Functionalized N,N-Diphenylamines as Potent and Selective EPAC2 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00477

N,N-Diphenylamines were discovered as potent and selective EPAC2 inhibitors. A study was conducted to determine the structure-activity relationships in a series of inhibitors of which several compounds displayed submicromolar potencies. Selectivity over t

Full text of DOI:10.1021/acsmedchemlett.5b00477

Letter
pubs.acs.org/acsmedchemlett
Functionalized N,N‑Diphenylamines as Potent and Selective EPAC2
Inhibitors
Christopher T. Wild,†,§ Yingmin Zhu,‡,§ Ye Na,† Fang Mei,‡ Marcus A. Ynalvez,† Haiying Chen,†
Xiaodong Cheng,*,‡ and Jia Zhou*,†
†Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas
77555, United States
‡Department of Integrative Biology and Pharmacology and Texas Therapeutics Institute, University of Texas Health Science Center,
Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: N,N-Diphenylamines were discovered as potent and selective
EPAC2 inhibitors. A study was conducted to determine the structure−activity
relationships in a series of inhibitors of which several compounds displayed
submicromolar potencies. Selectivity over the related EPAC1 protein was also
demonstrated. Computational modeling reveals an allosteric site that is distinct
from the cAMP binding domain shared by both EPAC isoforms, providing a
theory with regards to subtype selectivity.
KEYWORDS: Exchange proteins directly activated by cAMP (EPAC) inhibitors, diphenylamines, Buchwald−Hartwig amination,
structure−activity relationship
he intracellular effects of cyclic adenosine monophosphate
(cAMP) are mediated by intracellular effector proteins
T
with evolutionary conserved cAMP binding domains (CNBD).1
Prior to the discovery of exchange proteins directly activated by
cAMP (EPAC) in 1998, cAMP-mediated signaling events were
believed to be transduced largely by protein kinase A (PKA).2,3
The discovery of EPAC has changed the landscape of cAMP-
signaling research. Evidence continues to emerge underscoring
the importance of EPAC and its complex relationship to
PKA.4−6 Upon binding cAMP, EPAC proteins activate the Ras
superfamily small GTPases Rap1 and Rap2.2,3 To date, many
studies have shown the physiological and pathophysiological
significance of EPAC proteins7 and their involvement in
cancer,8,9 bacterial and viral infections,10,11 energy homeostasis
and obesity,12,13 as well as cardiac functions.14,15 Given that
mammalian EPAC proteins exist as two structurally homolo-
gous but functionally nonredundant isoforms, EPAC1 and
EPAC2,16,17 the development of isoform selective EPAC
inhibitors as molecular probes and potential therapeutics is
urgently needed. Herein we report the design, synthesis,
pharmacological characterization, and molecular docking results
of functionalized N,N-diphenylamines as potent and selective
EPAC2 inhibitors.
Figure 1. Structures of cAMP, HTS hits ESI-05 (1) and ESI-10 (2),
and designed N,N-diphenylamines as a new class of EPAC2 inhibitors.
