3-Acrylamide-4-aryloxyindoles: Synthesis, biological evaluation and metabolic stability of potent and selective EP3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.12.112

A series of potent and selective EP₃ receptor antagonists are described. Utilizing a pharmacophore model developed for the EP₃ receptor, a series of 3,4-disubstituted indoles were found to be efficient ligands for this target. These compounds showed high selectivity over IP, FP and other EP receptors. An optimized molecule 7c featured a sound profile and potency in the functional rat and human platelet aggregation assays.

Full text of DOI:10.1016/j.bmcl.2008.12.112

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1528–1531
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Acrylamide-4-aryloxyindoles: Synthesis, biological evaluation and metabolic
stability of potent and selective EP3 receptor antagonists
Nian Zhou a, Wayne Zeller a, Jun Zhang a, Emmanuel Onua a, Alex S. Kiselyov a, Jose Ramirez b,
Guðrún Palsdottir b, Guðrún Halldorsdottir b, Þorkell Andrésson b, Mark E. Gurney b, Jasbir Singh a,
*
a Medicinal Chemistry Department, 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 potent and selective EP3 receptor antagonists are described. Utilizing a pharmacophore model
developed for the EP3 receptor, a series of 3,4-disubstituted indoles were found to be efficient ligands for
this target. These compounds showed high selectivity over IP, FP and other EP receptors. An optimized
molecule 7c featured a sound profile and potency in the functional rat and human platelet aggregation
assays.
Received 2 October 2008
Revised 20 December 2008
Accepted 31 December 2008
Available online 8 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Prostanoid receptor antagonists
EP3 receptor antagonists
Peri-substituted indoles
Acylsulfonamide
The prostanoids constitute a family of endogenous signaling li-
gands arising from phospholipase A2-mediated release of aracha-
donic acid followed by its further transformation through a series
of synthases. The prostanoid family includes prostaglandins D2,
leases PGE2 locally and promotes vicinal platelet aggregation
through the platelet EP3 receptor.11b
A series of reports from the Merck–Frosst labs12 have docu-
mented the synthesis, biological activity and selectivity of three
different series of ortho-substituted cinnamyl and dihydrocinnam-
yl derivatives as highly potent and selective human EP3 receptor
antagonists.
E2, F2 , I2 and thromboxane A2. These ligands preferentially binds
a
the series of G-coupled protein receptors DP1 and CRTH(DP2), EP1
through EP4, FP, IP and TP, respectively.1,2 Activation of these
receptors controls biological homeostasis.3 Of these, the EP3 recep-
tor has been shown to play key roles in sodium and water reab-
sorption in kidney tubules,4 smooth muscle contraction of the GI
tract,3 acid secretion,5 uterine contraction during fertilization and
implantation,6 fever generation7 and hyperalgesia.8 Eight isoforms
In a preceding paper in this series we described a series of 3-
thioaryl ether (A) and 3-sulfonylaryl indoles (B) which were potent
and selective antagonists of the EP3 receptor (Fig. 1).13 In order to
further expand the diversity of the EP3 receptor antagonists and to
improve their physico-chemical and pharmacokinetic properties
we investigated other chemotypes.14 Several fused bicyclo[4.3.0]
of the human EP3 receptor have been detailed (EP3I through EP3VI
,
EP3e and EP3f) which are distinguished through unique cytoplasmic
C-terminus ends ranging from 6 to 66 amino acids in length.9
Recently, we have identified DNA variants of the gene encoding
for the EP3 receptor that confer a significantly increased risk for
development of peripheral arterial disease (PAD).10 Utilizing EP3
KO mice, it has been shown that the stimulatory effects of PGE2
on a platelet aggregation are exerted specifically through the EP3
receptor.11a Based on this evidence we reasoned that the develop-
ment of potent and selective EP3 antagonists may provide a novel
therapeutic entry into the treatment of PAD through modulation of
platelet aggregation. A subsequent report from the Fabre lab dem-
onstrated that inflammation/rupturing of the existing plaque re-
O
S
N
O
O
S
Ar1
H
O
O
Ar1
O
O
N
H
Ar2
X
D
A
Ar2
X
N
R
N
H
A (X = S)
B (X = SO2)
C X = O
A-D = CH=CH
* Corresponding author. Tel.: +1 630 783 4915; fax: +1 630 783 4646.
