Identification and characterization of small molecules as potent and specific EPAC2 antagonists

Article abstract of DOI:10.1021/jm3014162

EPAC1 and EPAC2, two isoforms of exchange proteins directly activated by cAMP (EPAC), respond to the second messenger cAMP and regulate a wide variety of intracellular processes under physiological and pathophysiological circumstances. Herein, we report the chemical design, synthesis, and pharmacological characterization of three different scaffolds (diarylsulfones, N,N-diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective EPAC2 antagonists have been identified including 20i (HJC0350), which has an apparent IC₅₀ of 0.3 μM for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP. Compounds 1 (ESI-05), 14c (HJC0338), and 20i, selected from each series, have exhibited no inhibition of EPAC1-mediated Rap1-GDP exchange activity at 25 μM, indicating that they are EPAC2-specific antagonists. Moreover, live-cell imaging studies using EPAC1, EPAC2, or PKA FRET sensor also demonstrate that 20i functions as an EPAC2 specific antagonist.

Full text of DOI:10.1021/jm3014162

Article
pubs.acs.org/jmc
Identification and Characterization of Small Molecules as Potent and
Specific EPAC2 Antagonists
Haijun Chen,† Tamara Tsalkova,†,‡ Oleg G. Chepurny,§ Fang C. Mei,†,‡ George G. Holz,§,∥
Xiaodong Cheng,*,†,‡ and Jia Zhou*,†
‡
†Department of Pharmacology and Toxicology and Sealy Center for Structural Biology and Molecular Biophysics, University of
Texas Medical Branch, Galveston, Texas 77555, United States
∥
§Department of Medicine and Department of Pharmacology, State University of New York (SUNY), Upstate Medical University,
Syracuse, New York 13210, United States
S
* Supporting Information
ABSTRACT: EPAC1 and EPAC2, two isoforms of exchange proteins directly activated by cAMP
(EPAC), respond to the second messenger cAMP and regulate a wide variety of intracellular processes
under physiological and pathophysiological circumstances. Herein, we report the chemical design,
synthesis, and pharmacological characterization of three different scaffolds (diarylsulfones, N,N-
diarylamines, and arylsulfonamides) as highly potent and selective antagonists of EPAC2. Several selective
EPAC2 antagonists have been identified including 20i (HJC0350), which has an apparent IC50 of 0.3 μM
for competing with 8-NBD-cAMP binding of EPAC2 and is about 133-fold more potent than cAMP.
Compounds 1 (ESI-05), 14c (HJC0338), and 20i, selected from each series, have exhibited no inhibition
of EPAC1-mediated Rap1-GDP exchange activity at 25 μM, indicating that they are EPAC2-specific antagonists. Moreover, live-
cell imaging studies using EPAC1, EPAC2, or PKA FRET sensor also demonstrate that 20i functions as an EPAC2 specific
antagonist.
antagonists to probe the functions of EPAC in relevant
signaling pathways and that act as potential therapeutics with
unique targets.16−18
INTRODUCTION
■
Identification and development of small-molecule probes with
high potency and specificity represent a major effort for
chemical biologists to explore and validate the roles of the
proteins in a broader biological context.1 The most common
and versatile second messenger, cyclic adenosine mono-
phosphate (cAMP), regulates a striking number of physio-
logical and pathophysiological processes through the signaling
networks that interfere with many disease states including
inflammatory disorders, immune deficiencies, and cancer.2
Initially, the effects of cAMP were believed to be transduced by
protein kinase A (PKA) and cAMP-gated ion channels.
However, the contribution of more recently discovered cAMP
target exchange proteins directly activated by cAMP (EPAC) is
becoming more and more appreciated.3−7 EPAC proteins are
now assumed to be implicated in a number of cellular processes
that researchers had previously thought to be associated only
with PKA, including insulin secretion, neurotransmitter release,
integrin-mediated cell adhesion, cell survival and apoptosis,
gene transcription, and chromosomal integrity.8−10 Therefore,
advances in the understanding the role of EPAC proteins as
regulators of cAMP-dependent signaling are critical for further
elucidating the mechanism of cAMP signaling. cAMP analogues
that selectively activate EPAC have been developed to help
reveal the ability of EPAC to modulate ongoing physiological
signaling.11−13 Nevertheless, novel EPAC-specific antagonists
are desperately needed as diverse approaches to help
elucidation of the mechanisms by which cAMP fulfills its
function via EPAC.14,15 To this end, we direct our efforts on
identification of novel small molecules as selective EPAC
EPAC is a cAMP-activated guanine nucleotide exchange
factor (cAMP-GEF) for the small GTP-binding proteins Rap1
and Rap2.3,4 There are two isoforms of EPAC, EPAC1 and
EPAC2, which are coded by two independent genes with
distinct tissue distributions in mammals. EPAC1 is ubiquitously
expressed in all tissues with high levels of expression in the
kidney, while EPAC2 is detectable most notably in the central
nervous system, adrenal gland, and pancreas. Given that
EPAC1 and EPAC2 share extensive sequence homology,
developing EPAC1- or EPAC2-specific antagonists with the
capability of discriminating the functions of EPAC1 and
EPAC2 is quite essential in this field.
To identify new chemical probes with high capability of
specifically inhibiting EPAC, a Maybridge Hitfinder compound
library of 14 400 structurally diverse small molecules has been
screened using a fluorescence-based high-throughput screening
(HTS) assay.16−18 This assay was based on a fluorescent cyclic
nucleotide analogue 8-NBD-cAMP, the binding of which to
purified full-length EPAC2 protein could lead to a dose-
dependent increase in fluorescent signal, while cAMP or
EPAC2 antagonists could compete with their binding and
decrease the fluorescent signal in a dose-dependent manner.16
After analysis of the EPAC2 inhibition activity, some of the
compounds screened from the library were applied to detect
Received: September 28, 2012
Published: January 3, 2013
© 2013 American Chemical Society
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a
their selectivity using a secondary functional assay that could
evaluate their cAMP-mediated EPAC1 GEF activity in the
purified recombinant full-length EPAC1 protein. Two HTS hits
1 (ESI-05) and 2 (ESI-10) have been identified (Figure 1),
Scheme 1. Synthesis of the Diarylsulfone Scaffolds
Figure 1. Structures of cAMP and HTS hits: 1 (ESI-05) and 2 (ESI-
10).
which inhibit cAMP-mediated EPAC2 GEF activity with IC50
values of 0.5 and 18 μM, respectively. The hit compound 1 is
exhibited as a selective antagonist of EPAC2 without apparent
activity toward EPAC1, while 2 is not exclusively specific for
EPAC2.18b
In continuation of our efforts to identify novel potent and
EPAC-specific antagonists,17 we have designed, synthesized,
and characterized three different series of new molecules,
namely, diarylsulfones, N,N-diarylamines, and arylsulfonamides
based on the two HTS hits 1 and 2 as our lead molecules.
Herein, we describe our findings on their potency and
specificity as well as the structure−activity relationship (SAR)
results of the new analogues.
a
Reagents and conditions: (a) AlCl3, 25 °C, 69−96%; (b) Pd(dppf)-
Cl2, KOAc, ArB(OH)2, THF/EtOH/H2O, 80 °C, 70%; (c) BBr3,
DCM, 0−25 °C, 92%. (d) For 11a and 11b: DIAD, PPh3, THF, 25
°C, 77−90%. For 11c and 11d: (i) DIAD, PPh3, THF, 25 °C, 87−
90%; (ii) TFA, DCM, 0 °C, 98−99%.
RESULTS AND DISCUSSION
a
■
Scheme 2. Synthesis of the N,N-Diarylamine Scaffold
Chemistry. Our previous medicinal chemistry effort and
molecular dockings have demonstrated that appropriate
hydrophobic pharmacophores of the molecules play a crucial
role for their potency and EPAC-isoform specificity.17 There-
fore, in our initial drug design and SAR studies, we have
attempted to retain 2,4,6-trimethylphenyl ring of hit 1 as a
privileged fragment to investigate the new molecules. As shown
in Scheme 1, the diarylsulfone derivatives 5a−c and 8a,b were
prepared via Friedel−Crafts sulfonylation of 3a−c with 2,4,6-
trimethylbenzene-1-sulfonyl chloride 4 or that of 7 with
substituted benzenesulfonyl chlorides 6a,b in 69−96% yields.
