Structure-activity relationships of 2-substituted phenyl-N-phenyl-2-oxoacetohydrazonoyl cyanides as novel antagonists of exchange proteins directly activated by cAMP (EPACs)

Article abstract of DOI:10.1016/j.bmcl.2017.10.056

Exchange proteins directly activated by cAMP (EPACs) are critical cAMP-dependent signaling pathway mediators that play important roles in cancer, diabetes, heart failure, inflammations, infections, neurological disorders and other human diseases. EPAC spe

Full text of DOI:10.1016/j.bmcl.2017.10.056

Accepted Manuscript
Structure-activity relationships of 2-substituted phenyl-N-phenyl-2-oxoaceto-
hydrazonoyl cyanides as novel antagonists of exchange proteins directly acti-
vated by cAMP (EPACs)
Zhiqing Liu, Yingmin Zhu, Haiying Chen, Pingyuan Wang, Fang C. Mei, Na
Ye, Xiaodong Cheng, Jia Zhou
PII:
S0960-894X(17)31054-5
https://doi.org/10.1016/j.bmcl.2017.10.056
BMCL 25385
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
20 September 2017
19 October 2017
22 October 2017
Please cite this article as: Liu, Z., Zhu, Y., Chen, H., Wang, P., Mei, F.C., Ye, N., Cheng, X., Zhou, J., Structure-
activity relationships of 2-substituted phenyl-N-phenyl-2-oxoacetohydrazonoyl cyanides as novel antagonists of
exchange proteins directly activated by cAMP (EPACs), Bioorganic & Medicinal Chemistry Letters (2017), doi:
https://doi.org/10.1016/j.bmcl.2017.10.056
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Bioorganic & Medicinal Chemistry Letters
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Structure-activity relationships of 2-substituted phenyl-N-phenyl-2-
oxoacetohydrazonoyl cyanides as novel antagonists of exchange proteins directly
activated by cAMP (EPACs)
a,1
b,1
a
a
b
a
Zhiqing Liu , Yingmin Zhu , Haiying Chen , Pingyuan Wang , Fang C. Mei , Na Ye , Xiaodong
b,
a,
Cheng *, and Jia Zhou *
a
Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Galveston,
Texas 77555, United States
b
Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, The University of Texas Health Science Center, 7000 Fannin St #1200,
Houston, Texas 77030, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Exchange proteins directly activated by cAMP (EPACs) are critical cAMP-dependent signaling
pathway mediators that play important roles in cancer, diabetes, heart failure, inflammations,
infections, neurological disorders and other human diseases. EPAC specific modulators are
urgently needed to explore EPAC’s physiological function, mechanism of action and therapeutic
applications. On the basis of a previously identified EPAC specific inhibitor hit ESI-09, herein
we have designed and synthesized a novel series of 2-substituted phenyl-N-phenyl-2-
oxoacetohydrazonoyl cyanides as potent EPAC inhibitors. Compound 31 (ZL0524) has been
discovered as the most potent EPAC inhibitor with IC50 values of 3.6 µM and 1.2 µM against
EPAC1 and EPAC2, respectively. Molecular docking of 31 onto an active EPAC2 structure
predicts that 31 occupies the hydrophobic pocket in cAMP binding domain (CBD) and also
opens up new space leading to the solvent region. These findings provide inspirations for
discovering next generation of EPAC inhibitors.
Keywords:
Exchange proteins directly activated by cAMP
EPAC
Antagonist
Molecular docking
2
017 Elsevier Ltd. All rights reserved.
2
Cyclic adenosine monophosphate (cAMP, 1), generated from
adenosine triphosphate (ATP) by adenylyl cyclase, is a second
messenger for intracellular signal transduction in many different
organisms. cAMP-mediated signaling events were considered to
be transduced largely by protein kinase A (PKA) until the
discovery of exchange proteins directly activated by
cAMP(EPACs)/cAMP regulated guanine nucleotide exchange
mainly found in CNS, pancreatic islets and adrenal gland.
