Lead optimization studies of cinnamic amide EP2 antagonists

Article abstract of DOI:10.1021/jm5000672

Prostanoid receptor EP2 can play a proinflammatory role, exacerbating disease pathology in a variety of central nervous system and peripheral diseases. A highly selective EP2 antagonist could be useful as a drug to mitigate the inflammatory consequences of EP2 activation. We recently identified a cinnamic amide class of EP2 antagonists. The lead compound in this class (5d) displays anti-inflammatory and neuroprotective actions. However, this compound exhibited moderate selectivity to EP2 over the DP1 prostanoid receptor (～10-fold) and low aqueous solubility. We now report compounds that display up to 180-fold selectivity against DP1 and up to 9-fold higher aqueous solubility than our previous lead. The newly developed compounds also display higher selectivity against EP4 and IP receptors and a comparable plasma pharmacokinetics. Thus, these compounds are useful for proof of concept studies in a variety of models where EP2 activation is playing a deleterious role.

Full text of DOI:10.1021/jm5000672

Article
pubs.acs.org/jmc
Open Access on 04/28/2015
Lead Optimization Studies of Cinnamic Amide EP2 Antagonists
Thota Ganesh,* Jianxiong Jiang, Myung-Soon Yang, and Ray Dingledine
Department of Pharmacology, School of Medicine, Emory University, 1510 Clifton Road, Atlanta, Georgia 30322, United States
S
* Supporting Information
ABSTRACT: Prostanoid receptor EP2 can play a proinflammatory
role, exacerbating disease pathology in a variety of central nervous
system and peripheral diseases. A highly selective EP2 antagonist
could be useful as a drug to mitigate the inflammatory consequences
of EP2 activation. We recently identified a cinnamic amide class of
EP2 antagonists. The lead compound in this class (5d) displays anti-
inflammatory and neuroprotective actions. However, this compound
exhibited moderate selectivity to EP2 over the DP1 prostanoid
receptor (∼10-fold) and low aqueous solubility. We now report
compounds that display up to 180-fold selectivity against DP1 and up
to 9-fold higher aqueous solubility than our previous lead. The newly
developed compounds also display higher selectivity against EP4 and
IP receptors and a comparable plasma pharmacokinetics. Thus, these
compounds are useful for proof of concept studies in a variety of models where EP2 activation is playing a deleterious role.
resulting in elevation of cytoplasmic cAMP concentration,
which initiates downstream events mediated by protein kinase
A (PKA)32,33 or exchange protein activated by cAMP
(Epac).34−36
INTRODUCTION
■
Inflammation plays a pathogenic role in a variety of acute and
chronic neurodegenerative diseases such as status epilepticus
(SE), epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer’s
disease (AD), Parkinson’s disease (PD), and traumatic brain
injury (TBI).1−8 Cyclooxygenase 2 (COX-2) is induced during
and after brain injury and is a major contributor to the
inflammation and disease progression in a variety of central
nervous system (CNS) diseases.9−12 COX-2 inhibitors have
been widely explored for suppression of pain and inflammation
in variety of peripheral diseases, for example, in patients with
arthritis.13,14 However, COX-2 inhibitors cause adverse
cardiovascular effects by reducing activation of a downstream
prostanoid receptor subtype IP.15−18 As a result, two COX-2
inhibitors, rofecoxib (Vioxx) and valdecoxib (Bextra), were
withdrawn from the U.S. market. Moreover, it is not yet clear
that COX-2 inhibitors could provide a benefit to patients with
chronic inflammatory neurodegenerative diseases such as
epilepsy and AD.19−26 Thus, future anti-inflammatory therapy
should be targeted through a specific proinflammatory
prostanoid synthase or receptor to blunt the inflammation
and neuropathology in CNS diseases rather than to block the
entire COX-2 signaling.
COX-2 catalyzes the synthesis of prostaglandin-H2 (PGH2)
from arachidonic acid, which is transformed into five
prostanoids, PGD2, PGE2, PGF2, PGI2 and TXA2, by cell
specific synthases. These prostanoids activate nine receptors,
DP1, DP2, EP1, EP2, EP3, EP4, FP, IP, and TP. Each of these
receptors can play protective as well as harmful roles in a variety
of CNS and peripheral pathophysiologies.27−29 EP2 receptor
has emerged as an important biological target for drug
discovery to treat a variety of CNS and peripheral diseases.30,31
When activated by PGE2, EP2 stimulates adenylate cyclase
The EP2 receptor is widely expressed in both neurons and
glia in the brain and plays a “yin−yang” nature of protective as
well as deleterious role.31 For example, in some chronic
neurodegenerative disease models, EP2 activation appears to
promote inflammation and neurotoxicity. Deletion of the EP2
receptor reduces oxidative damage and amyloid-β burden in a
mouse model of AD.37 EP2 deletion also attenuates neuro-
toxicity by α-synuclein aggregation in mouse model of PD.38
Moreover, EP2 deletion improves motor strengths and the
survival of the ALS mouse.39 Furthermore, mice lacking EP2
receptors have shown less cerebral oxidative damage produced
by the activation of innate immunity.40 In vitro, microglia
cultures from mice lacking EP2 have shown enhanced amyloid-
β phagocytosis and are less sensitive to amyloid-β induced
neurotoxicity.41 Despite a wealth of information available from
EP2 gene knockout studies, results from pharmacological
inhibition of EP2 are limited because the antagonists for EP2
receptors have only been created recently by Pfizer42 and us.43
Earlier, we reported identification of a cinnamic amide class of
EP2 antagonists by using a high-throughput screening
method.43 A limited structure−activity relationship study
(SAR) concluded that this class of compounds displays high
potency to EP2 receptor but moderate selectivity to EP2 over
another prostanoid receptor, DP1. The lead compound in this
class, 5d (aka TG6-10-1), displays about 10-fold selectivity to
EP2 over DP1 and poor aqueous solubility (27 μM). However,
Received: January 13, 2014
Published: April 28, 2014
© 2014 American Chemical Society
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5d demonstrated robust neuroprotective and anti-inflammatory
effects in a pilocarpine model of status epilepticus when
administered in three doses beginning 4 h after mice entered
into status epilepticus.44 A key to advance this class of
compounds for preclinical studies in a variety of neuro-
degenerative disease models is to improve their EP2 selectivity,
aqueous solubility, and in vivo pharmacokinetics. In the present
study we report the synthesis of 45 new analogues and their
structure−activity relationships and show that improvements
are made in terms of selectivity, solubility, and metabolic
stability in liver microsomes. Two compounds, 6a and 6c,
display about 4- to 18-fold higher selectivity against DP1
receptor and 5- to 8-fold higher aqueous solubility than the
previous best compound 5d.
homologous to DP1 (44%) and IP receptors (40%).45 Earlier,
compounds 5a and 5d were tested against other prostanoid
receptors. Although they displayed high selectivity to EP2 over
EP1, EP3, EP4, FP, IP, and TP receptors, they showed only
moderate selectivity (∼10-fold) to DP1 receptor.44,46 None of
the earlier set of 27 compounds were more selective over DP1
than 5a and 5d (not shown). Furthermore, compounds 5a and
5d displayed low aqueous solubility (45 and 27 μM,
respectively) (Table 1). Thus, our initial goal was to identify
compounds with enhanced selectivity and aqueous solubility.