The target compound 9 was obtained by Suzuki coupling
reaction of 8a with 2-fluoropyridine-5-boronic acid in the
presence of Pd(dppf)Cl2 catalyst in 70% yield. Generation of
10 was achieved in 92% yield by demethylation of 8b using
boron tribromide. Compounds 11a−d were produced in 77−
90% overall yields by Mitsunobu reaction of 10, followed by
subsequent Boc-deprotection with trifluoroacetic acid if
necessary (e.g., 11c,d).
a
Reagents and conditions: (a) Pd2(dba)3, NaOtBu, ( )-BINAP,
toluene, 120 °C, 62−93%; (b) EtOH, reflux, 65%.
treated with various sulfonyl chlorides 19a−h in the presence of
60% NaH as base to give the corresponding arylsulfonamides
20a−i in 30−96% yields. The synthesis of indole and azaindole
derivatives 22a−c and 24a−d was accomplished by the
treatment of various indoles 21a,b or azaindoles 23a−d with
2,4,6-trimethylbenzene-1-sulfonyl chloride 4 in a similar
fashion. The indole ester 22b was saponified to generate the
carboxylic acid 22c in 66% yield.
The synthetic route to N,N-diarylamine derivatives is
outlined in Scheme 2. N,N-Diarylamine analogues 14a−c
were prepared from substituted anilines 12a−c by palladium-
catalyzed cross-coupling with 2,4,6-mesityl bromide 13 in 62−
93% yields. The cyclization reaction of α-bromoketone 15 with
substituted thiourea 16 afforded N-phenylthiazoleamine 17 in
65% yield.
In Vitro Evaluation of EPAC2 Inhibition. All compounds
have been evaluated for their inhibitory activity against the
recombinant fusion protein EPAC2 using 8-NBD-cAMP as the
Scheme 3 illustrates the synthesis of diversely substituted
arylsulfonamide analogues. Substituted pyrroles 18a,b were
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a
diarylsulfones and N,N-diarylamine scaffolds for competing
with 8-NBD-cAMP in binding EPAC2. Among these new
diarylsulfone analogues, compound 5a is the most potent
antagonist with an IC50 of 0.7 μM. When one aryl ring of the
diarylsulfones is substituted with too bulky groups including 4-
pentyl, 4-cyclohexyl, and 4-O-piperidinyl, these compounds
such as 5b−c, 9, and 11a−d display very low to no activity.
When the methyl group in one ring of hit 1 is replaced by iodo,
methoxy, or hydroxyl, compounds 8a,b and 10 exhibit a slightly
lower potency with IC50 of 4, 1.9, and 13.5 μM, respectively.
Among N,N-diarylamine derivatives, compound 14a is a new
analogue of hit 2 with the 4-amino group of one phenyl ring
replaced by the 4-methyl substituent and the 5-amino-
benzenesulfonic acid moiety replaced by the 2,4,6-trimethyl-
phenyl fragment. 14a demonstrates a substantially enhanced
activity with an IC50 of 3.8 μM when compared with the hit
lead 2. Interestingly, analogues 14b,c with the 3,5-dichloro or
2,5-dichloro exhibit a significantly better potency with IC50
values of 0.9 and 0.4 μM, respectively, than the 4-methyl
derivative 14a. Compound 17 containing a thiazole ring as one
of the aromatic moieties was also explored and was found to
have no activity.
Scheme 3. Synthesis of the Arylsulfonamide Scaffold
On the basis of the structural features of hits 1 and 2 as well
as the preliminary SAR results from the above efforts, we have
designed and investigated a series of arylsulfonamides as a new
chemical entity of EPAC2 antagonists. The findings of the
arylsulfonamide analogues are summarized in Table 2.
Compound 20a with a 2-ethylpyrrole moiety displays a high
potency of EPAC2 inhibition with an IC50 of 0.5 μM.
Analogues 20b−h with 2,4,6-trimethylphenyl moiety replaced
by other substituted phenyl groups exhibit apparently
dampened activities in comparison with 20a. Interestingly,
the 4-trifluoromethyl substituent on the phenyl ring of analogue
a
Reagents and conditions: (a) 60% NaH, THF, 0−25 °C, 30−96%;
(b) 2 N LiOH/H2O, MeOH/H2O, 25 °C, 66%.
artificial substrate to determine IC50 values, while cAMP
competes with 8-NBD-cAMP in binding EPAC2 with an IC50
of 40 μM.16 Table 1 shows apparent IC50 values of the
Table 1. Apparent IC50 Values of the Diarylsulfones and N,N-Diarylamine Scaffolds for Competing with 8-NBD-cAMP in
Binding EPAC2
a
compd
R1
R2
X
IC50 (μM)
relative potency (RA)
cAMP
1
40
1
4-methyl
4-NH2
2′,4′,6′-trimethyl
2′-SO3H-4′-NH2
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
2′,4′,6′-trimethyl
SO2
NH
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
NH
NH
NH
NH
0.5
80
2
18
2.2
5a
2,4,5-trimethyl
4-pentyl
0.7
57.1
<0.13
<0.13
10
5b
>300
>300
4
5c
4-cyclohexyl
4-iodo
8a
8b
4-methoxy
1.9
21.1
<0.13
3.0
9
4-(2-fluoro-5-pyridinyl)
4-hydroxyl
>300
13.5
>300
>300
>300
>300
3.8
10
11a
11b
11c
11d
14a
14b
14c
17
4-O-cyclohexyl
4-O-piperidinyl-1-Boc
4-O-piperidinyl
4-O-2-ethylamino
4-methyl
<0.13
<0.13
<0.13
<0.13
10.5
44.4
100
3,5-dichloro
0.9
2,5-dichloro
0.4
>300
<0.13
a
RA = IC50,cAMP/ IC50,compound
.
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Table 2. Apparent IC50 Values of the Arylsulfonamide Scaffold for Competing with 8-NBD-cAMP in Binding EPAC2
a
a
compd
R1
R2
IC50 (μM)
relative potency (RA)
compd
IC50 (μM)
relative potency (RA)
20a
20b
20c
20d
20e
20f
2-ethyl
2-ethyl
2-ethyl
2-ethyl
2-ethyl
2-ethyl
2-ethyl
2-ethyl
2,4-dimethyl
2,4,6-trimethyl
4-methyl
0.5
4
80
22a
1.2
33.3
<0.13
3.7
10
22b
22c
>300
10.8
3.8
4-methoxy
8
5
4-chloro
8
5
24a
10.5
4.5
4-trifluoromethyl
2-methyl
>300
5.3
4.7
1.3
0.3
<0.13
7.5
8.5
30.8
133
24b
24c
8.9
12.6
2.4
3.2
20g
20h
20i
3,5-dimethyl
2,4-dimethyl
2,4,6-trimethyl
24d
cAMP
16.7
1
40
a
RA = IC50,cAMP/ IC50,compound
.
20e results in a dramatic loss of EPAC inhibitory activity.
Compound 20i bearing the 2,4-dimethylpyrrole fragment
displays a remarkable activity with an IC50 of 0.3 μM, which
is about 133-fold more potent than cAMP (Table 2, Figure 2).
scaffolds have been evaluated in suppressing cAMP-mediated
EPAC1 and EPAC2 GEF activities using purified recombinant
full-length EPAC1 and EPAC2 proteins (Figure 3).16,19,20
These three analogues have been shown to be able to inhibit
EPAC2 GEF activity to basal levels at 25 μM in the presence of
equal concentration of cAMP. All of these compounds were
found not to inhibit EPAC1-mediated Rap1-GDP exchange
activity at 25 μM in the presence of equal concentration of
cAMP, indicating that they are EPAC2-specific antagonists. To
further investigate their specificity for EPAC over PKA, we
performed counterscreening assays that measure type I and
type II PKA holoenzyme activities.16,21,22 All these analogues
have demonstrated that they do not significantly alter cAMP-
induced type I and type II PKA holoenzymes activation at 25
μM, while the selective PKA inhibitor H89, the positive control,
blocked the type I or II PKA activities completely. These results
suggest that compounds 1, 14c (HJC0338), and 20i
(HJC0350) are EPAC2-specific antagonists that selectively
block cAMP-induced EPAC2 activation but do not inhibit
cAMP-mediated PKA activation. It should be emphasized that
while these compounds exert no activity toward PKA, their
pharmacological properties should be further evaluated to
ensure that they do not disrupt other components of the cyclic
nucleotide signaling pathways, such as the cyclic-nucleotide
regulated ion channels (CNG and HCN channels), phospho-
diesterases, or adenylyl cyclases.