EPAC1 and EPAC2 are structurally homologous but functionally
nonredundant. A number of studies have revealed that EPAC
proteins are critically involved in a variety of human diseases
such as cancer, inflammation, bacterial and viral infections,
central nervous system disorders, energy homeostasis and
8
-14
obesity, and cardiac functions.
1
-4
factor (cAMP-GEF). These two intracellular receptor families
mediated the major physiological effects of cAMP in mammalian
cells through the cAMP binding domain (CBD) which acts as a
Given the physiological and pathophysiological significance
of EPAC proteins, the development of pharmacological EPAC
modulators is needed. Most EPAC agonists are derivatives of
cAMP, while non-cyclic nucleotide ligands usually display
EPAC inhibitory activities, except a very recently reported small
1
5
5
, 6
16
molecular switch for controlling various cellular activities. The
identification of EPACs opens new avenues for cAMP signaling
7
17
research. Unlike PKA, EPAC proteins do not have kinase
molecule partial agonist with modest potency. Cheng, et al
activity and activate the Ras superfamily small GTPases Rap1
and Rap2 in response to the generation of intracellular cAMP.
There are two isoforms of mammalian EPACs, EPAC1 which is
more ubiquitously expressed and EPAC2 which is
developed a sensitive and robust fluorescence-based high
18
throughput (HTS) assay, which led to the discovery of a series
1
9
of non-cyclic nucleotide EPAC inhibitors. After extensive
modifications by our team, dihydropyrimidine 2 (HJC0198, Fig.
1
) was obtained with an IC50 value of 4.0 µM against EPAC2. It
—
——
selectively blocks cAMP-induced EPAC activation without
Corresponding authors. Jia Zhou, Tel.: +1-409-772-9748; fax: +1-409-772-9648; e-mail: jizhou@utmb.edu. Xiaodong Cheng, Tel.: +1-713-
00-7487; fax: +1-713-500-7456; e-mail: xiaodong.cheng@uth.tmc.edu.
*
5

affecting cAMP-mediated PKA activation at the concentration of
transformed into corresponding diazo salts and coupled with 8 in
the presence of NaOAc to produce compounds 6 and 10~18 in
good yields.
20
2
5 µM. Diphenylamine 3 (MAY0132, Fig. 1), also developed
by our team, exhibited potent and selective EPAC2 inhibitory
activity with an IC50 value of 0.4 µM and inhibited cAMP-
mediated EPAC2 GEF activity with IC50 of 1 µM, while
displaying no significant inhibition against EPAC1 at 100
2
1,22
µM.
Subtype selective compound will not only help
understanding diverse functions of EPACs but also may
contribute to therapeutic safety in the future, thus further
modifications on compound 3, a good EPAC2 selective inhibitor,
are undergoing in our lab. Compound 4 ((R)-CE3F4, Fig. 1),
Scheme 1. Synthetic route to access compounds 6 and 10~18. Reagents and
2
3
identified by Courileau and coworkers via HTS screening,
displayed EPAC1 inhibitory activity with an IC50 value of 4.2
3 3 2
conditions: (a) CH CN, CH Li, -78 C̊ , 1h, 64%; (b) i): 10% HCl, NaNO ,
H
2
O, rt.; ii): NaOAc, EtOH, rt., 67% to quant. for two steps.
2
4
µM with 10-fold selectivity over EPAC2. In our assays, its
inhibitory activities against EPAC1 and EPAC2 are 5.5 and 17
µM, respectively. Compound 5 (ESI-09, Fig. 1) is another
promising hit discovered by our team, displaying micromolar
The second series of compounds were designed to replace
tert-butyl group on phenyl ring B, and the synthetic protocol is
depicted in Scheme 2. 3,5-Dichloroaniline 19 was transformed
into its corresponding diazo salt and coupled with different 3-
oxo-3-phenylpropanenitriles 21 in the presence of NaOAc to give
compounds 22~33 in moderate to good yields. All the structures
25-27
inhibitory activities against both EPAC1 and EPAC2.
