Activating Effect of 3-Benzylidene Oxindoles on AMPK: From Computer Simulation to High-Content Screening

Article abstract of DOI:10.1002/cmdc.202000579

AMP-activated protein kinase (AMPK) is currently the subject of intensive study and active discussions. AMPK performs its functions both at the cellular level, providing the switch between energy-consuming and energy-producing processes, and at the whole

Full text of DOI:10.1002/cmdc.202000579

Accepted Article
Title: Activating effect of 3-benzylidene oxindoles on AMPK: from
computer simulation to high-content screening
Authors: Daria S Novikova, Tatyana A Grigoreva, Gleb S Ivanov, Gerry
Melino, Nickolai A Barlev, and Vyacheslav G Tribulovich
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000579
Link to VoR: https://doi.org/10.1002/cmdc.202000579
01/2020

10.1002/cmdc.202000579
ChemMedChem
FULL PAPER
Activating effect of 3-benzylidene oxindoles on AMPK: from
computer simulation to high-content screening
Daria S. Novikova,*[a] Tatyana A. Grigoreva,[a] Gleb S. Ivanov,[a, c] Gerry Melino,[b] Nickolai A. Barlev,[c]
and Vyacheslav G. Tribulovich[a]
[a]
D. S. Novikova, 0000-0002-5310-4570, Dr. T. A. Grigoreva, 0000-0003-1271-0328, Dr. G. S. Ivanov, Dr. V. G. Tribulovich, 0000-0001-7723-4962
Laboratory of Molecular Pharmacology
Saint Petersburg State Institute of Technology (Technical University)
Moskovskii pr. 26, 190013 Saint Petersburg, Russia
E-mail: dc.novikova@gmail.com
[b]
[c]
Prof. Dr. G. Melino, 0000-0001-9428-5972
Department of Experimental Medicine and Surgery
University of Rome Tor Vergata
Via Montpellier 1, 00133 Rome, Italy
Dr. G. S. Ivanov, Prof. Dr. N. A. Barlev, 0000-0001-7111-2446
Laboratory of Regulation of Gene Expression
Institute of Cytology RAS
Tikhoretskii pr. 4, 194064 Saint Petersburg, Russia
Supporting information for this article is given via a link at the end of the document.
Abstract: AMP-activated protein kinase (AMPK) is currently the
subject of intensive study and active discussions. AMPK performs its
functions both at the cellular level, providing the switch between
energy-consuming and energy-producing processes, and at the
whole body level, particularly, regulating certain aspects of higher
nervous activity and behavior. Control of such a ‘main switch’
compensates dysfunctions and associated diseases. In the present
paper, we studied the binding of 3-benzylidene oxindoles to the
kinase domain of the AMPK α-subunit, which is thought to prevent
its interaction with the autoinhibitory domain and thus result in the
AMPK activation. For this purpose, we developed the cellular test
system based on the AMPKAR plasmid, which implements the
FRET effect, synthesized a number of 3-benzylidene oxindole
compounds and simulated their binding to various sites of the kinase
domain. The most probable binding site for the studied compounds
was established by the correlation of calculated and experimental
data. The obtained results allow to analyze various classes of AMPK
activators using virtual screening and HCS.
therapeutic approach to the treatment of type 2 diabetes. So,
metformin, whose beneficial effect is partially contributed by the
activation of liver AMPK and suppression of gluconeogenesis,
as well as increased glucose uptake in peripheral tissues, is the
first-choice drug for type 2 diabetes patients.[5]
AMPK is a heterotrimeric complex consisting of three subunits:
α-subunit contains the main activation site, threonine residue (in
the literature Thr172), and is responsible for the catalytic
functions of the complex; regulatory β-subunit acts as
a
backbone of the complex, and also is involved in carbohydrate
metabolism; regulatory γ-subunit is an energy sensor of the
complex, having two competitive binding sites for adenine
nucleotides (AMP/ADP/ATP).[6]
Despite significant therapeutic interest in AMPK as a target[7]
and intensive study of its activation mechanisms in the last
decade,[6a, 8] a limited number of effective approaches have been
developed to the design of compounds that activate the
kinase.[9] One can distinguish (1) targeting of AMP-binding sites,
causing allosteric activation of AMPK;[10] (2) development of
compounds targeted at an alternative allosteric site (so-called
ADaM site), identified by high-throughput screening;[11] (3)
blocking the function of the autoinhibitory domain (AID) of the
kinase.[12] The use of the latter approach is complicated by the
lack of complete data on the structure of AMPK. X-ray structural
data and NMR studies of individual domains allow us to fill in the
gaps of the structural map of AMPK, as it was for the region of
the autoinhibitory domain, whose structure has not been solved
Introduction
Currently, AMP-activated protein kinase (AMPK) is considered
as one of the key players in maintaining energy homeostasis.
Numerous studies show that AMPK is the main regulator of
energy balance at both the cellular and whole-body levels.[1]
Since the relevance of diseases associated with energy
metabolism disturbances has been rapidly increased in recent
decades, the regulation of the kinase activity of AMPK has
become current therapeutic approach for the treatment and
prevention of the considered group of diseases.[2]
AMPK functions as the energy regulator through the activation
under energy stress. Being activated, AMPK blocks energy-
consuming processes, such as protein synthesis, and activates
processes aimed at energy production, which allows it to
maintain the energy status of the organism at a certain level.[3] It
owes its name to AMP, its endogenous activator, whose
concentration sharply increases under stress and energy
depletion.[4] Currently, the AMPK activation underlies the
for
a
long time.[13] However, in order to bring together
experimental data on the structure of subunits within the
complex, it is often necessary to use computer simulation, which
allows to make certain conclusions about the functioning of the
kinase complex as a whole with a certain degree of the reliability.
