Discovery of 3-(2-aminoethyl)-5-(3-phenyl-propylidene)-thiazolidine-2,4- dione as a dual inhibitor of the Raf/MEK/ERK and the PI3K/Akt signaling pathways

Article abstract of DOI:10.1016/j.bmcl.2010.06.030

A thiazolidine-2,4-dione derivative, 3-(2-aminoethyl)-5-(3-phenyl- propylidene)-thiazolidine-2,4-dione (2), was identified as a dual inhibitor of the Raf/MEK/ extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades. The discovered compound inhibited cell proliferation, induced early apoptosis, and arrested cells in G₀/G₁ phase in human leukemia U937 cells. These results indicate its potential as a new lead compound to develop novel dual signaling pathway inhibitors and anticancer agents.

Full text of DOI:10.1016/j.bmcl.2010.06.030

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4526–4530
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3-(2-aminoethyl)-5-(3-phenyl-propylidene)-thiazolidine-2,4-dione
as a dual inhibitor of the Raf/MEK/ERK and the PI3K/Akt signaling pathways
Qianbin Li a, Jingde Wu a, Hui Zheng b, Kai Liu a, Tai L. Guo c, Yuying Liu b, Scott T. Eblen b, Steven Grant d,
Shijun Zhang a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298-0540, USA
b Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC, USA
c Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA
d Departments of Internal Medicine and Biochemistry, Virginia Commonwealth University, Richmond, VA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A thiazolidine-2,4-dione derivative, 3-(2-aminoethyl)-5-(3-phenyl-propylidene)-thiazolidine-2,4-dione
(2), was identified as a dual inhibitor of the Raf/MEK/ extracellular signal-regulated kinase (ERK) and
the phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades. The discovered compound inhibited cell
proliferation, induced early apoptosis, and arrested cells in G0/G1 phase in human leukemia U937 cells.
These results indicate its potential as a new lead compound to develop novel dual signaling pathway
inhibitors and anticancer agents.
Received 10 May 2010
Accepted 4 June 2010
Available online 8 June 2010
Keywords:
Dual pathway inhibitor
Raf/MEK/ERK
Published by Elsevier Ltd.
PI3K/Akt
Thiazolidine-2,4-dione
Dysregulated signaling pathways have been implicated to
promote cancer cell survival and growth, in which the Raf/MEK/
extracellular signal-regulated kinase (ERK) cascade and the phos-
phatidylinositol 3-kinase (PI3K)/Akt cascade are the best charac-
terized. The Raf/MEK/ERK pathway is one of the evolutionarily
conserved mitogen-activated protein kinase (MAPK) pathways that
play critical roles in driving proliferation and preventing apopto-
sis.1 Upon activation by growth factors, serum, cytokines and
osmotic stresses, ERK can phosphorylate and regulate multiple
substrates such as cytoskeletal proteins, kinases and transcription
factors within various cellular compartments.2 These events in
turn result in gene expression changes and alteration in cell prolif-
eration, differentiation and survival. This pathway has received
particular attention in the past 15 years as substantial evidence
has shown that aberrant activation of this pathway at different lev-
els is involved in the oncogenesis of various human cancers, espe-
cially in melanoma, breast cancers, ovarian cancers and human
leukemias.3,4 Numerous structurally diverse molecules have been
developed by targeting the Raf/MEK/ERK pathway in search for po-
tential medications for various human cancers and have been
extensively reviewed in recent articles.3,5,6
pathway in human cancers with the possible exception of the
p53 and retinoblastoma pathway.7 Upon stimulation by growth
factors and cytokines, PI3K is recruited to the plasma membrane
and subsequently converts phosphatidylinositol-3,4-bisphosphate
(PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3) that
will in turn recruits and activates a serine/threonine kinase Akt to-
gether with 30-phosphoinositide-dependent kinase (PDK). Signals
through this cascade regulate many fundamental cellular functions
such as cell growth, proliferation, survival, apoptosis, and metabo-
lism through a variety of downstream effectors including proapop-
totic and antiapoptotic factors, mTOR, glycogen synthase kinase-3
(GSK-3), and p53, among others.7–9 Phosphatase and tensin homo-
log deleted on chromosome 10 (PTEN), a negative regulator of
PI3K/Akt signaling by specifically dephosphorylating PIP3 has been
detected to lose its activity by either genetic or epigenetic modifi-
cations in many primary and metastatic human cancers.9,10 Muta-
tions and/or activation of PI3K and Akt have been detected in
various human cancers.9,11,12 Therefore, it is logical to target this
pathway to develop potential treatment agents, and indeed small
molecule inhibitors including PI3K inhibitors, Akt inhibitors and
mTOR inhibitors have been developed and/or approved as treat-
ment agents for various human cancers.13
PI3K/Akt signaling pathway is another signaling cascade that
has been implicated to be crucial in cancer development. Genomic
aberrations in this pathway are prevalent compared to any other
Notably, these two signaling pathways intimately and coopera-
tively link with each other, rather than exclusively, to regulate
apoptosis and the survival of transformed cells.4,14 Both signaling
pathways can phosphorylate and regulate many common down-
stream effectors involved in the regulation of cell survival and
* Corresponding author. Tel.: +1 804 6288266; fax: +1 804 8287625.
E-mail address: szhang2@vcu.edu (S. Zhang).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.06.030

