Discovery and characterization of carbamothioylacrylamides as EP2 selective antagonists

Article abstract of DOI:10.1021/ml400112h

Prostanoid receptor EP2 is emerging as a novel target for development of anti-inflammatory drugs for the treatment of chronic neurodegenerative and peripheral diseases; however, the availability of EP2 antagonist probes for exploration of peripheral disease models is very limited. We now report identification and characterization of a novel chemical class of compounds that show nanomolar potency and competitive antagonism of the EP2 receptor. A compound in this class, TG6-129, showed prolonged plasma half-life and did not cross the blood-brain barrier. This compound also suppressed the induction of inflammatory mRNA markers in a macrophage cell line upon activation of EP2. Thus, this compound could be useful as a probe for a variety of peripheral chronic inflammatory diseases such as rheumatoid arthritis and chronic obstructive pulmonary disease, in which EP2 appears to play a pathogenic role.

Full text of DOI:10.1021/ml400112h

Letter
pubs.acs.org/acsmedchemlett
Discovery and Characterization of Carbamothioylacrylamides As EP2
Selective Antagonists
Thota Ganesh,*,†,§ Jianxiong Jiang,*,†,§ Rangaiah Shashidharamurthy,‡ and Ray Dingledine†
†Department of Pharmacology, School of Medicine, Emory University, Atlanta, Georgia 30322, United States
‡Department of Pharmaceutical Sciences, PCOM-SOP, GA Campus, Suwanee, Georgia 30024, United States
S
* Supporting Information
ABSTRACT: Prostanoid receptor EP2 is emerging as a novel
target for development of anti-inflammatory drugs for the
treatment of chronic neurodegenerative and peripheral
diseases; however, the availability of EP2 antagonist probes
for exploration of peripheral disease models is very limited. We
now report identification and characterization of a novel
chemical class of compounds that show nanomolar potency
and competitive antagonism of the EP2 receptor. A compound
in this class, TG6-129, showed prolonged plasma half-life and
did not cross the blood−brain barrier. This compound also
suppressed the induction of inflammatory mRNA markers in a macrophage cell line upon activation of EP2. Thus, this
compound could be useful as a probe for a variety of peripheral chronic inflammatory diseases such as rheumatoid arthritis and
chronic obstructive pulmonary disease, in which EP2 appears to play a pathogenic role.
KEYWORDS: Inflammation, EP2 receptor, EP2 antagonist, cytokines, acrylamides, macrophage
nflammation is an early event during the development of
activation.14−16 Thus, future anti-inflammatory strategy could
Imany chronic peripheral diseases including rheumatoid involve specific prostanoid receptor inhibition rather than
generic block of the entire COX-2 cascade.
arthritis, chronic obstructive pulmonary disease, and inflamma-
tory bowel disease.1−4 Inflammation is also a key driver of many
central nervous system disorders such as Alzheimer’s and
Parkinson’s disease, amyotrophic lateral sclerosis, and epi-
lepsy.5−7 Elevation of cyclooxygenase-2 (COX-2), inducible
nitric oxide synthase (iNOS), inflammatory cytokines, and
chemokines occurs during the early stages of the development
of these diseases.1−7 COX-2 has been extensively explored as a
biological target for the development of anti-inflammatory
therapy.8,9 In the past, COX-2 inhibitors including rofecoxib
(Vioxx) and valdecoxib (Bextra) were approved for the
treatment of acute pain and inflammation in arthritis patients.