Figure 2. Relative potency of EPAC2 antagonist 20i (HJC0350),
showing dose-dependent competition of EPAC2 antagonists with 8-
NBD-cAMP in binding to EPAC2: closed circles, 20i; closed squares,
cAMP.
To test whether 20i is capable of modulating EPAC
activation in living cells and to further validate that 20i is an
EPAC2 isoform-specific antagonist, we tested the compound
using HEK293 cells stably expressing an EPAC2- or EPAC1-
based fluorescence resonance energy transfer (FRET) sensor,
EPAC2-FL or EPAC1-FL.23 As expected, stimulation of
HEK293/EPAC2-FL cells by 8-(4-chlorophenylthio)-2′-O-
methyladenosine 3′,5′-cyclic monophosphate acetoxymethyl
ester (007-AM), a membrane permeable EPAC selective
cAMP analogue, led to a decrease of FRET measured as an
increase of the 485/535 nm FRET ratio (Figure 4A).
Pretreatment of HEK293/EPAC2-FL cells with 10 μM 20i
fully blocked the 007-AM induced decrease of FRET (Figure
4B). In contrast, 20i was found to be incapable of blocking the
007-AM induced decrease of FRET changes in HEK293/
EPAC1-FL (Figure 4C and Figure 4D). By use of HEK293 cells
stably expressing the PKA sensor AKAR3,24 it was also possible
to demonstrate the stimulatory action of 8-Br-cAMP-AM at
Analogues 22a−c and 24a−d have been investigated to replace
the pyrrole scaffold with the indole or azaindole moieties.
Among these new molecules, the indole analogue 22a and the
7-azaindole analogue 24d display an almost retained activity of
EPAC2 inhibition in contrast with pyrrole derivatives.
Analogues 22c and 24a−c are less active with a moderate
potency. An ester group on indole ring of 22b results in a
dramatic loss of activity, while the saponification of the ester
22b to carboxylic acid 22c can significantly regain the inhibitory
activity of EPAC2. Taken together, the preliminary SAR results
support that appropriate hydrophobic pharmacophores of the
molecules play a crucial role for their activity targeting the
EPAC proteins, and more potent ligands may be achieved
through further systematic and extensive structural optimiza-
tions.
Selectivity for EPAC2. To investigate the EPAC-isoform
specificity, the inhibitory potencies of three most potent
compounds 1, 14c, and 20i selected from three different
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studies support that EPAC proteins may represent promising
targets for the design of new therapies for various human
disorders such as diabetes, heart disease, and cancer.25−29
Therefore, these compounds may not only be valuable
pharmacological tools to explore physiological and pathological
processes related to signaling pathways that are regulated by
EPAC proteins but also have great potential to be developed as
novel molecular therapeutics for relevant human diseases.
Further structural optimization of these scaffolds based upon
our SAR findings and in vivo pharmacological evaluation of
selected EPAC antagonists in disease models are currently
underway.
EXPERIMENTAL SECTION
■
General Chemistry Information. All commercially available
starting materials and solvents were reagent grade and used without
further purification. Reactions were performed under a nitrogen
atmosphere in dry glassware with magnetic stirring. Preparative
column chromatography was performed using silica gel 60, particle size
0.063−0.200 mm (70−230 mesh, flash). Analytical TLC was carried
out employing silica gel 60 F254 plates (Merck, Darmstadt, Germany).
Visualization of the developed chromatograms was performed with
detection by UV (254 nm). NMR spectra were recorded on a Bruker
600 (1H, 600 MHz; 13C, 150 MHz) spectrometer. H and 13C NMR
1
spectra were recorded with TMS as an internal reference. Chemical
shifts were expressed in ppm, and J values were given in Hz. High-
resolution mass spectra were obtained using a Thermo Fisher LTQ
Orbitrap Elite mass spectrometer. Parameters include the following:
nano ESI spray voltage was 1.8 kV; capillary temperature was 275 °C,
and the resolution was 60 000; ionization was achieved by positive
mode. Melting points were measured on a Thermo Scientific
electrothermal digital melting point apparatus and uncorrected.
Purities of final compounds were established by analytical HPLC,
which was carried out on a Shimadzu HPLC system (model CBM-20A
LC-20AD SPD-20A UV/vis). HPLC analysis conditions were as
follows: Waters μBondapak C18 (300 mm × 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 (for 11c and 11d, the same
mobile phase with 0.1% TFA). All biologically evaluated compounds
are >95% pure.
1,3,5-Trimethyl-2-(2,4,5-trimethylbenzenesulfonyl)benzene
(5a). A mixture of mesitylsulfonyl chloride (219 mg, 1.0 mmol), 1,2,4-
trimethylbenzene (125 mg, 1.05 mmol), and AlCl3 (266 mg, 2.0
mmol) in DCM (5 mL) was stirred for 2 h at room temperature. The
mixture was then poured into 10 mL of 5% HCl (aq) and extracted by
DCM (30 mL). The organic phase was washed by aqueous KHCO3
solution, brine and dried over anhydrous Na2SO4. The resulting
solution was evaporated, and the residue was purified by silica gel
column chromatography (hexane/EtOAc = 10/1) to give the desired
product as a white solid (290 mg, 96%): mp 147−148 °C; HPLC
purity 98.4% (tR = 25.33 min). 1H NMR (600 MHz, DMSO-d6) δ 7.70
(s, 1H), 7.11 (s, 1H), 7.05 (s, 2H), 2.38 (s, 6H), 2.27 (s, 6H), 2.25 (s,
3H), 2.07 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 143.0, 142.3,
138.5, 138.4, 134.4, 134.3, 133.7, 133.3, 132.0, 128.2, 21.8, 20.5, 19.1,
18.9, 17.8. HRMS (ESI) calcd for C18H23O2S 303.1413 (M + H)+,
found 303.1407.
Figure 3. Specificity of EPAC antagonists 1, 14c, and 20i. (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 μM cAMP; closed triangles up, EPAC1
with 25 μM cAMP and 25 μM 1; open circles, EPAC1 with 25 μM
cAMP and 25 μM 14c; closed circles, EPAC1 with 25 μM cAMP and
25 μM 20i. (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 μM cAMP; closed
triangles up, EPAC2 with 25 μM cAMP and 25 μM 1; open circles,
EPAC2 with 25 μM cAMP and 25 μM 14c; closed circles, EPAC2
with 25 μM cAMP and 25 μM 20i.
PKA, as measured as an increase FRET with a corresponding
decrease of the 485/535 nm FRET ratio (Figure 5A). This
action of 8-Br-cAMP-AM to activate PKA was not inhibited by
20i (Figure 5B). These live-cell results are in complete
agreement with our biochemical data and further validate that
20i is an effective EPAC2-isoform specific antagonist.
1,3,5-Trimethyl-2-(4-pentylbenzenesulfonyl)benzene (5b).
Compound 5b was prepared in 90% yield by a procedure similar to
that used to prepare compound 5a. The title compound was obtained
CONCLUSION
■
In summary, this work presents three series of new molecules
that exhibit outstanding selectivity and potency for the
inhibition of EPAC2 through a preliminary hit-to-lead
optimization process. Analogues 14c and 20i have been
identified as the most potent EPAC2-specific antagonists with
IC50 values of 0.4 and 0.3 μM, which are about 100- and 133-
fold more potent than cAMP, respectively. Accumulating
1
as a pale yellow oil. HPLC purity 97.2% (tR = 26.16 min). H NMR
(600 MHz, CDCl3) δ 7.68 (d, 2H, J = 7.2 Hz), 7.25 (d, 2H, J = 6.6
Hz), 6.93 (s, 2H), 2.63 (t, 2H, J = 7.2 Hz), 2.49 (s, 6H), 2.28 (s, 3H),
1.57−1.62 (m, 2H), 1.28−1.32 (m, 4H), 0.88 (t, 2H, J = 6.6 Hz). 13C
NMR (150 MHz, CDCl3) δ 148.4, 143.3, 140.9, 140.1, 134.3, 132.3,
128.9, 126.4, 35.9, 31.5, 30.8, 22.9, 22.6, 21.1, 14.1. HRMS (ESI) calcd
for C20H27O2S 331.1726 (M + H)+, found 331.1734.
