RETRACTED ARTICLE: Development of novel adenosine monophosphate-activated protein kinase activators

Article abstract of DOI:10.1021/jm901773d

In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)γ-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we condu

Full text of DOI:10.1021/jm901773d

2552 J. Med. Chem. 2010, 53, 2552–2561
DOI: 10.1021/jm901773d
Development of Novel Adenosine Monophosphate-Activated Protein Kinase Activators
Jih-Hwa Guh,†,‡ Wei-Ling Chang,† Jian Yang,† Su-Lin Lee,† Shuo Wei,† Dasheng Wang,† Samuel K. Kulp,† and
Ching-Shih Chen*,†
†Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, 336 Parks Hall, 500 West 12th Avenue, Columbus, Ohio
43210, and ‡School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
Received November 30, 2009
In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor
(PPAR)γ-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and
suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidine-
dione-based focused compound library to identify novel agents with these dual pharmacological
activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homologue
of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase,
respectively) and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator
of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human
macrophages were used to screen this compound library, which led to the identification of compound 53
(N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-tri-
fluoro-methyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced
suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore,
compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells,
indicating that it is not a cell line-specific event.
Introduction
responses by inhibiting the production of inflammatory cyto-
kines, especially interleukin (IL)-6, in macrophages.5,6 To-
gether, these findings suggest that AMPK represents a
therapeutically relevant target for the treatment of type II
diabetes, the metabolic syndrome, and cancer.7-10
The functional role of adenosine monophosphate-activated
protein kinase (AMPKa) in regulating energy homeostasis
and insulin sensitivity at both cellular and whole body levels
has been well recognized.1-3 In response to stimuli such as
exercise, cellular stress, and adipokines, this cellular fuel-
sensing enzyme induces a series of metabolic changes to
balance energy consumption, including stimulation of glucose
and fatty acid uptake, fatty acid oxidation, and mitochondrial
biogenesis, and inhibition of glycogen synthesis, via multiple
downstream signaling pathways controlling nutrient uptake
and energy metabolism. More recently, accumulating evi-
dence suggests a link between AMPK and cancer cell growth
and survival in light of its ability to activate tuberous sclerosis
complex 2, a tumor suppressor that negatively regulates
protein synthesis by inhibiting mammalian homologue of
target of rapamycin (mTOR).4 From a mechanistic perspec-
tive, AMPK integrates growth factor signaling with cellular
metabolism through the negative regulation of mTOR. In
addition, AMPK has been reported to suppress inflammatory
A number of antidiabetic agents, including the stable AMP
analogue 5-aminoimidazole-4-carboxamide ribose (AICAR),
the thiazolidinedione family of PPARγ agonists, and metfor-
min, are accepted pharmacological activators of AMPK
although via distinct mechanisms.11-13 These AMPK activa-
tors mediate indirect AMPK activation by mimicking AMP
or altering cellular energy status9 but suffer from low potency
and/or “off-target” effects. For example, AICAR, after being
converted to its monophosphate, mimics AMP to activate
AMPK and its upstream kinase LKB1.14 However, it exhibits
an IC50 (the half maximal inhibitory concentration) in the
mM range and has the potential to activate other AMP-
sensitive enzymes.15 More recently, a number of novel direct
AMPK activators have been discovered, some of which
exhibited promising in vitro and/or in vivo activities via the
stimulation of AMPK function.16-19
In light of the unique ability of the thiazolidinedione family
of PPARγ agonists to mediate PPARγ-independent activa-
tion of AMPK, we hypothesized that these agents could be
pharmacologically exploited to develop potent AMPK acti-
vators by dissociating these two pharmacological activities. In
this study, weconducteda two-tiered screening ofanin-house,
thiazolidinedione-based focused compound library to identify
novel agents that, at low μM concentrations, exhibited the
ability to activate AMPK and to inhibit IL-6 production
independently of PPARγ in human THP-1 macrophages.
*To whom correspondence should be addressed. Phone: (614)-688-
4008. Fax: (614)-688-8556. E-mail: chen.844@osu.edu.
aAbbreviations: AICAR, 5-aminoimidazole-4-carboxamide ribose;
AMPK, adenosine monophosphate-activated protein kinase; ELISA, en-
zyme-linked immunosorbent assay (ELISA); FBS, fetal bovine serum; IC50,
the half maximal inhibitory concentration; JNK, Jun N-terminal kinase
(JNK); IL-6, interleukin-6; LPS, lipopolysaccharide; mTOR, mammalian
homologue of target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide); p70S6K, p70 ribosomal protein S6 kinase;
PMA, phorbol 12-myristate 12-acetate; PPARγ, peroxisome proliferator-
activated receptor γ; PPRE, PPAR response element; Stat3, signal trans-
ducer and activator of transcription 3.
r
pubs.acs.org/jmc
Published on Web 02/19/2010
2010 American Chemical Society

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2553
Figure 1. (A) Schematic representation of the two-tiered screening of the benzylidene-thiazolidinedione-based focused compound library to
identify lead AMPK activators. (B) General synthetic procedure for series A-C compounds. Reaction conditions: Series A: a, K2CO3/R1-Br;
b, LAH, THF; c, (CF3SO2)2O, pyridine, CH2Cl2; d, K2CO3, DMF; e, AcOH, piperidine, ethanol/reflux. Series B: a, AcOH, piperidine, ethanol/
reflux; b, K2CO3, DMF. Series C: a, K2CO3, DMF; b, pyridine, CH2Cl2; c, LAH, dry THF, 0 °C; d, MnO2, CH2Cl2, reflux; d, piperidine,
EtOH, reflux; e, AcOH, piperidine, ethanol/reflux.
