Antidiabetic and antiobesity effects of ampkinone (6f), a novel small molecule activator of AMP-activated protein kinase

Article abstract of DOI:10.1021/jm100565d

Adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK) has emerged as an attractive target molecule for the treatment of metabolic disorders, including obesity and type 2 diabetes. In this study, we identified a novel small molecule, ampkinone (

Full text of DOI:10.1021/jm100565d

J. Med. Chem. 2010, 53, 7405–7413 7405
DOI: 10.1021/jm100565d
Antidiabetic and Antiobesity Effects of Ampkinone (6f), a Novel Small Molecule Activator
of AMP-Activated Protein Kinase
Sangmi Oh,† Sung Jin Kim,§, Jung Hwan Hwang,^ Hyang Yeon Lee,† Min Jeong Ryu,§, Jongmin Park,† Soung Jung Kim,§,
Young Suk Jo,§, Yong Kyung Kim,§, Chul-Ho Lee,^ Ki Ryang Kweon, Minho Shong,*,§, and Seung Bum Park*,†,‡
†Department of Chemistry, ‡Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Korea,
§Department of Internal Medicine, and Department of Biochemistry, Chungnam National University School of Medicine, Daejeon 301-721,
Korea, and ^Laboratory of Experimental Animals, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea
Received May 10, 2010
Adenosine 5’-monophosphate (AMP) activated protein kinase (AMPK) has emerged as an attractive target
molecule for the treatment of metabolic disorders, including obesity and type 2 diabetes. In this study, we
identified a novel small molecule, ampkinone (6f ), as an indirect AMPK activator, which was derived from
the small molecule library constructed by diversity-oriented synthesis. Ampkinone stimulated the phosphor-
ylation of AMPK via the indirect activation of AMPK in various cell lines. Ampkinone-mediated activation
of AMPK required the activity of LKB1 and resulted in increased glucose uptake in muscle cells. In addition,
ampkinone-treated DIO mice significantly reduced total body weight and overall fat mass. Histological
examination and measurement of lipid parameters showed that ampkinone effectively improved metabolic
abnormalities in the DIO mice model. Our results demonstrate that ampkinone, a small molecule with a
privileged benzopyran substructure, has a potential as a new class of therapeutic agent for antidiabetic and
antiobesity treatment via the indirect stimulation of AMPK.
Introduction
AMP.8 AMPK is activated during muscle contraction and
exercise by the phosphorylation of threonine 172 by LKB19
and Ca2þ/calmodulin-dependent kinase kinase (CaMKK).10
Once AMPK is activated, it stimulates glucose uptake in
skeletal muscle through independent pathways in insulin-resis-
tant conditions.11 Enhanced glucose uptake induced by AMPK
is achieved, as least in part, by enhanced GLUT4 translocation
to the plasma membrane for fusion and docking.12 Several
studies have reported that the AMP analogue, 5-aminoimid-
azole-4-carboxamide-1-β-D-ribofuranoside (AICAR), and other
small-molecule activators of AMPK increase glucose uptake
in vitro13 and in vivo.14 Although their mechanisms of action
are still incompletely understood, evidence indicates that
metformin15 and thiazolidinediones (TZDs)16 act through
activation of AMPK in type 2 diabetes. Furthermore, AMPK
is the target molecule of two adipose tissue-derived hormones,
leptin17 and adiponectin,18 which are major regulators of
energy metabolism and glucose homeostasis. These accumu-
lated findings have made AMPK an attractive therapeutic
target for the treatment of type 2 diabetes and obesity.
The global incidence of type 2 diabetes is increasing rapidly,
with higher rates of morbidity and mortality, resulting in a
significant financial and social burden worldwide.1 Type 2
diabetes is characterized by insulin resistance and hyperglyce-
mia, which lead to chronic complications in both small and large
blood vessels.2,3 Therefore, numerous studies have focused on
the development of therapeutics able to maintain normal levels
of blood glucose by increasing glucose clearance in peripheral
tissues such as skeletal muscle and adipose tissue.4,5 Recent
studies have shown that impaired handling of cellular energy
homeostasis is closely associated with insulin resistance, result-
ing in type 2 diabetes, metabolic syndrome, hypertension, and
increased cardiovascular risk.6 Therefore, molecules that reg-
ulate cellular energy metabolism hold promise as drug targets
for the development of therapeutics for diabetes and obesity.7
AMPa-activated protein kinase (AMPK) is a heterotrimeric
complex comprising catalytic R and regulatory β/γ subunits
that senses low energy status by monitoring the ratio of ATP to
In this study, we identified a small-molecule activator of
AMPK, ampkinone (6f), as an effective therapeutic agent
for the treatment of obesity and type 2 diabetes. This novel
skeleton (6) is a tetracyclic structure embedded with a privileged
benzopyran substructure, derived from 2500-member small
molecules library constructed in-house using diversity-oriented
synthesis strategy to prepare skeletally diverse small molecules
to mimic natural product-like or druglike chemical structures.19
This collection of druglike small molecules containing more
than 40 unique molecular frameworks can increase the possi-
bility of identifying a novel modulator of biological system such
as AMPK.20 Ampkinone, a small molecule with this molecular
*To whom correspondence should be addressed. For M.S.: phone,
(þ)82-42-280-7161; fax, (þ)82-42-280-7995; e-mail, minhos@cnu.ac.kr.