Herein,
we describe the discovery of a series of novel EPAC inhibitors
such as compound 31, which displays enhanced potency against
EPAC proteins in comparison with chemical lead 5.
1
and purity of synthesized compounds were confirmed by H
1
3
31
NMR, C NMR and HR-MS.
Scheme 2. Synthetic route of compounds 22~33. Reagents and conditions: (a)
0% HCl, NaNO , H O, rt; (b) NaOAc, EtOH, rt., 37% to quant.
1
2
2
Compounds were then evaluated for their abilities to inhibit
EPAC1 and EPAC2-mediated Rap1b-bGDP exchange activity
using purified recombinant full-length EPAC1 and EPAC2
32
proteins. For promising compounds, the IC50 values against
both EPAC1 and EPAC2 were determined (Table 1 and Table 2).
Fig. 1. The structures of cAMP and representative EPAC antagonists
including newly identified 31 (ZL0524) reported in this paper.
Compound
5 was used as the reference compound for
comparison with IC50 values of 10.8 µM and 4.4 µM against
EPAC1 and EPAC2, respectively. Based on our previous SAR
study, 3-Cl or 3-CF on phenyl ring A which resides in a
3
While previously reported optimizations on compound 5 focused
2
8
on the phenyl ring A (Fig. 2) and substituents on isoxazole ring,
other alternatives to replace isoxazole ring were never explored.
Herein, we propose to replace the isoxazole with its bioisosteric
substituted phenyl ring B (Fig. 2) to explore new chemical space
hydrophobic pocket P1 is favorable for EPAC binding. Thus, for
most newly designed compounds, we chose to retain these
substituents. Compared to compound 6, compound 10 with an
additional 5-Cl substituent has 2.5-fold improvement (IC50 = 5.4
µM) on EPAC1 inhibitory activity and 11-fold improvement
(IC50 = 2.5 µM) on EPAC2 inhibitory activity. In addition,
substituent at 5-position is more favorable than that with a same
group at 4-positon on this phenyl ring A (compound 11 vs 12,
and compound 16 vs 17). Triple substituents at 3,4,5-position
displayed slightly decreased EPAC inhibition (compounds 13
and 18), but compound 18 has a 4.6-fold EPAC2/EPAC1
2
9
and improve EPAC inhibitory potency. Docking studies of
compound 5 into the cAMP binding domain of active EPAC2
suggested that 3-chloro phenyl ring A and tert-butylisoxazole
occupy two hydrophobic pockets and play important roles for
1
5
their interaction.
Compound 6 was synthesized and gave an
IC50 value of 13.3 µM, showing the phenyl bioisosteric
replacement was a viable route forward.
3
selectivity. 3,5-Di CF substituted compound 14 showed similar
EPAC inhibitory activities to those of compound 10.
Table 1. IC50 values of substituted 2-(4-(tert-butyl)phenyl)-N-
phenyl-2-oxoacetohydrazonoyl cyanides for inhibiting
EPAC1/2 GEF activity.
Fig. 2. Design strategy of 2-substituted phenyl-N-phenyl-2-oxoaceto
hydrazonoyl cyanides using a bioisosteric replacement approach.