E-mail address: jsingh@decode.com (J. Singh).
Figure 1. 3,4-Disubstituted indole analogs.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.112

N. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1528–1531
1529
aromatic scaffolds afforded the proper spatial arrangement for the
pharmacophoric entities in the targeted molecules. Of these scaf-
folds we inquired whether the acidic and lipophilic appendages
of the previously disclosed 3,4-disubstituted indoles could be
interchanged off of the indole ring and retain the EP3 binding affin-
ity experience. A priori, this exchange seem reasonable expecting
the acidic and lipophilic appendages would project from indole
core at a similar dihedral angle and thus the EP3 receptor could
be occupied with a compound of similar topology. In the current
disclosure, we outline the synthesis and the biological evaluation
of 3,4-disubstituted indole analogs C.
The target 3-acrylsulfonamide-4-aryloxy indole analogs, re-
ported in Table 1, were synthesized from 4-bromo-1-methyl indole
2 (Scheme 1). The 4-aryloxy group was introduced by Ullmann-type
diaryl ether formation using CuI/N,N-dimethylglycine as a cata-
lyst.15 Formylation16 of 3 with DMF/POCl3 afforded the correspond-
ing 4-formyl-3-aryloxy indoles 4 in 33–95% yield. Wittig–Horner
reaction of 4 provided the 4-acryl ester-3-aryloxy indoles 5. Esters
5 were saponified to afford free acids 6 in 40–90% yield after extrac-
tion under acidic conditions. EDCI-mediated coupling of com-
pounds 6 with the appropriate sulfonamide at room temperature
resulted in the corresponding acylsulfonamides 7–9 (20–60% yield).
Table 1
IC50 for hEP3 receptor binding affinity for selected compounds
F
F
O
S
O
S
O
S
O
O
O
O
O
Ar1
F
F
O
N
Ar2
N
Ar2
N
H
H
H
O
F
O
O
N
N
N
7a-r
9a-l
8a-j
Compound
Ar1
Ar2
EP3 receptor binding (nM)
Normal buffer
10% human seruma
Fold-shift in 10% human serumb
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
4,5-Dichlorothiophene
3,4-Difluorophenyl
2,4,5-Trifluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
3,4-Dichlorophenyl
2,4-Dichlorophenyl
3,5-Dichlorophenyl
2,4-Difluorophenyl
2,5-Difluorophenyl
2,6-Difluorophenyl
3,5-Difluorophenyl
3-Fluorophenyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
4.6
7.4
9.2
9.4
18.4
15.7
11.8
54.1
8.7
3.5
6.5
5.9
44.9
17.5
11.9
21.9
100.8
5.1
89.6
15.9
28..0
182.3
1682
35.0
19.4
2.1
3.1
19.4
91.3
2.2
92.9
7.8
1542
321.3
115.3
156.7
709.6
156.7
115.5
646.4
1100
PD
28.5
37.0
32.7
24.3
120.6
3.5
6.6
2-Fluorophenyl
4-Chlorophenyl
4-Methoxyphenyl
Pentafluorophenyl
54.3
50.2
—
PD
—
8a
8b
8c
8d
8e
8f
8g
8h
8i
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Difluorophenyl
3,4-Dichlorophenyl
2,4-Dichlorophenyl
4-Chlorophenyl
3,4-Difluorophenyl
2,4-Difluorophenyl
3-Chloro-4-fluorophenyl
4-Chloro-3-fluorophenyl
4-Chloro-2-fluorophenyl
2-Chloro-4-fluorophenyl
3-Methoxyphenyl
12.4
11.8
27.3
4.7
18.5
26.9
23.4
7.2
50.8
23.7
776.2
195.9
PD
4.1
2.0
28.4
41.9
—
81.5
3.0
612.4
124.0
145.8
PD
129.8
216.5
26.3
17.2
10.5
—
68.7
35.3
13.8
4.4
1.9
8j
8k
8l
6-Quinolinyl
2-Quinoxalinyl
6.1
9a
9b
9c
9d
9e
9f
9g
9h
9i
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
2,4,5-Trifluorophenyl
3,4-Dichlorophenyl
2,4-Dichlorophenyl
4-Chlorophenyl
2.6
3.2
3.0
3.8
84.8
9.7
6.7
4.4
18.1
5.1
8.5
7.2
45.2
16.7
392.9
209.5
PD
61.1
53.9
155.7
316.3
PD
17.1
5.2
131.7
55.0
PD
3,4-Difluorophenyl
2,4-Difluorophenyl
3-Chloro-4-fluorophenyl
4-Chloro-3-fluorophenyl
4-Chloro-2-fluorophenyl
2-Chloro-4-fluorophenyl
3-Methoxyphenyl
6-Quinolinyl
6.3
8.0
35.6
17.5
PD
19.8
17.7
9j
9k
9l
167.8
128.2
2-Quinoxalinyl
a
PD = partial displacement of [3H]-PGE2 in the radioligand assay at 20
Fold-shift value is ratio of IC50 in the presence and absence of human serum.
