3,5-Disubstituted-thiazolidine-2,4-dione analogs as anticancer agents: Design, synthesis and biological characterization

Article abstract of DOI:10.1016/j.ejmech.2011.10.031

A series of 2,5-disubstituted-thiazolidine-2,4-dione analogs based on the newly identified lead 1, a potential anticancer agent via the inhibition of the Raf/MEK/extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades, were synthesized and biologically characterized. A new lead structure, 15, was identified to have improved anti-proliferative activities in U937 cells, to induce apoptosis in U937, M12 and DU145 cancer cells, and to arrest U937 cells at the S-phase. Furthermore, Western blot analysis demonstrated a correlation of the anti-proliferative activity and blockade of the Raf/MEK/ERK and PI3K/Akt signaling pathways. Collectively, these results strongly encourage further optimization of 15 as a new lead with multi-target properties to develop more potent compounds as anticancer agents.

Full text of DOI:10.1016/j.ejmech.2011.10.031

European Journal of Medicinal Chemistry 47 (2012) 125e137
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
3,5-Disubstituted-thiazolidine-2,4-dione analogs as anticancer agents: Design,
synthesis and biological characterization
Kai Liu a, Wei Rao a, Hardik Parikh a, Qianbin Li a, Tai L. Guo b, Steven Grant c, Glen E. Kellogg a,
,
Shijun Zhang a
*
a Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA
b Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA
c Department of Internal Medicine and Massey Cancer Center, 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 series of 2,5-disubstituted-thiazolidine-2,4-dione analogs based on the newly identified lead 1,
a potential anticancer agent via the inhibition of the Raf/MEK/extracellular signal regulated kinase (ERK)
and phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades, were synthesized and biologically
characterized. A new lead structure, 15, was identified to have improved anti-proliferative activities in
U937 cells, to induce apoptosis in U937, M12 and DU145 cancer cells, and to arrest U937 cells at the S-
phase. Furthermore, Western blot analysis demonstrated a correlation of the anti-proliferative activity
and blockade of the Raf/MEK/ERK and PI3K/Akt signaling pathways. Collectively, these results strongly
encourage further optimization of 15 as a new lead with multi-target properties to develop more potent
compounds as anticancer agents.
Received 16 June 2011
Received in revised form
12 October 2011
Accepted 14 October 2011
Available online 21 October 2011
Keywords:
Thiazolidine-2,4-dione
Anticancer
Signaling pathways
Apoptosis
Ó 2011 Elsevier Masson SAS. All rights reserved.
Multitarget molecule
1. Introduction
signaling cascades are the most commonly up-regulated in human
cancers [2e8]. More importantly, these two signaling pathways
Cancer has surpassed heart disease as the leading cause of death
in the United States in people younger than 85 and it is expected
that 1.53 million cases of cancer will be diagnosed in the United
States in 2010, among which more than 570,000 are expected to die
[1]. In addition to the human cost, more than $72 billion is spent
annually on cancer treatment, exacerbating problems with the
overextended U.S. health care economy. While many chemothera-
peutic strategies for cancer treatment have been proposed, tested
and in some cases implemented in the past few decades, these
diseases remain tenacious and deadly. Therefore, there is
a desperate need to develop treatments with novel mechanisms to
combat this disease.
have been shown to function cooperatively to promote trans-
formed cancer cell survival. For example, crosstalk and a feedback
regulation between the Raf/MEK/ERK and PI3K/Akt pathways have
been demonstrated recently, indicating the potential of interrup-
tion of more than one signaling pathway to optimize cytotoxic
effects and to reduce drug resistance [5,9e14]. To further support
this, several studies have demonstrated the synergistic effects in
triggering cancer cell death by concomitant interruption of these
two pathways both in vitro and in vivo using a combination regimen
[12e18]. Collectively, these results implicate that development of
novel compounds that can co-target the Raf/MEK/ERK and PI3K/Akt
signaling pathways may represent an innovative strategy to
provide clinically beneficial pharmacotherapy for human cancer.
Thiazolidine-2,4-dione (TZD) and rhodanine analogs have been
recognized as the privileged templates in drug design and
discovery [19,20] and numerous compounds containing the TZD
ring have been developed as potential anticancer agents, such as
the PI3K inhibitor GSK1059615 and its analogs [21]. More inter-
estingly, some TZD analogs such as the anti-diabetic drug trogli-
tazone have been shown to exhibit anticancer activities through
the inhibition of the Raf/MEK/ERK signaling pathway [22]. Rhoda-
nine compounds have been referred to as Pan Assay INterference
Survival signaling pathways under growth factor loops have
been implicated in cancer development, progression, and metas-
tasis, among which the Raf/MEK/extracellular signal regulated
kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt
* Corresponding author. Department of Medicinal Chemistry, School of Phar-
macy, Virginia Commonwealth University, 800 E. Leigh Street, Suite 205, Richmond,
VA 23298-0540, USA. Tel.: þ1 804 6288266; fax: þ1 804 8287625.
E-mail address: szhang2@vcu.edu (S. Zhang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.031

126
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
compounds (PAINs) by one group [23]. TZD compounds, differing
by only one atom from rhodanine (S to O), can produce completely
different pharmacological effects [24] and the success of TZD
analogs in preclinical and clinical trials may well support the
potential of such molecules as lead structures in developing anti-
cancer agents. Recently, we identified a TZD analog 1 with potential
anticancer activity mainly through the inhibition of the Raf/MEK/
ERK and PI3K/Akt signaling pathways (Fig. 1) [25,26]. Herein, we
report the structureeactivity relationship (SAR) studies of 1 to shed
light on the structural features that are critical to their biological
activity. One new lead compound with improved potency was
identified to inhibit the growth of multiple cancer cell lines via the
inhibition of the Raf/MEK/ERK and PI3K/Akt signaling cascades, to
induce apoptosis as well as to interfere with the cell cycle
transition.
the aromatic ring in biological activity. Compounds 17e19 were
designed to evaluate whether the primary amine is critical or
a functional group with similar H-bond interactions will retain
the activity. Compounds 20 (racemic) and 21 were designed to
investigate the role of the exocyclic double bond and the TZD
ring.
The synthesis began with the preparation of various aldehydes
as shown in Scheme 1. Briefly, substituted benzaldehydes (22), N-
Boc-indole-3- or -5-carbaldehyde (23 or 24), or cyclo-
propanecarbaldehyde (25) were converted to their Meldrum’s acid
derivatives 26 followed by reduction and hydrosilylation to give
various aldehydes 29 [27]. The synthesis of cyclohexyl-
propionaldehyde and 3-pyridin-3-yl-propionaldehyde was ach-
ieved through the Swern oxidation of 3-cyclohexyl-propanol 27
and 3-pyridin-3-yl-propanol 28, respectively. Then, Knoevenagel
condensations [28] of aldehydes 29 with 31, 32, 33 or 34 that were
synthesized from the alkylation reactions of TZD with (2-bromo-
ethyl)-carbamic acid tert-butyl ester, 2-bromoethanol, 2-bromo-
acetic acid tert-butyl ester, or 4-(2-bromo-ethyl)-morpholine fol-
lowed by removal of Boc or tert-butyl ester group afforded
compounds 2e8 and 11e19. The synthesis of 20 was achieved
through PdeC catalyzed hydrogenation of lead 1.
The synthesis of 9 and 10 was shown in Scheme 2. The reaction
of 4-iodo-phenylamine 35 with methanesulfonyl chloride afforded
36, which after Heck coupling reaction with allyl alcohol under the
catalysis of palladium (II) acetate gave aldehyde 37. Similarly,
aldehyde 40 was synthesized from 38. Then, Knoevenagel
condensations of 37 and 40 with 31 followed by removal of Boc
group yielded compounds 9 and 10, respectively.
2. Molecular design and chemistry
In order to obtain information of structural features in 1 that
are critical to the anticancer activity, compound 1 was divided
into three domains for structural modification and optimization:
1) phenyl ring domain; 2) ethylamine domain; and 3) TZD
domain. As shown in Fig. 1, compounds 2e10 were designed to
evaluate how the electrostatic and steric nature of the substitu-
ents on the phenyl ring will influence the biological activity.
Compounds 11e13 were designed to examine whether the bio-
isosteric replacement of benzene ring with pyridine or indole ring
will be tolerated or improve activity. The design and character-
ization of compounds 14e16 will help shed light on the roles of
O
N
O
NH2
S
1
O
O
R
NH2
N
N
NH2
S
S
R
O
O
O
2 R=
3 R=
4 R=
17 R= -CH2OH
9 R=
OCH3
10 R=
N
NH2
NH2
S
NHSO2CH3
SO2NH2
-COOH
N
18 R=
19 R=
20
O
OEt
O
11 R=
12 R=
O
N
N
OEt
O
5 R=
21
N
H
EtO
6 R=
13 R=
N
H
NO2
14 R=
7 R=
H
15 R=
16 R=
NO2
8 R=
Cl
Fig. 1. Lead compound 1 and newly designed analogs.