Rational drug design is based on the consistent study of such
relationships as disease–target, structure of target–ligand and,
finally, structure–activity of the ligand. For the study of the last
two relationships, the computer simulation of the ligand–receptor
interaction and high-content screening (HCS) can be used as an
effective set of tools. In this paper, we attempted to determine
the binding site for the compounds activating AMPK by blocking
1
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10.1002/cmdc.202000579
ChemMedChem
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the autoinhibitory domain, and to establish
a relationship
between experimental data on the activity of these compounds
towards AMPK and calculated results based on the interaction
simulation of the studied compounds with the active site of the
target. The specificity of 3-(benzylidene)indolin-2-one structure,
which provides the possibility of binding to lipophilic areas of
proteins, places increased demands on targeting of these
compounds. The ability of a number of 3-(benzylidene)indolin-2-
one derivatives to bind to the lipophilic cavity of the MDM2
protein, leading to activation of the p53 signaling pathway, was
established.[14] Correct application of the structure–activity
relationship allows one to develop selective[15] or multitarget
small molecules based on indolin-2-one scaffold.[16]
Figure 1. FRET biosensor based on cyan (CFP) and yellow (YFP) fluorescent
protein.
Results and Discussion
The autoinhibitory domain is one of the most interesting
structural elements of AMPK used for drug development. The
mobility of the autoinhibitory domain is functionally essential for
the activation-inactivation process of the kinase complex. Being
located within the catalytic α-subunit, the autoinhibitory domain
directly affects the kinase function of AMPK. It was shown that
the construct consisting of only the kinase and autoinhibitory
domains is inactive, while the deletion of a small region of the
autoinhibitory domain converts the catalytic α-subunit into the
permanently active state.[17] The search for small molecule
compounds that block intramolecular protein-protein interaction
between the autoinhibitory and kinase domains seems to be a
promising approach in the development of active and selective
compounds for the AMPK activation.
The choice of an experimental model that quantitatively fixes the
effect of a small molecule compound on the target is a required
step in the rational drug design in addition to the druggable
target suitable for the design of drugs. For this study, we chose
the FRET (Förster resonance energy transfer) method.
Determination of the kinase activity using FRET biosensors
introduced into living cells is a fairly common method in
molecular biology.[18] It is based on the phenomenon of energy
transfer between two chromophores, in which fluorescence of
the donor chromophore is quenched, while the acceptor
chromophore emits fluorescence in the long-wave region. The
closure of the chromophores is required for the transfer, which is
achieved by the presence of special protein sensors sensitive to
the studied process (Fig. 1). The most common chromophore
pair is cyan (CFP) and yellow (YFP) fluorescent protein.
The first FRET biosensor, designed specifically for the study the
AMPK activation process, was AMPKAR (AMPK activity
reporter).[19] This construct consists of a modified version of cyan
fluorescent protein (eCFP) as the donor fluorophore, the FHA1
domain[20] that recognizes threonine residue phosphorylation
(ligand domain), the protein substrate of AMPK (sensory
domain) and a modified version of yellow fluorescent protein
(Venus) as the acceptor fluorophore. The protein sequence
GSGEGSTKMRRVAT*LVDLGTGGSEL (where T* is threonine,
phosphorylated by AMPK), detected in the screening of the
peptide library and showed specificity for AMPK, was used as
the AMPK substrate. The construct obtained by the authors was
used to study the process of the AMPK activation in a single cell
(Single Cell Analysis) using modern microfluidic technique.
The degree of the AMPK activation is usually assessed either by
the phosphorylation degree of the main substrates of the
kinase[21] or by physiological effects such as decreased
lipogenesis,[10] increased glucose uptake,[22] decreased glucose
production[23] and others. In the case of AMPKAR, one can most
accurately assess the AMPK activity without the need to
consider additional pathways of phosphorylation and
dephosphorylation of AMPK substrates or complicated
mechanisms of integral effects.[24]
We tried to adapt the described system for HCS assays using
the Operetta High-Content Imaging System, which allows
conducting a wide range of such studies.[25] The FRET biosensor
was used to obtain a cellular test system for evaluating the
ability of small molecule compounds to activate AMPK. H1299
(human non-small cell lung cancer) cell line was laid in the basis
of the test system. Non-small cell carcinoma is the most
common type among lung cancers and represented by LKB1
positive cells, which are used in AMPK-related studies.[26]
H1299 cells, which stably express the AMPKAR construct, were
obtained by lentiviral infection. The most stable clones with the
maximum fluorescence signal were recruited as the test system
for AMPK activation. To validate the sensitivity of modified cell
line under HCS conditions known activators were used: AMP,
endogenous AMPK activator, and 5-aminoimidazole-4-
carboxamide riboside (AICAR), a synthetic AMPK activator,
which reproduces the effects of extracellular AMP.[27] According
to the published data, AMP causes up to 10-fold activation of the
AMPK complex depending on the subunit composition. It is
believed that activation of intracellular AMPK through increasing
the concentration of extracellular AMP is achieved due to
dephosphorylation by 5′-nucleotidase (CD73), the use of
adenosine
transport
and
subsequent
intracellular
monophosphorylation.[28] AICAR is thought to be an incomplete
AMP mimetic, and it is incapable of showing the same activating
effect in vivo as AMP.[29] The treatment with these activators at
the concentration of 1 mM allowed to observe the activating
effect (Fig. 2). The small value of the change in the YFP to CFP
signal ratio (ΔFRET) is due to the specificity of the FRET effect,
which is the energy transfer through the space and is inversely
related to the sixth degree of the distance between the sensors.