lM (highest concentration).
b

1530
N. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1528–1531
Ar2
Ar2
O
Br
Br
O
O
H
i
ii
iii
N
H
N
O
N
N
2
3
4
1
O
S
Ar1
O
O
O
O
OH
vi
N
Ar2
Ar2
Ar2
H
O
O
O
iv
v
N
N
N
7-9
5
6
Scheme 1. Reagents and conditions: (i) NaH, CH3I, DMF, rt, 3 h, 100%; (ii) Ar2–OH, CuI, N,N-dimethylglycine HCl salt, Cs2CO3, dioxane, 105 °C, 2–3 d; (iii) DMF, POCl3, 50 °C,
1 h, then aq NaOH, 33–95% from 2; (iv) NaH, triethyl phosphonoacetate, THF, rt, 10 min then compound 4, THF, 60 °C, 16 h; (v) aq, NaOH, THF, MeOH, 50 °C, 3 h, 40–90% from
4; (vi) Ar1–SO2–NH2, DMAP, EDCI, CH2Cl2, rt, 16 h, 20–60%.
The SAR data and diversity of Ar1/Ar2 groups from the previ-
Table 2
ously reported 1,7- and 3,4-peri-substituted indole series13,17
Selectivity of the selected compounds for hEP3 versus other prostanoid receptors
served as starting point for evaluating SAR for the current series.
An analog with the best Ar1/Ar2 group from series A (Ar1 = 4,5-di-
chloro-2-thiophene, Ar2 = naphthyl)13 was prepared providing
analog 7a. Compound 7a exhibited essentially identical activity
in the hEP3 radioligand binding assay in normal buffer
(IC50 = 4.6 nM) versus the corresponding compound from series A
(IC50 = 9.7 nM). This data strongly supported our hypothesis that
the acidic and lipophilic appendages could be interchanged be-
tween the 3,4-peri-disubstitued indole series A and C with reten-
tion of hEP3 binding affinity.
Compound
Prostanoid panel binding (lM)
hEP3
EP1
EP2
EP4
IP
FP
7b
8a
8b
8f
8h
9a
9b
9f
0.0074
0.0124
0.0096
0.0269
0.0072
0.0026
0.0032
0.0097
0.0061
1.42
>10
3.86
ND
>10
5.32
ND
12.5
4.2
9.6
>10
>10
7.68
9.3
>10
10
12.5
20.4
14.6
>10
>10
13.0
>10
>10
10
1.88
ND
ND
4.1
>10
ND
ND
ND
ND
3.59
2.4
1.97
1.82
3.19
4.64
3.57
3.16
2.23
>10
1.37
To optimize the Ar1 (acylsulfonamide) group for the current ser-
ies we elected to retain the 2-naphthyl substituent (Ar2). Our pre-
vious studies have consistently shown that the derivatives
endowed with this moiety were potent and isoform selective EP3
antagonists.13,17 Results from the human EP3 receptor binding13
in both normal buffer and in the presence of 10% human serum18
are given in Table 1 (7a–r). Gratifyingly, except for the Ar1 = 4-
MeO–C6H4, all derivatives displayed EP3 receptor activity in the
low nM range. Except for 7c, ortho-substituted Ar1 groups showed
significant plasma protein binding in the presence of human ser-
um. SAR studies suggested that in this series binding affinity in
the absence or presence of plasma proteins (IC50) followed the in-
crease of the electron deficiency19 for the Ar1. For example, com-
parison of data for the pair of molecules 7d/7p, 7b/7g and 7j/7h,
suggested that fluorine substituent(s) were generally favored over
chlorine group(s). Based on this initial set of SAR data, we priori-
tized compounds 7b and 7c that demonstrated superior EP3 bind-
ing affinity and low plasma protein binding as prototypes for
further investigation. In addition, we expected the fluorine substi-
tuted phenyl derivatives to provide overall better physico-chemi-
cal properties. Thus, the next round of SAR was conducted with
the Ar1 = 3,4-difluorophenyl (8a–l) and 2,4,5-trifluorophenyl (9a–
l).