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Figure 4. Effects of 20i on 007-AM mediated cellular activation of EPAC. For HEK293 cells stable expression EPAC2-FL (A, B) or EPAC1-FL (C,
D), it was demonstrated that 10 μM 20i blocked EPAC2 but not EPAC1 activation in response to 10 μM 007-AM.
2-(4-Cyclohexylbenzenesulfonyl)-1,3,5-trimethylbenzene
(5c). Compound 5c was prepared in 75% yield by a procedure similar
to that used to prepare compound 5a. The title compound was
obtained as a white solid (mp 102−103 °C). HPLC purity 99.8% (tR =
26.29 min). 1H NMR (600 MHz, CDCl3) δ 7.69 (d, 2H, J = 8.4 Hz),
7.27 (d, 2H, J = 8.4 Hz), 6.93 (s, 2H), 2.60 (s, 6H), 2.52−2.55 (m,
1H), 2.27 (s, 3H), 1.82−1.83 (m, 4H), 1.73−1.75 (m, 1H), 1.34−1.41
(m, 4H), 1.22−1.26 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 153.3,
143.2, 141.0, 140.0, 134.3, 132.2, 127.4, 126.4, 44.6, 34.2, 26.7, 26.0,
22.9, 21.0. HRMS (ESI) calcd for C21H27O2S 343.1726 (M + H)+,
found 343.1730.
2-(4-Iodobenzenesulfonyl)-1,3,5-trimethylbenzene (8a). A
mixture of 4-iodobenzenesulfonyl chloride (302 mg, 1.0 mmol),
mesitylene (120 mg, 1.0 mmol), and AlCl3 (150 mg, 1.2 mmol) in
DCM (5 mL) was stirred for 2 h at room temperature. The mixture
was then poured into 10 mL of 5% HCl (aq) and extracted by DCM
(30 mL). The organic phase was washed by aqueous KHCO3, brine
and dried over anhydrous Na2SO4. The resulting solution was
evaporated, and the residue was purified by silica gel column
chromatography (hexane/EtOAc = 10/1) to give the desired product
(266 mg, 69%) as a white solid (mp 123−124 °C). HPLC purity
96.0% (tR = 23.82 min). 1H NMR (600 MHz, CDCl3) δ 7.82 (d, 2H, J
= 8.4 Hz), 7.48 (d, 2H, J = 7.8 Hz), 6.95 (s, 2H), 2.58 (s, 6H), 2.30 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 143.9, 143.4, 140.2, 138.3, 133.5,
132.5, 127.8, 100.0, 23.0, 21.2. HRMS (ESI) calcd for C15H16IO2S
386.9910 (M + H)+, found 386.9915.
2-Fluoro-5-[4-(2,4,6-trimethylbenzenesulfonyl)phenyl]-
pyridine (9). To a solution of 8a (77 mg, 0.2 mmol) and 2-
fluoropyridine-5-boronic acid (28 mg, 0.2 mmol) in THF/EtOH/H2O
(1 mL/1 mL/1 mL) were added KOAc (59 mg, 0.6 mmol) and then
Pd(dppf)Cl2 (16 mg, 0.02 mmol). The resulting mixture was
deoxygenated via five vacuum/N2-refill cycles. The mixture was stirred
at 80 °C for 18 h and was then concentrated under vacuum. The
residue was partitioned between EtOAc (50 mL) and H2O (20 mL).
The organic layer was separated and washed with brine (10 mL), dried
over anhydrous Na2SO4, filtered, and concentrated to give an oily
residue. This residue was purified with silica gel column (hexane/
EtOAc = 3/1) to obtain 9 (50 mg, 70%) as a red solid (mp 150−151
1
°C). HPLC purity 95.1% (tR = 22.50 min). H NMR (600 MHz,
CDCl3) δ 8.39−8.41 (m, 1H), 7.95−7.98 (m, 1H), 7.88 (d, 2H, J = 8.4
Hz), 7.62−7.65 (m, 2H), 7.03 (d, 1H, J = 8.4 Hz), 6.96 (s, 2H), 2.61
(s, 6H), 2.30 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 164.5, 162.9,
146.3, 143.8, 143.3, 141.0, 140.2, 140.1, 133.6, 133.2, 132.4, 127.6,
127.2, 110.1, 109.9, 23.0, 21.1. HRMS (ESI) calcd for C20H19FNO2S
356.1115 (M + H)+, found 356.1120.
4-(2,4,6-Trimethylbenzenesulfonyl)phenol (10). To a solution
of 8b (350 mg, 1.2 mmol) in 10 mL of DCM was added 1 N BBr3/
DCM (1.45 mL, 1.45 mmol) at 0 °C. The resulting mixture was stirred
at room temperature for 16 h. The solution was diluted with EtOAc
(50 mL), washed with H2O (10 mL) and brine (10 mL). The organic
layer was dried over anhydrous Na2SO4 and then concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 3/1) to give the desired product
(306 mg, 92%) as a white solid (mp 180−181 °C). HPLC purity
99.4% (tR = 19.46 min). 1H NMR (600 MHz, CDCl3) δ 7.64 (d, 2H, J
= 8.4 Hz), 6.93 (s, 2H), 6.86 (d, 2H, J = 8.4 Hz), 6.23 (bs, 1H), 2.58
(s, 6H), 2.29 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 160.0, 143.4,
140.0, 135.1, 134.4, 132.4, 128.8, 115.8, 23.0, 21.1. HRMS (ESI) calcd
for C15H17O3S 277.0893 (M + H)+, found 277.0898.
2-(4-Methoxybenzenesulfonyl)-1,3,5-trimethylbenzene (8b).
Compound 8b was prepared in 86% yield by a procedure similar to
that used to prepare compound 8a. The title compound was obtained
as a white solid (mp 132−133 °C). HPLC purity 98.9% (tR = 21.80
1
min). H NMR (600 MHz, CDCl3) δ 7.72 (d, 2H, J = 8.4 Hz), 6.93
(d, 2H, J = 7.2 Hz), 6.92 (s, 2H), 3.84 (s, 3H), 2.60 (s, 6H), 2.28 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 162.9, 143.2, 140.0, 135.4, 134.7,
132.3, 128.6, 114.2, 55.7, 23.0, 21.1. HRMS (ESI) calcd for C16H19O3S
291.1049 (M + H)+, found 291.1056.
2-(4-Cyclohexyloxybenzenesulfonyl)-1,3,5-trimethylben-
zene (11a). To a solution of 10 (50 mg, 0.18 mmol) and Ph3P (58
mg, 0.22 mmol) in THF (5 mL) was added cyclohexanol (36 mg, 0.36
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(tR = 17.63 min). 1H NMR (600 MHz, CDCl3) δ 8.55 (bs, 1H), 7.70
(d, 2H, J = 7.2 Hz), 6.92 (d, 2H, J = 9.0 Hz), 6.91 (s, 2H), 4.67 (s,
1H), 3.28 (t, 2H, J = 8.4 Hz), 3.12−3.14 (m, 2H), 2.57 (s, 6H), 2.27
(s, 3H), 2.17 (t, 2H, J = 7.8 Hz), 2.02−2.04 (m, 2H). 13C NMR (150
MHz, CDCl3) δ 159.8, 143.4, 139.8, 136.1, 134.2, 132.3, 128.7, 115.6,
68.9, 40.2, 27.2, 22.9, 21.0. HRMS (ESI) calcd for C20H26NO3S
360.1628 (M + H)+, found 360.1631.
2-[4-(2,4,6-Trimethylbenzenesulfonyl)phenoxy]ethylamine
(11d). Compound 11d was prepared in 85% yield (two steps) by a
procedure similar to that used to prepare compounds 11a and 11c.