In addition, it should be noted that the activating effect strongly
depends on the initial (basal) level of the AMPK activity.
2
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The prospects of the approach to the AMPK activation by
blocking the function of the autoinhibitory domain have been
considered for a long time. The first advance in this area is
compound PT1, which was identified by screening the library of
3600 compounds and showed the ability to activate the single
catalytic α-subunit beyond the heterotrimeric AMPK complex.[30]
Due to an extremely low bioavailability of PT1, efforts were
made to develop similar small molecule compounds. Thus 3-
benzylidene oxindoles were designed, which are the most
interesting among the developed activators of this type showing
significant activity towards AMPK.[31] However, the AMPK
binding site has not been established for this class of
compounds yet.
In order to develop a computational model for the search and
optimization of AMPK activators, which can act as autoinhibitory
domain blockers, we tried to determine the binding site for 3-
benzylidene oxindole compounds. As starting point we used the
most active 3-benzylidene oxindole compound, which is often
referred in the literature as C24 (or YLF-466D). The ability to
Figure 2. Response of AMPK activation cell model under the treatment with
AMP and AICAR at the concentration of 1 mM. Curves ‘AMP single cell’ and
‘AICAR single cell’ correspond to the FRET effect measured for an individual
cell. Curves ‘AMP average’ and ‘AICAR average’ correspond to the average
FRET effect of 8 fields within the well.
highly activate AMPK in both in vitro and in vivo experiments has
32]
been demonstrated for this compound.[31,
We performed
We measured the FRET effect for both individual cells, which
were monitored throughout the experiment, and in the HCS
mode by the average signal of 8 fields within the well of a 96-
well plate. As can be seen from Fig. 2, both modes of the study
demonstrate a high degree of similarity, which indicates a
correct transfer of the single cell technique to HCS.
molecular docking of C24 to the catalytic α-subunit of AMPK.
The docking area, further referred to as the αA site, was chosen
based on available X-ray diffraction data and data concerning
the effects of proteolytic mutations on the autoinhibitory domain
interactions,[8, 17b, 33] and also using the results of our simplified
Table 1. Calculated lipophilicity (logP), scoring function (GoldScore) of binding to different sites and experimental activating effect (ΔFRET) measured in the cell
model for series of 3-benzylidene oxindole compounds.
lopP
GoldScore
αB site
41.92
39.98
39.27
39.49
39.48
40.73
41.4
Compound
ΔFRET
ChemOffice
2.36
AlogPS 2.1
2.80
3.33
3.58
3.60
3.54
3.50
4.10
3.93
4.54
4.54
3.90
4.50
4.46
4.87
5.62
5.62
αA site
40.53
42.50
38.83
38.91
38.80
41.54
40.39
34.93
37.63
37.19
43.53
45.21
46.03
46.25
47.44
48.33
αAB site
41.36
43.82
43.97
41.55
44.36
44.45
43.75
44.41
45.62
46.33
50.45
50.81
51.63
54.51
56.98
54.66
3a
3b
1.04±0.02
1.01±0.02
1.17±0.02
1.08±0.02
1.12±0.02
1.16±0.03
1.21±0.02
1.26±0.03
1.35±0.03
1.47±0.02
1.53±0.03
1.55±0.03
1.71±0.03
1.89±0.04
2.00±0.04
1.83±0.04
2.78
3c
2.92
3d
2.92
3e
3.19
3f
2.92
3g
3.48
3h
3.57
42.26
44.59
41.81
45.54
45.82
46.09
47.74
49.27
48.62
3i
4.12
3j
4.12
3k
4.39
3l
4.94
3m
3n
4.94
5.59
3o (C24)
3p
6.15
6.15
3
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Figure 3. Proposed sites of C24 interaction with catalytic subunit of AMPK (PDB id 4QFG). Blue surface corresponds to αA site, red surface corresponds to αB
site. The residue separating two binding sites (Arg72) is given in thin tubes.
molecular dynamics calculations.[34] 4QFG structure, which is a
heterotrimeric inactive AMPK complex, was used as a protein
Tyr275, which suggested the need to search for alternative sites
of the ligand–receptor interaction (Fig. 3). The expansion of the.
model. To assess binding affinity, the GoldScore fitness function, docking area revealed an additional site of strong interaction of
which earlier showed good accuracy of prediction when
correlating with experimental data, was used.[35]
The primary docking of compound C24 to the proposed binding
site αA showed a significant GoldScore value (Table 1).
However, the ligand interaction diagram showed the presence of
a small number of lipophilic contacts limited by Pro74, Tyr80 and
C24 with the catalytic subunit, which we called as the αB site
(Table 1). In this case, the ligand–receptor interaction was
mainly hydrophobic, which largely corresponds to the lipophilic
nature of C24 (Fig. 3). The lipophilic potential of
4-chlorodiphenylmethane fragment of compound C24 is realized
through the interaction with Ile65, Leu68, Phe71, Leu79, Tyr80
Scheme 1. Synthesis of 3-benzylidene oxindole compounds. Reagents and conditions: (a) pyrrolidine, toluene, reflux; (b) aluminum isopropoxide, pyridine, THF,
40°C; (c) bromoacetic acid methyl ester, NaH, THF, rt; (d) methyl 3-(bromomethyl)benzoate, NaH, THF, rt; (e) LiOH, H2O/THF, rt.
