1,7-Disubstituted oxyindoles are potent and selective EP3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.02.028

A series of novel 1,7-disubstituted oxyindoles were shown to be potent and selective EP₃ receptor antagonists. Variation of substitution pattern at the C-3 position of indole enhanced in vitro metabolic stability of the resulting derivatives. Series 27a-c showed >1000-fold selectivity over a panel of prostanoid receptors including IP, FP, EP₁, EP₂ and EP₄. These agents also featured low CYP inhibition and good activity in the functional rat platelet aggregation assay.

Full text of DOI:10.1016/j.bmcl.2010.02.028

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2658–2664
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,7-Disubstituted oxyindoles are potent and selective EP3 receptor antagonists
Nian Zhou a, Alexandre M. Polozov a, Matthew O’Connell a, James Burgeson a, Peng Yu a, Wayne Zeller a,
Jun Zhang a, Emmanuel Onua a, Jose Ramirez b, Gudrun A. Palsdottir b, Gudrun V. Halldorsdottir b,
Thorkell Andresson b, Alex S. Kiselyov a, Mark Gurney a,b, Jasbir Singh a,
*
a deCODE Chemistry, 2501 Davey Road, Woodridge, IL 60517, USA
b deCODE genetics, Inc. Reykjavik, Iceland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,7-disubstituted oxyindoles were shown to be potent and selective EP3 receptor antag-
onists. Variation of substitution pattern at the C-3 position of indole enhanced in vitro metabolic stability
of the resulting derivatives. Series 27a–c showed >1000-fold selectivity over a panel of prostanoid recep-
tors including IP, FP, EP1, EP2 and EP4. These agents also featured low CYP inhibition and good activity in
the functional rat platelet aggregation assay.
Received 10 December 2009
Revised 5 February 2010
Accepted 8 February 2010
Available online 25 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Prostanoids generated in vivo from arachidonic acid act through
specific membrane-bound G protein-coupled receptors (GPCRs)
and play an essential role in vascular homeostasis, including regu-
Previously, we have reported identification and SAR of potent,
isoform selective hEP3 receptor antagonists derived from 1,7-
disubstituted indoles. In our hands, these molecules displayed
strong platelet aggregation in both ex vivo and in vivo settings
(Fig. 1, structure A).5 During structure–activity relationship (SAR)
studies we noticed that basic Ar1 or Ar2 functionalities lead to a sig-
nificant reduction of hEP3 affinity.6 In this Letter, we followed the
original pharmacophore hypothesis4 and studied indolone deriva-
tives with the specific focus to improve physicochemical properties
of the resultant derivatives. We have previously reported that the
nonaromatic hexahydro-indolone core did furnish potent hEP3
antagonists.7 However, in spite of good potency in the binding
lation of platelet function. Specifically, PGE2 preferentially binds to
1
a family of receptors referred to as EP1–4
.
Low levels of PGE2
potentiate platelet aggregation while at higher concentrations this
effect is inhibited.2 Furthermore, studies utilizing knock-out mice
showed that the stimulatory effects of PGE2 on platelet aggregation
are exerted specifically through the EP3 receptor.3 Notably, EP3
receptors are not expressed in the arterial wall. Therefore, selective
EP3 receptor antagonists are expected to be anti-thrombotic agents
that do not increase risk of bleeding.4
R2
R1
R1
R1
R3
R3
O
R3
O
N
N
N
Ar1
Ar1
Ar1
HN
S O
O
HN
O
HN
S O
O
Ar2
S O
Ar2
Ar2
O
O
O
2
1
Indole series A
Figure 1. Proposed indolone series of derivatives 1 and 2.
* Corresponding author. Tel.: +1 630 904 2218.
E-mail addresses: JSingh@deCODE.com, Jasbir@JasinDS.com (J. Singh).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.028

N. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2658–2664
2659
assay, this series showed somewhat lower activity in the functional
rat platelet aggregation assay.8 It was anticipated that the indolone
templates, which retain the aromatic characteristics of the indole
series A would provide improved activity in the functional assay.
Moreover, the indolone analogs are expected to exhibit enhance
GI permeability and PK characteristics due to keto–enol tautomer-
ization of the carbonyl moiety. Chemical feasibility of this chemo-
type also allowed for the additional diversity at the C-3 position.