The first series of compounds were designed to keep the tert-
butyl phenyl group B intact and the synthetic route is depicted in
Scheme 1. Starting material methyl 4-(tert-butyl)benzoate (7)
Rap1b-bGDP
Rap1b-bGDP
EPAC2
IC50 (µM)
Entry
R
EPAC1 a
was reacted with CH
3
CN in the presence of CH Li to give 3-(4-
3
IC50 (µM)
30
(
tert-butyl)phenyl)-3-oxopropanenitrile (8). Anilines 9 were

a
IC50 (µM)
IC50 (µM)
5
6
-
10.8 ± 1.6
13.3 ± 3.0
5.4 ± 0.7
12.4 ± 0.9
6.4 ± 1.4
24.4 ± 5.7
5.5 ± 0.7
6.1 ± 1.0
5.6 ± 0.9
50.8 ± 14.6
4.4 ± 0.5
28.4 ± 5.6
2.5 ± 0.6
3-Cl
10
22
23
24
25
26
4-tert butyl
4-Cl
5.4 ± 0.7
>300
2.5 ± 0.6
b
1
0
1
2
3
4
5
6
7
8
3,5-di Cl
3-Cl, 4-F
3-Cl, 5-F
3,4,5-tri F
ND
b
1
1
1
1
1
1
1
1
ND
3-Cl
>300
ND
ND
ND
ND
3.4 ± 0.6
15.8 ± 3.8
6.3 ± 0.9
12.4 ± 3.9
5.4 ± 1.1
ND
4-OCF
3
40.9 ± 6.8
29.3 ± 6.2
>300
4-CO Me
2
3,5-di CF
3-CF , 4-Cl
3
4-Ph
3
27
28
3,4-di OMe
>300
ND
ND
3-Cl, 5-CF
3
3
4-Piperidin-1-ylmethyl
39.4 ± 4.7
3-Cl, 4-CF
29
30
4-Morpholinomethyl
4-Cyclohexane
14.6 ± 5.7
32.4 ± 9.5
ND
ND
3,4,5-tri Cl
13.9 ± 3.6
3.0 ± 0.4
a
b
The values are the mean ± SE, n = 3. ND: not determined.
3
1
3
.6 ± 0.3
1.2 ± 0.1
The first round of optimization illustrated that all compounds
obtained via bioisosteric replacement of tert-butyl isoxazole ring
with tert-butyl phenyl group retained both EPAC1 and EPAC2
inhibitory activities, while nearly half of them displayed more
potent inhibitory activities than reference compound 5. 3,5-di Cl
substituents on phenyl ring A are favored and compound 10
exhibited the most potent EPAC1 and EPAC2 inhibitory
activities. Thus, we kept this fragment intact and further modified
substituents on phenyl ring B which occupies the other
hydrophobic pocket P2 (Table 2). We investigated 3-Cl (22) and
3
3
2
3
11.3 ± 1.4
19.6 ± 6.7
2.5 ± 0.5
ND
a
b
The values are the mean ± SE. ND: not determined.
4
-Cl (23) first, but both are detrimental to EPAC inhibition. It
appears that the bulk size of substituents on phenyl ring B is
critical. We then explored electron donating group 4-OCF (24)
and electron withdrawing group 4-CO Me (25). Interestingly,
3
2
neither of them showed increased inhibition against EPAC1
compared to compound 10. Introduction of 4-Ph and 3,4-di OMe
substituents on phenyl ring B led to compounds 26 and 27 which
have a complete loss of EPAC inhibitory activities. The various
rings including piperidine (28), morpholine (29) and cyclohexane
(
30) were also investigated, but none of them displayed more
potent EPAC inhibition than compound 10. However,
compounds 31~33 with fused rings (e.g. 1,2,3,4-
tetrahydronaphthalene, naphthalene and quinoline) all displayed
improved EPAC inhibitory activities when compared to afore-
mentioned compounds that have different substituents on phenyl
ring B. Among them, compound 31 exhibited the most potent
EPAC inhibitory activities with IC50 values of 3.6 µM and 1.2
µM against EPAC1 and EPAC2, respectively. In Fig. 3, examples
of dose-response curves are shown for compounds 5, 6, 14 and
Fig. 3. A) Relative inhibitory activity for EPAC1-mediated Rap1b-bGDP
exchange. B) Relative inhibitory activity for EPAC2-mediated Rap1b-bGDP
exchange. Dose-dependent inhibition of EPAC1/2 GEF activity by
compounds 5, 6, 14, and 31, in the presence of 20 µM cAMP. Relative GEF
activity were presented as normalized reaction rate constant (means ± SEM, n
3
1.
Table 2. IC50 values of substituted 2-phenyl-N-(3,5-
dichlorophenyl)-2-oxoacetohydrazonoyl
inhibiting EPAC1/2 GEF activity.
cyanides
for
=3) described in the method.
