Comparison between two classes of selective EP3 antagonists and their biological activities

Article abstract of DOI:10.1016/j.bmcl.2006.08.025

Two different series of very potent and selective EP₃ antagonists have been reported: a novel series of ortho-substituted cinnamic acids [Belley, M., Gallant, M., Roy, B., Houde, K., Lachance, N., Labelle, M., Trimble, L., Chauret, N., Li, C.,

Full text of DOI:10.1016/j.bmcl.2006.08.025

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5639–5642
Comparison between two classes of selective EP3 antagonists
and their biological activities
*
´ `
Michel Belley, Chi Chung Chan, Yves Gareau, Michel Gallant, Helene Juteau,
Karine Houde, Nicolas Lachance, Marc Labelle, Nicole Sawyer, Nathalie Tremblay,
`
Sonia Lamontagne, Marie-Claude Carriere, Danielle Denis, Gillian M. Greig,
Deborah Slipetz, Robert Gordon, Nathalie Chauret, Chun Li,
Robert J. Zamboni and Kathleen M. Metters
Merck Frosst Centre for Therapeutic Research, PO Box 1005, Pointe Claire-Dorval, Que., Canada H9R 4P8
Received 6 June 2006; accepted 2 August 2006
Available online 22 August 2006
Abstract—Two different series of very potent and selective EP3 antagonists have been reported: a novel series of ortho-substituted
cinnamic acids [Belley, M., Gallant, M., Roy, B., Houde, K., Lachance, N., Labelle, M., Trimble, L., Chauret, N., Li, C., Sawyer,
`
N., Tremblay, N., Lamontagne, S., Carriere, M.-C., Denis, D., Greig, G. M., Slipetz, D., Metters, K. M., Gordon, R., Chan, C. C.,
Zamboni, R. J. Bioorg. Med. Chem. Lett. 2005, 15, 527] and the acylsulfonamides of ortho-(arylmethyl)cinnamates. [(a) Juteau, H.,
`
Gareau, Y., Labelle, M., Sturino, C. F., Sawyer, N., Tremblay, N., Lamontagne, S., Carriere, M.-C., Denis, D., Metters, K. M.
Bioorg. Med. Chem. 2001, 9, 1977; (b) Juteau, H., Gareau, Y., Labelle, M., Lamontagne, S., Tremblay, N., Carriere, M.-C., Denis,
`
D., Sawyer, N., Metters, K. M. Bioorg. Med. Chem. Lett. 2001, 11, 747] The structural differences between the two series, along with
their biological activity in vivo, in vitro, and metabolism, are analyzed. Some of those compounds, including hybrids containing the
best structural features of both series, possess Ki as low as 0.6 nM on the EP3 receptor.
Ó 2006 Elsevier Ltd. All rights reserved.
Previously, our group had disclosed two novel series of
potent and selective EP3 antagonists which might be
potentially useful as analgesics and for the treatment
of inflammatory condition such as in arthritis.1 They
were consisting of ortho-substituted cinnamic acids
1–31 (Table 1) and acylsulfonamides derived from
ortho-(arylmethyl)cinnamic acids such as compounds
7b–c2 (Table 2). These two classes of compounds were
discovered using two very distinct approaches: com-
pounds 1–3 were found by varying the substitution pat-
tern around the general structure of EP1 antagonists,
while 7a–c were discovered by SAR studies starting
from a lead compound found by screening our sample
collection.2b Comparing their structures, three differenc-
es can be noted: (1) the styrene group in 1 is replaced by
a naphthalene, (2) a carboxylic acid is converted to an
acylsulfonamide, and (3) the presence of an additional
benzyl ether substituent on compounds 1–3. Herein,
we wish to report the effect of each of these modifica-
tions on the biological activity of the compounds, along
with the biological profile of hybrids containing the best
structural features from each series.
