Synthesis and pharmacological evaluation of 2-aryloxy/arylamino-5- cyanobenzenesulfonylureas as novel thromboxane A2 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2013.04.033

New series of original 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas were synthesized and evaluated as thromboxane A₂ receptor (TP receptor) antagonists. A functional pharmacological test was used, which consisted of measuring the inhibition

Full text of DOI:10.1016/j.ejmech.2013.04.033

European Journal of Medicinal Chemistry 65 (2013) 32e40
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of 2-aryloxy/arylamino-
5-cyanobenzenesulfonylureas as novel thromboxane A2 receptor
antagonists
,
Sylvie-Mireille Bambi-Nyanguile a, Julien Hanson a d, Annie Ooms b, Lutfiye Alpan c,
,
Philippe Kolh b, Jean-Michel Dogné c, Bernard Pirotte a
*
a Laboratory of Medicinal Chemistry, Drug Research Center, University of Liege, 1, Avenue de l’Hôpital, Sart-Tilman, B-4000 Liege, Belgium
b Department of Cardiovascular Research, University of Liege, Belgium
c Department of Pharmacy, Namur Thrombosis and Hemostasis Center e Narilis, University of Namur, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium
d Molecular Pharmacology, GIGA-Signal Transduction, University of Liege, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of original 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas were synthesized and evalu-
ated as thromboxane A2 receptor (TP receptor) antagonists. A functional pharmacological test was used,
which consisted of measuring the inhibition of intracellular calcium mobilization in a model of
Received 12 December 2012
Received in revised form
10 April 2013
Accepted 13 April 2013
Available online 23 April 2013
mammalian cell line that specifically over-expressed the individual TP
cyanobenzenesulfonylureas exhibited virtually identical affinity and/or functional activity than 2-
aryloxy-5-cyanobenzenesulfonylureas for both TP and TP , but some 2-aryloxy-substituted com-
pounds showed increased selectivity for TP relative to TP . Several compounds were found to be as
a or TPb isoforms. 2-Arylamino-5-
a
b
b
a
Keywords:
potent as the 2-arylamino-5-nitrobenzenesulfonylurea reference compound BM-573, supporting the
view that the bioisosteric replacement of the nitro group by a cyano group was tolerated.
TP receptor antagonist activity of the most promising molecules was confirmed in a platelet aggre-
gation assay using the TP receptor agonist U-46619 as a proaggregant.
Three compounds (7e, 7h and 8h) were identified as leads for further non-clinical pharmacological
and toxicological studies.
Thromboxane receptor antagonists
Antiplatelet agents
2-Aryloxy/arylamino-5-
cyanobenzenesulfonylureas
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
stimulatory role of TXA2 in the proliferation of vascular smooth
muscle cells and mitogenesis [17,18]. TXA2 exerts its action through
Thromboxane A2 (TXA2) is a lipid mediator belonging to a group
of bioactive compounds called prostanoids and results from
arachidonic acid metabolism by the cyclooxygenase pathway [1e3].
This metabolite is formed by the action of thromboxane synthase
on prostaglandin endoperoxide H2 (PGH2) and causes vasocon-
striction, bronchoconstriction, and platelet aggregation [4e8]. This
short-live lipid mediator is mainly produced by platelets, macro-
phages, and lung parenchyma [9]. It has been reported that high
plasma levels of this metabolite is implicated in the pathogenesis of
thrombotic diseases, including myocardial infarction, unstable
angina, pulmonary embolism, atherosclerosis, thrombosis [10e14];
preeclampsia [15], septic shock, asthma and pulmonary hyperten-
sion [16]. Furthermore, several studies pointed out a significant
the specific G-protein-coupled prostanoid receptors called TP re-
ceptors [2,19]. These human TP receptors exist in two isoforms, TP
and TP [20,21], which are identical for their first 328 amino
acids and differ exclusively in their carboxyl-terminal cytoplasmic
tail (C-tail) regions, due to alternative splicing in exon 3 of the TP
gene [21,22].
TheyexhibitidenticalligandbindingandcouplesimilarlytoGq/G11
[23,24], G16 and G12 [25], but oppositely regulate adenylyl cyclise [26].
TPb isan alternative mRNA splicingvariantwith an extended carboxyl
a
b
terminus. The differences in intracellular termini may influence
desensitization, internalization, and G protein coupling [2,27,28].
However, only the TP
noteworthy that the TP
liferation and migration and invasion by bladder cancer cells.
Moreover, TP receptor expression was increased in tissue obtained
a
isoform is translated in platelets [29]. It is
b
isoform, but not TP , promoted cell pro-
a
b
Abbreviations: TXA2, thromboxane A2; TXS, thromboxane synthase; GPCR,
G-protein coupled receptor; TP receptor, thromboxane A2 receptor; PLC,
phospholipase C; IP3, inositol 1,4,5-triphosphate.
from bladder cancer patients [30].
The design and synthesis of selective isoform-specific TP recep-
tor antagonists is of great interest notonly in therapeutics but also as
pharmacological tools to decipher TP isoform specific roles [7,31].
* Corresponding author. Tel.: þ32 43664365; fax: þ32 43664362.
E-mail address: b.pirotte@ulg.ac.be (B. Pirotte).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.033

S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
33
In this study, we describe the synthesis of a series of novel 5-
cyanobenzenesulfonylureas structurally related to previously
described 5-nitrobenzenesulfonylureas [32] and outline their
pharmacological evaluation as TP receptor antagonists. We also
describe the antiplatelet activity of the most interesting compounds.