For S.B.P.: phone, (þ)82-2-880-9090; fax, (þ)82-2-884-4025; e-mail,
sbpark@snu.ac.kr.
a Abbreviations: AMP, adenosine 5’-monophosphate; AMPK, adenosine
5’-monophosphate activated protein kinase; AKN, ampkinone; CaMKK,
Ca2þ/calmodulin-dependent kinase kinase; DIO, diet-induced obese;
GLUT4, glucose transporter 4; AICAR, 5-aminoimidazole-4-carboxa-
mide-1-β-D-ribofuranoside; TZDs, thiazolidinediones; ACC, acetyl-CoA
carboxylase; DOS, diversity-oriented synthesis; DDQ, 2,3-dichloro-5,6-
dicyanobenzoquinone; SAR, structure-activity relationship; CLSM, con-
focal laser scanning microscopy; IPGTT, intraperitoneal glucose tolerance
test; ITT, insulin tolerance test; NEFA, nonesterified fatty acids; HMG-
CoA, 3-hydroxy-3-methylglutaryl coenzyme A.
r
2010 American Chemical Society
Published on Web 09/28/2010
pubs.acs.org/jmc

7406 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Oh et al.
framework (6), stimulated the activation and phosphorylation
of AMPK in cell lines originating from muscle, fat, and liver.
Phosphorylation of acetyl-CoA carboxylase (ACC), a major
downstream target molecule of AMPK, confirmed that amp-
kinone stimulates functional activation of AMPK via the
phosphorylation at Thr172 in cultured cells. We also demon-
strated the enhanced cellular glucose uptake monitored by
fluorescent glucose bioprobe, Cy3-Glc-R,21 and cross-checked
with 3H-labeled 2-deoxy-D-glucose. Consistent with the in vitro
data presented here, administration of ampkinone to diet-
induced obese (DIO) mice up-regulated the activity of AMPK
in the liver and muscle and enhanced insulin sensitivity.
Furthermore, histological analysis confirmed that ampkinone
treatment accelerated the consumption of lipids via increased
oxidation in the liver and adipose tissues, similar to other small
molecule activators of AMPK.22 Collectively, the results dem-
onstrate that ampkinone is a new small-molecule activator
of AMPK that may be effective for the treatment of type
2 diabetes and obesity.
Scheme 1. Synthetic Scheme of Benzopyran-Embedded Molec-
ular Framework (6)a
a Reagents and conditions: (a) cyclopentanone or acetone, pyrroli-
dine, EtOH, reflux; (b) trifluoromethanesulfonic anhydride, 2,6-di-tert-
butyl-4-methylpyridine, CH2Cl2, 0 ꢀC; (c) vinylboronic acid dibutyl
ester, Pd(PPh3)4, Na2CO3, EtOH/toluene/H2O, 70 ꢀC; (d) maleimide
derivatives, toluene, reflux; (e) DDQ, toluene, reflux.
Table 1. Activation of AMPK (pT172) and ACC (pS79) phosphoryla-
tion induced by individual compounds in the focused library
Results and Discussion
% activitya
Cpd.
R
R1 R2
R3
Identification of Ampkinone, a Novel Small-Molecule
Activator of AMPK. We previously reported synthetic methods
for the concise and efficient library construction using diversity-
oriented synthesis (DOS) strategy, which generated novel
scaffolds embedded with privileged substructures via various
chemical transformations.20 To identify a novel small-molecule
activator of AMPK, we evaluated an in-house small molecule
collection constructed by these DOS pathways. After extensive
bioassays using Western blot analysis, we identified a new
molecular framework (6) that stimulates the phosphorylation
of AMPK. The aromatized benzopyran moiety in 6 was shown
to be essential for AMPK activation because other types of
benzopyran-fused tetracycles such as monoene (5), hydroge-
nated compound (S7), and allylic alcohol (S8) were inactive
toward AMPK (Figure S1 of Supporting Information). On the
basis of initial screening results, a focused small molecule library
was constructed by a series of reactions designed to produce 6:
[1] the cyclization of hydroxyacetophenones (1) with acetone or
cyclopentanone, [2] triflation of carbonyl moiety on substituted
chroman-4-ones (2), [3] palladium-mediated vinylation of vinyl
triflate intermediates (3), [4] Diels-Alder reaction of bicyclic
dienes (4) with substituted maleimides, and [5] subsequent aro-
matization of benzopyran-containing tetracyclic monoenes (5)
with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (Scheme 1).
Using a 15-member focused library (6a-o) with various struc-
tural modifications, we conducted a structure-activity relation-
ship (SAR) study via Western blot analysis (see Table 1). On the
basis of our SAR study, 2-(4-benzoylphenyl)-6-hydroxy-7-me-
thoxy-4,4-dimethylchromeno[3,4-e]isoindole-1,3-dione (6f) de-
monstrated the most efficient activation of AMPK and sub-
sequent functional phosphorylation of ACC (Figure S2). The
compound activity on AMPK phosphorylation was slightly
attenuated when benzophenone moiety at the R3 position of
molecular framework 6 was replaced with simpler moieties such
as acetylphenyl group (6e) and phenyl (6c), but it was completely
abolished when hydrogen (6a), methyl (6b), and benzyl group
(6d) were introduced at the R3 position. The introduction of
bulky substituents such as biphenyl-4-yl (6g), biphenyl-3-yl (6i),
and 4-benzylphenyl group (6j) diminished the activity in Western
blot analysis; however, 9-oxo-9H-fluoren-3-yl moiety (6h) some-
what preserved its activity because of its structural similarity to
benzophenone. In addition, cyclopentyl moiety at the R position
AMPK ACC
6a
6b
6c
6d
6e
6f
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
OMe OH
OMe OH
OMe OH
OMe OH
OMe OH
OMe OH
OMe OH
OMe OH
OMe OH
OMe OH
H
Me
158
205
273
165
264
322
160
249
175
169
92
123
145
254
194
384
295
114
154
201
129
91
Ph
Bn
p-Acetylphenyl
4-Benzoylphenyl
Biphenyl-4-yl
9-Oxo-9H-fluoren-3-yl
Biphenyl-3-yl
4-Benzylphenyl
6g
6h
6i
6j
6k
6l
OMe OH 4-(Benzo[d ]oxazol-2-yl)phenyl
OMe
F
H
H
H
4-Benzoylphenyl
4-Benzoylphenyl
4-Benzoylphenyl
4-Benzoylphenyl
159
186
203
113
154
156
92
6m
6n
H
6o -(CH2)4- OMe OH
a Relative phosphorylation activity induced by the treatment of
individual compounds at 10 μM in the Western blot analysis.