This Letter describes EP3 receptor binding affinities, liver micro-
some stability, prostanoid selectivity and rat platelet aggregation
studies of the novel 1,7-substituted oxyindole series represented
by the generic structures 1 and 2 (Fig. 1).
a diverse aryl sulfonamides to furnish the respective derivatives 6.
Alkylation of the indole nitrogen with benzylic halides provided
1,7-disubstituted indoles 7a–p that were converted to the targeted
oxyindoles 8a–p via oxidation with DMSO/concentrated HCl/acetic
acid system. These compounds, as shown, imply presence of a chi-
ral center. However, due to keto–enol tautomerism this center will
be expected to equilibrate under physiological conditions.
In a subsequent synthetic effort, we prepared a 3,3-disubsti-
tuted oxyindole series. Isatin core was selected as a convenient
starting point as the C-3 position may be derivatized by reduction,
reductive amination and addition reactions to rapidly produce the
targets of interest (Schemes 2 and 3). The key oxyindole molecules
from this series were produced from the isatin intermediates 15
and 16.
First, we prepared a series of 3-methyl oxyindoles.
a-Methyl
substituted oxyindoles (R1 = CH3, R2 = H) were accessed via oxida-
tion of the corresponding 3-methyl-indoles. Starting 7-bromo-3-
methylindole (3a, R3 = H) was subjected to Heck olefination condi-
tions with methyl acrylate to afford 4 (Scheme 1). Compound 4
was saponified to yield acrylic acid 5 followed by its coupling with
The synthesis of compound 16 commenced with the reaction of
2-bromoaniline (9) with chloral hydrate and hydroxylamine to
afford the intermediate hydroxylamine (10).9 Heating of 10 in
the concentrated H2SO4 afforded 7-bromoisatin (11).9 Direct Heck
R3
R3
R3
c
a
b
N
N
H
N
H
H
Br
5
O
OH
R3
4
O
O
3
R3
R3
O
e
d
N
N
Ar1
N
H
Ar1
H
H
N
O
H
N
N
O
Ar2
O
S
O
Ar2
O
S
O
Ar2
O
S
O
O
8a-p
7a-p
6
Scheme 1. Reagents and conditions: (a) methyl acrylate, Et3N, Pd(OAc)2, tri-o-tolylphosphine, 100 °C, 4 h, sealed tube, (82%); (b) aq NaOH, THF, CH3OH, room temperature,
16 h (100%); (c) Ar2SO2NH2, EDCI, DMAP, CH2Cl2, rt, 2–16 h, 80–94%; (d) NaH, DMF or THF, ArCH2X, rt, 16 h, 20–85%; (e) DMSO, HCl–AcOH, rt, 2–5.5 h, 54–91%.
HO
O
O
N
O
O
O
c
a
a
b
O
O
O
O
N
H
N
N
N
NH2
H
H
H
Br
Br
Br
Br
O
O
13
9
11
12
10
O
O
O
O
O
g
O
O
f
O
e
d
N
N
N
O
Ar1
Ar1
Ar1
N
Ar1
O
OH
O
NH-SO2-Ar2
O
O
O
O
14
15
16
Ar1 = 2,4-Dichlorophenyl, 2-Naphthyl, 3,4-Difluorophenyl
Scheme 2. Reagents and conditions: (a) Cl3CCHOÁH2O, NH2OHÁHCl, reflux, 30 min, then H2SO4, 50–80 °C, 30 min, 44%, two steps; (b) ethylene glycol, pTsOH, benzene, reflux,
23 h, 90%; (c) methyl acrylate, Pd(OAc)2, (o-tolyl)3P, Et3N, sealed tube, 100 °C, 6 h, 81%; (d) ArCH2X, K2CO3, DMF, 50 °C, 3 h, 77–84%; (e) concentrated HCl, MeOH, 90 °C, 3 h,
77–83%; (f) NaOH, CH3OH, H2O, 50 °C, 4 h, 78–83%; (g) Ar2SO2NH2, EDCI, DMAP, CH3CN, rt, 8–70 h, 60–85%.