The aim of this study is to investigate if the replacement of the
nitro group by a cyano group has an impact on the in vitro phar-
macological activity observed with nitrobenzenesulfonylureas as
test, which consisted of measuring the inhibition of intracellular
calcium ([Ca2þ]i) mobilization in a model of mammalian cell line
that specifically over-expressed the individual TPa or TPb isoforms
(HEK 293), in presence of the TXA2-mimetic compound U-46619
(7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo-
[2.2.1]hept-5-yl]-5-heptenoic acid) [7]. Calcium is the adequate
second messenger to evaluate since TP
a or TPb each couples to Gq/
phospholipase (PL) activation, leading to inositol 1,4,5-
C
TP
a
and TP
b
antagonists. Due to potential toxicity with nitro-
trisphosphate (IP3) -mediated intracellular calcium mobilization
from intracellular stores [23,24]. The intracellular calcium mobili-
zation was measured with cells preloaded with fluorescent dye
Fluo-4, whose fluorescence increases upon binding with calcium
benzenic derivatives, a further investigation will be the assessment,
by means of appropriate in vitro and in vivo tests, of the acute and/
or chronic toxicity of the cyanobenzenic compounds compared to
the nitrobenzenic compounds.
ion released in response to the TXA2 mimetic U-46619 (1
mM).
Briefly, cells preloaded in Fluo-4/AM were stimulated with 1
mM
ligand (U-46619) for dose response studies, using concentrations of
the target compounds ranging from 10ꢀ9 M to 10ꢀ5 M. Represen-
tative data from at least three independent experiments are plotted
2. Chemistry
The cyano-substituted compounds evaluated in this work were
synthesized according to a synthetic process inspired from the one
previously described for nitro-substituted analogues [32]. The
starting compound was the commercially available 4-fluoro-3-
nitrobenzonitrile (1), which was reduced with stannous chloride
in the presence of sodium hydrogenocarbonate in ethyl acetate to
give 3-amino-4-fluorobenzonitrile (2). This latter reacted with so-
dium nitrite in acidic medium, and the resulting diazonium salt
solution was mixed with a solution of sulphur dioxide in acetic acid
in the presence of cuprous ions to generate 2-fluoro-5-
cyanobenzenesulfonyl chloride (3) according to the Meerwein
variation of the Sandmeyer reaction [32,33]. The decomposition of
as changes in intracellular Ca2þ concentration mobilized ( [Ca2þ
D ]
i
(mM)) as a function of time (s) upon ligand stimulation. Concen-
trationeresponse curves were determined, and the IC50, defined as
the concentration of compound required to inhibit 50% of U-46619
induced [Ca2þ]i mobilization, were calculated. A selectivity index
ratio, defined as the IC50 TPa/IC50 TPb, was also calculated.
Results were compared to those observed with the reference
compound, BM-573 (Fig. 1), a well-known nitrobenzenesulfonylurea
previously reported to be a potent thromboxane A2 receptor antago-
nist and a thromboxane synthase inhibitor, and to prevent human
platelet aggregation induced byarachidonicacid or the TXA2-mimetic
compound U-46619 [36].
compound
3 into a solution of ammonia gave 2-fluoro-5-
Secondly, the ability of the most interesting tested compounds
to prevent human platelet aggregation in response to U-46619 was
also evaluated. In this test, briefly, anticoagulated blood was
immediately centrifuged at 100ꢁg for 10 min at room temperature
and platelet rich plasma collected. The remaining fraction was
centrifuged at 2000ꢁg to obtain platelet poor plasma. Platelet ag-
gregation was determined by light absorbance using a platelet
cyanobenzenesulfonamide (4), which was the common interme-
diate giving access to the target compounds (Scheme 1).
The nucleophilic substitution of the fluorine atom of 4 by
various phenols and amines gave access to intermediates 5 and 6.
The last step consisted of sulfonamide group deprotonation of in-
termediates 5 or 6 followed by their reaction with the appropriate
isocyanate in DMF, providing the target compounds 7 and 8
(Schemes 2 and 3).
aggregometer with constant magnetic stirring. U-46619 (1 mM), the
stable analogue of TXA2, was used as an agent to induce an irre-
versible aggregation.
The structures of the compounds prepared were confirmed by
elemental and spectral analyses.
3. Pharmacological evaluation
4. Results and discussion
The aim of this study is to investigate if the replacement of the
nitro group by a cyano group has an impact on the in vitro phar-
macological activity observed with nitrobenzenesulfonylureas as
In this study, a series of 33 original 2-aryloxy/arylamino-5-
cyanobenzenesulfonylureas (7aeu and 8ael) was synthesized ac-
cording to Schemes 1e3.
TP
a
and TP
b
antagonists. Antagonistic activity and platelet aggre-
First, we have characterized the antagonistic potency of these
gation were studied as previously described [7,24,34,35].
First, we have characterized the potency of the new synthetic
cyanobenzenesulfonylureas by using a functional pharmacological
new synthetic cyanobenzenesulfonylureas on the TP
forms of the human TXA2 receptors (see Table 1). All compounds
evaluated (7aeu) and (8aej) exhibited high activity as TP and TP
a and TPb iso-
a
b
F
F
F
F
O
O
O
O
S
NO2
NH2
S
NH2
Cl
CN
CN
CN
CN
Scheme 1. Synthesis of 4-fluoro-3-sulfamoylbenzonitrile (4).

34
S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
X
X
O
F
O
O
O
O
O
O
O
O
S
S
S
R
NH2
NH2
N
N
H
H
CN
CN
CN
Scheme 2. General procedure for the preparation of N-alkyl-N’-[2-(aryloxy)-5-cyanobenzenesulfonyl]ureas (7aeu).
receptor antagonists. The bridging atom (O or NH in series 7aeu or
8aej, respectively) between the cyanobenzene nucleus and the
second ring did not affect the affinity of the molecules for the TP
receptors.
activity probably resulted from the steric hindrance due to the
substituent in the ortho position of the aromatic ring forcing the
phenyl ring to adopt another spatial orientation. Further studies
will be needed to confirm this hypothesis.
In order to establish structureeactivity relationships (SAR) and
to evaluate selectivity for TP receptor isoforms within this family of
compounds, we first varied the position of the methyl group on the
aromatic ring (2-, 3- or 4-position), and we addressed the influence
of various alkyl (isopropyl, tert-butyl and n-pentyl) side chains R for
each position in the two series of molecules (arylamino- and
aryloxy-substituted compounds). Compounds characterized by a 3-
methylphenyl or a 4-methylphenyl group and a tert-butyl or a n-
pentyl hydrocarbon chain in R (7e, 7f, 7h, 7i, 8h and 8i) were
characterized by potent antagonistic activity in the two series as
shown in Table 1. One possible explanation for this is that the
spatial arrangement of meta or para methyl position on the aro-
matic ring allows a better positioning in the active site of the re-
ceptor (at the level of a hydrophobic pocket). Most compounds
characterized by an isopropyl moiety in R exerted a weak activity
compared with the other substituents.