In the ortho-(arylmethyl)cinnamate series, it was report-
ed that the acylsulfonamides 7b–c were 20 to 60 times
more potent than the parent cinnamic acid 7a (Table
2).2 To determine whether the same boost in activity
could be applied to the cinnamic acids 1–3, a small num-
ber of compounds having potencies ranging from 9 to
126 nM on the EP3 receptor were derivatized to their
corresponding thiophenesulfonamides.3 These com-
pounds were synthesized using reactions (g–h) in
Scheme 1 or via a coupling reaction with the cinnamic
acid in the presence of EDCI.2a,4
Keywords: EP3 antagonist; Acylsulfonamides; Cinnamic acids; Metab-
olism; Rat paw edema; Rat paw hyperalgesia; Anti-inflammation;
Analgesic.
*
Corresponding author. Tel.: +1 514 428 3075; fax: +1 514 428
4900; e-mail: belley@merck.com
Table 1 shows the potency of all these acylsulfonamides
on the different PGE2 receptors compared with their
parent cinnamic acids. The data for the best compound
Present address: Merck and Co. Inc, 126 Lincoln Avenue, PO Box
2000, Rahway, NJ 07065-0900, USA.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.025

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M. Belley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5639–5642
Table 1. Affinities (Ki, lM)a of a series of cinnamic acids and their acylsulfonamide analogs to the different prostanoid E2 receptor subtypes
Y
O
Y
O
Y
O
Ar2
R'
R'
R'
2
2
4
2
R
R
R
1, 4
3, 6
Ar1
2, 5
Compound
R
R0
Y
EP1
19
2.3
>100
8.5
>100
2.6
>20
1.5
6.7
23
6.7
EP2
EP3-III
EP4
1a, 2a
4a, 5a
1b, 2b
4b, 5b
1c, 2c
4c, 5c
1d, 2d
4d, 5d
1e
2-BnO
2-BnO
H
Me
Me
H
OH
NHSO2-2-thienyl
OH
0.86
2.1
0.009
0.010
0.10
2.0
0.96
4.4
1.4
8.0
H
H
NHSO2-2-thienyl
OH
0.005
0.068
0.0024
0.008
0.0016
0.003
0.065
0.056
1.5
6.2
4-MeO
4-MeO
4-BnO
4-BnO
H
2.5
6.5
H
NHSO2-2-thienyl
OH
1.0
MeO
MeO
Me
Me
Me
0.75
8.7
0.61
0.84
0.62
0.44
1.0
NHSO2-2-thienyl
OH
OH
2-(2,6-Cl2Bn)O
2-BnO
2-BnO
5.4
3
0.94
1.6
6
NHSO2-2-thienyl
a Ki determinations are averages based on at least two experiments.
Table 2. Affinities (Ki, lM)a of a series of (naphthylmethyl)cinnamic acids and their acylsulfonamide analogs to the different prostanoid E2 receptor
subtypes
O
Y
7
R
Compound
R
Y
EP1
EP2
12
8.6
EP3-III
EP3-III (0.05% HSA)
EP4
13
7a
7b
7c
7d
7e
7f
H
OH
>100
0.047
0.40
0.28
H
NHSO2-2-thienyl
NHSO2-(2-MeO-5-BrPh)
OH
9.6
0.0013
0.0006
0.0032
0.0006
0.0009
1.3
0.89
>15
H
39
>100
>50
>40
0.0069
0.046
0.0024
0.0007
BnO
BnO
BnO
NHSO2-2-thienyl
NHSO2-(2-MeO-5-BrPh)
3.2
>100
8.0
>100
1.1
8.0
a Ki determinations are averages based on at least two experiments.
COY
CO2Me
OH
Br
a-d
e, f
B
OH
+
Br
HO
BnO
BnO
g, h
7d
Y = OH
7e, f Y = NHSO2Ar
Scheme 1. Reagents and conditions: (a) NaH, DMF, PhCH2Br, 0 °C to rt, 1 h; (b) BuLi, THF, ꢀ78 °C; (c) B(Oi-Pr)3, rt, 16 h; (d) HCl, 81% yield
(four steps); (e) Pd(Ph3P)4, aq Na2CO3, toluene, 100 °C, 4 h, 73% yield; (f) NaOH, MeOH/THF/water, 90% yield; (g) (COCl)2, DMF, CH2Cl2; (h)
ArSO2NH2, Et3N, CH2Cl2/THF 1:1, 30–60% yield.
in the cinnamic acid series 1e are also given for compar-
ison. In general, very potent and selective EP3 antago-
nists were obtained when the acylsulfonamide
derivatives were prepared. The potency improvement
could be correlated with the position and/or absence
of a benzyl ether substituent on the aromatic ring Ar2.