Q. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4526–4530
4527
apoptosis such as CREB, Bad, Bim and caspase 9, among others.4
Accumulating evidence has strongly suggested crosstalk and the
possible existence of a feedback regulation loop between these
two pathways. For example, most recently, studies have demon-
strated the activation of Raf/MEK/ERK cascade upon the treatment
with mTOR inhibitor in patients with metastatic cancers as well as
in cancer cell lines and prostate cancer animal model, which
strongly suggests feedback regulation loops in and crosstalk be-
tween the Raf/MEK/ERK and PI3K/Akt cascades.15 This phenome-
non may contribute to drug resistance to inhibitors targeting
single cascade. Another elegant study also supported this notion
by showing frequent activation of Raf/MEK/ERK and PI3K/Akt cas-
cades in advanced human prostate cancer.16 More importantly,
several elegant studies have demonstrated synergistic effects in
triggering cancer cell death by concomitant interruption of these
two pathways both in vitro and in vivo,15–19 which indicates that
more clinically beneficial pharmacotherapies may be obtained by
co-targeting these two pathways simultaneously. However, to
our knowledge, all the combined targeted therapies use a mixture
of two individual inhibitors for the Raf/MEK/ERK and PI3K/Akt
pathways and no single small molecule has been reported or devel-
oped to inhibit these two pathways simultaneously. The use of
dual pathway inhibitors may provide certain advantages over sin-
gle pathway inhibitors in the following aspects: (1) enhanced po-
tency and reduced risk of drug resistance; (2) reduced toxicity
and improved patient compliance. Thus, the design and develop-
ment of such dual inhibitors would provide the cancer research
community with novel chemical tools and potential newer anti-
cancer agents.
its analogs began with the synthesis of thiazolidine-2,4-dione
intermediates 7. As shown in Scheme 1, alkylation of thiazoli-
dine-2,4-dione 6 with Boc protected 2-bromoethaneamine affor-
ded 7, which on Knoevenagel condensation reaction with
propionaldehyde or 3-phenylpropionaldehyde to afford 2 or 3,
respectively.22 Reduction of 2 under catalytic hydrogenation condi-
tions yielded saturated analog 4 in good yield. Acetylation of 2 with
acetic anhydride afforded compound 5.
The activation of the Raf/MEK/ERK pathway and the PI3K/Akt
pathway has been shown to play multiple important roles in the
proliferation and apoptosis of hematopoietic cells.4 Furthermore,
it has been shown that interruption of this process by MEK and
Akt inhibitors leads to a dramatic increase in mitochondrial dam-
age and apoptosis in human leukemia cells. Additionally, human
leukemia cells have been shown to be good models to assay new
potential ERK inhibitors in our laboratory.20 Therefore, human leu-
kemia U937 cells were initially employed to evaluate 2 and its ana-
logs for their inhibitory effects on cell viability with the MTS [(3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium] assay. The known MEK inhibitor
PD184352 was used as positive control.23 As shown in Figure 2A,
2 exhibited significant inhibition on U937 cell viability at higher
concentrations (15, 20, and 25
than 10 M. PD184352 and 2 showed comparable potencies at
15, 20, and 25 M concentrations. Notably, compounds 3, 4, and
lM) but not at concentrations less
l
l
5 did not show any inhibitory effects at tested concentrations,
which may suggest that the phenylpropylidene, primary amine
and aromatic ring are important structural determinants for activ-
ities (Fig. 2B). The inhibitory effects of 2 were further confirmed in
multiple cancer cell lines including OVCAR5, SKOV3, PC3, DU145,
and HT-29 cells (data not shown). The MTS assay only examines
the metabolically healthy cells, but cannot distinguish whether
the cells are actively dividing or quiescent. In contrast, the [3H]-
thymidine incorporation assay assesses actively dividing cells in
a sample by detecting DNA synthesis. Therefore, 2 and PD184352
were further evaluated using [3H]-thymidine incorporation assays
Molecules containing the thiazolidine-2,4-dione moiety such as
the anti-diabetic drug troglitazone have been recently reported to
have anticancer activities through inhibition of the Raf/MEK/ERK
signal cascade.20 In our effort to design and discover novel tem-
plates targeting the Raf/MEK/ERK signaling cascade, we have em-
barked on development of the thiazolidine-2,4-dione derivatives
as potential substrate-specific ERK inhibitors.21 From our recent
studies, we discovered that the structural extension of benzylidene
in compound 1 to alkylidene shifted the biological target from ERK
to their upstream activators. Herein, we report the discovery of a
new thiazolidine-2,4-dione compound, 3-(2-aminoethyl)-5-(3-
in U937 cells. As shown in Figure 2C, at 10
inhibited the [3H]-thymidine incorporation, while PD184352 did
not. At 30
M, 2 almost completely inhibited [3H]-thymidine
lM, 2 significantly
l
incorporation, and PD184352 also exhibited significant inhibition
at this concentration. Taken together, these results may indicate
that 2 may induce early apoptosis of U937 cells through inhibiting
phenyl-propylidene)-thiazolidine-2,4-dione 2, as
a novel lead
structure for developing dual pathway inhibitors of the Raf/MEK/
ERK and PI3K/Akt pathways and anticancer agents (Fig. 1).
DNA synthesis at 10 lM but not affecting cell viability.
Compound 2 contains several structural features that may con-
tribute to its functional activities, such as the phenylpropylidene
double bond which can act as a Michael addition reaction acceptor,
the primary amine in the ethylamine tail for ionic interactions, and
the aromatic ring for hydrophobic interactions. Therefore, com-
pounds 3, 4, and 5 were designed along with 2 to shed light on
its potential binding interaction features. The synthesis of 2 and
To get insight into the signaling pathways that are possibly in-
volved in 2’s functional activities, Western blot analysis was then
performed in U937 cells. As shown in Figure 3A, PD184352 signif-
icantly inhibited the phosphorylation of both ERK and its down-
stream substrate Rsk1 at as low as
3 lM concentration.
Compound 2 also significantly inhibited the phosphorylation of
O
O
O
NHBoc
O
R
a
b
N
N
NH
NH2
O
S
S
O
S
O
O
7
O
6
N
O
N
O
NH2
NH2
NH2
S
S
S
3 R = CH3
2 R = Ph
O
S
2
1
3
5
c
O
O
N
NH2
S
4
N
O
N
O
O
NH2
4
O
O
d
O
N
O
NH
S
5
N
O
NH
S
O
Scheme 1. Synthesis of designed compounds. Reagents: (a) BocNHCH2CH2Br, TBAl,
acetone; (b) (i) RCHO, piperidine, EtOH; (ii) HCl/dioxane; (c) H2, Pd–C, MeOH; (d)
Ac2O, CH2Cl2.
Figure 1. Chemical structures of designed compounds.

