5-Cyano-6-oxo-1,6-dihydro-pyrimidines as potent antagonists targeting exchange proteins directly activated by cAMP

Article abstract of DOI:10.1016/j.bmcl.2012.04.082

Exchange proteins directly activated by cAMP (Epac) are a family of guanine nucleotide exchange factors that regulate a wide variety of intracellular processes in response to second messenger cAMP. To explore the structural determinants for Epac antagonist properties of high throughput screening (HTS) hit ESI-08, pyrimidine 1, a series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues have been synthesized and evaluated for their activities for Epac inhibition. Structure-activity relationship (SAR) analysis led to the identification of three more potent Epac antagonists (6b, 6g, and 6h). These inhibitors may serve as valuable pharmacological probes for further elucidation of the physiological functions and mechanisms of Epac regulation. Our SAR results and molecular docking studies have also revealed that further optimization of the moieties at the C-6 position of pyrimidine scaffold may allow us to discover more potent Epac-specific antagonists.

Full text of DOI:10.1016/j.bmcl.2012.04.082

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4038–4043
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Cyano-6-oxo-1,6-dihydro-pyrimidines as potent antagonists targeting
exchange proteins directly activated by cAMP
Haijun Chen a, Tamara Tsalkova a,b, Fang C. Mei a,b, Yaohua Hu a,b, Xiaodong Cheng a,b, , Jia Zhou a,
⇑
⇑
a Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA
b Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Exchange proteins directly activated by cAMP (Epac) are a family of guanine nucleotide exchange factors
that regulate a wide variety of intracellular processes in response to second messenger cAMP. To explore
the structural determinants for Epac antagonist properties of high throughput screening (HTS) hit ESI-08,
pyrimidine 1, a series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues have been synthesized and
evaluated for their activities for Epac inhibition. Structure–activity relationship (SAR) analysis led to
the identification of three more potent Epac antagonists (6b, 6g, and 6h). These inhibitors may serve
as valuable pharmacological probes for further elucidation of the physiological functions and mecha-
nisms of Epac regulation. Our SAR results and molecular docking studies have also revealed that further
optimization of the moieties at the C-6 position of pyrimidine scaffold may allow us to discover more
potent Epac-specific antagonists.
Received 14 February 2012
Revised 16 April 2012
Accepted 17 April 2012
Available online 26 April 2012
Keywords:
Pyrimidine analogues
Exchange proteins directly
activated by cAMP (Epac)
Antagonists
Ó 2012 Elsevier Ltd. All rights reserved.
Structure–activity relationship (SAR)
The cyclic adenosine monophosphate (cAMP) signaling
pathway, widely viewed as the ‘prototypic’ second messenger
signaling pathway, regulates a myriad of biological processes,
including cardiac and smooth contraction, insulin secretion, neuro-
transmitter release, learning and memory, inflammation, cell
growth, cell differentiation, and cell apoptosis.1–5 The effects of
cAMP in mammalian cells are attributed to the activation of two
intracellular cAMP targets: the classic cAMP-dependent protein
kinase (PKA) and the more recently discovered exchange protein
directly activated by cAMP (Epac, a.k.a. cAMP-GEF).6–8 By far the
best-studied aspect of cAMP signaling involves cAMP-mediated
activation of PKA. Since the discovery of the Epac proteins in
1998,9 significant progress has been made toward elucidating the
molecular mechanism of Epac activation. These new findings have
altered the prevailing viewpoints about cAMP signaling that previ-
ously had been ascribed mainly to PKA, and the contribution of Epac
is now more and more appreciated.10,11 Epac proteins are guanine
nucleotide exchange factors for Ras-like small GTPases, Rap1 and
Rap2. There are two isoforms of Epac, Epac1, and Epac2, which have
been implicated in a number of cellular processes.12–15 Epac pro-
teins exert their diverse biological functions alone, in concert with
PKA, and/or even in opposed to PKA.16 Therefore, it remains a great
challenge to understand the complexity of Epac activation/inhibi-
tion within the cAMP signaling pathway and to provide insights
into the molecular mechanisms of Epac in the regulation of multiple
effectors and biological functions.