51
(6o) or various substituents at the R1 and R2 positions (6l-n)
caused a significant reduction in the compound activity on
AMPK phosphorylation. On the basis of this screening result,
we classified 6f as a novel small molecule activator of AMPK and
designated this compound ampkinone (AKN) (Figure 1A).
AKN Stimulates Phosphorylation of AMPK in Muscle-,
Adipose-, and Liver-Derived Cell Lines in a Dose- and Time-
Dependent Manner. To investigate the effect of AKN on
AMPK activation, we utilized cell lines originating from cardiac
and skeletal muscle, adipose tissue, and liver. Immunoblot
analysis of a time course assay conducted in L6 skeletal muscle
cells revealed that the AKN-mediated phosphorylation of
AMPK (Thr172) was increased at 10 min, reached a maximum
at 1 h, and persisted for 6 h (Figure 1B). Phosphorylation of
ACC (Ser79), an intracellular substrate of AMPK, was also
increased by treatment with AKN on a timeline similar to that
of AMPK. Immunocomplex kinase assays with the SAMS
peptide revealed a 2.7-fold increase in AMPK activity upon
treatment with 10 μM AKN. Treatment with 1 mM AICAR as
a positive control for AMPK activation induced a 3.2-fold
increase in AMPK activity (Figure 1C). We next examined the
dose-dependent effect of AKN on AMPK activation. The
phosphorylation of AMPK and ACC was dose-dependently
increased in various cell lines, such as C2C12 skeletal muscle
cells, 3T3-L1 adipose-like mouse embryonic fibroblasts, and

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7407
Figure 1. AKN stimulates the phosphorylation of AMPK (pT172) and ACC (pS79) in a time- and dose-dependent manner. (A) Chemical
structure of AKN (6f). (B) AKN-induced phosphorylation of AMPK (pT172) and ACC (pS79). Western blot analysis of lysates from L6 cells
treated with AKN (10 μM) for the indicated times was performed with antibodies against the indicated proteins. (C) Total AMPK activity in
cells treated with AKN or DMSO was measured using a synthetic SAMS peptide substrate and [γ-32P]ATP. (D) L6 cells, C2C12 myocytes, 3T3-
L1 preadipocytes, and HepG2 hepatic cells were incubated for 1 h with the indicated concentrations of AKN. (E) EC50 was determined by
Western blotting in various concentrations (0-20 μM) and data analysis with PRISM3 program. Immunoblots were performed using anti-
AMPK (pT172), anti-AMPK, anti-ACC (pS79), and anti-ACC antibodies. (/) P<0.05 vs corresponding control.
Figure 2. AKN predominantly stimulates AMPK activation via the LKB1 pathway. (A) AMP to ATP ratio was not significantly changed
upon treatment with AKN. (B) LKB1-deficient HeLa cells and HeLa cells transfected with a pcDNA3.1 plasmid encoding LKB1-tagged
hemagglutinin (HA) were exposed to AKN (10 μM) for the indicated time periods after 6 h of serum starvation. Immunoblot analysis was then
performed with antibodies against AMPK (pT172), AMPK, and HA. (C) The effect of the CaMKK inhibitor, STO-609, on AKN-mediated
AMPK activation following 1 h of treatment in Hela cells. (D) L6 cells were exposed to AKN (10 μM) or AICAR (1 mM) for 1 h in the presence
or absence of compound C (20 μM), and immunoblots were performed with anti-ACC (pS79), anti-ACC, and anti-actin as a loading control.
HepG2 human hepatoma cells as well as L6 cells (Figure 1D).
These data indicate that AKN stimulates the phosphorylation
and activity of AMPK in multiple cell lines originating from
muscle, fat, and liver. The estimated EC50 of AKN is 4.3 μM in
L6 cells (Figure 1E and Figure S3).
AKN-Mediated AMPK Phosphorylation Requires the
Upstream Kinases, LKB1 and CaMKK. We next determined
whether AKN could directly activate AMPK. 32P incorpora-
tion by isolated AMPK into the SAMS peptide was not
different in AKN-treated samples compared to the DMSO
control (data not shown). This suggests that AKN does not
directly activate purified AMPK in a cell-free condition.
Then we investigated whether the cellular levels of AMP,
ADP, or ATP can be perturbed upon treatment with AKN.
As shown in Figure 2A, the treatment with AKN at 10 μM
caused no significant alteration in AMP to ATP ratio in
L6 cells (Figure S4).