Collectively, results from Table 1 highlighted the following general
trend in the choice of the R substituent for optimal activity on both
isoforms TPa and TPb: tert-butyl > n-pentyl > isopropyl. For com-
pounds with a tert-butyl or an n-pentyl chain, the best positions for a
methyl group on the phenyl ring were positions 3 and 4.
The presence of a cyano group allows the molecule to exhibit a
similar profile compared to the previously described nitro-
benzenesulfonylureas [7,32] because this group confers a similar
electron withdrawing impact on the molecule for a better interac-
tion with the receptors. Regarding the difference of affinity for one
of the two isoforms, there was no clear difference with the com-
pounds from the arylamino series. However, with compounds from
the aryloxy series, we found a better affinity for TP
b with several
compounds. Nevertheless this difference was not significant
(p < 0.05), the higher isoform selectivity ratio (IC50 TPa/IC50 TPb)
being 2.5.
The presence of a 2-methylphenyl group appeared to be less
favourable for the antagonistic potency compared to the presence
of the 3- and 4-methyl-substituted phenyl group. This decrease of
The results obtained from this study with cyanobenzenesulfo-
nylureas do not fully correlate with those obtained with aryloxy-
substituted nitrobenzenesulfonylureas for which a difference of

S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
35
X
X
NH
CN
F
NH
CN
O
O
O
O
O
O
O
S
S
S
R
NH2
NH2
N
N
H
H
CN
Scheme 3. General procedure for the preparation of N-alkyl-N’-[2-(arylamino)-5-cyanobenzenesulfonyl]ureas (8ael).
Table 1
Estimated IC50 values for the inhibition of [Ca2þ]i mobilization mediated by either
activity between the two isoforms was observed. Some compounds
were characterized by an isoform selectivity ratio of 16 or 17 sug-
gesting an increased selectivity for TPb relative to TPa [7].
We next investigated the effect of introducing a halogen atom
on the aromatic ring (X ¼ F, Cl, Br, I) by preparing compounds 7jeu
and 8jel. These compounds exhibited a weaker antagonistic po-
TPa or TPb upon stimulation by U-46619 (1 mM). First series: Influence of the po-
sition of the methyl substituent in R1 and of the R2 alkyl side chain (18 compounds).
X
Y
Z
O
O
O
tency, with IC50 values comprised between 4 and 10
mM in our
S
R
assays (Table 2). From the data summarized in Table 2, it can be
concluded that the best antagonistic potency on TP receptors by
these series of compounds was observed with compounds bearing
a tert-butyl moiety at R. The most interesting compounds of
this series were 7k, 7n, 7q, 7t and 8k with IC50 values between 4
N
N
H
H
Compound
X
R
Y
Z
Inhibition of U-46619-mediated
and 6 mM.
[Ca2þ]i mobilization (IC50
,
m
M)a
Regarding the selectivity for one of the two isoforms of TP re-
ceptors, no difference was observed in affinity for one of them
(p < 0.05). The selectivity ratio varied between 0.9 and 1.2. We then
confirmed the activity of selected compounds on human platelets
by assessment of their ability to inhibit platelet aggregation in
response to the TXA2 mimetic U-46619. The results obtained at
TP TP
a
b
Ratiob
BM-573
7a
7b
7c
7d
7e
7f
7g
7h
7i
8a
8b
8c
8d
8e
8f
8g
8h
8i
4-Methyl tert-Butyl NH NO2 0.70 ꢂ 0.08 0.62 ꢂ 0.05 1.1
2-Methyl Isopropyl
2-Methyl tert-Butyl
2-Methyl Pentyl
O
O
O
O
O
O
O
O
O
CN 8.68 ꢂ 0.20 6.13 ꢂ 0.34 1.4
CN 3.69 ꢂ 0.18 1.47 ꢂ 0.10 2.5
CN 2.24 ꢂ 0.12 1.78 ꢂ 0.11 1.3
CN 2.38 ꢂ 0.10 1.69 ꢂ 0.15 1.4
CN 1.05 ꢂ 0.09 0.69 ꢂ 0.04 1.5
CN 1.33 ꢂ 0.11 0.68 ꢂ 0.07 2.0
CN 3.05 ꢂ 0.10 1.35 ꢂ 0.09 2.3
CN 0.75 ꢂ 0.07 0.68 ꢂ 0.03 1.1
CN 0.90 ꢂ 0.08 0.63 ꢂ 0.05 1.4
3-Methyl Isopropyl
3-Methyl tert-Butyl
3-Methyl Pentyl
4-Methyl Isopropyl
4-Methyl tert-Butyl
4-Methyl Pentyl
1
mM with compounds 7aei and 8aei are summarized in Fig. 2. All
H3C
2-Methyl Isopropyl NH CN 7.24 ꢂ 0.19 10.12 ꢂ 0.24 0.7
NH
O
2-Methyl tert-Butyl NH CN 2.75 ꢂ 0.21 4.78 ꢂ 0.75 0.6
O
S
O
2-Methyl Pentyl
NH CN 3.19 ꢂ 0.14 7.85 ꢂ 0.95 0.4
3-Methyl Isopropyl NH CN 9.50 ꢂ 0.71 9.11 ꢂ 0.14 1.0
N
N
H
3-Methyl tert-Butyl NH CN 1.59 ꢂ 0.08 2.20 ꢂ 0.09 0.7
H
3-Methyl Pentyl
NH CN 2.95 ꢂ 0.16 2.33 ꢂ 0.13 1.3
4-Methyl Isopropyl NH CN 4.11 ꢂ 0.19 4.22 ꢂ 0.23 0.9
4-Methyl tert-Butyl NH CN 0.85 ꢂ 0.07 1.04 ꢂ 0.09 0.8
NO2
4-Methyl Pentyl
NH CN 1.04 ꢂ 0.10 2.06 ꢂ 0.15 0.5
a
Fig. 1. Structure of the previously described nitrobenzenic sulfonylurea as throm-
boxane A2 receptor antagonist (BM-573).