2660
N. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2658–2664
F
F
F
F
F
O
F
a
c
b
O
O
O
O
N
N
N
N
Ar1
Ar1
Ar1
Ar1
O
NH
S
O
O
O
O
OH
O
O
O
Ar2
18a-c
15a-c
17a-c
19a-u
Ar1 = 2,4-Dichlorophenyl, 2-Naphthyl, 3,4-Difluorophenyl
Scheme 3. Reagents and conditions: (a) DAST, CH2Cl2, rt, 18 h, 90–99%; (b) NaOH, CH3OH, H2O, 50 °C, 4 h, 45–91%; (c) Ar2SO2NH2, EDCI, DMAP, CH3CN, rt, 16–72 h, 62–80%.
coupling of bromoisatin 11 with methyl acrylate did not yield the
anticipated 7-isatin acrylate presumably due to the significant side
reactions involving active C-3 carbonyl group of isatin. Thus, com-
pound 11, was converted to the respective ethylene acetal 12.10
This protection facilitated both Heck coupling to furnish 13 and a
subsequent N-alkylation to result in compound 14. This four-step
conversion of 11 to 14 followed by its deprotection afforded the
acrylate 15 in a 52% yield. Saponification of methyl ester 15 pro-
vided acrylic acid 16.
Conversion of 15 into our second set of desired target com-
pounds involved treatment with DAST in CH2Cl2 to provide gem-di-
fluoro derivatives 17a–c (Scheme 3). Saponification of 17a–c
afforded the corresponding acrylic acids 18a–c. Ar1 = 2,4-dichloro-
phenyl, 2-naphthyl or 3,4-difluorophenyl substituents were se-
lected as optimal from our earlier SAR studies of unsaturated
analogs.6,7 EDCI-mediated coupling of 18a–c with aryl sulfona-
mides provided targeted molecules 19a–u in a 62–80% overall
yields.
OH
N
OH
O
O
N
Ar1
Ar1
O
NH
S
O
NH
S
O
O
O
O
Ar2
Ar2
22a-h
21d, e, f, g, j, k, l, m
Scheme 5. Reagents and conditions: Na2CO3, 37% aq CH2O, dioxane, rt, 18–60 h,
22–64%.
p showed high binding affinity for the EP3 receptor in normal buf-
fer providing low nanomolar and/or sub-nanomolar IC50S. When
the receptor binding assay was performed in the presence of 10%
human serum (HS), series 8 analogs, showed low-to-moderate fold
shift (<10Â) in binding affinities with the exception of Ar2 as 2-
thiophenyl (8a and 8g) or Ar1 as 3,4-difluorophenyl (8n–p) indicat-
ing their low potential for protein plasma binding. Several com-
pounds from this series were further evaluated for stability using
liver microsome incubations (Table 1). Unfortunately, these dis-
played low metabolic stability in both human and rodent liver
microsomes. Incorporation of the fluorine atom at the C-5 position
of indole core improved metabolic stability albeit at the expense of
the plasma binding properties, as these analogs only displayed par-
tial activity in the receptor binding assay in the presence of human
serum.
Methylene oxyindoles 21a–q were accessed in a 23–94% overall
yields by a two-step sequence involving reduction of 16a–c with
hydrazine7 to oxyindoles 20a–c followed by their coupling with
the respective aryl sulfonamides (Scheme 4).
Several selected compounds 21 underwent
a-bis-hydroxyme-
thylation in basic formalin solution to provide molecules 22
(Scheme 5).
Saponification of compounds 14a and 14b afforded acrylic acids
23a and 23b, respectively (Scheme 6). A standard acylsulfonamide
coupling protocol described above (Scheme 1) afforded target com-
pounds 24a–c in 65–89% yields.
Acylsulfonamide formation using compound 16 afforded
isatin derivatives 25a–f. Compound 26 was made through the
condensation of compound 25a with 2-mercaptoethylamine. A
The most compelling initial biological data for the series 8 were
obtained from the rat platelet aggregation studies, the respective
IC50 values ranging from single digit to double digit nanomolar
(Table 2). This further supported our notion that indolone core
would provide improved functional activity. These results trig-
gered additional structural modifications within this chemotype
series of
a-hydroxy oxyindoles (27a–c) were produced by reduc-
tion of the corresponding isatin derivatives 25a–e (Scheme 7).