Synthesis and Further Structure−Activity Relation-
ship Study on Cinnamic Amide Analogues. The scaffold
5a (Figure 1) possess four obvious sites for structural
modification: (i) trimethoxyphenyl group, (ii) acrylamide
moiety, (iii) ethylene linker, (iv) a methyl group on the indole
ring. Earlier, we had designed a compound 5d with CF3 in place
of CH3 on the indole ring, with a premise that the fluorine
atom(s) often enhances ADME properties.47 Indeed, this
transformation enhanced metabolic stability (Table 3) and
brain and plasma PK properties.44 However, the CF3 analogue
(5d) was about 7-fold less potent for EP2 in comparison to the
CH3 analogue 5a (Table 2). In the present study, to examine
whether three methoxyl groups on the phenyl ring are
important for bioactivity, we have synthesized several
derivatives that have reduced number of methoxyl groups or
were completely substituted with other substituents as shown
in Scheme 1. The synthesis is carried out starting from
RESULTS AND DISCUSSION
■
First Generation Cinnamic Amide EP2 Antagonists
Show Poor Aqueous Solubility, Poor in Vitro Liver
Microsomal Stability, and Moderate Plasma Half-Life.
We previously synthesized 27 compounds around initial high-
throughput screening hit 5a (aka TG4-155) (Figure 1) for
Scheme 1. Synthesis of First Generation 1-Indole Cinnamic
a
Amide EP2 Antagonists
Figure 1. Optimization strategy. Structures of representative first
generation EP2 antagonists. Regions marked are explored for SAR
study.
structure−activity relationship study. Several derivatives from
this set showed potent EP2 inhibition with Schild KB values at
the low nanomolar level, and they also displayed excellent
selectivity against EP4 and β-AR receptors.43 To examine the
druglike properties within the class, 10 potent compounds
(EP2 Schild KB < 20 nM) were selected and subjected to
metabolic stability in microsomal fractions of mouse and
human liver at two different concentrations (1 and 10 μM). A
majority of these compounds were found to be labile in these
liver fractions with <15 min half-life at 10 μM concentration43
except one compound 5d, which showed >15 min half-life in
mouse and human liver microsomes at 10 μM (Table 3).
Moreover, compound 5d showed improved brain-to-plasma
ratio (1.7) and plasma half-life (1.7 h) in a pharmacokinetic
(PK) study in C57BL/6 mice, in comparison to initial hit
compound 5a.44 Although 5d has been used for initial proof of
concept studies, the plasma half-life should be improved for
testing in a wider variety of preclinical models.
a
Reagents and conditions: (a) NaH, bromoacetonitrile, DMF, 75%;
(b) lithium aluminum hydride (LAH), tetrahydrofuran (THF), 32−
57%; (c) cinnamic acid drivative (4), 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDCI), dimethylaminopyr-
idine (DMAP), CH2Cl2, 75−80%.
commercially available 2-methylindole or 2-trifluoromethylin-
doles (1a−c), which on treatment with bromoacetonitrile
provided intermediates (2a−c), which then were subjected to
lithium aluminum hydride to reduce cyanide to amine,
providing advanced intermediates 3a−c in poor to moderate
yields. In an effort to improve the yield of amines, we explored
other methods of cyanide reduction using a variety of reducing
The structural identity among the prostanoid receptor family
is very limited. EP1, EP2, EP3, and EP4 share a common
endogenous ligand PGE2 for their activation, but they only
share 20−30% structural homology.45 In contrast, EP2 is more
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Figure 2. List of additional active/inactive first generation cinnamic amide derivatives synthesized and used for SAR study.
agents (see Supporting Information Table S1). These methods
provided limited success, and they often resulted in an
unwanted indole-dimer product as a major constituent. The
classical lithium aluminum hydride (LAH) reduction method
provided only 32−57% yield of the required amine products
(Supporting Information Table S1 and discussion in the
Supporting Information text). These amines were coupled to
3,4,5-trimethoxycinnamic acid derivatives (4a−c) to provide
final products (5a−f) (Scheme 1). As shown in Table 1, newly
synthesized derivatives are tested by using a cAMP-derived TR-
FRET assay48 at single concentration (1 μM) to observe a
rightward shift of PGE2 (an EP2 agonist) concentration−
response curve in a C6G cell line that overexpresses human
EP2 receptors (see Experimental Methods for details). From
this a Schild KB value (a concentration required to cause a 2-
fold rightward shift of agonist EC50) is calculated assuming a
Schild slope of 1.07, which is the mean slope determined from
four concentration (0.1, 0.3, 1, and 3 μM) Schild plots carried
out on a dozen compounds in this series. A similar procedure is
carried out with human DP1 receptors at a single compound
concentration of 10 μM and used to rank-order the analogues
based on EP2 potency and selectivity against DP1.
The structure−activity relationship (SAR) study indicates a
3,5-dimethoxycinnamic amide derivative (5b) and a compound
in which three methoxyls are substituted with two methyl
groups and a fluorine (5c) display similar EP2 potency, in
comparison to 5a. These derivatives show improved selectivity
against DP1 (5b displays 24-fold, and 5c displays 60-fold)
(Table 1). We have earlier shown that a single methoxycin-
namic amide derivative (6q, Figure 2) exhibited 7-fold less
potency (KB = 16.5 nM) on EP2 in comparison to parent 5a.43
We now synthesized compounds with single methoxyl group at
ortho and meta positions. The m-methoxy derivative (6r,
Figure 2) is about 2-fold less potent than p-methoxy derivative
(6q), but the o-methoxy derivative (6s, Figure 2) displayed a
similar potency to 6q (Table 1). All of these single methoxy
derivatives are about 5- to 12-fold less potent than a trimethoxy
derivative (5a) or a dimethoxy derivative (5b). Similar exercises
on CF3 analogue 5d, for example, substitution of three
methoxyls with a single methoxyl group (5e) or two methyl
groups and a fluorine (5f), reduced the EP2 activity by 16-fold
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in comparison to 5d (Table 1). Taken together, these results
indicate that three methoxyl groups on the phenyl ring are not
absolutely essential for EP2 activity.
In parallel to indole-1 derivatives (Scheme 1), we also
explored synthesis of several indole-3 derivatives (Scheme 2) as
EP2, compound 5h with one less methoxyl group has equal
potency to a previously described 1-indole isomer with two
methoxyl derivative TG4-166,43 indicating that both indole
positional isomers are equally active to pursue further.