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Q. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4526–4530
Figure 2. Inhibition of cell proliferation using MTS assay (A and B) and [3H]-thymidine incorporation (C) in human leukemia U937 cells.
ERK and Rsk1 at 10 and 30
l
M concentrations, therefore being
To further evaluate whether other signaling pathways that are
involved in apoptosis and survival regulation are affected by 2,
we next examined the levels of p-Akt, p-p38 and p-JNK upon treat-
slightly less potent than PD184352. However, when the p-MEK le-
vel was evaluated, it is notable that 2 dose-dependently decreased
the p-MEK level in U937 cells while treatment with PD184352 re-
sulted in a dose-dependent increase in the p-MEK levels, which is
consistent with the reported negative feedback mechanism in the
Raf/MEK/ERK pathway.24 This might indicate that 2 targets either
an upstream activator of MEK in the Raf/MEK/ERK signaling path-
way or it inhibits MEK via a different mechanism. When com-
pounds 3, 4, and 5 were evaluated, no inhibitory effects on p-
MEK and p-ERK were observed (Fig. 3B), which is consistent with
their activities in cell viability assays. These limited structure–
activity relationship (SAR) data further highlight the importance
of the phenylpropylidene, the primary amine, and the aromatic
ring for activity.
ment with PD184352 and 2 in U937 cells. Notably, 2 at 25 lM sig-
nificantly and consistently suppressed the p-Akt level while the
specific MEK inhibitor PD184352 exhibited no inhibitory effects
on p-Akt (Fig. 3C). Surprisingly, PD184352 increased the total Akt
level at tested concentrations. Compound 2 also increased the total
Akt level at tested concentrations except for 25 lM. At this stage, it
is not clear at what level (PI3K or PDK) 2 targets the PI3K/Akt cas-
cade to reduce the p-Akt level. Further structural modifications of 2
in our laboratory have demonstrated consistent p-MEK and p-Akt
suppression and studies have been undertaken to identify target
protein(s), to elucidate the mode of action, and to increase its
potencies. Furthermore, both 2 and PD184352 exhibited minimal
Figure 3. Western blot analysis of compound 2 and its analogs in U937 cells.