Chemistry
(ELISA) (Figure 3A). The first-tier screening of individual
compounds at 10 μM netted eight active agents (8, 12, 21, 31,
42, 49, 53, and 54), which were classified into three structural
series (Figure 1A). A further examination of the ability of
these agents at 1 μM to block the LPS-stimulated production
of IL-6 identified compound 53 as the optimal agent. General
procedures for the synthesis of series A-C compounds are
depicted in Figure 1B.
Previously, we reported that introduction of a double bond
adjoining the thiazolidinedione ring abrogated the PPARγ
ligand property of troglitazone and ciglitazone (Figure 1A).15
To abolish the PPARγ activity, we used the unsaturated
derivatives of troglitazone and ciglitazone 61 (Δ2TG)20 and
62 (Δ2CG)21 as scaffolds to develop a focused compound
library consisting of 60 compounds (1-60; Figure 2). Cell-
based assays pertinent to the activation status of AMPK and
mTOR [i.e., phosphorylation of AMPK and p70 ribosomal
protein S6 kinase (p70S6K), respectively] and IL-6/IL-6
receptor signaling [i.e., IL-6 production and signal transducer
and activator of transcription 3 (Stat3) phosphorylation,
respectively] in lipopolysaccharide (LPS)-stimulated THP-1
macrophages were used to screen this compound library via
Western blotting and enzyme-linked immunosorbent assay
Results
Proof-of-Concept That Thiazolidinediones Can Be Structu-
rally Optimized to Develop Potent AMPK Activators. In this
study, we used phorbol 12-myristate 12-acetate (PMA)-
differentiated THP-1 cells, a cell line model mimicking many
characteristic features of primary macrophages,22 to exam-
ine the effect of thiazolidinedione derivatives on AMPK

2554 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Guh et al.
Figure 2. Chemical structures of compounds 1-60 in the thiazolidinedione-based focused compound library.
activation and LPS-induced mTOR activation and IL-6
secretion into the medium. In addition, the phosphorylation
of p70S6K and Stat3 were monitored as markers for the
activation status of mTOR and IL-6 receptor signaling
pathways, respectively, in drug-treated cells (Figure 3A).
Consistent with a recent report that high doses of ciglitazone
(g100 μM) were required to activate AMPK,23 our data
indicate that ciglitazone at 10 μM exhibited no appreciable
effect on the levels of p-AMPK or p-p70S6K relative to
the LPS-treated control after 6 h of treatment (Figure 3B).
In contrast, its PPARγ-inactive counterpart 62 at the
same concentration was effective in elevating the level of
p-AMPK, accompanied by a parallel decrease in p70S6K
phosphorylation. Nevertheless, ciglitazone displayed a
several-fold higher potency than 62 in inhibiting LPS-
stimulated IL-6 production (Figure 3C), suggesting that
the anti-IL6 activity of ciglitazone was primarily attributable
to a PPARγ-dependent mechanism. This reduction in IL-6
production was not due to drug-induced cell death as neither
agent inhibited the viability of THP-1 cells within the dose
range examined. Moreover, the ability of ciglitazone and 62,
at 10 μM, to suppress LPS-activated IL-6 receptor signaling
was evident by reduced Stat3 phosphorylation relative to the
control (Figure 2B).
Screening of an In-House, Benzylidene-Thiazolidinedione-
Based Focused Compound Library to Identify Effective
AMPK Activators with High Potency in Suppressing IL-6
Production. On the basis of the above finding, we used 61 and
62 as scaffolds to generate a focused compound library
consisting of 60 derivatives with diverse structures in which
62 was blindly embedded as a control (compound 33) during
the screening process (Figure 2). These compounds along
with ciglitazone, each at 10 μM, were assessed for their
abilities to activate AMPK and to inhibit IL-6 production
in LPS-stimulated THP-1 cells (Figure 4). Consistent with
the earlier finding, 10 μM 62 exhibited modest activities in
AMPK activation and against IL-6 secretion, in conjunction
with the inhibition of the phosphorylation of p70S6K and
Stat3, while 10 μM ciglitazone was effective in inhibiting IL-6
production without affecting AMPK phosphorylation sta-
tus. Of the other compounds examined, eight derivatives (8,
12, 21, 31, 44, 49, 53, 54) exhibited substantially higher

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2555
Figure 3. (A) Schematic representation of the role of AMPK as a negative regulator of mTOR- and IL-6/IL-6 receptor-mediated signaling
pathways. (B) Western blot analysis of the effects of ciglitazone and 62, each at 10 μM, on the phosphorylation of AMPK, p70S6K, and Stat3 in
LPS-treated THP-1 cells relative to that on LPS-treated and untreated (Ctr) THP-1 macrophages in 10% FBS-containing medium after 6 h of
treatment. (C) Left panel, ELISA analysis of the inhibitory effects of ciglitazone (CG) and 62 at the indicated concentrations on LPS-stimulated
IL-6 production in THP-1 macrophages in 10% FBS-containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). Right panel,
the corresponding effects on the viability of THP-1 cells by MTT assays (N = 6).