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
127
O
O
a
R
O
R
H
O
O
26
R'
22 R=
b
23 R=
N
O
27 R=
28 R=
Boc
c
29
HO
R
R
H
24 R=
25 R=
N
Boc
N
O
17 R=CH2OH
18 R=COOH
e
e
N
O
O
S
R
19 R=
N
O
d
O
N
O
NH
S
R
O
S
f
O
R
N
O
30
N
O
NH2
S
31 R=CH2NHBoc
32 R=CH2OH
33 R=COOtBu
NH2
S
O
20
2 R=4-methoxyphenyl
3 R=4-ethoxyphenyl
4 R=3-ethoxyphenyl
5 R=2-ethoxyphenyl
6 R=4-nitrophenyl
11 R=3-pyridinyl
12 R=3-indolyl
13 R=5-indolyl
14 R=H
15 R=cyclohexyl
16 R=cyclopropyl
34 R=
N
O
7 R=3-nitrophenyl
8 R=4-chlorophenyl
Scheme 1. Reagents and conditions: a) Meldrum’s acid, piperidine, EtOH; b) i. NaBH4, AcOH, CH2Cl2; ii. PhSiH3, Et3N, THF; c) DMSO, (COCL2), Et3N, CH2Cl2; d) BrCH2CH2NHBoc, or
BrCH2CH2OH, or 2-bromo-acetic acid tert-butyl ester, or 4-(2-bromo-ethyl)-morpholine, K2CO3, TZD, TBAI, acetone; e) i. piperidine EtOH, reflux, ii. 1,4-DioxaneeHCl or TFA/CH2Cl2;
f) Pd/C, H2, MeOH.
As shown in Scheme 3, coupling reaction of mono Boc-protected
1,2-diaminoethane with trimellitic anhydride 41 followed by
reduction with BH3 in THF afforded 43 in good yield. Esterification
of 43 with diethyl chlorophosphate gave intermediate 44, which
after SuzukieMiyaura cross-coupling reaction with benzenebor-
onic acid in the presence of palladium (II) acetate and PPh3 fol-
lowed by the removal of Boc group afforded 21 in high yield [29].
role of the exocyclic double bond in the activity of 1 as this double
bond is incorporated into the benzene ring in 21. Notably, 15
exhibited enhanced growth inhibition compared to 1 (w3 fold
increase) in U937 cells. This may suggest an important role of steric
effects at this specific domain as the phenyl ring in 1 is planar while
the cyclohexane ring in 15 is non-planar with possible chair, boat,
or other transitional conformations. This notion was further sup-
ported by the fact that 16 with a cyclopropane ring exhibited
decreased growth inhibition activity in U937 cells. Compounds 2e5
with electron-donating substitutions on the phenyl ring of 1
exhibited comparable potency to 1. On the other hand, compounds
with electron-withdrawing substitutions on the phenyl ring
exhibited decreased growth inhibition of U937 cells (6e8)
compared to 1. Interestingly, introduction of sulfonamide substi-
tutions at the para-position of 1 led to a total loss of activity as
demonstrated by 9 and 10. This may indicate that protein interac-
tions introduced by the sulfonamide moieties other than steric
effects are not favored at this specific domain as 3 with an ethoxy
group exhibited comparable potency to 1. This is somewhat echoed
by the decreased potency of compounds 11e15 with the bio-
isosteric replacement of phenyl ring with a pyridine or an indole
ring, respectively.
3. Results and discussion
3.1. Growth inhibition of human leukemia U937 cells
The activation of the Raf/MEK/ERK and PI3K/Akt signaling
pathways has been shown to play multiple roles in the proliferation
and apoptosis of hematopoietic cells including U937 cells [5].
Furthermore, U937 cell line has been shown to be a good model in
characterizing our compounds in our previous work [25,26].
Therefore, compounds 1e21 were first tested for their inhibitory
effects on the growth of U937 cells to identify active compounds. As
shown in Table 1, no growth inhibition was observed for
compounds 14 and 20 at concentrations up to 30 mM highlighting
the essential role of the aromatic ring and the exocyclic double
bond to the biological activity. 21 did not show inhibitory effects on
the growth of U937 cells either, which further highlights the critical
Replacement of the NH2 of the ethylamine with carboxyl (18) or
tertiary amino (19) groups led to significant decrease or total loss of
O
O
O
H3C
S
CHO
O
O
b
N
RHN
I
HN
NH2
NH2
S
S
H3C
N
H
O
O
35 R=H
37
9
d
a
36 R=CH3SO2
N
CHO
S
b
H2NO2S
RO2S
c
I
H2NO2S
O
10
38 R=Cl
39 R=NH2
40
Scheme 2. Reagents and conditions: a) CH3SO2Cl, Et3N, CH2Cl2; b) Allyl alcohol, NaHCO3, TBACl, Pd(OAc)2, DMF; c) NH4OH; d) i. 13, piperidine EtOH, reflux; ii. 1,4-DioxaneeHCl.

128
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
3.2. Effects of 15 on the Raf/MEK/ERK and PI3K/Akt signaling
pathways
O
O
R
HOOC
c
a
N
O
NHBoc
To confirm whether the growth inhibition of U937 cells was
O
O
41
mediated through the inhibition of the Raf/MEK/ERK and PI3K/
Akt signaling cascades as seen in 1, 15 was examined by Western
blot analysis in U937 cells. As shown in Fig. 2A, 15 consistently
suppressed the expression of the p-MEK, p-ERK and p-Akt levels,
critical components of these two signaling pathways, at
42 R=COOH
43 R=CH2OH
b
O
O
O
d
(EtO)2PO
N
N
a concentration as low as 3 mM, indicating that signaling blockage
NH2
NHBoc
and growth inhibition in U937 cells were correlated. This notion
is further supported by the results of compounds 1 [26] and 20,
which showed decreased suppression or no suppression of the
expression of these proteins compared to 15 (Fig. 2B and C), thus
suggesting that 15 has potential as a new lead for developing
more potent dual-pathway inhibitors. The level of p-4EBP1,
a downstream substrate of these two signaling pathways, was
also consistently suppressed. 15 did not inhibit other signaling
pathways such as p38 and JNK signaling cascades (data not
shown). To evaluate whether the anti-proliferative and signaling
cascade inhibitory effects of 15 are cell specific, 15 was tested in
androgen-insensitive prostate PC-3, DU145 and M12 cells. As
shown in Fig. 3A, 15 exhibited growth inhibition on all three
cancer cell lines comparable to the results from U937 cells. More
importantly, 15 also consistently inhibited the PI3K/Akt and Raf/
MEK/ERK signaling pathways as seen in the suppressed level of p-
ERK and p-Akt in DU145 cells (Fig. 3B).
Next, to get preliminary target information, 15 was tested
against a 52-kinase panel. As shown in Table 2, 15 significantly
inhibited MEK1 and moderately inhibited PI3K consistent with
the effects on p-MEK, p-ERK and p-Akt in our cell-based assays.
The fact that the p-Raf level was not affected by treatment with
15 (Fig. 2A) may also support MEK as its potential target protein.
Interestingly, 15 also inhibited 50-adenosine monophosphate-
activated protein kinase (AMPK). To confirm the inhibitory
effects on AMPK, Western blot analysis was performed in U937
cells. Surprisingly, AMPK is activated upon treatment with 15
with an enhanced p-AMPK level (Fig. 2A). Discrepancies in results
O
O
21
44
Scheme 3. Reagents and conditions: a) NH2CH2CH2NHBoc THF, Et3N; b) BH3, THF; c)
diethyl chlorophosphate, Et3N, THF, DMAP; d) i. Benzeneboronic acid, Pd(OAc)2, PPh3,
K3PO4, toluene; ii. 1,4-DioxaneeHCl.
Table 1
Growth inhibition of U937 cells by 1e21 (mM).
Compound
IC50 ꢀ SEM
Compound
IC50 ꢀ SEM
29.5 ꢀ 0.12
27.7 ꢀ 0.11
>30
3.4 ꢀ 0.043
14.1 ꢀ 0.010
11.3 ꢀ 0.031
>30
1
2
3
4
5
6
7
8
10.4 ꢀ 0.025
9.4 ꢀ 0.015
9.6 ꢀ 0.040
10.0 ꢀ 0.13
11.3 ꢀ 0.17
>30
24.4 ꢀ 0.21
21.2 ꢀ 0.089
>30
12
13
14
15
16
17
18
19
20
21
>30
>30
>30
9
10
11
>30
>30
activity, which highlights the important role of primary amine.
Interestingly, replacement of the NH2 with an OH (17) can maintain
the biological activity in U937 cells. These results may suggest that
H-bond interactions instead of ionic interactions with certain acidic
amino acid residues in the target protein(s) are important for bio-
logical activity while steric effects are not favored at this specific
site.
Fig. 2. U937 cells were treated with 15 (A), or 1 (B), or 20 (C) for 1 h, after which cell lysates were analyzed by Western blot using corresponding primary antibodies. The image of
Western blot represents the results from one of three independent experiments.