In the absence of that substituent (4b/5b and 4c/5c vs
1b/2b and 1c/2c) the increase of activity is equivalent
to what is found in the ortho-(arylmethyl)cinnamate
series (compounds 7a–b, Table 2). In the presence of a
benzyl ether group in position 2 (1a/2a and 3 vs 4/5a
and 6), equipotent acylsulfonamides were obtained.

M. Belley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5639–5642
5641
The boost of potency observed with the thiophenesulf-
onamide substitution is not additive with the effect of
introducing a benzyl ether in position 2 of Ar2.
was 65% metabolized.1 The major metabolites of 7b and
7c were isolated and partially identified by HPLC/MS
and NMR. The major metabolic pathways are: (1)
hydroxylation of the methylene linker, (2) oxidation of
the phenyl group to a phenol, (3) oxidation of the naph-
thalene moiety to an ortho-dihydronaphthalenediol, and
(4) hydrolysis of the sulfonamide to the cinnamic acid
7a. Demethylation of the methoxyphenyl portion of
the molecule was also observed for 7c. In human hepa-
tocytes, however, the metabolism of 7b and 7c is reduced
and almost exclusively limited to the hydrolysis of the
acylsulfonamide to the cinnamic acid 7a (45% for 7b
and 36% for 7c, with the remainder being the parent
drug).
On the other hand, incorporating a benzyl ether in posi-
tion 4 gives acylsulfonamide analogs which are 5 times
more active than their corresponding parent acids (4d/
5d vs 1d/2d). This result led us to believe that the addi-
tion of a benzyl ether group in position 6 of the (naph-
thylmethyl)cinnamic acid and acylsulfonamide series
might improve their potency as well. Compounds 7d–f
were thus prepared as described in Scheme 1, with the
key step involving a palladium catalyzed cross-coupling
reaction between methyl (bromomethyl)cinnamate and
a naphthaleneboronic acid.2a
But even with their enhanced metabolic stability
in vitro, the plasma levels and anti-inflammatory
activity of compounds 7 are similar to compounds
1a/2a and 1e (Table 3) in adult rats. They possess sim-
ilar bioavailabilities, clearances, and Cmax, with 7
showing a slightly longer half-life. However, in the
young rats used in the hyperalgesia model, the plasma
levels of compounds 7 are higher. When dosed po in
1% methocel, 7b and 7c, but not 7d, inhibited carra-
geenan-induced rat paw edema7 in a dose-dependent
manner, but their effect on carrageenan-induced
hyperalgesia7 is very variable, with ED50 ranging from
3.3 to >30 mg/kg. Their analgesic activity seems to be
not related to their potency in vitro, nor to their
plasma levels at the time of the assessment of hyperal-
gesia (1 h post-dosing; 6th row of Table 3).
Table 2 shows the effect of adding that benzyl ether
group on the activity of the (naphthylmethyl)cinnamates
as selective EP3 antagonists. The most striking result is
the 15-fold improvement in activity observed with the
cinnamic acid 7d (vs 7a), which brings its potency to a
level that is closer to the acylsulfonamide derivatives.
However, addition of the benzyl group on the acylsulf-
onamides 7b and 7c gives compounds that are equipo-
tent (7e and 7f).
Finally, a comparison of the Ki of the compounds 1b/2b,
4b/5b, 1d/2d, and 4d/5d with their respective analogs 7a,
7b, 7d, and 7e shows that the modification of the styryl
group of the former compounds to a naphthalene unit is
giving approximatively a 3-fold improvement of the
potency on the EP3 receptor, along with an appreciable
boost of selectivity.