potencies relative to 62 in the overall assessment of all of these
markers (Figure 4; only those with greater than 80% inhibi-
tion of IL-6 production were selected). Again, none of these
agents caused significant suppression of THP-1 cell viability
(Figure 4B, lower panel), indicating that the inhibition of IL-6
production was not due to cell death. It is interesting to note
that some of the agents in the library showed activities in
AMPK activation but lacked effects on suppressing IL-6
production (e.g., compounds 4 and 5) or vice versa (e.g.,
compounds 9, 19, 51, 52, 55, and 59), suggesting the involve-
ment of alternative mechanisms in their modes of action.
To demonstrate that the drug-induced inhibition of IL-6
production was independent of PPARγ, we examined the
ability of these eight agents versus ciglitazone to transacti-
vate PPARγ by using the PPAR response element (PPRE)
luciferase reporter assay. In THP-1 cells transiently trans-
fected with a reporter construct (PPRE-x3-TK-Luc), cigli-
tazone at 10 μM significantly increased luciferase activity
(P < 0.001) (Figure 5A). In contrast, none of the eight agents
examined showed appreciable activity in PPARγ activation.
Compound 53 Represents the Lead Agent in AMPK Activa-
tion and IL-6 Repression. The activities of these candidate
compound 53 in activating AMPK is about two orders-of-
magnitude higher than that of AICAR (Figure 6C).
To establish the mechanistic link between AMPK activa-
tion and anti-IL-6 activity, we examined the effect of the
dominant-negative (DN) inhibition of AMPK via the ecto-
pic expression of the K45R kinase-dead mutant (Figure 7A).
Relative to the pCMV control, transient transfection of
differentiated THP-1 cells with the DN-AMPK mutant
significantly enhanced LPS-induced IL-6 production and
reversed the inhibitory effect of compound 53 (10 μM)
(Figure 7B). This finding suggests that AMPK activation is
essential to the ability of compound 53 to suppress LPS-
stimulated IL-6 production.
We further assessed the effect of compound 53 on the
phosphorylation of AMPK and p70S6K in C-26 colon adeno-
carcinoma cells, a cell model commonly used for studying
cancer cachexia.24 Similar to that observed in THP-1 cells,
compound 53 mediated robust increases in the p-AMPK level
in dose- and time-dependent manners, accompanied by parallel
decreases in p-p70S6K (Figure, 7C). It is noteworthy that this
AMPK activation occurred almost immediately following drug
treatment. Together, these findings confirmed that this drug
effect was not a cell line-specific event.
ꢀ
agents vis-a-vis ciglitazone in blocking IL-6 release were
further assessed at 1 μM. As shown, compound 53 showed
the highest potency, exceeding that of ciglitazone, followed
by 54 and 49 (Figure 5B, upper panel), all three of which
possess largely shared structural motifs with variations in
substituents. Again, this inhibition of IL-6 production was
not due to cell death as none of these agents exhibited a
significant effect on THP-1 cell viability (lower panel).
Compound 53 inhibited LPS-stimulated IL-6 production in
a dose-dependent manner, with an IC50 of approximately 1 μM
(Figure 6A), paralleling its effect on the intracellular IL-6
mRNA expression (Figure 6B) and the phosphorylation levels
of AMPK and p70S6K (Figure 6C). Moreover, the potency of
Discussion
Recent evidence suggests that AMPK serves as a metabolic
checkpoint by integrating growth factor signaling with cellu-
lar metabolismthrough the negative regulation of mTOR.7-10
This unique functional role underscores the therapeutic value
of targeting AMPK activation in different diseases ranging
from insulin resistance to cancer through the regulation of
energy metabolism. For example, a recent study demonstrates
that AICAR was effective in suppressing the growth of
EGFR-activated glioblastoma cells by inhibiting cholesterol
and fatty acid biosynthesis.25

2556 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Guh et al.
ꢀ
Figure 4. (A) Western blot analysis of the effects of compounds 1-60 vis-a-vis ciglitazone (CG), each at 10 μM, on the phosphorylation of
AMPK, p70S6K, and Stat3 in LPS-treated THP-1 cells relative to that in LPS only treated (L) and untreated (Ctr) THP-1 macrophages in 10%
ꢀ
FBS-containing medium after 6 h of treatment. (B) Upper panel, ELISA analysis of the inhibitory effects of compounds 1-60 vis-a-vis
ciglitazone (CG), each at 10 μM, on LPS-stimulated IL-6 production in THP-1 macrophages in 10% FBS-containing medium after 6 h of
treatment. Columns, mean; bars, SD (N = 3). Lower panel, the corresponding effects on the viability of THP-1 cells by MTT assays (N = 6).