4
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Figure 4. Curves of AMPK activation under the treatment with C24 at different
concentrations used to determine the value of EC50 for the developed cell
model.
Figure 6. Correlation of experimental and calculated data on the interaction of
3-benzylidene oxindoles with different sites of AMPK α-subunit.
obtained series of 3-benzylidene oxindoles was evaluated in the
cellular test system; the average values of the change in the
FRET effect under the treatment of the studied compounds are
given in Table 1. A more detailed study of the activating effect of
C24 yielded the EC50 value (Fig. 4).
To confirm that the observed FRET effect is mediated by AMPK,
in vitro experiments by Western blot were carried out. In this
case, the ability of the compounds to both stimulate AMPK
phosphorylation at the activation site and to phosphorylate the
main downstream targets was evaluated. The results obtained
(Fig. 5) were in good agreement with those obtained in the
cellular test system.
All the compounds and considered interaction sites were used in
the docking study (Table 1). Even though the minimum activity
was predicted for propiophenone derivative using the αA site,
the GoldScore values for more lipophilic derivatives were not
consistent with the data of the biological experiment. Docking to
the αB site showed significantly better agreement with the
and Val82. The carboxyl group forms a hydrogen bond with
Lys69.
To develop a working model reflecting the relationship between
experimental and calculated data for autoinhibitory domain
blockers, we designed a series of 3-benzylidene oxindole
compounds with different activating potential and studied their
activity using the cellular test system.
The simplest active compound of 3-benzylidene oxindoles is
compound 3a. Its synthesis is carried out by condensation of 2-
oxindole with acetophenone, subsequent alkylation of the
nitrogen atom with methyl bromoacetate and alkaline hydrolysis
(Scheme 1).[36] Despite considerable structural similarity with
compound 3a, synthesis of compound C24 is carried out
according to
a
more complicated scheme[31] due to low
conversion upon condensation of 4-chlorobenzophenone with
2-oxindole. However, we developed a method of condensation,
which allows to obtain benzophenone derivatives according to
the same scheme.[37] Therefore, we synthesized several new
compounds between 3a and C24 with intermediate activity
according to Scheme 1. The lipophilicity of molecule binding
fragments was the main varying parameter when changing the
structure from 3а to C24 (Table 1). The target activity of the
experimental data (Fig. 6). Obviously, the absence of
a
correlation between calculated and experimental data for the αA
site may lead to the loss of highly active compounds during
virtual screening of large libraries of compounds, as well as to
the choice of incorrect directions for the optimization of the
structure of small molecule compounds.
Figure 5. Western blot analysis of 3-benzylidene oxindole compounds
(incubation for 1 h at the concentration of 0.1 mM). 2-Deoxyglucose (2DG) and
AICAR were used as reference AMPK activators at the concentration of 20
mM and 2.5 mM, respectively.
Figure 7. Cartoon representation of KD-AID dimer (PDB id 4RED).
5
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Figure 8. Proposed αAB site of C24 interaction with catalytic subunit of AMPK (PDB id 4RED) and ligand interaction diagram.
The search for resolved structures of the kinase domain (KD) of
AMPK led us to the 4RED structure, which represents not full-
length AMPK complex, but the KD-AID dimer.[33] This dimer of
the α-subunit is folded so that the autoinhibitory domain of one
subunit interacts with the kinase domain of another (Fig. 7). The
tendency to dimerization observed in 4RED, probably,
determines the lack of the activity of a single α-subunit, which is
in an inactive state due to the fixed conformation.
nature of the interaction of 3-benzylidene oxindoles with the
kinase domain of AMPK was established.
The obtained data allow both to optimize the studied series of
3-benzylidene oxindoles and to analyze diverse libraries of
compounds using virtual screening and HCS.
Experimental Section
Changes in the kinase domain structure of the 4RED monomer,
associated with an untwisting of the α-helix in the Ile65–Leu70
region, result in a change in the tertiary structure of the
hydrophobic binding cavity compared to 4QFG. The hybrid
binding site αAB, which combines the properties of both αA and
αB sites, considered previously, is formed (Fig. 8).
Using the 4RED structure and the proposed binding site for our
virtual experiments, we obtained good correlation between the
calculated and experimental data (Fig. 6). The computer
simulation of C24 binding showed the presence of a large
number of lipophilic contacts for phenyl fragments of the
compound: Tyr18, Ala43, Val44, Pro76, Val94, Tyr97, Val98,
and hydrogen bonding of the carboxyl group with Ser99 (Fig. 8).
The observed mode of C24 binding quite fully realizes both its
lipophilic potential and the binding ability of the carboxyl group.
Computer simulation
Molecular docking of the studied compounds was performed on a HP Z8
G4 (nVidia Quadro RTX 5000) workstation using the CCDC GOLD Suite
v5.2.2 software package.[38] The X-ray crystallographic structures of the
AMPK kinase domain were obtained from the Protein Data Bank (PDB id
4QFG[8] and 4RED[33]). The structure of the kinase domain was prepared
for docking studies by adding hydrogen atoms with the standard
geometry. The binding affinity of the studied compounds was evaluated
by the GoldScore fitness function. The binding poses that correspond to
the highest values of GoldScore were visualized to confirm the accuracy
of the binding. The GoldScore values obtained for different active sites
were used to construct correlations between experimental and calculated
data.