Results are expressed as mean ꢂ standard deviation of three separate experiments.
b
IC50TPa/IC50TPb.

36
S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
Table 2
compounds tested inhibited platelet aggregation at 10 mM (results
Estimated IC50 values for the inhibition of [Ca2þ]i mobilization mediated by either
not shown), which was in line with their antagonistic potency
showed on TP receptors. The concentrationeresponses curves were
obtained for the most interesting compounds giving a significant
TP or TP upon stimulation by U-46619 (1 M). Second series: Influence of the
a
b
m
position of the halogen atom on the phenyl ring at R1 and of the hydrocarbon side
chain R2 (15 compounds).
activity at 1 mM (7b, 7e, 7h, 8b, 8e and 8h) and the IC50 values for
X
inhibition of U-46619 induced platelet aggregation were reported
in Table 3. For the 4-methylphenyl/tert-butyl compounds 7h and
8h, the IC50 value was found to be approximately the same as that
of BM-573, the reference compound.
It should be highlighted that all the compounds evaluated in the
pharmacological assessment models exhibited promising results in
terms of measurement of inhibition of [Ca2þ]i mobilization, and
inhibition of U-46619 induced platelet aggregation. Compounds 7e,
7h and 8h were the most potent antagonists for U-46619 induced
platelet aggregation since their IC50 value was respectively 0.705,
Y
Z
O
O
O
S
R
N
N
H
H
Compound
X
R
Y
Z
Inhibition of U-46619-mediated,
[Ca2þ]i mobilization (IC50 M)a
,
m
0.649 and 0.504
mM. Finally, it is noteworthy that these three
TP TP
Ratiob
a
b
compounds were also found to be among the most potent TP re-
ceptor antagonists.
BM-573
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
8j
8k
8l
4-Methyl tert-Butyl NH NO2 0.70 ꢂ 0.08 0.62 ꢂ 0.05 1.1
4-Fluoro Isopropyl
4-Fluoro tert-Butyl
4-Fluoro Pentyl
O
O
O
O
O
O
O
O
O
O
O
O
CN 9.62 ꢂ 0.28 9.30 ꢂ 0.80 1.0
CN 4.35 ꢂ 0.20 3.89 ꢂ 0.12 1.2
CN 9.43 ꢂ 0.50 9.53 ꢂ 0.12 0.9
CN 9.46 ꢂ 0.17 8.49 ꢂ 0.50 1.1
CN 5.64 ꢂ 0.13 5.27 ꢂ 0.70 1.1
CN 8.64 ꢂ 0.17 8.72 ꢂ 0.30 0.9
CN 9.47 ꢂ 0.90 9.13 ꢂ 0.35 1.0
CN 5.88 ꢂ 0.10 5.31 ꢂ 0.14 1.1
CN 8.52 ꢂ 0.17 9.54 ꢂ 0.70 0.9
CN 9.52 ꢂ 0.12 9.49 ꢂ 0.33 1.0
CN 5.52 ꢂ 0.82 5.54 ꢂ 0.05 0.9
CN 9.52 ꢂ 0.01 9.49 ꢂ 0.05 1.0
In conclusion, we have demonstrated that 2-aryloxy/arylamino-5-
cyanobenzenesulfonylureas are TXA2 receptor antagonists and that
their activity is closely related to the presence of both the 4-
methylphenyl group and a tert-butyl or a n-pentyl hydrocarbon
chain on the terminal nitrogen atom of the urea function. We didn’t
observe a difference of activity on the two isoforms in the arylamino
series. The aryloxy compounds were characterized by an isoform
selectivity ratio comprised between 0.9 and 2.5. Thus, no marked
difference in selectivity between the two isoforms was observed.
Selected 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas pre-
vented human platelet aggregation induced by the TXA2-mimetic
compound U-46619. Three lead compounds (7e, 7h and 8h) were
selected for further in vitro and in vivo pharmacological and toxico-
logical studies.
4-Chloro Isopropyl
4-Chloro tert-Butyl
4-Chloro Pentyl
4-Bromo Isopropyl
4-Bromo tert-Butyl
4-Bromo Pentyl
4-Iodo
4-Iodo
4-Iodo
Isopropyl
tert-Butyl
Pentyl
4-Fluoro Isopropyl NH CN 8.17 ꢂ 0.38 7.83 ꢂ 0.44 1.04
4-Fluoro tert-Butyl NH CN 4.64 ꢂ 0.07 4.91 ꢂ 0.47 0.95
4-Fluoro Pentyl
NH CN 6.78 ꢂ 0.21 6.45 ꢂ 0.81 1,05
a
Results are expressed as mean ꢂ standard deviation of three separate experiments.
b
IC50TPa/IC50TPb.
5. Experimental section
5.1. Chemistry
All commercial chemicals (SigmaeAldrich, Belgium) and solvents
are reagent grade and were used without further purification unless
otherwise stated. Melting points were determined with a Büchie
Tottoli capillary apparatus and are uncorrected. The 1H and 13C NMR
spectra were recorded on a Bruker Avance (500 and 125 MHz
respectively) instrument using d6-DMSO as the solvent with TMS
(tetramethylsilane) as an internal standard; chemical shifts were
reported in
d values (ppm) relative to that of internal TMS. The ab-
breviations s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quadruplet,
m ¼ multiplet, and b ¼ broad were used throughout. Elemental
analyses (C, H, N, and S) were realized on a Thermo Scientific Flash EA
1112-elemental analyzer and were within 0.4% of the theoretical
values. High-resolution mass spectra (positive electrospray
Table 3
Estimated IC50 values for the inhibition of platelet Aggregation induced, by U-46619
(1 mM).