Thus, this study was aimed at the pharmacological exploi-
tation of thiazolidinediones to develop novel AMPK activa-
tors. Although AMPK is a highly conserved sensor of cellular
energy status, its functional role varies in a cell context- and
cell type-specific manner. Here, we used differentiated THP-1
macrophages as a cellular platform to conduct drug screening
in light of the pivotal role of AMPK, as a negative regulator of
mTOR, in promoting the anti-inflammatory phenotype of
macrophages by suppressing the production of inflammatory
cytokines such as IL-6.6 By screening an in-house, thiazolidi-
nedione-based focused compound library, this cell-based
assay identified compound 53 as the lead agent with low μM
potency in activating AMPK and inhibiting LPS-induced
IL-6 secretion in THP-1 cells. We further demonstrated that this
drug-induced suppression of LPS-stimulated IL-6 production
was attributable to AMPK activation, which contrasts with
the PPARγ-dependent mechanism of ciglitazone. Neverthe-
less, it was found that a number of the agents examined,
although inactive in AMPK activation, exhibited significant
effect on IL-6 production (compounds 9, 19, 51, 52, 55, and
59). From a mechanistic perspective, separation of these two
pharmacological activities suggests diversity in the mode of
action among these pharmacological agents, which warrants
investigation. The premise is manifest by the ability of a
number of small-molecule agents to modulate IL-6 produc-
tion through distinct mechanisms. For example, luteolin, a
flavonoid, reduced LPS-induced IL-6 production by inhibit-
ing the Jun N-terminal kinase (JNK)-activator protein 1
pathway,26 while chloroquine-mediated inhibition of IL-6
expression was associated with reduced mRNA stability and
mRNA levels.27 In addition, the bisphosphonate zoledronic
acid was also reported to downregulate IL-6 gene expression
in prostate cancer cells although the underlying mechanism is
unclear.28 In contrast, many therapeutic agents are associated
with the upregulation of IL-6 expression, including paclitaxel
through the activation of the JNK and Toll-like receptor
4 signaling pathways29 and the multikinase inhibitor sunitinib
through a yet-to-be-identified mechanism.30 Consequently,
understanding the mechanism of this AMPK-independent
induction will shed light onto the regulation of IL-6
production.
Recently, two direct, small-molecule activators of AMPK,
4-hydroxy-3-(20-hydroxy-biphenyl-4-yl)-6-oxo-6,7-dihydro-
thieno[2,3-b]pyridine-5-carbonitrile (63, A-769662) and 5-{5-
[5-(2-nitro-4,5-dimethyl-phenyl)-furan-2-ylmethylene]-4-
oxo-thiazolidin-2-ylidene-amino}-2-chloro-benzoic acid (64,
PT1), were discovered, each of which exhibits a unique mode
of activation.16-19 Evidence suggests that allosteric binding of
compound 63 to the AMPK γ subunit stabilizes a conforma-
tion of AMPK that inhibits dephosphorylation at the Thr-
172,18 while compound 64 antagonizes AMPK autoinhibition
by binding to the R subunit near the autoinhibitory domain.17
Although the mode of protein-ligand recognition remains
unclear, molecular docking of compound 64 into AMPK R1
suggests that the binding is mainly attributable to electrostatic
interactions.17 As compound 53 contains many electron-rich
moieties as compounds 63 and 64 do, molecular modeling
analysis was carried out to compare the electrostatic potential

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2557
ꢀ
Figure 5. (A) Effects of compounds 8, 12, 31, 44, 49, 53, and 54 vis-a-vis ciglitazone (CG), each at 10 μM, on PPARγ activation in
differentiated THP-1 cells. THP-1 cells were transiently transfected with the PPRE-x3-TK-Luc reporter vector and then exposed to individual
agents or DMSO vehicle in 10% FBS-supplemented RPMI 1640 medium for 48 h. Analysis of luciferase activity was carried out as described in
the Experimental Section. Columns, mean; bars, SD (N = 6). (B) Upper panel, ELISA analysis of the inhibitory effects of compounds 8, 12, 21,
ꢀ
31, 44, 49, 53, and 54 vis-a-vis ciglitazone (CG), each at 1 μM, on LPS-stimulated IL-6 production in THP-1 macrophages in 10% FBS-
containing medium after 6 h of treatment. Columns, mean; bars, SD (N = 3). Lower panel, the corresponding effects on the viability of THP-1
cells by MTT assays (N = 6).
similarity to that of compound 64, and, to a lesser extent,
compound 63. This finding suggests that compound 53 might
mediate AMPK activation through an allosteric binding
mechanism similar to that of compound 63 or 64, which
constitutes the current focus of this investigation.
As AMPK represents a therapeutically relevant target for
the treatment of the metabolic syndrome and cancer,7-9 there
is a growing interest in the development of novel pharmaco-
logical activators for this fuel-sensing enzyme.16-19 However,
questions remain regarding the potential adverse effects of
sustained pharmacological activation of AMPK, especially in
the liver and skeletal muscle.9 In the face of this challenge,
understanding the functional role of AMPK isozymes in
different tissues as a prelude to designing isozyme-specific
activators and/or tissue-selective delivery represents an urgent
issue.