To distinguish the signaling pathway activated by Epac proteins,
membrane-permeable cAMP analogues with high affinity and spec-
ificity for Epac proteins are needed. The 8-pCPT-20-O-Me-cAMP
(a.k.a. 007), a selective agonist for Epac proteins, is very useful in
unraveling the role of Epac in a wide variety of biological processes,
ranging from exocytosis of insulin to the regulation of calcium
channels and permeability of the vascular endothelium.17–21 While
007 has become a widely used tool in Epac-related research, its
application is greatly limited by its low membrane permeability be-
cause of the negatively charged phosphate group. To mitigate this
issue, an AM (acetoxymethyl)-ester (007-AM) has been introduced
to mask the negatively charged group.22 The ester appendage of
007-AM can be intracellularly removed by esterase to generate
007. Nevertheless, 007 can also bind and inhibit phosphodiester-
ase-1, -2, and -6, and thus indirectly increase intracellular cAMP
or/and 30-50-cyclic guanosine monophosphate (cGMP) levels, and
eventually activate PKA, PKG and/or cyclic nucleotide gated chan-
nels.23 Meanwhile, accumulating evidence demonstrated that Epac
proteins may represent new mechanism-based therapeutic targets
for the treatment of various human disorders such as diabetes,
heart disease and cancer.24–28 Therefore, development of Epac
antagonists as useful pharmacological probes to dissect the physio-
logical functions that Epac proteins play in the overall cAMP-med-
iated signaling remains a major challenge in the research field.29
In an attempt to identify novel molecular antagonists that are
capable of specifically inhibiting Epac, an automated, fluores-
cence-based high throughput screening (HTS) assay has been
⇑
Corresponding authors. Tel.: +1 409 772 9656; fax: +1 409 772 7050 (X.C.);
tel.: +1 409 772 9748; fax: +1 409 772 9818 (J.Z.).
E-mail addresses: xcheng@utmb.edu (X. Cheng), jizhou@utmb.edu (J. Zhou).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.082

H. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4038–4043
4039
developed by our team using a fluorescent cyclic nucleotide analog,
O
O
NC
R1
8-NBD-cAMP (8-(2-[7-Nitro-4-benzofurazanyl]aminoethylthio)
adenosine-30,50-cyclic monophosphate). This competitive assay
which is performed in a ‘mix and measure’ format, is based upon
a dramatic dose-dependent change in fluorescence signal of the
binding of 8-NBD-cAMP to Epac2.30 A Maybridge Hitfinder com-
pound library of 14,400 chemically diversified small molecules
has been screened. ESI-08 (1) has been identified as an Epac
antagonist (Fig. 1) which is capable of completely inhibiting both
S
OMe
NH
SH
a
NC
R1
H
+
+
H2N
NH2
O
3
N
5a-f
2a-f
4
O
NC
R1
NH
b
N
S
R2
Epac1 and Epac2 activity at 25 lM in the presence of equal molar
6a-i
concentration of cAMP (unpublished data). Herein, we report the
synthesis and the exploration of the structure–activity relation-
ship (SAR) of the 5-cyano-6-oxo-1,6-dihydro-pyrimidine ana-
logues as potent antagonists targeting Epac. Molecular
modeling studies of these novel Epac inhibitors were also per-
formed using AutoDock Vina docking approach.31
6j
6k
R3
c
O
NC
O
H
NH
N
NH2
a
3
+
+
H
N
N
H
R3
NH
Using the HTS hit ESI-08 as a starting point, structural modifica-
tions of three major sites on the 5-cyano-6-oxo-1,6-dihydro-
pyrimidine scaffold have been explored. The synthesis of various
pyrimidine analogues is outlined in Scheme 1. The reaction of
methyl cyanoacetate with the corresponding aldehydes 2a–f and
thiourea in the presence of piperidine refluxing in EtOH afforded
the pyrimidine thiones (or thiouracils) 5a–f.32 Selective S-alkyl-
ation of 5a–f with the appropriate benzyl halide in the presence
of K2CO3 in acetone gave the desired products 6a–j.33 Removal of
the Boc group from 6j with trifluoroacetic acid yielded the desired
compound 6k. Condensation of 2a with methyl cyanoacetate 3 and
appropriate phenylguanidines 7a–b provided analogues 8a–b.