Compound
Inhibition of platelet aggregation induced
by 1 M U-46619 IC50 values (
M)a
m
m
BM-573
7b
7e
7h
8b
0.509 ꢂ 0.006
0.966 ꢂ 0.051
0.705 ꢂ 0.035
0.649 ꢂ 0.006
0.971 ꢂ 0.021
0.726 ꢂ 0.072
0.504 ꢂ 0.016
8e
8h
Fig. 2. Inhibition of ex vivo platelet aggregation induced by U-46619. Platelets were
incubated with 1
M of tested sample or 0.1% DMSO at 37 ꢃC for 1 min, then U-46619
(1
M) was added to trigger the aggregation. Values are presented as means ꢂ SD, n ꢄ 2.
a
m
Results expressed as mean (standard deviation of at least three determinations
(n ꢄ 3).
m

S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
37
ionization) were obtained using ToF MS and ToF MS/MS scan modes
5.1.11. General procedure for the preparation of 4-arylamino-3-
sulfamoylbenzonitriles (6aed)
with MS-TOF equipment. All reactions were routinely checked by
thin layer chromatography on silica gel 60F254 Merck and visualiza-
tion was accomplished with UV light (254 nm).
4-Fluoro-3-sulfamoylbenzonitrile 4 (2 g, 0.01 mol) and the
appropriate amine (4 equivalents; 0.04 mol) were dissolved in
dioxane (10 mL) and the mixture was refluxed for 20e36 h. When
the reaction was finished, the mixture was evaporated under
reduced pressure. The residue was dissolved in an aqueous 0.5 N
NaOH solution. This mixture was treated with diethyl ether
(30 mL) and the aqueous layer was separated and adjusted to pH 1
with 12 N hydrochloric acid. The precipitate, which appeared,
was collected by filtration, washed with water and dried (Yield:
60e80%).
5.1.1. 3-Amino-4-fluorobenzonitrile (2)
4-Fluoro-3-nitrobenzonitrile (1) (2 g, 0.012 mol) in ethyl acetate
(50 mL) was supplemented with stannous chloride (11.4 g,
0.06 mol). The reaction mixture was stirred for 30 min and then a
saturated solution of sodium hydrogenocarbonate (100 mL) was
added. This mixture was treated with ethyl acetate (300 mL) and
the aqueous layer was separated. The ethyl acetate layer was
evaporated under reduced pressure. The crude product was dis-
solved in methanol, and ice was added; the resulting precipitate
was collected by filtration (Yield: 30e60%).
The following compounds were obtained according to this
procedure.
5.1.12. 4-(4-Methylphenylamino)-3-sulfamoylbenzonitrile (6a)
Mp: 159e161 ꢃC.
5.1.2. 4-Fluoro-3-sulfamoylbenzonitrile (4)
3-Amino-4-fluorobenzonitrile (2) (2 g, 0.015 mol) in acidic
medium (28 mL HCl 6 N) reacted with sodium nitrite (1.4 g in
2 mL of water). The resulting solution of diazonium salt was
mixed with a solution of sulphur dioxide in acetic acid in the
presence of CuCl2 (0.8 g in 2 mL of water) to generate 3-
chlorosulfonyl-4-fluorobenzonitrile (3) according to the Meer-
wein variation of Sandmeyer reaction. Compound 3 was poured
into a solution of ammonium hydroxide and gently heated,
resulting in the synthesis of intermediate 4-fluoro-3-
sulfamoylbenzonitrile (4) (Yield: 40e65%).
5.1.13. 4-(3-Methylphenylamino)-3-sulfamoylbenzonitrile (6b)
Mp: 160e162 ꢃC.
5.1.14. 4-(2-Methylphenylamino)-3-sulfamoylbenzonitrile (6c)
Mp: 145e147 ꢃC.
5.1.15. 4-(4-Fluorophenylamino)-3-sulfamoylbenzonitrile (6d)
Mp: 208e210 ꢃC.
5.1.16. General procedure for the preparation of N-alkyl-N’-[2-
(arylamino/aryloxy)-5-cyanobenzenesulfonyl]ureas (7aeu and 8ael)
The appropriate 4-arylamino/aryloxy-3-sulfamoylbenzonitrile 5
or 6 (0.01 mol) and 1.1 equivalent of NaOH were dissolved in a
mixture of methanol and water (70/30) (10 mL). The reaction
mixture was gently stirred during 10 min and then evaporated
under reduced pressure. The resulting solid was suspended in
acetone (10 mL) and evaporated under reduced pressure. The crude
product was dissolved in dimethylformamide (5 mL) and gently
refluxed. The appropriate isocyanate (2 equivalents; 0.02 mol) was
added to the mixture. At the end of the reaction monitored by tlc
(0.1e1 h), the reaction mixture was evaporated under reduced
pressure and acidified with hydrochloric acid (2 N). The resulting
precipitate was collected by filtration, washed with distilled water
and dried. The crude product was then crystallized in methanol/
water (50/50) (Yields: 75e90%).
5.1.3. General procedure for the preparation of 4-aryloxy-3-
sulfamoylbenzonitriles (5aeg)
The appropriate methylphenol (0.05 mol) or halophenol
(0.05 mol) was deprotonated in presence of 1.1 equivalent of
NaOH (in aqueous solution). Crystals of methyl/halophenolates
were provided after evaporation under reduced pressure. 4-
Fluoro-3-sulfamoylbenzonitrile (4) (0.01 mol), and potassium
carbonate (7 equivalents) in acetonitrile (10 mL) were added and
the mixture was refluxed for 16e30 h. At the end of the reaction
and after cooling, the mixture was acidified with hydrochloric
acid (12 N) and filtered. The filtrate was evaporated under
reduced pressure. The crude product was dissolved in methanol,
and ice was added; the resulting precipitate was collected by
filtration (Yield: 60e85%).
The following compounds were obtained according to this
procedure.
The 1H and 13C NMR spectral data of compounds 7aeu and 8ael
are reported in the Supplementary material available online.
5.1.4. 4-(4-Methylphenoxy)-3-sulfamoylbenzonitrile (5a)
Mp: 186e188 ꢃC.
5.1.17. N-Isopropyl-N0-[2-(2-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7a)
5.1.5. 4-(3-Methylphenoxy)-3-sulfamoylbenzonitrile (5b)
Mp: 161e163 ꢃC.