Consequently, we hypothesized that AKN might stimulate
AMPK phosphorylation through the activation of upstream
signaling pathway. LKB1 and CaMKK are two primary AMPK

7408 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Oh et al.
Figure 3. AKN stimulates glucose uptake through AMPK but not Akt. (A) C2C12 and (B) L6 myocytes were incubated for 1 h with AKN
(10 μM) or insulin (100 nM). Next, (A, B) Cy3-Glc-R (DMSO-treated = closed circles; AKN-treated = closed squares; insulin-treated =
closed triangles) or (C) 2-deoxy-D-glucose-1,2-3H(N) was added to culture media for the indicated time periods (A) or 10 min (B, C), and
glucose uptake was measured. (D) The effect of the AMPK inhibitor compound C (20 μM, 30 min preincubation) and (E) the PI3K inhibitor
wortmannin (1 μM, 30 min preincubation) on AKN- (10 μM), insulin- (100 nM), or AICAR (1 mM)-mediated glucose uptake following 1 h of
treatment in L6 myocytes: (/) P < 0.05; (//) P < 0.01 vs corresponding control; (#) P < 0.05 vs corresponding treated groups (AKN, insulin,
and AICAR).
kinases.9,10 For the mechanistic understanding, we investigated
whether one or both of these kinases were required for the
activation of AMPK by AKN. As confirmed by the data shown
in Figure 2B, HeLa cells do not express LKB1.23 Therefore,
HeLa cells were transfected with hemagglutinin (HA)-tagged
LKB1 for 24 h to induce the expression of exogenous LKB1 and
then treated with AKN for the indicated time. In mock-trans-
fected cells, treatment with AKN slightly increased AMPK
phosphorylation from 60 to 360 min, while AMPK phosphor-
ylation in LKB1-transfected cells was noticeably higher. These
data indicate that LKB1 activity is required for robust AKN-
mediated AMPK phosphorylation, though the slight increase in
AMPK phosphorylation in mock-transfected cells suggested the
involvement of another upstream kinase. Accordingly, HeLa
cells were preincubated with STO-609, a specific inhibitor of
CaMKK.24 Treatment of STO-609 in mock-transfected HeLa
cells completely suppressed the phosphorylation of AMPK
induced by AKN (Figure 2C), indicating that LKB1 and
CaMKK mediate the activation of AMPK by AKN. Finally,
to determine whether phosphorylation of ACC upon treatment
with AKN is associated with AMPK, L6 cells were preincubated
with the AMPK-specific inhibitor, compound C, followed by
treatment with AKN for 1 h. As shown in Figure 2D, AKN-
induced ACC phosphorylation was significantly diminished
through the specific inhibition of AMPK with compound C.
Taken together, these data confirm that AKN stimulates the
phosphorylation of AMPK neither by direct activation as in the
case of AICAR, an AMP analogue, nor by the consequence of
altered cellular AMP/ATP ratio. In addition, it was also con-
firmed that both kinases, LKB1 and CaMKK, are required for
full activation of AKN-stimulated phosphorylation of AMPK.
AKN Increases the Cellular Uptake of Glucose in C2C12
and L6 Myocytes. AMPK plays a crucial role as an energy
sensor in metabolic tissues and stimulates glucose uptake in
skeletal muscle.8 To determine the effect of AKN on cellular
glucose uptake, we utilized the fluorescent glucose bioprobe,
Cy3-Glc-R, which enters the cell through competition with
D-glucose via glucose transporters (GLUTs) (Figure S5).21
Compared to glucose uptake assay with radioisotope-labeled
deoxy-D-glucose using scintillation counter, this method
allows the real-time monitoring of cellular glucose uptake
under experimental conditions using fluorescent microscopy
or confocal laser scanning microscopy (CLSM). As shown in
Figure 3A, a 2.3-fold enhancement in cellular glucose uptake
was observed by CLSM in C2C12 myocytes treated with
insulin. Under identical experimental settings, we measured
a 3.2-fold enhancement in cellular glucose uptake upon
treatment with AKN. Enhanced cellular glucose uptake
was also monitored by fluorescent microscopy following
treatment with AKN, insulin, and AICAR (Figure 3B and
Figure S6), and a dose-dependent enhancement of fluores-
cent intensity was observed in cells treated with AKN
(Figure S7). These data were confirmed by the conventional
glucose uptake assay with 2-deoxy-D-glucose-1,2-3H(N) using
scintillation counter (Figure 3C).
Insulin-dependent PI3K/Akt signaling and insulin-inde-
pendent AMPK signaling are two of the pathways that
regulate cellular glucose uptake.25 To understand the cellular
mechanisms underlying this process, we investigated the
involvement of both pathways in AKN-enhanced glucose
uptake by inhibiting either the AMPK pathway or the PI3K/
Akt pathway with compound C and wortmannin, respec-
tively. Cellular glucose uptake was stimulated by AKN,
AICAR, and insulin in C2C12 myocytes. The inhibition of
AMPK with compound C resulted in a complete inhibition
of enhanced glucose uptake in cells treated with AKN and
AICAR (Figure 3D). However, the enhanced glucose uptake
induced by insulin was not affected by the inhibition of

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7409
Figure 4. AKN activates the AMPK signaling pathway and enhances glucose homeostasis in vivo. (A) The effect of AKN on the
phosphorylation of AMPK and ACC in vivo. Vehicle (PEG400, n = 7) or 10 (mg/kg)/d AKN (n = 7) were administered subcutaneously
to DIO mice. After administration of AKN for 1 month, immunoblot analysis of tissue lysates from liver and muscles was performed with
antibodies against the indicated proteins after fasting for 24 h. (B) Serum insulin and (C) blood glucose levels were determined using the
ALPCO insulin EIA kit and a glucometer (Accu Check, Roche), respectively. (D) The intraperitoneal glucose tolerance test (IPGTT) and (E)
insulin tolerance test (ITT) were performed in DIO mice treated with vehicle (0, n = 7) or 10 (mg/kg)/d AKN (9, n = 7) for 3 weeks and
4 weeks, respectively: (/) P < 0.05; (//) P < 0.01 vs the corresponding vehicle control group.