Conclusion
In light of the high potency of compound 53 in activating
Figure 6. (A) ELISA analysis of the dose-dependent effect of
AMPK and inhibiting IL-6 production, it serves as a useful
compound 53 on LPS-stimulated IL-6 production in THP-1 macro-
agent to investigate the effects of modulating these two
phages in 10% FBS-containing medium after 6 h of treatment.
signaling effectors in the therapeutic intervention of different
diseases in cell and animal models. In addition, characteriza-
tion of its mechanism in AMPK activation will shed light onto
the functional regulation of AMPK, which might lead to the
identification of additional AMPK activators.
Columns, mean; bars, SD (N = 3). (B) RT-PCR analysis of the
dose-dependent suppressive effect of compound 53 on the mRNA
levels of IL-6 in LPS-treated THP-1 macrophages in 10% FBS-
containing medium after 6 h of treatment. Columns, mean; bars, SD
(N = 3). (C) Western blot analysis of the dose-dependent effect of
compound 53 relative to 0.5 mM AICAR on the phosphorylation
levels of AMPK and p70S6K in LPS-treated THP-1 macrophages in
10% FBS-containing medium after 6 h of treatment.
Experimental Section
Chemical reagents and organic solvents were purchased from
Sigma-Aldrich (St. Louis, MO) unless otherwise mentioned.
Nuclear magnetic resonance spectra (1H NMR) were measured
on a Bruker DPX 300 model spectrometer. Chemical shifts (δ)
were reported in parts per million (ppm) relative to the TMS
peak. Electrospray ionization mass spectrometry analyses
were performed with a Micromass Q-Tof II high-resolution
of these compounds (Figure 7D). The electron densities of
individual areas are colored in red and blue to indicate posi-
tive and negative electrostatic potentials, respectively, and
changes in electrostatic potential from positive to negative are
seen in transition from red to blue. As shown, the electrostatic
potential map of compound 53 exhibited some degree of

2558 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Guh et al.
Figure 7. (A) Western blot analysis of the expression levels of AMPK in THP-1 macrophages transiently transfected with the dominant
negative (DN)-AMPK plasmid or pCMV empty vector. (B) Protective effect of ectopic expression of DN-AMPK on LPS-stimulated IL-6
production in THP-1 macrophages with or without cotreatment with 10 μM compound 53. Columns, mean; bars, SD (N = 3). (C) Western blot
analysis of the dose- and time-dependent effects of compound 53 on the phosphorylation levels of AMPK and p70S6K in C-26 adenocarcinoma
cells in 10% FBS-containing medium. (D) The surface electrostatic potentials and structures of compound 53 versus compounds 63 and 64.
electrospray mass spectrometer. The purities of all tested com-
pounds are higher than 95% by elemental analyses, which were
performed by Atlantic Microlab, Inc. (Norcross, GA), and were
reported to be within 0.4% of calculated values. Flash column
chromatography was performed using silica gel (230-400 mesh).
The structures of the eight lead candidates could be divided into
three series, i.e., A (8, 12, and21), B(31 and44), andC(49, 53, and
54) (Figure 1A). The general procedures for the synthesis of series
A-C compounds are described in Figure 1B. Compounds of
series A and B were synthesized according to slight modifications
of previously reported procedures,20,21 and the synthesis of the
series C active compounds (49, 53, and 54) is illustrated by the
synthesis of compound 53 as an example.
5-[3-Bromo-4-(6-ethoxy-2,5,7,8-tetramethyl-chroman-2-ylme-
thoxy)-benzylide-ne]-thiazolidine-2,4-dione (8). 1H NMR (300
MHz, CDCl3), δ 1.42 (t, J = 7.2 Hz, 3H), 1.51(s, 3H), 1.92-
2.03 (m, 1H), 2.05-2.23 (m, 10H), 2.58-2.72 (m, 2H), 3.74 (q,
J = 7.2 Hz, 2H), 4.14 (q, J = 9.3 Hz, 2H), 7.00 (d, J = 8.4 Hz,
1H), 7.41 (dd, J = 2.4, 8.4 Hz, 1H), 7. 71 (d, J = 2.4 Hz, 1H),
7.75 (s, 1H), 8.65 (s, 1H). HRMS exact mass of C26H28BrNO5S
(M þ Na)þ, 568.0769 amu; observed mass of (M þ Na)þ,
568.0786 amu. Anal. calcd C 57.14, H 5.16, O 14.64; found C
57.23, H 5.26, O 14.66.
5-[4-(6-Butoxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-3-
methoxy-benzyli-dene]-thiazolidine-2,4-dione (12). 1H NMR
(300 MHz, CDCl3), δ 0.99 (t, J = 7.2 Hz, 3H), 1.45 (s, 3H),
1.52-1.66 (m, 2H), 1.73-1.85 (m, 2H), 1.89-1.99 (m, 1H),
2.02-2.23 (m, 10H), 2.60-2.69 (m, 2H), 3.63 (t, J = 6.6 Hz,
2H), 3.90 (s, 3H), 4.04 (d, J = 9.3 Hz, 1H), 4.12 (d, J = 9.3 Hz,
1H), 6.96-7.10 (m, 3H), 7.80 (s, 1H), 8.58 (br, 1H). HRMS exact
mass of C29H35NO6S (M þ Na)þ, 548.2083 amu; observed mass
of (M þ Na)þ, 548.2095 amu. Anal. calcd C 66.26, H 6.71, O
18.26; found C 66.32, H 6.80, O 18.29.
