Development of novel alkene oxindole derivatives as orally efficacious AMP-activated protein kinase activators

Article abstract of DOI:10.1021/ml400028q

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is emerging as a promising drug target for its regulatory function in both glucose and lipid metabolism. Compound PT1 (5) was originally identified from high throughput screening as a small molecu

Full text of DOI:10.1021/ml400028q

Letter
pubs.acs.org/acsmedchemlett
Development of Novel Alkene Oxindole Derivatives As Orally
Efficacious AMP-Activated Protein Kinase Activators
Li-Fang Yu,† Yuan-Yuan Li,† Ming-Bo Su, Mei Zhang, Wei Zhang, Li-Na Zhang, Tao Pang,
Run-Tao Zhang, Bing Liu, Jing-Ya Li, Jia Li,* and Fa-Jun Nan*
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, P. R. China
S
* Supporting Information
ABSTRACT: Adenosine 5′-monophosphate-activated protein
kinase (AMPK) is emerging as a promising drug target for its
regulatory function in both glucose and lipid metabolism.
Compound PT1 (5) was originally identified from high
throughput screening as a small molecule activator of AMPK
through the antagonization of the autoinhibition in α subunits.
In order to enhance its potency at AMPK and bioavailability,
structure−activity relationship studies have been performed
and resulted in a novel series of AMPK activators based on an
alkene oxindole scaffold. Following their evaluation in pharmacological AMPK activation assays, lead compound 24 was
identified to possess improved potency as well as favorable pharmacokinetic profile. In the diet-induced obesity (DIO) mouse
model, compound 24 was found to improve glucose tolerance and alleviate insulin resistance. The in vitro and in vivo data for
these alkene oxindoles warrant further studies for their potential therapeutic medications in metabolic associated diseases.
KEYWORDS: AMPK activator, alkene oxindole, DIO mouse model, insulin sensitivity, diabetes
denosine 5′-monophosphate-activated protein kinase
A(AMPK) is an evolutionarily conserved serine/threonine
protein kinase, which exists as a heterotrimer comprising a
catalytic α-subunit and two regulatory β and γ subunits.
Different combinations of the three subunits encoded by seven
genes (α1, α2; β1, β2; γ1, γ2, and γ3) result in different αβγ
heterotrimers. The α2 subunit is highly abundant in skeletal
muscle, heart, and liver, while AMPK α1 subunit is relatively
evenly distributed across rat heart, liver, kidney, brain, spleen,
lung, and skeletal muscle.1 The β1 and γ1 subunits are
expressed universally, while β2, γ2, and γ3 subunits are
relatively more confined to muscle or heart. AMPK acts as a
cellular energy sensor and regulator, balancing the energy at
both the cellular level and the whole body. In mammals, the
activity of AMPK is regulated by predominant upstream kinases
liver kinase B1 (LKB1) and calcium/calmodulin-dependent
protein kinase kinase (CaMKKα/β) through the phosphor-
ylation of a conserved threonine (Thr 172 in rat).2,3 Once
activated by hypoxia, pressure overload, or hypertrophy, AMPK
recovers intracellular energy balance either by stimulating the
activity of catabolic pathways that generate ATP, such as fatty
acid oxidation, or inhibiting the activity of ATP-consuming
anabolic pathways, such as the synthesis of fatty acids,
cholesterol, glycogen, and proteins.4 Recent reports have
demonstrated that AMPK activation plays a central role in
the regulation of body weight, systemic glucose homeostasis,
lipid metabolism, and mitochondrial biogenesis. The two
endogenous adipokines, leptin and adiponectin exhibit
beneficial effects on glucose and lipid metabolism by activating
AMPK.5−7 A number of pharmacological agents including two
major classes of existing antidiabetic drugs biguanides and
thiazolidinediones were found to exert their effects at least
partially by activating AMPK.8,9 Therefore, activators of AMPK
represent a promising therapeutic approach for the treatment of
type 2 diabetes and metabolic syndromes.