AMPK. In contrast, the glucose uptake pattern in insulin-
treated cells was reversed only after pretreatment with
wortmannin (Figure 3E). Consistent with this data, immu-
noblot analysis revealed that Akt phosphorylation was
not increased above basal levels by treatment with AKN
(Figure S8). This demonstrates that AKN-stimulated glucose
uptake results primarily from increased AMPK activation
and not via PI3K/Akt signaling.
food intake was observed between the groups (Figure 5A).
Interestingly, by week 3, the body weights of AKN-treated
DIO mice were significantly decreased, resulting in a ∼5%
decrease compared to the initial weight. Conversely, the
weights of vehicle-treated DIO mice increased slightly over
the study period (Figure 5B). The changes in body weight
were the result of changes in fat mass, which were signifi-
cantly decreased in AKN-treated mice compared to vehicle-
treated mice (Figure 5C). In agreement with this observation,
Oil-red O staining of liver and H & E staining of gonadal fat
revealed dramatic differences between AKN- and vehicle-
treated mice in terms of fat mass and fat area, respectively
(Figure 5D). Finally, although triglyceride levels in serum
were not affected upon treatment with either AKN or vehicle
(Figure 5F), other lipid parameters such as cholesterol and
nonesterified fatty acids (NEFA) were significantly decreased
in AKN-treated DIO mice compared to control groups
(Figure 5E and 5G). These data indicate that AKN also
has antiobesity effects.
AKN Enhances Glucose Dispersal and Insulin Sensitivity
via Activation of AMPK in Vivo. We next examined the in
vivo effect of AKN in DIO mice fed a diet containing 60% fat
for 8 weeks after microsomal stability test of this compound
(Figure S9). DIO mice were injected subcutaneously for 4 weeks
with 10 (mg/kg)/d of AKN in PEG400. Phosphorylation of
AMPK was confirmed by immunoblot analysis of proteins
from the liver and muscle of DIO mice treated with vehicle or
AKN. Consistent with our in vitro data, AKN-treated DIO
mice had increased levels of phosphorylated AMPK/ACC,
suggesting that AKN is an effective activator of AMPK in liver
and muscle (Figure 4A). Interestingly, we observed that serum
insulin and glucose levels in AKN-treated DIO mice were lower
than those in the control groups (Figure 4B and Figure 4C).
Therefore, we investigated the glucose dispersal rates in both
groups. As shown in Figure 4D, elevated blood glucose induced
by intraperitoneal injection was more rapidly lowered in AKN-
treated DIO mice than control mice. These findings indicate that
AKN effectively enhanced insulin sensitivity in animal models
(Figure 4E). Taken together, these data support that AKN is a
novel small-molecule AMPK activator with antidiabetic effects.
AKN Accelerates the Consumption of Accumulated Fat in
DIO and Reduces Total Body Weight. We next sought to
investigate the potential antiobesity effects of AKN. AKN
(10 (mg/kg)/d) was administered to DIO mice for 30 days via
subcutaneous injection, and food intake and body weight
were monitored every 3 days. No significant difference in
Conclusion
In this paper, we have described ampkinone, a novel small-
molecule activator of AMPK with potential antidiabetic and
antiobesity effects. Through extensive immunoblotting assays
using a 2500-member small molecule collection constructed
by diversity-oriented synthesis strategy,20 we identified a novel
molecular framework (6) with a privileged benzopyranyl sub-
structure. We then conducted a structure-activity relationship
(SAR) study using a 15-member focused library with a tetracyclic
skeleton (6) and identified 6f as the best candidate for further in
vitro and in vivo evaluation. This compound, which we desig-
natedampkinone (AKN), is an effective small-molecule activator
of AMPK. We confirmed the in vitro effects of AKN on the func-
tional activation of AMPK through active phosphorylation of

7410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Oh et al.
Figure 5. AKN treatment ameliorates metabolic symptoms in DIO mice. (A) Food intake and (B) the body weight of DIO mice groups, either
vehicle-treated (0, n= 7) or AKN-treated (9, n= 7), were monitored during the subcutaneous administration of 10 (mg/kg)/d AKN for 1 month. (C)
The weights of adipose tissues (GF, gonadal fat; MF, mesenteric fat; PF, perirenal fat) and other organs were compared between DIO mice treated with
vehicle (0, n = 7) and those treated with 10 (mg/kg)/d AKN (9, n = 7) for 1 month. (D) Oil-red O staining of liver cells from DIO mice treated
with vehicle (upper, left) or 10 (mg/kg)/d AKN (upper, right) and H & E staining of gonadal fat from DIO mice treated with vehicle (lower, left) or
10 (mg/kg)/d AKN (lower, right). (E-G) Lipid parameters of serum from DIO mice after subcutaneous administration of vehicle (0, n = 7) or
10 (mg/kg)/d AKN (9, n = 7) for 1 month: (/) P < 0.05; (//) P < 0.01 vs the corresponding vehicle control group.