Compound 5j has about 3-fold less potency to equivalent 5c.
Moreover, incorporation of two fluorine atoms on the indole
phenyl ring to block the ortho and para (fifth, seventh)
positions to the ring nitrogen (5i) maintained EP2 potency of
the parent 5a. This result is consistent with our previous
observation, where one fluorine atom at (fifth position) para to
indole nitrogen on the phenyl ring yielded an equally potent
compound to 5a.43 Nonetheless, all of these derivatives 5g,h
showed EP2 Schild KB values less than that of the previous lead
compound 5d. These 3-indole derivatives (5g−j) also displayed
improved selectivity (45- to 80-fold) against DP1 (Table 1). To
determine whether indole ring can be replaced with other
structurally equivalent rings, a benzofuran derivative 5k was
synthesized starting from 3-bromo-2-methylfuran as shown in
Supporting Information Scheme S1. This analogue (5k)
showed 138-fold reduced potency compared to its indole
equivalents 5a and 5g. We then synthesized and tested an
indazole derivative 6t (Figure 2), but this derivative completely
lost EP2 potency. Moreover, we also examined other scaffolds
such as indolin-2-one (5p) and phenyethyl and phenylpropyl
groups (5q, 5r) (Figure 2). However, these derivatives showed
very weak (300- to 1000-fold less) potencies (Table 1). In our
earlier study we had shown that a 2-methylpiperidine ring (in
Scheme 2. Synthesis of Isomeric Indole-3 Cinnamic Amide
a
Analogues for SAR Study
a
Reagents and conditions: (a) cinnamic acid derivative (4a or 4c or
4d), EDCl, DMAP, CH2Cl2, 70−80%.
positional isomers. As shown in Scheme 2, commercially
available 2-(2-methyl-1H-indol-3-yl)ethanamines (3k, l) were
coupled to cinnamic acid derivatives (4a,c,d) to synthesize
compounds 5g−j. Isomeric 3-indole analogues displayed Schild
potencies similar to those of the parent series shown in Scheme
1. In particular, compound 5g has equal potency to 5a against
a
Scheme 3. Synthesis of More Aqueous Soluble First Generation Cinnamic Amide EP2 Antagonists
a
Reagents and conditions: (a) NaH, bromoacetonitrile, DMF, 75%; (b) morpholine or 4-amino-1-methylpiperidine, Na(OAc)3BH, AcOH, 60−
75%; (c) LAH, THF, 3e, 55%, 3f, 20%; (d) cinnamic acid (4a), EDCl, DMAP, CH2Cl2, 75%.
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a
Scheme 4. Synthesis of Aqueous Soluble Hydroxyethyl Ether Cinnamic Amide Derivatives
a
Reagents and conditions: (a) 2-tert-butyldimethylsilyloxyethanol, PPh3, DIAD,THF, reflux, 36 h, 70%; (b) 1 N NaOH, THF, 2 N HCl, 801%; (c)
3a, 3b, or 3k, EDCl, DMAP, DCM/DMF (5:1), 80%.
place of indole ring) derivative 6j (Figure 2) displayed
complete loss of activity.43 Taken together, these results
suggest that the indole (1- or 3-positional isomers) ring is
crucial for higher EP2 potency but substitution on the indole
rings is allowable.
The second strategy that we explored to improve the
aqueous solubility of the scaffold 5a and 5d is a substitution of
methoxyl groups with one or two hydroxyalkyl groups on the
phenyl ring. The synthesis of these analogues is shown in
Scheme 4. First commercially available 3,4-dihydroxycinnamic
acid ethyl ester (2k) was subjected to Mitsunobu reaction50
with 2-tert-butyldimethylsilyloxyethanol to get bis-tert-butyldi-
methylsilyloxy ether (2l), which on treatment with 1 N NaOH
in refluxing tetrahydrofuran and then quenching with 2 N HCl
(in one pot) provided precursor acid (2m). This acid was
coupled individually to indole amine 3a, 3b, or 3k to provide
final products (6a−c) with two pendent hydroxyethyl ether
moieties. As we predicted, compounds 6a and 6c have 3- to 5-
fold higher aqueous solubility (Table 1) in comparison to
parent compound 5a when measured by nephelometry in PBS
buffer in 1% DMSO.51 Similarly, a trifluoromethylindole
compound (6b) has shown 2.5-fold higher solubility (67
μM) in comparison to its parent compound 5d (Table 1).
Moreover, we also synthesized several compounds with only
one hydroxyethyl ether or hydroxypropropyl ether moieties
(6d−g). These derivatives also showed about 1.2- to 1.4-fold
higher solubility than the parents 5a and 5d (Table 1).
Although several compounds from Schemes 1 and 2 (5a−d,
5g−j) showed high EP2 potency and improved DP1 selectivity,
they displayed poor aqueous solubility (Table 1). We explored
two strategies to improve the aqueous solubility in this class of
compounds. First, we functionalized the indole ring at second
and third positions (see Figure 1 for number illustration) with
more polar functional groups that should enhance the solubility
of the scaffold. As shown in Scheme 3, the synthesis is initiated
with 2-formylindole (1d), which on treatment with bromoa-
cetonitrile provided 2d, which then on reductive amination49
with morpholine and 4-amino-1-methylpiperidine provided 2e
and 2f. These compounds were reduced with lithium aluminum
hydride to get 3e and 3f, which were then coupled to 3,4,5-
trimethoxycinnamic acid (4a) to provide final products (5l,
5m). Similarly, 3-substituted indoles (5n−o) with more
solubilizing functional groups were synthesized as shown in
Supporting Information Scheme S2. We anticipated that these
derivatives 5l−o could be transformed into their hydrochloride
salt (HCl) forms to improve the solubility as needed. Indeed,
these derivatives (5l−o) and their HCl salts forms (not shown)
have improved solubility in the range of 75−180 μM (Table 1)
compared to parents 5a and 5d, but they failed to show a
strong EP2 antagonistic activity at 1 μM (Table 1), suggesting
these regions on the indole ring are not flexible for structural
modification.
Some compounds with improved aqueous solubility have
high EP2 potency. For example, compounds 6a, 6d, and 6e
displayed similar EP2 potency (Schild KB of 11.4−13.6 nM).
These derivatives are about 5-fold less potent than 5a but are
slightly more potent than 5d. Among these three, 6a, a bis-
hydroxyethyl ether compound, exhibited 44-fold selectivity to
DP1, but monohydroxyethyl ether derivatives 6d, 6e, and 6f
displayed <10-fold selectivity (Table 1). Likewise, compound
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compounds thus far. Moreover, compound 6f, which has two
extra methoxyl groups in addition to a hydroxyethyl ether unit,
has nearly equal EP2 potency to 5a, but a CF3 analogue 6g
showed 3.2-fold less potency than its parent 5d (Table 1). In
contrast to bis-hydroxyethyl ether derivatives 6a−c, the
monohydroxyethyl ether derivatives 6d−g showed a modest
selectivity (5- to 8-fold) over DP1 (Table 1).