In our previous Letter4,7 we have reported that Ar1 = 2,4-dichlo-
rophenyl, 2-naphthyl and 3,4-difluoro-phenyl groups displayed
good hEP3 receptor affinity. As shown in Table 1, compounds 8a–
O
b
a
O
O
O
N
N
N
Ar1
Ar1
Ar1
O
NH
S
O
OH
O
OH
O
O
Ar2
20a-c
16a-c
21a-q
Ar1 = 2,4-Dichlorophenyl, 2-Naphthyl, 3,4-Difluorophenyl
Scheme 4. Reagents and conditions: (a) NH2NH2ÁH2O, 60 °C, 18 h, 73%; (b) Ar2SO2NH2, EDCI, DMAP, CH2Cl2 (or CH3CN or THF), rt, 16–72 h, 23–94%.

N. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2658–2664
2661
O
O
O
O
O
O
O
O
N
O
N
N
Ar1
Ar1
Ar1
O
O
O
OH
O
NH
S
O
O
Ar2
23a, 23b
14a
24a Ar2 = 4,5-Dichlorothiophenyl
24b Ar2 = 2-Thiophenyl
a) Ar1 = 2,4-Dichlorophenyl
b) and c) Ar1 = 2-Naphthyl
24c Ar2 = 4,5-Dichlorothiophenyl
Scheme 6. Reagents and conditions: (a) LiOH, CH3OH, THF, H2O, 50 °C, 4 h, 67–95%; (b) Ar2SO2NH2, EDCI, DMAP, CH2Cl2, rt, 16–72 h, 65–89%.
OH
O
N
Ar1
O
c
O
a
O
NH
S
O
O
O
O
N
N
Ar2
27a-c
Ar1
Ar1
S
O
NH
S
O
OH
NH
O
O
O
Ar2
b
N
S
16
25a-f
Cl
Cl
O
NH
S
O
O
Cl
26
Cl
Scheme 7. Reagents and conditions: (a) Ar2SO2NH2, EDCI, DMAP, CH2Cl2, rt, 16–72 h, 38–77%; (b) HSCH2CH2NH2, TsOHÁH2O, 40%; (c) NaBH4, MeOH, H2O, 0 °C, 1 min, 57–82%.
aimed at the enhanced liver microsome stability. We speculated
that liver microsome instability most likely originated from the
CYP-mediated oxidation of the C-3 benzylic position of oxyindole.
In order to both improve metabolic stability and physiochemi-
cal parameters of the oxyindole derivatives, we further pursued
modification at the C-3 of oxyindole core specifically by varying
R1/R2 moieties. gem-Disubstituted derivatives including 22 and
24 showed both reduced EP3 receptor affinity in normal buffer
and high fold shifts with a 10% HS. Compound 26 featuring a pro-
tonizable nitrogen displayed good EP3 receptor affinity but it suf-
fered from high protein binding as well.
Molecules 25a–f exhibited single to double digit nanomolar
binding affinities and moderate fold shifts in the presence of HS.
Analogs 27a–c afforded high affinity for the EP3 receptor, a prom-
ising inhibition profile in the rat platelet aggregation assay
(Table 2) and reduced potential for the plasma protein binding.
Importantly, compounds 27a–c were considerably more stable
towards human and rodent liver microsomes treatment. Three spe-
We next focused on the
a,a-difluoro derivatives 19. As shown in
Table 2, many of these compounds retained high binding affinity
for the EP3 receptor and showed relatively low fold shift in the
receptor binding assays in the presence of 10% HS. Furthermore,
series 19 molecules showed good metabolic stability for human,
rat and mouse liver microsomes (Table 1), however they suffered
a setback in the rat platelet aggregation assay showing reduced
activity with the IC50 values exceeding 800 nM. Compound 19 also
displayed tendency for a mild CYP inhibition, most notably for the
CYP3A4 (BFC used as a substrate). Consequently, these series were
not pursued further.
Next, we investigated
a-methylene oxyindole series 21. With
the exception of 21b and 21h (Ar2 = 2-thiophene), all compounds
from this class exhibited IC50 <3 nM for the EP3 receptor in normal
buffer. In addition, except for the compounds featuring Ar1 = 3,4-
difluorophenyl, other derivatives from this chemotype displayed
low fold shifts in the presence of 10% HS following the trend ob-
served for the series 8 and 19. Considering combined evidence,
we focused on preparing oxyindole derivatives with Ar1 = 2,4-
dichlorophenyl or 2-naphthyl.4,7 Analogs from series 21 gave the
poorest results in the rat platelet aggregation assay (Table 2) and
thus these were not pursued further.
cific a-hydroxy oxyindole analogs (27a–c) furnished high selectiv-
ity for the EP3 receptor in the panel of other prostanoid receptors
(Table 3). These agents did not inhibit CYP isoforms including
1A2, 2D6 and subset of 3A4 isozymes. However, they did affect
the 3A4 site that metabolizes DBF and showed relatively high inhi-
bition of CYP2C9 (Table 4).