The COX-2 inhibitors have also been used in an attempt to
prevent the progression of neurodegenerative disease such as
Alzheimer’s disease (AD).10 However, although these agents
have shown efficacy in AD animal models, they have not shown
any benefit to patients with AD. Clinical studies from chronic
use of COX-2 inhibitors for pain and inflammatory diseases
resulted in cardiovascular side effects. Consequently, Vioxx and
Bextra were withdrawn from the United States market.11
COX-2 synthesizes five prostanoids PGD2, PGE2, PGF2,
PGI2, and TXA2, which activate nine G protein-coupled
receptors (EP1−EP4, DP1−DP2, FP, IP, and TP) and several
of these receptors play protective as well as deleterious roles.12
For example, IP activation can be cardioprotective, whereas
EP2 activation can be both pro- and anti-inflammatory.13
Studies now conclude that COX-2 inhibition mediated toxicity
is due to reduced levels of PGI2 leading to reduced IP receptor
Prostanoid receptor EP2 is activated by PGE2 and is
emerging as an important biological target for anti-inflamma-
tory therapy.17−20 EP2 is positively coupled though Gαs to
cyclic AMP (cAMP) production. When activated by PGE2, EP2
stimulates adenylate cyclase resulting in elevation of cAMP
concentration, which initiates multiple downstream events
mediated by protein kinase A (PKA) or exchange protein
activated by cAMP (Epac).21,22 We have developed a set of cell
based TR-FRET assays to identify EP2 selective antagonists.23
By using these assays in conjunction with high-throughput
screening of 262,300 compounds we have previously identified
an acrylamide class of selective antagonists.24,25 From the same
screen, we have also found a compound 1 (PubChem, SID
17503974) as a hit with IC50 = 1.6 μM at saturating
concentrations (1 μM) of PGE2 used for the activation of
EP2 receptors in C6 glioma (C6G) cells. Interestingly, this
compound up to 40 μM has shown no inhibition of EP4 or β2-
adrenergic receptors under agonist saturated conditions (Figure
1A). Compound 1 possesses a carbamothioylacrylamide moiety
flanked by phenylacryl-amide on one side and a thiadiazole-
sulfonamide on the other side. Initially, we asked whether this
scaffold is worth pursuing for further characterization because
of structural similarity to thioureas, which are known to
Received: March 20, 2013
Accepted: May 20, 2013
© XXXX American Chemical Society
A
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We have resynthesized compound 1, as shown in Scheme 1.
Commercially available p-nitrobenzene-sulfonyl chloride (A)
and thiadiazole-2-amine (B) were coupled to form intermediate
C, which on metal catalyzed reduction using tin chloride
provided p-aminobenzene-sulfonamide D. The final step was
carried out analogous to the reported procedure in a one-pot
reaction,28 where ammonium thiocyanate was treated with 4-
fluorocinnamyl chloride (E) in refluxing acetone for 15 min
followed by D for 1 h to synthesize the product (henceforth
referred to as TG6-129). Both synthesized and commercially
available materials have shown greater than 95% purity by
1HNMR and LCMS analysis (see Supporting Information for
characterization data), and both show bioactivity in the EP2
screen (see below).
We then demonstrated that TG6-129 inhibits PGE2 induced
EP2 receptor activation in a concentration-dependent manner
(Figure 1B). In this assay, it displayed a competitive antagonism
of EP2 receptors as shown by Schild regression analysis with KB
of 8.8 nM and slope of 1.2 (Figure 1C). There are four Gαs
coupled prostanoid receptors: DP1, EP2, EP4, and IP. Among
these, DP1 has the closest sequence homology as EP2, followed
by IP, then EP4, although both EP2 and EP4 share a common
endogenous ligand PGE2 for their activation.13 To evaluate the
selectivity of TG6-129 for EP2 over other Gαs-coupled
prostanoid receptors, we created C6G-cell lines overexpressing
DP1, EP4, and IP receptors and used them for counter
screening. Compound TG6-129 showed 1660-fold selectivity
against DP1, 443-fold for EP4, and 22-fold selectivity against IP
receptors (Figure 1D), indicating it is an EP2 selective
antagonist and could be a useful probe for in vitro and in
vivo biochemical studies.
To develop structure−activity relationships (SAR) for
compound TG6-129, a limited set of 8 commercially available
analogues has been purchased and tested in EP2, EP4, and β2-
AR assays. The EP2 Schild KB value, which represents the
concentration required to cause a 2-fold increase of the EC50 of
the agonist PGE2, is used for SAR comparisons below. As
shown in Table 1, removal of fluorine (compound 2) resulted
in 2-fold less potency on EP2 in comparison to parent TG6-
129. Incorporation of a methoxy group as in 3 in place of
fluorine caused 4-fold less potency. Replacement of the whole
cinnamoyl group with thiocinnamoyl (4 vs 2) reduced the
potency 2-fold, but 4-fold less to parent compound TG6-129.
Replacement of a 4-methoxycinnamicamide with a 4-methox-
yamide (5 vs 3) improved the activity by 1.5-fold, but in
comparison to the parent, it is still 2.6-fold less. This result
indicates the acrylamide moiety is not absolutely essential and
may be replaced with amides for further SAR study.