The S-alkylation reaction of 5a with bromoacetic acid 9 afforded
the intermediate 10. Compound 10 was then condensed with the
appropriate anilines in the presence of EDCI and DIPEA to form
11a–c. The structures and purity of all synthesized compounds
were confirmed by 1H and 13C NMR, HR-MS and HPLC analysis.34
Synthesized 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues
5a, 6a–k, 8a–b, 10 and 11a–c were screened for their relative
binding affinity of Epac2 by using dose-dependent titrations to
compete the binding of 8-NBD-cAMP to Epac2.30 Apparent IC50
values of these analogues are summarized in Table 1. As illustrated
in Table 1, the pharmacophore moieties at the 2-position of the
pyrimidine scaffold have demonstrated very crucial to the activity,
and an appropriate hydrophobic group such as S-benzyl moiety is
required to achieve the activity of these analogues to target Epac.
For example, compound 5a with a mercapto group at the 2-posi-
tion that is not alkylated with a hydrophobic group displayed no
7a-b
2a
8a-b
O
O
NC
NC
NH
NH
b
OH
Br
OH
+
N
S
N
SH
O
O
5a
9
10
O
N
NC
NH
H
N
d
S
R4
O
11a-c
Scheme 1. Synthesis of the 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues.
Reagents and conditions: (a) piperidine, EtOH, reflux; 5a R1 = cyclohexyl, 51%; 5b
R1 = isopropyl, 55%; 5c R1 = cyclopropyl, 18%; 5d R1 = cyclopentyl, 62%; 5e R1 = 1-
methyl-piperidin-4-yl, 69%; 5f R1 = 1-Boc-piperidin-4-yl, 59%; 8a R3 = H, 48%; 8b
R3 = 4-Cl, 49%; (b) various benzyl halides, K2CO3, acetone, rt; 6a 93%; 6b 87%; 6c
91%; 6d 94%; 6e 95%; 6f 75%; 6g 80%; 6h 92%; 6i 65%; 6j 77%; (c) TFA, DCM, 0 °C,
92%; (d) EDCI, DIPEA, various anilines, DCM, rt; 11a 91%; 11b 80%; 11c 82%.
activity (IC50 >300 lM). Given that the hydrophobic group is
important for the potency and the hit (1) itself has a 2,5-dimethyl
substituent on the phenyl ring, analogues 6a–e with substituents
of proton, methyl, dimethyl, or trimethyl groups at various posi-
tions of the aromatic ring were initially explored and compared.
Among compounds 6a–e, 6c and 6e demonstrated a moderate po-
tency against Epac2 with IC50 values of 15.7 and 11.6
tively. The 3,5-dimethyl analogue 6b, with an IC50 value of
7.0 M, showed a slightly better activity than the hit ESI-08 (1),
a corresponding 2,5-dimethyl analogue. Compounds 6a and 6d
with the R2 of either 4-methyl or H displayed a much lower
potency.
The effects of various alkyl substituents at the C-6 position of
the pyrimidine ring have also been investigated. As shown in Table
1, compound 6g with the 6-cyclohexyl replaced by 6-cyclopropyl
lM, respec-
NH2
NH2
N
N
N
N
N
N
l
S
N
O
N
O
O
P
O
P
NaO
HO
Cl
O
O
O
OMe
O
OH
8-pCPT-2'-O-Me-cAMP (007)
Epac agonist
cAMP
NH2
moiety displayed enhanced activity with an IC50 value of 4.0
when compared with the hit ESI-08 (1). 6-Cyclopentyl derivative
6h showed a similar potency with an IC50 value of 5.9
lM
N
N
O
N
O
lM
S
NC
(Fig. 2). In contrast, 6f and 6i with an isopropyl or a N-methyl
piperidyl were found significantly less active, with IC50 values of
12.7 and 127 lM, respectively. Interestingly, compound 6k with
6-(piperidin-4-yl) group, which was initially designed with an at-
tempt to improve the aqueous solubility, showed no activity
(IC50 >300 lM), indicating that a basic moiety at this position
seems inappropriate. Taken together, these results suggest that a
moderate hydrophobic group at the C-6 position of the pyrimidine
scaffold is important to achieve the potent inhibition activity. In
NH
N
Me
O
O
N
S
O
O
P
Cl
O
O
OMe
1: high-throughput screen hit
Epac antagonist: ESI-08
8-pCPT-2'-O-Me-cAMP-AM (007-AM)
Epac agonist
Figure 1. The structures of cAMP, Epac agonists: 20-O-Me-cAMP analogs 007 and
007-AM, Epac antagonist: HTS hit 1 (ESI-08).