ACC, its specific substrate, in multiple cell lines derived from
metabolic tissues.
strong activation of AMPK for 6 h, which was confirmed by
cell viability assays (Figures S10 and S11). Furthermore,
OXPHOS activity in L6 myocytes was not affected by treat-
ment with AKN, indicating that AKN-mediated AMPK
activation did not result from inhibition of the respiratory
chain.28 Therefore, AKN could be ideal for investigating the
physiological effects of AMPK up-regulation without causing
oxidative stress.
AMPK is an important energy sensor in mammalian cells.
Its activity is induced in response to high levels of AMP and/
or low ATP. Under these conditions, AMP binds to the
γ-subunit of AMPK and induces a conformational change
that allows Thr172 of the R-subunit to be phosphorylated by
AMPK kinases.8 AMPK is activated by upstream kinases,
including LKB19 and CaMKK.10 We determined that both
kinases, LKB1 and CaMKK, are required for full activation
of AKN-stimulated phosphorylation of AMPK. On the basis
of our findings in LKB1- and mock-transfected HeLa cells,
AKN-mediated AMPKactivation was inducedmainly via the
up-regulation of LKB1. Although AKN-mediated AMPK
phosphorylation is primarily mediated by LKB1, the activa-
tion of AMPK is also slightly stimulated by CaMKK upon
treatment of AKN evidenced by immunoblot analysis with
specific inhibitors. Both LKB1 and CaMKK function as
AMPK kinases in different cell lines and tissues, suggesting
that they play different roles in the regulation of AMPK.
However, the molecular mechanism of AKN-mediated
AMPK activation remains to be elucidated.
It has been well established that activation of AMPK
stimulates glucose uptake byincreasing GLUT4translocation
to the cell surface.26 Interestingly, AMPK-mediated glucose
uptake occurs through a different mechanism than in the
insulin-signaling pathway.27 As shown in Figure 3, AKN
strongly increased glucose uptake in C2C12 myocytes. This
effect was completely eliminated by the inhibition of AMPK
with compound C but not by the inhibition of PI3K via
wortmannin. In addition, AKN treatment did not induce
phosphorylation of Akt in L6 cells. These data suggest that
AKN-mediated glucose uptake is dependent on AMPK, not
Akt. Inaddition, our results demonstrated thatAKN does not
induce cell cycle arrest or associated cell apoptosis even after
Abnormal AMPK activity has been implicated in the
progression of obesity and diabetes.8 Several reports suggest
that activation of AMPK enhances insulin sensitivity and
increases glucose cellular uptake in vitro and in vivo.4,5,7 In
this study, activation of AMPK by AKN enhanced insulin
sensitivity and induced glucose uptake in various cultured cell
lines and DIO mice, resulting in a reduction in serum insulin
and glucoselevels(Figure 4). Moreover, the activityofACC, a
key enzyme in lipid accumulation and synthesis, is negatively
regulated by activation of AMPK. Similar to other AMPK
stimulators,16,29 AKN treatment resulted in a mild reduc-
tion in body weight and a concomitant reduction in adipose
tissue weight. Furthermore, hepatic steatosis can result from
abnormal activity of 3-hydroxy-3-methylglutaryl coenzyme A
reductase (HMG-CoA reductase) and ACC. These two en-
zymes are regulated by AMPK and primarily responsible for
hepatic cholesterol and fatty acid synthesis.30 Our data sug-
gested that AKN-mediated AMPK activation in DIO mice
leads to a reduction in hepatic steatosis and decreased choles-
terol and free fatty acids in the serum. However, we need to
identify the real target protein regulating AKN-mediated
AMPK activation in order to understand the exact regulating
mechanism of these antidiabetic and antiobesity effects found
in vitro and in vivo. Overall, our results demonstrate that the
small-molecule AKN indirectly stimulates the activation of
AMPK and has a potential as a therapeutic agent for the
treatment of diabetes and obesity.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7411
Experimental Section
150 mM NaCl) containing 0.1% Tween 20 and 5% nonfat dry
milk and incubated with primary antibodies diluted in blocking
buffer overnight at 4 ꢀC. The membranes were washed and
incubated with the appropriate secondary antibodies for 1 h
at room temperature. HRP-conjugated secondary antibodies were
detected using ECL detection reagents (Amersham Biosciences,
Buckinghamshire, U.K.).
Chemical Synthesis. All compounds were synthesized from
commercially available starting materials, 2-hydroxyacetophe-
none derivatives, and the purity of each compound was over
95%. See Supporting Information for details.
Compound 6f, Ampkinone. Yellow solid (62% from com-
pound 5f). 1H NMR (500 MHz, CDCl3): δ 8.04 (d, J = 8.0 Hz,
1H), 7.96(d, J= 8.5 Hz, 2H), 7.93 (d, J= 8.0 Hz, 1H), 7.85 (d, J =
7.5 Hz, 2H), 7.64-7.60 (m, 3H), 7.51 (t, J = 7.5 Hz, 2H), 7.20 (d,
J = 8.5 Hz, 1H), 6.67 (d, J = 8.5 Hz, 1H), 5.52 (s, 1H), 3.96 (s, 3H),
2.00 (s, 6H). 13C NMR (125 MHz, CDCl3): δ 195.9, 166.8, 166.4,
149.4, 140.6, 140.1, 137.6, 137.2, 137.0, 135.6, 135.1, 132.9, 131.4,
131.1, 130.3, 128.6, 128.0, 127.1, 126.4, 123.7, 115.1, 114.8, 105.8,
80.3, 56.5, 27.3. HRMS (FAB) m/z calculated for C31H24NO6
[M þ H]þ: 506.1603. Found: 506.1604.