5-Aminoimidazole-4-carboxamide riboside (AICAR) (Figure
1) has been widely used as an AMPK activator via conversion
into 5-amino-4-imidazolecarboxamide ribotide (ZMP) by
Figure 1. Structures of selected AMPK activators.
Received: January 19, 2013
Accepted: March 24, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
a
adenosine kinase inside the cell to regulate cellular metabolism.
Currently, pharmaceutical companies and academic bodies are
actively involved in the discovery and development of small-
molecule AMPK activators. Four research groups have
published AMPK activators, which were originally identified
in enzymatic assays and exhibited promising in vitro and/or in
vivo activities. The thienopyridone A-769662 (2) identified by
Abbott Laboratories was the first reported compound to
stimulate partially purified rat liver AMPK (EC50 = 0.8 μM) and
Scheme 1
inhibit fatty acid synthesis in primary rat hepatocytes (IC50
=
3.2 μM).10 Treatment of ob/ob mice with compound 2
decreased the plasma glucose, body weight gain, and both the
plasma and liver triglyceride levels. 5-(5-Hydroxy-isoxazol-3-
yl)-furan-2-phosphonic acid (3) was identified by Metabasis
Therapeutics as a tool compound for AMPK activator through
the screening of a library of AMP mimetics that showed high
potency for AMPK (EC50 = 6 nM vs AMP EC50 = 6 μM).11
Compound 4 was found to stimulate recombinant AMPK
activity (EC50 = 0.3 μM) and showed translational potential for
breast cancer therapy.12 From our own research institute,
compound PT1 (5) was originally identified as a novel small
molecule that activates AMPK through antagonizing the
autoinhibition in α subunits.13 It activated the inactive forms
of AMPK α2398 (α2, residues 1−398) and α1394 (α1, residues
1−394) with moderate activity (EC50 = 12 and 9 μM).
Compound 5 was found to stimulate the phosphorylation of
AMPK and acetyl-CoA carboxylase (ACC) and dose-depend-
ently decreased the lipid content in HepG2 cells. However,
compound 5 was not active in vivo (unpublished data),
probably due to the insufficient potency and poor bioavail-
ability. Here, we present our efforts in the optimization of this
reported hit compound 5 that led to the identification of alkene
oxindole derivatives as novel AMPK activators that activates
AMPK in vitro and more importantly showed beneficial
metabolic effects on diet induced obese (DIO) mouse model.
Following the discovery of hit compound 5 from our library
screening against AMPK, we devised a synthetic route for this
compound in order to confirm its activity (Scheme 1). Initially,
Meerwein arylation of 4,5-dimethyl-2-nitro-phenylamine 6 with
furan-2-carboxylic acid afforded the acid, which was sub-
sequently reduced with borane-THF and then oxidized with
manganese dioxide to furnish the aldehyde 7. Methylrhodanine
8 was reacted with 5-amino-2-chloro-benzoic acid to afford the
thiazolidinone 9, which in turn was condensed with the
aldehyde 7 to afford compound 5. Utilizing this synthetic route,
compounds 12−18 were prepared starting from different
aminobenzenes as shown in Table 1. The synthesized
compounds were initially characterized using an inactive α1394
form to measure AMPK activation under typical assay
conditions containing 2 μL of compound (20 μg/mL)
dissolved in dimethyl sulfoxide, in which compound 5 reached
the maximum activation.
a
Reagents and conditions: (a) i. NaNO2, 4 N aq. HCl; ii. furan-2-
carboxylic acid, CuCl; (b) i. BH3·THF, THF; ii. MnO2, CHCl3; (c)
EtOH, reflux; (d) 7, NaOAc, AcOH, reflux; (e) 3-bromomethyl-
benzoic acid methyl ester, NaH, DMF; (f) i. LiOH, dioxane, H2O; ii.
N2H4.H2O, reflux; (g) 3-nitro-phenyl-furan-2-carbaldehyde, pyridine,
EtOH, reflux.