Mp: 191e193 ꢃC. Anal (C18H19N3O4S) theoretical: N, 11.25; C,
57.89; H, 5.13; S, 8.59. Found: N, 11.06; C, 57.85; H, 5.30; S, 8.56.
EI/HRMS (m/z) [M þ H]þ: 374.1171. Calcd for [C18H20N3O4S]þ:
374.1175.
5.1.6. 4-(2-Methylphenoxy)-3-sulfamoylbenzonitrile (5c)
Mp: 176e178 ꢃC.
5.1.18. N-tert-Butyl-N0-[2-(2-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7b)
5.1.7. 4-(4-Fluorophenoxy)-3-sulfamoylbenzonitrile (5d)
Mp: 189e191 ꢃC.
Mp: 136e138 ꢃC. Anal (C19H21N3O4S) theoretical: N, 10.85; C,
58.90; H, 5.46; S, 8.27. Found: N, 10.58; C, 58.94; H, 5.83; S, 8.16. EI/
5.1.8. 4-(4-Chlorophenoxy)-3-sulfamoylbenzonitrile (5e)
Mp: 210e212 ꢃC.
HRMS (m/z) [M
388.1331.
þ
H]þ: 388.1369. Calcd for [C19H22N3O4S]þ:
5.1.9. 4-(4-Bromophenoxy)-3-sulfamoylbenzonitrile (5f)
Mp: 205e207 ꢃC.
5.1.19. N-Pentyl-N0-[2-(2-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7c)
Mp: 115e117 ꢃC. Anal (C20H23N3O4S) theoretical: N, 10.47;
C, 59.83; H, 5.77; S, 7.99. Found: N, 10.43; C, 59.92; H, 5.70;
S, 7.73.
5.1.10. 4-(4-Iodophenoxy)-3-sulfamoylbenzonitrile (5g)
Mp: 212e214 ꢃC.

38
S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
5.1.20. N-Isopropyl-N0-[2-(3-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7d)
5.1.32. N-Isopropyl-N0-[2-(4-bromophenoxy)-5-cyanobenzenesulfonyl]
urea (7p)
Mp: 186e188 ꢃC. Anal (C18H19N3O4S) theoretical: N, 11.25; C,
57.89; H, 5.13; S, 8.59. Found: N, 11.35; C, 57.67; H, 5.19; S, 8.56. EI/
HRMS (m/z) [M þ H]þ: 374.1204. Calcd for [C18H20N3O4S]þ: 374.1175.
Mp: 160e162 ꢃC. Anal (C17H16BrN3O4S) theoretical: N, 9.59; C,
46.59; H, 3.68; S, 7.32. Found: N, 9.40; C, 46.68; H, 3.75; S, 7.41.
5.1.33. N-tert-Butyl-N0-[2-(4-bromophenoxy)-5-cyanobenzenesulfonyl]
urea (7q)
5.1.21. N-tert-Butyl-N0-[2-(3-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7e)
Mp 179e180 ꢃC. Anal (C18H18BrN3O4S) theoretical: N, 9.29; C,
47.80; H, 4.01; S, 7.09. Found: N, 9.19; C, 47.89; H, 4.12; S, 7.13.
Mp: 135e137 ꢃC. Anal (C19H21N3O4S) theoretical: N, 10.85; C,
58.90; H, 5.46; S, 8.27. Found: N, 10.55; C, 59.79; H, 5.79; S, 8.25. EI/
HRMS (m/z) [M þ H]þ: 388.1341. Calcd for [C19H22N3O4S]þ: 388.1331.
5.1.34. N-Pentyl-N0-[2-(4-bromophenoxy)-5-cyanobenzenesulfonyl]
urea (7r)
5.1.22. N-Pentyl-N0-[2-(3-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7f)
Mp: 170e172 ꢃC. Anal (C19H20BrN3O4S) theoretical: N, 9.01; C,
48.93; H, 4.32; S, 6.88. Found: N, 8.91; C, 48.97; H, 4.38; S, 6.91.
Mp: 171e173 ꢃC. Anal (C20H23N3O4S) theoretical: N, 10.47; C,
59.83; H, 5.77; S, 7.99. Found: N, 10.27; C, 59.67; H, 5.70; S, 7.90.
5.1.35. N-Isopropyl-N0-[2-(4-iodophenoxy)-5-cyanobenzenesulfonyl]
urea (7s)
5.1.23. N-Isopropyl-N0-[2-(4-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7g)
Mp: 174e176 ꢃC. Anal (C17H16IN3O4S), theoretical: N, 8.66; C,
42.07; H, 3.32; S, 6.61. Found: N, 8.57; C, 42.21; H, 3.35; S, 6.52.
Mp: 218e220 ꢃC. Anal (C18H19N3O4S) theoretical: N, 11.25; C,
57.89; H, 5.13; S, 8.59. Found: N, 11.14; C, 57.82; H, 5.12; S, 8.54.
5.1.36. N-tert-Butyl-N-[2-(4-iodophenoxy)-5-cyanobenzenesulfonyl]
urea (7t)
5.1.24. N-tert-Butyl-N0-[2-(4-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7h)
Mp: 183e185 ꢃC. Anal (C18H18IN3O4S) theoretical: N, 8.42; C,
43.30; H, 3.63; S, 6.42. Found: N, 8.50; C, 43.29; H, 3.61; S, 6.50.
Mp: 175e177 ꢃC. Anal (C19H21N3O4S) theoretical: N, 10.85; C,
58.90; H, 5.46; S, 8.27. Found: N, 11.21; C, 58.97; H, 5.47; S, 8.01. EI/
HRMS (m/z) [M þ H]þ: 388.1350. Calcd for [C19H22N3O4S]þ:
388.1331.
5.1.37. N-Pentyl-N0-[2-(4-iodophenoxy)-5-cyanobenzenesulfonyl]
urea (7u)
Mp: 159e161 ꢃC. Anal (C19H20IN3O4S) theoretical: N, 8.19; C,
44.45; H, 3.93; S, 6.25. Found: N, 8.01; C, 44.53; H, 3.95.55; S, 5.99.
