Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.02.034

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced

Full text of DOI:10.1016/j.bmcl.2010.02.034

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2639–2643
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel N-acylsulfonamide analogs as potent and selective EP3
receptor antagonists
*
Masaki Asada , Tetsuo Obitsu, Atsushi Kinoshita, Yoshihiko Nakai, Toshihiko Nagase, Isamu Sugimoto,
Motoyuki Tanaka, Hiroya Takizawa, Ken Yoshikawa, Kazutoyo Sato, Masami Narita, Shuichi Ohuchida,
Hisao Nakai, Masaaki Toda
Minase Research Institute, Ono Pharmaceutical Co., Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
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 N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity
and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated
for their inhibitory effect on PGE2-induced uterine contraction in pregnant rats. Among those tested, a
series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding car-
boxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR)
are also discussed.
Received 29 October 2009
Revised 8 February 2010
Accepted 9 February 2010
Available online 13 February 2010
Keywords:
Prostaglandin
Ó 2010 Published by Elsevier Ltd.
EP3 receptor
Antagonist
N-Acylsulfonamide
N-Acyl (3,4-difluorobenzene)sulfonamide
O
H
N
It is well known that prostaglandin E2 (PGE2) has diverse biolog-
ical activities mediated by four receptor subtypes, namely EP1-4
receptors. Recent studies using knockout mice have suggested that
the EP3 receptor is involved in a large number of pharmacological
actions of PGE2 including hyperalgesia,1 pyrexia,2 uterine contrac-
tion,3 gastric acid secretion,4 platelet aggregation,5 and thrombo-
sis.6 Thus, identification of a potent and selective EP3 receptor
antagonist may prove to be an attractive starting point from which
to design clinically useful drugs. In one of our preceding papers,7
we reported the discovery of the carboxylic acid analog 1 as a
new chemical lead of a selective EP3 receptor antagonist.
S
O
O
O
HO
O
OH
2 (Sulprostone)
O
O
O
S
Br
F
O
O
O
S
N
H
S
N
Cl
O
H
An N-acylsulfonamide group, a functionality often found in var-
ious drugs, is a well known bioisostere of a carboxylic acid because
of its similar acidity. A few EP3 receptor ligands possessing the N-
acylsulfonamide group are illustrated in Figure 1. Sulprostone (2)8
is a well-known EP3 (and EP1) receptor agonist possessing an N-
acylmethanesulfonamide group. In addition, researchers at Merck
have reported EP3 receptor antagonists with N-acylbenzenesulf-
onamide groups, exemplified by 3.9 The aforementioned informa-
tion prompted us to replace the carboxylic acid of our initial lead
1 with various N-acylsulfonamide groups which were expected
to be able to obtain more potent affinity for the EP3 receptor, as
illustrated in Figure 2. Recently, researchers at de CODE have also
N
Cl
Cl
Cl
4 (DG-041)
3
Figure 1. N-Acylsulfonamide containing EP3 receptor ligands.
O
O
O
COOH
S
R
N
H
O
O
N
N
N
N
1
* Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: m.asada@ono.co.jp (M. Asada).
Figure 2. N-Acylsulfonamide analogs as a novel EP3 receptor antagonist.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.02.034