Table 1. EP2 Bioactivity, DP1 Selectivity, and Aqueous
Solubility of Cinnamic Amide Analogues
a
KB (nM)
compd
TG4-155
EP2
2.4
DP1
SI (DP1/EP2) solubility (μM)
5a
5b
5c
5d
5e
5f
34.5
83
14.4
24
45
TG7-23
TG7-98
TG6-10-1
TG7-2
3.4
<25
<25
27
3.4
210
166
ND
ND
110
255
175
900
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
505
2820
7450
108
67.1
19.9
198
ND
ND
ND
752
283
265
ND
ND
ND
66
60
We also synthesized compounds 6k−m containing a 2-
dimethylaminoethoxy ether group on the phenyl ring as shown
in Scheme 5. The qikprop (Schrodinger Inc.) predicted ADME
properties (see Supporting Information Table S2) suggest that
these derivatives may display similar solubilities (due to basic
nitrogen) to hydroxyethyl ethers (6a−g) and may show
improved metabolic stability and brain penetration properties
because the pendent tertiary amine group is masked by
hydrophobic methyl groups. Indeed, compounds 6k−m have
enhanced solubility (180, 306, and 90 μM, respectively) (Table
1). However, these derivatives have reduced EP2 potencies
(Table 1) in comparison to their hydroxyethyether equivalents
6d−f. Given their reduced potency and modest selectivity
against DP1 (Table 1), they are not tested for liver metabolism
and brain-permeation properties. A future study will address
whether incorporation these basic amine functionality at meta
or ortho positions improves EP2 potency and selectivity against
DP1.
17.8
9.3
305
ND
ND
45
ND
ND
43
TG7-13
TG7-74
TG7-76
TG7-96
TG7-186
TG7-122
TG7-6
306
5g
5h
5i
2.4
4.9
52
41
3.3
53
<25
<25
ND
91
5j
11.3
80
5k
5l
333
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
44
>1000
>1000
>1000
>1000
>1000
>1000
667
5m
5n
5o
5p
5q
5r
5s
5t
TG7-9
180
75
TG7-21
TG7-138
TG7-109
TG7-91
TG7-95
TG8-116
TG7-133
TG7-89
TG4-156
TG7-149
TG7-128
TG7-97
TG7-103
TG8-4
110
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
153
67
>1000
>1000
>1000
214
In our earlier study,43 we briefly explored the linker unit for
structural modification and learned that extension of two-
carbon ethylamino chain (see Figure 1) to three-carbon
propylamino chain resulted in 775-fold less EP2 potency, and
saturating the double bond of acrylamide as in 5v (Figure 2)
reduced the potency by 90-fold compared with 5a (Table 1). In
the present study, we synthesized compounds with one-carbon
methylamino linker such as 5s,t (Figure 2), but these analogues
showed complete loss of potency (Table 1). We also
synthesized an analogue by reversing the amide (5u, Figure
2), which also killed EP2 potency. However, saturation of the
double bond and then addition of an amino group (5w), or
reducing the length of acrylamide to single methylphenyl (5x)
(Figure 2), reduced the potency by 180- and 160-fold,
respectively, in comparison to 5a. Given the limited availability
of synthetic methods to modify the ethylene linker (Figure 1),
only two derivatives with a carboxymethyl ester group on the
ethylamine linker (5y,z) have been synthesized, but these
compounds were inactive on EP2. Moreover, to determine
whether the amide is absolutely essential for EP2 potency, we
synthesized an ester analogue 6h (Figure 2). This analogue
showed about 140-fold less potency than 5a, suggesting that the
potency in the scaffold arises not just from the acrylamide in
the linker. Furthermore, we synthesized a cyclopropylamide
analogue 6i, which showed 100-fold less potency than 5a.
Taken together, these results suggest an ethylamine linker, one
side attached to the indole ring and other side attached to the
acrylamide, is optimal for bioactivity, but acrylamide moiety is
not solely responsible for the activity; thus, it may be
expendable.
5u
5v
5w
5x
5y
5z
6a
6b
6c
6d
6e
6f
410
680
>1000
>1000
11.4
TG8-16
TG8-21
TG8-23
TG8-32
TG8-27
TG8-30
TG7-209
TG7-273
TG-109-1
TG8-57
TG8-53
TG8-56
TG7-291
TG7-294
TG8-17-1
TG4-94-1
TG8-117
TG8-118
TG8-122
260
10
41.1
181
7.9
235
68
13.6
11.8
5.6
66
3.7
5.3
66
6g
6h
6i
58.3
3.4
35
340
ND
ND
ND
8.9
ND
ND
ND
180
306
90
236
6j
>1000
84.5
6k
6l
74.6
3.8
6m
6n
6o
6p
6q
6r
6s
6t
137
2
>1000
>1000
>1000
16.5
ND
ND
ND
4
ND
ND
ND
ND
ND
ND
152
29.2
ND
ND
ND
ND
ND
ND
13.0
>1000
a
Schild KB values are calculated using the formula log(dr − 1) = log XB
− log KB, where dr (dose ratio) = fold shift in EC50 of PGE2 by the test
compound, XB is antagonist concentration [1 μM]. KB value indicates
a concentration required to produce a 2-fold rightward shift of PGE2
concentration−response curve. The values are the mean of two to four
independent measurements run in duplicate. The solubility of the
compounds is measured in PBS buffer (pH 7.4) with 1% DMSO by
nephelometry.51 ND = not determined.
To minimize the conformational freedom arising from the
ethylamine linker and to minimize the exposure of the linker
unit to metabolizing enzymes, we synthesized derivatives with
constrained and bulkier internal cyclic rings 6n−p as shown in
Supporting Information Scheme S3. Compounds 6n−p were
inactive suggesting that the acyclic ethylene amide is essential
for high EP2 potency. It is worth mentioning that 6n−p are
chiral compounds; we have synthesized only racemic forms,
6c, which showed 17-fold less potency than 5a, 2.3-fold less
than 5d, showed very high selectivity (180-fold) to DP1. This
compound is the second most soluble in this whole class of
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a
Scheme 5. Synthesis of 2-Dimethylaminoalkyl Ether Cinnamic Amide Derivatives
a
Reagents and conditions: (a) MeOH, H2SO4 (drops), reflux, quantitative; (b) dimethylaminoethanol, PPh3, DIAD, THF, 70%; (c) 1 N NaOH,
THF, reflux, quantitative (reagent grade salt); (d) 3a, EDCl, DMAP, DMF, 70%.