work.22 Herein, we present the synthesis and biological activity
of N,N-diphenylamines that are structurally related to both
HTS hits ESI-05 (1) and ESI-10 (2) (Figure 1). The general
approach as depicted in Figure 1 was to retain the 2,4,6-
trimethylphenyl (mesityl) moiety or A-ring of 1, a favorable
hydrophobic fragment of EPAC antagonists identified in
previous studies.7 The incorporation of various electron
withdrawing and donating groups with differing substitution
patterns in the B-ring of the diphenyl amine was used to
Our previous high-throughput screening (HTS) campaign
led to the discovery of several validated hits with specific EPAC
inhibitory activity and was subsequently followed by extensive
medicinal chemistry optimizations.9,18−21 Among the identified
“hits”, the diarylsulfone and arylsulfonamide scaffolds related to
ESI-05 (1, Figure 1) were explored to elucidate structure−
activity relationships (SARs) as described in our previous
Received: December 10, 2015
Accepted: March 28, 2016
Published: March 28, 2016
© 2016 American Chemical Society
460
DOI: 10.1021/acsmedchemlett.5b00477
ACS Med. Chem. Lett. 2016, 7, 460−464

ACS Medicinal Chemistry Letters
Letter
explore the B-ring electronic requirements to optimize EPAC2
inhibitory activity and selectivity. To this end, a one-step
synthetic strategy based on the Buchwald−Hartwig amination
protocol7 was utilized as a facile entry to potential EPAC2
inhibitors (Scheme 1, 3−32). B-ring fragments were chosen
Table 1. IC50 Values of Functionalized N,N-Diphenylamine
Scaffolds in 8-NBD-cAMP EPAC2 Binding Assay
a
Scheme 1. Synthesis of N,N-Diphenylamine Scaffold
a
compd
R
IC50 (μM)
b
cAMP
see Figure 1
40
b
1
2
see Figure 1
0.5
b
see Figure 1
18
b
HJC0338
3
2,5-dichloro
0.4
3,4-dichloro
1.1 0.3
0.7 0.2
0.7 0.3
5.7 1.3
8.7 1.7
2.9 0.5
3.3 0.6
2.9 0.5
1.8 0.3
0.5 0.2
1.4 0.4
0.5 0.1
0.4 0.1
1.5 0.4
1.6 0.4
5.8 1.1
1.1 0.3
3.0 0.5
2.7 0.5
1.0 0.2
2.2 0.3
1.8 0.3
1.7 0.2
1.8 0.3
0.6 0.1
1.8 0.4
2.0 0.4
>300
4
2,3-dichloro
5
2,4-dichloro-6-methyl
3,5-difluoro
6
7
3,4-difluoro
8
3-chloro-5-fluoro
4-chloro-3-fluoro
3-chloro-4-fluoro
3-chloro-4-methyl
3-chloro-2-methyl
5-chloro-2-methyl
3-chloro-5-trifluoromethyl
4-chloro-3-trifluoromethyl
3-bromo
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
a
Reagents and conditions: (a) Pd2(dba)3, NaOtBu, ( )-BINAP,
toluene, 120 °C, 11−97%; (b) 48% HBr (aq), 110 °C, 50%; (c) H2,
Pd/C, MeOH, 74%.
4-bromo
based on our previous findings,17,19,22,23 commercial availability,
and coupling capacity in the described protocol. The
demethylation of methoxy and reduction of nitro groups
were achieved utilizing standard protocols to afford 33 and 34,
respectively. The synthetic protocols and detailed character-
ization data are reported in the Supporting Information (SI).
A previously described EPAC2-based assay utilizing a
fluorescent cyclic nucleotide analogue 8-NBD-cAMP was
used to quantify the test ligands’ capacity to prevent 8-NBD-
cAMP binding to EPAC2.17,18 The binding of 8-NBD-cAMP to
purified EPAC2 leads to a dose-dependent increase in
fluorescent signal, which can be reversed by cAMP or EPAC2
antagonists. Therefore, fluorescence intensity can be used to
quantify the potency of the synthesized compounds as the IC50
values (Table 1). Our previous report disclosed HJC0338
(Table 1), a diphenylamine that was able to prevent 8-NBD-
cAMP binding to EPAC2. This provided the rationale to
further investigate a larger number of analogues.22 Therefore,
dichloro analogues were synthesized and assessed to have
promising activity similar to previously reported compounds,
e.g., 2,3-dichloro derivative (4, IC50 = 0.7 μM).22 Incorporation
of a methyl into the B-ring proved beneficial as shown by 12 (3-
chloro-2-methyl, IC50 = 0.5 μM). Incorporation of fluorine,
bromine, and trichloro/trifluoro provided little to no increase
in activity (6−10, 16−21). Inclusion of trifluoromethyl resulted
in active compounds. As an example, 15 demonstrated EPAC2
inhibition (IC50 = 0.4 μM). While incorporation of a single
methyl, trifluoromethyl, ethyl, dimethyl, and other hydrophobic
moieties showed virtually no gains in inhibitory activity (22−
26, 28−30). The 3,5-bis(trifluoromethyl) derivative 27 and
2,4,6-trimethyl derivative 31 both displayed promising
inhibitory activities (IC50 = 0.6 and 0.4 μM, respectively).