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M. Asada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2639–2643
Table 2
disclosed several series of novel EP3 receptor antagonists possess-
ing N-acylsulfonamide groups.10 Among them, DG-041 (4)11 has
been advanced into clinical trials as a therapeutic agent for periph-
eral occupied arterial disease (PAOD). In this paper, we report the
identification of a series of novel N-acylsulfonamide analogs as
highly potent and selective EP3 receptor antagonists.
Effects of the N-acylsulfonamide moiety on the binding affinity and the antagonist
activity for the EP3 receptor
O
O
O
S
Ar
N
H
O
As shown in Table 1, several N-acylsulfonamide analogs, which
were designed based on 1, were synthesized and evaluated for
their in vitro activities. The binding affinity and the antagonist
activity were evaluated according to the same protocol as de-
scribed in our previous papers.7,12 All antagonist activities were
evaluated in the presence of 1% bovine serum albumin (BSA).
Replacement of the carboxylic acid of 1 with an N-acylmethane-
sulfonamide and an N-acylbenzenesulfonamide afforded 5 and 6,
respectively. Compound 5 showed equipotent binding affinity rel-
ative to 1 with reduced antagonist activity, while compound 6
showed increased potency with regard to both the binding affinity
and antagonist activity. The N-benzenesulfonyl moiety of 6 was
found to be a more optimized sub-structure for binding to the
EP3 receptor. However, compound 6 showed unexpectedly weak
antagonist activity compared with its potent binding affinity rela-
tive to the carboxylic acid analog 1 because of its presumed in-
crease in protein interaction.13 Replacement of the methylene
moiety adjacent to the carbonyl group of 6 with NH afforded 7
which exhibited 30-fold less potent binding affinity and 5.9-fold
less potent antagonist activity relative to 6. The reverse N-acylsulf-
onamide analog 8 showed 280-fold less potent binding affinity and
less than 70-fold weaker antagonist activity relative to 6.
Further effort to optimize the N-acylsulfonamide moiety was
continued as shown in Table 2. Replacement of the N-benzene-
sulfonyl moiety of 6 with the N-heteroarylsulfonyl moiety affor-
ded 9–12. N-Pyridinesulfonyl analogs 9 and 10 exhibited nearly
equipotent binding affinity and antagonist activity relative to 6.
N-(5-Methylfuran)-2-sulfonyl analog 11 and N-(4-methylthia-
zole)-2-sulfonyl analog 12 also showed retained binding affinity
and antagonist activity. Effect of the substituent on the benzene-
sulfonamide moiety of 6 was investigated. As summarized in
Table 2, N-(3,4-disubstituted benzene)sulfonyl analogs 13–16
had remarkable improvement in their antagonist activity relative
to 6. Introduction of one or two substituents into the benzene
N
N
Compd
Ar
Binding Ki (nM)
Function IC50 (nM)
140
*
6
0.50
N
*
*
9
1.6
190
N
10
11
1.0
150
88
O
N
*
*
0.58
12
13
0.35
0.43
110
10
S
Cl
Cl
*
*
14
15
0.45
0.22
7.8
5.5
Cl
Cl
*
*
*
F
F
16
17
0.086
0.065
1.2
18
F
CN
nucleus of the N-benzenesulfonyl moiety of 6 seemed to be effec-
tive for increasing the antagonist activity. Among them, N-(3,4-
difluorobenzene)sulfonyl analog 16 was found to exhibit the most
potent antagonist activity with an IC50 value of 1.2 nM. Compound
16 showed 110 and 480-fold more potent antagonist activity rel-
ative to non-substituted analog 6 and our lead compound 1,
respectively. N-(3-Cyanobenzene)sulfonyl analog 17 was also
more potent relative to 6. Although introduction of heteroaro-
matic rings is tolerated, the EP3 receptor prefers hydrophobic
parts such as the N-benzenesulfonyl moiety.
Table 1
Activity profiles of 1 and its N-acylsulfonamide analogs
X
O
N
N
Optimization of the pyrazol-1-ylmethyl side chain or the ether
side chain of 16 and the corresponding in vivo evaluations were
shown in Table 3. The in vivo activity was evaluated for their inhib-
itory effect on PGE2-induced uterine contraction in pregnant rats,
according to the protocol described in our previous paper.12,14 All
the test compounds 18–26 listed in Table 3 were found to exhibit
excellent subtype selectivity for the EP3 receptor similar to the re-
sults for 16. Compound 16 showed potent in vivo efficacy in a dose
dependent manner, with 43% inhibition at 0.3 mg/kg id and 76%
inhibition at 1 mg/kg id. Replacement of the 2-(2-naphthyl)ethyl-
oxy moiety of 16 with a N-(1-naphthyl)methylcarboxyamide moi-
ety afforded 18 with equipotent in vitro activity relative to 16,
while it showed relatively weak in vivo efficacy. Replacement of
the naphthalene ring of 16 with a benzene ring afforded 19 with
equipotent binding affinity and 49-fold less potent antagonist
activity. Compound 19 showed dose-dependent in vivo efficacy,
but its potency was approximately threefold less than 16. In an ef-
fort to improve the potency of 19, compounds 20–22 were synthe-
Compd
X
Binding Ki (nM)
Function IC50 (nM)
580
COOH
1
21
*
O
O
O
S
5
6
22
>10,000
140
N
*
*
H
O
O
O
O
S
S
0.50
N
H
O
O
7
8
15
820
N
H
N
H
*
*
O
O
O
S
140
>10,000
N
H