4-{2-[2-Bromo-4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phe-
noxymethyl]-2,5,7,8-tetramethyl-chroman-6-yloxy}-butyroni-
trile (21). 1H NMR (300 MHz, CDCl3), δ 1.49 (s, 3H),
1.94-2.23 (m, 13H), 2.58-2.74 (m, 4H), 3.76 (t, J = 5.7 Hz,
2H), 4.00-4.16 (m, 2H), 6.97 (d, J = 8.7 Hz, 1H), 7.40 (dd, J =
2.1, 8.7 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 7.74 (s, 1H), 8.36 (br,
1H). HRMS exact mass of C28H29BrN2O5S (M þ Na)þ,
607.0878 amu; observed mass of (M þ Na)þ, 607.0882 amu.
Anal. calcd C 57.44, H 4.99, O 13.66; found C 57.54, H 5.04, O
13.72.
5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-(1-methyl-cy-
clohexylmethyl)-thiazolidine-2,4-dione (31). 1H NMR (300
MHz, CDCl3) 0.95 (s, 3H), 1.46-1.56 (m, 10H), 3.64 (s, 2H),

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2559
6.08-6.38 (br, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.4 Hz,
1H), 7.69 (s, 1H), 7.83 (s, 1H). HRMS exact mass of C19H20-
F3NO3S, (M þ Na)þ, 422.1014 amu; found: 422.1012 amu.
Anal. calcd C 57.13, H 5.05, O 12.02; found C 57.38, H 5.04, O
12.16.
5-(4-Hydroxy-3-trifluoromethyl-benzylidene)-3-propyl-thiazo-
lidine-2,4-dione (44). 1H NMR (300 MHz, CDCl3) 0.97 (t, J =
7.5 Hz, 3H), 1.60-1.78 (m, 2H), 3.74 (t, J = 7.5 Hz, 2H), 6.19
(br, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.69
(s, 1H), 7.83 (s, 1H). HRMS exact mass of C14H12F3NO3S, (M þ
Na)þ, 331.3112 amu; found: 331.3124 amu. Anal. calcd C 50.75,
H 3.65, O 14.49; found C 50.84, H 3.68, O 14.54.
N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-
5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoromethyl-benzenesul-
fonamide (53). Step a. Trifluoro-methanesulfonic acid 1-methyl-
cyclohexylmethyl ester (i) was prepared from 1-methyl-
cyclohexanecarboxylic acid as previously described.21 A mix-
ture of i (0.5 mmol), 2,4-thiazolidinedione (0.6 mmol) and
K2CO3 (0.7 mmol) were dissolved in DMF (3 mL), heated to
80 °C with stirring for 4 h, poured into water, extracted with
ethyl acetate (10 mL) three times, and concentrated. The residue
was purified by flash column chromatography to afford 3-(1-
methyl-cyclohexylmethyl)-thiazolidine-2,4-dione (ii) in 50%
yield.
Step b. To a mixture of methyl 4-aminobenzoate (1.51 g,
10 mmol) and pyridine (0.97 mL) in dry methylene chloride
(100 mL), 4-nitro-3-trifluoromethylbenzenesulfonyl chloride
(2.89 g, 10 nmol) in dry methylene chloride (20 mL) was added
slowly and washed, in tandem, with 1N HCl, water, 10%
Na2CO3 aqueous solution, and brine. The organic layer was
dried, filtered, and concentrated. The residue was purified by
chromatography (EtOAc-hexane, 1:5) to give 4-(4-nitro-3-tri-
fluoromethyl-phenyl-sulfamoyl)-benzoic acid methyl ester (iii)
as colorless crystal with 86% yield. 1H NMR (300 MHz, CDCl3)
δ 3.79 (s, 3H), 7.25 (d, J = 5.58 Hz, 2H), 7.85 (d, J = 6.72 Hz,
2H), 7.94 (d, J = 8.43 Hz, 1H), 8.16 (d, J = 8.64 Hz, 1H), 8.30
(s, 1H).
(s, 1H), 11.12 (br, 1H). HRMS exact mass of C25H24F3N3O6S2,
(M þ Na)þ, 606.0956 amu; found: 606.0974 amu. Anal. calcd C
51.45, H 4.15, O 16.45; found C 51.72, H 4.20, O 16.54.
4-Methoxy-N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-
thiazolidin-5-yli-denemethyl]-phenyl}-benzenesulfonamide (49).
1H NMR (300 MHz, CDCl3/MeOD-d4) 0.96 (s, 3H), 1.22-
1.62 (m, 10H), 3.66 (s, 2H), 3.91 (s, 3H), 7.02 (d, J = 8.7 Hz, 2H),
7.55 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 7.87(d, J = 8.1
Hz, 2H), 7.88 (s, 1H). HRMS exact mass of C26H28N2O5S2, (M
þ Na)þ, 535.1337 amu; found: 535.1352 amu. Anal. calcd C
60.92, H 5.51, O 15.60; found C 60.72, H 5.60, O 15.54.