Incorporation of the electron donating 3-methoxy, 3-hydroxy,
and 3-amino groups (33−34) provided no gain in activity.
Based on the obtained SAR, hydrophobicity of both phenyl
rings remains essential for inhibitory activity, consistent with
our previously reported EPAC2 antagonists.17,19
3-fluoro
3,4,5-trichloro
3,4,5-trifluoro
2,4,6-trifluoro
2,3-dimethyl
2,4-dimethyl
2,5-dimethyl
3,5-dimethyl
3,4-(CH2CH2CH2)−
3,5-bis(trifluoromethyl)
3-ethyl
3-trifluoromethyl
3-(mesitylethynyl)phenyl
2,4,6-trimethyl
3-methoxy
0.4 0.1
10.7 3.9
46.0 7.9
34.0 8.7
3-hydroxy
3-amino
a
b
SEM, n ≥ 3. See ref 22.
Several promising compounds, 12, 15, 27, and 31 (Figure 2),
were screened for selectivity between EPAC2 and EPAC1. A
previously described17 in vitro guanine nucleotide exchange
factor (GEF) functional assay was used to assess selectivity
given that the 8-NBD-cAMP binding assay is only useful for
EPAC2, not EPAC1 under HTS format (Table 2, Figure 3).
Compounds 12, 15, 27, and 31 do not display measurable
EPAC1 activity under the doses tested in the reported assay
(no effect at up to 100 μM) yet display low micromolar
inhibitory activity at EPAC2. However, ESI-09, a nonselective
EPAC 1/2 antagonist, displayed EPAC1 and EPAC2 inhibitory
activity under the same conditions (Table 2, Figure 3).18 This
suggests that the aforementioned compounds represent potent
and highly selective EPAC2 inhibitors. Interestingly, while each
compound inhibited EPAC2 GEF activity to the same extent
(>80% activity ablation, Figure 3), the same compounds vary in
their capacity to prevent 8-NBD-cAMP binding to EPAC2
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DOI: 10.1021/acsmedchemlett.5b00477
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ACS Medicinal Chemistry Letters
Letter
Figure 4. Domains of EPAC proteins. All EPAC family members
possess an N-terminal autoinhibitory regulatory region and a C-
terminal catalytic region. The regulatory region contains a Dishevelled,
Egl-10, and Pleckstrin (DEP) domain and up to two cyclic adenosine
monophosphate (cAMP) nucleotide-binding domains (CNBD). While
EPAC1 contains only one CNBD and is truncated at the N-terminus,
EPAC2 contains two cAMP binding domains (CNBD-A and CNBD-
B), which form an interface in the tertiary structure of the
autoinhibited apo-EPAC2 (PDB# 2BYV).
Figure 2. Relative potency of EPAC2 inhibitors 12, 15, 27, 31, and
cAMP; dose-dependent inhibition of 8-NBD-cAMP binding to
EPAC2; open circles, 12; open squares, 15; open triangles, 27; closed
circles, 31; ×, cAMP (n ≥ 3).
Table 2. IC50 Values of Select N,N-Diphenylamines in
cAMP-Mediated Guanine Nucleotide Exchange Factor
(GEF) Activity Assay of EPAC2 or EPAC1
exposes the catalytic region responsible for Rap activation.
However, EPAC2 has an additional cAMP binding domain
(CNBD-A) that binds cAMP with a low affinity and is not
required for EPAC2 regulation by cAMP.4 Interestingly, in the
reported autoinhibitory apo-EPAC2 structure, CNBD-A and
CNBD-B are oriented toward each other forming an interface
that blocks both cAMP cavities (Figure 4).24,25 EPAC1 lacks
this interface, given that it only contains the CNBD-B.