a
Table 2. EP2 Potency, Selectivity against EP4 and IP Receptors, and Cytotoxicity of Selected EP2 Antagonists
KB(EP2),
nM
KB(EP4),
μM
selective index
EP4/EP2
KB(IP),
μM
selective index
IP/EP2
cytotoxicity CC50,
therapeutic index
CC50/KB(EP2)
compd
μM
5a (TG4-155)
5c (TG7-98)
5d (TG6-10-1)
5g (TG7-74)
5h (TG7-76)
5j (TG7-186)
6a (TG8-4)
2.4
11.4
41.0
11.2
4.3
4750
12100
630
62
25800
6630
475
172
368
81
71700
108 000
4550
3.4
22.5
8.45
12.7
42.7
21.7
1.57
240
30.0
210
85.0
95.9
17.8
2.4
1790
300
5310
8720
1920
138
59.5
246
317
92.3
126
81.7
36.6
43.3
31.2
24800
50200
28000
8100
4.9
1.46
3.5
11.3
11.4
41.1
13.6
11.8
3.7
310
7.13
9.5
625
6c (TG8-21)
6d (TG8-23)
6e (TG8-32)
6f (TG8-27)
6g (TG8-30)
230
5840
2200
1780
23000
164
3060
7.58
5.96
7.49
7.93
560
6000
505
3100
2020
136
11700
535
58.3
a
EP2, EP4, and IP Schild KB values are average of two to three independent experiments run in duplicate. CC50 values are the average of two
measurements run in triplicate. CC50 = critical concentration required to kill 50% cells.
and we did not make any effort to make them in
enantiomerically enriched form because of their weak or nil
potency.
mediates cytosolic Ca2+ signaling, suggesting that these
receptors could play different, often opposite, roles in
pathophysiology.27−29 On the other hand, although DP1
receptor is not activated by PGE2, it has the highest structural
homology to EP2 and is known to exert proinflammatory
effects similar to those of EP2 in certain conditions.27−29 EP2
receptor also shares a 40% structural homology to the IP
receptor. IP receptor activation is shown to play an important
role in cardioprotection.15,17 Thus, it is crucial to establish
selectivity for the novel antagonists to EP2 over DP1, EP4, and
IP, for preclinical and clinical studies. Previously synthesized
first generation analogues43,44and several other newly synthe-
sized derivatives showed modest selectivity to DP1 (Table 1).
But derivatives 5c, 5g−j, 6a, 6c showed >44-fold selectivity to
EP2 over DP1. So we selected these derivatives for selectivity
testing against EP4 and IP receptors. We created cell lines that
overexpress EP4 receptors, or IP receptors on C6-glioma cells,
and developed a cAMP-derived TR-FRET assay using agonists
PGE2 (for EP4) and iloprost (for IP), similar to EP2 assay (see
Experimental Methods for details). The results show that the
new analogues display micromolar Schild KB values for EP4 and
IP receptors (Table 2), with high selectivity indexes. For
Overall, SAR indicates that in the 1- or 3-indole rings, a CH3
at second position is optimal for EP2 potency. A small
structural change at the second position, for example, a CF3
group, reduces EP2 potency by 7−18 times (cf. 5a vs 5d; 5e vs
6q). An acrylamide group is optimal for high EP2 potency but
may be removed. Modifications to the amide group and to the
ethylamine linker reduce or eliminate EP2 potency. But three
methoxyl groups on the phenyl ring could be substituted with a
variety of other groups to maintain high EP2 potency.
Novel Analogues Show High EP2 Selectivity over
Other Prostanoid Receptors. As indicated briefly in the
previous section, there are nine prostanoid receptors in the
family: DP1, DP2, EP1, EP2, EP3, EP4, FP, IP, and TP. These
receptors are widely distributed in organs and cell types and are
activated by endogenous prostanoids (PGD2, PGE2, PGF2,
PGI2, and TXA2). Among these receptors, EP1, EP2, EP3, and
EP4 share a common endogenous ligand PGE2 for their
activation. EP2 and EP4 are positively coupled to cAMP
signaling, whereas EP3 inhibits cAMP production and EP1
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a
Table 3. Liver Microsomal Stability and in Vivo Pharmacokinetic Properties of Selected Compounds
% of parent compound remaining
at 60 min vs T = 0 min
human liver
microsomes
mouse liver
microsomes
mouse in vivo pharmacokinetics properties
compd
1 μM 10 μM 1 μM 10 μM route of administration (dose, mg/kg) Cmax (ng/mL) AUClast (h·ng/mL) T1/2 (plasma) (h) B/P
5a (TG4-155)
0.1
10.6
0.2
4.8
iv (3)
ip (3)
nd
2400 350
738 207
ND
749 24
457 64
ND
0.45
0.58
ND
1.6
0.3
5c (TG7-98)
20.9
2.3
24.0
39.9
18.3
2.0
13.0
23.1
ND
1.8
5d (TG6-10-1)
ip (5)
po (10)
nd
115 44
248 61
ND
453 49
475 60
ND
1.8
1.6
5g (TG7-74)
5h (TG7-76)
5j (TG7-186)
6a (TG8-4)
0.8
5.8
0
0.2
ND
ND
ND
1.49
1.44
ND
ND
ND
ND
<0.1
<0.1
ND
12.4
0.4
18.6
21.4
43.8
0.2
0.1
1.7
0.1
nd
ND
ND
51.7
8.7
nd
ND
ND
16.2
ip (5)
po (10)
ND
1510 142
128 23
ND
1050 58
197 26
ND
6c (TG8-21)
58.5
70.4
7.1
19.3
a
Male or female C57Bl/6 mice were used for in vivo pharmacokinetic study. Formulation used: 5% DMA, 50% PEG400, and 45% saline for
compound 5a; 10% DMSO, 50% PEG400, 40% sterile water for 5d; and 2.5% DMA, 12.5% propylene glycol, and 85% phosphate-buffered saline
(pH 7.4) for 6a. DMA = N,N-dimethylacetamide. Cmax is the maximum observed concentration that occurs at Tmax. T1/2 is the terminal half-life. AUC
= area under the curve from time zero to the time of the last measurable observation (AUClast). B/P = brain to plasma ratio, calculated from drug
concentrations in plasma and brain tissue at 1 and 2 h. ND = not determined.
example, 5c displayed 12100-fold selectivity against EP4 and
over 6000-fold selectivity against IP receptor. Compound 5g
also displayed high selectivity to EP2 over EP4 (1790-fold) and
IP (5310-fold). Likewise compounds 5h and 5j showed 300-
and 310-fold selectivity to EP4 and 8720- and 1920-fold
selectivity to IP receptor (Table 2). However, these derivatives
showed weak aqueous solubility (<25 μM). Compounds with
improved aqueous solubility, for example, 6a, displayed 625-
fold selectivity against EP4 and 138-fold selectivity against IP;
6c displayed 230-fold selectivity to EP4 and greater than 5000-
fold selectivity against IP. Likewise, compounds 6d−f also
displayed good selectivity against EP4 and IP receptor (Table
2), but these latter derivatives showed poor selectivity against
DP1 (Table 1). Compound 6g has slightly less selectivity to
EP4 (136-fold) and IP (164-fold) (Table 2). We also tested
these selective antagonists in a cell viability assay against C6
glioma cells, and these derivatives have insignificant toxicity
with in vitro therapeutic indexes over several orders of
magnitude (Table 2).