The lipophilicity (lopP) of the studied compounds was calculated using
ChemOffice software package and AlogPS 2.1 online service.[39]
Conclusions
Chemistry
In summary, the cell model based on the AMPKAR plasmid was
developed and validated for HCS. The experiments were carried
out in living cells, the FRET effect arising from the treatment with
small molecule compounds was measured in real time. The
ability of 3-benzylidene oxindoles to bind to the kinase domain of
the AMPK α-subunit for preventing its interaction with the
autoinhibitory domain was simulated. Structures of the kinase
domain obtained from the Protein Data Bank (PDB id 4QFG and
4RED) were used as receptors for the considered ligand–
receptor system. The preferred binding site of the α-subunit for
the studied small molecule ligands was determined by the
correlation of quantitative parameters of computer simulation
and experimental data on the AMPK activation. The lipophilic
2-Oxindoles obtained according to the known procedure[40] were used as
starting compounds. Other starting compounds and reagents used were
commercially available. Synthesis of the studied compounds was carried
out in accordance with Scheme 1.
The reactions were monitored by TLC on Silica gel 60 F254 plates (Merk)
in hexane-ethyl acetate. Purification of the products was carried out using
an Isolera Four flash chromatograph on SNAP KP-Sil 100g cartridges
(Biotage) using hexane-ethyl acetate eluent.
1H and 13C NMR spectra were recorded on a Bruker Avance III 400 (400
MHz) device in DMSO-d6. Mass spectra were recorded on a LCMS-2020
(Shimadzu) with a single quadrupole detector under positive mode;
electrospray ionization (ESI).
6
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General procedure for synthesis of 3-benzylidene oxindoles 1a-g.
2-Oxindole or substituted oxindole (0.075 mol) was suspended in 200 mL
of toluene. The corresponding ketone (0.09 mol, 1.2 equiv.), 16.5 mL of
pyrrolidine (0.15 mol, 2 equiv.) were added to the suspension. The
reaction mixture was refluxed with the Dean-Stark trap for 0.5–4 hours.
The reaction was monitored by TLC. Then the mixture was cooled, the
solvent was evaporated under reduced pressure. The residue was
treated with a small amount of ethyl acetate and then filtered off, or
recrystallized from n-hexane/ethyl acetate, or purified by flash
chromatography using n-hexane/ethyl acetate eluent.
2H), 7.20 (dd, J = 8.4, 2.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 5.87 (d, J =
1.9 Hz, 1H), 4.54 (s, 2H), 2.76 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 169.82, 167.19, 157.83, 142.29, 140.41, 129.88, 129.33, 128.23,
126.50, 125.73, 123.81, 122.29, 122.07, 110.46, 41.32, 23.00; MS (ESI)
m/z: 328.1 [M + H]+.
(E)-2-(6-chloro-2-oxo-3-(1-phenylethylidene)indolin-1-yl)acetic acid
(3d): yellow crystalline solid in 91% yield; mp 213–214°C; 1H NMR (400
MHz, DMSO-d6) δ (ppm): 7.58–7.48 (m, 3H), 7.36 (d, J = 7.1 Hz, 2H),
7.16 (s, 1H), 6.72 (d, J = 8.3 Hz, 1H), 5.98 (d, J = 8.3 Hz, 1H), 4.55 (s,
2H), 2.73 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 169.81,
167.52, 156.58, 142.99, 142.48, 133.22, 129.81, 129.25, 126.64, 123.50,
121.91, 121.35, 121.07, 109.45, 41.43, 22.99; MS (ESI) m/z: 328.1 [M +
H]+.
Analytical data for compounds 1a-g are given in Supporting Information.
General procedure for synthesis of 3-benzylidene oxindoles 1h-j.
2-Oxindole (4 g, 0.03 mol) was dissolved in dried and freshly distilled
THF, the corresponding benzophenone (0.036 mol, 1.2 equiv.), pyridine
(4.8 mL, 0.06 mol, 2 equiv.) and aluminum isopropoxide (18.4 g, 0.09 mol,
3 equiv.) were added. The reaction mixture was heated to 40°C and kept
overnight. Then the reaction mixture was quenched with 3% Na2CO3
solution, extracted with ethyl acetate, dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The product (or mixture of products)
was isolated by flash chromatography with n-hexane/ethyl acetate eluent.
(E)-2-(5-bromo-2-oxo-3-(1-phenylethylidene)indolin-1-yl)acetic acid
(3e): yellow crystalline solid in 89% yield; mp 245–247°C; 1H NMR (400
MHz, DMSO-d6) δ (ppm): 7.61–7.52 (m, 3H), 7.36 (dd, J = 7.7, 1.5 Hz,
2H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.01 (d, J =
1.9 Hz, 1H), 4.53 (s, 2H), 2.76 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 169.76, 167.07, 157.82, 142.29, 140.77, 131.01, 129.85, 129.31,
126.50, 125.12, 124.26, 121.98, 113.53, 110.95, 41.29, 22.98; MS (ESI)
m/z: 372.0 [M + H]+.
Analytical data for compounds 1h-j are given in Supporting Information.
(E)-2-(3-(1-(4-chlorophenyl)ethylidene)-2-oxoindolin-1-yl)acetic acid
(3f): yellow crystalline solid in 93% yield; mp 230–232°C; 1H NMR (400
MHz, DMSO-d6) δ (ppm): 7.57 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz,
2H), 7.14 (t, J = 7.5 Hz, 1H), 6.90 (d, J = 7.8 Hz, 1H), 6.68 (t, J = 7.5 Hz,
1H), 6.15 (d, J = 7.7 Hz, 1H), 4.49 (s, 2H), 2.73 (s, 3H); 13C NMR (100
MHz, DMSO-d6) d (ppm): 169.94, 167.43, 153.76, 141.75, 141.50,
133.75, 129.84, 128.93, 123.12, 122.45, 122.00, 121.80, 109.02, 41.21,
22.70; MS (ESI) m/z: 328.1 [M + H]+.
