2,3-Diarylthiophenes as selective EP1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.12.005

The synthesis and the EP₁ receptor binding affinity of 2,3-diarylthiophene derivatives are described. The evaluation of the structure-activity relationship (SAR) in this series led to the identification of compounds 4, 7, and 12a, which exhibit high affinity for the human EP ₁ receptor and a selectivity greater than 100-fold against the EP₂, EP₃, EP₄, DP, FP, and IP receptors and greater than 25-fold versus the TP receptor. These three antagonists present good pharmacokinetics in rats and significant differences in the way they are distributed in the brain.

Full text of DOI:10.1016/j.bmcl.2004.12.005

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1155–1160
2,3-Diarylthiophenes as selective EP1 receptor antagonists
*
`
ˆ ´
Yves Ducharme, Marc Blouin, Marie-Claude Carriere, Anne Chateauneuf, Bernard Cote,
Danielle Denis, Richard Frenette, Gillian Greig, Stacia Kargman, Sonia Lamontagne,
Evelyn Martins, Franc¸ois Nantel, Gary OÕNeill, Nicole Sawyer,
Kathleen M. Metters and Richard W. Friesen
´
Merck Frosst Centre for Therapeutic Research, PO Box 1005, Pointe Claire-Dorval, Quebec, Canada H9R 4P8
Received 3 November 2004; revised 24 November 2004; accepted 2 December 2004
Available online 7 January 2005
Abstract—The synthesis and the EP1 receptor bindingaffinity of 2,3-diarylthiophene derivatives are described. The evaluation of the
structure–activity relationship (SAR) in this series led to the identification of compounds 4, 7, and 12a, which exhibit high affinity
for the human EP1 receptor and a selectivity greater than 100-fold against the EP2, EP3, EP4, DP, FP, and IP receptors and greater
than 25-fold versus the TP receptor. These three antagonists present good pharmacokinetics in rats and significant differences in the
way they are distributed in the brain.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Prostanoids are important lipid mediators involved in a
broad spectrum of physiological and pathophysiological
events.1 They exert their effects through specific interac-
tions with prostanoid receptors, which are all members
of the G-protein coupled receptor superfamily.2 In par-
ticular, prostaglandin E2 (PGE2), the most abundant
mammalian prostaglandin, elicits its effects primarily
through interaction with four distinct receptors desig-
nated EP1, EP2, EP3 and EP4.3 Recent studies with
EP1 receptor knockout mice suggest that this receptor
is involved in PGE2-induced allodynia4 and in acute
inflammatory pain.5 The involvement of the EP1 recep-
tor in pain is further supported by the demonstrated effi-
cacy of the EP1 selective antagonist ONO-8711 in rat
models of allodynia,6 postoperative pain7, and neuro-
pathic pain.8 Another EP1 antagonist, ZD6416, is re-
ported to attenuate secondary esophageal hyperalgesia
in man.9 Further studies with knockout mice and the
antagonists ONO-8711 and ONO-8713 suggest a role
for the EP1 receptor in colon10, and breast11 carcinogen-
esis, blood pressure regulation,5 adaptive gastric cyto-
protection,12 and diabetic nephropathy.13 Previous
reports from this laboratory described the SAR of vari-
ous prostanoid analogs14 and of a family of dibenzazo-
cinone derivatives15 with the human EP1 receptor.
Previous work also permitted the identification of 2,3-
diarylthiophene 1 as a prototypical EP1 receptor antag-
onist.16 Compound 1 binds with good affinity to the EP1
receptor (Ki = 15 nM) and is shifted 15-fold to a Ki of
220 nM in the presence of 2% human serum albumin
(HSA). The selectivity ratio of 1 is at least 100-fold
against the EP2, EP4, FP, and IP receptors. Its selectivity
versus the EP3, DP, and TP receptors is 67-, 11-, and 10-
fold, respectively. While this antagonist presents an
acceptable pharmacokinetic profile in rats (F = 51%;
Cmax = 18 lM at 0.5 h, 10 mg/kg P.O., t1/2 = 3 h), only
a modest brain–blood ratio of 0.1 is measured in the
same species.
S
Cl
OH
O
O
1
In order to evaluate the in vivo pharmacology associ-
ated with EP1 receptor antagonism, we wished to dis-
cover antagonists presenting
selectivity over the other prostanoid receptors. We also
wanted to increase the brain–blood ratio since it is
a higher degree of
Keywords: Prostaglandin E2; EP1 Receptor antagonist.
*
Correspondingauthor. Tel.: +1 514 428 8621; fax: +1 514 428 4900;
e-mail: yves_ducharme@merck.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.005

