Discovery of pyridones as oral AMPK direct activators

Article abstract of DOI:10.1021/ml400157g

AMP-activated protein kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme that is activated in shortage of energy and suppressed in its surfeit. AMPK activation stimulates fatty acid oxidation, enhances insulin sensitivity, alleviates hypergl

Full text of DOI:10.1021/ml400157g

Letter
pubs.acs.org/acsmedchemlett
Discovery of Pyridones As Oral AMPK Direct Activators
Olivier Mirguet,*,† Stephane Sautet, Catherine-Anne Clement, Jerome Toum, Frederic Donche,
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́
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̂
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Celine Marques, Emilie Rondet,‡ Mathieu Pizzonero,‡ Benjamin Beaufils, Yann Dudit, Pascal Huet,
Lionel Trottet, Pascal Grondin, Jean-Marie Brusq, Eric Boursier, Yannick Saintillan, and Edwige Nicodeme
́
Centre de Recherches Franco̧ is Hyafil, GlaxoSmithKline R&D, 25 avenue du Quebec, 91140 Villebon-sur-Yvette, France
S
* Supporting Information
ABSTRACT: AMP-activated protein kinase (AMPK) is an
evolutionarily conserved fuel-sensing enzyme that is activated
in shortage of energy and suppressed in its surfeit. AMPK
activation stimulates fatty acid oxidation, enhances insulin
sensitivity, alleviates hyperglycemia and hyperlipidemia, and
inhibits proinflammatory changes. Thus, AMPK is a well-
received therapeutic target for type 2 diabetes and other
metabolic disorders. Here, we will report the discovery of
pyrrolopyridone derivatives as AMPK direct activators. We will
illustrate the synthesis and structure−activity relationships of
the series as well as some pharmacokinetic results. Some
compounds exhibited encouraging oral exposure and were evaluated in a mouse diabetic model. Compound 17 showed oral
activity at 30 mg/kg on blood glucose.
KEYWORDS: AMPK direct activator, pyridone, P-gp substrate, type 2 diabetes
lipogenic transcription factor, has been found following AMPK
stimulation.
AMPK, a well-known heterotrimeric serine/threonine-protein
kinase, is a key player in regulating energy metabolism. Because
of the different existing subunit isoforms, 12 different AMPK
complexes can theoretically exist. AMPK has been established
as a sensor and regulator of cellular energy homeostasis.1,2
Allosteric activation of this kinase due to rising AMP levels
occurs in states of cellular energy depletion. The resulting
serine/threonine phosphorylation of target enzymes leads to an
adaptation of cellular metabolism to the low energy state. The
net effect of AMPK activation induced changes is inhibition of
ATP consuming processes and activation of ATP generating
pathways and therefore regeneration of ATP stores. Examples
of AMPK substrates include acetyl-CoA-carboxylase (ACC)
and HMG-CoA-reductase.3 Phosphorylation and therefore
inhibition of ACC leads to a decrease in fatty acid synthesis
(ATP-consuming) and at the same time to an increase in fatty
acid oxidation (ATP-generating). Phosphorylation and result-
ing inhibition of HMG-CoA reductase leads to a decrease in
cholesterol synthesis. Other substrates of AMPK include
hormone sensitive lipase,4 glycerol-3-phosphate acyltransfer-
ase,5 and malonyl-CoA decarboxylase,6 some of which are
potential drug targets for components of metabolic syndrome.
Additional processes that are believed to be regulated through
AMPK activation, but for which the exact AMPK substrates
have not been identified, include stimulation of glucose
transport in skeletal muscle and expressional regulation of
key genes in fatty acid and glucose metabolism in liver.1,2,7 For
example, decreased expression of glucose-6-phosphatase,8 a key
enzyme in hepatic glucose production, and SREBP-1c,9 a key
The activation of AMPK is deemed to be an attractive
pharmacological approach for the treatment of type 2 diabetes
and other metabolic disorders. More recently, AMPK has also
been suggested as a potential target for pharmacological
intervention in cancer.10 A plethora of small molecules have
been reported to activate AMPK, but most of them in an
indirect manner. Only a few were described to directly activate
AMPK by allosteric activation of its catalytic activity but also by
protection against AMPK-Thr172 dephosphorylation11,12 in-
cluding Metabasis AMP mimetic compound 213 and Abbott
compound A769662 1.14 Unfortunately, A769662 1 was found
to activate only AMPK β1-containing heterotrimers,15 and
neither compound 2 or A769662 1 are useful as oral drug to
explore the effects of AMPK activation utility in long-term
treatments.
