cancer cells.
In contrast, 1 is less efficient than 6b in inhibiting proteasome
activity and it is cytotoxic, at least partly through the activation of
pro-apoptotic processes. Compound 1, i.e., glycyrrhetinic acid,
displays in vitro growth inhibitory activity in various cancer cells
with an efficiency that is about ten times weaker than that
displayed by 6b. We are currently developing specifically for-
mulated forms of 6b to proceed with in vivo experiments aimed at
investigating the anticancer activity of 6b as a single agent or in
association with conventional cytotoxic chemotherapeutics.
Figure 4. Determination of activating versus inhibiting effects of the 10
compounds in this study (the same as those used for proteasome-related
studies; see Figure 3A) on PPAR-γ. Each compound has been analyzed
at its IC50 in vitro growth inhibitory concentration in the human U373
GBM cell line (see Table 1 and its legend) treated for 6 h with the
compound. The data are presented as mean ( SD values calculated in
triplicate for each experimental condition. Determination of the effects
of the 10 compounds after 6 h of treatment of human U373 GBM cells.
vitro growth inhibitory concentration; Table 1). Compound 6b
could therefore be considered as a novel GA derivative that acts
both as a proteasome inhibitor and as a kinase inhibitor, as
detailed below. It must be noticed that, apart from 2 and 6b, the
remaining compounds under study for which their effects on the
proteasome have been analyzed display activating rather than
inhibiting effects on the proteasome.
Before analyzing the antikinase profile of 6b, we investigated
its effects on PPARs because 1 and several of its derivatives can
also target PPARs.21ꢀ24 PPAR agonists, particularly thiazolidi-
nedione derivatives, are promising therapeutic agents because
they exert both a direct anticancer and a broad spectrum of anti-
stromal, antiangiogenic, and immuno-modulating activities.25,76
However, the great majority of reports from the literature clearly
indicate that PPAR-targeting anticancer agents must display overall
agonistic and not antagonistic activity for PPAR-γ.77ꢀ80 Chinthar-
lapalli et al.21 and Jutooru et al.15 demonstrated PPAR-γ activation
with 1 derivatives in colon and pancreatic cancer cells, respectively.
Thus, we wanted to confirm that 6b does not antagonize PPAR-γ
activity before further deciphering the mechanisms of action
through which it displays its cytostatic activity in cancer cells. The
data illustrated in Figure 4B confirm that 6b (and also 2) did not
antagonize PPAR-γ activity, whereas the remaining compounds,
including the reference compounds 828 and 9,24 antagonized PPAR-
γ activity when assayed at their IC50 in vitro growth inhibitory
concentration (Table 1).
Together, these data thus highlight the fact that 6b, which is a
novel GA (1) derivative, displays marked in vitro activity both in
apoptosis-resistant and apoptosis-sensitive cancer cells and that
this cytostatic activity is partly mediated through the inhibition of
the three catalytic units of the proteasome. As previously
indicated in the Introduction, 1 and/or several 1 derivatives
are also known to target various kinases, including the following:
phosphatidylinositol-3-kinase (PI3ꢀK) and p42 and p38 mitogen-
activated protein kinase (MAPK),15 src,16 protein kinase C
(PKC),17 VEGF receptor 2 (VEGFR2), mTOR kinase, ribosomal
’ EXPERIMENTAL SECTION
Chemistry. General Methods. Before their use, the solvents were
distilled and dried using standard methods, i.e., THF and ether from Na/
benzophenone, and CH2Cl2 from P2O5. The 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance 300 spectrometer in CDCl3
using the residual isotopic solvent CHCl3 and CH3OH, respectively, as
references for δH = 7.26 ppm, δC = 77.16 ppm, δH = 3.31 ppm, δC =
49.00 ppm. The following abbreviations are used for spin multiplicity: s,
singlet; d, doublet; t, triplet; q, quadruplet, qt, quintuplet; m, multiplet;
br, broad. 1H NMR and 13C NMR spectra were assigned by comparison
with NMR spectra of oleanane compounds previously described in the
literature.84,85 Routine thin-layer chromatography (TLC) was per-
formed on silica gel plates (Silica gel GF254 from VWR), and visualiza-
tion was performed using UV. Flash column chromatography was
carried out using silica gel at moderate pressure (spherical particle size
60ꢀ200 μm from MP Biomedicals). HPLC analyses were performed
with an Agilent 1100 series HPLC system (Agilent, Diegem, Belgium).
The chromatographic system was an RX-C8 (5 μm) (4.6 mm ꢁ
250 mm) (Agilent, Diegem, Belgium) using the same mobile phase for
all compounds: MeOHꢀwater 90:10 (0.1% TFA) 20 min. The detec-
tion system was an Agilent Diode Array Detector G1315B (monitoring
wavelength given for each compound) (Agilent, Diegem, Belgium). The
purity given was measured by this HPLC method at 249 nm. How-
ever, we are aware that this method does not take into account the
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cocrystallized solvents that could be detected with NMR. High-
resolution mass spectra (positive mode) were recorded by direct
infusion in a 6520 series electrospray ion source (ESI)ꢀquadrupole
time-of-flight (Q-TOF) mass spectrometer (Agilent, Palo Alto, CA,
USA). The error between the observed mass and the calculated mass is
expressed in ppm. Below 3 ppm, the compounds were considered to
have the predicted formula. The infrared spectra were recorded as KBr
pellets on a Perkin-Elmer 1750 FT-spectrophotometer (Perkin-Elmer,
Waltham, MA, USA) and the wavelengths are expressed in cmꢀ1. The
monoprotected ethylenediamine, i.e., H2NCH2CH2NHBoc, was
synthesized according to procedures previously described in the
literature.29 The purity of each synthesized compound was verified
by means of 1H NMR, HRMS, and HPLC-UV analyses and reached a
level >95% (Table 1).
Procedure for the Synthesis of N-(2-Aminoethyl)-glycyrrhetinamide
(2). Compound 5 (450 mg, 0.735 mmol) was dissolved in DCM (9 mL)
at 0 ꢀC under stirring. Trifluoroacetic acid (1 mL, 13 mmol) was added,
and the reaction was stirred at 0 ꢀC for 3 h. The reaction mixture was
evaporatedbelow50 ꢀC and then pouredinto dry sodiumbicarbonate and
ice, extracted with dichloromethane, dried over Na2SO4, filtered, and
concentrated to produce the corresponding amine in quantitative yield.