1156
Y. Ducharme et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1155–1160
S
Cl
OH
O
OBn
1
X
Cl
Loss of affinity
for EP1 receptor
OBn
R
Cl
O
R =
X = F, CF3, NO2,
CH3, COCH3,
SO2CH3
N
N
OBn
<5 X
O
O
O
R =
R =
X = CN, CO2CH3,
5 - 25 X
OBn
OBn
CH3
X = Ph, C(CH3)2OH,
O
Cl
Cl
CO2H
OBn
BnO
S
Br
>25 X
O2
S
OBn
Figure 1. SAR overview at the 3-thienyl position.
probable that centrally mediated EP1 agonism plays a
role in certain types of pain.4 We now disclose the results
of a SAR study directed toward the optimization of ana-
logs of antagonist 1 in terms of EP1 bindingaffinity,
selectivity, functional activity, and pharmacokinetics.
An extensive SAR study was conducted on the structure
of compound 1. Modifications made at the 3-thienyl po-
sition at best permitted preservation of the bindingaffin-
ity for the EP1 receptor (Fig. 1). Surrogates for the
central thiophene ringwere identified since small lipo-
philic rings such as cyclopentene and pyrrole are toler-
ated (Fig. 2). However, no significant effects on
selectivity were observed as a result of these structural
modifications. In contrast, modifications at the 2-thienyl
position proved more fruitful. Three classes of deriva-
Human prostanoid receptor bindingaffinities were
determined as described previously by Abramovitz
et al.17 The functional antagonist properties of com-
18
pounds were evaluated usinga cell-based assay.
S
Cl
OH
O
OBn
1
Loss of affinity for
EP1 receptor
S
CH3
O
S
<5 X
N
N
N
N
N
H H
5 - 25 X
CH3
N
N
O
O
N
S
N
N
S
>25 X
N
N
S
Figure 2. SAR overview at the central ring.

Y. Ducharme et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1155–1160
1157
Table 1. Benzoic acid analogs
Cl
EP1 Ki (nM)
EP1 2% HSA
Ki (nM) (shift)
Functional assay
Kb (nM)
TP Ki (nM)
(selectivity)
Rat PK
half-life (h)
Ar
Ar
OBn
S
1
15
4
220 (15X)
70 (18X)
90 (30X)
280 (45X)
66
7
150 (10X)
100 (25X)
230 (75X)
140 (25X)
3
CO2H
S
S
CO2H
2
3
<0.5
<0.5
4
CO2H
CO2H
3
4
N
N
S
4
6
1
Each Ki value is an average of at least three experiments.
tives were identified with improved bindingaffinity,
selectivity and/or pharmacokinetic profiles.
logs, shows good pharmacokinetics in the same species
(F = 73%; Cmax = 20 lM at 4 h, 10 mg/kg P.O.,
t1/2 = 4 h). The brain–blood ratio (0.1) is modest for
this compound.
First is a class of benzoic acid analogs (Table 1). The
benzoic acid derivative 2 and the nicotinic acid 3 both
show higher affinity for the EP1 receptor in the binding
assay compared to the phenylacetic acid analog 1. This
behavior is translated in the functional assay where
antagonists 2 and 3 are 10- to 16-fold more potent than
phenylacetic acid 1. Unfortunately, these two com-
A second class of analogs, the trifluoromethylketone hy-
drates was discovered by preparingcarboxylic acid sur-
rogates. For example, in the nicotinic acid series, the
carboxylic acid moiety found in antagonist 3 was re-
placed by various functional groups (Table 2). While
substitution by a bis(trifluoromethyl)methanol (5) or a
cyanohydrin (8) function was detrimental to binding
affinity, equipotent ligands were obtained by replacing
the carboxylic acid group by a 2,2,2-trifluoroethan-1-ol
(6) or a trifluoromethylketone hydrate moiety (7). This
pounds are plagued with short half-lives in rats (t1/2
<
0.5 h). The best representative of this class is picolinic
acid derivative 4, which binds with high affinity to
the human EP1 receptor (Ki = 6 nM) and is shifted 45-
fold to a Ki of 280 nM in the presence of 2% HSA. Picol-
inic acid 4 is very potent in the functional assay with a
Kb of 1 nM. The selectivity ratio of antagonist 4 is at
least 100-fold against the EP2, EP3, EP4, DP, FP, and
IP receptors and 25-fold versus the TP receptor. Antag-
onist 4 binds with high affinity to the rat EP1 receptor
(Ki = 12 nM) and, in contrast to other benzoic acid ana-
last analogbinds with high affinity to the human EP
1
receptor (Ki = 4 nM), is not shifted in presence of 2%
HSA (Ki = 5 nM) and presents a selectivity greater than
100-fold against the EP2, EP3, EP4, DP, FP, and IP
receptors. Its selectivity versus the TP receptor is 55-
fold. Compound 7 is potent (Kb = 70 nM) in the
Table 2. Trifluoromethylketone hydrate as a carboxylic acid bioisostere
EP1 Ki (nM)
EP1 2% HSA
Ki (nM) (shift)
Functional assay
Kb (nM)
TP Ki (nM)
(selectivity)
Rat PK
Half-life (h)
S
X
Cl
X
OBn
N
O
3
5
6
7
8
3
260
3
90 (30X)
—
4
—
—
70
—
230 (75X)
200 (0.8X)
120 (40X)
220 (55X)
360 (15X)
<0.5
—
1
OH
F3C CF3
OH
CF3
6 (2X)
5 (1X)
160 (7X)
OH
HO CF3
4
2
OH
CN
23
—
OH
Each Ki value is an average of at least three experiments.