N-{4-[3-(1-Methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-
5-ylidenemethyl]-phenyl}-4-nitro-benzenesulfonamide (54). 1H
NMR (300 MHz, d-DMSO), δ 0.82 (s, 3H), 1.21-1.45 (m,
10H), 3.51 (s, 2H), 7.24 (d, J = 8.55 Hz, 2H), 7.51(d, J = 8.55
Hz, 2H), 7.78 (s, 1H), 8.04 (d, J = 9.00 Hz, 2H), 8.36 (d, J = 8.97
Hz, 2H), 11.12 (br, 1H). HRMS exact mass of C24H25N3O6S2,
(M þ Na)þ, 538.1083 amu; found: 538.1092 amu. Anal. calcd C
55.91, H 4.89, O 18.62; found C 55.98, H 4.98, O 18.76.
Cells and Cell Culture. THP-1 monocytic cells were purchased
from the American Type Culture Collection (Rockville, MD)
and maintained with L-glutamine-containing RPMI 1640 sup-
plemented with 10% fetal bovine serum (FBS), 0.25% glucose,
0.01% sodium pyruvate, 50 μM 2-mercaptoethanol, and 0.1
mL/mL penicillin/streptomycin/L-glutamine. Differentiation of
THP-1 monocytes into macrophages was carried out by expo-
sure to PMA (50 nM) in the aforementioned RPMI 1640
medium for 24 h. Colon-26 (C-26) adenocarcinoma cells were
a kind gift of Dr. Denis Guttridge (The Ohio State University).
The C-26 cells were maintained in RPMI 1640 medium
supplemented with 5% FBS and 1% penicillin/streptomycin.
All cell types were cultured at 37 °C in a humidified incubator
containing 5% CO2.
Cell Viability Assay. Cell viability was determined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
(MTT) assay. Cells were seeded in 96-well plates (5000 cells/
well) and incubated in 10% FBS-supplemented medium for 24 h
and were then treated with individual agents for 72 h. Drug-
containing medium was then replaced with MTT (0.5 mg/mL in
RPMI 1640), followed by incubation at 37 °C for 2 h. After
removal of medium, the reduced MTT dye was solubilized in
200 μL/well DMSO, and absorbance at 570 nm was measured.
ELISA. IL-6 release by differentiated THP-1 macrophages in
response to 10 ng/mL of LPS was analyzed by using the IL-6
ELISA kit (Cayman Chemical Co., Ann Arbor, MI) according
to the manufacturer’s instruction in triplicate. The effect of each
test compound on LPS-stimulated IL-6 release is presented as
percent inhibition and was calculated using the following for-
mula: percentage inhibition = 100% ꢀ {1 - [(O.D. of sample -
O.D. of control)/(O.D. of LPS - O.D. of control)]}.
Western Blotting. THP-1 cells were lysed in SDS-sample
buffer after washing with iced-PBS buffer and then heated at
95 °C for 20 min. Protein extracts were prepared using M-PER
mammalian protein extraction reagent (Pierce, Rockford, IL)
containing freshly added 1% phosphatase and protease inhibi-
tor cocktails (Calbiochem). After centrifugation of lysates at
13000g for 10 min, supernatants were collected and the protein
concentration in each sample was determined by protein assay
(Bio-Rad). Protein extracts were then suspended in 2ꢀ SDS
sample buffer, separated by electrophoresis in 10% SDS-poly-
acrylamide gels, and transferred to nitrocellulose membranes
using a semidry transfer cell. The transblotted membrane was
washed twice with Tris-buffered saline containing 0.1% Tween-
20 (TBST). After blocking with TBST containing 5% nonfat
milk for 1 h, the membrane was incubated with primary anti-
bodies: p172Thr-AMPK, AMPK, p705Tyr-Stat3 and Stat3 (Cell
Signaling Technology; Beverly, MA), p389Thr-p70S6K and
p70S6K (Santa Cruz Biotechnology; Santa Cruz, CA), each at
1:1000 dilution in 1% TBST-nonfat milk at 4 °C overnight.
After incubation with the primary antibody, the membrane was
Step c. To a solution of compound iii (3.26 g, 8.06 mmol) in
dry THF (50 mL), LAH pallet (0.46 g, 12.10 mmol) was added at
0 °C. The resulting reaction mixture was stirred for 4 h,
quenched by the addition of water (5 mL), concentrated, diluted
with ethyl acetate (50 mL), and washed, in tandem, with 1N
HCl, water, 10% Na2CO3 aqueous solution, and brine. The
organic layer was dried, filtered, and concentrated. The residue
was purified by silica gel chromatography (ethyl acetate-
hexane, 3:7) to afford 4-hydroxymethyl-N-(4-nitro-3-trifluoro-
methyl-phenyl)-benzenesulfonamide (iv) as light-yellow solid
with 71% yield. 1H NMR (300 MHz, CDCl3) δ 2.06 (br, 1H),
4.75 (s, 2H), 6.96 (br, 1H), 7.59 (d, J = 10.17 Hz, 2H), 7.84 (d,
J = 8.19 Hz, 2H), 7.88 (d, J = 8.43 Hz, 1H), 8.03 (d, J = 8.43
Hz, 1H), 8.16 (s, 1H).