Therefore, it is conceivable that a small molecule can prevent
the binding of cAMP (or the 8-NBD-cAMP probe used in the
described assay) to EPAC2 by bridging CNBD-A and CNBD-B
domains. This would promote stabilization of the auto-
inhibitory, versus the active state, a transition that is known
to be highly dynamic for EPAC.25−30 This interaction is not
possible for EPAC1 as it lacks the interface. This idea is
supported by the fact that our original HTS hit 1 loses EPAC2
inhibitory activity upon deletion of CNBD-A.31 Additionally,
15 (1 μM) acted as a noncompetitive inhibitor of cAMP-
mediated EPAC2 GEF activity by producing a rightward-shift
in cAMP dose−response and reduction in maximum activation
(see SI, Figure S1), suggesting that these, and similar scaffolds,
may access an allosteric site such as the interface of CNBD-A
and CNBD-B. Noncompetitive/allosteric inhibition of EPAC1
has been proposed in previous reports of ligands that act at the
hinge region between CNBD-B and REM (Figure 4). However,
the hinge region is highly conserved in both EPAC 1 and 2.32,33
Since compounds 12, 15, 27, and 31 selectively inhibit EPAC2
but not EPAC1, it is reasonable to suggest that the
aforementioned compounds interact at the interface of both
cAMP binding domains in the EPAC2 protein. Therefore,
molecular docking to the apo-EPAC2 at the CNBD-A and
CNBD-B interface was conducted with ligands 12, 15, 27, and
31. Lowest energy poses for each are overlaid in Figure 5
showing a well-defined binding pocket between the cAMP
binding domains. A zoomed view of 15 and apo-EPAC2
(Figure 6) predicts a key cation−π interaction between the
electron rich A ring and LYS 42 of CNBD-A and an H-bond
interaction between the N,N-diphenylamine N−H and the
backbone amide carbonyl of HIS 335 of CNBD-B. Therefore,
the privileged mesityl fragment and N−H bridge the gap
between CNBD-A and CNBD-B in EPAC2, presumably
stabilizing the autoinhibited state, a binding mode that is not
possible in EPAC1 provided the absence of CNBD-A. In fact,
all but one of the compounds can be docked to this interface
with similar results indicating that a basic structural require-
ment for this predicted binding pose is the diphenylamine
IC50 (μM)
compd
EPAC2
EPAC1
a
12
15
27
31
3.6 1.0
1.0 0.1
1.3 0.2
1.7 0.3
NE
NE
NE
NE
b
ESI-09
4.4 0.5
10.8 0.6
a
b
NE (no effect at up to 100 μM). See ref 18.
Figure 3. Relative cAMP-mediated EPAC2 GEF activity in the
presence of 12, 15, 27, and 31, and ESI-09; open red circles, 12; open
squares, 15; open triangles, 27; closed circles, 31; open orange
hexagons, ESI-09 (n = 3).
(Figure 2). Compound 27 led to a decrease in 8-NBD-cAMP
binding of approximately 60%, while the other compounds
prevent roughly 80−90% of 8-NBD-cAMP binding. However,
all four compounds display nearly the same inhibitory activity
in the functional assay. This suggests that the tested EPAC2
inhibitors may not only prevent 8-NBD-cAMP binding but also
promote a conformation that decreases EPAC2 GEF activity.
To gain insight with regards to EPAC2 selectivity, molecular
docking studies were performed using the Schrodinger Drug
̈
Discovery Suite to investigate predicted binding modes of the
N,N-diphenylamine scaffold and apo-EPAC2 (PDB# 2BYV; see
Experimental Methods in the SI).24 Both EPAC1 and EPAC2
contain a conserved cAMP binding domain that binds cAMP
with high affinity (CNBD-B, Figure 4). Binding of cAMP to the
CNBD-B results in a conformational change of EPAC that
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ACS Medicinal Chemistry Letters
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EPAC1 while exhibiting submicromolar potency at EPAC2. To
the best of our knowledge, compounds 12, 15, 27, and 31 are
comparable with the most potent of any synthesized EPAC2
selective inhibitors reported to date. These compounds, with
decent physicochemical profiles (i.e., avg ligand efficiency =
0.41. avg tPSA = 13, avg Mw = 268, avg ClogP = 5.4; see SI,
Table S1), may be useful in facilitating our efforts in uncovering
the isoform-specific roles of EPAC proteins. Further studies to
improve the drug-likeness (e.g., lipophilicity and solubility) of
these molecules while maintaining activity/selectivity are
currently underway.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00477.