4040
H. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4038–4043
Table 1
Apparent IC50 values of the 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues for competing with 8-NBD-cAMP in binding Epac2
O
O
O
NC
NC
NC
R1
NH
SH
NH
NH
OH
Y
N
N
S
N
X
R2
O
10
6a-k, 8a-b, 11a-c
5a
Entry
R1
X
Y
R2
IC50
(lM)
Relative potency (RA)a
cAMP
1
48
8.4
1
5.7
Cyclohexyl
S
CH2
2,5-Dimethyl
5a
6a
6b
6c
6d
6e
6f
6g
6h
6i
6k
8a
8b
10
11a
11b
11c
>300
38.7
7.0
15.7
30.5
11.6
12.7
4.0
<0.16
1.2
6.9
3.1
1.6
4.1
3.8
12
8.1
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Isopropyl
Cyclopropyl
Cyclopentyl
1-Methyl-piperidin-4-yl
Piperidin-4-yl
Cyclohexyl
S
S
S
S
S
S
S
S
S
S
NH
NH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
—
4-Methyl
3,5-Dimethyl
2,4-Dimethyl
H
2,4,6-Trimethyl
2,5-Dimethyl
2,5-Dimethyl
2,5-Dimethyl
2,5-Dimethyl
2,5-Dimethyl
H
5.9
127
0.4
>300
>300
>300
>300
>300
>300
>300
<0.16
<0.16
<0.16
<0.16
<0.16
<0.16
<0.16
Cyclohexyl
—
4-Cl
Cyclohexyl
Cyclohexyl
Cyclohexyl
S
S
S
CH2C(O)NH
CH2C(O)NH
CH2C(O)NH
H
2,4,6-Trimethyl
4-Methyl
a
RA = IC50, cAMP/IC50, compound
(Fig. 3).30,35,36 These two analogues were shown to be able to inhi-
bit Epac2 GEF activity to basal levels at 25 M concentration in the
l
presence of equal concentration of cAMP. Compound 6h was found
to also inhibit Epac1-mediated Rap1-GDP exchange activity at
25 lM in the presence of equal concentration of cAMP, while com-
pound 6g is more Epac2-specific (Fig. 3). From these results it ap-
pears that smaller alkyl substituent on the C-6 position of the
pyrimidine ring may be more beneficial for the specificity of Epac2.
These findings suggest that further optimization on the moieties at
the C-6 position of pyrimidine scaffold may provide a great poten-
tial to develop new and more Epac2-specific inhibitors.
To further characterize the relative potency of these Epac antag-
onists, we have performed counter-screening assays that measure
type I and II PKA holoenzyme activities, respectively.30 As shown in
Figure 4, compounds ESI-08, 6g (HJC0198) and 6h (HJC0197) at
25 lM have been found not to alter cAMP-induced type I and II
PKA holoenzymes activation while H89, a selective PKA inhibitor,
blocked the type I or II PKA activities completely. These results sug-
gest that compounds ESI-08, 6g (HJC0198) and 6h (HJC0197) are
Epac-specific inhibitors that selectively block cAMP-induced Epac
activation, but do not inhibit cAMP-mediated PKA activation.
Epac proteins are also known to activate the Akt/PKB signaling
pathways.16 To determine if our newly synthesized Epac specific
inhibitors are capable of blocking Epac1- or Epac2-mediated Akt
activation, the phosphorylation statuses of T308 and S473 of Akt
in HEK293 cells ectopically expressing Epac1 or Epac2 were mon-
itored using anti-phospho-Akt antibodies. As shown in Figure 5,
pretreatment of HEK293/Epac1 and HEK293/Epac2 cells with
Figure 2. Relative potency of Epac2 antagonists. Dose-dependent competition of
Epac2 antagonists with 8-NBD-cAMP in binding to Epac2: closed circles, 6g
(HJC0198); closed triangles up, 6h (HJC0197); closed diamonds, 1 (ESI-08); open
triangles down, 6a (HJC0167); closed squares, cAMP.
addition, the replacement of the S-benzyl group at the 2-position of
the pyrimidine scaffold with either phenylamino (8a) or 4-chloro-
phenylamino (8b) led to a dramatic loss of the inhibitory activity
(IC50 >300 lM). This is also true for analogues 10 and 11a–c, in
which S-benzyl moiety was replaced with either 2-sulfanyl acetic
acid or 2-sulfanyl acetamides, indicating that 2-benzylsulfanyl
group is a crucial hydrophobic pharmacophore for this type of mol-
ecules to achieve the activity targeting Epac2.
The in vitro SAR results allowed us to select compounds exhib-
iting a high Epac2 inhibitory activity for further investigation.