Figure 1. Selective inhibition of EP2 receptor by a carbamothioyla-
crylamide. (A) Compound TG6-129 (SID 17503974) showed robust
inhibition of PGE2 (1 μM)-induced cAMP accumulation in C6G-EP2
cells with an IC50 = 1.6 μM, without affecting prostaglandin EP4 and
β2-adrenergic receptors. Data were normalized as percentage
inhibition of maximum control response; points represent mean
SEM (n = 3). (B) TG6-129 inhibited PGE2-induced EP2 receptor
activation in a concentration-dependent manner. The PGE2 EC50s
were 0.79, 1.86, 15.2, and 275 nM, respectively, in the presence of
vehicle, 0.01, 0.1, or 1 μM TG6-129. Data were normalized as percent
maximum control (vehicle) response; points represent mean SEM
(n = 4). (C) TG6-129 displayed competitive antagonism of the EP2
receptor as shown by Schild regression analysis, with KB = 8.8 nM and
slope = 1.2. (D) TG6-129 was tested for inhibition of all 4 human
Gαs-coupled prostanoid receptors. KB values: 14,600, 8.8, 3900, and
199 nM for DP1, EP2, EP4, and IP receptors, respectively.
Scheme 1. Synthesis of (E)-N-((4-(N-(5-Ethyl-1,3,4-
thiadiazol-2-yl)sulfamoyl)carbamothioyl)-3-(4-
a
fluorophenyl)acrylamide (TG6-129)
a
Reagents and conditions: (i) pyridine, 95%; (ii) tin-chloride
We have also included analogues in which the thiadiazole
ring was replaced with methoxy-pirimidine (6) or methoxy-
piridazine (7), and in which the methoxy-pirimidines were
substituted with amides rather than acryl amides on the left side
(8−9). Compounds 6 and 7 showed only about 2- and 1.5-fold
less potency in comparison to the parent, respectively,
indicating the thiadiazole ring may be replaced with other
heterocycles for further SAR study. However, the amide
derivatives 8 and 9 were 12−15-fold less potent than parent.
Nonetheless, all these compounds (2−9) have shown nano-
molar Schild KB values for EP2 receptors.
dihydrate in ethyl acetate at reflux, 70%; (iii) ammoniumthiocyanate
in acetone at reflux, 80%.
potentially form adducts with glutathione in in vitro and in vivo
systems.26 While the formation of adducts (a.k.a., bioactivation)
is not a stumbling-block for compound development, this
process could have widespread effects on other proteins. We
have searched the PubChem database to find whether
compound 1 (SID 17503974) belongs to a promiscuous class
of compounds, but interestingly, we found it was only active in
19 bioassays out of 575 tested. It showed an IC50 of 10−100
μM in most of the assays, except in an assay to identify
inhibitors of S. aureus NadD (saNadD), where it has an IC50 of
4.5 μM.27 Thus, this compound may not be a promiscuous hit.
Analogues 2−9 were then subjected to a selectivity test using
C6G-cells overexpressing the EP4 receptors. Interestingly, a
majority of them have shown low to high micromolar Schild KB
for EP4 receptors, except compound 8, which showed
B
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a
Table 1. EP2 Potency, Selectivity, and Cytotoxicity of Carbamothioylacrylamide Derivatives
a
EP2, EP4, IP, and DP1 KB values are averages from 2 to 5 independent experiments. In vitro half-maximal cytotoxic concentration (CC50) values
are from one experiment run in quadruplicate measurements.
submicromolar Schild KB value. As shown in Table 1, the
selectivity index against EP4 is excellent for five compounds 2,
3, 5, 6, and 7 in the range 340- to 2000-fold, but marginal for
three compounds 4, 8, and 9 (78-, 7-, and 24-fold, respectively).
The compounds 2−5 also showed high selectivity index (311−
1300-fold) against DP1 receptor; compounds 6 and 7 showed
no activity to DP1 (Table 1). However, a majority of these
derivatives only show 10−75-fold selectivity index against IP
receptor, except compound 6, which showed 180-fold
selectivity, and compound 2, which was only 3.5-fold selective
for EP2 over IP receptor. Unlike our other class of EP2
antagonists, which showed high selectivity to IP receptor (240−
300-fold) and low selectivity to DP1 (∼10-fold),24,25 the
present class of carbamothioylacrylamides (TG6-129 and 2−9)
show the opposite: high selectivity to DP1 and low selectivity
to IP (Table 1) suggesting a somewhat different nature of
binding to the receptors. We also determined the in vitro
cytotoxicity for these compounds by measuring the cell viability
of C6G-cells after incubation for 48 h. All the test compounds
showed low cytotoxicity with a CC50 ≥ 96 μM (Table 1). The
high selectivity against EP4 and DP1 and moderate selectivity
to IP receptors and insubstantial cytotoxicity in vitro prompted
us to characterize this scaffold for further development.
We first determined the aqueous solubility of the compound
TG6-129 in PBS buffer at pH 7.0 by nephelometry.29 It was
soluble at 71 μg/mL (145 μM) indicating a moderate solubility.