Table 1. Initial measurement of AMPK Activation Using
Inactive α1394 Form
a
1
The overlapping broad signals in the H NMR spectra of 2-
imino-4-thiazolidinones 5 and 12−18 are consistent with the
previous reports that these compounds exist either as amino or
the imino tautomer in the solution state.14−17 In order to
eliminate this tautomerization, a synthetic route was devised
from the known compound 10 to access compound 11
(Scheme 1). The activity dramatically decreased when
compared to the original compound 5 (Table 1). In the
subsequent structure−activity relationships (SAR) studies, the
replacement of the central 2-imino-4-thiazolidinone with 3-
alkylideneoxindole ring system was attempted since it
a
b
See Experimental Section, Supporting Information. Activation
c
relative to compound 5, 20 μg/mL. The EC50 values of 5 and 19
were determined (Supporting Information Figure 1): 5, EC50 = 9.6
μM; 19, EC50 = 2.1 μM.
B
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a
represents an important substructure found in naturally
occurring bioactive indole alkaloids. Accordingly, 3-alkylide-
neoxindole 19 was designed and synthesized as shown in
Scheme 2. N-Alkylation of isatin 20 with 3-bromomethyl-
Table 2. Activation of the AMPK Heterotrimer α2β1γ1.
a
Scheme 2
a
Reagents and conditions: (a) phenyl isocyanate, EtMgBr; (b) 3-
bromomethyl-benzoic acid methyl ester, Cs2CO3, (nBu)4N+I−, DMF;
(c) ArI, cat. Pd(OAc)2, NaOAc, DMF; (d) LiOH, 1,4-dioxane, or
THF, H2O; (e) i. DPPA, Et3N, PhH; ii. 1,1-dibromo-3-methyl-but-1-
n
ene, BuLi, THF; (f) 4-chlorobenzeneboronic acid, Pd(OAc)2, PPh3,
CsF, THF.
benzoic acid methyl ester afforded the intermediary ester.
Hydrolysis of methyl ester and reduction of carbonyl group
with hydrazine hydrate provided the oxindole. Aldol con-
densation of 21 with 3-nitro-phenyl-furan-2-carbaldehyde
afforded 19. Gratifyingly, compound 19 increased the AMPK
activation by 3-fold at the initial tested dose of 20 μg/mL
(Table 1) compared to compound 5. A dose−response curve
was then calculated for 19 (see Supporting Information), where
it was shown that this compound activated the recombinant
AMPK α1394 with an EC50 value of 2.1 μM. The potency of 19
in the activation of AMPK α1394 is improved about 5-fold
compared to that of compound 5 (EC50 = 9.6 μM). As
nitroaromatic moiety is often associated with toxicity via
producing the potentially hazardous nitroanion radical, nitroso
intermediate, and N-hydroxy derivative in medicinal chem-
istry,16−18 replacement with halogens was attempted as
exemplified by 3-chloro-4-fluoro substitution. In a similar
manner, compound 20 was synthesized using 3-chloro-4-
fluorophenyl-furan-2-carbaldehyde. The two novel oxindoles 19
and 20 were tested using a scintillation proximity assay for their
ability to activate the bacterial expressed isoform of AMPK
α2β1γ1 as described in Table 2. Both of them exhibited
enhanced potency and efficacy compared to compound 5.
Furthermore, it was found that the potency and efficacy were
retained with the biaryl substitutions of the alkene oxindole
(23, EC50 = 5.9 μM, efficacy 105%) compared to the diaryls
(22, EC50 = 4.8 μM, efficacy 122%).
a
b
See Experimental Section, Supporting Information. Efficacy relative
c
to AMP. Not active, defined as EC50 > 10 μM or efficacy < 50%
activation of AMP.