Compounds 6g (HJC0198) and 6h (HJC0197) have therefore been
selected for further evaluation in suppressing cAMP-mediated
Epac1 and Epac2 GEF activities to determine their specificity using
purified recombinant full-length Epac1 and Epac2 proteins
10 lM of HJC0197 (6h) and HJC0198 (6g) for 5 min before the
administration of 007-AM, a membrane permeable Epac selective
agonist, completely blocked Epac1 and Epac2-mediated Akt phos-
phorylation. These results demonstrate that in addition to inhibit-
ing Epac1 and Epac2 biochemically, HJC0197 (6h) and HJC0198
(6g) can also suppress Epac1 and Epac2 function in cells.
Molecular docking studies were performed to investigate the
conformation and the required spatial relationship between the
pyrimidine scaffold and Epac2 protein.37 Since our robust HTS

H. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4038–4043
4041
Figure 4. Effects of Epac antagonists ESI-08, 6g (HJC0198) and 6h (HJC0197) on
type I and II PKA activities. Relative Type I (filled bars) and II (open bars) PKA
holoenzyme activities in the presence of 100
H89 or 25 M ESI-08 or 25 M 6g (HJC0198) or 25
presented in the format of means and standard deviations (n = 3).
l
M cAMP plus vehicle control, 25
lM
l
l
lM 6h (HJC0197). Data are
Figure 5. Effects of Epac antagonists on Epac-mediated Akt/PKB phosphorylation in
HEK293/Epac cells. HEK293/Epac1 and HEK293/Epac2 cells with or without
pretreatment of 10
were stimulated with 10
l
M Epac antagonists (HJC0197 and HJC0198, respectively)
M 007-AM. Cell lysates were subjected to Western blot
l
analyses using anti-phospho-Ser473-specific (PKB-P473) and anti-phospho-
Thr308-specific (PKB-P308) PKB antibodies.
pyrimidine scaffold are crucial to target Epac. These docking find-
ings may also help us understand why analogues with the moder-
ate hydrophobic substituent at the C-6 position have better
potency while analogue 6k with 6-(piperidin-4-yl) group displays
no activity. Moreover, this binding mode is further stabilized by
the occurrence of one hydrogen bond between the oxygen of car-
bonyl group in the pyrimidine ring with the residue Lys450. Taken
together, these studies suggest that optimization of the moieties at
the C-6 position and 5-cyano of the pyrimidine scaffold may pro-
vide further opportunities to improve the binding affinity and
specificity of the pyrimidine derivatives.
Figure 3. Specificity of Epac antagonists 6g and 6h. (A) cAMP-mediated Epac1 GEF
activity measured in the presence or absence of Epac antagonists: open squares,
Epac1 alone; closed squares: Epac1 in the presence of 25
Epac1 with 25 M cAMP and 25 M 6g; closed circles, Epac1 with 25
25 M 6h. (B) cAMP-mediated Epac2 GEF activity measured in the presence or
absence of Epac antagonists: open squares, Epac2 alone; closed squares: Epac2 in
the presence of 25 M cAMP; open circles, Epac2 with 25 M cAMP and 25 M 6g;
closed circles, Epac2 with 25 M cAMP and 25 M 6h. Similar results were obtained
from two independent experiments.
l
M cAMP; open circles,
l
l
lM cAMP and
l
l
l
l
l
l
assay is particularly sensitive for searching compounds that di-
rectly compete with 8-NBD-cAMP in binding to Epac2, we pre-
dicted that our compounds may bind to the cAMP binding
domain (CBD) of Epac2. AutoDock Vina docking data indeed re-
vealed that our compounds could fit nicely into the functional
cAMP binding packet of Epac2.38 To further characterize the bind-
ing pose, we selected analogue 6h as a case study for our theoret-
ical investigation. As depicted in Figure 6A & B, the molecular
docking results showed that the position and size of substituted
groups played a key role in the binding of the compound with
Epac2. The hydrophobic S-benzyl moiety interacts with the hydro-
phobic residues of Val447, Phe403, Lys405 and Leu406.
Meanwhile, the cyclopentyl moiety at the C-6 position of the
pyrimidine ring was also found to form hydrophobic interactions
with Phe367, Ala407, Ala412, Pro413, Ala415, Ala416 and Ile418.