Our early discovery program led us to investigate the
pyridone core starting from A769662, 1. We focused our efforts
on replacing the thiophene ring of the Abbott compound 1 by
5-memberred ring heterocycles in order to improve oral
absorption and selectivity profile over the AMPK heterotrimers.
Our initial efforts allowed us to identify the pyrrolopyridone
2 as a new scaffold for AMPK direct activation (Chart 1).16
Shown in Scheme 1 is the general synthetic route for
preparing the pyrrolopyridones discussed in this letter.17 3-
Received: April 24, 2013
Accepted: May 17, 2013
Published: May 17, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
chemistry efforts from 2 was to introduce oral bioavailability by
reducing blood clearance and improve absorption for this series
of compounds. We first tried to replace the phenol in R2 with a
methoxy group to try to reduce clearance; potency dropped
dramatically whatever the position on the phenyl ring is
(compounds 9 to 11). When substituents on compounds 2 and
10 were mixed, compound 12 was found to be potent with a
reduced blood clearance of 2.4 mL/min/kg. Probably due to its
very low permeability, oral bioavailability of 12 was still very
low. Introduction of a chlorine atom at position 2 of the pyrrole
ring (compound 13) led to a nice increase of potency giving us
the most potent compound of the series. This extra substituent
did not improve permeability nor blood clearance, but oral
exposure of 13 started to be of interest for such a potent
activator. Replacement of the guaiacol moiety by a tolyl group
(compound 14) gave a less potent compound but with an
improved pharmacokinetic profile. Compound 14 exhibited a
very low blood clearance and a slightly enhanced permeability
that resulted in a very nice oral exposure and an oral
bioavailability of 14%. The top phenyl ring could be replaced
with a thiophene that gave a potent compound 15. Again,
despite a low permeability, this compound exhibited a nice oral
exposure and an oral bioavailability of 21%. All the compounds
from this series showed very high plasma protein binding
(>99%) that could be attributed in part to the very acidic
nature of the 3-cyanopyridone ring (pKa for compound 15 was
measured lower than 2). We thought of modulating the pKa of
the ring by replacing the cyano group with a phenyl ring.
Compound 16, having a substituted phenyl moiety attached to
the pyrrole ring displayed, reduced potency and had no real
improvement in plasma protein binding compared to the
previous compounds. Nevertheless, it showed a nice perme-
ability that could come from a higher pKa of the pyridone ring,
resulting in an oral bioavailability of 50%. When the guaiacol
moiety was introduce on this template, compound 17 exhibited
an improved potency compared to compound 16. Acidic pKa
was measured at 6.0 for this compound proving our hypothesis
on the cyano group driving the pKa and thus permeability
down. Mouse pharmacokinetics was found to be very
encouraging for 17 displaying a very low blood clearance and
an oral bioavailability of 15%. Finally, introduction of a
carboxylic acid on the meta position of the right-hand side
phenyl ring (compound 18) gave a real improvement in
potency along with a loss of permeability. This resulted in a
very poor pharmacokinetic profile compared to the previous
compound.
Chart 1. Identification of Pyrrolopyridones As AMPK Direct
Activators
a
Scheme 1. General Synthetic Route for Pyrrolopyridones
a
Reagents and conditions: (a) AcCl, Et3N, DCM, RT; (b)
R2PhB(OH)2, Cu(OAc)2, pyridine or Et3N, air, DCM, RT; (c)
HCl, EtOH, reflux; (d) R1CH2COCl, Et3N, DCM, RT or
R1CH2CO2H, Et3N, coupling reagent, acetonitrile, RT; (e) NaH or
KHMDS in THF or t-BuOK in DMSO.