The title compound was obtained a pale red oil. HPLC purity 97.7%
1
(tR = 16.68 min). H NMR (600 MHz, CDCl3) δ7.64 (d, 2H, J = 7.2
Hz), 6.89 (s, 2H), 6.88 (d, 2H, J = 7.2 Hz), 5.40−5.48 (bs, 2H), 4.02−
4.03 (m, 2H), 3.10−3.12 (m, 2H), 2.52 (s, 6H), 2.23 (s, 3H). 13C
NMR (150 MHz, CDCl3) δ 161.6, 143.4, 139.8, 135.7, 134.2, 132.3,
128.5, 114.7, 67.1, 40.0, 22.8, 21.1. HRMS (ESI) calcd for
C17H22NO3S 320.1315 (M + H)+, found 320.1319.
p-Tolyl-(2,4,6-trimethylphenyl)amine (14a). NaOtBu (115 mg,
1.2 mmol), Pd2(dba)3 (92 mg, 0.1 mmol), and ( )-BINAP (124 mg,
0.2 mmol) were placed into a flask and dissolved into distilled toluene
(5 mL). To this solution were added mesityl bromide (995 mg, 5.0
mmol) and p-tolylamine (107 mg, 1.0 mmol) dropwise with stirring at
room temperature, and the mixture was refluxed at 120 °C for 24 h.
After the mixture was cooled, 10 mL of 5% HCl (aq) was added and
extracted with EtOAc (50 mL). The combined organic layer was
washed with NaHCO3 and dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 10/1) to give the desired product
as a pale yellow oil (210 mg, 93%). HPLC purity 99.4% (tR = 24.58
min). 1H NMR (600 MHz, CDCl3) δ 7.11 (d, 2H, J = 7.8 Hz), 7.09 (s,
2H), 6.57 (d, 2H, J = 7.8 Hz), 5.12 (s, 1H), 2.47 (s, 3H), 2.40 (s, 3H),
2.33 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 144.4, 136.1, 135.7,
135.1, 129.8, 129.3, 127.1, 113.5, 21.0, 20.5, 18.3. HRMS (ESI) calcd
for C16H20N 226.1590 (M + H)+, found 226.1593.
(3,5-Dichlorophenyl)-(2,4,6-trimethylphenyl)amine (14b).
Compound 14b was prepared in 68% yield by a procedure similar
to that used to prepare compound 14a. The title compound was
obtained as a pale yellow solid (mp 99−100 °C). HPLC purity 98.8%
(tR = 27.53 min). 1H NMR (600 MHz, CDCl3) δ 7.00 (s, 2H), 6.73 (s,
1H), 6.37 (s, 2H), 5.21 (s, 1H), 2.36 (s, 3H), 2.20 (s, 6H). 13C NMR
(150 MHz, CDCl3) δ 148.8, 136.7, 136.4, 135.7, 133.9, 129.6, 117.7,
111.3, 21.0, 18.2. HRMS (ESI) calcd for C15H16Cl2N 280.0654 (M +
H)+, found 280.0653.
Figure 5. Effects of 20i on 8-Br-cAMP-AM mediated cellular
activation of PKA. For HEK293 cells stable expression AKAR3, it
was demonstrated that 10 μM 20i did not affect PKA activation in
response to 10 μM 8-Br-cAMP-AM.
mmol) and DIAD (44 mg, 0.22 mmol). The reaction mixture was
stirred at room temperature for 16 h, and then it was partitioned
between EtOAc (50 mL) and H2O (20 mL). The organic layer was
washed with brine (10 mL), dried with anhydrous Na2SO4, and
concentrated to give the crude product. This residue was purified with
silica gel column (hexane/EtOAc = 7/1) to afford the desired product
as a colorless oil (50 mg, 77%). HPLC purity 99.7% (tR = 25.64 min).
1H NMR (600 MHz, CDCl3) δ 7.69 (d, 2H, J = 7.2 Hz), 6.91 (s, 2H),
6.91 (d, 2H, J = 6.6 Hz), 4.30 (s, 1H), 2.60 (s, 6H), 2.27 (s, 3H),
1.93−195 (m, 2H), 1.75−1.77 (m, 2H), 1.47−1.57 (m, 3H), 1.29−
1.39 (m, 3H). 13C NMR (150 MHz, CDCl3) δ 161.4, 143.1, 139.9,
134.8, 134.8, 132.3, 128.6, 115.6, 75.8, 31.6, 25.6, 23.7, 23.0, 21.1.
HRMS (ESI) calcd for C21H27O3S 359.1675 (M + H)+, found
359.1680.
(2,5-Dichlorophenyl)-(2,4,6-trimethylphenyl)amine (14c).
Compound 14c was prepared in 62% yield by a procedure similar
to that used to prepare compound 14a. The title compound was
obtained as a pale yellow solid (mp 91−93 °C). HPLC purity 99.5%
(tR = 27.32 min). 1H NMR (600 MHz, CDCl3) δ 7.22 (d, 1H, J = 8.4
Hz), 6.98 (s, 2H), 6.62 (dd, 1H, J1 = 7.8 Hz, J2 = 1.8 Hz), 6.14 (d, 1H,
J = 2.4 Hz), 5.67 (s, 1H), 2.33 (s, 3H), 2.16 (s, 6H). 13C NMR (150
MHz, CDCl3) δ 143.8, 136.9, 136.5, 133.9, 133.8, 130.1, 129.6, 117.8,
117.2, 112.1, 21.1, 18.1. HRMS (ESI) calcd for C15H16Cl2N 280.0654
(M + H)+, found 280.0656.
(4,5-Dimethylthiazol-2-yl)-(2,4,6-trimethylphenyl)amine
(17). To a solution of (2,4,6-trimethylphenyl)thiourea (49 mg, 0.25
mmol) in EtOH (5 mL) was added 3-bromobutan-2-one (38 mg, 0.25
mmol). The mixture was stirred at 90 °C for 1 h. The solution was
diluted with EtOAc (30 mL) and washed with H2O (10 mL) and brine
(10 mL). The organic layer was dried over Na2SO4 and then
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane/EtOAc = 3/1) to give the
desired product (40 mg, 65%) as a pale yellow solid (mp 180−182
4-[4-(2,4,6-Trimethylbenzenesulfonyl)phenoxy]piperidine-
1-carboxylic Acid tert-Butyl Ester (11b). Compound 11b was
prepared in 90% yield by a procedure similar to that used to prepare
compound 11a. The title compound was obtained as a colorless oil.
HPLC purity 99.9% (tR = 24.54 min). 1H NMR (600 MHz, CDCl3) δ
7.71 (d, 2H, J = 9.0 Hz), 6.93 (s, 2H), 6.91 (d, 2H, J = 8.4 Hz), 4.52−
4.54 (m, 1H), 3.65−3.69 (m, 2H), 3.33−3.37 (m, 2H), 2.60 (s, 6H),
2.29 (s, 3H), 1.90−1.93 (m, 2H), 1.73−1.77 (m, 2H), 1.46 (s, 9H).
4-[4-(2,4,6-Trimethylbenzenesulfonyl)phenoxy]piperidine
(11c). To a solution of 11b (128 mg, 0.28 mmol) in DCM (5 mL)
was added TFA (1 mL) at 0 °C. The mixture was stirred at 0 °C for 1
h. The reaction mixture was concentrated, and the residue was
partitioned between EtOAc (50 mL) and 1 N NaHCO3 (10 mL). The
organic layer was washed with brine (10 mL), dried with anhydrous
Na2SO4, and concentrated to give the crude product. This residue was
purified with silica gel column (DCM/MeOH = 10/1) to provide 11c
(100 mg, 99%) as a white solid (mp 95−97 °C). HPLC purity 99.5%
1
°C). HPLC purity 97.5% (tR = 22.67 min). H NMR (600 MHz,
CDCl3) δ 7.70−8.00 (bs, 1H), 6.94 (s, 2H), 2.30 (s, 3H), 2.27 (s, 6H),
2.09 (s, 3H), 2.02 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.4,
143.1, 137.4, 136.9, 135.5, 129.5, 113.2, 21.1, 18.2, 14.5, 11.1. HRMS
(ESI) calcd for C14H19N2S 247.1263 (M + H)+, found 247.1267.