We have published data in the past that supported the
hypothesis stating that serum protein binding could de-
crease the effectiveness of a compound in vivo.8 Addi-
tion of human serum albumin (HSA) in the enzymatic
assay can help assess this effect. Most of the compounds
of Table 2 showed a variable decrease of potency in the
presence of HSA. However, this loss of potency is greatly
decreased for analogs containing the benzyl ether substi-
tuent (7d–f). Yet, even if the activity of 7f is not modified
by HSA, it is still inactive in hyperalgesia. We have ana-
lyzed other parameters such as their binding affinities to
the other prostanoid receptors9 and their CSF and brain
levels in vivo, but we could not find any good
correlations.
In our functional assay measuring the inhibition of
c-AMP production in HEK (EBNA) cells5 (Table 3),
compounds 1a/2a, 1e, and 7 all behave as full EP3
antagonists.
Metabolism studies of the EP3 selective antagonists 7
demonstrate that they are more stable in vitro than com-
pounds 1–2.1 After incubations with rat hepatocytes6 for
3 h, 43% of 7b and 30% of 7c were recovered intact,
compared with 8% for the mixture of compounds 1a
and 2a. With rat liver microsomes,6 7b was 35% metab-
olized after a typical 1-h incubation, while compound 1a
Table 3. Inhibition of c-AMP production and biological profile of selected EP3 antagonists in ratsa
1a/2a
1e
5.8
7a
7b
3.3
7c
0.6
72
1.0
7d
7f
c-AMP assay, Kb (nM)
Bioavailabilityb (%)
Clearanceb (ml/min)
32
97
0.7
74
16
144
12
59
3.1
10
2 h
b
t1/2 (2–6 h)
b
2 h
25 (30 min)
3 h
21 (30 min)
4 h
3 h
28 (30 min)
Cmax (lM)
Rat paw edema (ED50, mg/kg)
22 (30 min)
50 (1 h)
3.3
32
0.9
1.4d
20
5.1
2.5
5.8
1.2
5.0
6.2
>10
11e
4.6
Concentration at 1 h post-dosingc (lM)
Rat paw hyperalgesia (ED50, mg/kg)
5.4
30
8
>30
3.3
a Compounds dosed as their sodium salts.
b Compounds dosed at 10 mg/kg po in 0.5% methocel (except for 7d in 25% b-cyclodextrin) and 5 mg/kg iv in 5% dextrose or 25% b-cyclodextrin in
male Sprague–Dawley rats weighing 300–400 g.
c Compounds dosed at 10 mg/kg po in 0.5% methocel in the 100 g rats used for the hyperalgesia assay.
d Sum of 1.2 lM for 1a and 0.24 lM for 2a; ratio of 1a/2a given po was 1.3:1.
e Dosed at 5 mg/kg po.

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M. Belley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5639–5642
C.; Denis, D.; Metters, K. M. Bioorg. Med. Chem. 2001, 9,
1977; (b) Juteau, H.; Gareau, Y.; Labelle, M.; Lamonta-
Several splice variants of the EP3 receptor, defined by
different C-terminal cytoplasmic tails, have been discov-
ered.10 Their localization in the body and their physio-
logic significance remain uncertain. The antagonists
described here may bind in a different manner to these
splice variants and it is also possible that only binding
to a specific splice variant may be effective for the reduc-
tion of fever and/or hyperalgesia.
`
gne, S.; Tremblay, N.; Carriere, M.-C.; Denis, D.; Sawyer,
N.; Metters, K. M. Bioorg. Med. Chem. Lett. 2001, 11,
747.
3. Most of the compounds reported in Table 1 are mixtures
of two isomers, but we have shown previously that the
difference of activity between these isomers is 2-fold on the
EP3 receptor,1 and the variation in the ratio of isomers
should not significantly change the results described
herein.
4. Pelletier, J. C.; Hesson, D. P. Synlett 1995, 1141.
5. Boie, Y.; Stocco, R.; Sawyer, N.; Slipetz, D. M.;
Ungrin, M. D.; Neuscha¨fer-Rube, F.; Puschel, G. P.;
¨
Metters, K. M.; Abramovitz, M. Eur. J. Pharmacol.