General Procedure for 18 β-Glycyrrhetinamides (3, 5). 18-β-Glycyr-
rhetinic acid (200 mg, 0.43 mmol) was dissolved in DMF (10 mL)
at room temperature under stirring. N,N0-Dicyclohexylcarbodiimide
(175.3 mg, 0.85 mmol), 1-hydroxy-1H-benzotriazole (86.1 mg, 0.64
mmol), and diisopropylethylamine (444 μL, 2.55 mmol) were added
successively to the reaction mixture. After 30 min, the solution was
cooled at 0 ꢀC, and the primary amine was added dropwise. The reaction
mixture was brought to room temperature and then was stirred over-
night. The reaction mixture was concentrated under vacuum and water
was added. After dichloromethane extraction, the organic layer was dried
over anhydrous Na2SO4, filtered, and concentrated under vacuum. N,
N0-Dicyclourea was eliminated by trituration in acetonitrile. The organic
layer was evaporated under vacuum. The crude product was then
chromatographed on silica (cyclohexane; ethyl acetate).
concentrated under vacuum. The residual solid was then chromato-
graphed on silica (DCM; methanol), with the exception of compounds
6b and 7b, which were purified through trituration in Et2O.
N-(2-{3-[3,5-Bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrheti-
namide (6b). Yield: 36%. Rf = 0.30 (DCM; MeOH 95:5). RP-HPLC:
purity = 99%, tR = 8.40 min 1H NMR characteristic protons (HSQC, 300
MHz, CD3OD): δ (ppm) = 8.02 (s, 2H, ArꢀH35, H39), 7.42 (s, 1H, Ar-
H37), 5.61 (s, 1H, CH-12), 3.62ꢀ3.22 (m, 4H, NHCH2CH2NH), 3.15
(dd, 1H, J = 7.0 Hz, 5.1 Hz, CH-3), 2.66 (dt, 1H, J = 13.0 Hz, 3.0 Hz, CH-
1), 2.37 (s, 1H, CH-9), 1.38 (s, 3H, Me-H27), 1.10 (s, 3H, Me-H29),
1.05 (s, 3H, Me-H25), 0.98 (s, 3H, Me-H23), 0.94 (s, 3H, Me-H26),
0.78 (s, 3H, Me-H24), 0.74 (s, 3H, Me-H28). 13C NMR (HSQC, 75
MHz, CD3OD): δ (ppm) = 202.4 (C11), 179.4 (C30), 172.6 (C13),
157.6 (C33), 143.4 (C34), 133.0 (q, J = 33.0 Hz, C36/C38), 128.9
(C12), 124.8 (q, J = 270.1 Hz, CF3), 118.8 (q, J = 3.0 Hz, C35/C39),
115.3 (q, J = 7.0 Hz, C37), 79.4 (C3), 63.1 (C9), 56.2 (C5), 49.3 (C18),
46.6 (C14), 44.9 (C20), 44.5 (C8), 42.6 (C19), 41.1 (C31/C32*), 40.6
(C31/C32*), 40.3 (C1), 40.2 (C4), 38.7 (C22), 38.3 (C10), 33.7 (C7),
32.9 (C17), 31.9 (C21), 29.6 (C29), 29.1 (C28), 28.7(C23), 27.8 (C2),
27.6 (C15/C16*), 27.4 (C15/C16*), 23.7 (C27), 19.1 (C26), 18.6
(C6), 16.8 (C25), 16.3 (C24). IR (KBr): ν (cmꢀ1): 3360 (OH), 2932
(CH aliphatic), 1643 (CdO), 1544, 1467, 1386. Mp: 201 ꢀC. HRMS
(ESI-QTOF) calcd for (6b) C41H55F6N3O4 (M+H+) 768.4170, obsd
768.4167, error: 0.29 ppm.
Pharmacology. Determination of in Vitro Anticancer Activity.
The histological types and origin of the eight cancer cell lines are detailed
in the legend of Table 1. The cells were cultured in RPMI (Invitrogen,
Merelbeke, Belgium) media supplemented with 10% heat inactivated
fetal calf serum (Invitrogen). All culture media were supplemented with
4 mM glutamine, 100 μg/mL gentamicin, and penicillinꢀstreptomycin
(200 U/mL and 200 μg/mL) (Invitrogen).
The overall growth level of human cancer cell lines was determined
using a colorimetric MTT (3-[4,5-dimethylthiazol-2yl-diphenyl tetra-
zolium bromide, Sigma, Belgium) assay as detailed previously.30ꢀ33
Each experimental condition was performed in six replicates.
Procedure for the Synthesis of 3e from 3d. Compound 3d (200 mg,
0.331 mmol) was dissolved in acetone (20 mL) at 0 ꢀC under stirring.
Jones reagent (150 μL) was added dropwise until the reaction mixture
became orange. After 45 min, methanol was added until the solution
turned dark green. The reaction was poured into a mixture of ice and
water (20 mL), and then acetone and methanol were removed under
reduced pressure. The aqueous residue was extracted three times with
10 mL DCM. The combined organic extracts were washed first with
water (10 mL) and then with brine (10 mL) and dried over Na2SO4. The
solvent was evaporated under vacuum, leading to a solid that corre-
sponds to compound 3e (98% yield).
General Procedure for 18 β-Glycyrrhetindiamides (4). Compound 2
(100.8 mg, 0.20 mmol) was dissolved in 4 mL of anhydrous DCM. N,N0-
Dimethylaminopyridine (48.7 mg, 0.40 mmol) was then added to this
solution. After cooling at 0 ꢀC, the acyl chloride (0.20 mmol) was added
dropwise to the reaction mixture, which was then stirred at room
temperature for 12 h. The mixture was concentrated under vacuum.