Molecular docking study of compound 31 was performed
using the Schrödinger Small-Molecule Drug Discovery Suite
taking advantage of known X-ray crystal structures of activated
3
3
EPAC2 protein. The model shows that compound 31 occupies
the CBD of EPAC2 and forms hydrogen bonds with Arg448 and
the cyano-hydrazine substituent (Fig. 4A). The fused
tetrahydronaphthalene ring B lays in the same pocket as tert-
butyl isoxazole ring of ESI-09 does and is surrounded by
Rap1b-bGDP
EPAC1
Rap1b-bGDP
EPAC2
Entry
R or X

hydrophobic Ala416, Leu406, Val447, Val386, etc. Interestingly,
ring A of compound 31 extends to the opposite direction (the
solvent region) compared to ESI-09 (Fig. 4B). This docking
mode suggests that there exists a large space deep pocket
remaining to be explored in the CBD domain of EPAC protein
for the discovery of next generation ligands.
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and synthetic ease suitable for scale up. Among those new
molecules, compound 31 (ZL0524) was the most potent EPAC
inhibitory activities with IC50 values of 3.6 µM and 1.2 µM
against EPAC1 and EPAC2, respectively. Docking studies of
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1. Spectra data of the representative compounds: (E)-2-(4-(tert-
3
3
Butyl)phenyl)-N-(3,5-dichlorophenyl)-2-oxoacetohydrazonoyl cyanide (10).
1
Yellow solid, 69%. H NMR (300 MHz, DMSO-d
6
) δ 12.43 (s, 1H), 7.81 (d,
Acknowledgements
1
3
J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.31 (s, 2H), 1.33 (s, 9H). C NMR
75 MHz, DMSO-d ) δ 187.29, 166.62, 156.14, 145.04, 135.30, 133.76,
30.36, 125.25, 123.96, 115.50, 115.30, 111.49, 35.28, 31.29. HRMS (ESI):
(
6
This work was supported by grants R01 GM106218, R01
AI111464, R01 GM066170 and R35 GM122536 from the
National Institutes of Health. We want to thank Drs. Lawrence C.
Sowers at the Department of Pharmacology as well as Dr.
Tianzhi Wang at the NMR core facility of UTMB for the NMR
spectroscopy assistance.
1
+
m/z (M + Na) calcd for C19
2 3
H17Cl N NaO: 396.0646, found: 396.0670. (E)-2-
(
4-(tert-Butyl)phenyl)-N-(3-chloro-5-fluorophenyl)-2-oxoacetohydrazonoyl
1
cyanide (12). Yellow solid, quant. H NMR (300 MHz, MeOD) δ 7.88 (d, J =
8
6
.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.21 (s, 1H), 7.04 (d, J = 10.2 Hz, 1H),
13
.94 (d, J = 8.4 Hz, 1H), 1.39 (s, 9H). C NMR (75 MHz, MeOD) δ 187.07,
References and notes
164.99, 161.71, 156.55, 144.61, 135.93, 135.77, 133.19, 129.84, 124.80,
1
12.00, 111.96, 111.60, 111.26, 109.70, 101.86, 101.50, 34.61, 30.09. HRMS
1
.
X. Cheng, Z. Ji, T. Tsalkova, F. Mei, Acta Biochim. Biophys. Sin.
+
(
(
ESI): m/z (M + H) calcd for C19
H18ClFN
3
O: 358.1122, found: 358.1123.
(
Shanghai) 2008, 40, 651-662.
E)-N-(3,5-Bis(trifluoromethyl)phenyl)-2-(4-(tert-butyl)phenyl)-2-

1
oxoacetohydrazonoyl cyanide (14). Yellow solid, 67%. H NMR (300 MHz,
1
3
MeOD) δ 7.88 (m, 4H), 7.69 (s, 1H), 7.62 – 7.56 (m, 2H), 1.40 (s, 9H).