2-Ethyl-1-(2,4,6-trimethylbenzenesulfonyl)-1H-pyrrole
(20a). To a solution of 2-ethyl-1H-pyrrole (∼80% purity) (24 mg,
0.25 mmol) and mesitylsulfonyl chloride (110 mg, 0.5 mmol) in 5 mL
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Journal of Medicinal Chemistry
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of THF was added 60% NaH (24 mg, 0.6 mmol) at 0 °C. The
resulting mixture was stirred at room temperature for 16 h. The
solution was diluted with EtOAc (50 mL), washed with 1 N HCl (aq)
(10 mL) and brine (10 mL). The organic layer was dried over
anhydrous Na2SO4 and then concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(hexane/EtOAc = 10/1) to give the desired product as a pale yellow
solid (20 mg, 36%): mp 74−75 °C; HPLC purity 95.2% (tR = 25.07
CDCl3) δ 139.6, 139.4, 137.5, 135.5, 124.3, 122.4, 111.2, 111.0, 21.4,
20.6, 12.9. HRMS (ESI) calcd for C14H18NO2S 264.1053 (M + H)+,
found 264.1056.
1-(2,4-Dimethylbenzenesulfonyl)-2-ethyl-1H-pyrrole (20h).
Compound 20h was prepared in 76% yield by a procedure similar
to that used to prepare compound 20a. The title compound was
obtained as a white solid (mp 75−77 °C). HPLC purity 98.3% (tR =
24.40 min). 1H NMR (600 MHz, CDCl3) δ 7.46 (d, 1H, J = 7.2 Hz),
7.33−7.35 (m, 1H), 7.10−7.12 (m, 2H), 6.22−6.24 (m, 1H), 6.04−
6.06 (m, 1H), 2.55 (q, 2H, J = 7.2 Hz), 2.47 (s, 3H), 2.39 (s, 3H), 1.13
(t, 3H, J = 7.2 Hz). 13C NMR (150 MHz, CDCl3) δ 144.7, 137.9,
137.4, 135.5, 133.6, 128.7, 127.1, 122.9, 110.6, 110.1, 21.4, 20.3, 19.8,
12.6. HRMS (ESI) calcd for C14H18NO2S 264.1053 (M + H)+, found
264.1058.
2,4-Dimethyl-1-(2,4,6-trimethylbenzenesulfonyl)-1H-pyrrole
(20i). Compound 20i was prepared in 58% yield by a procedure
similar to that used to prepare compound 20a. The title compound
was obtained as a pale red solid (mp 98−100 °C). HPLC purity 95.7%
(tR = 25.20 min). 1H NMR (600 MHz, CDCl3) δ 7.01 (s, 1H), 6.95 (s,
2H), 5.77 (s, 1H), 2.49 (s, 6H), 2.31 (s, 3H), 2.00 (s, 3H), 1.99 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 143.8, 140.2, 133.8, 132.2, 130.2,
119.7, 119.2, 114.5, 23.4, 21.1, 12.6, 11.8. HRMS (ESI) calcd for
C15H20NO2S 278.1209 (M + H)+, found 278.1205.
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-indole (22a). Com-
pound 22a was prepared in 84% yield by a procedure similar to that
used to prepare compound 20a. The title compound was obtained as a
white solid (mp 110−112 °C). HPLC purity 98.9% (tR = 24.73 min).
1H NMR (600 MHz, CDCl3) δ 7.59−7.61 (m, 1H), 7.54−7.55 (m,
1H), 7.34−7.35 (m, 1H), 7.17 (s, 2H), 6.93 (s, 2H), 6.61−6.62 (m,
1H), 2.52 (s, 6H), 2.26 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
144.1, 140.3, 134.7, 133.1, 132.5, 130.3, 126.8, 124.2, 122.8, 121.5,
112.5, 106.5, 22.7, 21.1. HRMS (ESI) calcd for C17H18NO2S 300.1053
(M + H)+, found 300.1057.
1
min). H NMR (600 MHz, CDCl3) δ 7.29−7.30 (m, 1H), 6.95 (s,
2H), 6.13−6.14 (m, 1H), 5.96−5.98 (m, 1H), 2.46 (s, 6H), 2.34−2.38
(m, 2H), 2.31 (s, 3H), 1.07 (t, 3H, J = 7.2 Hz). 13C NMR (150 MHz,
CDCl3) δ 144.0, 140.2, 136.5, 133.5, 132.3, 122.3, 110.0, 109.1, 22.4,
21.2, 19.3, 12.4. HRMS (ESI) calcd for C15H20NO2S 278.1209 (M +
H)+, found 278.1205.
2-Ethyl-1-(toluene-4-sulfonyl)-1H-pyrrole (20b). Compound
20b was prepared in 30% yield by a procedure similar to that used to
prepare compound 20a. The title compound was obtained as a
1
colorless oil. HPLC purity 98.0% (tR = 22.25 min). H NMR (600
MHz, CDCl3) δ 7.64 (d, 2H, J = 8.4 Hz), 7.28 (d, 2H, J = 8.4 Hz),
7.27 (d, 1H, J = 8.4 Hz), 6.20 (t, 1H, J = 3.0 Hz), 5.99 (d, 1H, J = 3.0
Hz), 2.68 (q, 2H, J = 7.2 Hz), 2.40 (s, 3H), 1.16 (t, 3H, J = 7.2 Hz).
13C NMR (150 MHz, CDCl3) δ 144.8, 137.5, 136.7, 130.1, 126.9,
122.4, 111.3, 111.1, 21.7, 20.6, 12.8. HRMS (ESI) calcd for
C13H16NO2S 250.0896 (M + H)+, found 250.0900.
2-Ethyl-1-(4-methoxybenzenesulfonyl)-1H-pyrrole (20c).
Compound 20c was prepared in 38% yield by a procedure similar
to that used to prepare compound 20a. The title compound was
obtained as a pale red solid (mp 73−74 °C). HPLC purity 99.5% (tR =
21.63 min). 1H NMR (600 MHz, CDCl3) δ 7.72 (d, 2H, J = 10.2 Hz),
7.28 (t, 1H, J = 1.2 Hz), 6.95 (d, 2H, J = 11.4 Hz), 6.19 (t, 1H, J = 3.0
Hz), 5.99 (d, 1H, J = 1.8 Hz), 3.85 (s, 3H), 2.69 (q, 2H, J = 7.8 Hz),
1.16 (t, 3H, J = 7.2 Hz). 13C NMR (150 MHz, CDCl3) δ 163.7, 137.4,
131.1, 129.2, 122.2, 114.7, 111.2, 111.0, 55.8, 20.6, 12.8. HRMS (ESI)
calcd for C13H16NO3S 266.0845 (M + H)+, found 266.0849.
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-indole-5-carboxylic
Acid Methyl Ester (22b). Compound 22b was prepared in 84% yield
by a procedure similar to that used to prepare compound 20a. The
title compound was obtained as a white solid (mp 131−133 °C).
HPLC purity 99.4% (tR = 23.80 min). 1H NMR (600 MHz, CDCl3) δ
8.30 (s, 1H), 7.88 (d, 1H, J = 9.0 Hz), 7.64 (d, 1H, J = 3.6 Hz), 7.40
(d, 1H, J = 8.4 Hz), 6.95 (s, 2H), 6.70 (d, 1H, J = 3.6 Hz), 3.90 (s,
3H), 2.51 (s, 6H), 2.28 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
167.3, 144.6, 140.4, 137.3, 132.8, 132.6, 130.0, 128.0, 125.6, 125.1,
123.9, 112.3, 107.1, 52.2, 22.7, 21.2. HRMS (ESI) calcd for
C19H20NO4S 358.1108 (M + H)+, found 358.1114.
1-(4-Chlorobenzenesulfonyl)-2-ethyl-1H-pyrrole (20d). Com-
pound 20d was prepared in 76% yield by a procedure similar to that
used to prepare compound 20a. The title compound was obtained as a
pale red solid (mp 61−62 °C). HPLC purity 95.6% (tR = 22.68 min).