Amino-pyrroles 3 were first protected as acetates 4 using acetyl
chloride in the presence of triethylamine in DCM at RT. A
Chan−Lam coupling using standard conditions was then
performed to introduce the para-substituted phenyl ring leading
to compounds 5 in good yields. Deprotection in acidic
conditions of the acetate gave the amino-pyrroles 6 that were
then subjected to peptide-coupling conditions to lead to amides
7. Finally, cyclization of compounds 7 in the presence of strong
base afforded the desired pyrrolopyridones 8. In some cases,
the R2 substituent is further elaborated either from compound
6 or 8 using know chemistries as shown in the Supporting
Information section.
As shown in Table 1, compound 2 is a potent AMPK direct
activator of the α1β1γ1 heterotrimer slightly less potent than
A769662, 1. Compound 2 was directly assessed in mice
pharmacokinetic experiment in order to determine the
potential of the series. The compound was thus dosed at 30
mg/kg po and gave better blood exposure and oral
bioavailability than 1 but displayed a higher blood clearance.
Having identified pyrrolopyridones as a new interesting
template activating AMPK, the main objective of the medicinal
Despite improvements on most parameters, finding potent
AMPK activators with high oral absorption remained
challenging. Given the low permeability of this series of
compounds, we then assessed their susceptibility against P-
glycoprotein (P-gp). P-gp is an ATP-dependent efflux pump
with broad substrate specificity that could be the cause of their
absorption issue along with their poor permeability.
We selected compounds 13, 15, and 17 for further evaluation
in mice pharmacokinetics where the compounds (5 mg/kg for
13 and 15; 30 mg/kg for 17) were coadministered with P-gp
inhibitor GF12091818 (30 mg/kg). Figure 1 shows a marked
improvement of mean blood AUC for the 3 compounds under
those conditions suggesting that compounds of this series are
P-gp substrate. The low permeability compound 13 shows a 10-
fold increase in blood exposure when codosed with GF120918.
Compound 15 exhibited the same level of improvement in oral
exposure. Compound 17, probably due to a reduced affinity to
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Table 1. SAR Results for Pyrrolopyridones 2−18
a
Hum. rec. AMPK α1β1γ1 (Invitrogen) was used in a FRET assay format (ZLyte, Invitrogen). pEC200 = −log(compound concentration leading to a
b
2-fold AMPK activity increase). All data are means of at least 2 independent experiments. Permeability: Pampa (Parallel Artificial Membrane
Permeability Assay). Solubility: soluble fraction of oral formulation (see below) following its manufacture by ball milling from solid powder (sol
max. 500 mg/mL). Mouse PK: po @ 5 mg/kg in suspension in HPMC K100 0.5%/Tween 80 0.1% in buffer pH 7 and iv @ 1 mg/kg in solution in
c
d
DMSO/hydroxypropyl-β-cyclodextrin 20% in buffer pH 7 (5:95). po @ 30 mg/kg in suspension in HPMC K100 0.5%/Tween 80 0.1% in buffer pH
e
7. po @ 30 mg/kg in suspension in HPMC K100 0.5%/Tween 80 0.1% in buffer pH 7.
P-gp, shows only a 2-fold increase in blood AUC (see
Supporting Information Table S1 for detailed data).
heterotrimers we tested. Despite the fact that compound 13 is
more potent on the β1-containing heterotrimers, it is activating
α2β2γ1 and α2β2γ3 with a pEC200 = 7.3 for both heterotrimers
(see Supporting Information for complete heterotrimers data).
Even compound 14, a weaker compound on the α1β1γ1
AMPK heterotrimer, was able to modestly activate some β2-
Having identified compounds from the pyridone series that
were compatible with an oral route, we turned our efforts on
their AMPK heterotrimers selectivity. As explained earlier in
this letter, in order to explore the AMPK pathway in vivo, a
compound activating all or most AMPK heterotrimers seems
attractive. Compound A769662, 1, has been described as β1-
selective AMPK heterotrimers,15 and we assessed compounds
from the pyrrolopyridone series on the seven AMPK
heterotrimers that were available to us (Figure 2). Compounds
13, the most potent compound of the series on the α1β1γ1
AMPK heterotrimer, proved to activate all seven AMPK
containing heterotrimers (α2β2γ1 and α2β2γ3 with a pEC200
=
5.5, but for α1β2γ1, pEC200 < 4.5). Compounds 15 and 17
were also tested on β2-containing heterotrimer α2β2γ1 and
found active (see Supporting Information).