Figure 5. Lowest energy docking pose overlay of 12, 15, 27, and 31
(gray stick) with autoinhibited apo-EPAC2 (ribbon, PDB code:
2BYV); key residues LYS 42 and HIS 335 (blue stick); CNBD-A
highlighted with orange ellipse; CNBD-B highlighted with light blue
ellipse.
Detailed experimental procedures, spectroscopic data of
all compounds, and methods of biological evaluation
(PDF)
AUTHOR INFORMATION
Corresponding Authors
*(X.C.) Tel: (713) 500-7487. Fax: (409) 500-7465. E-mail:
xiaodong.cheng@uth.tmc.edu.
■
*(J.Z.) Tel: (409) 772-9748. Fax: (409) 772-9648. E-mail:
jizhou@utmb.edu.
Author Contributions
§These authors contributed equally to this work and should be
considered as co-first authors.
Funding
This work was supported by grants R01 GM066170,
GM106218, and F31 DA038922-01A1 from the National
Institutes of Health, a training fellowship from the Keck Center
for Interdisciplinary Bioscience Training of the Gulf Coast
Consortia (GCC) (NIGMS grant T32 GM089657), and the
Center for Addiction Research (NIDA grant T32 DA07287).
Figure 6. Zoomed-view of lowest energy docking pose of 15 (gray
stick/wire-mesh) with autoinhibited apo-EPAC2 (ribbon, PDB #
2BYV); key residues LYS 42 (blue stick) from CNBD-A and HIS 335
amide (green stick) from CNBD-B.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
scaffold. An exception, compound 30, is not able to access the
proposed allosteric site due to its size, consistent with the fact
that compound 30 was ineffective in preventing 8-NBD-cAMP
binding (Table 1). Moreover, ligands with strong electron
donating groups (−OMe, −OH, −NH2; 32−34) consistently
display IC50 values weaker than the other compounds tested.
Given that the docking indicates that the N,N-diphenylamine
N−H interacts with the amide carbonyl of HIS 335 of CNBD-
B, it is conceivable that −OMe, −OH, and −NH2 disrupt the
avidity of this predicted H-bonding interaction due to their π-
donating properties through the aromatic ring.
In summary, a series of N,N-diphenylamine derivatives have
been designed, synthesized, and evaluated systematically as
EPAC2-specific inhibitors. Molecular docking was further used
to provide insight into potential binding modes. Specifically, an
allosteric site unique to EPAC2 was identified and the predicted
ligand−protein interactions were used to rationalize the
observed SAR data and should stimulate new strategies toward
the inhibition of EPAC proteins. Compounds 12 (CTW0151),
15 (MAY0132), 27 (CTW0181), and 31 (CTW0167)
displayed no substantial activity at the highly homologous
We would like to thank Drs. Lawrence C. Sowers and Jacob A.
Theruvathu at the Department of Pharmacology and Dr.
Tianzhi Wang at the NMR core facility of UTMB for the NMR
spectroscopy assistance.
ABBREVIATIONS
■
EPAC, exchange protein directly activated by cAMP; cAMP,
cyclic adenosine monophosphate; PKA, protein kinase A; HTS,
high-throughput screening; SARs, structure−activity relation-
ships; GEF, guanine nucleotide exchange factor; CNBD, cyclic
nucleotide binding domain; 8-NBD-cAMP, 8-(2-[7-nitro-4-
benzofurazanyl]aminoethylthio)adenosine-3′,5′-cyclic mono-
phosphate; Pd2(dba)3, tris(dibenzylidene-acetone)dipalladium-
(0); NaOtBu, sodium tert-butoxide; ( )-BINAP, 2,2′-bis-
(diphenylphosphino)-1,1′-binaphthyl
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