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
129
Fig. 3. A. M12, PC-3 and DU145 cells were treated with 15 for 24 h and the cell viability was analyzed by MTS; B. DU145 cells were treated with 15 at indicated concentrations for
24 h. Lysates from cultures were analyzed by Western blotting using corresponding primary antibodies; C. U937 cells were treated with 15 with or without the presence of AICA
(320
m
M) for 72 h, then growth inhibition was analyzed by MTS assay. Data were expressed as mean percentage viability (n ¼ 6). Error bars represent standard error of mean (SEM);
D. U937 cells were treated with 15 in the presence of 1
183754 ꢀ 43264 cpm/20,000 cells for 1 h and 24 h control, respectively). **P < 0.01 indicates significant differences from control group analyzed by one-way ANOVA.
m
Ci [3H]-thymidine. Cells were collected and the radioactivity was counted (3H-thymidine incorporation is 3428 ꢀ 338 and
from isolated kinase assays compared to results obtained against
whole cell assays may exist due to assay conditions; thus, we
believe that assay results from human cancer cells may deliver
the most meaningful information. To further confirm 150s acti-
vation of AMPK, the effects of AICA (5-amino-4-imidazole car-
boxamide) on the growth inhibition of U937 cells by 15 were
AMPK and this inhibition precipitates the lethal effects of 15 in
U937 cells. As shown in Fig. 3C, the addition of AICA (320 M)
m
enhanced the growth inhibition of U937 cells by 15. Since AICA
does not produce cytotoxic effects by itself at the tested
concentration (data not shown), clearly 15 activates AMPK, which
is consistent with the Western blot results (Fig. 2A). It has been
documented that activation of AMPK leads to the suppression of
mTOR [33], the direct downstream substrate of Akt. Therefore,
the activating effects of 15 on AMPK suggest additional benefits of
our proposed approach in treating cancer. 15 also inhibited CaMK
tested.
AICA
is
metabolized
to
AICAR
(5-amino-4-
imidazolecarboxamide ribonucleotide) and has been shown to
activate AMPK [30e32]. Addition of AICA would be expected to
mitigate the growth inhibition of U937 cells by 15 if 15 inhibits
Table 2
Selectivity of 15.a
Kinase
% Inhibition
Kinase
% Inhibition
Kinase
% Inhibition
Kinase
% Inhibition
ABL
6
3
8
c-TAK1
DYRK1a
Erk1
Erk2
FGFR1
FLT3
FYN
GSK3b
HGK
IGF1R
INSR
IRAK4
KDR
6
ꢁ5
21
11
3
38
0
0
ꢁ1
ꢁ5
ꢁ2
ꢁ1
ꢁ28
LCK
LYN
4
0
3
PI3Ka
PIM2
PKA
45
38
ꢁ3
16
ꢁ8
0
AKT1
AKT2
AMPK
AurA
BTK
CAMK2
CAMK4
CDK2
CHK1
CHK2
Ckld
MAPKAPK2
MARK1
MEK1
MEK2
MET
MSK1
MST2
P38a
76
ꢁ4
12
60
71
ꢁ2
11
8
0
PKCb2
PKCz
PKD2
PKGa
PRAK
ROCK2
RSK1
SGK1
SRC
69
27
ꢁ1
6
ꢁ2
4
3
ꢁ1
2
14
2
ꢁ13
P70S6K
PAK2
PDK1
5
ꢁ5
10
ꢁ2
c-Raf
ꢁ1
SYK
9
a
The effects of 15 on the indicated protein kinases were determined through the Rapid KinaseAdvisor profiling service from Caliper Life Sciences. Values represent the
M of the test compound and are the average of two independent experiments (n ¼ 2).
percent inhibition of each kinase at 10
m

130
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
Fig. 4. Docking studies of 15 into MEK1 and PI3Ka.
(Ca2þ/calmodulin-dependent protein kinases 2/4) in this
screening test. Since CaMKs are involved in the upstream of both
the Raf/MEK/ERK and PI3K/Akt signaling pathways, immunoblot
assays of certain protein levels may not reflect the true interac-
tions of 15 with CAMKs and further studies will be required to
confirm the inhibitory effects of 15 on CaMKs. Taken together, the
results suggest that 15 may function as a multi-target molecule
that exhibits anti-proliferative activity mainly through the Raf/
MEK/ERK and PI3K/Akt signaling pathways.
treatment, 15 exhibited significant inhibition on DNA synthesis of
U937 cells at all tested concentrations. However, 15 did not show
cytotoxic effects at lower concentrations (0.3 and 1
mM) when
tested using MTS assay. These results appear to indicate that 15 is
able to inhibit the synthesis of DNA without significant lethal
effects on U937 cells after a short-term treatment.
To further evaluate its effects on other human cancer cells, 15
was tested against the National Cancer Institute (NCI)’s 60 cancer
cell lines (data not shown). As anticipated, 15 consistently inhibited
the cancer cells at GI50 values (50% of growth inhibition) ranging
from 1.40
mMe5.10 mM, which indicates its potential as a lead
3.3. Effects of 15 on the DNA synthesis of U937 cells
compound for broad-spectrum anticancer agents.
In order to examine the effects on short-term DNA synthesis and
to confirm the anti-proliferative activity, 15 was tested in U937 cells
using [3H]-thymidine incorporation assay. As shown in Fig. 3D, 15
significantly inhibited the DNA synthesis at as early as 1 h treat-
3.4. Molecular modeling studies
Since 15 exhibits dual-inhibition of MEK1 and PI3K through the
ment at higher concentrations (10 and 30
mM). After 24 h
kinase screening, it would be of interest to get some information
Fig. 5. 15 induces apoptosis in U937, M12 and DU145 cells. A. U937 cells were treated with 15 for indicated time, after which the cells were stained with Annexin V/PI and analyzed
by flow cytometry; B. Indicated cancer cells were treated with 15 (20 M) for 24 h. The cells were then stained with Annexin V/PI and analyzed by flow cytometry. The upper right
m
represents late apoptosis as detected by annexin V/Pi staining; the lower right represents early apoptosis as detected by only annexin V staining; the total percentage from upper
right and lower right was referred as the percentage of apoptosis. The image represents the results from one of two independent experiments.

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
131
Fig. 6. Effects of 15 and PD184352 on the cell cycle of U937 cells. Cells were treated with 15 or PD184352 at indicted concentrations for 24 h and stained with PI. Then the cells were
analyzed by flow cytometry.
about the nature of the interactions of this compound with these
enzymes. Thus,15 was initially docked into the ATP binding pockets
of MEK1 and PI3K using GOLD 3.0 [34] as the binding sites of 15 on
these two kinases are unknown. Briefly, the 3D structures of MEK1
at 10 and 15 mM respectively) were also consistent with the results
from apoptosis assay (Fig. 5A). On the other hand, treatment of
U937 cells with the MEK inhibitor PD184352 [40] arrested U937
cells at the G2/M phase, an event accompanied with significant
decrease of the S-phase population. The different effects exhibited
by PD184352 and 15 in cell cycle may be due to their having
different inhibitory effects on the PI3K/Akt signaling pathway.
Further studies such as evaluation of the levels of cyclins, p21 and
p27 are warranted to better understand the mechanistic informa-
tion of 15 on cell cycle progress.
(PDB ID 1s9j [35]) and PI3Ka (PDB ID 3hhm [36]) were modeled by
adding hydrogens and minimizing using Sybyl 7.2 (1000 iterations,
Gasteiger-Hückel charges, gradient of 0.05 kcal molꢁ1 Åꢁ1). 15 was
docked into the ATP binding pockets (50 positions per ligand; no
early termination; no constraints) and the docked positions were
scored using HINT [37]. The best docked positions were selected for
minimization (2500 iterations, gradient of 0.005 kcal/mol Å). The
docking results suggest that 15 fits nicely into the ATP binding
pocket of both MEK1 and PI3K. As shown in Fig. 4, 15 interacts with
the MEK1 through H-bond interactions of the ethylamine with the
Asp190, Asn195 and Asp208 residues. The two carbonyl groups of
TZD also interact with Lys97 and Ser194 through H-bonds. The
cyclohexane ring of 15 fits into a hydrophobic pocket that consists
of Leu74, Val82, Ala95 and Leu197. Similarly, the ethylamine
interacts with Glu768 and Glu798 of PI3K through H-bond inter-
actions and the cyclohexane ring interacts with the residues of
Ile800, Ile848 and Ile932 through hydrophobic interactions. When
ERK2 (PDB ID 3erk) was examined as a control, no favorable
interactions were observed for 15 in the ATP binding pocket (data
not shown). Taken together, the preliminary docking studies also
support the results of 15 concluded from the anti-proliferation
studies in U937 cells.
4. Conclusion
In summary, a series of TZD analogs of 1 were designed and
synthesized. Biological characterization of these analogs in
human leukemia U937 cells established that steric interaction at
the aromatic domain, the exocyclic double bond, and the H-bond
interaction at the ethylamine domain are critical to the growth
inhibition of cancer cells. One new lead compound, 15, was
identified to have improved anti-proliferative activity in U937
cells. More importantly,
a close correlation was confirmed
between the anti-proliferative activity and blockade of the Raf/
MEK/ERK and PI3K/Akt signaling pathways. Compound 15 may
execute its anti-proliferative activity through apoptotic effects in
U937, M12 and DU145 cancer cells. Furthermore, 15 arrested
U937 cells at the S-phase. In addition, it was discovered that 15
inhibits both MEK and PI3K, the critical kinases involved in the
signal transduction of the Raf/MEK/ERK and PI3K/Akt cascades.
Collectively, these results strongly suggest the potential of 15 as
3.5. Effects of 15 on the apoptosis and cell cycle of U937 cells
a
new lead compound with multi-target properties and
The Raf/MEK/ERK and PI3K/Akt signaling cascades play
important roles in the regulation of apoptosis and the G1-S and
G2-M transition in the cell cycle [5,38,39]. To get preliminary
mechanistic information for 150s anti-proliferative activity, the
apoptotic effects of 15 on U937, M12 and DU145 cells were
evaluated. As shown in Fig. 5A, 15 induced apoptosis at as early as
3 h in U937 cells (23.4% increase) and longer exposure to 15
(18 h) significantly induced apoptosis in U937 cells (43.8%
encourage further optimization of 15 to develop more potent and
effective analogs as anticancer agents.
5. Experimental
5.1. Chemistry
increase) at 10
m
M concentration. 15 (20
m
M) significantly induced
Reagents and solvents were obtained from commercial
suppliers and used as received unless otherwise indicated. All
reactions were carried out under inert atmosphere (N2) unless
otherwise noted. Reactions were monitored by thin-layer chro-
matography (TLC) (pre-coated silica gel 60 F254 plates, EMD
Chemicals) and visualized with UV light or by treatment with
phosphomolybdic acid. Flash chromatography was performed on
silica gel (200e300 mesh, Fisher Scientific) using solvents as indi-
cated. 1H NMR and 13C NMR spectra were routinely recorded on
Bruker ARX 400 spectrometer. The NMR solvent used was CDCl3 or
DMSO-d6 as indicated. Tetramethylsilane (TMS) was used as
apoptosis in M12 (71.4% increase) while moderately induced
apoptosis in DU145 cells (22.5% increase) after 24 h treatment
(Fig. 5B). Furthermore, we have confirmed that reactive oxygen
species (ROS) are not involved in the lethal effects of 15 on these
cancer cells (data not shown).
When the cell cycle of U937 cells was evaluated (Fig. 6), 15 dose-
dependently arrested U937 cells at the S-phase (23.6% and 31.4%
increase at 10 and 15 mM, respectively), an event accompanied with
decrease of the G1/G0 and G2/M populations. The apoptotic effects
as measured by the sub-diploid apoptotic fraction (49.9% and 64.3%