AMPK Activity Assay. Total AMPK activity was measured
using a synthetic SAMS peptide substrate and [γ-32P]ATP, as
described previously.31 Briefly, 500 μg of protein extract was
incubated with anti-AMPK-R1 and R2 antibodies for 2 h at 4 ꢀC.
Protein A/G sepharose and agarose were added, and the mix-
tures were incubated for 3 h at 4 ꢀC. After the samples were
washed three times with RIPA buffer, the activity was assessed
in AMPK reaction buffer containing 20 mM HEPES-NaOH
(pH 7.0), 0.4 mM dithiothreitol, and 0.01% Brij-35, with or
without 300 μM AMP. The immune complexes were added to
23 μL of reaction buffer per assay, and then 7 μL of SAMS
substrate peptide (HMRSAMSGLHLVKRR, 100 μM final
concentration) and 10 μL of ATP mixture (an aliquot of
1 μCi/μL: 90 μL of 75 mM magnesium chloride, 500 μM
unlabeled ATP in 20 mM MOPS, pH 7.2, 25 mM β-glycerophos-
phate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM
dithiothreitol) were added, and the mixture was incubated at
30 ꢀC for 15 min. After 35 μL was spotted onto the center of
P81 paper, the paper was washed three times with 0.75%
phosphoric acid and once with acetone for 5 min. The samples
were read using a scintillation counter as described previously.32
Glucose Uptake Assay with 2-Deoxy-D-glucose-1,2-3H(N).
L6 myocytes were cultured in 12-well plates and incubated in
low glucose medium for 16 h. The cells were washed in Kreb’s
Ringer phosphate (KRPH) buffer (136 mM NaCl, 20 mM
HEPES, pH 7.4, 5 mM sodium phosphate, pH 7.4, 4.7 mM
KCl, 1 mM MgSO4, and 1 mM CaCl2) and then treated with the
appropriate drugs in KRPH buffer for 1 or 3 h. Next, the cells
were incubated with 0.5 μCi 2-deoxy-D-glucose-1,2-3H(N) for
10 min. After being washed three times with ice-cold phosphate
buffered saline (PBS), the cells were lysed in 500 μL of 0.2 N
NaOH and 2-deoxy-D-glucose-1,2-3H(N) and counted in 5 mL
of Ready Safe liquid scintillation cocktail (Beckman Coulter,
USA) using a Packard 1900TR scintillation counter. When
inhibitors were used, the cells were pretreated with the inhibitors
for 1 h before application of the drugs.
Animal Models. All animals were maintained and used in
accordance with the guidelines of the Institutional Animal Care
and Use Committee of the Chungnam National University
School of Medicine. C57BL/6J mice were obtained from the
Jackson Laboratory and housed individually in a room main-
tained at 25 ꢀC on a 12/12 h light/dark schedule. For diet-
induced obesity (DIO) mice, 4-week-old male C57BL/6J mice
were fed a high-fat diet (HFD, Research Diets, 60% calories
from fat) ad libitum for 8 weeks. Ampkinone in polyethylene
glycol (PEG400) or vehicle was administered subcutaneously at
10 mg/kg body weight per day for 1 month. Body weight and
food intake were measured every 3 days. All animals were given
an insulin tolerance test (ITT) and sacrificed at 30 days. Liver
weight and fat masses were measured, sectioned, and stained
with Oil-red O and H&E.
Antibodies and Reagents. Antibodies against total AMPK,
phospho-AMPK (Thr172), total ACC, phospho-ACC (Ser79), total
Akt, and phospho-Akt (Ser473) were purchased from Cell Signaling
Technology (Beverly, MA). Antibodies against AMPK-R1 and
AMPK-R2 were obtained from Upstate Biotechnology (Lake
Placid, NY). The AMPK activator, AICAR, and the AMPK
inhibitor, compound C, were purchased from Merck Research
Laboratories (NJ). 2-Deoxy-D-glucose-1,2-3H(N) (25-50 Ci/mmol)
and adenosine 50-triphosphate, [γ-32P] (3000 Ci/mmol) were pur-
chased from PerkinElmer Inc. (Shelton, CT), and STO-609 was
purchased from Calbiochem (La Jolla, CA). All cell culture media
and supplements were obtained from Gibco-BRL Life Technologies
(Gaithersburg, MD). All other reagents were purchased from Sigma
Chemical Co. (St. Louis, MO).
Cell Culture. L6 muscle cells and all cell lines were cultured in
Dulbecco’s modified Eagle medium (DMEM) containing 10%
fetal bovine serum (FBS) and antibiotic solution (100 units/mL
penicillin, 100 g/mL streptomycin) at 37 ꢀC in an atmosphere of
5% CO2 and 95% air. Plasmids containing the recombinant
HA-LKB1 gene were transfected using Lipofectamin and Lipo-
fectamin Plus (Invitrogen) according to the manufacturer’s
instructions. Cells were grown to 70% confluence and deprived
of serum for 6 h prior to treatments. Differentiation of L6 and
C2C12 myocytes was induced by DMEM supplemented with
2% horse serum, and the medium was replaced every 2 days.