The residual solid was dissolved in 20 mL of DCM and the organic layer
was washed twice with 20 mL of 1 M HCl. The aqueous layer was then
extracted twice with 10 mL of DCM. The combined organic layers were
washed twice with 20 mL of 1 M NaOH, and the aqueous layers
were extracted twice with 10 mL of DCM. The combined organic layers
were dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was then chromatographed on
silica (DCM; methanol).
Molecular Docking. Docking was conducted using the Glide
program (version 4.5),86 which positions the 3D structure of flexible
ligands in the 3D structure of rigid or partially flexible receptors.
Docking experiments were performed on the X-ray crystallographic
structure of the 20S proteasome from Saccharomyces cerevisae (PDB
code 3D29).51 The 20S proteasome is highly conserved from yeast to
human.87 The protein structures were prepared using the Protein
Preparation Wizard in the Schr€odinger software graphical user interface
Maestro. Glide XP docking protocol and scoring function were used to
dock in the rigid receptors and to score the poses of the different ligands.
The default settings of Glide version 4.5 were used for the remaining
parameters. For each docking run, the binding zone was defined as a
cube of length 40 Å. This cube was centered on the former positions of
the removed ligand cocrystallized with the protein in each proteolytic
site. The initial 3D structures of the ligands were generated with the
CORINA program.88
Biochemical Analyses Aimed at Characterizing Anti-Pro-
teasome Activity. The substrates used for performing this assay were
Suc-LLVY-Glo, Z-LRR-Glo, and Z-nLPnLD-Glo for the chymotrypsin-
like, trypsin-like, and caspase-like assay, respectively. U373 GBM cells
were incubated at a concentration of 5000 cells/mL for 48 h in three
different 96-microwell plates (one plate for each catalytic site). U373
GBM cells were then treated with each compound at their IC50 in vitro
growth inhibitory concentration (see Table 1) for two days. Cells were
then washed with PBS (Invitrogen, Merelbeke, Belgium) before adding
digitonin at 0.025% to rapidly and selectively permeate the cytoplasmic
membranes.89 Proteasome-Glo reagent (Promega Corporation, USA),
specific for a catalytic site, was added in each well sample. Because the
light of the aminoluciferine released was proportional to the rate of
General Procedure for (Thio)Ureido-glycyrrhetinamides (6, 7).
Compound 2 (90.9 mg, 0.18 mmol) was dissolved in 4 mL of anhydrous
THF. The desired isocyanate or thioisocyanate (0.18 mmol) was added.
The reaction mixture was stirred at room temperature for 20 h and was
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proteasome cleavage of the substrate, after 10 min of dark incubation,
the bioluminescence of each well was measured with an ActiveGlo LR-
100 luminometer (DSLabs, USA). Each experiment was conducted in
triplicate. These cells’ bioluminescence assay was normalized by a
concomitant cell viability assay (using the MTT colorimetric assay as
detailed before) performed using strictly identical conditions.
’ AUTHOR INFORMATION
Corresponding Author
*Tel.: +32 477 62 20 83; E-mail: rkiss@ulb.ac.be.
PPAR-γ Activity Assay. U373 glioblastoma cells were treated with
each compound at their IC50 in vitro growth inhibitory concentration
(see Figure 4 and its legend) for 6 h. After these incubations, cells were
collected and nuclear extracts were purified according to the manufac-
turer’s instructions (Cayman Chemical; Complete Transcription Factor
Assay Kit). In brief, cells were detached with trypsin and centrifuged at
300 g for 5 min. The pellets were resuspended in an ice-cold PBS/
Phosphatase inhibitor solution for 5 min. This step was repeated once.
Extracts were resuspended in ice-cold hypotonic buffer and incubated
for 15 min. To break the cytoplasmic membrane, cells were treated with
Nonidet p-40 (Sigma-Aldrich, Bornem, Belgium). The extracts were
centrifuged and the cytoplasmic fraction was saved for other applica-
tions. The pellet was resuspended in an extraction buffer containing
protease inhibitor and incubated with gentle rocking for 30 min on ice.
Extracts were centrifuged at 14 000 g for 10 min at 4 ꢀC. The supernatant
contained the nuclear fraction. Nuclear extracts were loaded in 96 well
plates and incubated overnight at 4 ꢀC. The primary antibody (from the
PPAR-γ; Complete Transcription Factor Assay Kit) was added and
incubated for one hour at room temperature. The wells were washed
5 times and a goat antirabbit HRP conjugated secondary antibody
was added and incubated for one hour at room temperature. The wells
were washed 5 times. Developing solution was added for 45 min and
absorbance was measured at 450 nm with a spectrophotometer (Biorad
model 680XR, Biorad, Nazareth, Belgium).
’ ACKNOWLEDGMENT
Q-TOF was acquired thanks to grants from the Fonds National
de la Recherche Scientifique (FRS-FNRS) no 34553.08 and from
the Universitꢀe Libre de Bruxelles (ULB-FER2007). Marina Bury
is the holder of a grant FRIA from the FNRS and Cꢀedric Delporte
is a PhD student from the FNRS. Robert Kiss is a director of
research with the FNRS. Jean-Paul Becker is a postdoctoral fellow
^
supported by the F.R.S.-FNRS. Martine Prꢀevost is ‘Maitre de
Recherche’ at the FNRS. We thank Prof. M. Luhmer and Dr. R.
D’Orazio from CIREM (ULB) for NMR spectra recording, Dr. P.
Van Antwerpen for the HRMS analysis from Analytical Platform of
the Pharmacy Faculty (ULB), and V. Megalizzi for the settingup of
the proteasome test.
’ ABBREVIATIONS
ATCC: American Type Culture Collection; DSMZ: Deutsche
Sammlung von Mikroorganismen and Zellkulturen; ECACC:
European Collection of Cell Culture; GA: Glycyrrhetinic acid; GBM:
Glioblastoma; MTT: 3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide; NSCLC: Non-small-cell lung carcinoma;
PPARs: Peroxysome proliferator-activated receptors
Flow Cytometry Determination of Apoptosis and Cell Cycle Kinetics.