132
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
internal standard. The purity of target compounds was determined
by elemental analysis (Atlantic Microlab, Inc.).
5.2.10. 5-(N-Boc-indole-5-methylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione
1H NMR (300 MHz, CDCl3):
d 8.56 (s, 1H), 8.55 (s, 1H), 8.22e8.19
5.2. Procedure A. Preparation of various intermediates 26 through
Meldrum’s acid
(d, J ¼ 9.0 Hz, 1H), 8.04e8.00 (dd, J ¼ 9.0 Hz, 1.8 Hz, 1H), 7.67e7.66
(d, J ¼ 3.9 Hz, 1H), 6.68e6.67 (d, J ¼ 3.9 Hz, 1H), 1.82 (s, 6H), 1.69 (s,
9H).
To a stirred solution of various substituted benzaldehydes or N-
Boc-indole-carbaldehydes orcyclopropanecarbaldehyde(10.0 mmol)
and Meldrum’s acid (10.0 mmol) in EtOH (20.0 mL) was added
piperidine (onedrop), then the resulting solutionwasheatedto reflux
for 5 h and cooled to room temperature. The reaction mixture was
filtered to give a light yellow solid which was purified by flash chro-
matography (hexane/acetone ¼ 5/1) to give intermediate 26.
5.3. Procedure B. Preparation of various aldehydes 29 through
Meldrum’s acid derivative 26
To a solution of various intermediates 26 (1.0 mmol) and acetic
acid (1.5 mL) in dichloromethane (DCM) (9 mL) at 0 ꢂC was added
NaBH4 (4.2 mmol) portion wise and the resulting solution was
stirred at room temperature for 1 h. The reaction was then diluted
with DCM (30.0 mL) and washed with brine and water. The organic
layer was dried over anhydrous Na2SO4 and concentrated under
vacuum. The residue was purified by flash chromatography
(Hexane/acetone ¼ 5/2) to give a light yellow oil which was treated
with Et3N (2.0 mmol) followed by phenylsilane (3.0 mmol) in THF
(10.0 mL). The resulting solution was stirred for 2 h at room
temperature and quenched with H2O. The reaction mixture was
diluted with ethyl ether (30.0 mL) and washed with brine and
water. The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by
flash chromatography (Hexane/acetone ¼ 5/1) to give 29 as light
yellow oil. Most of the aldehydes synthesized through this proce-
dure were directly added to the Knoevenagel condensation reac-
tions as described in Procedure E without further purification and
characterization.
5.2.1. 5-(3-Cyclopropyl-methylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione
1H NMR (400 MHz, CDCl3):
3.25e3.20 (m, 1H),1.75 (s, 6H),1.47e1.45 (m, 2H), 1.11e1.09 (m, 2H).
d
7.21e7.18 (d, J ¼ 11.9 Hz, 1H),
5.2.2. 5-(3-(4-Methoxyphenyl)-methylene)-2,2-dimethyl-1,3-dioxane-
4,6-dione
1H NMR (300 MHz, CDCl3):
d 8.38 (s, 1H), 8.24e8.21 (d,
J ¼ 9.3 Hz, 2H), 6.99e6.97 (d, J ¼ 9.3 Hz, 2H), 3.91 (s, 3H), 1.79 (s,
6H).
5.2.3. 5-(3-(4-Ethoxyphenyl)-methylene)-2,2-dimethyl-1,3-dioxane-
4,6-dione
1H NMR (300 MHz, CDCl3):
d
8.37 (s, 1H), 8.23 (d, J ¼ 9.3 Hz, 2H),
6.97 (d, J ¼ 9.3 Hz, 2H), 4.15 (q, J ¼ 7.0 Hz, 2H), 1.78 (s, 6H), 1.47 (t,
J ¼ 6.9 Hz, 3H).
5.3.1. 3-Cyclopropyl-propionaldehyde
5.2.4. 5-(3-(3-Ethoxyphenyl)-methylene)-2,2-dimethyl-1,3-dioxane-
4,6-dione
1H NMR (400 MHz, CDCl3):
(m, 2H), 1.57e1.51 (m, 2H), 0.71e0.67 (m, 1H), 0.48e0.43 (m, 2H),
0.07e0.04 (m, 2H).
d
9.80 (t, J ¼ 1.8 Hz, 1H), 2.55e2.51
1H NMR (400 MHz, CDCl3):
d 8.39 (s, 1H), 7.80 (s, 1H), 7.52e7.51
(d, J ¼ 7.7 Hz,1H), 7.38 (t, J ¼ 8.1 Hz,1H), 7.12e7.10 (d, J ¼ 8.2 Hz,1H),
4.12e4.07 (q, J ¼ 7.0 Hz, 2H), 1.81 (s, 6H), 1.44 (t, J ¼ 7.0 Hz, 3H).
5.3.2. N-Boc-indole-3-yl-propionaldehyde
1H NMR (400 MHz, CDCl3):
d
9.87 (s, 1H), 8.13e8.11 (d, J ¼ 7.6 Hz,
5.2.5. 5-(3-(2-Ethoxyphenyl)-methylene)-2,2-dimethyl-1,3-dioxane-
4,6-dione
1H), 7.52e7.50 (d, J ¼ 7.6 Hz, 1H), 7.37 (s, 1H), 7.34e7.30 (m, 1H),
7.27e7.23 (m,1H), 3.04 (t, J ¼ 7.5 Hz, 2H), 2.86 (t, J ¼ 7.5 Hz, 2H),1.66
(s, 9H).
1H NMR (400 MHz, CDCl3):
d
8.76 (s, 1H), 7.94e7.91 (d, J ¼ 7.8 Hz,
1H), 7.48 (t, J ¼ 7.9 Hz, 1H), 6.99 (t, J ¼ 7.4 Hz, 1H), 6.94e6.92 (d,
J ¼ 8.4 Hz, 1H), 4.17e4.11 (q, J ¼ 7.0 Hz, 2H), 1.82 (s, 6H), 1.46 (t,
J ¼ 7.0 Hz, 3H).
5.4. Procedure C. Preparation of aldehydes 29 through Swern
oxidation reaction
5.2.6. 5-(3-(4-Nitrophenyl)-methylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione
d 8.46 (s, 1H), 8.32e8.29 (d,
J ¼ 9.3 Hz, 2H), 8.08e8.05 (d, J ¼ 9.3 Hz, 2H), 1.84 (s, 6H).
DMSO (14.0 mmol) was added dropwise to a stirred solution of
oxalyl chloride (5.0 mmol) in DCM (20.0 mL) at ꢁ78 ꢂC and the
resulting reaction mixture was stirred for 20 min at this tempera-
ture. Then 3-cyclohexyl-propanol or 3-pyridinepropanol (4.0 mmol)
was added dropwise and stirred for 1 h followed by the addition of
Et3N (1.0 mL). The reaction was gradually brought to room temper-
ature and H2O was added. The separated DCM phase was washed
with brine and purified by flash chromatography (Hexane/ethyl
acetate (EtOAc) ¼ 10/1) to give a colorless oil.
1H NMR (300 MHz, CDCl3):
5.2.7. 5-(3-(3-Nitrophenyl)-methylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione
1H NMR (400 MHz, CDCl3):
d
8.83 (t, J ¼ 1.8 Hz, 1H), 8.46 (s, 1H),
8.40e8.37 (d, J ¼ 8.2 Hz, 1H), 8.28e8.26 (d, J ¼ 7.9 Hz, 1H), 7.68 (t,
J ¼ 8.0 Hz, 1H), 1.84 (s, 6H).
5.4.1. 3-Cyclohexyl-propionaldehyde
5.2.8. 5-(3-(4-Chlorophenyl)-methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione
1H NMR (400 MHz, CDCl3):
2.45e2.41 (dt, J ¼ 7.5, 1.9 Hz, 2H), 1.71e1.55 (m, 5H), 1.51e1.49 (m,
2H), 1.26e1.11 (m, 4H), 0.93e0.86 (m, 2H). 13C NMR (100 MHz,
CDCl3): 203.1, 41.5, 37.2, 33.0, 29.3, 26.4, 26.2.
d
9.77e9.76 (t, J ¼ 1.9 Hz 1H),
1H NMR (400 MHz, CDCl3):
d 8.37 (s, 1H), 8.04e8.02 (d,
J ¼ 8.7 Hz, 2H), 7.47e7.45 (d, J ¼ 8.7 Hz, 2H), 1.81 (s, 6H).
5.2.9. 5-(N-Boc-indole-3-methylene)-2,2-dimethyl-1,3-dioxane-4,6-
dione
5.5. Procedure D. Preparation of 3-substituted thiazoldine-2,4-
diones 31e34
1H NMR (400 MHz, CDCl3):
d 9.67 (s, 1H), 8.55 (s, 1H), 8.82 (s,
1H), 8.27e8.25 (d, J ¼ 8.0 Hz, 1H), 7.89e7.86 (d, J ¼ 8.0 Hz, 1H),
A
mixture of thiazolidine-2,4-dione (68.0 mmol), various
745e7.42 (m, 1H), 1.79 (s, 6H), 1.72 (s, 9H).
bromide compounds (80.0 mmol), K2CO3 (92.0 mmol), TBAI