materials.19 Initially, commercially available ethynyl benzene
was reacted with phenyl isocyanate in the presence of ethyl
magnesium bromide to form the unsaturated amide. This
intermediate was subsequently reacted with 3-bromomethyl-
benzoic acid methyl ester using cesium carbonate as a base and
catalytic amount of n-tetrabutylammonium iodide. Palladium-
(II) acetate-catalyzed coupling with appropriate aryl iodides
using N,N-dimethylformamide as a solvent and NaOAc as a
base at 110 °C afforded the key alkene oxindoles. Hydrolysis of
an ester group gave the desired final compounds. Compounds
29−31 were synthesized utilizing the synthetic routes shown in
Scheme 2, exemplified by compound 29. Curtius rearrange-
ment of 2-iodobenzoic acid using diphenylphosphoryl azide
and triethylamine in benzene afforded the intermediate
isocyanate. 1,1-Dibromo-3-methyl-but-1-ene was reacted with
n-butyl lithium under Corey−Fuchs condition to give the
intermediary alkynyl anion in situ, which was then coupled with
the isocyanate to give the unsaturated amide. This intermediate
was subsequently reacted with 3-bromomethyl-benzoic acid
methyl ester using cesium carbonate as a base and catalytic
Encouraged by the improvement of the AMPK activation of
alkene oxindoles compared to the thiazolidinones, a subsequent
broader range of structural modifications were carried out. The
SAR studies were mainly focused on the 3 positions (Table 2;
R1, R2, and R3). Compounds 23, 25−28, and 32−34 were
synthesized utilizing the same synthetic routes as described for
24 shown in Scheme 2. The synthetic approach was based on a
highly efficient palladium-catalyzed synthesis of asymmetrically
substituted alkene oxindoles from readily accessible starting
C
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amount of n-tetrabutylammonium iodide. The key palladium-
catalyzed Heck-carbocyclization/Suzuki-coupling reaction be-
tween iodide 38 and benzeneboronic acid in the presence of
catalytic amount of Pd(PPh3)4 afforded the alkene oxindole,
followed by ester hydrolysis. This reaction proceeded smoothly
when CsF or copper thiophene-2-carboxylic acid was used as a
base.20,21 The configuration of this series of compounds was
confirmed by the crystal structure (methyl ester of compound
29) as shown in Scheme 2.
stimulated glucose uptake dose-dependently, which was
blocked in the dominant-negative mutant of AMPK (DN-
AMPK). The results are presented as the mean SE: *, P <
0.05; **, P < 0.01; and ***, P < 0.001 (n = 3 replicated assays).
Additionally, no significant off-target effects of 24 were
observed toward two closely related kinases in the AMPK
family: MARK2 and MELK, as well as Aurora A, GSK3β, and
IKKβ.
In vitro metabolism studies of compounds 24 showed no
detectable metabolism at both mouse and rat liver microsomes.
The hepatic clearance rate of 24 was 8.6 mL/min/kg in human
liver microsomes. Assays of the compound’s inhibitory
potential toward multiple CYP450 enzymes including
CYP3A4, CYP2C9, CYP2D6, CYP2C19, and CYP1A2,
revealed no significant inhibition, suggesting that 24 will not
alter the metabolism of other exnobiotics or endogenous
compounds that are substrates for the CYP isoforms tested.
Cardiotoxicity associated with the inhibition of human ether-a-
go-go-related gene was also evaluated, where no obvious
inhibition was seen (IC50 > 20 μM). To further explore the
bioavailability of alkene oxindole series of activators, compound
24 was selected for full in vivo pharmacokinetic studies on
Wistar rats. Compound was intravenously and orally adminis-
tered to rats with the dose of 2 and 10 mg/kg, respectively. The
plasma samples were collected after dosing, and the drug
concentrations in plasma were determined by a developed LC−
MS/MS method. After an intravenous dose of 2 mg/kg, the
mean terminal half-life (t1/2) value of 24 was 3.94 h, and the
mean values of AUC0−24 h and AUC0−∞ were about 17.3 and
17.5 μg·h/mL (Supporting Information Table 1 and Figure 2).
After an oral dose of 10 mg/kg of 24, plasma concentrations in
rats reached peaks with a mean value of 3.27 μg/mL at 4.0 h
post dose. The mean t1/2 value was about 4 h. The mean values
of AUC0−24 h and AUC0−∞ of 24 were 28.9 and 29.5 μg·h/mL,
respectively. On the basis of the AUC0−∞ values after being
intravenously and orally administered, the oral bioavailability of
24 in rat was estimated to be 33.7%. The favorable
pharmacokinetic data indicated the possible oral efficacy of
these compounds in vivo.