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Y. Ducharme et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1155–1160
Table 3. Amide and sulfonamide derivatives
Cl
Ar
EP1 Ki (nM)
EP1 2% HSA
Ki (nM) (shift)
Functional assay
Kb (nM)
TP Ki (nM)
(selectivity)
Rat PK
half-life (h)
Ar
OBn
S
CO2H
O
2
4
9
70 (18X)
17 (2X)
5 (1X)
7
97
100 (25X)
<0.5
<1
<1
<1
2
S
S
S
9
1200 (130X)
N
H
N
H
N
10
11
12a
4
—
>21,000 (>5000X)
>21,000 (>400X)
390 (50X)
O
H
N
S
52
8
72 (1.4X)
18 (2X)
—
O2
S
SO2NH2
100
Each Ki value is an average of at least three experiments.
functional EP1 cell-based assay. Also, antagonist 7
shows good pharmacokinetics in rats (F = 42%;
Cmax = 5.5 lM at 6 h, 20 mg/kg P.O., t1/2 = 2 h) and a
higher brain–blood ratio of 1.
bioavailable in rats but presents an acceptable half-life
(F = 9%; Cmax = 0.4 lM at 30 min, 20 mg/kg P.O.,
t1/2 = 2 h) and a high brain–blood ratio of 7.
Synthesis
Benzamide and sulfonamide analogs, such as com-
pounds 9–12, form the third class identified (Table 3).
The benzamides 9 and 10 are less protein shifted and
more selective than their carboxylic acid counterparts,
but formulation difficulties due to insolubility and poor
pharmacokinetics reduced their utility. Sulfonamide 12a
binds with high affinity to the human EP1 receptor
(Ki = 8 nM) and is shifted only 2-fold to a Ki of 18 nM
in the presence of 2% HSA. The selectivity ratio of
12a is at least 100-fold against the EP2, EP3, EP4, DP,
FP, and IP receptors and 50-fold versus the TP receptor.
Compound 12a is potent (Kb = 100 nM) in the func-
tional EP1 cell-based assay. Antagonist 12a is modestly
The synthesis of these antagonists relied heavily on Stille
and Suzuki couplingreactions. The preparation of the
required buildingblocks is presented in Scheme 1.
Reduction of ethyl 6-bromo-2-pyridinecarboxylate with
diisobutylaluminum hydride in tetrahydrofuran fol-
lowed by treatment with triisopropylsilyl trifluorome-
thanesulfonate afforded silyl ether 13b in 98% yield.
Benzyl ether 13c was obtained quantitatively by treat-
ment of 2-bromo-4-chlorophenol with benzyl bromide.
Subsequent addition of n-butyllithium followed by tri-
isopropyl borate afforded boronic acid 16 in 73% yield.
Palladium-catalyzed stannylation of ethyl 5-bromonico-
Br
Cl
N
Br
Cl
N
CO2Et
1) DIBAL-H
2) TIPSOTf
98%
OTIPS
13b
Cl
B(OH)2
OBn
Br
Br
BnBr
K2CO3
100%
nBuLi
B(OiPr)3
H3O+
OH
OBn
16
13c
73%
Me Sn
CO2Et
3
Br
CO2Et
(Me3Sn)2
Pd(PPh3)4
100%
N
N
17
Scheme 1. Preparation of buildingblocks.