Analyses of apoptosis and cell cycle kinetics were performed with the
APO AF detection kit (BD Pharmingen, Erembodegem-Aalst, Belgium)
according to the manufacturer’s instructions and based on a procedure
we previously described.36 The cancer cells were treated for 48 or 72 h
with their respective IC50 in vitro growth inhibitory concentrations,
10 μM for U373 and A549 cancer cells (6b), and 80 and 100 μM for
U373 and A549 cancer cells (1). TUNEL labeling of U373 GBM and
A549 NSCLC cells was performed for 1 h at 37 ꢀC followed by
propidium iodide (PI) staining (5 μg/mL) in the presence of RNase.
Stained cells were analyzed on a CellLab Quanta SC flow cytometer
(Beckman Coulter; Analis, Suarlee, Belgium).
Kinase Activity Determination. We originally prepared ProQinase
(Freiburg, Germany) with 6b as a stock solution in 100% DMSO, and
aliquots were further diluted with water in 96 well microliter plates
immediately before use. For each kinase assay, 5 μL from a 2 ꢁ 105 M/
10% DMSO compound solution were transferred into the assay plates.
The final volume of the assay was 50 μL. The final assay concentration of
6b was 1 μM. A radiometric protein kinase assay (33PanQinase Activity
Assay) was used to measure the kinase activity of the 333 protein kinases
under study, as detailed previously.90
’ REFERENCES
(1) Obolentseva, G. V.; Litvinenko, V. I.; Ammosov, A. S.; Popova,
T. P.; Sampiev, A. M. Pharmacological and therapeutic properties of
licorice preparations. Pharm. Chem. J. 1999, 33, 427–434.
(2) Abe, H.; Ohya, N.; Yamamoto, K. F.; Shibuya, T.; Arichi, S.;
Odashima, S. Effects of glycyrrhizin and glycyrrhetinic acid on growth
and melanogenesis in cultured B16 melanoma cells. Eur. J. Cancer Clin.
Oncol. 1987, 23, 1549–1555.
(3) Okamoto, H.; Yoshida, D.; Mizusaki, S. Inhibition of 12-O-
tetradecanoylphorbol-13-acetate-induced induction in Epstein-Barr
virus early antigen in Raji cells. Cancer Lett. 1983, 19, 47–53.
(4) Hibasami, H.; Iwase, H.; Yoshioka, K.; Takahashi, H. Glycyr-
rhetic acid (a metabolic substance and aglycon of glycyrrhizin) induces
apoptosis in human hepatoma, promyelotic leukemia and stomach
cancer cells. Int. J. Mol. Med. 2006, 17, 215–219.
(5) Isbrucker, R. A.; Burdock, G. A. Risk and safety assessment on
the consumption of licorice root (Glycyrrhiza sp.), its extract and
powder as a food ingredient, with emphasis on the pharmacology and
toxicology of glycyrrhizin. Regul. Toxicol. Pharmacol. 2006, 46, 167–192.
(6) Zhang, M.-Z.; Xu, J.; Yao, B.; Yin, H.; Cai, Q.; Shrubsole, M. J.;
Chen, X.; Kon, V.; Zheng, W.; Pozzi, A.; Harris, R. C. Inhibition of
11beta-hydroxysteroid dehydrogenase type II selectively blocks the
tumor COX-2 pathway and suppresses colon carcinogenesis in mice
and humans. J. Clin. Invest. 2009, 119, 876–885.
(7) Satomi, Y.; Nishino, H.; Shibata, S. Glycyrrhetinic acid and
related compounds induce G1 arrest and apoptosis in human hepato-
cellular carcinoma HepG2. Anticancer Res. 2005, 25, 4043–4047.
(8) Hawthorne, S.; Gallagher, S. Effects of glycyrrhetinic acid and
liquorice extract on cell proliferation and prostate-specific antigen
secretion in LNCaP prostate cancer cells. J. Pharm. Pharmacol. 2008,
60, 661–666.
(9) Lee, C. S.; Yang, J. C.; Kim, Y. J.; Jang, E. R.; Kim, W.; Myung,
S. C. 18beta-glycyrrhetinic acid potentiates apoptotic effect of trichos-
tatin A on human epithelial ovarian carcinoma cell lines. Eur. J.
Pharmacol. 2010, 649, 354–361.
Statistical Analyses. Data obtained from independent groups were
compared by the nonparametric KruskalꢀWallis (Dunn’s procedure),
MannꢀWhitney U, or Spearman Rank Correlation tests. The statistical
analysis was performed using Statistica software (Statsoft, Tulsa, OK).
’ ASSOCIATED CONTENT
S
Supporting Information. The characterization data for all
b
compounds that have been synthesized and biologically evaluated
in this study (HPLC, 1H NMR, 13C NMR, mass spectra, etc.) and
the detailed biological and biochemical protocols.This material is
available free of charge via the Internet at http://pubs.acs.org.
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dx.doi.org/10.1021/jm200285z |J. Med. Chem. 2011, 54, 6501–6513

Journal of Medicinal Chemistry
ARTICLE
(10) Salvi, M.; Fiore, C.; Armanini, D.; Toninello, A. Glycyrrhetinic
acid-induced permeability transition in rat liver mitochondria. Biochem.
Pharmacol. 2003, 66, 2375–2379.
(11) Lee, C. S.; Kim, Y. J.; Lee, M. S.; Han, E. S.; Lee, S. J. 18beta-
Glycyrrhetinic acid induces apoptotic cell death in SiHa cells and
exhibits a synergistic effect against antibiotic anti-cancer drug toxicity.
Life Sci. 2008, 83, 481–489.
(12) Schwarz, S.; Csuk, R. Synthesis and antitumour activity of
glycyrrhetinic acid derivatives. Bioorg. Med. Chem. 2010, 18, 7458–7474.
(13) Anuchapreeda, S.; Leechanachai, P.; Smith, M. M.; Ambudkar,
S. V.; Limtrakul, P. N. Modulation of P-glycoprotein expression and
function by curcumin in multidrug-resistant human KB cells. Biochem.
Pharmacol. 2002, 64, 573–582.
(14) Mayur, Y. C.; Peters, G. J.; Prasad, V. V.; Lemo, C.; Sathish,
N. K. Design of new drug molecules to be used in reversing multidrug
resistance in cancer cells. Curr. Cancer Drug Targets 2009, 9, 298–306.