Q. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4526–4530
4529
Figure 4. Apoptotic effects of compound 2 and PD184352 on U937 cells.
effects on the level of p-p38 and p-JNK. These results may indicate
that 2 has specific dual inhibition towards the Raf/MEK/ERK and
the PI3K/Akt signaling pathways. Further studies are warranted
to evaluate its target specificity.
the lethal effects induced by 2 at this concentration (Fig. 4A) are
mainly through early apoptosis while preserving the metabolic
functions of U937 cells. At higher concentrations (15, 20 and
25 lM), 2 dose-dependently increased both cell death and the
The Raf/MEK/ERK cascade and the PI3K/Akt cascade have been
demonstrated to play important roles in the regulation of apopto-
sis.4 In addition, mitochondria have been shown to play an impor-
tant role in cell death and the loss of mitochondria membrane
potential is an early event in mitochondrially mediated apopto-
sis.25 Therefore, we next studied the effects of 2 in inducing apop-
tosis in U937 cells using 7-aminoactinomycin D (7-AAD) combined
with the 3,30-dihexyloxocarbocyanine iodide (DiOC6) uptake to
measure the extent of mitochondria membrane potential change
number of cells exhibiting mitochondria membrane potential loss
in U937 cells. Together with the results from MTS assay (Fig. 2A),
these results indicate that 2 may inhibit U937 cell proliferation
through both apoptotic and necrotic pathways. On the other hand,
PD184352 only exhibited moderate lethal effects on U937 cells in
7-AAD assay and minimal effects on mitochondria membrane po-
tential loss at higher concentrations (15, 20, 25 lM), which sug-
gests that PD184352 inhibits U937 cell growth mainly through
necrotic mechanism. The superior effects of 2 in inducing apoptosis
as compared to PD184352 in U937 cell under these experimental
conditions might be due to its dual inhibition of the Raf/MEK/
ERK and PI3K/Akt pathways compared to PD184352’s inhibition
of only the Raf/MEK/ERK pathway.
Both Raf/MEK/ERK pathway and PI3K/Akt pathway have been
shown to regulate the G1–S and G2–M transition in the cell cy-
cle.28,29 Since 2 demonstrated dual inhibition of these two signal-
(D
w
m) in U937 cells by flow cytometry.26,27 As shown in Figure
4A and B, 2 at 10 M moderately induced U937 cell death
l
(17.2%) and slightly increased the number of U937 cells exhibiting
mitochondria membrane potential loss (13%) indicating early
apoptosis induction. However, 2 exhibited significant inhibitory ef-
fects on DNA synthesis without affecting the cell viability in U937
cells at this concentration (Fig. 2A and C). This may indicate that
Figure 5. Effects of compound 2 and PD184352 (25 lM) on U937 cell cycle.

4530
Q. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4526–4530
ing pathways, growth inhibition of various cancer cells, and the
induction of apoptosis in U937 cells, it would be of value to evalu-
ate whether it has effects on the cell cycle of U937 cells. As shown
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Supplementary data (details for synthetic procedures, analytical
data, and biological studies for compounds 2–5) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.06.030.