1H NMR (600 MHz, CDCl3) δ 7.68 (d, 2H, J = 7.2 Hz), 7.47 (d, 2H, J
= 7.2 Hz), 7.27 (d, 1H, J = 3.6 Hz), 6.23 (t, 1H, J = 3.6 Hz), 6.02 (d,
1H, J = 3.6 Hz), 2.68 (q, 2H, J = 7.2 Hz), 1.18 (t, 3H, J = 7.2 Hz). 13C
NMR (150 MHz, CDCl3) δ 140.4, 138.1, 137.6, 129.8, 128.3, 122.4,
111.9, 111.6, 20.7, 12.8. HRMS (ESI) calcd for C12H13ClNO2S
271.0350 (M + H)+, found 270.0355.
2-Ethyl-1-(4-trifluoromethyl-benzenesulfonyl)-1H-pyrrole
(20e). Compound 20e was prepared in 58% yield by a procedure
similar to that used to prepare compound 20a. The title compound
was obtained as a pale red solid (mp 55−57 °C). HPLC purity 96.0%
(tR = 22.82 min). 1H NMR (600 MHz, CDCl3) δ 7.86 (d, 2H, J = 7.8
Hz), 7.76 (d, 2H, J = 7.8 Hz), 7.28−2.30 (m, 1H), 6.25 (d, 1H, J = 1.8
Hz), 6.03−6.05 (m, 1H), 2.68 (q, 2H, J = 7.2 Hz), 1.18 (t, 3H, J = 7.8
Hz). HRMS (ESI) calcd for C13H13F3NO2S 304.0614 (M + H)+,
found 304.0602.
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-indole-5-carboxylic
Acid (22c). To a solution of 22b (72 mg, 0.2 mmol) in MeOH/H2O
(4 mL/1 mL) was added 2 N LiOH (0.4 mL, 0.8 mmol). The mixture
was stirred at room temperature for 16 h. The solution was diluted
with EtOAc (30 mL), washed with 1 N HCl (aq) (10 mL) and brine
(10 mL). The organic layer was dried over Na2SO4 and then
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane/EtOAc = 1/1) to give the
desired product (45 mg, 66%) as a pale yellow solid (mp 223−224
2-Ethyl-1-(toluene-2-sulfonyl)-1H-pyrrole (20f). Compound
20f was prepared in 73% yield by a procedure similar to that used
to prepare compound 20a. The title compound was obtained as a pale
1
°C). HPLC purity 98.4% (tR = 14.89 min). H NMR (600 MHz,
DMSO-d6) δ 12.80 (bs, 1H), 8.28 (s, 1H), 7.83−7.86 (m, 2H), 7.40
(d, 1H, J = 8.4 Hz), 7.15 (m, 2H), 6.94 (d, 1H, J = 3.6 Hz), 2.44 (s,
6H), 2.27 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 167.3, 144.7,
139.6, 136.3, 132.5, 131.9, 129.7, 128.5, 125.6, 125.4, 123.7, 111.8,
107.6, 21.9, 20.5. HRMS (ESI) calcd for C18H18NO4S 344.0951 (M +
H)+, found 344.0954.
1
red oil (mp 55−57 °C). HPLC purity 98.3% (tR = 22.42 min). H
NMR (600 MHz, CDCl3) δ 7.50 (d, 2H, J = 7.2 Hz), 7.31−7.36 (m,
3H), 6.24−6.26 (m, 1H), 6.07−6.09 (m, 1H), 2.55 (q, 2H, J = 7.2
Hz), 2.54 (s, 3H), 1.13 (t, 3H, J = 7.2 Hz). 13C NMR (150 MHz,
CDCl3) δ 138.6, 138.0, 137.6, 133.7, 132.9, 128.3, 126.5, 123.0, 110.7,
110.3, 20.3, 20.0, 12.6. HRMS (ESI) calcd for C13H16NO2S 250.0896
(M + H)+, found 250.0900.
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-pyrrolo[3,2-b]-
pyridine (24a). Compound 24a was prepared in 96% yield by a
procedure similar to that used to prepare compound 20a. The title
compound was obtained as a white solid (mp 105−107 °C). HPLC
1-(3,5-Dimethylbenzenesulfonyl)-2-ethyl-1H-pyrrole (20g).
Compound 20g was prepared in 67% yield by a procedure similar
to that used to prepare compound 20a. The title compound was
obtained as a pale red solid (mp 67−70 °C). HPLC purity 98.2% (tR =
23.09 min). 1H NMR (600 MHz, CDCl3) δ 7.36 (s, 2H), 7.29 (s, 1H),
7.19 (s, 1H), 6.21 (t, 1H, J = 3.0 Hz), 6.00 (s, 1H), 2.70 (q, 2H, J = 7.2
Hz), 2.35 (s, 6H), 1.17 (t, 3H, J = 7.2 Hz). 13C NMR (150 MHz,
1
purity 99.8% (tR = 21.84 min). H NMR (600 MHz, CDCl3) δ 8.48
(d, 1H, J = 3.6 Hz), 7.74−7.75 (m, 1H), 7.71 (d, 1H, J = 8.4 Hz),
7.09−7.11 (m, 1H), 6.94 (s, 2H), 6.81 (d, 1H, J = 3.0 Hz), 2.50 (s,
6H), 2.25 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 148.1, 145.8,
144.7, 140.4, 132.6, 132.5, 129.6, 128.3, 119.8, 118.7, 107.8, 22.7, 21.1.
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HRMS (ESI) calcd for C16H17N2O2S 301.1005 (M + H)+, found
301.1010.
reflected by a decrease in absorbance at 340 nm. The kinase reaction
mixture (100 μL) contained 50 mM Mops (pH 7.0), 10 mM MgCl2, 1
mM ATP, 1 mM PEP, 0.1 mM NADH, 8 U of pyruvate kinase, 15 U
of lactate dehydrogenase, a fixed amount of type I or type II PKA
holoenzyme, and 0.1 mM cAMP, with or without 25 μM test
compound. Reactions were pre-equilibrated at room temperature and
initiated by adding the kemptide substrate (final concentration, 0.26
mM). PKA activities measured in the presence of 25 μM H89, a
selective PKA inhibitor, were used as positive controls of PKA
inhibition.
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-pyrrolo[3,2-c]-
pyridine (24b). Compound 24b was prepared in 84% yield by a
procedure similar to that used to prepare compound 20a. The title
compound was obtained as a white solid (mp 119−120 °C). HPLC
purity 99.7% (tR = 22.77 min). 1H NMR (600 MHz, CDCl3) δ 8.88 (s,
1H), 8.33 (d, 1H, J = 6.0 Hz), 7.54 (d, 1H, J = 3.6 Hz), 7.29 (d, 1H, J
= 5.4 Hz), 6.95 (s, 2H), 6.68 (d, 1H, J = 3.6 Hz), 2.50 (s, 6H), 2.26 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 144.8, 144.3, 143.6, 140.4, 139.0,
132.6, 132.3, 127.2, 126.4, 107.6, 105.1, 22.6, 21.1. HRMS (ESI) calcd
for C16H17N2O2S 301.1005 (M + H)+, found 301.1009.
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-pyrrolo[2,3-c]-
pyridine (24c). Compound 24c was prepared in 87% yield by a
procedure similar to that used to prepare compound 20a. The title
compound was obtained as a white solid (mp 132−134 °C). HPLC
purity 99.8% (tR = 21.77 min). 1H NMR (600 MHz, CDCl3) δ 8.69 (s,
1H), 8.35 (d, 1H, J = 5.4 Hz), 7.73 (d, 1H, J = 3.6 Hz), 7.48 (d, 1H, J
= 5.4 Hz), 6.96 (s, 2H), 6.63 (d, 1H, J = 3.0 Hz), 2.53 (s, 6H), 2.28 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 144.9, 142.1, 140.6, 135.8, 135.1,
132.8, 132.4, 131.8, 130.0, 115.9, 105.5, 22.7, 21.2. HRMS (ESI) calcd
for C16H17N2O2S 301.1005 (M + H)+, found 301.1011.
FRET-Based Assay for EPAC1, EPAC2, or AKAR3 Activation.