The intrinsic PK parameters for the
a-hydroxy oxyindole 27a
following iv dosing to mouse and rat are shown in Table 5.
Although the half-life was similar, the clearance and volume of dis-
tribution was lower in rat than that observed in mouse.

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N. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2658–2664
Table 1
Binding affinities (IC50s) for compound series 8, 19, 21, 22, 24, 25, 26 and 27 in normal buffer and 10% human serum, and liver microsome stabilities (% parent remaining after
30 min incubation) for human (HLM), rat (RLM), mouse (MsLM), dog (DLM) and monkey (MkLM)
Ar1
O
R1
R2
N
Ar2
O
H
N S
O
O
Cmpd
R1
R2
Ar1
Ar2
hEP3 NBa (nM)
hEP3 HSb (nM)
Metabolic stabilityc
HLM
64.7
RLM
MsLM
C3-monomethyl analogs
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8n
8o
8p
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Thiophenyl
2.4
3.4
0.4
0.9
0.5
0.5
3.5
4.4
0.6
0.3
0.4
0.6
0.9
1.4
4.0
3.8
237
0.2
0.2
0.2
0.2
1.1
0.2
11.6
201.8
0.3
0.2
0.3
0.1
2.7
5.5
7.2
16.5
1.3
1.3
5.0
660
23.9
3.3
9.8
6.8
4.3
96.5
3.2
20
5.4
4,5-Dichlorothiophenyl
3,4-Difluorophenyl
3,4-Dichlorophenyl
3-Chlorophenyl
2,4,5-Trifluorophenyl
2-Thiophenyl
4,5-Dichlorothiophenyl
3,4-Difluorophenyl
2,4,5-Trifluorophenyl
3-Chlorophenyl
3,4-Chlorophenyl
3,4-Dichlorophenyl
2,4,5-Trifluorophenyl
3-Chlorophenyl
4,5-Dichlorothiophenyl
2-Thiophenyl
2,4,5-Trifluorophenyl
3,4-Difluorophenyl
3,4-Chlorophenyl
3-Chlorophenyl
62.1
16
49.5
48.4
43.5
48.2
23.6
22.4
48
7.26
4.85
32.6
67.7
8.0
13.7
24.8
36.4
17.6
13.1
10.3
5.1
5.63
5.83
2.8
4.1
2-Naphthyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
95.8
10.7
39.0
64.0
PDd
5.6
6.5
5.5
7.7
251
64.7
20.8
PD
9.3
5.0
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
19n
19o
19p
19q
19r
19s
19t
19u
94.4
94.8
100
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
74.6
74.4
73.1
66.6
77.4
80.1
70.4
62.2
60.6
71
82.8
31.2
4-Chlorophenyl
4-Fluorophenyl
4,5-Dichlorothiophenyl
2-Thiophenyl
2,4,5-Trifluorophenyl
3,4-Difluorophenyl
3,4-Chlorophenyl
3-Chlorophenyl
79.3
71.8
80.7
68.8
68.1
79.4
64.5
54.2
45
62.1
21
47.9
47.1
41.3
21.6
4.5
1.9
4-Fluorophenyl
4-Chlorophenyl
27.2
125.1
979.9
720.9
251.1
PD
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
4,5-Dichlorothiophenyl
2,4,5-Trifluorophenyl
3,4-Difluorophenyl
3,4-Chlorophenyl
3-Chlorophenyl
F
F
F
F
416.3
C3-methylene (unsubstituted) series
21a
21b
21c
21d
21e
21f
21g
21h
21i
21j
21k
21l
21m
21n
21o
21p
21q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
4,5-Dichlorothiophenyl
2-Thiophenyl
3-Chlorophenyl
2.9
56.0
0.5
0.2
0.7
1.2
1.3
23.6
0.3
0.4
0.5
0.6
0.9
0.3
0.4