General procedure for synthesis of N-substituted 3-benzylidene
oxindoles 2a-p. Compound 1a-j (0.01 mol) was dissolved in 50 mL THF
and cooled on ice, 0.5 g sodium hydride (60%, 0.0125 mol, 1.25 equiv.)
was added under stirring. The mixture was kept for 30 min at rt. Than
methyl bromoacetate or methyl 3-(bromomethyl)benzoate (0.014 mol, 1.4
equiv.) dissolved in 10 mL THF was added dropwise. The reaction
mixture was stirred for 3 h, the solvent was then evaporated under
reduced pressure, 50 mL water was added to the residue. The resulting
precipitate was filtered off, dried, and the product was isolated by flash
chromatography.
(E)-2-(5-chloro-3-(1-(4-chlorophenyl)ethylidene)-2-oxoindolin-1-
yl)acetic acid (3g): yellow crystalline solid in 90% yield; mp 226–228°C;
1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.66 (d, J = 8.4 Hz, 2H), 7.44 (d,
J = 8.3 Hz, 2H), 7.23 (dd, J = 8.4, 2.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H),
5.97 (d, J = 1.9 Hz, 1H), 4.54 (s, 2H), 2.74 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 169.78, 167.08, 156.30, 140.96, 140.53, 134.06,
129.96, 128.84, 128.47, 125.77, 123.57, 122.33, 122.16, 110.64, 41.34,
22.85; MS (ESI) m/z: 362.1 [M + H]+.
Analytical data for compounds 2a-p are given Supporting Information.
General procedure for synthesis of acids 3a-p. Compound 2a-p (0.01
mol) in 50 mL THF was added to lithium hydroxide (2.4 g, 0.1 mol)
dissolved in 50 mL water. The reaction mixture was stirred for 10-30 min
(for 2h-j and 2n-p –12 h) at rt, the organic solvent was then distilled off.
The mixture was acidified with 15% HCl, the precipitate was filtered off,
washed with 50 mL water and dried. The crude product was purified by
recrystallization from ethyl acetate.
2-(3-(diphenylmethylene)-2-oxoindolin-1-yl)acetic acid (3h): yellow
crystalline solid in 95% yield; mp 244–246°C; 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 7.53–7.48 (m, 3H), 7.34–7.25 (m, 7H), 7.08 (t, J = 7.6 Hz,
1H), 6.74 (d, J = 7.8 Hz, 1H), 6.60 (t, J = 7.6 Hz, 1H), 6.09 (d, J = 7.7 Hz,
1H), 4.07 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d (ppm): 165.72,
153.01, 144.00, 141.80, 140.38, 129.95, 129.68, 129.38, 129.28, 128.84,
128.73, 128.07, 124.74, 122.66, 122.60, 121.01, 109.41, 43.54; MS (ESI)
m/z: 356.2 [M + H]+.
(E)-2-(2-oxo-3-(1-phenylethylidene)indolin-1-yl)acetic
acid
(3a):
yellow crystalline solid in 94% yield; mp 213–215°C; 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 7.57–7.47 (m, 3H), 7.35 (d, J = 6.7 Hz, 2H), 7.12 (t, J
= 7.6 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 6.62 (t, J = 7.6 Hz, 1H), 6.04 (d, J
= 7.7 Hz, 1H), 4.50 (s, 2H), 2.75 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ (ppm): 169.97, 167.55, 155.40, 142.83, 141.64, 129.75, 129.02, 128.77,
126.67, 122.78, 122.47, 122.23, 121.65, 108.92, 41.19, 22.90; MS (ESI)
m/z: 294.1 [M + H]+.
(E)-2-(3-((4-chlorophenyl)(phenyl)methylene)-2-oxoindolin-1-
yl)acetic acid (3i): yellow crystalline solid in 94% yield; mp 216–218°C;
1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.59 (d, J = 8.3 Hz, 2H), 7.38–
7.33 (m, 5H), 7.30–7.25 (m, 2H), 7.20 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.9
Hz, 1H), 6.75 (t, J = 7.7 Hz, 1H), 6.29 (d, J = 7.7 Hz, 1H), 4.44 (s, 2H);
13C NMR (100 MHz, DMSO-d6) d (ppm): 169.86, 165.89, 152.67, 142.90,
140.22, 139.87, 134.42, 130.98, 130.08, 129.85, 129.72, 129.26, 128.23,
124.23, 122.86, 122.50, 121.91, 109.27, 41.30; MS (ESI) m/z: 390.1 [M +
H]+.
(E)-2-(2-oxo-3-(1-phenylpropylidene)indolin-1-yl)acetic acid (3b):
yellow crystalline solid in 95% yield; mp 197–198°C; 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 7.55–7.45 (m, 3H), 7.28 (d, J = 6.7 Hz, 2H), 7.08 (t, J
= 7.7 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H), 6.56 (t, J = 7.7 Hz, 1H), 5.95 (d, J
= 7.7 Hz, 1H), 4.45 (s, 2H), 3.28 (q, J = 7.4 Hz, 2H), 1.07 (t, J = 7.4 Hz,
3H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 169.97, 167.17, 160.79,
141.80, 141.07, 129.68, 128.96, 128.81, 127.11, 122.56, 122.44, 122.28,
121.65, 108.92, 41.20, 27.76, 12.21; MS (ESI) m/z: 308.2 [M + H]+.