HEK293 cells stably transfected with genetically encoded FRET
biosensors for EPAC1 (EPAC1 FL), EPAC2 (EPAC2 FL), or PKA
(AKAR3) were used in this study. EPAC1 FL and EPAC2 FL were
generated by sandwiching the full-length human EPAC1 or EPAC2
proteins with a Cerulean variant of cyan fluorescent protein (CFP) at
their N-termini and a Venus variant of yellow fluorescent protein
(YFP) at their C-termini.23 Activation of EPAC1 or EPAC2 in
response to cAMP was measured as a decrease of FRET, and it was
plotted as an increase of the CFP/YFP emission ratio (i.e., 485/535
nm emission ratio). The function core of AKAR3 contains a PKA
substrate motif and a phosphoamino acid binding domain connected
by a flexible linker, which is flanked by a variant of CFP at its N-
terminus and a variant of YFP at its C-terminus.24 Activation of
AKAR3 in response to cAMP was measured as an increase of FRET,
and it was plotted as a decrease of the 485/535 nm emission ratio. To
test the effect of 20i on EPAC1, EPAC2, or PKA activation in live
cells, single-cell suspensions of EPAC1-C9, EPAC2-C1, and AKAR3-
C12 clones were plated onto 96-well clear-bottom assay plates (Costar
3904) coated with rat tail collagen (Invitrogen). The clones were
maintained in DMEM (25 mM glucose, 10% FBS) for 24 h in order to
allow the cell cultures to become 80−90% confluent. Cells were
pretreated with various concentrations of 20i or vehicle control before
being activated by membrane permeable cAMP analogues. Fluo-
rescence spectra were recorded as described previously32 using a
FlexStation 3 microplate reader controlled using SoftMax Pro software
(Molecular Devices) with the excitation wavelength set at 435/9 nm
(455 nm cutoff) and the emission wavelength set at 485/15 nm (CFP)
or 535/15 nm (YFP). The time course of the change of FRET ratio
was plotted after exporting data to Origin, version 7.5 (OriginLab).
1-(2,4,6-Trimethylbenzenesulfonyl)-1H-pyrrolo[2,3-b]-
pyridine (24d). Compound 24d was prepared in 87% yield by a
procedure similar to that used to prepare compound 20a. The title
compound was obtained as a white solid (mp 139−141 °C). HPLC
1
purity 99.6% (tR = 22.81 min). H NMR (600 MHz, CDCl3) δ 8.22
(d, 1H, J = 4.2 Hz), 7.85 (d, 1H, J = 3.0 Hz), 7.83 (d, 1H, J = 7.8 Hz),
7.08−7.11 (m, 1H), 6.93 (s, 2H), 6.57 (d, 1H, J = 3.0 Hz), 2.71 (s,
6H), 2.27 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 147.6, 144.7,
144.0, 141.3, 132.8, 132.1, 129.3, 126.9, 122.4, 118.6, 103.8, 23.0, 21.2.
HRMS (ESI) calcd for C16H17N2O2S 301.1005 (M + H)+, found
301.0981.
Biological Evaluation Methods. Primary Screen Assay.
Fluorescence intensity of 8-NBD-cAMP in complex with EPAC2 has
been used as the readout in the primary screen assay. Briefly, 50 nM
EPAC2 solution was prepared in 20 mM Tris buffer, pH 7.5,
containing 150 mM NaCl, 1 mM EDTA, and 1 mM DDT. 8-NBD-
cAMP was added to EPAC2 solution up to 60 nM from stock solution
in water. Sample was dispensed into 96-well plate (100 μL/well), and
test compounds were added (1 μL/well) from 96-well mother plates.
Test compounds were added from 10 mM stock solutions in DMSO.
Samples with cAMP addition (1 μL/well from 30 mM stock solution
in water) and no additions were used as a positive and a negative
control. Fluorescence intensity signal from 8-NBD probe was recorded
at room temperature before and after tested compounds were added
using SpectraMaxM2 microplate reader (Molecular Devices, Silicon
Valley, CA, U.S.) with excitation/emission wavelengths set at 470/540
nm.
Secondary Confirmation Assay. Measurement of in vitro
guanine nucleotide exchange factor (GEF) activity of EPAC was
adapted from a well-known fluorescence-based assay using a
fluorescent guanine nucleotide analogue30 and used as a functional
confirmation assay for the compounds identified from primary screen.
Briefly, 0.2 μM Rap1B (1−167) loaded with the fluorescent GDP
analogue (Mant-GDP) was incubated with EPAC in 50 mM Tris
buffer, pH 7.5, containing 50 mM NaCl, 5 mM MgCl2, 1 mM DTT,
and a 100-fold molar excess of unlabeled GDP (20 μM) in the
presence of 25 μM tested compound and 25 μM cAMP. Exchange of
Mant-GDP by GDP was measured as a decrease in fluorescence
intensity over time using a FluoroMax-3 spectrofluorometer with
excitation/emission wavelengths set at 366/450 nm. Typically, decay
in the fluorescence intensity was recorded over a time course of 6000 s
with data points taken every 60 s.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For X.C.: phone, (409) 772-9656; fax, (409) 772-7050; e-
mail, xcheng@utmb.edu. For J.Z.: phone, (409) 772-9748; fax,
(409) 772-9818; e-mail, jizhou@utmb.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grants P30DA028821,
R21MH093844 (J.Z.), R01GM066170, and R21NS066510
(X.C.) from the National Institutes of Health, R. A. Welch
Foundation Chemistry and Biology Collaborative Grant from
Gulf Coast Consortia (GCC) for Chemical Genomics, and
John Sealy Memorial Endowment Fund. We thank Dr. Jin
Zhang of Johns Hopkins School of Medicine for providing the
EPAC1, EPAC2, and AKAR3 FRET reporters. We also thank
Drs. Lawrence C. Sowers, Carol L. Nilsson, Jacob A.
Theruvathu, and Huiling Liu for mass spectrometry assistance
Counter Screening Assay. Kinase activities of the type I and type
II PKA holoenzymes were measured spectrophotometrically in a 96-
well plate with a coupled enzyme assay as described previously.31 In
this assay, the formation of the ADP is coupled to the oxidation of
NADH by the pyruvate kinase/lactate dehydrogenase reactions so the
reaction rate can be determined by following the oxidation of NADH,
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Bos, J. L. Ligand-mediated activation of the cAMP-responsive guanine
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and Dr. Tianzhi Wang at the NMR core facility of University of
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ABBREVIATIONS USED
■
EPAC, exchange proteins directly activated by cAMP; SAR,
structure−activity relationship; cAMP, cyclic adenosine mono-
phosphate; 8-NBD-cAMP, 8-(2-[7-nitro-4-benzofurazanyl]-
aminoethylthio)adenosine 3′,5′-cyclic monophosphate; GDP,
guanosine diphosphate; PKA, protein kinase A; FRET,
fluorescence resonance energy transfer; GEF, guanine nucleo-
tide exchange factor; GTP, guanosine triphosphate; Rap, Ras-
related protein; HTS, high-throughput screening; 007-AM, 8-
(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic mono-
phosphate acetoxymethyl ester; AKAR3, A-kinase activity
reporter 3; 8-Br-cAMP-AM, 8-bromoadenosine 3′,5′-cyclic
monophosphate acetoxymethyl ester; TLC, thin layer chroma-
tography; UV, ultraviolet; TMS, tetramethylsilane; HRMS,
high-resolution mass spectrometry; HPLC, high-performance
liquid chromatography; TFA, trifluoroacetic acid; DCM,
dichloromethane; EtOAc, ethyl acetate; DMSO, dimethyl
sulfoxide; EDTA, ethylenediaminetetraacetic acid; DDT,
dichlorodiphenyltrichloroethane; ADP, adenosine diphosphate;
NADH, nicotinamide adenine dinucleotide; PEP, phospho-
(enol)pyruvate; H89, N-[2-[[3-(4-bromophenyl)-2-propenyl]-
amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride; CFP,
cyan fluorescent protein; YFP, yellow fluorescent protein;
DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine
serum
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Cheng, X. Isoform-specific antagonists of exchange proteins directly
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(19) Tsalkova, T.; Blumenthal, D. K.; Mei, F. C.; White, M. A.;
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(22) Yu, S.; Mei, F. C.; Lee, J. C.; Cheng, X. Probing cAMP-
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NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published ASAP on January 15, 2013, a
correction was made to the Acknowledgments. The revised
version was reposted January 31, 2013.
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