Immunoblot Analysis. Cells were serum-starved for 6 h before
drug treatments and exposed to drugs for the times and concentra-
tions indicated. To prepare total proteins, cells were extracted in
RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM
EDTA, 5 mM EGTA, 5 mM sodium fluoride, 2 mM sodium
orthovanadate, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS],
1 mM phenylmethylsulfonyl fluoride [PMSF], and protein inhibitor
cocktail [Roche Diagnostics, Heidelberg, Germany]). The protein
concentration of the lysate was determined using the Bio-Rad dye-
binding microassay. For immunoblotting, 40 μg of cell lysate was
separated by SDS-polyacrylamide gel electrophoresis (PAGE).
Proteins were transferred onto Hybond-ECL nitrocellulose mem-
branes (Amersham Biosciences, Buckinghamshire, U.K.). The
membranes were blocked with TBS (10 mM Tris-HCl, pH 7.4,
Glucose Uptake Assay with Fluorescent Glucose Bioprobe
(Cy3-Glc-r). C2C12 myocytes were cultured on a microscope
cover glass in a 35 mm cell culture dish. After 2 days, the cell
culture medium was removed and changed to low glucose
medium (without FBS) for 4 h. The cells were incubated in
glucose-depleted media (DMEM, no glucose, GIBCO) with
100 nM insulin, 10 μM AKN, and 1 mM AICAR for 1 h. Next,
the medium was replaced with Cy3-Glc-R (10 μM in glucose-
depleted DMEM) for 30 min. After the sample was washed three
times with ice-cold phosphate buffered saline (PBS), the cover-
slip was loaded on the caster of a fluorescence microscope
(Olympus IX71). During microscopic observation, the cells were
kept in a chamber maintained at 37 ꢀC. To monitor glucose
uptake with a confocal laser scanning microscope (CLSM, Carl
Zeiss-LSM510), the cover glass was loaded on the caster of the
CLSM after pretreatment with the appropriate drugs for 1 h.
After injection of 5 μM of Cy3-Glc-R in glucose-depleted
DMEM into the chamber, a fluorescent image was taken every
7 s, digitized, and saved on the computer for analysis. The
temperature of the chamber was maintained at 37 ꢀC.
Analysis of AMP and ATP Concentration. 1ꢀ104 cells were
seeded in 96-well plate and attached for 4 h. Cells were treated
with AKN for 1 h at the indicated concentrations. ATP mea-
surement was performed by standard protocol offered by
ATPlite 1step kit (Perkin-Elmer). For the quantitative analysis
of cellular AMP to ATP ratio, 1ꢀ106 cells were extracted in
800 μL of the extraction buffer (80% methanol) with sonication.

7412 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Oh et al.
Then the extracts were centrifuged at 13 000 rpm for 20 min at
4 ꢀC. The supernatants were stored at a -80 ꢀC deep freezer until
quantitative analysis was performed with HPLC-MS/MS.
Electrospray ionization mass spectrometry was operated in
positive ion mode using MDS Sciex API4000 triple quadrupole
mass spectrometer (Applied Biosystems, Ontario, CA) followed
by chromatographic separation with Agilent 1100 series HPLC
system (Agilent Technologies, Palo Alto, CA) and XTerra MS
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C
18 2.1 ꢀ 150 mm, 3.5 μm column (Waters, Milford, MA, USA).
Microsomal Stability Assay. AKN was incubated with liver
microsomes (human, dog, rat, and mouse) in potassium phos-
phate buffer. The microsomal protein concentration in the assay
is 1 mg/mL, and the final percent of DMSO is 0.2%. Micro-
somal activity was initiated by the addition of NADPH and
stopped either immediately or after 15 min for initial screening
or at 15, 30, and 60 min for a more precise estimate of clearance.
The corresponding loss of parent compound was determined by
LC/MS. The % remaining of compound was calculated in
comparison with the initial quantity at time zero. Half-life time
was then calculated on the basis of first-order reaction kinetics
of the % remaining of compound.
Glucose Tolerance and Insulin Stimulation Tests. After
3 weeks, glucose tolerance tests were performed in DIO mice
fasted for 16 h as described previously.33 At week 4, the mice
were fasted for 4 h and ITTs were performed as described
previously.34 The animals were injected intraperitoneally with
glucose (1 g/kg body weight) or human insulin (1 international
unit (IU)/kg body weight), and blood glucose levels were
measured.
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Serum Analysis. Insulin levels in the serum of mice treated
with vehicle or AKN were measured using a mouse insulin
ELISA kit (ALPCO Diagnostics) according to the manufac-
turer’s instructions. Total cholesterol, triglycerides (TG), and
nonesterified free fatty acids (NEFA) in aliquoted plasma
samples were determined using an automated blood chemistry
analyzer (Hitachi 7150; Tokyo, Japan).
Statistical Analysis. Results were expressed as the mean (
standard deviation. Differences between groups were analyzed
for statistical significance using Student’s t test and analysis of
variance (ANOVA). P < 0.05 was considered significant.
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Acknowledgment. This study was supported by (1) the
National Research Foundation of Korea (NRF); (2) the
WCU program through the NRF funded by the Korean
Ministry of Education, Science, and Technology (MEST);
and (3) the MarineBio Technology Program funded by the
Ministry of Land, Transport and Maritime Affairs (MLTM),
Korea. S.O., S.J.K., H.Y.L., and J.P. are grateful for the
fellowship awarded by the BK21 program, MEST, Korea. H.
Y.L. and J.P. are also grateful for the predoctoral fellowship
awarded of the Seoul Science Fellowship.
€
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Supporting Information Available: Experimental procedures,
spectroscopic characterization data of all new compounds, and
results of biological studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
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