New Selective EP2 Antagonists Show Improved
Microsomal Stability. Having several potent and selective
EP2 antagonists in hand, we asked whether any of these
compounds show improved metabolic stability in human and
mouse liver microsomes in comparison to previous lead
compound 5d. Compound 5a, which showed 0.2% remaining
at 60 min (at 1 μM concentration) in mouse liver microsomes,
exhibited an in vivo plasma half-life of ∼30 min. Compound 5d
with 2% remaining at 60 min had 1.6 h in vivo plasma half-life
in mouse (Table 3), suggesting that in vitro liver metabolism
may be correlated to in vivo plasma half-life in this class. Thus,
we examined novel compounds that showed enhanced
selectivity in comparison to previous lead 5d for liver
microsomal stability. Compound 5c showed high stability in
both liver fractions, but this compound exhibited poor aqueous
solubility; thus, it was not selected for further exploration.
Likewise, 3-indole isomeric derivatives 5g−j also showed poor
stability in liver fractions (Table 3). Interestingly, a 5-fold more
aqueous soluble compound 6a showed nearly similar stability in
both liver fractions in comparison to 5d. Furthermore,
compound 6c which is about 8-fold more soluble than 5d
displayed 3-fold improved stability in mouse liver fractions. It is
also more stable in human liver microsomal fractions (Table 3),
suggesting that these two compounds are suitable for in vivo
pharmacokinetic study.
ADME Characterization and Pharmacokinetic Studies
of Selected EP2 Antagonists. We examined a number of
these derivatives to estimate their ADME properties by qikprop
software (Schrodinger Inc.). As shown in Supporting
Information Table S2, compounds 6a, 6c, and 6f possess
solubility and permeability properties in the suggested range for
95% of the known drugs. However, because of the free hydroxyl
group, they may encounter some resistance in crossing the
blood−brain barrier (BBB) because the predicted values for 6a
and 6c are lower in comparison to 5d, a compound
experimentally determined to be highly brain permeable.
Nonetheless, we have selected compound 6a and subjected it
to in vivo pharmacokinetics study in C57Bl6 mice. As shown in
Table 3, this compound displayed more than an hour plasma
half-life. However, its brain penetration property is poor
compared to previous lead 5d (Table 3), consistent with
qikprop predictions (Supporting Information Table S2).
Blood−brain barrier (BBB) is composed of a network of
endothelial cells, astroglia, pericytes, and a basal lamina. The
capillary of endothelium of the brain is sealed by tight
junctions, produced by the interaction of several trans-
membrane proteins.52,53 Interaction of these junctional proteins
blocks the entry of polar solutes from blood along the
paracellular pathways and so denies access to brain interstitial
fluid. However, small molecules with less than 500 molecular
weight and high lipophilicity can pass through this barrier by
passive transport mechanism. Small molecules could also enter
into brain by other mechanisms (e.g., active transport).54,55
Endothelial cells also express a variety of efflux pumps on their
surface, which play a role in export of small molecules into
brain. Compound 5d (our earlier lead) and 6a display <500
molecular weight, but 5d is more lipophilic than 6a based on its
poor aqueous solubility (Table 1) and predicted log P (Table
2). On the other hand, compound 6a is more polar (5-fold
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Figure 3. Competitive antagonism of EP2 receptor by novel acrylamide analogues. (A−C) Compounds 6a (TG8-4), 6c (TG8-21), and 6f (TG8-27)
inhibited PGE2-induced human EP2 receptor activation in a concentration dependent manner. (D) Schild regression analysis is performed to
determine the modality of antagonism by these compounds. Schild KB values for each compound are shown in inset of part D. Data were normalized
as percentage of maximum response; points represent the mean SEM (n = 4). We observed about 1.1- to 1.8-fold higher KB values from dose−
response test in comparison to KB values derived from single concentration (1 μM) tests (presented in Table 2). These changes are within the limits
of assay variability.
more aqueous soluble) with two free hydroxyl groups readily
available to form hydrogen bonds. However, we do not yet
know whether low levels of 6a in brain are due to poor passive
diffusion or extrusion by efflux pumps. A future study will
address this question by synthesis and testing of additional
hydroxyl group masked derivatives (e.g., methoxy ethers).
Nevertheless, 6a displayed a 2-fold higher potency and 4-fold
higher selectivity against DP1 and 4-fold higher aqueous
solubility than 5d; thus, this compound should be useful for
exploring in in vitro and in vivo proof of concept studies in a
variety of peripheral disease models where EP2 plays a
deleterious role.56−60
Novel Analogues Show Competitive Mechanism of
Inhibition. We previously demonstrated that compounds 5a
and 5d and other analogues in this class exhibit a competitive
antagonism of EP2.43,44 All of these derivatives had only
methoxyl groups on the phenyl ring. In this study, to determine
whether the compounds containing hydroxyethyl ether
moieties (6a−g) also exhibit a similar mechanism of action,
we selected three derivatives 6a, 6c, and 6f and tested them in
concentration response against PGE2 EC50 on EP2 receptors.
As illustrated in Figure 3D, a linear regression of log(dr − 1) on
log XB with slope of unity characterizes a competitive
antagonism. Schild KB values are derived by the equation
log(dr −1) = log XB − log KB, where dr = dose ratio (i.e., the
fold shift in EC50), XB is [antagonist], and KB indicates the
antagonist concentration required for a 2-fold rightward shift in
the PGE2 concentration−response curve. A lower KB value
indicates a higher inhibitory potency. The selected three
compounds induced a concentration-dependent, parallel right-
ward shift in the PGE2 concentration−response curve (Figure
3A−C). Schild regression analyses demonstrated that these
compounds have a competitive mechanism of antagonism on
EP2 with Schild KB 14.8 nM for 6a, 47.1 nM for 6c, and 6.7 nM
for 6f. Thus, the mechanism is competitive in general for this
class of EP2 antagonists presented in this study.
In conclusion, we have synthesized 45 new cinnamic amide
EP2 antagonists to optimize the selectivity against DP1 and IP
receptors and to improve aqueous solubility and pharmacoki-
netics properties. Two compounds, namely, 6a (TG8-4) and 6c
(TG8-21), emerged as selective EP2 antagonists (with 44- to
180-fold selectivity against DP1), with more aqueous solubility
(153 and 235 μM) and more stability in vitro in pooled human
and mouse liver microsomes in comparison to previous lead
compound 5d (TG6-10-1). But in vivo pharmacokinetics
properties still need to be optimized within the class to be
useful for in vivo preclinical studies. However, the new
analogues 6a and 6c could serve as tools for in vitro proof of
concept studies.