(Z)-2-(3-((4-chlorophenyl)(phenyl)methylene)-2-oxoindolin-1-
yl)acetic acid (3j): yellow crystalline solid in 95% yield; mp 195–196°C;
1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.55–7.50 (m, 3H), 7.42 (d, J =
8.5 Hz, 2H), 7.36–7.32 (m, 2H), 7.31 (d, J = 8.5 Hz, 2H), 7.19 (t, J = 7.6
Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.69 (t, J = 7.6 Hz, 1H), 6.19 (d, J = 7.7
Hz, 1H), 4.44 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 169.85,
166.00, 152.55, 142.90, 141.01, 138.93, 133.91, 131.86, 129.81, 129.73,
(E)-2-(5-chloro-2-oxo-3-(1-phenylethylidene)indolin-1-yl)acetic acid
(3c): yellow crystalline solid in 92% yield; mp 243–245°C; 1H NMR (400
MHz, DMSO-d6) δ (ppm): 7.61–7.52 (m, 3H), 7.36 (dd, J = 7.8 Hz, 1.5 Hz,
7
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10.1002/cmdc.202000579
ChemMedChem
FULL PAPER
128.83, 128.27, 124.38, 122.96, 122.55, 121.82, 109.25, 41.31; MS (ESI)
Biology
m/z: 390.1 [M + H]+.
For the development of a cellular test system suitable for testing the
ability of small molecule compounds to activate AMPK, the AMPKAR
construct kindly provided by Lewis Cantley (Addgene plasmid #35097)
was used.[19] The construct was cloned into the pEYFP-N2 vector, which
was transfected into H1299 cells (non-small cell lung cancer); expressing
cells were selected with Genicitin (G-418, Gibco). The most stable clones
with the maximum fluorescence at 477 nm were used as a basis for the
cellular test system. Transfected cells were cultured in DMEM (Gibco)
supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin,
and 100 μg/mL streptomycin at 37°C in an atmosphere of 5% CO2.
(E)-3-((2-oxo-3-(1-phenylethylidene)indolin-1-yl)methyl)benzoic acid
(3k): yellow crystalline solid in 93% yield; mp 193–194°C; 1H NMR (400
MHz, DMSO-d6) δ (ppm): 7.93 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.60 (d, J
= 7.5 Hz, 1H), 7.57–7.45 (m, 4H), 7.38 (d, J = 7.1 Hz, 2H), 7.09 (t, J = 7.6
Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 6.62 (t, J = 7.6 Hz, 1H), 6.03 (d, J = 7.7
Hz, 1H), 5.06 (s, 2H), 2.79 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 167.61, 167.56, 155.81, 142.85, 141.30, 137.86, 132.29, 131.64,
129.72, 129.45, 129.03, 128.81, 128.49, 126.74, 122.80, 122.63, 122.44,
121.77, 109.14, 42.55, 23.01; MS (ESI) m/z: 370.2 [M + H]+.
(E)-3-((5-chloro-2-oxo-3-(1-phenylethylidene)indolin-1-
To evaluate the activity of the studied compounds, the cells were plated
in FluoroBrite DMEM medium (Gibco) supplemented with 10% FBS and
2 mM L-glutamine on a 96-well plate with opaque black walls (Greiner).
The studied compounds were dissolved in DMSO prior to the experiment.
Detection of the fluorescent signal was performed on the Operetta High-
Content Imaging System (Perkin Elmer); three channels were used for
the detection of FRET signal (excitation filter 410–430 nm, emission filter
520–560 nm), CFP fluorescence (excitation filter 410–430 nm, emission
filter 460–500 nm) and YFP fluorescence (excitation filter 490–510 nm,
emission filter 520–560 nm). The first time point corresponded to the
untreated cells; immediately after the measurement of the first time point
the solution of the studied compounds was added to each well to the final
concentration of 1 mM (6 wells per compound). The same amount of
DMSO was used as a control (6 control wells). The plate was kept at
37°C in an atmosphere of 5% CO2 during the experiment. The
experiment was performed in triplicate.
yl)methyl)benzoic acid (3l): yellow crystalline solid in 92% yield; mp
217–219°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.91 (s, 1H), 7.86 (d,
J = 7.7 Hz, 1H), 7.60–7.54 (m, 4H), 7.49 (t, J = 7.6 Hz, 1H), 7.40 (dd, J =
7.7, 1.4 Hz, 2H), 7.17 (dd, J = 8.4, 2.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H),
5.88 (d, J = 2.0 Hz, 1H), 5.06 (s, 2H), 2.81 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 167.54, 167.26, 158.21, 142.32, 140.02, 137.52,
132.25, 131.65, 129.85, 129.50, 129.35, 128.89, 128.48, 128.28, 126.57,
125.88, 124.07, 122.46, 122.10, 110.56, 42.62, 23.11; MS (ESI) m/z:
404.2 [M + H]+.
(E)-3-((3-(1-(4-chlorophenyl)ethylidene)-2-oxoindolin-1-
yl)methyl)benzoic acid (3m): yellow crystalline solid in 95% yield; mp
240–242°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.91 (s, 1H), 7.85 (d,
J = 7.7 Hz, 1H), 7.63–7.58 (m, 3H), 7.48 (t, J = 7.9 Hz, 1H), 7.44 (d, J =
8.3 Hz, 2H), 7.12 (t, J = 7.7 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.70 (t, J =
7.7 Hz, 1H), 6.12 (d, J = 7.7 Hz, 1H), 5.06 (s, 2H), 2.77 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 167.58, 167.49, 154.23, 141.54, 141.38,
137.77, 133.74, 132.23, 131.78, 129.84, 129.43, 129.03, 128.82, 128.47,
123.11, 122.59, 122.19, 121.95, 109.28, 42.56, 22.83; MS (ESI) m/z:
404.2 [M + H]+.