Then, we wanted to know whether this scaffold is stable in liver
microsomal fractions. On the basis of the potency and
selectivity, we have selected two compounds: compound
TG6-129 represents a thiadiazole-sulfonamide and compound
6 represents a methoxypyrimidine-sulfonamide, both of which
have shown high selectivity and low cytotoxicity (Table 1).
These two compounds therefore were subjected to in vitro liver
metabolism in microsomal fractions of pooled mouse or human
livers at 1 and 10 μM. As shown in Figure 2, compound TG6-
129 was slightly more stable in comparison to compound 6 in
both mouse and human liver microsomes, with 40−60% of
both compounds remaining at 60 min. Midazolam was used as
C
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Figure 2. Metabolic stability study of carbamothioylacrylamide
compounds in liver microsomes. Two representative carbamothioyla-
crylamide compounds (TG6-129 and compound 6) were tested for
metabolic stability at 1 or 10 μM in pooled mouse (A) or human (B)
liver microsomes with midazolam as a control. The metabolic stability
of compounds was expressed as half-life (t1/2) in liver microsomes.
Data are shown as mean SEM (n = 3).
Figure 3. Pharmacokinetics of TG6-129. The compound was
administered to mice by i.p. (5 mg/kg) or p.o. (10 mg/kg) route
for pharmacokinetics. Compound concentrations in plasma (ng/mL)
were measured at different time points. Data are shown as mean
SEM (n = 3 mice per time point).
Figure 4. EP2 receptor activation induces macrophage activation. (A)
P388D1 macrophages were pretreated with TG6-129 (10 μM) or
vehicle for 1 h, then followed by incubation with EP2 selective agonist
butaprost (10 μM) for 2 h. The mRNA levels of the indicated
inflammatory mediators were measured by qRT-PCR. (B) EP2 mRNA
levels in P388D1 macrophages stimulated with butaprost for 2 h (n =
3−4; ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA with
post hoc Bonferroni test). Data are shown as mean SEM.
a reference in these tests for comparison, which showed
expected half-lives (<15 min) in both liver fractions. These
results prompted us to examine pharmacokinetics.
On the basis of its higher stability in liver fractions, we have
chosen TG6-129 for pharmacokinetic study in mice.
Compound TG6-129 was dosed into female C57BL/6 mice
at 10 mg/kg by oral gavage or 5 mg/kg intraperitoneally in a
vehicle consisting of 10% DMSO, 70% PEG400, and 20%
sterile water. At five time points, starting from 0.5 to 8 h after
administration, blood was collected and processed for
preparation of plasma samples. Brain samples were also
collected at 1 and 2 h time points for brain permeability
analysis. As shown in Figure 3, TG6-129 was readily absorbed
into plasma. About 12 (by oral route) and 30 μM (by ip route)
concentrations are observed at 30 min. These concentrations
are 1 to 3 orders of magnitude higher than its EP2 KB value
(Figure 1). Although the calculated half-life in plasma is 2.7 h,
its concentration at 8 h by both routes of administration is
about 200−500-fold higher than its EP2 Schild KB value,
indicating prolonged availability of therapeutic levels in plasma.
Interestingly, analysis of brain samples revealed that at 5 mg/kg
by ip or 10 mg/kg by oral gavage no detectable amount is
found. However, at 10 mg/kg ip, very little compound at 1 h
was detected in the brain tissue with a brain-to-plasma ratio of
0.02. Prolonged plasma half-life and exclusion from the brain
will be particularly attractive for using this compound as a
probe for chronic peripheral disease models, in contrast to the
other class of highly brain permeant EP2 antagonists we
recently reported.24,25
Recently, Pfizer has reported a novel class of selective EP2
antagonists with requisite plasma pharmacokinetics.30 While
the Pfizer lead compound may well be useful for anti-
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ASSOCIATED CONTENT
■
S
* Supporting Information
Contains experimental methods including details of synthesis
and characterization data and assay protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: tganesh@emory.edu (T.G.); jjiang3@emory.edu (J.J.).
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Author Contributions
§T.G., J.J., R.S., and R.D. designed the research; T.G., J.J., and
R.S. performed the research; T.G., J.J., and R.D. wrote the
manuscript. T.G. and J.J. contributed equally to this work.
Funding
This work was supported by NIH/NINDS grants
K99NS082379 (to J.J.) and U01NS058158 (to R.D.), and the
Epilepsy Foundation (to J.J.).
Notes
The authors declare no competing financial interest.
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