A potent and selective AMPK activator that inhibits de novo lipogenesis

Article abstract of DOI:10.1021/ml100143q

AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that regulates cellular energy metabolism by affecting energy-consuming pathways such as de novo lipid biosynthesis and glucose production as well as energy-producing pathways such as lipid ox

Full text of DOI:10.1021/ml100143q

pubs.acs.org/acsmedchemlett
A Potent and Selective AMPK Activator That Inhibits de
Novo Lipogenesis
,†
‡
§
ꢀ
Jorge E. Gomez-Galeno,* Qun Dang, Thanh H. Nguyen, Serge H. Boyer, Matthew P. Grote,
Zhili Sun, Mingwei Chen, William A. Craigo, Paul D. van Poelje,^ Deidre A. MacKenna,#
Edward E. Cable,r Paul A. Rolzin,O Patricia D. Finn, Bert Chi, David L. Linemeyer,þ
Scott J. Hecker,§ and Mark D. Erion‡
Metabasis Therapeutics, Inc., La Jolla, California 92037
ABSTRACT AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that
regulates cellular energy metabolism by affecting energy-consuming pathways
such as de novo lipid biosynthesis and glucose production as well as energy-
producing pathways such as lipid oxidation and glucose uptake. Accordingly,
compounds that activate AMPK represent potential drug candidates for the
treatment of hyperlipidemia and type 2 diabetes. Screening of a proprietary library
of AMP mimetics identified the phosphonic acid 2 that bears little structural
resemblance to AMP but is capable of activating AMPK with high potency
(EC50 = 6 nM vs AMP EC50 = 6 μM) and specificity. Phosphonate prodrugs of 2
inhibited de novo lipogenesis in cellular and animal models of hyperlipidemia.
KEYWORDS 5-(5-Hydroxy-isoxazol-3-yl)-furan-2-phosphonic acid, AMPK activator,
lipogenesis, hepatocytes
iseases associated with obesity, such as type 2 dia-
betes, hypertension, and dyslipidemia, are growing
worldwide health concerns. These conditions are
5-Aminoimidazole-4-carboxamide riboside (AICAR) is a
nucleoside that undergoes intracellular phosphorylation to
the corresponding 50-mono-, di-, and triphosphate (Chart 1).
AICAR-monophosphate (ZMP) is an AMP mimetic that binds
to AMP binding sites on various intracellular proteins8,9 and
induces the associated functional response such as inhibi-
tion of fructose-1,6-bisphosphatase and the activation of
glycogen phosphorylase (GPPase).10 While ZMP is a mod-
estly potent and poorly selective activator of AMPK, it has
been used in hundreds of studies attempting to elucidate the
role of AMPK in energy homeostasis.11 -16 In addition, it has
been suggested that AMPK activation may contribute to the
glucose-lowering activity of marketed diabetes drugs
metformin17 and the thiazolidinediones.18,19 These fin-
dings as well as the report of a specific AMPK activator20,21
(A-769662, 1) suggest that a small molecule activator of
AMPK may be a viable approach to treat diseases associated
with dysregulation of energy homeostasis.
Herein, we disclose the discovery of compound 2, a potent
and selective activator of human AMPK. We also report the
synthesis of esterase-sensitive phosphonate prodrugs of 2
and demonstrate the abilityof these compounds to inhibitde
novo lipogenesis in rat hepatocytes and in rodents.
Compound 2 was identified as a potent activator of AMPK
after screening a proprietary library containing ca. 1200
AMP mimetics. Its synthesis is depicted in Scheme 1.