The obtained images were processed using Harmony 3.1 software
(Perkin Elmer). The cells were defined by intense fluorescence in the
YFP fluorescence channel. The FRET effect was calculated by the ratio
of mean FRET fluorescence intensity to mean CFP fluorescence
intensity; eight most representative fields were processed in each well.
All the obtained values were normalized. The activating effect of the
studied compounds was assessed by the maximum change in the FRET
effect (ΔFRET), observed after 50 min of the experiment.
3-((3-(diphenylmethylene)-2-oxoindolin-1-yl)methyl)benzoic
acid
(3n): yellow crystalline solid in 91% yield; mp 230–232°C (decomp.); 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 7.90 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H),
7.55 (t, J = 7.4 Hz, 1H), 7.53–7.50 (m, 3H), 7.47 (t, J = 7.7 Hz, 1H), 7.38–
7.31 (m, 7H), 7.13 (t, J = 7.7 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 6.66 (t, J =
7.6 Hz, 1H), 6.16 (d, J = 7.7 Hz, 1H), 4.97 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 167.55, 166.16, 154.63, 142.45, 141.56, 140.18,
137.79, 132.17, 131.69, 130.19, 129.69, 129.51, 129.43, 129.19, 128.91,
128.77, 128.49, 128.16, 123.94, 123.06, 122.99, 121.81, 109.30, 42.66;
MS (ESI) m/z: 432.2 [M + H]+.
Western blot analysis in H1299 cells untreated or treated with
compounds (0.1 mM for 1 h) was performed on cell lysates centrifuged
and resolved by SDS-PAGE. The separated proteins were transferred to
a PVDF membrane and immunoblotted with specific antibodies according
to the manufacturer’s recommendations. The following antibodies were
used in the work: phospho-AMPK alpha-1 (Thr172) polyclonal antibody
(PA5-17831, Invitrogen), AMPK alpha-1 polyclonal antibody (PA5-29615,
Invitrogen), phospho-Acetyl-CoA Carboxylase (Ser79) (D7D11)
monoclonal antibody (#11818, Cell Signaling), anti-Acetyl Coenzyme A
Carboxylase monoclonal antibody (ab45174, Abcam), phospho-Raptor
(Ser792) polyclonal antibody (PA5-17116, Invitrogen), Raptor
monoclonal antibody (H.558.9) (MA5-15051, Invitrogen), anti-GAPDH
antibody (ab9484, Abcam). The primary antibodies were detected using
Peroxidase conjugated anti-Rabbit IgG antibodies (A9169, Sigma-
Aldrich) and anti-Mouse IgG (Fab specific)-Peroxidase antibodies (A9917,
Sigma-Aldrich). The chemiluminescence intensity was evaluated using
Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) in the
ChemiDoc Imaging System (BioRad).
(E)-3-((3-((4-chlorophenyl)(phenyl)methylene)-2-oxoindolin-1-
yl)methyl)benzoic acid, C24 (3o): yellow crystalline solid in 88% yield;
mp 204–206°C (decomp.); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.91
(s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.60–7.54 (m, 3H), 7.47 (t, J = 7.7 Hz,
1H), 7.40–7.30 (m, 7H), 7.15 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H),
6.73 (t, J = 7.5 Hz, 1H), 6.29 (d, J = 7.7 Hz, 1H), 4.97 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 167.55, 166.04, 152.98, 142.55, 140.27,
139.91, 137.75, 134.48, 132.22, 131.59, 131.16, 130.32, 129.81, 129.44,
129.36, 128.79, 128.53, 128.24, 124.29, 122.96, 122.81, 122.00, 109.43,
42.66; MS (ESI) m/z: 466.2 [M + H]+.
(Z)-3-((3-((4-chlorophenyl)(phenyl)methylene)-2-oxoindolin-1-
yl)methyl)benzoic acid (3p): yellow crystalline solid in 89% yield; mp
211–213°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.92 (s, 1H), 7.85 (d,
J = 7.7 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.54 – 7.50 (m, 3H), 7.47 (t, J =
7.7 Hz, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.39 – 7.33 (m, 4H), 7.14 (t, J = 7.7
Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 6.20 (d, J = 7.6
Hz, 1H), 4.97 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d (ppm): 167.55,
166.16, 152.83, 142.57, 141.07, 138.99, 137.74, 134.02, 132.22, 132.08,
131.64, 129.84, 129.77, 129.74, 129.44, 129.00, 128.81, 128.57, 128.27,
124.47, 123.07, 122.87, 121.91, 109.41, 42.71; MS (ESI) m/z: 466.2 [M +
H]+.
Acknowledgements
This work was financially supported by the Russian Science
Foundation (project no. 16-13-10358). The authors also thank M.
A. Gureev for calculations and A. V. Garabadzhiu for useful
discussion.
8
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10.1002/cmdc.202000579
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FULL PAPER
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10.1002/cmdc.202000579
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The binding site of 3-benzylidene oxindoles within the AMPK kinase domain was established by correlating calculated and
experimental activities. Such binding to the kinase domain leads to the AMPK activation. The cell model based on the FRET effect
was used under HCS conditions to evaluate the target activity.
10
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