1997, 340, 227.
6. Li, C.; Chauret, N.; Trimble, L. A.; Nicoll-Griffith, D. A.;
Silva, J. M.; Macdonald, D.; Perrier, H.; Yergey, J. A.;
Parton, T.; Alexander, R. P.; Warrellow, G. J. Drug
Metab. Dispos. 2001, 29, 232.
In summary, we have compared here the activity and
biological profile in vivo of two different series of very
potent and selective EP3 antagonists: the ortho-substi-
tuted cinnamic acids 1–3 and the acylsulfonamides of
ortho-(arylmethyl)cinnamates 7. Compounds 1a/2a, 1e,
and 7b–c all showed good anti-inflammatory properties,
as measured by the rat paw edema assay. Curiously, the
analgesic activity of 1a/2a, 1e, and 7a–f varies a lot from
one compound to the next and seems not to be propor-
tional to their potency on the EP3 receptor, nor to their
plasma concentration. Further studies will be needed to
really understand why very potent and selective EP3
antagonists are not always efficacious in the rat paw
hyperalgesia model.
7. Chan, C.-C.; Boyce, S.; Brideau, C.; Ford-Hutchinson, A.
W.; Gordon, R.; Guay, D.; Hill, R. G.; Li, C.-S.; Mancini,
J.; Penneton, M.; Prasit, P.; Rasori, R.; Riendeau, D.;
Roy, P.; Tagari, P.; Vickers, P.; Wong, E.; Rodger, I. W.
J. Pharm. Exp. Ther. 1995, 274, 1531.
8. (a) Labelle, M.; Gareau, Y.; Dufresne, C.; Lau, C. K.;
Belley, M.; Jones, T. R.; Leblanc, Y.; McAuliffe, M.;
McFarlane, C. S.; Metters, K. M.; Ouimet, N.;
Perrier, H.; Rochette, C.; Sawyer, N.; Slipetz, D.;
Xiang, Y. B.; Wang, Z.; Pickett, C. B.; Ford-Hutch-
inson, A. W.; Young, R. N.; Zamboni, R. J. Bioorg.
Med. Chem. Lett. 1995, 5, 2551; (b) Jones, T. R.;
Labelle, M.; Belley, M.; Champion, E.; Charette, L.;
Evans, J.; Ford-Hutchinson, A. W.; Gauthier, J.-Y.;
Lord, A.; Masson, P.; McAuliffe, M.; McFarlane, C.
S.; Metters, K. M.; Pickett, C.; Piechuta, H.; Rochette,
C.; Rodger, I. W.; Sawyer, N.; Young, R. N.;
Zamboni, R. J.; Abraham, W. M. Can. J. Physiol.
Pharmacol. 1995, 73, 191.
Acknowledgments
The authors thank Renee Aspiotis for proofreading this
manuscript and Jose Silva for the preparation of rat and
human hepatocytes.
References and notes
1. Belley, M.; Gallant, M.; Roy, B.; Houde, K.; Lachance,
N.; Labelle, M.; Trimble, L.; Chauret, N.; Li, C.; Sawyer,
9. The Ki for the IP, TP, FP, and DP receptors for the
compounds included in Table 3 are all above 0.5 lM, with
the exception of 7d which shows a Ki of 0.082 lM on the
DP receptor.
`
N.; Tremblay, N.; Lamontagne, S.; Carriere, M.-C.;
Denis, D.; Greig, G. M.; Slipetz, D.; Metters, K. M.;
Gordon, R.; Chan, C. C.; Zamboni, R. J. Bioorg. Med.
Chem. Lett. 2005, 15, 527.
10. Breyer, R. M.; Bagdassarian, C. K.; Myers, S. A.; Breyer,
M. D. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 661.
2. (a) Juteau, H.; Gareau, Y.; Labelle, M.; Sturino, C. F.;
`
Sawyer, N.; Tremblay, N.; Lamontagne, S.; Carriere, M.-