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
133
(6.8 mmol) in acetone (100.0 mL) was stirred at 40 ꢂC for 10 h. The
reaction mixture was cooled to room temperature and filtered
through a short bed of celite and the filtrate was concentrated
under vacuum. The residue was purified by flash chromatography
(Hexane/EtOAc ¼ 4/1 to 2/1) to give a white solid.
(C16H21ClN2O3S$0.25H2O) Calcd. C: 53.18%, H: 6.00%, N: 7.75%;
found C: 53.11%, H: 5.98%, N: 7.88%.
5.6.4. 3-(2-Aminoethyl)-5-(3-(2-ethoxyphenyl)propylidene)
thiazolidine-2,4-dione 5
1H NMR (400 MHz, DMSO-d6):
d 8.05 (brs, 3H), 7.21e7.16 (m,
5.5.1. tert-Butyl 2-(2,4-dioxo-thiazolidin-3-yl)-ethylcarbamate 31
2H), 7.08e7.05 (t, J ¼ 7.6 Hz, 1H), 6.96e6.94 (d, J ¼ 7.7 Hz, 1H),
6.89e6.85 (dt, J ¼ 1.0, 7.4 Hz, 1H), 4.08e4.02 (q, J ¼ 7.0 Hz, 2H),
3.85e3.82 (t, J ¼ 6.0 Hz, 2H), 3.01 (m, 2H), 2.81e2.78 (t, J ¼ 7.4 Hz,
2H), 2.52e2.50 (m, 2H), 1.40e1.36 (t, J ¼ 7.0 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6): 167.6, 164.5, 156.4, 137.7, 129.8, 128.1, 127.8,
125.1, 120.2, 111.5, 63.1, 36.6, 31.4, 28.2, 14.7. Anal.
(C16H21ClN2O3S$0.25H2O) Calcd. C: 53.18%, H: 6.00%, N: 7.75%;
found C: 53.23%, H: 6.08%, N: 7.75%.
1H NMR (300 MHz, CDCl3):
d
3.94 (s, 2H), 3.78e3.74 (t, J ¼ 6.0 Hz,
2H), 3.40e3.34 (t, J ¼ 6.0 Hz, 2H), 1.43(s, 9H).
5.5.2. 3-(2-Hydroxy-ethyl)thiazolidine-2,4-dione 32
1H NMR (400 MHz, CDCl3):
d 3.99 (s, 2H), 3.86e3.81 (m, 4H).
5.5.3. (2,4-Dioxo-thiazolidin-3-yl)-acetic acid tert-butyl ester 33
1H NMR (300 MHz, CDCl3):
4.25 (s, 2H), 4.02 (s, 2H), 1.46 (s,
9H).
d
5.6.5. 3-(2-Aminoethyl)-5-(3-(4-nitrophenyl)propylidene)
thiazolidine-2,4-dione 6
5.5.4. 3-(2-Morpholino-ethyl)thiazolidine-2,4-dione 34
1H NMR (400 MHz, CDCl3):
1H NMR (400 MHz, DMSO-d6):
d
8.19e8.17 (d, J ¼ 8.7 Hz, 2H),
d
3.95 (s, 2H), 3.77e3.74 (t, J ¼ 6.4 Hz,
7.88 (brs, 3H), 7.58e7.56 (d, J ¼ 8.7 Hz, 2H), 7.06e7.03 (t, J ¼ 7.5 Hz,
1H), 3.83e3.80 (t, J ¼ 5.8 Hz, 2H), 3.04e2.99 (m, 4H), 2.65e2.59 (m,
2H). 13C NMR (100 MHz, DMSO-d6): 167.5, 164.5, 148.7, 146.1, 136.7,
129.7, 125.6, 123.5, 36.8, 32.7, 32.0. Anal. (C16H16F3N3O6S) Calcd. C:
44.14%, H: 3.70%, N: 9.65%; found C: 43.93%, H: 3.78%, N: 9.70%.
2H), 3.66e3.63 (t, J ¼ 4.6 Hz, 4H), 2.57e2.54 (t, J ¼ 6.4 Hz, 2H),
2.49e2.47 (t, J ¼ 4.6 Hz, 4H).
5.6. Procedure E. Knoevenagel condensations of aldehydes 29 with
31e34 followed by the removal of Boc or tert-butyl ester group
5.6.6. 3-(2-Aminoethyl)-5-(3-(3-nitrophenyl)propylidene)
A solution of aldehyde 29 or propionaldehyde (0.5 mmol), 3-
substituted-thiazolidine-2,4-dione (0.5 mmol) and piperidine (1
drop) in MeOH (3.0 mL) was stirred at room temperature overnight.
The solvent was removed under reduced pressure and the residue
was purified by flash chromatography (Hexane/acetone ¼ 10/1).
Products with Boc or tert-butyl ester group were treated with HCl
(4.0 M in 1,4-dioxane) in EtOAc or trifluoroacetic acid in DCM to
remove the protecting groups.
thiazolidine-2,4-dione 7
1H NMR (400 MHz, DMSO-d6):
d 8.18 (s, 1H), 8.11e8.08 (d,
J ¼ 7.8 Hz,1H), 7.87 (brs, 3H), 7.77e7.75 (d, J ¼ 7.8 Hz,1H), 7.64e7.60
(t, J ¼ 7.9 Hz, 1H), 7.08e7.04 (t, J ¼ 7.5 Hz, 1H), 3.83e3.80 (t,
J ¼ 5.8 Hz, 2H), 3.03e2.99 (m, 4H), 2.66e2.60 (m, 2H). 13C NMR
(100 MHz, DMSO-d6): 167.6, 164.5, 147.9, 142.7, 136.8, 135.3, 129.9,
125.6, 123.1, 121.3, 36.8, 32.3, 32.2. Anal. (C16H16F3N3O6S) Calcd. C:
44.14%, H: 3.70%, N: 9.65%; found C: 43.21%, H: 3.65%, N: 9.40%.
5.6.1. 3-(2-Aminoethyl)-5-(3-(4-methoxyphenyl)propylidene)
5.6.7. 3-(2-Aminoethyl)-5-(3-(4-chlorophenyl)propylidene)
thiazolidine-2,4-dione 2
thiazolidine-2,4-dione 8
1H NMR (400 MHz, DMSO-d6):
d
8.04 (brs, 3H), 7.11e7.08 (d,
1H NMR (400 MHz, DMSO-d6):
d 7.89 (brs, 3H), 7.37e7.35 (d,
J ¼ 8.6 Hz, 2H), 6.96e6.92 (t, J ¼ 7.4 Hz,1H), 6.80e6.78 (d, J ¼ 8.6 Hz,
2H), 3.78e3.75 (t, J ¼ 6.0 Hz, 2H), 3.67(s, 3H), 2.95e2.92 (t,
J ¼ 6.0 Hz, 2H), 2.72e2.69 (t, J ¼ 7.4 Hz, 2H), 2.47e2.41 (m, 2H). 13C
NMR (100 MHz, DMSO-d6): 167.6, 164.5, 157.7, 137.4, 132.1, 129.2,
125.2, 113.8, 54.9, 36.6, 32.9, 32.1. Anal. (C15H19ClN2O3S$0.25H2O)
Calcd. C: 51.87%, H: 5.66%, N: 8.06%; found C: 51.57%, H: 5.53%, N:
8.34%.
J ¼ 7.8 Hz, 2H), 7.30e7.28 (d, J ¼ 7.8 Hz, 2H), 7.04e7.00 (t, J ¼ 7.5 Hz,
1H), 3.83e3.80 (t, J ¼ 5.9 Hz, 2H), 3.05e3.02 (t, J ¼ 5.9 Hz, 2H),
2.86e2.83 (t, J ¼ 5.9 Hz, 2H), 2.58e2.52 (m, 2H). 13C NMR (100 MHz,
DMSO-d6): 167.6, 164.5, 139.3, 137.1, 130.8, 130.2, 128.3, 125.4, 36.8,
32.5, 32.2. Anal. (C16H16ClF3N2O4S) Calcd. C: 45.24%, H: 3.80%, N:
6.59%; found C: 45.06%, H: 3.66%, N: 6.61%.
5.6.8. 3-(2-Aminoethyl)-5-(3-pyridin-3-yl-propylidene)
5.6.2. 3-(2-Aminoethyl)-5-(3-(4-ethoxyphenyl)propylidene)
thiazolidine-2,4-dione 11
thiazolidine-2,4-dione 3
1H NMR (400 MHz, DMSO-d6):
d
8.90e8.89 (d, J ¼ 1.2 Hz, 1H),
1H NMR (400 MHz, DMSO-d6):
d
8.05 (brs, 3H), 7.09e7.07 (d,
8.79e8.77 (d, J ¼ 5.1 Hz, 1H), 8.50e8.48 (d, J ¼ 8.1 Hz, 1H), 8.21 (brs,
3H), 7.99e7.96 (dd, J ¼ 8.0, 5.6 Hz,1H), 7.06 (t, J ¼ 7.5 Hz,1H), 3.84 (t,
J ¼ 6.0 Hz, 2H), 3.07 (t, J ¼ 7.3 Hz, 2H), 3.02e2.98 (m, 2H), 2.68e2.63
(q, J ¼ 7.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): 167.5, 164.4, 145.1,
141.9, 140.3, 140.0, 136.0, 126.6, 126.1, 38.9, 36.5, 31.6, 29.7. Anal.
(C13H17Cl2N3O2S) Calcd. C: 44.35%, H: 4.92%, N: 11.94%; found C:
44.35%, H: 4.85%, N: 11.90%.
J ¼ 8.6 Hz, 2H), 6.94 (t, J ¼ 7.4 Hz, 1H), 6.79e6.76 (d, J ¼ 8.6 Hz,
2H), 3.94e3.89 (q, J ¼ 7.0 Hz, 2H), 3.76 (t, J ¼ 6.0 Hz, 2H), 2.93 (m,
2H), 2.70 (t, J ¼ 7.4 Hz, 2H), 2.47e2.41 (m, 2H), 1.23 (t, J ¼ 7.0 Hz,
3H). 13C NMR (100 MHz, DMSO-d6): 167.6, 164.5, 157.0, 137.4,
132.0, 129.3, 125.2, 114.3, 62.9, 36.6, 33.0, 32.1, 14.6. Anal.
(C16H21ClN2O3S) Calcd. C: 53.85%, H: 5.93%, N: 7.85%; found C:
53.96%, H: 6.06%, N: 7.68%.
5.6.9. 3-(2-Aminoethyl)-5-(3-indol-3-yl-propylidene)thiazolidine-
5.6.3. 3-(2-Aminoethyl)-5-(3-(3-ethoxyphenyl)propylidene)
2,4-dione 12
thiazolidine-2,4-dione 4
1H NMR (400 MHz, CDCl3):
d 8.03 (brs, 1H), 7.58e7.56 (d,
1H NMR (400 MHz, DMSO-d6):
d
8.06 (brs, 3H), 7.22e7.18 (t,
J ¼ 7.8 Hz, 1H), 7.38e7.36 (d, J ¼ 8.1 Hz, 1H), 7.22e7.11 (m, 3H),
7.01e7.00 (d, J ¼ 2.1 Hz, 1H), 3.74 (t, J ¼ 6.3 Hz, 2H), 3.02 (t,
J ¼ 7.3 Hz, 2H), 2.93 (t, J ¼ 6.3 Hz, 2H), 2.68e2.62 (q, J ¼ 7.4 Hz, 2H).
13C NMR (100 MHz, CDCl3): 168.1, 165.3, 138.3, 136.4, 127.1, 125.6,
122.3, 121.6, 119.5, 118.6, 114.4, 111.3, 44.5, 40.0, 32.4, 23.6. Anal.
(C16H18ClN3O2S) Calcd. C: 54.62%, H: 5.16%, N: 11.94%; found C:
54.59%, H: 5.18%, N: 11.90%.
J ¼ 7.9 Hz, 1H), 7.04e7.01 (t, J ¼ 7.4 Hz, 1H), 6.81 (s, 1H),
6.78e6.75 (m, 2H), 4.03e3.98 (q, J ¼ 7.0 Hz, 2H), 3.85e3.82 (t,
J ¼ 6.0 Hz, 2H), 3.02e2.99 (t, J ¼ 6.0 Hz, 2H), 2.82e2.79 (t,
J ¼ 7.4 Hz, 2H), 2.58e2.52 (m, 2H), 1.33e1.30 (t, J ¼ 7.0 Hz, 3H).
13C NMR (100 MHz, DMSO-d6): 167.6, 164.5, 158.6, 141.8, 137.3,
129.4, 125.2, 120.4, 114.5, 112.1, 62.8, 36.6, 32.9, 32.5, 14.6. Anal.