These results are in full agreement with our established SAR data
that hydrophobic groups at the C-6 position and 2-position of the
In summary, a series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine
derivatives have been synthesized and biologically evaluated as a
novel class of Epac antagonists. We have identified important
structural features of the small molecule inhibitors targeting Epac.
The SAR results and docking studies indicate that further modifica-
tion at the C-6 position of the pyrimidine scaffold can tune the ori-
ginal hit 1 to achieve a more specific Epac2 antagonist. The
pharmacological probes identified such as 6g (HJC0198) and 6h
(HJC0197) will facilitate our efforts in elucidating the physiological
functions of Epac and mechanisms of cAMP-mediated cell signal-
ing. These probes will also have potential for the development of
novel therapeutics against human diseases associated with dys-
function of specific cAMP-signaling components. Further studies
of a systematic lead optimization on the C-6 position and 5-cyano
of the pyrimidine scaffold by appropriate modifications of length,
size, and chemical nature of the substituents are undergoing and
will be reported in due course.

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5. Follin-Arbelet, V.; Hofgaard, P. O.; Hauglin, H.; Naderi, S.; Sundan, A.; Blomhoff,
R.; Bogen, B.; Blomhoff, H. K. BMC Cancer 2011, 11, 301.
6. Smith, F. D.; Scott, J. D. Eur. J. Cell Biol. 2006, 85, 585.
7. Cheng, X.; Ji, Z.; Tsalkova, T.; Mei, F. C. Acta. Biochim. Biophys. Sin. (Shanghai)
2008, 40, 651.
8. Kawasaki, H.; Springett, G. M.; Mochizuki, N.; Toki, S.; Nakaya, M.; Matsuda, M.;
Housman, D. E.; Graybiel, A. M. Science 1998, 282, 2275.
9. de Rooij, J.; Zwartkruis, F. J.; Verheijen, M. H.; Cool, R. H.; Nijman, S. M.;
Wittinghofer, A.; Bos, J. L. Nature 1998, 396, 474.
10. Gloerich, M.; Bos, J. L. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 355.
11. Grandoch, M.; Roscioni, S. S.; Schmidt, M. Br. J. Pharmacol. 2010, 159, 265.
12. Breckler, M.; Berthouze, M.; Laurent, A. C.; Crozatier, B.; Morel, E.; Lezoualc’h, F.
Cell. Signal. 2011, 23, 1257.
13. Sehrawat, S.; Cullere, X.; Patel, S.; Italiano, J., Jr.; Mayadas, T. N. Mol. Biol. Cell.
2008, 19, 1261.
14. Honegger, K. J.; Capuano, P.; Winter, C.; Bacic, D.; Stange, G.; Wagner, C. A.;
Biber, J.; Murer, H.; Hernando, N. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 803.
15. Zieba, B. J.; Artamonov, M. V.; Jin, L.; Momotani, K.; Ho, R.; Franke, A. S.; Neppl,
R. L.; Stevenson, A. S.; Khromov, A. S.; Chrzanowska-Wodnicka, M.; Somlyo, A.
V. J. Biol. Chem. 2011, 286, 16681.
16. Mei, F. C.; Qiao, J.; Tsygankova, O. M.; Meinkoth, J. L.; Quilliam, L. A.; Cheng, X. J.
Biol. Chem. 2002, 277, 11497.
17. Holz, G. G.; Chepurny, O. G.; Schwede, F. Cell. Signal. 2008, 20, 10.
18. Rangarajan, S.; Enserink, J. M.; Kuiperij, H. B.; de Rooij, J.; Price, L. S.; Schwede,
F.; Bos, J. L. J. Cell Biol. 2003, 160, 487.
19. Enserink, J. M.; Christensen, A. E.; de Rooij, J.; van Triest, M.; Schwede, F.;
Genieser, H. G.; Døskeland, S. O.; Blank, J. L.; Bos, J. L. Nat. Cell Biol. 2002, 4,
901.
20. Kang, G.; Joseph, J. W.; Chepurny, O. G.; Monaco, M.; Wheeler, M. B.; Bos, J. L.;
Schwede, F.; Genieser, H. G.; Holz, G. G. J. Biol. Chem. 2003, 278, 8279.
21. Adamson, R. H.; Ly, J. C.; Sarai, R. K.; Lenz, J. F.; Altangerel, A.; Drenckhahn, D.;
Curry, F. E. Am. J. Physiol. Heart Circ. Physiol. 2008, 294, H1188.