Given the compelling data from both the in vitro efficacy and
pharmacokinetics studies, compound 24 was further inves-
tigated in an animal model. We chose the insulin-resistant DIO
mouse model, which is commonly used as an obese rodent
model of type 2 diabetes. Metformin, the front-line drug for the
treatment of type 2 diabetes, served as a positive control in the
experiments. The DIO mice were administered compound 24
(15, 50, and 150 mg/kg) or metformin (150 mg/kg) by daily
oral gavage for 4 weeks. All three dose treatment groups
displayed apparent reductions in plasma triglyceride (Table 3).
The magnitude of the reduction in plasma triglyceride was
similar to the reduction caused by metformin. Glucose
tolerance test demonstrated that both 50 and 150 mg/kg of
24 markedly improved glucose tolerance, whereas the lower
dose (15 mg/kg) showed a tendency toward improvement. The
area under the curve for the 50 and 150 mg/kg of 24 treatment
group was attenuated by 30% when compared with the vehicle
group; the extent of this effect was similar to that of metformin.
Insulin tolerance test revealed that treatment with all three
doses of 24 strikingly alleviated insulin resistance in DIO mice
when compared to vehicle group. This result suggests that 24
treatment in vivo improved glucose tolerance and alleviated
insulin resistance. We next measured the expression levels of
genes related to fatty acid oxidation and mitochondrial
The alkene oxindole derivatives were screened for their
ability to activate the AMPK heterotrimer α2β1γ1, and the
EC50 and efficacy values were obtained (Table 2). Different
electron withdrawing and donating groups were selected for
modification at R1 position. The R2 and R3 were maintained
throughout the series as a phenyl and benzyl-3-carboxylic acid,
respectively. For the R1 position, a 2- or 4-chloro substitution
enhanced the potency (EC50 = 1.8 and 1.2 μM) compared to 3-
chloro-4-fluoro substituted 23 (EC50 = 5.9 μM). The 3-pyridyl
group (28) also retained the activity with an EC50 value of 3.2
μM and efficacy value of 96%. When 4-chloro substitution at
the R1 position was deleted, the AMPK activity was abolished
(31). Maintaining R1 position as a 4-chlorophenyl and R3 as a
benzyl-3-carboxylic acid, some structural variations were
explored at the R2 position. A cyclohexyl group at the R2
position (30) was found to retain the efficacy but with a 4-fold
decrease in potency compared to 24. An isopropyl group (29)
resulted in about 10-fold lower potency (EC50 = 9.9 μM) and
2-fold higher efficacy compared to 24. Deletion of the
carboxylic acid moiety at the benzyl ring (34) resulted in an
approximately 40% decrease in the efficacy compared to 24.
Substitution of the benzyl-3-carboxylic acid (24) with benzyl-3-
carboxamide (33) resulted in a slight decrease of the potency
(1.2 vs 4.5 μM) but accompanied with about 50% increase of
efficacy. Exchanging the phenyl central core at R3 by a pyridinyl
(32) led to the decrease of both the potency and efficacy.
After observing the activation of AMPK on a molecular level,
its effect in cells was next investigated in L6 myoblasts. The
phosphorylation of AMPK and its downstream substrate ACC
was monitored as markers for the activation status of AMPK
pathways. Western blotting analysis (Figure 2a) showed that
compound 19, 22, and 24 activated the cellular AMPK activity
in a dose-dependent manner, and this effect was confirmed by
the dose-dependently increased phosphorylation of ACC.
Because the activation of AMPK in muscle has been linked
to the stimulation of glucose uptake, we next assessed this effect
(Figure 2b). Treatment of L6 myotubes with 24 for 3 h
Figure 2. Efficacy on cultured L6 myotubes.