134
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
5.6.10. 3-(2-Aminoethyl)-5-(5-indol-3-yl-propylidene)thiazolidine-
2,4-dione 13
5.7. Preparation of 3-(2-aminoethyl)-5-(3-phenyl-propyl)
thiazolidine-2,4-dione 20
1H NMR (400 MHz, CDCl3): 8.15 (brs, 1H), 7.45 (s, 1H), 7.34e7.32
(d, J ¼ 8.3 Hz, 1H), 7.20 (t, J ¼ 2.9 Hz, 1H), 7.14 (t, J ¼ 7.6 Hz, 1H),
7.03e7.01 (dd, J ¼ 8.3 Hz, 1.6 Hz, 1H), 6.51e6.50 (m, 1H), 3.74 (t,
J ¼ 6.3 Hz, 2H), 2.96e2.92 (m, 4H), 2.62e2.57 (q, J ¼ 7.6 Hz, 2H); 13C
NMR (100 MHz, CDCl3): 168.1, 165.3, 138.1, 134.7, 131.3, 128.2, 125.6,
124.6, 122.7, 120.0, 111.2, 102.4, 44.5, 40.0, 34.3, 34.1. Anal.
(C16H18ClN3O2S) Calcd. C: 54.62%, H: 5.16%, N: 11.94%; found C:
54.70%, H: 5.10%, N: 11.87%.
Compound 1 (55.2 mg, 0.2 mmol) and 10% Pd/C (50.0 mg) in
MeOH (10.0 mL) was hydrogenated (60 psi) at room temperature
for 15 h. The reaction was filtered through a short bed of celite and
the filtrate was concentrated under reduced pressure to give 20 as
white solid. 1H NMR (400 MHz, DMSO-d6):
d 8.07(brs, 3H),
7.31e7.17 (m, 5H), 4.57e4.54 (dd, J ¼ 4.5, 8.9 Hz, 1H), 3.76e3.73 (t,
J ¼ 6.1 Hz, 2H), 3.01e2.93 (m, 2H), 2.65e2.61 (m, 2H), 2.13e2.05 (m,
1H), 1.91e1.75 (m, 2H), 1.65e1.57 (m, 1H); 13C NMR (100 MHz,
DMSO-d6): 174.7, 171.6, 141.3, 128.3, 128.2, 125.8, 49.6, 36.5, 34.5,
31.2, 28.3. Anal. (C14H19ClN2O2S) Calcd. C: 53.41%, H: 6.08%, N:
8.90%; found C: 53.38%, H: 6.03%, N: 8.79%.
5.6.11. 3-(2-Aminoethyl)-5-propylidenethiazolidine-2,4-dione 14
1H NMR (400 MHz, DMSO-d6):
d 8.27 (brs, 3H), 7.12e7.08 (t,
J ¼ 7.6 Hz, 1H), 3.93e3.90 (t, J ¼ 6.0 Hz, 2H), 3.07 (m, 2H), 2.33e2.26
(m, 2H), 1.17e1.13 (t, J ¼ 7.5 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
167.6, 164.7, 139.4, 124.3, 36.5, 24.6, 12.1. Anal. (C8H13ClN2O2S)
Calcd. C: 40.59%, H: 5.54%, N: 11.83%; found C: 40.39%, H: 5.42%, N:
11.60%.
5.7.1. N-(4-Iodo-phenyl)-methanesulfonamide 36
To a stirred solution of 4-iodo-phenylamine 17 (1.1 g, 5.0 mmol)
and Et3N (0.8 mL, 5.5 mmol) in DCM (20.0 mL) was added meth-
anesulfonyl chloride (0.4 mL, 5.5 mmol) slowly. The reaction was
stirred for 2 h, then water was added. The organic layer was
extracted by 1 M NaOH and the aqueous phase was washed with
DCM, and then was acidified with 1 M HCl to pH ¼ 2e3. The
aqueous phase was then extracted with DCM (2 ꢃ 20 mL) and dried
over anhydrous Na2SO4. After filtration, the solvent was removed
under educed pressure to give a white solid (492.0 mg). 1H NMR
5.6.12. 3-(2-Aminoethyl)-5-(3-cyclohexyl-propylidene)
thiazolidine-2,4-dione 15
1H NMR (400 MHz, DMSO-d6):
d 8.16 (brs, 3H), 7.05e7.01 (t, 1H),
3.87e3.84 (t, J ¼ 6.0 Hz, 2H), 3.03e3.00 (t, J ¼ 6.0 Hz, 2H), 2.26e2.21
(q, J ¼ 7.6 Hz, 2H), 1.70e1.60 (m, 5H), 1.42e1.37 (q, J ¼ 7.3 Hz, 2H),
1.27e1.10 (m, 4H), 0.92e0.83 (m, 2H). 13C NMR (100 MHz, DMSO-
d6): 167.6, 164.6, 138.5, 124.7, 36.5, 34.6, 34.7, 32.5, 28.6, 26.0, 25.6.
Anal. (C14H23ClN2O2S) Calcd. C: 52.73%, H: 7.27%, N: 8.79%; found C:
52.87%, H: 7.17%, N: 8.79%.
(400 MHz, CDCl3):
J ¼ 8.6 Hz, 2H), 3.01 (s, 3H).
d
7.68e7.66 (d, J ¼ 8.7 Hz, 2H), 6.99e6.97 (d,
5.8. Procedure F. Preparation of N-(4-{3-[3-(2-aminoethyl)-2,4-
5.6.13. 3-(2-Aminoethyl)-5-(3-cyclopropyl-propylidene)
dioxo-thiazolidin-5-ylidene]-propyl}-phenyl)-methanesulfonamide
thiazolidine-2,4-dione 16
9
1H NMR (400 MHz, DMSO-d6):
d
8.13 (brs, 3H), 7.06 (t, J ¼ 7.7 Hz,
1H), 3.83 (t, J ¼ 6.0 Hz, 2H), 3.00 (m, 2H), 2.32e2.26 (q, J ¼ 7.4 Hz,
2H), 1.43e1.37 (q, J ¼ 7.1 Hz, 2H), 0.72e0.68 (m, 1H), 0.42e0.38 (m,
2H), 0.06e0.03 (m, 2H). 13C NMR (100 MHz, DMSO-d6): 167.6,164.6,
138.1, 124.9, 36.6, 32.2, 31.6, 10.4, 4.5. Anal. (C11H17ClN2O2S) Calcd.
C: 47.73%, H: 6.19%, N: 10.12%; found C: 47.67%, H: 6.19%, N: 10.12%.
A mixture of 36 (178.0 mg, 0.6 mmol), allyl alcohol (0.06 mL,
0.9 mmol), NaHCO3 (125.0 mg, 1.5 mmol), Pd(OAc)2 (7.0 mg), TBACl
(180.0 mg), and 4 Å sieves (100.0 mg) in DMF (2.0 mL) was stirred at
room temperature for 48 h. The mixture was poured into water,
extracted with diethyl ether, and dried over anhydrous Na2SO4.
After removal of solvents, the residue was reacted with 31
following procedure E to give (2-{5-[3-(4-methanesulfonylamino-
phenyl)-propylidene]-2,4-dioxo-thiazolidin-3-yl}-ethyl)-carbamic
acid tert-butyl ester as an oil (70.0 mg). 1H NMR (400 MHz, CDCl3):
5.6.14. 3-(2-Hydroxyethyl)-5-(3-phenyl-propylidene)thiazolidine-
2,4-dione 17
1H NMR (400 MHz, CDCl3):
d 7.33e7.31 (m, 2H), 7.24e7.18 (m,
3H), 7.11 (t, J ¼ 7.7 Hz, 1H), 3.91 (t, J ¼ 5.0 Hz, 2H), 3.84e3.83 (m,
2H), 2.86 (t, J ¼ 7.5 Hz, 2H), 2.59e2.53 (q, J ¼ 7.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3): 168.2, 165.5, 139.8, 137.8, 128.7, 128.3, 126.6,
125.7, 60.4, 44.1, 33.9, 33.4. Anal. (C14H15NO3S$0.10H2O) Calcd. C:
60.24%, H: 5.49%, N: 5.02%; found C: 60.13%, H: 5.47%, N: 4.84%.
d
7.20e7.15 (m, 4H), 7.05 (t, J ¼ 7.6 Hz, 1H), 6.83 (s, 1H), 4.79 (brs,
1H), 3.81 (t, J ¼ 5.9 Hz, 2H), 3.38e3.37 (m, 2H), 2.99 (s, 3H), 2.83 (t,
J ¼ 7.5 Hz, 2H), 2.55e2.50 (q, J ¼ 7.6 Hz, 2H), 1.41 (s, 9H).
To the solution of (2-{5-[3-(4-methanesulfonylamino-phenyl)-
propylidene]-2,4-dioxo-thiazolidin-3-yl}-ethyl)-carbamic acid tert-
butyl ester (70.0 mg, 0.2 mmol) in ethyl acetate (3.0 mL) was added
4 M HCl in 1,4-dioxane (2.0 mL). The solution was stirred at room
temperature for 5 h and filtered to give 9 as a light yellow solid. 1H
5.6.15. [5-(3-Phenyl-propylidene)thiazolidine-2,4-dione-3-yl]-
acetic acid 18
1H NMR (400 MHz, CDCl3):
d
7.32e7.29 (m, 2H), 7.24e7.23 (m,
NMR (400 MHz, DMSO-d6): d 9.66 (s, 1H), 8.03 (brs, 3H), 7.23e7.20
1H), 7.20e7.17 (m, 2H), 7.14 (t, J ¼ 7.7 Hz, 1H), 4.46 (s, 2H), 2.86 (t,
J ¼ 7.5 Hz, 2H), 2.59e2.54 (q, J ¼ 7.6 Hz, 2H). 13C NMR (100 MHz,
CDCl3): 171.5, 167.0, 164.0, 139.7, 138.6, 128.7, 128.3, 126.6, 125.4,
41.4, 33.8, 33.5. Anal. (C14H13NO4S) Calcd. C: 57.72%, H: 4.50%, N:
4.81%; found C: 57.42%, H: 4.51%, N: 4.74%.
(d, J ¼ 8.5 Hz, 2H), 7.16e7.13 (d, J ¼ 8.5 Hz, 2H), 7.02 (t, J ¼ 7.5 Hz,
1H), 3.83 (t, J ¼ 5.9 Hz, 2H), 3.01e3.00 (m, 2H), 2.94 (s, 3H), 2.80 (t,
J ¼ 7.3 Hz, 2H), 2.55e2.50 (q, J ¼ 7.3 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6): 167.5, 164.4, 137.2, 136.4, 135.9, 129.0, 125.3, 120.2, 36.5,
32.6, 32.2. Anal. (C15H20ClN3O4S2) Calcd. C: 44.38%, H: 4.97%, N:
10.35%; found C: 44.12%, H: 4.91%, N: 10.20%.
5.6.16. 3-(2-Morpholin-ethyl)-5-(3-phenyl-propylidene)
thiazolidine-2,4-dione 19
5.8.1. 4-Iodo-benzenesulfonamide 39
1H NMR (400 MHz, CDCl3):
d
7.32e7.18 (m, 5H), 7.08e7.05 (t,
To the ammonium hydroxide (5.0 mL) was added 4-iodo-ben-
zenesulfonyl chloride (454.0 mg, 1.5 mmol). The resulting mixture
was stirred at room temperature for 2 h and extracted with DCM
(2 ꢃ 20.0 mL). The combined organic phase was dried over anhy-
drous Na2SO4, and concentrated to give 39 as a white solid
J ¼ 7.6 Hz, 1H), 3.81e3.78 (t, J ¼ 6.4 Hz, 2H), 3.65e3.62 (t, J ¼ 4.6 Hz,
4H), 2.88e2.84 (t, J ¼ 7.5 Hz, 2H), 2.58e2.54 (m, 4H), 2.49e2.47 (t,
J ¼ 4.5 Hz, 4H). 13C NMR (100 MHz, CDCl3): 167.6,164.9,139.9, 136.9,
128.7, 128.3, 126.6, 126.0, 67.0, 55.2, 53.5, 38.6, 33.9, 33.4. Anal.
(C18H22N2O3S) Calcd. C: 62.40%, H: 6.40%, N: 8.09%; found C:
62.39%, H: 6.53%, N: 7.90%.
(392.0 mg). 1H NMR (400 MHz, DMSO-d6):
J ¼ 8.6 Hz, 2H), 7.60e7.58 (d, J ¼ 8.6 Hz, 2H), 7.45 (s, 2H).
d 7.98e7.96 (d,