EXPERIMENTAL METHODS
■
Chemistry General. Proton NMR spectra were recorded in
solvent in CDCl3 on a Varian Inova 400 (400 MHz) instrument. Thin
layer chromatography was performed on precoated, aluminum-backed
plates (silica gel 60 F254, 0.25 mm thickness) from EM Science and was
visualized by UV lamp. Column chromatography was performed with
silica gel cartridges on Teledyne-ISCO machine. An Agilent LCMS
instrument was used to measure purity of the products. Elemental
analyses were performed by Atlantic Microlab Inc. (Norcross, GA).
Chemicals and drugs PGE2, BW245C, iloprost, and rolipram were
purchased from Cayman Chemical.
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General Procedure for Synthesis of 2-(2-Substituted-1H-
indol-1-yl)acetonitriles (2) from Indoles (1).61 A solution of 2-
(trifluoromethyl)-1H-indole (1b) (0.5 g, 2.7 mmol) in DMF (2.5 mL)
was added to a suspension of NaH (160 mg, 1.5 equiv) in DMF (3
mL) at 0 °C, and the resulting reaction mixture was stirred for 30 min.
Then bromoacetonitrile (0.27 mL, 1.5 equiv) in DMF (2.5 mL) was
introduced into the above mixture at 0 °C, and then the mixture was
brought to room temperature overnight. Water (20 mL) was added to
quench the reaction. Then the product was extracted with ethyl ether
(30 mL × 3). Organics were washed with water, brine, dried over
Na2SO4, and concentrated. The crude mass on silica gel chromatog-
raphy, eluting with 0−10% ethyl acetate, furnished 2b (865 mg, 71%
yield; 85% based on recovered starting material).
1H), 7.07 (dd, J = 6.8, 1.6 Hz, 1H), 7.04 (d, J = 2 Hz, 1H), 6.87 (d, J =
8.4 Hz, 1H), 6.26 (d, J = 16 Hz, 1H) 4.24 (q, J = 6.8 Hz, 2H), 4.07 (q,
J = 5.6 Hz, 4H), 3.97 (t, J = 5.6 Hz, 4H), 1.30 (t, J = 7.2 Hz, 3H), 0.88
(two singlets, 18H), 0.08 (two singlets, 12H). LCMS (ESI): >95%
purity at λ = 254 nm. MS m/z, 525 [M + H]+.
Step 2. To a solution of 2l (375 mg, 0.71 mmol) in THF (10 mL) was
added 1 N NaOH (2.13 mL, 2.13 mmol, 3 equiv), and the resulting
reaction was refluxed for 48 h. The reaction mixture was cooled and
neutralized with 1 N HCl (10 mL) to pH 4. Then the product was
extracted with ethyl acetate (25 mL × 3). Organics were dried over
Na2SO4 and concentrated to dryness under vacuum to furnish 2m
(190 mg, quantitative yield), which was used for next step without
further purification.
2-(2-Trifluoromethyl-1H-indol-1-yl)acetonitrile (2b). 1H NMR
(CDCl3): δ 7.70 (d, J = 8 Hz, 1H), 7.44 (m, 2H), 7.28 (t × d, J =
7.2, 1.6 Hz, 1H), 7.04 (s, 1H), 5.1 (s, 2H). LCMS (ESI): >95% purity
at λ = 254 nm. MS m/z, 225 [M + H]+. See Supporting Information
for synthesis and characterization data for compounds 2a and 2c−h.
General Procedure for Synthesis 2-(2-Substituted-1H-indol-
1-yl)ethanamines (3) from Acetonitriles (2). To a solution of 2b
(855 mg, 3.81 mmol) in THF (30 mL) was added LAH (1 M, 9.54
mmol, 2.5 equiv), dropwise at 0 °C, and the resulting reaction mixture
was brought to room temperature overnight. Methanol (2 mL) was
slowly added to quench the reaction at −78 °C, followed by 1 N
NaOH (3 mL) at room temperature. The product was extracted with
ethyl ether (30 mL × 3). Organics were washed with water, brine and
dried over Na2SO4 and concentrated. The crude mass was subjected to
silica gel chromatography, eluting with 0−5% methanol in dichloro-
methane to provide 3b (490 mg, 56% yield).
(E)-3-(3,4-Bis(2-hydroxyethoxy)phenyl)acrylic Acid (2m). 1H
NMR (DMSO-d6): δ 12.0 (bs, 1H), 7.46 (d, J = 15.6 Hz, 1H), 7.29
(d, J = 2 Hz, 1H), 7.14 (m, 1H), 6.96 (d, J = 8 Hz, 1H), 6.38 (d, J = 16
Hz, 1H) 4.0 (m, 4H), 3.68 (bs, 4H). LCMS (ESI): >95% purity at λ =
254 nm. MS m/z, 267 [M − H]. See Supporting Information for
synthesis and characterization for other carboxylic acid derivatives 2o−
q.
(E)-3-(3,4-Bis(2-hydroxyethoxy)phenyl)-N-(2-(2-methyl-1H-indol-
1-yl)ethyl)acrylamide (6a, TG8-4). This compound was prepared
1
from 2m and 3a in 80% yield by the method described for 5d. H
NMR (CDCl3): δ 7.46 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 16 Hz, 1H),
7.26 (d, J = 9.6 Hz, 1H), 7.07 (t, J = 6.8 Hz, 1H), 7.04 (t × d, J = 8.4, 2
Hz, 2H), 6.81 (d, J = 8 Hz, 1H), 6.12 (d, J = 15.6 Hz, 1H), 4.25 (t, J =
6 Hz, 2H), 4.04 (q, J = 4 Hz, 4H), 3.87 (q, J = 4.4 Hz, 4H), 3.62 (t, J =
5.6 Hz, 2H), 2.34 (s, 3H). LCMS (ESI): >97% purity at λ = 254 nm.
MS m/z, 425 [M + H]+. HRFABMS: calcd for C24H28N2O5Na,
447.189 04; found 447.189 76.
2-(2-(Trifluoromethyl)-1H-indol-1-yl)ethanamine (3b). 1H NMR
(CDCl3): δ 7.66 (d, J = 8 Hz, 1H), 7.44 (dd, J = 8.4, 0.8 Hz, 1H), 7.34
(t × d, J = 7.6, 0.8 Hz, 1H), 7.17 (t × d, J = 7.4, 1.2 Hz, 1H) 6.94 (s,
1H), 4.28 (t, J = 6.8 Hz, 2H) 3.12 (t, J = 6.8 Hz, 2H) 2.45 (s, 3H).
LCMS (ESI): >97% purity at λ = 254 nm. MS m/z, 229 [M + H]+. See
Supporting Information for synthesis and characterization data for 3a
and 3c−h.