9g
IC50 (lM) values reported are from receptor binding assays with displacement of
the respective radioligand.
ND = value not determined.
Table 3
In vitro metabolic stability of selected 3-acrylamide-4-aryloxy indoles in the
microsomal assays
Compound
Microsome metabolic stabilitya
Human
Rat
Mouse
Dog
69.7
90.4
80.6
Monkey
7b
7c
7f
8a
8b
8f
63.6
91.2
79.0
58.8
74.7
91.5
113
24.6
32.0
33.3
14.9
30.6
32
62
59
ND
ND
ND
69
ND
ND
ND
ND
ND
ND
53.8
ND
ND
ND
ND
104
ND
ND
ND
8h
9b
40
39.1
74.7
a
Each compound at 5 lM was incubated with liver microsomes representing
0.8 mg/mL protein concentration and the percent parent remaining at 30 min, as
determined by LC–MS/MS, is reported.
In order to enhance both solubility and formulability of the tar-
geted compounds, we introduced heterocyclic substituents with
basic nitrogen(s) (e.g., quinolinyls 8k and 9k and quinoxalinyls 8l
and 9l). Although these molecules afforded single digit nM potency
in the normal buffer binding assay, they suffered from the signifi-
cant plasma protein binding.
A number of potent molecules from the SAR studies exhibited
sound selectivity against a panel of prostanoid receptors (generally
>100- to 5000-fold) (Table 2).
observed for the pair 7b/7c and compound 8b in all species exam-
ined with the exception of rat.
Both compounds 7b and 7c (Table 3), were subsequently evalu-
ated in a functional rat platelet aggregation assay. The molecules
exhibited an EC50 value of 12.8 and 11.6 nM, respectively, which
compares favorably, with potency of previously reported com-
pounds.13,20 Compound 7c also exhibited an EC50 value of 8.1 nM
in a human platelet aggregation inhibition assay.
Table 3 provides a summary of metabolic stability for the prior-
itized analogs in liver microsomes. Good metabolic stability was
Selected compounds were evaluated in a functional cell-based
assays with CHO-KI cells transfected with the EP3II receptor to con-

N. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1528–1531
1531
6. Krall, J. F.; Barrett, J. D.; Jamgotchian, N.; Korenman, S. G. J. Endocrinol. 1984,
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160
120
80
FSK
FSK+PGE2
40
Cmpd (8b) EC50 (nM) = 26.1
0
0.0001 0.001
0.01
0.1
1
10
100
1000 10000 100000
12. (a) Gallant, M.; Belley, M.; Carriere, M.-C.; Chateauneuf, A.; Denis, D.; Lachance,
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C.; Chateauneuf, A.; Denis, D.; Gareau, Y.; Godbout, C.; Greig, G.; Juteau, H.;
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j.bmcl.2008.12.027.
µM
Figure 2. Dose–response of compound (8b) on CHO-K1 cells transfected with hEP3D
receptor in normal buffer.