Compounds 13, 15, and 17 were selected for further in vivo
profiling given their additional PK profiles. Compounds 13 and
15 were evaluated in a chronic oral treatment in ob/ob mice for
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reduction of blood glucose, respectively) at the dose of 100
mg/kg (P < 0.001) when administered with the P-gp inhibitor
highlighting the potential of our AMPK activators for blood
glucose control. Compound 17 having a better oral exposure
was dosed orally for 5 days bid at 10 and 30 mg/kg without P-
gp inhibitor. Blood glucose was monitored 12 h after the last
gavage, and compound 17 was found to decrease it significantly
by 53 mg/dL (17% reduction of blood glucose) at the dose of
30 mg/kg (P < 0.05, Figure 3). We only observed a trend for
blood glucose reduction (13%) at 10 mg/kg that was not
statistically significant.
The objectives of this study were to improve the
pharmacokinetics of pyridone compounds in order to obtain
active AMPK direct activators in vivo and explore the effects of
AMPK activation utility in longer term treatments. After
structural modification of the thiophene ring, a novel series of
pyrrolopyridones was found to activate directly AMPK
heterotrimers with a pan profile. In regard to its adequate
oral PK profile, compound 17 was further tested in the animal
model of diabetes and showed activity at 30 mg/kg on blood
glucose. All of the data suggest that we have identified a series
of compounds useful to explore AMPK direct activation in vivo.
Figure 1. Influence of P-gp inhibition on mean blood AUC of
pyrrolopyridones 13, 15, and 17.
ASSOCIATED CONTENT
* Supporting Information
■
S
All experimental procedures and compounds characterization,
material and methods about DMPK, in vivo experiment
protocol, and AMPK heterotrimers profiling. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(O.M.) Tel: +33169296074. E-mail: oliviermirguet@yahoo.fr.
■
Present Addresses
†Institut de Recherche Servier, 11 rue des Moulineaux, 92150
Suresnes, France.
‡Galapagos, SASU, 102 Avenue Gaston Roussel, 93230
Romainville, France.
Author Contributions
Figure 2. AMPK heterotrimers profiling for pyridones 13 and 14.
All authors have given approval to the final version of the
manuscript.
Notes
5 days at 30 and 100 mg/kg bid in combination with
GF120918. P-gp inhibitor GF120918 by itself has no effect on
blood glucose (Figure 3). Both compounds 13 and 15 were
able to reduce blood glucose of 55 mg/dL (18% and 17%
The authors declare the following competing financial
interest(s): All authors were GlaxoSmithKline full-time employ-
ee at the time when this study was performed.
All studies involving the use of animals were conducted after
review by the GlaxoSmithKline (GSK) Institutional Animal
Care and Use Committee and in accordance with the GSK
Policy on the Care, Welfare and Treatment of Laboratory
Animals.
ACKNOWLEDGMENTS
Yann Lamotte, Delphine Delannee
Bouillot, Van-Loc Nguyen, Nicolas Faucher, Olivier Pineau,
■
́
, Alain Laroze, Anne
́
Alain Daugan, and Nerina Dodic are warmly acknowledged for
their technical assistance and helpful discussions during the
course of this study.
ABBREVIATIONS
■
AMPK: adenosine 5′-monophosphate-activated protein kinase;
ATP: adenosine-5′-triphosphate; ACC: acetyl-coenzyme A
carboxylase; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme
Figure 3. Compounds 13, 15, and 17 orally reduce blood glucose in
ob/ob mice.
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A; SREBP-1c: sterol regulatory element-binding proteins-1c; P-
gp: P-glycoprotein; AUC: area under the curve; FRET:
fluorescence resonance energy transfer; Pampa: parallel artificial
membrane permeability; PK: pharmacokinetic; HPMC:
hydroxypropyl methylcellulose
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