D
frequently associated with impaired regulation of glucose
and lipid metabolism, leading to increased cardiovascular
risks. Current therapies for the treatment of dyslipidemia
and type 2 diabetes are only moderately effective, and even
in combination, many patients still do not meet the recom-
mended guidelines.1 Therefore, new pharmacological ap-
proaches that can address one or multiple components
related to these diseases are under investigation. One ap-
proach that has recently elicited considerable interest is the
activation of 50-AMP-activated protein kinase (AMPK).2-5
AMPK is a heterotrimeric kinase expressed in a variety of
tissues, particularly the liver, brain, and skeletal muscle. It
has been dubbed a “metabolic master switch”6 that puta-
tively regulates fatty acid synthesis, sterol synthesis, and
glucose production.7 AMPK acts as an energy sensor that is
activated by AMP under conditions where the intracellular
ratio of AMP to ATP is increased, such as hypoxia, exercise, or
glucose deprivation. As a result of this activation, processes
where ATP is consumed (e.g., fatty acid synthesis, gluconeo-
genesis, and cholesterol synthesis) are inhibited through
targets downstream of AMPK, while pathways where ATP
is generated (e.g, fatty acid oxidation, ketogenesis, and
glycolysis) are stimulated, resulting in restoration of energy
homeostasis. Because of its important role in the regulation
of lipid and carbohydrate metabolism, activators of AMPK
could potentially provide a multifaceted approach to the
treatment of metabolic diseases.
Received Date: June 18, 2010
Accepted Date: August 23, 2010
Published on Web Date: August 30, 2010
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Chart 1
Scheme 2
dichloromethane (Scheme 2). However, attempts to remove
the ethyl groups in phosphonate 8 under a variety of reaction
conditions failed to yield phosphonic acid 2, generating
instead a complex mixture of products.
While the diethyl phosphonate 8 was not an AMPK
activator, it was a useful tool compound that allowed us to
evaluate some of the properties of the 5-hydroxyisoxazole
portion of the molecule. The hydroxyisoxazole ring of 8
exists in the keto form in CDCl3, as determined by proton
NMR, which shows a 2H signal at 3.81 ppm corresponding to
the methylene group (C-4) of the isoxazolone ring. However,
in DMSO-d6, also 8 exists as the enol form, as indicated by
the presence of two 1H signals at 5.60 and 4.20 ppm.
Compound 9, the 13C-labeled version of 8, was similarly
generated by using 5-13C-Meldrum's acid. Consistent with
Scheme 1a
1
the results observed for compound 8, the H NMR for 9 in
CDCl3 showed a 2H signal (d, J = 138 Hz) at 3.84 ppm. The
13CNMR spectrum of 9 in the same solvent showed a signal at
33.85 ppm. When 9 was dissolved in DMSO-d6, it showed two
signals at 77.27 and 34.25 ppm, indicating the presence of
both the enol and the keto form, respectively, in this solvent.
The addition of triethylamine to this solution caused the
complete disappearance of these two signals and the appear-
ance of a single peak at 67.11 ppm, suggesting that conversion
to the triethylammonium enolate of the hydroxyisoxazole ring
in DMSO-d6 had occurred. In an aqueous solution of sodium
hydroxide (pH > 10), the 13C NMR of 9 showed a single peak
at 71.37 ppm, again indicating the presence of the enolate
form of 9 in this highly basic medium. The 13C NMR spectrum
of 9 in pH 7.4 phosphate buffer also shows a single peak at
71.37 ppm, indicating that it exists almost exclusively as the
enolate under these conditions. This keto-enol tautomeriza-
tion is evidence of the relatively high acidity of the 5-hydro-
xyisoxazole group. Indeed, potentiometric titration of 8 in
mixtures of methanol/water provided pKa values of 3.23.
Similarly, potentiometric titration of 2 in the same solvent
revealed pKa values of 3.32, 3.93, and 6.94. Consistent with
these findings, the log D7.4 for 2 was found to be -4.3.
The highly anionic nature of 2 was an impediment for its
transport across cell membranes and likely was responsible
for its lack of effect in cell-based and in vivo models; thus,
prodrug strategies were explored27 to improve cell penetra-
tion for compound 2.
a Reagents and conditions: (a) TMSBr, CH3CN, room temperature.