Step d. A reaction mixture of compound iv (2.00 g, 5.31
mmol) and MnO2 (2.34 g, 26.57 mmol) in chloroform (100 mL)
was refluxed overnight, concentrated, diluted with ethyl acetate,
filtered, and concentrated. The residue was purified by silica gel
chromatography (ethyl acetate-hexane, 1:7) to yield N-(4-formyl-
phenyl)-4-nitro-3-trifluoromethyl-benzenesulfonamide (v) as
1
light-yellow solid in 88% yield. H NMR (300 MHz, CDCl3)
δ 7.28 (d, J = 6.76 Hz, 2H), 7.88 (d, J = 6.72 Hz, 2H), 7.97 (d,
J = 8.43 Hz, 1H), 8.17 (d, J = 8.64 Hz, 1H), 8.31 (s, 1H).
Step e. A reaction mixture of compound ii (0.73 g, 3.21 mmol),
compound vi (1.25 g, 3.21 mmol), and catalytic amount of
piperidine in ethyl alcohol (50 mL) was refluxed overnight,
concentrated, dissolved in ethyl acetate (50 mL), neutralized
with acetic acid, washed with water and brine, dried, and
concentrated. The residue was purified by silica gel chromato-
graphy (ethyl acetate-hexane, 1:7) to give afforded compound
53 as yellow solid in 72% yield. 1H NMR (300 MHz, DMSO-d)
δ 0.80 (s, 3H), 1.20-1.43 (m, 10H), 3.47 (s, 2H), 7.24 (d, J = 7.65
Hz. 2H), 7.52 (d, J = 8.28 Hz, 2H), 7.78 (s, 1H), 8.26 (s, 1H), 8.31

2560 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Guh et al.
washed three times with TBST for a total of 30 min, followed by
incubation with horseradish peroxidase-conjugated goat anti-
mouse IgG (diluted 1:2500) for 1 h at room temperature. After
three thorough washes with TBST for a total of 30 min, the
immunoblots were visualized by enhanced chemiluminescence.
Transient Transfection. Transfection of THP-1 cells with
the K45R kinase-dead, dominant-negative AMPK plasmid
(Addgene, Cambridge, MA) or empty vector was performed
by electroporation using the Human Monocyte Nucleofector
Kit of the Amaxa Nucleofector System (Amaxa Biosystems,
Cologne, Germany) according to a published procedure.31
Analysis of PPARγ Activation. The PPRE-x3-TK-Luc repor-
ter vector containing three copies of the PPAR response ele-
ments preceding the thymidine kinase promoter-luciferase
construct was used for detection of PPARγ activation.32 Differ-
entiated THP-1 macrophages were plated at the density of
1 ꢀ 105 cells/per well in 24-well plates and then transiently trans-
fected by nucleofection with the PPRE-x3-TK-Luc reporter
plasmid followed by exposure to 10 μM ciglitazone or its
derivatives in triplicate in the presence of LPS for 48 h. Cells
were lysed with passive lysis buffer (Promega), and aliquots of
the lysates (50 μL) were transferred to 96-well plates and mixed
with 100 μL of luciferase substrate (Promega). Luciferase
activity was determined by using the MicroLumatPlus LB96 V
luminometer (Berthold Technologies, Oak Ridge, TN) with the
WinGlow software package.
Total RNA Isolation and RT-PCR Analysis of IL-6 Expres-
sion. Total RNA was extracted from drug- or vehicle-treated
THP-1 macrophages with TRIzol (Invitrogen, Carlsbad, CA)
and then reverse-transcribed to cDNA using the Omniscript RT
Kit (Qiagen, Valencia, CA). The sequences of the primers used
for RT-PCR were as follows:
IL-6: forward, 50-GAGAAAGGAGACATGTAACAA-
GAGT-30, reverse 50-GCGCAGA-ATGAGATGAGTTGT-30;
β-actin: forward, 50-TCTACAATGAGCTGCGTGTG-30, re-
verse, 50-GGTCAGGATCTTCATGAGGT-30. The PCR pro-
ducts were separated by electrophoresis on 1% agarose gels and
visualized by ethidium bromide staining.
Molecular Model Experiment. Molecular structures of com-
pound 53, 63, and 64 were initially subjected to 1000 steps of
Monte Carlo simulation using Merck Molecular Force Field
program available in Spartan 08 (Wavefuction, Inc., Irvine, Ca;
http://www.wavefun.com/). The minimum conformation
reached by the simulation was then fully optimized at a density
function theory level of B3LYP/6-31G* with Gaussian 03
(Gaussian, Inc., Wallingford, CT; http://www.gaussian.com/).
All the fully optimized structures were confirmed by normal-
mode analysis; no negative frequencies were found. Computa-
tions for electrostatic potential and density were carried out for
each optimal structure by potential population analysis under
Hartree-Fock/6-31G* function theory by using Gaussian 03.
The electrostatic potential map for each compound was gener-
ated by Molecular Operation Environment 2008.10 (Chemical
Computing Group, Montreal, Canada; http://www.chemcomp.
com/) and are presented with the electrostatic potential mapped
onto the electron density.
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