5.1.25. N-Pentyl-N0-[2-(4-methylphenoxy)-5-cyanobenzenesulfonyl]
urea (7i)
5.1.38. N-Isopropyl-N0-[2-(2-methylphenylamino)-5-cyanobenzenesul
fonyl]urea (8a)
Mp: 161e163 ꢃC. Anal (C20H23N3O4S) theoretical: N, 10.47; C,
59.83; H, 5.77; S, 7.99. Found: N, 10.49; C, 59.75; H, 5.88; S, 8.01.
Mp: 150e152 ꢃC. Anal (C18H20N4O3S) theoretical: N, 15.04; C,
58.05; H, 5.41; S, 8.61. Found: N, 15.29; C, 57.94; H, 5.52; S, 8.53. EI/
HRMS (m/z) [M þ H]þ: 373.1355. Calcd for [C18H21N4O3S]þ:
373.1334.
5.1.26. N-Isopropyl-N0-[2-(4-fluorophenoxy)-5-cyanobenzenesulfonyl]
urea (7j)
Mp: 179e181 ꢃC. Anal (C17H16FN3O4S), theoretical: N, 11.13; C,
54.10; H, 4.27; S, 8.50. Found: N, 11.10; C, 54.21; H, 4.24; S, 8.32.
5.1.39. N-tert-Butyl-N0-[2-(2-methylphenylamino)-5-cyanobenzenesul
fonyl]urea (8b)
5.1.27. N-tert-Butyl-N0-[2-(4-fluorophenoxy)-5-cyanobenzenesulfonyl]
urea (7k)
Mp: 165e167 ꢃC. Anal (C19H22N4O3S) theoretical: N, 14.50; C,
59.05; H, 5.74; S, 8.30. Found: N, 14.55; C, 59.07; H, 5.90; S, 8.33.
Mp: 152e154 ꢃC. Anal (C18H18FN3O4S), theoretical: N, 10.74; C,
55.23; H, 4.64; S, 8.19. Found: N, 10.62; C, 55.42; H, 4.74; S, 8.31. EI/
HRMS (m/z) [M þ H]þ: 392.1073. Calcd for [C18H19FN3O4S]þ:
392.1080.
5.1.40. N-Pentyl-N0-[2-(2-methylphenylamino)-5-cyanobenzenesulfonyl]
urea (8c)
Mp: 159e161 ꢃC. Anal (C20H24N4O3S) theoretical: N, 13.99; C,
59.98; H, 6.04; S, 8.00. Found: N, 14.19; C, 60.14; H, 6.25; S, 8.26.
5.1.28. N-Pentyl-N0-[2-(4-fluorohenoxy)-5-cyanobenzenesulfonyl]urea
(7l)
5.1.41. N-Isopropyl-N0-[2-(3-methylphenylamino)-5-cyanobenzenesul
fonyl]urea (8d)
Mp: 186e188 ꢃC. Anal (C19H20FN3O4S) theoretical: N, 10.36; C,
56.28; H, 4.97; S, 7.91. Found: N, 10.42; C, 56.43; H, 4.77; S, 7.98.
Mp: 136e138 ꢃC. Anal (C18H20N4O3S) theoretical: N, 15.04; C,
58.05; H, 5.41; S, 8.61. Found: N, 15.12; C, 58.07; H, 5.66; S, 8.51. EI/
HRMS (m/z) [M þ H]þ: 373.1338. Calcd for [C18H21N4O3S]þ:
373.1334.
5.1.29. N-Isopropyl-N0-[2-(4-chlorophenoxy)-5-cyanobenzenesulfonyl]
urea (7m)
Mp: 168e170 ꢃC. Anal (C17H16ClN3O4S) theoretical: N, 10.67; C,
51.84; H, 4.09; S, 8.14. Found: N, 10.45; C, 51.72; H, 4.15; S, 8.24.
5.1.42. N-tert-Butyl-N0-[2-(3-methylphenylamino)-5-cyanobenzenesul
fonyl]urea (8e)
5.1.30. N-tert-Butyl-N0-[2-(4-chlorophenoxy)-5-cyanobenzenesulfonyl]
urea (7n)
Mp: 138e140 ꢃC. Anal (C19H22N4O3S) theoretical: N, 14.50; C,
59.05; H, 5.74; S, 8.30. Found: N, 14.56; C, 58.98; H, 6.01; S, 8.12. EI/
HRMS (m/z) [M þ H]þ: 387.1485. Calcd for [C19H23N4O3S]þ:
387.1491.
Mp: 154e156 ꢃC. Anal (C18H18ClN3O4S) theoretical: N, 10.30; C,
53.01; H, 4.45; S, 7.86. Found: N, 9.99; C, 53.03; H, 4.42; S, 7.64.
5.1.31. N-Pentyl-N0-[2-(4-chloroxyphenoxy)-5-cyanobenzenesulfonyl]
urea (7o)
5.1.43. N-Pentyl-N0-[2-(3-methylphenylamino)-5-cyanobenzenesulfonyl]
urea (8f)
Mp: 167e169 ꢃC. Anal (C19H20ClN3O4S) theoretical: N, 9.96; C,
54.09; H, 4.78; S, 7.60. Found: N, 10.05; C, 54.17; H, 4.55; S, 7.44.
Mp: 125e126 ꢃC. Anal (C20H24N4O3S) theoretical: N, 13.99; C,
59.98; H, 6.04; S, 8.00. Found: N, 13.87; C, 59.81; H, 6.21; S, 7.89. EI/

S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
39
HRMS (m/z) [M þ H]þ: 401.1643. Calcd for [C20H25N4O3S]þ:
(F ꢀ Fmin)/(Fmax ꢀ F), assuming a Kd of 385 nM for Fluo-4. The re-
401.1647.
sults (IC50) presented are the concentration required to inhibit 50%
of the normal rise of [Ca2þ]i upon stimulation with 1
mM U-46619,
determined in the absence of any compounds. The IC50s were
calculated by nonlinear regression analysis (GraphPad Prism soft-
ware) from at least three concentrationeresponse curves.