(15) Jutooru, I.; Chadalapaka, G.; Chintharlapalli, S.; Papineni, S.;
Safe, S. Induction of apoptosis and nonsteroidal anti-inflammatory drug-
activated gene 1 in pancreatic cancer cells by a glycyrrhetinic acid
derivative. Mol. Carcinog. 2009, 48, 692–702.
(16) Chung, T. H.; Wang, S. M.; Chang, Y. C.; Chen, Y. L.; Wu, J. C.
18beta-glycyrrhetinic acid promotes src interaction with connexin43 in
rat cardiomyocytes. J. Cell Biochem. 2007, 100, 653–664.
(17) Kawakami, Z.; Ikarashi, Y.; Kase, Y. Glycyrrhizin and its
metabolite 18beta-glycyrhetinic acid in glycyrrhiza, a constituent herb
of yokukansan, ameliorate thiamine deficiency-induced dysfunction of
glutamate transport in cultured rat cortical astrocytes. Eur. J. Pharmacol.
2010, 626, 154–158.
(18) Pang, X.; Zhang, L.; Wu, Y.; Lin, L.; Li, J.; Qu, W.; Safe, S.; Liu,
M. Methyl 2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate (CDODA-
Me), a derivative of glycyrrhetinic acid, functions as a potent angiogen-
esis inhibitor. J. Pharmacol. Exp. Ther. 2010, 335, 172–179.
(19) Kao, T.; Shyu, M. H.; Yen, G. C. Glycyrrhizic acid and 18beta-
glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3beta signal-
ing and glucocorticoid receptor activation. J. Agric. Food Chem. 2010,
58, 8623–8629.
(20) Huang, L.; Yu, D.; Ho, P.; Qian, K.; Lee, K. H.; Chen, C. H.
Synthesis and proteasome inhibition of glycyrrhetinic acid derivatives.
Bioorg. Med. Chem. 2008, 16, 6696–6701.
(21) Chintharlapalli, S.; Papineni, S.; Jutooru, I.; McAlees, A.; Safe, S.
Structure-dependent activity of glycyrrhetinic acid derivatives as peroxi-
some proliferator-activated receptor gamma agonists in colon cancer
cells. Mol. Cancer Ther. 2007, 6, 1588–1598.
(28) Tatsuzaki, J.; Taniguchi, M.; Bastow, K. F.; Nakagawa-Goto, K.;
Morris-Natschke, S. L.; Itokawa, H.; Baba, K.; Lee, K.-H. Anti-tumor
agents: Novel glycyrrhetinic acid-dehydrozingerone conjugates as cyto-
toxic agents. Bioorg. Med. Chem. 2007, 15, 6193–6199.
(29) Fukuoka, S.; Kida, T.; Nakajima, Y.; Tsumagari, T.; Watanabe,
W.; Inaba, Y.; Mori, A.; Matsumura, T.; Nakano, Y.; Takeshita, K.
Thermo-responsive extraction of cadmium(II) ion with TPEN-NIPA
gel. Effect of the number of polymerizable double bond toward gel
formation and the extracting behavior. Tetrahedron 2010, 66,
1721–1727.
(30) Joseph, B.; Darro, F.; Bꢀeliard, A.; Lesur, B.; Collignon, F.;
Decaestecker, C.; Frydman, A.; Guillaumet, G.; Kiss, R. 3-aryl-2-
quinolone derivatives: synthesis and characterization of in vitro and
in vivo anti-tumor effects with emphasis on a new therapeutical target
connected with cell migration. J. Med. Chem. 2002, 45, 2543–2555.
(31) Ozap-Yaman, S.; de Hoog, P.; Amadei, G.; Pitiꢀe, M.; Gamez, P.;
Dewelle, J.; Mijatovic, T.; Meunier, B.; Kiss, R.; Reedijk, J. Platined
copper (3-clip-phen) complexes as effective DNA-cleaving and cyto-
toxic agents. Chem.—Eur. J. 2008, 14, 3418–3426.
(32) Lamoral-Theys, D.; Andolfi, A.; Van Goietsenoven, G.; Cim-
mino, A.; Le Calvꢀe, B.; Wauthoz, N.; Mꢀegalizzi, V.; Gras, T.; Bruyꢁere, C.;
Dubois, J.; Mathieu, V.; Kornienko, A.; Kiss, R.; Evidente, A. Lycorine,
the main phenanthridine Amaryllidaceae alkaloid, exhibits significant
antitumor activity in cancer cells that display resistance to proapoptotic
stimuli: An investigation of structure-activity relationship and mechan-
istic insight. J. Med. Chem. 2009, 52, 6244–6256.
(33) Ingrassia, L.; Lefranc, F.; Dewelle, J.; Pottier, L.; Mathieu, V.;
Spiegl-Kreinecker, S.; Sauvage, S.; El Yazidi, M.; Dehoux, M.; Berger, W.;
Van Quaquebeke, E.; Kiss, R. Structure-activity relationship analysis of
novel derivatives of narciclasine (an Amaryllidaceae isocarbostyril
derivative) as potential anticancer agents. J. Med. Chem. 2009, 52,
1100–1114.
(34) Branle, F.; Lefranc, F.; Camby, I.; Jeuken, J.; Geurts-Moespot,
A.; Sprenger, S.; Sweep, F.; Kiss, R.; Salmon, I. Evaluation of the
efficiency of chemotherapy in in vivo orthotopic models of human
glioma cells with and without 1p19q deletions and in C6 rat orthotopic
allografts serving for the evaluation of surgery combined with che-
motherapy. Cancer 2002, 95, 641–655.
(35) Mathieu, A.; Remmelink, M.; D’Haene, N.; Penant, S.; Gaussin,
J. F.; Van Ginckel, R.; Darro, F.; Kiss, R.; Salmon, I. Development of a
chemoresistant orthotopic human nonsmall cell lung carcinoma model
in nude mice. Cancer 2004, 101, 1908–1918.
(36) Mijatovic, T.; Mathieu, V.; Gaussin, J. F.; De Nꢁeve, N.;
Ribaucour, F.; Van Quaquebeke, E.; Dumont, P.; Darro, F.; Kiss, R.