Y. Ducharme et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1155–1160
1159
S
S
S
S
16
B(OH)2
NBS
Cl
ArBr
Ar
Pd(PPh3)4
83-98%
Pd(PPh3)4
73-80%
55-75%
Br
Ar
15a-c
Ar
14a-c
OBn
12a,b
13a-c
Ar
17
SO2NH2
S
a
b
Pd2(dba)3
AsPh3
75%
CO2Et
Ar
N
OTIPS
N
12c
Cl
c
BnO
Scheme 2. Palladium-catalyzed couplingreactions.
tinate resulted in the quantitative production of aryl-
stannane 17.
In conclusion, we have optimized a novel series of po-
tent EP1 receptor antagonists. The results of the SAR
study in this series led to the identification of antagonists
4, 7, and 12a, which exhibit high affinity for the human
EP1 receptor (Ki < 10 nM) and a selectivity greater than
100-fold against the EP2, EP3, EP4, DP, FP, and IP
receptors and greater than 25-fold versus the TP recep-
tor. These three antagonists present good pharmacoki-
netics in rats. Interestingly, antagonists 4, 7, and 12a
show significant differences in the way they are distrib-
uted in rats with brain to blood ratios of 0.1, 1, and 7,
respectively. The overall profiles of these antagonists
are complementary and appropriate for the pharmaco-
logical evaluation of EP1 antagonism. These results will
be reported in due course.
The elaboration of the tricyclic 2,3-diarylthiophenes is
shown in Scheme 2. First, Suzuki couplingbetween
the appropriate aryl bromides 13a–c and 3-thiophene-
boronic acid afforded the corresponding3-arylthiophenes
14a–c, which upon treatment with N-bromosuccinimide,
yielded the 2-bromo-3-arylthiophenes 15a–c. Further
Suzuki couplingreactions between intermediates 15a
and b with boronic acid 16 afforded 2,3-diarylthiophenes
12a and b. Alternatively, Stille couplingbetween bromo-
thiophene 15c and 3-pyridylstannane 17 gave access to
analog 12c.
The final transformations leadingto antagonists 4 and 7
are shown in Scheme 3. Deprotection of silyl ether 12b
with tetrabutylammonium fluoride (TBAF) followed
by oxidation of the resultingprimary alcohol with
potassium permanganate afforded antagonist 4 in 62%
yield. Reduction of nicotinate derivative 12c with so-
dium borohydride followed by oxidation with manga-
nese oxide afforded the correspondingaldehyde.
Reaction of this intermediate with (trifluoromethyl)tri-
methylsilane and TBAF followed by a further oxidative
treatment with manganese oxide gave access to trifluo-
romethylketone hydrate 7.
References and notes
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S
1) TBAF
2) KMnO4
62%
N
CO2H
Cl
Cl
12b
12c
OBn
4
1) NaBH4; 59%
2) MnO2; 72%
S
HO OH
CF3
3) CF3TMS/TBAF
4) MnO2; 87%
N
OBn
7
Scheme 3. Synthesis of antagonists 4 and 7.

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18. HEK cells were grown in DMEM, 10% (v/v) heat
inactivated fetal bovine serum, 100 units/ml penicillin-G
and 100 lg/ml streptomycin sulfate, 1 mM sodium pyru-
vate, 0.1 mg/ml hygromycin B, and 500 lg/ml G418 in a
humidified atmosphere at 37 ꢁC, 6% CO2. One day prior
to fluorescent imaging plate reader (FLIPR) functional
assay, hEP1 stably transfected HEK293 cells were seeded
at 35,000 cells/well. Agonist (PGE2) and antagonists were
prepared in DMSO at 200· concentration and stored at
À20 ꢁC. For the FLIPR assay, agonist and antagonist
drugplates were thawed and diluted 10-fold to 20 · in
Hanks Balanced Salt Solution (HBSS) containing20 mM
HEPES, pH 7.4 (HBSS/HEPES). FLIPR no wash dye
(Molecular Probes R8033) was diluted in HBSS/ HEPES.
Confluent cell monolayers were washed twice in HBSS/
HEPES buffer. HBSS/HEPES buffer and FLIPR dye were
added to cell monolayers and cells were incubated for 1 h
at 37 ꢁC in the presence of CO2 to enable dye loading. The
plates containingthe dye loaded cells, diluted antagonists
and agonist were placed in the FLIPR. Addition of
compound to cells and fluorescence readingwere per-
formed automatically by the FLIPR. Fluorescence was
monitored for 15 min (10 min antagonist preincubation
followed by 5min agonist stimulation). For each antago-
nist tested, a dose response curve (from 0.001 to 30 lM)
was first generated for PGE2. Three additional dose
response curves were generated in which cells were
preincubated in the presence of fixed concentrations of
antagonist for 10 min followed by challenge with increas-
ingconcentrations of PGE .
2