General Procedures for Synthesis of Cinnamic Amide Final
Products. To a solution of 3b (480 mg, 2.1 mmol) in dichloro-
methane (10 mL) were added (E)-3-(3,4,5-trimethoxyphenyl)acrylic
acid (4a) (504 mg, 1 equiv), 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide hydrochloride (EDCI) (523 mg, 1.3 equiv), and N,N-
dimethylaminopyridine (10 mg), and resulting reaction mixture was
stirred at room temperature for 8 h. The reaction was quenched with
water (10 mL), and the product was extracted with ethyl acetate (20
mL × 3). Organics were washed with 1% HCl (10 mL), saturated
NaHCO3 (10 mL), water (20 mL), brine solution (20 mL) and dried
over Na2SO4. The crude product was purified by silica gel
chromatography, eluting with 0−35% ethyl acetate in hexane to
provide 5d (700 mg, 74% yield).
(E)-N-(2-(2-(Trifluoromethyl)-1H-indol-1-yl)ethyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (5d). 1H NMR (CDCl3): δ 7.59 (d, J =
8 Hz, 1H), 7.54 (d, J = 8.4, Hz, 1H), 7.50 (d, J = 15.2 Hz, 1H), 7.27
(q, J = 7.2 Hz, 1H), 7.1 (t, J = 7. 2 Hz, 1H), 6.89 (s, 1H), 6. 63 (s, 2H),
6.4 (t, J = 6 Hz, 1H), 6. 25 (d, J = 15.2 Hz, 1H), 4.4 (t, J = 6.4 Hz,
1H), 3.8 (s, 3H), 3.76 (s, 6H), 3.69 (q, J = 6.4 Hz, 2H). LCMS (ESI):
>95% purity at λ = 254 nm. MS m/z, 449 [M + H]+. Anal. Calcd for
C23H23F3N2O4: C, 61.60; H, 5.17; N, 6.25. Found: C, 61.34; H, 5.10;
N, 6.16. See Supporting Information for characterization of 5a−c,e−z.
General Synthesis for 2-Hydroxyethyl- Or 2-
Dimethylaminoethylcinnamic Acids. Step 1. To a solution of
ethyl-3,4-dihydroxycinnamate (2k) (460 mg, 2.21 mmol), 2-tert-
butyldimethylsilyloxyethanol (2 mL, 9.52 mmol 4.3 equiv), and
triphenylphosphine (3.43 g, 13 mmol, 5.8 mmol) in THF (40 mL)
was added diisopropyl azodicarboxylate (2.4 mL, 12 mmol, 5.3 equiv)
dropwise at 0 °C. Then the resulting solution was refluxed for 36 h.
The volatiles were removed under vacuum and the crude product was
subjected to silica gel chromatography, eluting with 0−20% ethyl
acetate in hexane to furnish 2l (775 mg, 67%).
(E)-3-(3,4-Bis(2-hydroxyethoxy)phenyl)-N-(2-(2-methyl-1H-indol-
3-yl)ethyl)acrylamide (6c, TG8-21). This compound was prepared
1
from 2m and 3k in 80% yield by the method described for 5d. H
NMR (CDCl3 + MeOH-d4): δ 7.41 (d, J = 7.2 Hz, 1H), 7.34 (d, J =
15.6 Hz, 1H), 7.19 (d × t, J = 8.4, 0.8 Hz, 1H), 6.92 (m, 4H), 6.77 (d,
J = 8 Hz, 1H), 6.10 (d, J = 16 Hz, 1H), 3. 99 (t, J = 4 Hz, 4H), 3.81 (q,
J = 3.6 Hz, 4H), 3.48 (t, J = 6.8 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.27
(s, 3H). LCMS (ESI): >97% purity at λ = 254 nm. MS m/z, 425 [M +
H]+. HRFABMS calcd for C24H28N2O5Na, 447.189 04; found 447.188
89. See Supporting Information for synthesis and characterization data
for remaining compounds 6b,d−p.
Bioactivity Testing. Cell Culture. The rat C6 glioma (C6G) cells
stably expressing human DP1, EP2, EP4, or IP receptors were created
in the laboratory43,44,48 and grown in Dulbecco’s modified Eagle
medium (DMEM) (Invitrogen) supplemented with 10% (v/v) fetal
bovine serum (FBS) (Invitrogen), 100 U/mL penicillin and 100 μg/
mL streptomycin (Invitrogen), and 0.5 mg/mL G418 (Invitrogen).
Cell-Based cAMP Assay. Intracellular cAMP was measured with a
cell-based homogeneous time-resolved fluorescence resonance energy
transfer (TR-FRET) method (Cisbio Bioassays), as previously
described.43,44 The assay is based on generation of a strong FRET
signal upon the interaction of two molecules, an anti-cAMP antibody
coupled to a FRET donor (cryptate), and cAMP coupled to a FRET
acceptor (d2). Endogenous cAMP produced by cells competes with
labeled cAMP for binding to the cAMP antibody and thus reduces the
FRET signal. Cells stably expressing human DP1, EP2, EP4, or IP
receptors were seeded into 384-well plates in 30 μL of complete
medium (4000 cells/well) and grown overnight. The medium was
carefully withdrawn, and 10 μL of Hanks’ buffered salt solution
(HBSS) (Hyclone) containing 20 μM rolipram was added into the
wells to block phosphodiesterases. The cells were incubated at room
temperature for 0.5−1 h and then treated with vehicle or test
compound for 10 min before addition of increasing concentrations of
appropriate agonist: BW245C for DP1, PGE2 for EP2 and EP4, or
iloprost for IP. The cells were incubated at room temperature for 40
min and then lysed in 10 μL of lysis buffer containing the FRET
acceptor cAMP-d2, and 1 min later another 10 μL of lysis buffer with
anti-cAMP-cryptate was added. After a 60−90 min incubation at room
temperature, the FRET signal was measured by an Envision 2103
multilabel plate reader (PerkinElmer Life Sciences) with a laser
Ethyl (E)-3-(3,4-Bis(2-((tert-butyldimethylsilyl)oxy)ethoxy)-
1
phenyl)acrylate (2l). H NMR (CDCl3): δ 7.60 (d, J = 15.6 Hz,
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excitation at 337 nm and dual emissions at 665 and 590 nm for d2 and
cryptate (50 μs delay), respectively. The FRET signal was expressed as
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S
* Supporting Information
Synthesis schemes for compounds 5k,o,p, and 6n−p; a table of
the reagents and reaction conditions tested to improve yield of
starting materials 3 from 2; a table of qikprop calculations on
selected compounds; NMR and MS characterization data for
remaining compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1-404-727-7393. Fax: +1-404-727-0365. E-mail:
tganesh@emory.edu.
Author Contributions
T.G. and R.D. designed the research. T.G., J.J., and M.-S.Y.
performed the research. T.G. wrote the manuscript. R.D.
helped with the writing.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Alzheimer’s Drug Discovery
Foundation (T.G.), NIH/NINDS Grants K99/R00NS082379
(to J.J.) and U01NS058158 (to R.D.), NARSAD Young
Investigator Grant (to J.J.), and the Epilepsy Foundation (to
J.J.).
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