(b) PhCH2OC(dN(cyclohexyl))NH(cyclohexyl), DMF, toluene, 100 ꢀC.
(c) H2NOH HCl, NaOAc, THF, room temperature. (d) NCS, DMF, room
3
temperature. (e) PhCH2OCCH, Et3N, CH2Cl2, room temperature. (f) H2,
Pd(OH)2 on C, N,N-dicyclohexylamine, EtOH.
The diethyl aldehyde 38 was deprotected using bromotri-
methylsilane in acetonitrileat room temperature. The resulting
phosphonic acid 4 was reprotected as the dibenzyl phospho-
nate using the DCC adduct of benzyl alcohol in DMF/toluene.22
The crude aldehyde was converted to a mixture of isomeric
oximes (cis/trans, 34% yield over two steps) by treatment with
hydroxylamine hydrochloride. Chlorination of the inter-
mediate oximes with N-chlorosuccinimide (NCS) in DMF
yielded chlorooxime 5 (88%). [3 þ 2] Cycloaddition of 5
with benzyloxyacetylene23 regioselectively produced isoxazole
6. Hydrogenation of 6 using palladium hydroxyde on carbon
in ethanol in the presence of N,N-dicyclohexylamine gave
compound 2.
For the synthesis of esterase-sensitive prodrugs of 2, the
formal [3 þ 2] cycloaddition reaction using Meldrum's acid
was utilized. Scheme 3 illustrates the application of this
method to the synthesis of compound 12, a bis-pivaloyl-
oxymethyl (bis-POM) prodrug of 2. Alkylation of 4 with the
Alternatively, chlorooxime 7 (prepared from 3 using the
methods described above) was used to generate the diethyl-
phosphonate analogue 8 by reaction with Meldrum's acid in
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Scheme 3a
a Reagents and conditions: (a) Iodomethyl pivalate, N,N-diisopropyl ethylamine, acetonitrile, 23 ꢀC. (b) Hydroxylamine hydrochloride, THF, sodium
acetate, 23 ꢀC. (c) NCS, DMF, 23 ꢀC. (d) Meldrum's acid, Et3N, CH2Cl2, 23 ꢀC.
Table 1. In Vitro and in Vivo Inhibition of de Novo Lipogenesis
(DNL) by Phosphonate Prodrugs of 1
in vivo DNL
compound no.
R
EC50 (nM)a inhibitionb (%)
2
N/A
>10000
100 (r)
NDc
65
12
t-butyl
13 (m)
20 (r)
30 (r)
13
14
15
iso-propyl
cyclopentyl
O-ethyl
78
ND
73
42 (r)
17 (m)
Figure 1. Activation of human AMPK by AMP and 2. Activation
was determined by monitoring the phosphorylation of the SAMS
peptide by AMPK.
16
17
O-benzyl
609 (r)
34
73
O-iso-propyl
27 (r)
8 (m)
18
O-C(CH3)2 CH2CH3 390 (r)
ND
of rat AMPK, reaching a maximum of 51% of the activation
observed with AMP. In selectivity assessments, screening of
2 against a panel of 64 targets (MDS Pharma Services)
indicated no significant interactions with these targets at a
test concentration of 10 μM.
a EC50 for the inhibition of de novo lipogenesis in rat (r) or mouse (m)
hepatocytes. b After intraperitoneal administration of the compound at a
30 mg/kg dose to C57Bl/6 mice. c ND, not determined.
iodomethyl ester of pivalic acid in the presence of Hunig's
base generated 10. Chlorooxime 11 was generated from 10
in two steps: oxime formation by reaction with 1.05 equiv of
hydroxylamine hydrochloride and sodium acetate, followed
by conversion to 11 by reaction with NCS in DMF. Cycloaddi-
tion with Meldrum's acid gave the desired prodrug 12. This
method was used for the synthesis of additional prodrugs
(compounds 13-18, Table 1) where the phosphonic acid
was functionalized with other esterase sensitive groups.