22. Vliem, M. J.; Ponsioen, B.; Schwede, F.; Pannekoek, W. J.; Riedl, J.; Kooistra, M.
R.; Jalink, K.; Genieser, H. G.; Bos, J. L.; Rehmann, H. ChemBioChem 2008, 9,
2052.
23. Poppe, H.; Rybalkin, S. D.; Rehmann, H.; Hinds, T. R.; Tang, X. B.; Christensen, A.
E.; Schwede, F.; Genieser, H. G.; Bos, J. L.; Doskeland, S. O.; Beavo, J. A.; Butt, E.
Nat. Methods 2008, 5, 277.
24. Holz, G. G. Diabetes 2004, 53, 5.
25. Zhang, C. L.; Katoh, M.; Shibasaki, T.; Minami, K.; Sunaga, Y.; Takahashi, H.;
Yokoi, N.; Iwasaki, M.; Miki, T.; Seino, S. Science 2009, 325, 607.
26. Morel, E.; Marcantoni, A.; Gastineau, M.; Birkedal, R.; Rochais, F.; Garnier, A.;
Lompre, A. M.; Vandecasteele, G.; Lezoualc’h, F. Circ. Res. 2005, 97, 1296.
27. Metrich, M.; Lucas, A.; Gastineau, M.; Samuel, J. L.; Heymes, C.; Morel, E.;
Lezoualc’h, F. Circ. Res. 2008, 102, 959.
28. Lissitzky, J. C.; Parriaux, D.; Ristorcelli, E.; Verine, A.; Lombardo, D.; Verrando, P.
Cancer Res. 2009, 69, 802.
29. (a) Rehmann, H.; Schwede, F.; Døskeland, S. O.; Wittinghofer, A.; Bos, J. L. J. Biol.
Chem. 2003, 278, 38548; (b) Dao, K. K.; Teigen, K.; Kopperud, R.; Hodneland, E.;
Schwede, F.; Christensen, A. E.; Martinez, A.; Døskeland, S. O. J. Biol. Chem.
2006, 281, 21500; (c) Das, R.; Chowdhury, S.; Mazhab-Jafari, M. T.; Sildas, S.;
Selvaratnam, R.; Melacini, G. J. Biol. Chem. 2009, 284, 23682; (d) Selvaratnam,
R.; Chowdhury, S.; VanSchouwen, B.; Melacini, G. Proc. Natl. Acad. Sci. U.S.A.
2011, 108, 6133.
30. Tsalkova, T.; Mei, F. C.; Cheng, X. PLos One 2012, 7, e30441.
31. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455.
32. Abdou, I. M.; Strekowski, L. Tetrahedron 2000, 56, 8631.
33. Ram, V. J.; Goel, A.; Nath, M.; Srivastava, P. Bioorg. Med. Chem. Lett. 1994, 4,
2653.
Figure 6. Predicted binding mode and molecular docking of compound 6h into the
cAMP binding domain (CBD) of Epac2 protein. (A) Nitrogen, oxygen, sulfur and
phosphorus are shown in blue, red, orange and pale red, respectively. Compound 6h
is shown in big sticks and in green color. cAMP is shown in small sticks and in
yellow color. Protein residues likely to be involved in polar and hydrophobic
interactions are shown in sticks. Hydrogen bonds are indicated by dashed lines. (B)
Compound 6h is shown in yellow color while cAMP is shown in pink color.
34. Spectra data of the representative compounds: 4-Cyclopropyl-2-(2,5-
dimethylbenzylsulfanyl)-6-oxo-1,6-dihydro-pyrimidine-5-carbonitrile
(6g):
White solid, mp 235–236 °C. 1H NMR (600 MHz, CDCl3/CD3OD 1:2) d 6.89 (s,
1H), 6.86 (d, 1H, J = 7.8 Hz), 6.80 (d, 1H, J = 7.8 Hz), 4.15 (s, 2H), 2.12–2.08 (m,
1H), 2.11 (s, 3H), 2.10 (s, 3H), 1.14–1.12 (m, 2H), 1.05–1.02 (m, 2H). 13C NMR
(150 MHz, CDCl3/CD3OD 1:3) d 176.4, 165.6, 160.3, 135.6, 133.5, 132.0, 130.3,
130.3, 128.8, 114.7, 94.1, 33.3, 20.3, 18.3, 16.6, 11.4 (2C); ESI-MS (m/z): 645
(2M+Na)+; HRMS-ESI Calcd for C17H18N3OS: 312.1171 (M+H)+. Found:
Acknowledgements
This work was supported by Grants P30DA028821 (J.Z.),
R01GM066170 and R21NS066510 (X.C.) from the National Insti-
tute of Health, R.A. Welch Foundation Chemistry and Biology Col-
laborative Grant from Gulf Coast Consortia (GCC) for Chemical
Genomics, and John Sealy Memorial Endowment Fund. We thank
Drs. Lawrence C. Sowers, Carol L. Nilsson, Jacob A. Theruvathu
and Huiling Liu for mass spectrometry assistance and Dr. Tianzhi
Wang at the NMR core facility of UTMB for the NMR spectroscopy
assistance.