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
135
5.8.2. 4-{3-[3-(2-Aminoethyl)-2,4-dioxo-thiazolidin-5-ylidene]-
propyl}-benzenesulfonamide 10
J ¼ 5.6 Hz, 2H), 3.43e3.42 (m, 2H), 1.40e1.33 (m, 15H). 13C NMR
(100 MHz, DMSO-d6): 167.9, 132.7, 132.6, 131.9, 123.4, 121.9, 67.6,
65.1, 64.1, 38.2, 28.2, 16.1.
Compound 39 (283.0 mg, 1.0 mmol) was reacted with allyl
alcohol (0.1 mL, 1.5 mmol) followed by reaction with 31 following
procedure F to give (2-{2,4-dioxo-5-[3-(4-sulfamoyl-phenyl)-pro-
pylidene]-thiazolidin-3-yl}-ethyl)-carbamic acid tert-butyl ester as
5.8.6. 2-(2-Aminoethyl)-5-benzylisoindoline-1,3-dione 21
Pd(OAc)2 (1.1 mg, 1.0 mol%) and Ph3P (5.1 mg, 4.0 mol%) were
dissolved in toluene (1.0 mL) and transferred into flask containing
44 (0.2 g, 1.0 mmol), benzeneboronic acid (64.7 mg, 1.1 mmol) and
K3PO4 (0.1 g, 1.1 mmol). The vigorously stirred mixture was heated
to 90 ꢂC for 16 h under N2. The mixture was quenched with H2O
(10.0 mL) and extracted with EtOAc (2 ꢃ 20.0 mL). The combined
organic phase was washed with brine and H2O, then dried over
anhydrous Na2SO4. After removal of solvents under reduced pres-
sure, the residue was purified by flash chromatography (EtOAc/
hexane ¼ 3/7) to give Boc-protected 21. 1H NMR (400 MHz, CDCl3):
an oil (80.0 mg). 1H NMR (400 MHz, CDCl3):
d 7.88e7.86 (d,
J ¼ 8.2 Hz, 2H) 7.34e7.32 (d, J ¼ 8.2 Hz, 2H), 7.02 (t, J ¼ 7.6 Hz, 1H),
4.75 (brs, 1H), 3.80 (t, J ¼ 5.9 Hz, 2H), 3.37e3.36 (m, 2H), 2.93 (t,
J ¼ 7.5 Hz, 2H), 2.59e2.56 (q, J ¼ 7.6 Hz, 2H), 1.40 (s, 9H).
(2-{2,4-Dioxo-5-[3-(4-sulfamoyl-phenyl)-propylidene]-thiazo-
lidin-3-yl}-ethyl)-carbamic acid tert-butyl ester (80.0 mg,
0.2 mmol) was treated with HCl in 1,4-dioxane following Procedure
F to give 10 as a light yellow solid. 1H NMR (400 MHz, DMSO-d6):
d
8.06 (brs, 3H), 7.76e7.74 (d, J ¼ 8.3 Hz, 2H), 7.47e7.44 (d,
J ¼ 8.4 Hz, 2H), 7.30 (s, 2H), 7.02 (t, J ¼ 7.4 Hz, 1H), 3.83 (t, J ¼ 6.0 Hz,
2H), 3.03e2.98 (m, 2H), 2.93 (t, J ¼ 7.4 Hz, 2H), 2.61e2.56 (q,
J ¼ 7.3 Hz, 2H); 13C NMR (100 MHz, DMSO-d6): 167.4, 164.4, 144.4,
142.1, 136.8, 128.7, 125.7, 125.4, 36.5, 32.6, 32.1. Anal.
(C14H18ClN3O4S2$0.25H2O) Calcd. C: 42.42%, H: 4.70%, N: 10.60%;
found C: 42.63%, H: 4.74%, N: 10.24%.
d
7.76e7.74 (d, J ¼ 7.6 Hz, 1H), 7.66 (s, 1H), 7.54e7.52 (d, J ¼ 7.5 Hz,
1H), 7.33e7.16 (m, 5H), 4.80 (brs, 1H), 4.10 (s, 2H), 3.80 (t, J ¼ 5.4 Hz,
2H), 3.41e3.40 (m, 2H), 1.77 (s, 9H). 13C NMR (100 MHz, CDCl3):
139.2, 134.4, 132.6, 128.9, 128.8, 126.8, 123.8, 123.5, 42.2, 28.2.
Boc-protected 21 was dissolved in anhydrous EtOAc (4.0 mL),
and was added HCl solution (1.0 mL, 4 M in 1,4-dioxane). The
mixture was stirred for 2 h at room temperature and the precipitate
was filtered and washed with anhydrous diethyl ether to obtain 21
5.8.3. 2-(2-(tert-Butoxycarbonylamino)ethyl)-1,3-dioxoisoindoline-
5-carboxylic acid 42
as a white solid. 1H NMR (400 MHz, DMSO-d6):
d 8.07 (brs, 3H),
A mixture of N-tert-butoxycarbonyl-1,2-diaminoethane (2.0 g,
12.5 mmol), phthalic anhydride 41 (2.9 g, 15.0 mmol), and Et3N
(2.2 mL, 10.0 mmol) in THF (120.0 mL) was refluxed for 3 h. The
reaction mixture was concentrated, and the residue was purified by
flash chromatography (DCM/MeOH ¼ 9/1) to give 42 as a white
7.82e7.80 (d, 1H), 7.75e7.73 (m, 2H), 7.33e7.29 (m, 5H), 4.15 (s, 2H),
3.84e3.81 (t, J ¼ 5.9 Hz, 2H), 3.06e3.03 (t, J ¼ 5.9 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6): 167.9, 167.8, 148.8, 140.2, 134.4, 132.5, 129.8,
128.8,
126.4,
123.2,
123.1,
40.8,
37.3,
35.2.
Anal.
(C17H17ClN2O2$0.5H2O) Calcd. C: 62.67%, H: 5.57%, N: 8.60%; found
C: 62.99%, H: 5.27%, N: 8.69%.
crystal. 1H NMR (400 MHz, DMSO-d6):
d
13.7 (brs, 1H), 8.36e8.34
(d, J ¼ 7.6 Hz, 1H), 8.21 (s, 1H), 7.99e7.97 (d, J ¼ 7.6 Hz, 1H),
6.95e6.92 (s, 1H), 3.66e3.63 (m, 2H), 3.20e3.18 (m, 2H), 1.26 (m,
9H). 13C NMR (100 MHz, DMSO-d6): 167.1, 165.8, 155.7, 136.0, 135.2,
135.0, 132.3, 123.2, 122.7, 77.6, 38.2, 37.9, 28.0.
5.9. Biological assays
Human leukemia U937 cells and human prostate cancer DU145
and PC-3 cells were obtained from American Type Culture Collec-
tion (ATCC, Manassas, VA). Human prostate cancer M12 cells were
gift from Dr. Joye Ware from the Department of Pathology, VCU.
U937, PC-3, M12 and DU145 cells were cultured in RPMI 1640
medium (Life Technologies, Inc., Grand Island, NY) supplemented
with 10% (v/v) of heat-inactivated fetal bovine serum (FBS, Hyclone,
Logan, UT). All cells were maintained at 37 ꢂC in a fully humidified
atmosphere containing 5% CO2. Cells were rendered quiescent by
incubation for 16 h in FBS-free medium and were then growth
stimulated with phorbol 12-myristate 13-acetate (PMA, 200.0 nM)
for immunoblot assay. Alternatively, cells were treated with
compounds for indicated time in growing media and cell culture
lysates were analyzed by Western blot assay. The MEK inhibitor
PD0184352 was purchased from Upstate Biotechnology (Lake
Placid, NY). All reagents were prepared and used as recommended
by their suppliers.
5.8.4. tert-Butyl 2-(5-(hydroxymethyl)-1,3-dioxoisoindolin-2-yl)
ethylcarbamate 43
To a solution of 42 (588.0 mg, 1.8 mmol) in THF (10.0 mL) was
added BH3$THF (1.0 M THF solution, 2.1 mL, 2.1 mmol) at 0 ꢂC, and
the mixture was stirred at room temperature overnight. The reac-
tion was quenched by adding 2.0 mL of H2O. The mixture was
extracted with ethyl acetate (2 ꢃ 20.0 mL), washed with brine,
dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was purified by flash chromatography
(hexane/EtOAc ¼ 4/6) to give 43 as a white solid. 1H NMR (400 MHz,
CDCl3):
d
7.85e7.82 (d, J ¼ 7.7 Hz, 1H), 7.80 (s, 1H), 7.71e7.69 (d,
J ¼ 7.7 Hz, 1H), 4.85 (s, 2H), 3.83e3.81 (t, J ¼ 5.6 Hz, 2H), 3.44e3.41
(m, 2H), 1.34 (m, 9H). 13C NMR (100 MHz, DMSO-d6): 168.4, 156.0,
148.2, 132.5, 131.8, 131.0, 123.4, 121.3, 79.5, 64.2, 39.6, 38.1, 28.2.
5.8.5. tert-Butyl 2-(5-((diethoxyphosphoryloxy)-methyl)-1,3-
dioxoisoindolin-2-yl)-ethylcarbamate 44
5.9.1. Western blot assay
To a flask charged with 43 (0.2 g, 0.6 mmol), Et3N (0.1 mL,
1.0 mmol, 150 mol%), DMAP (7.6 mg, 0.06 mmol, 10 mol%) and THF
(10.0 mL) was added diethyl chlorophosphate (0.1 mL, 0.8 mmol,
120 mol%) over 30 min at 0 ꢂC. The resulting mixture was stirred for
16 h at room temperature, then quenched with EtOAc/H2O (1/1,
30.0 mL). The aqueous phase was extracted with EtOAc
(2 ꢃ 20.0 mL) and the combined organic phase was then washed
with saturated NaHCO3 (30.0 mL) and brine (30.0 mL), and dried
over anhydrous Na2SO4. After removal of solvents under reduced
pressure, the crude oil was purified by flash chromatography
(EtOAc/hexane ¼ 6/4) to give 44 as white solid. 1H NMR (400 MHz,
Cells (5 ꢃ 105/mL) were pretreated with compounds for 1 h and
then were lysed by sonication in 1x sample buffer [62.5 mM Tris
base (pH 6.8), 2% SDS, 50 mM DTT, 10% glycerol, 0.1% bromphenol
blue, and 5 mg/mL each chymostatin, leupeptin, aprotinin, pep-
statin, and soybean trypsin inhibitor] and boiled for 5 min. For
analysis of phospho-proteins, 1 mM of sodium orthovanadate and
sodium pyrophosphate was added to the sample buffer. Protein
samples were collected from the supernatant after centrifugation of
the samples at 12,800ꢃ g for 5 min, and protein was quantified
using Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Equal
amounts of protein (30.0
4e10% tris/glycine gel (Bio-Rad) and electrotransferred onto a PVDF
membrane (Bio-Rad). For blotting phospho-proteins, no SDS was
mg) were separated by SDS-PAGE on
CDCl3):
d
7.87e7.86 (d, J ¼ 7.7 Hz, 1H), 7.84 (s, 1H), 7.74e7.72 (d,
J ¼ 7.7 Hz, 1H), 5.18 (s, 2H), 4.17e4.11 (m, 4H), 3.85e3.82 (t,