Cardenolide-induced lysosomal membrane permeabilization demon-
strates therapeutic benefits in experimental human non-small cell lung
cancers. Neoplasia 2006, 5, 402–412.
(22) Chintharlapalli, S.; Papineni, S.; Konopleva, M.; Andreef, M.;
Samudio, I.; Safe, S. 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid and
related compounds inhibit growth of colon cancer cells through peroxi-
some proliferator-activated receptor gamma-dependent and -indepen-
dent pathways. Mol. Pharmacol. 2005, 68, 119–128.
(23) Papineni, S.; Chintharlapalli, S.; Safe, S. Methyl 2-cyano-3,11-
dioxo-18beta -olean-1,12-dien-30-oate is a peroxisome proliferator-
activated receptor-gamma agonist that induces receptor-independent
apoptosis in LNCaP prostate cancer cells. Mol. Pharmacol. 2008,
73, 553–565.
(37) Mathieu, V.; Pirker, C.; Martin de Lassalle, E.; Vernier, M.;
Mijatovic, T.; De Neve, N.; Gaussin, J. F.; Dehoux, M.; Lefranc, F.;
Berger, W.; Kiss, R. The sodium pump alpha-1 subunit: a disease
progression-related target for metastatic melanoma treatment. J. Cell
Mol. Med. 2009, 13, 3960–3972.
(38) Dumont, P.; Ingrassia, L.; Rouzeau, S.; Ribaucour, F.; Thomas,
S.; Roland, I.; Darro, F.; Lefranc, F.; Kiss, R. The Amaryllidaceae
isocarbostyril narciclasine induces apoptosis by activation of the death
receptor and/or mitochondrial pathways in cancer cells but not in
normal fibroblasts. Neoplasia 2007, 9, 766–776.
(24) Chadalapaka, G.; Jutooru, I.; McAlees, A.; Stefanac, T.; Safe, S.
Structure-dependent inhibition of bladder and pancreatic cancer cell
growth by 2-substituted glycyrrhetinic and ursolic acid derivatives.
Bioorg. Med. Chem. Lett. 2008, 18, 2633–2639.
(25) Michalik, L.; Wahli, W. PPARs mediate lipid signaling in
inflammation and cancer. PPAR Res. 2008, ID 134059, doi:10.1155/
2008/134059.
(39) Gerber, D. E.; Minna, J. D. ALK inhibition for non-small cell
lung cancer: from discovery to therapy in record time. Cancer Cell 2010,
18, 548–551.
(26) Bundscherer, A.; Reichle, A.; Hafner, C.; Meyer, S.; Vogt, T.
Targeting the tumor stroma with peroxisome proliferator activated
receptor (PPAR) agonists. Anticancer Agents Med. Chem. 2009, 9,
816–821.
(27) He, X.; Yu, S.; Dong, Y. Y.; Yan, F.; Chen, L. Preparation and
properties of a novel thermo-responsive poly(N-isopropylacrylamide)
hydrogel containing glycyrrhetinic acid. J. Mater. Sci. 2009, 44,
4078–4086.
(40) Qiu, Y.; Kung, H. J. Signaling network of the Btk family kinases.
Oncogene 2000, 19, 5651–5661.
(41) Dopeso, H.; Mateo-Lozano, S.; Mazzolini, R.; Rodrigues, P.;
Lagares-Tena, L.; Ceron, J.; Romero, J.; Esteves, M.; Landolfi, S.;
Hernandez-Losa, J.; Castano, J.; Wilson, A. J.; Ramon y Cajal, S.;
Mariadason, J. M.; Schwartz, S., Jr.; Arango, D. The receptor tyrosine
kinase EPHB4 has tumor suppressor activities in intestinal tumorigen-
esis. Cancer Res. 2009, 69, 7430–7438.
6511
dx.doi.org/10.1021/jm200285z |J. Med. Chem. 2011, 54, 6501–6513

Journal of Medicinal Chemistry
ARTICLE
(42) Herath, N. I.; Boyd, A. W. The role of Eph receptors and ephrin
ligands in colorectal cancer. Int. J. Cancer 2010, 126, 2003–2011.
(43) Smith, J. A.; Samayawardhena, L. A.; Craig, A. W. Fps/Fes
protein-tyrosine kinase regulats mast cell adhesion and migration
dowstrean of kit and beta1 integrin receptors. Cell Signal 2010, 22,
427–436.
(44) Saito, Y. D.; Jensen, A. R.; Salgia, R.; Posadas, E. M. Fyn: a novel
molecular target in cancer. Cancer 2010, 116, 1629–1637.
(45) Katoh, M. FGFR2 abnormalities underlies a spectrum of bone,
skin, and cancer pathologies. J. Invest. Dermatol. 2009, 129, 1861–1867.
(46) Scartozzi, M.; Bianconi, M.; Maccaroni, E.; Giamperi, R.;
Beradi, R.; Cascinu, S. Dalotuzumab, a recombinant humanized mAb
targeted against IGFR1 for the treatment of cancer. Curr. Opin. Mol.
Ther. 2010, 12, 361–371.
(47) Cockburn, J. G.; Richardson, D. S.; Gujral, T. S.; Mulligan, L. M.
RET-mediated cell adhesion and migration require multiple integrin
subunits. J. Clin. Endocrinol. Metab. 2010, 95, E342–E346.
(48) Lee, B. C.; Lee, T. H.; Zagozdzon, R.; Avraham, S.; Usheva, A.;
Avraham, H. K. Carboxyl-terminal Src kinase homologous kinase
negatively regulates the chemokine receptor CXCR4 through YY1 and
impairs CXCR4/CXCL12 (SDF-1alpha)-mediated breast cancer cell
migration. Cancer Res. 2005, 65, 2840–2845.
(49) Coniglio, S. J.; Zavarella, S.; Symons, M. H. Pak1 and Pak2
mediate tumor cell invasion through distinct signaling mechanisms. Mol.
Cell. Biol. 2008, 28, 4162–4172.