C
NMR (75 MHz, MeOD) δ 187.29, 156.50, 144.04, 133.30, 133.18, 132.86,
1
1
32.41, 131.97, 129.82, 128.53, 124.92, 124.70, 121.32, 117.71, 116.96,
16.90, 116.85, 116.80, 116.75, 116.23, 115.80, 115.76, 109.47, 34.57, 30.05.
+
HRMS (ESI): m/z (M + H) calcd for C21
H
18
F
6
N
3
O: 442.1354, found:
4
42.1355. (E)-2-(4-(tert-Butyl)phenyl)-N-(3-chloro-5-(trifluoromethyl)phenyl
1
)-2-oxoacetohydrazonoyl cyanide (16). Yellow solid, 76%. H NMR (300
MHz, DMSO-d ) δ 12.58 (s, 1H), 7.81 (d, J = 7.7 Hz, 2H), 7.56 (m, 5H), 1.32
6
1
3
(
s, 9H). C NMR (75 MHz, DMSO-d
6
) δ 187.37, 156.13, 144.79, 135.46,
1
1
33.68, 132.39, 131.95, 130.36, 125.16, 120.80, 120.00, 115.71, 111.78,
+
11.33, 35.23, 31.22. HRMS (ESI): m/z (M
+ Na) calcd for
C
20
H
17
F
3
N
3
ClNaO: 430.0910, found: 430.0956. (E)-N-(3,5-Dichlorophenyl)-
2
-oxo-2-(5,6,7,8-tetrahydronaphthalen-2-yl)acetohydrazonoyl cyanide (31).
1
Yellow solid, 59%. H NMR (300 MHz, DMSO-d
6
) δ 12.42 (s, 1H), 7.68 (s,
1
4
1
1
H), 7.58 (d, J = 8.0 Hz, 1H), 7.34 (s, 3H), 7.21 (d, J = 8.0 Hz, 1H), 2.81 (s,
1
3
H), 1.78 (s, 4H). C NMR (75 MHz, DMSO) δ 186.93, 144.98, 142.79,
36.83, 135.34, 133.44, 131.65, 129.19, 127.36, 123.94, 115.57, 115.23,
+
11.44, 29.39, 29.28, 22.95, 22.84. HRMS (ESI): m/z (M + H) calcd for
C
3
19
H
16
N
3
Cl
2. N. Ye, Y. Zhu, Z. Liu, F. C. Mei, H. Chen, P. Wang, X. Cheng, J. Zhou,
Eur. J. Med. Chem. 2017, 134, 62-71.
3. Molecular docking studies: The docking study was performed with
2
O: 372.0670, found: 372.0667.
3
Schrödinger Small-Molecule Drug Discovery Suite. The crystal structure of
EPAC2 (PDB code: 3CF6) was downloaded from RCSB PDB Bank and
prepared with Protein Prepared Wizard. During this step, hydrogens were
added, crystal waters were removed, and partial charges were assigned using
the OPLS-2005 force field. The 3D structures of ESI-09 and ZL0524 were
created with Schrödinger Maestro, and the initial lowest energy
conformations were calculated with LigPrep. For all dockings, the grid center
was chosen on the centroid of included ligand of PDB structure CBD-B site
and a 24 × 24 × 24 Å grid box size was used. All dockings were employed
with Glide using the XP protocol. Docking poses were incorporated into
Schrödinger Maestro for a visualization of ligand-receptor interactions and
overlay analysis.

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Graphical Abstract
Structure-Activity Relationships of 2-
Substituted Phenyl-N-Phenyl-2-
Leave this area blank for abstract info.
Oxoacetohydrazonoyl Cyanides as Novel
Antagonists of Exchange Proteins Directly
Activated by cAMP (EPACs)
a,1
b,1
a
a
b
a
b,
Zhiqing Liu , Yingmin Zhu , Haiying Chen , Pingyuan Wang , Fang C. Mei , Na Ye , Xiaodong Cheng *,
a,
and Jia Zhou *