Compound 2 was identified by screening a library
of proprietary AMP mimetics8 against both human and
rat AMPK by monitoring the phosphorylation of the
SAMS peptide, an extensively utilized substrate for AMPK.24
Compound 2 was found to be a full activator of human
AMPK, with an EC50 of 6.3 nM (Figure 1), which is >900-fold
AMPallostericallymodulates other key enzymes:It activates
GPPase (EC50 = 1.4 μM), while it inhibits FBPase (IC50
=
1.0 μM). ZMP is an AMP mimic that activates rat liver AMPK
with an EC50 = 84 μM. However, it is not specific toward
AMPK. For example, it activates GPPase with an EC50
=
11 5 μM and inhibits FBPase with an IC50 = 9.4 μM. In contrast,
2 had no activity against GPPase and FBPase at concentrations
of 100 μM, about 1000-fold its EC50 for activation of AMPK.
These results suggest that, unlike ZMP, 2 is a selective AMPK
activator.
Compounds 8 and 12 were not activators of the isolated
enzyme, suggesting that the phosphonic acid group was
essential for the in vitro activation of AMPK by 2. Acetyl-CoA
carboxylase (ACC) catalyzes the rate-limiting step in free fatty
acid biosynthesis. Phosphorylation of ACC by AMPK decreases
its activity, resulting in a reduction in the ACC product malonyl-
CoA and, correspondingly, in the inhibition of de novo lipogen-
esis. Because malonyl-CoA is a potent inhibitor of carnitine
more potent than the endogenous activator AMP (EC50
=
5.9 μM). The potency of 2 is similar against rat AMPK
(EC50 = 21 nM), but interestingly, it was a partial activator
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To determine whether the cellular effects of compounds
12-18 would translate to the in vivo setting, de novo
lipogenesis was measured in mice following treatment with
potential AMPK activators. Because preliminary results
showed that oral dosing of prodrugs 12-18 resulted in low
plasma levels of 2 as well as the corresponding prodrugs
(data not shown), the compounds were administered by
intraperitoneal injection. Compounds were dosed (generally
30-100 mg/kg) 1 h prior to intraperitoneal administration of
14C-sodium acetate in saline. One hour later, animals were
anesthetized, and plasma and liver were harvested. Newly
synthesized lipids and sterols were extracted using a mod-
ified Folch method28 and quantified relative to those in
vehicle-treated mice. At a dose of 30 mg/kg, compounds
12-17 achieved plasma concentrations of 200-300 μM and
inhibited de novo lipogenesis by more than 30% (Table 1).
In conclusion, we have described the synthesis and eva-
luation of 2, a furan phosphonic acid derivative containing
an acidic 5-hydroxyisoxazole group. Compound 2 activates
AMPK with an impressive EC50 < 10 nM and is the most
potent direct AMPK activator reported to date.29-31 Esterase-
sensitive prodrugs of 2 enhanced the intracellular delivery of
this highly anionic compound into hepatocytes. These tool
compounds inhibited de novo lipogenesis both in plated rat
hepatocytes and in mice. It should be noted that while 2 is
only a partial activator of rat liver AMPK, it was able to fully
inhibit de novo lipogenesis in rat hepatocytes. Given the high
specificity of compound 2 relative to ZMP, prodrugs of 2, for
example, 12-18, may be useful tools to elucidate the role
that AMPK activation plays in whole body energy homeo-
stasis. Additional studies are ongoing that utilize these and
related compounds in disease-relevant models to under-
stand the therapeutic potential of AMPK activators.