(50) Lee, D. H.; Goldberg, A. L. Proteasome inhibitors: valuable new
tools for cell biologists. Trends Cell Biol. 1998, 8, 397–403.
(51) Hines, J.; Groll, M.; Fahnestock, M.; Crews, C. M. Proteasome
Inhibition by Fellutamide B Induces Nerve Growth Factor Synthesis.
Chem. Biol. (Cambridge, MA, U. S.) 2008, 15, 501–512.
(52) de Bettignies, G.; Coux, O. Proteasome inhibitors: Dozens of
molecules and still counting. Biochimie 2010, 92, 1530–1545.
(53) Blackburn, C.; Gigstad, K. M.; Hales, P.; Garcia, K.; Jones, M.;
Bruzzese, F. J.; Barrett, C.; Liu, J. X.; Soucy, T. A.; Sappal, D. S.; Bump,
N.; Olhava, E. J.; Fleming, P.; Dick, L. R.; Tsu, C.; Sintchak, M. D.; Blank,
J. L. Characterization of a new series of non-covalent proteasome
inhibitors with exquisite potency and selectivity for the 20S beta
5-subunit. Biochem. J. 2010, 430, 461–476.
(54) Geurink, P. P.; Liu, N.; Spaans, M. P.; Downey, S. L.; van den
Nieuwendijk, A. M. C. H.; van der Marel, G. A.; Kisselev, A. F.; Florea,
B. I.; Overkleeft, H. S. Incorporation of fluorinated phenylalanine
generates highly specific inhibitor of proteasome’s chymotrypsin-like
sites. J. Med. Chem. 2010, 53, 2319–2323.
(64) Bruyꢁere, C.; Lonez, C.; Duray, A.; Cludts, S.; Ruysschaert, J. M.;
Saussez, S.; Yeaton, P.; Kiss, R.; Mijatovic, T. Considering Temozolo-
mide as a novel potential treatment for oesophageal cancer. Cancer 2011,
117, 2004–2016.
(65) Lefranc, F.; Brotchi, J.; Kiss, R. Possible future issues in the
treatment of glioblastomas, with a special emphasis on cell migration and
the resistance of migrating glioblastoma cells to apoptosis. J. Clin. Oncol.
2005, 23, 2411–2422.
(66) Landis-Piwowar, K. R.; Milacic, V.; Chen, D.; Yang, H.; Zhao,
Y.; Chan, T. H.; Yan, B.; Dou, Q. P. The proteasome as a potential target
for novel anticancer drugs and chemosensitizers. Drug Resist. Updates
2006, 9, 263–273.
(67) Guo, L.; Zhou, Y.; Sun, Y.; Zhang, F. Non-receptor tyrosine
kinase Etk regulation of drug resistance in small-cell lung cancer. Eur. J.
Cancer 2010, 46, 636–641.
(68) Chen, D.; Dou, Q. P. The ubiquitin-proteasome system as a
prospective molecular target for cancer treatment and prevention. Curr.
Protein Pept. Sci. 2010, 11, 459–470.
(69) Tsukamoto, S.; Yokosawa, H. Targeting the proteasome path-
way. Expert Opin. Ther. Targets 2009, 13, 605–621.
(70) Liggett, A.; Crawford, L. J.; Walker, B.; Morris, T. C. M.; Irvine,
A. E. Methods for measuring proteasome activity: Current limitations
and future developments. Leukemia Res. 2010, 34, 1403–1409.
(71) Lefranc, F.; Facchini, V.; Kiss, R. Pro-autophagic drugs: A novel
means to combat apoptosis-resistant cancers, with a special emphasis on
glioblastomas. Oncologist 2007, 12, 1395–1403.
(72) Wu, W. K.; Cho, C. H.; Lee, C. W.; Wu, K; Fan, D; Yu, J; Sung,
J. J. Proteasome inhibition: a new therapeutic strategy to cancer
treatment. Cancer Lett. 2010, 293, 15–22.
(73) Richardson, P. G.; Barlogie, B.; Berenson, J.; Singhal, S.;
Jagannath, S.; Irwin, D.; Rajkumar, S. V.; Srkalovic, G.; Alsina, M.;
Alexanian, R.; Siegel, D.; Orlowski, R. Z.; Kuter, D.; Limentani, S. A.;
Lee, S.; Hideshima, T.; Esseltine, D.-L.; Kauffman, M.; Adams, J.;
Schenkein, D. P.; Anderson, K. C. A phase 2 study of bortezomib
in relapsed, refractory myeloma. New Engl. J. Med. 2003, 348,
2609–2617.
(74) Richardson, P. G.; Briemberg, H.; Jagannath, S.; Wen, P. Y.;
Barlogie, B.; Berenson, J.; Singhal, S.; Siegel, D. S.; Irwin, D.; Schuster,
M.; Srkalovic, G.; Alexanian, R.; Rajkumar, S. V.; Limentani, S.; Alsina,
M.; Orlowski, R. Z.; Najarian, K.; Esseltine, D.; Anderson, K. C.; Amato,
A. A. Frequency, characteristics, and reversibility of peripheral neuro-
pathy during treatment of advanced multiple myeloma with bortezomib.
J. Clin. Oncol. 2006, 24, 3113–3120.
(55) Furet, P.; Imbach, P.; Noorani, M.; Koeppler, J.; Laumen, K.;
Lang, M.; Guagnano, V.; Fuerst, P.; Roesel, J.; Zimmermann, J.; Garcia
Echeverria, C. Entry into a new class of potent proteasome inhibitors
having high antiproliferative activity by structure-based design. J. Med.
Chem. 2004, 47, 4810–4813.
(75) Lonial, S.; Waller, E. K.; Richardson, P. G.; Jagannath, S.;
Orlowski, R. Z.; Giver, C. R.; Jaye, D. L.; Francis, D.; Giusti, S.; Torre,
C.; Barlogie, B.; Berenson, J. R.; Sunghal, S.; Schenkein, D. P.;
Esseltine, D. L. W.; Anderson, J.; Xiao, H.; Heffner, L. T.; Anderson,
K. C. Risk factors and kinetics of thrombocytopenia associated with
bortezomib for relapsed, refractory multiple myeloma. Blood 2005,
106, 3777–3784.