M. Asada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2639–2643
2641
Table 3
Activity profiles of a series of N-acyl 3,4-difluorobenzenesulfonamide analogs
O
O
O
F
F
S
N
H
X
Y
Compd
X
Y
Binding Ki (nM)
Function IC50 (nM)
In vivo (%inh.)a
0.3 mg/kg 1 mg/kg
EP1
EP2
EP3
EP4
N
N
*
*
16
18
19
20
2100
>10,000
>10,000
>10,000
>10,000
0.086
2400
1.2
1.4
59
43
76
30
51
33
*
*
O
N
N
H
N
5300
1300
560
0.13
0.20
0.54
1800
2500
3700
NTb
29
O
O
O
O
N
N
*
*
*
*
*
N
N
6.4
NTb
N
O
O
N
N
N
*
*
21
22
2800
260
>10,000
>10,000
0.27
0.12
>10,000
3500
51
NTb
NTb
28
50
N
N
*
O
3.8
N
*
23
24
>10,000
5000
>10,000
>10,000
8.2
1.2
>10,000
>10,000
24
47
NTb
NTb
32
35
*
*
*
O
O
O
N
*
O
25
26
>10,000
1700
>10,000
>10,000
0.51
0.16
2300
710
24
50
44
92
71
NC
N
O
*
0.40
*
O
O
*
a
All the values (%inh.) were determined as the mean values of twice of the experiments.
NT: not tested.
B
sized and evaluated. Replacement of the phenylethyloxy moiety of
19 with the (3-morpholinophenyl)ethyloxy, phenoxyethyloxy or
(N-methylanilino)ethyloxy moieties afforded 20–22, respectively.
Compound 20 showed slightly less potent binding affinity while
having 9.2-fold more potent antagonist activity relative to 19.
Compound 21 was nearly equipotency in both of the in vitro eval-
uations. Compound 22 displayed equipotent binding affinity with
an increase in antagonist activity. Regarding in vivo efficacy, 22
exhibited the same potency as 19 at a dose of 1 mg/kg id, while
20 and 21 showed less potency relative to 19.
of 16 with the more hydrophilic dimethylaminomethyl moiety
and N-morpholinomethyl moiety provided 23 and 24, respectively,
both of which resulted in reduced in vitro and in vivo potencies rel-
ative to 16. Introduction of more hydrophobic groups instead of the
hydrophilic groups afforded 25 and 26. Compound 25 possessing a
3-cyanophenoxymethyl moiety was found to show improved
in vivo efficacy relative to 16 compared with its reduced in vitro
activity. Compound 26 possessing a 3-pyridyloxymethyl moiety
was
a subnanomolar EP3 antagonist, with an IC50 value of
0.40 nM in the presence of 1% additive BSA and equipotent to 16
in term of in vivo efficacy. For comparison, evaluation of the
well-known standard compound 4 (DG-041) was carried out using
the same in vivo evaluation system as ours. The inhibitory effect of
4 and 25 for 4 h after their oral administration at 3 mg/kg exhibited
Further optimization was focused on the pyrazol-1-ylmethyl
side chain of 16. Replacement of the pyrazol-1-ylmethyl moiety
COOH
a
O
5-6 and 9-17
N
N
NH2
OH
a, b, c
1
O
O
COOH
N
N
N
N
d
O
a
28
29
18
N
HN
N
27
7
Scheme 1. Synthesis of 5–6 and 9–18. Reagents: (a) RSO2NH2, EDCÁHCl, DMAP,
Scheme 2. Synthesis of 7. Reagents: (a) PBr3, CH2Cl2; (b) NaN3, DMF; (c) H2, Pd/C,
MeOH; (d) PhSO2NCO, toluene.
DMF.