Table 4
Mouse iv exposure screen
Compound Average plasma
concentration (ng/mL)
Average brain
concentration (ng/g)
Ratio
plasma/
brain
7b
7c
3420
1518
55.7
29.1
61.4
52.2
15. Ma, D.; Cai, Q. Org. Lett. 2003, 5, 3799.
16. Tyson, F. T.; Shaw, J. T. J. Am. Chem. Soc. 1952, 74, 2273.
Compounds were dosed in NMRI mice, n = 3 per compound, at 2 mg/kg in 10%
solutol/PBS formulation. Samples were analyzed at 15 min.
17. 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., accepted for publication.
18. Ref. 12c lists data which includes determinations of binding affinity to the EP3
receptor in the presence of 0.05% HSA. This data indicates a propensity of these
compounds to strongly bind to serum proteins. Hence, we found it imperative
to routinely screen each strongly potent compound from the normal buffer
assay in the presence of 10% human serum to provide useful information on
the free fraction of the compound available for future in vivo examinations.
19. Previous studies have shown that strongly electron rich heteroaromatic Ar1
substituents of these acylsulfonamides suffer from poorer binding affinity to
the EP3 receptor in normal buffer in addition to exhibiting a high propensity to
suffer plasma protein binding. For an example, refer to structures 7g and 8g in
Ref. 13.
20. Two of our lead compounds, labeled 7aa and 8aa in Ref. 13, exhibited EC50 in
the rat platelet aggregation studies of 358 nM and 136 nM, respectively.
21. The hEP3II (also known as hEP3D) receptor isoform exhibited slightly higher
affinity for PGE2 and higher inhibitory efficiency compared to the other human
EP3 isoforms tested (data not shown). Therefore, hEP3II was selected for both
the primary binding screening assay and the functional cell-based assay.
22. Stably transfected CHO-K1 cells expressing the hEP3II receptor were plated into
96-well plates at a cell density of 105 cells/well and cultured overnight at 37 °C,
5% CO2 in culture media supplemented with 10% FIBS, 2% PS and 1 mg/mL
Geneticin. Cells were washed once with PBS and pre-incubated in fresh, serum-
and antibiotic-free medium containing 1 mM IBMX (3-isobutyl-1-
methylxanthine, Sigma) for 30 min at 37 °C. After pre-incubation, cells were
incubated with PGE2 (5 Â 10À9 M) and FSK (5 Â 10À6 M, Sigma), in absence or
presence of the testing compound at the appropriate concentrations (dose–
response 10À4 –10À13 M). Cells were then incubated for an additional 10 min at
37 °C. Reactions were terminated by aspiration of medium and addition of
firm EP3 receptor antagonism.21,22 A representative dose–response
curve is shown in Figure 2 for compound 8b (EC50 = 26.1 nM).
SAR studies and initial follow-up evaluation of the synthesized
3,4-disubstituted indoles prioritized the compounds 7b and 7c for
further studies. Both of these compounds gave relatively low brain
exposures while still showing relatively high plasma levels at
15 min post dose, plasma concentration of compounds (7b,
6.6 lM and 7c, 2.8 lM). These data favored our intended therapeu-
tic use as anti-thrombotic agents (Table 4).
The data presented here supported our hypothesis that indole
derived analogs representing peri-substitution pattern as repre-
sented by series A, B and C provides a sound approach to potent
and isoform selective hEP3 antagonists. For the 3-acrylamide-4-
aryloxyindole series, an optimized compound (7c) was selected
as both potent and selective hEP3 receptor antagonist with sound
functional activity in platelet aggregation studies.
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200 ll of lysis buffer 1B (cAMP EIA System kit, Amersham). cAMP levels were
determined using a commercially available cAMP EIA System kit (Amersham).
Raw OD values, were transformed into amount of cAMP (fmol/well) using
GraphPad Prism 3.02 for Windows (GraphPad Software). For EC50 calculations
in dose–response experiments, sigmoid non-linear regressions were
performed.
3. Watson, S. Arkinstall, Prostanoids in the G Protein-Linked Receptor Factsbook;
Academic Press, 1994. p 239.
4. Garcia-Perez, A.; Smith, W. L. J. Clin. Invest. 1984, 74, 63.
5. Chen, M. C. Y.; Amirian, D. A.; Toomey, M.; Sanders, M. J.; Soll, A. H.
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