(56) Simpson, C. D.; Anyiwe, K.; Schimmer, A. D. Anoikis resistance
and tumor metastasis. Cancer Lett. 2008, 272, 177–185.
(57) Savage, P.; Stebbing, J.; Bower, M.; Crook, T. Why does
cytotoxic chemotherapy cure only some cancers? Nat. Clin. Pract. Oncol.
2009, 6, 43–52.
(58) Wilson, T. R.; Johnston, P. G.; Longley, D. B. Anti-apoptotic
mechanisms of drug resistance in cancer. Curr. Cancer Drug Targets
2009, 9, 307–319.
(59) Bozec, A.; Peyrade, F.; Fischel, J. L.; Milano, G. Emerging
molecular targeted therapies in the treatment of head and neck cancer.
Expert Opin. Emerg. Drugs 2009, 14, 299–310.
(76) Blanquicett, C.; Roman, J.; Hart, C. M. Thiazolidinediones as
anti-cancer agents. Cancer Ther. 2008, 6, 25–34.
(77) He, B. C.; Chen, L.; Zuo, G. W.; Zhang, W.; Bi, Y.; Huang, J.;
Wang, Y.; Jiang, W.; Luo, Q.; Shi, Q.; Zhang, B. Q.; Liu, B.; Lei, X.; Luo,
J.; Luo, X.; Wagner, E. R.; Kim, S. H.; He, C. J.; Hu, Y.; Shen, J.; Zhou, Q.;
Rastegar, F.; Deng, Z. L.; Luu, H. H.; He, T. C.; Haydon, R. C.
Synergistic antitumor effect of the activated PPARgamma and
retinoid recptors on human osteosarcoma. Clin. Cancer Res. 2010, 16,
2235–2245.
(60) Han, S.; Roman, J. Targeting apoptotic signaling pathways in
human lung cancer. Curr. Cancer Drug Targets 2010in press.
(61) Soengas, M. S.; Lowe, S. W. Apoptosis and melanoma che-
moresistance. Oncogene 2003, 22, 3138–3151.
(78) Lee, J. Y.; Kim, J. K.; Cho, M. C.; Shin, S.; Yoon, D. Y.; Heo,
Y. S.; Kim, Y. Cytotoxic flavonoids as agonists of peroxysome prolif-
erator-activated receptor gamma on human cervical and prostate cancer
cells. J. Nat. Prod. 2010, 73, 1261–1265.
(62) Wong, H. H.; Lemoine, N. R. Pancreatic cancer: molecular
pathogenesis and new therapeutic targets. Nat. Rev. Gastroenterol.
Hepatol. 2009, 6, 412–422.
(63) D’Amico, T. A.; Harpole, D. H., Jr. Molecular biology of
esophageal cancer. Chest Surg. Clin. N. Am. 2000, 10, 451–469.
(79) Reka, A. K.; Kurapati, H.; Narala, V. R.; Bommer, G.; Chen, J.;
Standiford, T. J.; Keshamouni, V. G. Peroxisome proliferator-activated
receptor-gamma activation inhibits tumor metastasis by antagonizing
Smad3-mediated epithelial-mesenchymal transition. Mol. Cancer Res.
2010, 9, 3221–3232.
6512
dx.doi.org/10.1021/jm200285z |J. Med. Chem. 2011, 54, 6501–6513

Journal of Medicinal Chemistry
ARTICLE
(80) Voutsadakis, I. A. Peroxisome proliferator activated receptor-
gamma and the ubiquitin-proteasome system in colorectal cancer. World
J. Gastrointest. Oncol. 2010, 2, 235–241.
(81) Knight, Z. A.; Lin, H.; Shokat, K. M. Targeting the cancer
kinome through polypharmacology. Nat. Rev. Cancer 2010, 10, 130–137.
(82) Fedorov, O.; M€uller, S.; Knapp, S. The (un)target cancer
kinome. Nat. Chem. Biol. 2010, 6, 166–169.
(83) Gossage, L.; Eisen, T. Targeting multiple kinase pathways: a
change in paradigm. Clin. Cancer Res. 2010, 16, 1973–1978.
(84) Agrawal, P. K.; Jain, D. C. 13C NMR spectroscopy of oleanane
triterpenoids. Prog. NMR Spectrosc. 1992, 24, 1–90.
(85) Mahato, S. B.; Kundu, A. P. 13C NMR spectra of pentacyclic
triterpenoids ꢀ a compilation and some salient features. Phytochemistry
1994, 37 (6), 1517–1575.
(86) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra
precision Glide: Docking and scoring incorporating a model of hydro-
phobic enclosure for protein-ligand complexes. J. Med. Chem. 2006,
49, 6177–6196.
(87) Arrigo, A. P.; Tanaka, K.; Goldberg, A. L.; Welch, W. J. Identity
of the 19S ’prosome’ particle with the large multifunctional protease
complex of mammalian cells (the proteasome). Nature (London) 1988,
331, 192–194.
(88) Sadowski, J. A hybrid approach for addressing ring flexibility in
3D database searching. J. Comput.-Aided Mol. Des. 1997, 11, 53–60.
(89) Moravec, R. A.; O’Brien, M. A.; Daily, W. J.; Scurria, M. A.;
Bernad, L.; Riss, T. L. Cell-based bioluminescent assays for all three
proteasome activities in a homogeneous format. Anal. Biochem. 2009,
387, 294–302.
(90) Lamoral-Theys, D.; Pottier, L.; Kerff, F.; Dufrasne, F.; Prou-
tiꢁere, F.; Wauthoz, N.; Neven, P.; Ingrassia, L.; Van Antwerpen, P.;
Lefranc, F.; Gelbcke, M.; Pirotte, B.; Kraus, J. L.; Nꢁeve, J.; Kornienko, A.;
Kiss, R.; Dubois, J. Simple di- and trivanillates exhibit cytostatic proper-
ties toward cancer cells resistant to pro-apoptotic stimuli. Bioorg. Med.
Chem. 2010, 18, 3823–3833.
6513
dx.doi.org/10.1021/jm200285z |J. Med. Chem. 2011, 54, 6501–6513