D
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Notes
Table 3. Compound 24 Improves the Glucose and Lipid
Metabolism of DIO Mice after 4 Weeks of Treatment
a
The authors declare no competing financial interest.
compound
(mg/kg)
plasma TG
(mM)
f
g
GTT AUC
ITT AUC
ACKNOWLEDGMENTS
■
b
d
c
d
24 (15)
24 (50)
24 (150)
vehicle
2953.2 374.5
2820.0 147.5
8339.7 268.1
8165.6 142.4
7533.8 450.9
9262.5 385.8
0.52 0.02
0.52 0.02
0.54 0.02
0.62 0.02
We thank Drs. Li Chen and Yun He at Roche R&D Center
(China) for helpful comments.
c
c
c
e
d
2861.8 71.4
3533.8 70.5
2691.8 312.3
ABBREVIATIONS
h
c
i
d
■
met (150)
ND
0.50 0.02
a
d
f
b
c
The results are presented as the mean SE. #, p < 0.1. *, p < 0.05.
**, p < 0.01. ***, p < 0.001 compared with vehicle (n = 7−10).
GTT, glucose tolerance test; AUC, area under curve; TG, triglyceride.
AMPK, adenosine 5′-monophosphate (AMP)-activated protein
kinase; ATP, adenosine-5′-triphosphate; ACC, acetyl-CoA
carboxylase; LKB1, liver kinase B1; CaMKK, calcium/calm-
odulin-dependent protein kinase kinase; AICAR, 5-amino-
imidazole-4-carboxamide riboside; ZMP, 5-amino-4-imidazole-
carboxamide ribotide; SAR, structure−activity relationship;
DIO, diet induced obese; CPT1, carnitine palmitoyltransferase
1; CPT2, carnitine palmitoyltransferase 2; MCAD, medium
chain acyl CoA dehydrogenase; LCAD, long chain acyl CoA
dehydrogenase; PDK4, pyruvate dehydrogenase kinase isozyme
4; UCP2, uncoupling protein 2; FABP3, fatty acid binding
protein 3; PGC1α, peroxisome proliferator-activated receptor γ
coactivator 1α; CytoC, cytochrome c; COX5b, cytochrome c
oxidase subunit 5b
e
g
h
i
ITT, insulin tolerance test. Met, metformin. ND, not determined.
biogenesis in muscles. Compound 24 treatment significantly
upregulated the expression levels of genes related to fatty acid
uptake (such as fatty acid binding protein 3 (FABP3)) and fatty
acid oxidation (such as carnitine palmitoyltransferase 1 and 2
(CPT1 and CPT2), medium chain acyl CoA dehydrogenase
(MCAD), long chain acyl CoA dehydrogenase (LCAD),
pyruvate dehydrogenase kinase isozyme 4 (PDK4), and
uncoupling protein 2 (UCP2)) (Supporting Information Figure
3). Moreover, genes involved in mitochondrial biogenesis such
as peroxisome proliferator-activated receptor γ coactivator 1α
(PGC1α) and cytochrome c oxidase subunit 5b (COX5b) were
also upregulated following 24 treatment. These results suggest
that 24 likely improves insulin sensitivity by increasing fatty
acid oxidation in muscles.
The goals of this study were to improve the efficacy and
potency of hit compound 5, improve the pharmacokinetics in
order to obtain active lead compound in vivo, and better
understand the SAR. After initial structural modification of the
right side, the left side, and the linking part of compound 5, a
novel series of alkene oxindole derivatives were found to
stimulate the recombinant AMPK heterotrimers α2β1γ1, dose-
dependently stimulate AMPK and ACC phosphorylation, and
increase glucose uptake in L6 myotubes. Because of the
improved bioavailability of lead compound 24 and its in vitro
biological activity, the beneficial metabolic effects were
confirmed in insulin resistance DIO mice. Compound 24 was
further tested in preliminary ADME-Tox assays and showed
favorable profiles. The activity profiles shown by 24, at both
cellular and animal models, recommend an alkene oxindole
series of AMPK activators for further study in the quest for new
medications in metabolic diseases, particularly in the treatment
of diabetes.
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and analytical data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: fjnan@mail.shcnc.ac.cn (F.-J.N.); jli@mail.shcnc.ac.cn
(J.L.).
■
Author Contributions
†These authors contributed equally to this work.
Funding
This work was supported by National Natural Science
Foundation of China (Grants 30725049, 81125023 and
81273566).
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