136
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 125e137
included in the transfer buffer. The blots were blocked with 5% milk
in TBS-Tween 20 (0.1%) at room temperature for 1 h and probed
with the appropriate dilution of primary antibody overnight at 4 ꢂC.
The blots were washed twice in TBS-Tween 20 for 15 min and then
incubated with a 1:2000 dilution of horseradish peroxidase-
conjugated secondary antibody (Kirkegaard & Perry, Gaithers-
burg, MD) in 5% milk/PBS-Tween 20 at room temperature for 1 h.
After washing twice in TBS-Tween 20 for 15 min, the proteins were
visualized by Western Blot Chemiluminescence Reagent (NEN Life
Science Products, Boston, MA). Where indicated, the blots were re-
(SigmaeAldrich, St. Louis, MO) for 4 h at 4 ꢂC in the dark. Cell cycle
analysis was performed by flow cytometry using Verity Winlist
software (Topsham, ME) to determine the percentage of cells in the
G0/G1, S, G2/M and sub-G1 phases of the cell cycle.
5.9.6. Statistical analysis
Data are reported as mean ꢀ SEM of at least three independent
experiments. Statistical analysis was performed using one-way
ANOVA with Holm-Sidak post hoc test. Differences were consid-
ered significant at p < 0.05. Analyses were performed using Origin 8
software on a Windows platform.
probed with antibodies against a-tubulin (Calbiochem) to ensure
equal loading and transfer of proteins. The following antibodies
were used as primary antibodies: Cell Signaling, MA: phospho-p44/
42 MAPK (Thr202/Tyr204) antibody (1:1000, rabbit polyclonal),
p44/42 MAPK antibody (1:1000, rabbit polyclonal), phospho-
MEK1/2 (1:1000, rabbit polyclonal), MEK1/2 (1:1000, rabbit poly-
clonal), phospho-Akt (Ser473) antibody (1:1000, rabbit polyclonal),
phospho-AMPK (Thr172) antibody (1:1000, rabbit polyclonal),
Acknowledgments
We thank Dr. Joye Ware at Virginia Commonwealth University
for kindly providing the MC12 cells. The work was supported in part
by the PRIP award and new faculty start-up funds from Virginia
Commonwealth University.
AMPKa (1:1000, rabbit polyclonal); phospho-4EBP1 (Ser65)
(1:1000, rabbit polyclonal). Santa Cruz, CA: Akt1/2/3 (H-136)
(1:1000, rabbit polyclonal).
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