0.7
0.4
92.8
PD
9.4
4.4
3.3
3.3
5.4
PD
10.2
6.9
76.8
46.3
56.8
50.4
61.6
54.3
53.3
105
21
33.9
70.1
39.1
56.1
63.8
2,4,5-Trifluorophenyl
3,4-Difluorophenyl
3,4-Chlorophenyl
4,5-Dichlorothiophenyl
2-Thiophenyl
2,4,5-Trifluorophenyl
3,4-Difluorophenyl
3,4-Chlorophenyl
3-Chlorophenyl
3,4-Difluorophenyl
3,4-Chlorophenyl
3-Chlorophenyl
2,4,5-Trifluorophenyl
4,5-Dichlorothiophenyl
50.6
23.5
26.8
32.9
73.9
65.9
66.5
56.8
62.5
43
46
13.2
15.7
7.8
5.2
8.1
2-Naphthyl
29.8
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
47.8
55.4
35.5
42.0
55.4
C3-disubstituted analogs
22a
22b
22c
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
2-Naphthyl
2-Naphthyl
3,4-Difluorophenyl
4,5-Dichlorothiophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
2.3
11.6
45.4
243.1
186.9
PD

N. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2658–2664
2663
Table 1 (continued)
Cmpd
R1
R2
Ar1
Ar2
hEP3 NBa (nM)
hEP3 HSb (nM)
Metabolic stabilityc
HLM
RLM
MsLM
C3-spiro-analogs
24a
24b
24c
26
–OCH2CH2O–
–OCH2CH2O–
–OCH2CH2O–
2,4-Dichlorophenyl
2-Naphthyl
2-Naphthyl
4,5-Dichlorothiophenyl
2-Thiophenyl
4,5-Dichlorothiophenyl
4,5-Dichlorothiophenyl
3.8
13.0
7.9
PD
28.4e
37.8e
679.0
82.4
880
–SCH2CH2NH–
2,4-Dichlorophenyl
4.9
C3-oxo (isatin) analogs
25a
25b
25c
25d
25e
25f
R1 = R2 = O
1 = R2 = O
2,4-Dichlorophenyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
4,5-Dichlorothiophenyl
2-Thiophenyl
4,5-Dichlorothiophenyl
4-Fluorophenyl
3,4-Chlorophenyl
2,4,5-Trifluorophenyl
6.5
136.5
260.0
15.0
33.9
12.7
17.4
R
13.7
12.3
1.6
1.0
0.6
R1 = R2 = O
R
R
R
1 = R2 = O
1 = R2 = O
1 = R2 = O
2-Naphthyl
C3-hydroxy (a-hydroxy oxyindole) analogs
27a
27b
27c
OH
OH
OH
H
H
H
2-Naphthyl
2-Naphthyl
2-Naphthyl
4-Fluorophenyl
3,4-Difluorophenyl
2,4,5-Trifluorophenyl
0.9
1.6
0.6
19.4
7.6
9.4
97.7
89.3
95.6
103.3
87.9
99.7
83.4
72.7
73.5
a
b
c
NB: normal assay buffer.
HS: hEP3 IC50 in the presence of 10% human serum.
(1) Data shown as percent parent remaining at 30 min following incubation with liver microsomes. (2) HLM: human liver microsomes, RLM: rat liver microsomes, MsLM:
mouse liver microsomes, DLM: dog liver microsomes; MkLM: monkey liver microsomes. (3) DLM: dog liver microsomes data for selected compounds: 8g 81.8%, 8h 67.7%, 19a
83.3%, 21g 79.5%. (4) MkLM: monkey liver microsomes data for selected compounds: 8g 35.3%, 19a 67.3%, 21a 76.9%.
d
PD: Partial displacement of 3H-PGE2 in the in vitro assay.
These analogs were stable in the assay buffer in the absence of microsomes.
e
Table 2
Table 5
Rat platelet aggregation data for selected compounds
Pharmacokinetic parameters for compound 27a following iv dosing (6 mg/kg)
Compd no.