312.1158. HPLC analysis conditions: Waters
lBondapak C18 (300 ꢀ 3.9 mm);
flow rate 0.5 mL/min; UV detection at 270 and 254 nm; linear gradient from
10% acetonitrile in water to 100% acetonitrile in water in 20 min followed by
30 min of the last-named solvent; tR = 13.15 min; purity (by peak area) 97.3%.
4-Cyclopentyl-2-(2,5-dimethyl-benzylsulfanyl)-6-oxo-1,6-dihydropyrimidine
-5-carbonitrile (6h): White solid, mp 217–219 °C. 1H NMR (600 MHz, CDCl3) d
13.00 (bs, 1H), 7.15 (s, 1H), 7.08 (d, 1H, J = 7.8 Hz), 7.30 (d, 1H, J = 7.2 Hz), 4.47
(s, 2H), 3.48–3.44 (m, 1H), 2.34 (s, 3H), 2.25 (s, 3H), 2.08–2.05 (m, 2H), 1.92–
1.87 (m, 4H), 1.76–1.74 (m, 2H). 13C NMR (150 MHz, CDCl3) d 180.0, 165.9,
162.6, 136.0, 133.9, 132.4, 130.8, 130.8, 129.3, 114.2, 95.4, 46.0, 33.8, 32.7 (2C),
26.8 (2C), 20.9, 19.0; ESI-MS (m/z): 362 (M+Na)+; HRMS-ESI Calcd for
C19H22N3OS: 340.1484 (M+H)+. Found: 340.1472. HPLC analysis conditions
are same as described above; tR = 13.75 min; purity (by peak area) 98.7%.
35. Tsalkova, T.; Blumenthal, D. K.; Mei, F. C.; White, M. A.; Cheng, X. J. Biol. Chem.
2009, 284, 23644.
References and notes
1. Beavo, J. A.; Brunton, L. L. Nat. Rev. Mol. Cell Biol. 2002, 3, 710.
2. Houslay, M. D.; Kolch, W. Mol. Pharmacol. 2000, 58, 659.
3. Zaccolo, M.; Movsesian, M. A. Circ. Res. 2007, 100, 1569.
4. Bourne, H. R.; Lichtenstein, L. M.; Melmon, K. L.; Henney, C. S.; Weinstein, Y.;
Shearer, G. M. Science 1974, 184, 19.
36. Li, S.; Tsalkova, T.; White, M. A.; Mei, F. C.; Liu, T.; Wang, D.; Woods, V. L., Jr.;
Cheng, X. J. Biol. Chem. 2011, 286, 17889.
37. Rehmann, H.; Arias-Palomo, E.; Hadders, M. A.; Schwede, F.; Llorca, O.; Bos, J. L.
Nature 2008, 455, 124.

H. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4038–4043
4043
38. Molecular docking protocol: The pyrimidine analogues were docked with Epac2
using the X-ray structure of Epac2 (PDB code: 3CF6) and AutoDock Vina 1.1.2.
Water molecules and the ligand (Sp-cAMPS) within the crystal structure were
removed and polar hydrogens were added using AutoDockTools. In the
structures of the analogues, all bonds were treated as rotatable except for
the aromatic, amide, cyano and double bonds, whereas the protein was treated
as rigid. Docking runs were carried out using the standard parameters of the
program for interactive growing and subsequent scoring, except for the
parameters for setting grid box dimensions and center. For all of the docking
studies, a grid box size of 40 ꢀ 60 ꢀ 40 Å, centered at coordinates 11.0 (x),
ꢁ20.0 (y), and ꢁ70.0 (z) of the PDB structure. The binding affinities of the 6h
output structures ranged from ꢁ9.6 to ꢁ8.4 kcal/mol.