Figure 2. Western blot analysis of the phosphorylation of ACC by
13 or AICAR. Western blot analysis of phosphorylated ACC in cell
lysates from primary rat hepatocytes treated for 4 h with 1%
DMSO (line A), 1000 μM AICAR (line B), or compound 13 at 10, 3,
and 1 μM (lines C, D, and E, respectively).
palmitoyltransferase-1 (CPT-1), activation of AMPKalso results
in inactivation of CPT-1 and in an increase in free fatty acid
oxidation. In addition to its effects on fatty acid production and
metabolism, AMPK activation also results in the phosphoryla-
tion of HMGCoA reductase and, consequently, in the inhibition
of cholesterol biosynthesis.25,26
To evaluate the effect of 2 on de novo lipogenesis, plated
rat hepatocytes were incubated with the test compound and
then coincubated with 14C-sodium acetate as a lipogenic
substrate. De novo lipogenesis was evaluated by measuring
the radiolabeled counts coming from 14C-acetate incorpo-
rated into organic soluble lipids. Compound 2 did not affect
de novo lipogenesis in primary rat hepatocytes (data not
shown). The highly anionic nature of 2 suggested that its lack
of activity may have been due to poor cellular permeability.
Indeed, upon incubation of plated rat hepatocytes with 2, at
a concentration of 100 μM for up to 6 h, the intracellular
levels of 2 were below the limits of detection (55 nmol/g). To
improve cell permeability, the phosphonic acid group was
masked with esterase-sensitive prodrugs.27 Incubation of
plated rat hepatocytes with a 10 or 100 μM solution of 12 led to
254 ( 104 and 2151 ( 712 nmol/g intracellular concentrations
of 2, respectively, after a 2 h incubation period. The levels of 2
were maintained at >180 nmol/g for up to 8 h and detected as
early as 15 min (see the Supporting Information).
Shown in Table 1 are ester and carbonate prodrugs of 2
that inhibited de novo lipogenesis in plated rat hepatocytes
with EC50 values below 1 μM. Given that the prodrug 12 is not
an activator of isolated AMPK (data not shown), these results
are consistent with conversion to 2 by intracellular esterases.
It is clear from the data that relatively unhindered esters and
carbonates had very similar potencies in this assay, reflect-
ing similar rates of intracellular generation of 2 and subse-
quent AMPK activation in plated rat hepatocytes.
As evidence pointing to the activation of AMPK as the
source of the inhibition of the de novo lipogenesis, we
evaluated the ability of compound 13 to induce phosphor-
ylation of ACC. Primary plated rat hepatocytes were incu-
bated for a 4 h period with 13 (1, 3, and 10 μM) or AICAR
(1000 μM) as the positive control. Immunoblot analysis of
the lysate using a specific antibody that recognizes ACC
phosphorylated at serine-79 shows increased phosphoryla-
tion of ACC (Figure 2). There was no change in total ACC
protein levels following treatment with either of the agents
used, reprobing the blot with a total ACC antibody, or in total
protein loaded by staining the blot with Ponceau S (data not
shown). These results are consistent with the activation of
AMPKas the source for the inhibition of the de novo lipogenesis.
SUPPORTING INFORMATION AVAILABLE Synthesis and
characterization of new compounds reported and description of
biological protocols. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should
be addressed. Tel: 858-692-7759. E-mail: jorge.gomezgaleno@
gmail.com.
Present Addresses: † 7770 Regents Road, Suite 113-229, San
‡
Diego, California 92122. Merck Research Laboratories, Rahway,
New Jersey 07065. § Mpex Pharmaceuticals, Inc., San Diego, California
92121. ^ Pfizer, Inc., Groton, Connecticut 06340. # Regulus Therapeu-
tics, San Diego, California 92008. r Ferring Research Institute Inc., San
Diego, California 92121. O Takeda Pharmaceutical, San Diego, Califor-
nia 92121. þ Senomix, Inc., San Diego, California 92121.
ACKNOWLEDGMENT We thank Yaoming Wei, Michael C.
Matelich, Daniel D. Caspi, Jian Li, and Xuehong Song for their
scientific contributions.
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