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M. Asada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2639–2643
O
O
anion prepared from N-(t-butyl)methanesulfonamide in the pres-
ence of n-BuLi, followed by dehydration with methanesulfonyl
chloride in the presence of triethylamine afforded unsaturated sul-
fonamide 31. Catalytic hydrogenation of 31 in the presence of plat-
inum oxide followed by acidic deprotection of t-butyl group
resulted in saturated sulfonamide 32, N-benzoylation of which
was carried out to afford 8 according to the same procedure as de-
scribed above.
CHO
O
S
N
a, b
H
O
N
N
N
N
30
31
O
O
S
NH2
c,d
e
Compounds 19–26 were synthesized as outlined in Scheme 4.
Bromination of 7-methylcoumarin (33) followed by the substitu-
tion reaction with pyrazole afforded 35. Methanolysis of 35 with
sodium methoxide followed by catalytic hydrogenation afforded
a common intermediate 36 for etherification. O-Alkylation of 36
with an optional alcohol under Mitsunobu reaction conditions
afforded 37–40, respectively, alkaline hydrolysis of which followed
by the dehydrative condensation with 3,4-difluorobenzenesulfona-
mide provided 19–22, respectively. 7-Bromomethylcoumarin (34)
described above was converted into 41 by a substitution reaction
with potassium acetate, alkaline hydrolysis of which provided 42.
O-Alkylation of 42 with methoxymethyl chloride afforded 43.
Methanolysis of 43 followed by sodium borohydride reduction in
the presence of nickel chloride resulted in 44. O-Alkylation of 44
with 2-(naphthalene-2-yl)ethanol under Mitsunobu reaction con-
ditions afforded 45, acidic deprotection of which produced 46. O-
Methanesulfonylation of 46 afforded a common intermediate 47
for the preparation of the title compounds. Substitution reaction
of the methanesulfonate 47 with dimethylamine, morpholine, 3-
cyanophenol or 3-hydroxypyridine resulted in 48–51, respectively.
Alkaline hydrolysis of 48–51 followed by the condensation reac-
8
O
N
N
32
Scheme 3. Synthesis of 8. Reagents: (a) MeSO2NH(t-Bu), n-BuLi, THF; (b) MsCl, TEA,
ClCH2CH2Cl; (c) H2, PtO2, EtOH; (d) TFA, anisole; (e) PhCOOH, EDCÁHCl, DMAP, DMF.
70 6.5% and 66 12%, respectively. Both of them showed equipo-
tent efficacy in the in vivo assessment.
Synthesis of N-acylsulfonamide analogs listed in Tables 1–3 is
shown in Schemes 1–4. As shown in Scheme 1, preparation of N-
acylsulfonamides 5–6 and 9–18 was achieved using a dehydrative
condensation reaction of the corresponding carboxylic acid 1 or
2712 with an optional sulfonamide using EDCÁHCl in the presence
of DMAP. As shown in Scheme 2, compound 7 was synthesized
from previously reported alcohol 28,7 which was converted into
amine 29 in three steps. Reaction of 29 with benzenesulfonyl iso-
cyanate in toluene afforded 7. Synthesis of reverse N-acylsulfona-
mide 8 is shown in Scheme 3. Reaction of aldehyde 307 with an
COOMe
COOMe
e
f, g
c, d
b
O
R
O
O
OH
19 - 22
X
a
N
N
N
O
O
N
N
N
33 X = H
35
36
34 X = Br
37 R =
*
38 R =
39 R =
N
*
*
O
O
N
h
40 R =
*
COOMe
f, g
COOMe
k
c, j
A
23 - 26
O
MOMO
Y
OH
O
O
44
41 Y = OAc
42 Y = OH
43 Y = OMOM
45 A = OMOM
46 A = OH
f
i
l
m
47 A = OMs
n
48 A =
N
o
*
p
O
N
49 A =
N
O
q
*
*
CN
50 A =
51 A =
O
*
Scheme 4. Synthesis of 19–26. Reagents: (a) NBS, AIBN, CCl4; (b) pyrazole, NaH, DMF; (c) NaOMe, THF, MeOH; (d) H2, Pd–C, MeOH; (e) R–OH, DEAD, Ph3P, THF; (f) NaOHaq,
THF, MeOH; (g) 3,4-difluorobenzenesulfonamide, EDCÁHCl, DMAP, DMF; (h) AcOK, DMF; (i) MOMCl, i-Pr2NEt, ClCH2CH2Cl; (j) NaBH4, NiCl2Á6H2O, THF, MeOH; (k) 2-
(naphthalene-2-yl)ethanol, DEAD, Ph3P, THF; (l) HCl-dioxane, MeOH; (m) MsCl, Et3N, THF; (n) dimethylamine, K2CO3, DMF; (o) morpholine, K2CO3, DMF; (p) 3-cyanophenol,
NaH, DMF; (q) 3-hydroxypyridine, NaH, DMF.

M. Asada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2639–2643
2643
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respectively.
In conclusion, starting with further optimization of the carbox-
ylic acid of the previously reported EP3 antagonist 1, we identified
a series of novel N-acylsulfonamide analogs as more potent and
selective EP3 antagonists. Based on the SAR described above, the
optimized N-acylsulfonamide moieties were considered to play a
role of not only a bioisostere of the carboxylic acid but also another
interaction site through the arylsulfonamide moiety for the EP3
receptor. Among them, the N-(3,4-difluorobenzene)sulfonyl moi-
ety was found to be the most optimized one for the EP3 receptor
antagonist activity. A series of N-acyl 3,4-difluorobenzenesulfona-
mide analogs exhibited potent in vivo efficacy, which was indi-
cated as the inhibitory effect on the PGE2-induced uterine
contraction in pregnant rats. Compounds 16, 25, and 26 exhibited
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