EC50 (nM)
Species
t1/2 (h)
Cl_pred (ml/h/kg)
Vss, pred (ml/kg)
8b
8h
47
2
Mouse
Rat
2.10
1.75
4584
2510
5663
2405
8j
19
21c
21d
21e
21f
21g
21i
21j
21l
27a
27b
27c
290
361
137
316
27
1089
246
405
63
an optimized series of 3-hydroxy oxyindoles (27a–c) that afforded
high affinity for the human EP3 receptor, over >1000Â selectivity
across a panel of prostanoid receptors, good stability in the liver
microsomes assay, and low plasma binding potential. In addition,
these compounds exhibited good activity in the rat platelet aggre-
gation assay for EP3 antagonism. Detailed pharmacokinetic data
and in vivo pharmacology for the prioritized molecules will be re-
ported elsewhere.
87
69
Table 3
References and notes
IC50 (lM) activity for analogs from radioligand displacement assays across prostanoid
isoforms panel
1. (a) Abramovitz, M.; Adam, A.; Boie, Y.; Godbout, C.; Lamontagne, S.; Rochette,
C.; Sawyer, N.; Tremblay, N. M.; Belley, M.; Gallant, M.; Dufresne, C.; Gareau, Y.;
Ruel, R.; Juteau, H.; Labelle, M. Biochim. Biophys. Acta 2000, 1483, 285; (b)
Kiryiama, A.; Ushukubi, K.; Kobayashi, T.; Hirata, M.; Sugimoto, Y.; Narumiya, S.
Br. J. Pharmacol. 1997, 122, 217.
Compd
hEP2
hEP4
hFP
hIP
27a
27b
27c
>20
>20
>20
15.1
2.3
8.7
>20
>20
>20
>20
>20
>20
2. Gross, S.; Tilly, P.; Hentsch, D.; Vonesch, J. L.; Fabre, J. E. J. Exp. Med. 2007, 20,
311.
3. Fabre, J.; Nguyen, M.; Athirakul, K.; Coggins, K.; McNeish, J. D.; Austin, S.; Parise,
L. K.; FitzGerald, G. A.; Coffman, T. M.; Koller, B. H. J. Clin. Invest. 2001, 107, 603.
4. Singh, J.; Zeller, W.; Zhou, N.; Hategen, G.; Mishra, R.; Polozov, A.; Yu, P.; Onua,
E.; Zhang, J.; Zembower, D.; Kiselyov, A.; Ramírez, J.; Sigthorsson, G.; Bjornsson,
J.; Thorsteinnsdottir, M.; Andrésson, T.; Bjarnadottir, M.; Magnusson, O.;
Stefansson, K.; Gurney, M. ACS Chem. Biol. 2009, 4, 115.
5. Singh, J.; Zeller, W.; Zhou, N.; Hategen, G.; Mishra, R.; Polozov, A.; Yu, P.; Onua,
E.; Zhang, J.; Ramírez, J.; Sigthorsson, G.; Thorsteinnsdottir, M.; Kiselyov, A.;
Zembower, D.; Andrésson, Þ.; Gurney, M. J. Med. Chem. 2010, 53, 18.
6. For example, replacement of indole with 5-azaindole core led to over 100-fold
drop in activity versus the corresponding indole analog. Incorporation of (e.g.,
2-pyridyl methyl as Ar1 substituents also resulted in analogs with very poor
activity in the hEP3 receptor-binding assay.
Table 4
hCYP inhibition with compounds 27b–e
Compd
3A4a
3A4b
2C9
2C19
1A2
h2D6
27a
27b
27c
>10
>10
>10
3.06
2.12
2.33
1.04
1.02
0.8
4.89
6.18
5.38
>10
>10
>10
>10
>10
>10
a
Utilized BFC, 7-benzyloxy-4-trifluoromethylcoumarin, as substrate.
Utilized DBF, dibenzylfluorescein as substrate.
b
7. O’Connell, M.; Zeller, W.; Burgeson, J.; Mishra, R. K.; Ramirez, J.; Kiselyov, A. S.;
Andrésson, T.; Gurney, M. E.; Singh, J. Bioorg. Med. Chem. Lett. 2009, 19, 778.
8. In spite of excellent potency in the receptor binding assays (IC50 <10 nM), the
In conclusion, we have described SAR of 1,7-disubstituted oxy-
indoles where the C3 substituent modifications were guided by
both plasma protein binding and metabolic stability. We identified
hexa-hydroindolone analogs, that were evaluated, displayed low
lM potency
in the functional rat platelet aggregation assay.

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N. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2658–2664
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