5.1.44. N-Isopropyl-N0-[2-(4-methylphenylamino)-5-cyanobenzene
sulfonyl]urea (8g)
Mp: 136e138 ꢃC. Anal (C18H20N4O3S) theoretical: N, 15.04; C,
58.05; H, 5.41; S, 8.61. Found: N, 15.26; C, 57.97; H, 5.56; S, 8.26. EI/
HRMS (m/z) [M þ H]þ: 373.1338. Calcd for [C18H21N4O3S]þ:
373.1334.
5.3. Human in vitro platelet aggregation
The antiaggregant potency has been determined according to
the turbidimetric Born’s method [39]. The blood was drawn from 10
healthy donors of both genders. The subjects were free from
medication for at least 14 days. No significant differences in the
results were observed between the donors in our experiments.
Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were
prepared as previously described [32,40,41]. Platelet concentration
of PRP was adjusted to 250,000 cells/mL by dilution with PPP.
Platelet aggregation of PRP was studied using a double channel
aggregometer (Chronolog Corporation, Chicago, IL) connected to a
5.1.45. N-tert-Butyl-N0-[2-(4-methylphenylamino)-5-cyanobenzenesul
fonyl]urea (8h)
Mp: 139e141 ꢃC. Anal (C19H22N4O3S) theoretical: N, 14.50; C,
59.05; H, 5.74; S, 8.30. Found: N, 14.70; C, 59.08; H, 5.82; S, 8.21.
5.1.46. N-Pentyl-N0-[2-(4-methylphenylamino)-5-cyanobenzenesulfonyl]
urea (8i)
Mp: 134e136 ꢃC. Anal (C20H24N4O3S) theoretical: N, 13.99; C,
59.98; H, 6.04; S, 8.00. Found: N, 14.20; C, 59.96; H, 6.08; S, 8.16. EI/
HRMS (m/z) [M þ H]þ: 401.1649. Calcd for [C20H25N4O3S]þ:
401.1647.
linear recorder as previously described [42]. PRP (290
mL) was
added in a silanised cuvette and stirred (1000 rpm). Each com-
pound was diluted (1 mM) in a mixture of dimethyl sulfoxide-PBS
(DMSO/PBS) (30/70), and preincubated in PRP for 3 min at 37 ꢃC
before the aggregating agent was added. Compounds were tested at
various concentrations 10ꢀ5 M to 10ꢀ7 M. Platelet aggregation was
5.1.47. N-Isopropyl-N0-[2-(4-fluorophenylamino)-5-cyanobenzenesul
fonyl]urea (8j)
Mp: 190e192 ꢃC. Anal (C17H17FN4O3S) theoretical: N, 14.88; C,
54.25; H, 4.55; S, 8.52. Found: N, 15.01; C, 54.37; H, 4.38; S, 8.47.
initiated by addition of a fresh solution of U-46619 (1 mM final). To
evaluate platelet aggregation, the maximum increase in light
transmission was determined from the aggregation curve 6 min
after addition of the inducer. The substance concentration pre-
venting 50% of platelet aggregation (IC50) induced by U-46619 was
calculated by nonlinear regression analysis (GraphPad Prism soft-
ware) from at least three concentrationeresponse curves.
5.1.48. N-tert-Butyl-N0-[2-(4-fluorophenylamino)-5-cyanobenzenesul
fonyl]urea (8k)
Mp: 182e184 ꢃC. Anal (C18H19FN4O3S) theoretical: N, 14.35; C,
55.37; H, 4.91; S, 8.21. Found: N, 14.46; C, 55.49; H, 4.69; S, 8.25.
5.1.49. N-Pentyl-N0-[2-(4-fluorophenylamino)-5-cyanobenzenesulfonyl]
urea (8l)
5.4. Statistical analysis
Mp: 163e165 ꢃC. Anal (C19H21FN4O3S) theoretical: N, 13.85; C,
56.42; H, 5.23; S, 7.93. Found: N, 13.81; C, 56.51; H, 5.18; S, 7.84.
Results are expressed as the mean ꢂ standard deviation from at
least three determinations (n ¼ 3). Statistical differences between TP
isoforms have been determined using unpaired t-test between IC50s
values. p values of less than 0.05 were considered to be significant.
5.2. Calcium measurements
HEK.293TP
a
and HEK.293TP
b
cell lines, stably overexpressing
HA-tagged forms of TP
a
and TP
b in human embryonic kidney (HEK)
Acknowledgements
293 cells have been previously described [37]. Cells were routinely
grown in Dulbecco’s modified Eagle’s medium (DMEM) containing
10% foetal bovine serum (FBS). Measurement of [Ca2þ]i mobiliza-
This work was supported by The CoopérationTechnique Belge (CTB,
from Belgium). The authors gratefully acknowledge the technical
assistance of E. Goffin, J.C. Van Heugen, S. Counerotte andP. Neven. We
also thank Dr. P. de Tullio (Liege University) for his scientific advice.
tion either in HEK.293TP
a or HEK.293TPb cells was carried out
using fluorescent microplate reader Fluoroskan Ascent FL equipped
with two dispensers (thermo electron corporation, Finland) ac-
cording to modified method of Lin et al. [38].
Briefly, cells were trypsinized, washed twice with Krebs-HEPES
buffer (118 mM, NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4,
4.2 mM NaHCO3,11.7 mM
7.4), and incubated for 1 h with fluorescent dye Fluo-4/AM (5
Molecular Probes, Invitrogen, Merelbeke Belgium). Cells were then
rinsed three times with Krebs-HEPES buffer and 150 L of a sus-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.033.
D-glucose,1.3 mM CaCl2,10 mM HEPES, pH
mg/mL;
References
m
pension of cells in that buffer was loaded into each well of a 96-well
plate at a density of 150,000 cells/well. Cells were incubated 10 min
[1] J.S. Huang, S.K. Ramamurthy, X. Lin, G.C. Le Breton, Cell signalling through
thromboxane A2 receptors, Cell. Signalling. 16 (2004) 521e533.
[2] N. Nakahata, Thromboxane A2: physiology/pathology, cellular signal trans-
duction and pharmacology, Pharmacol. Ther. 118 (2008) 18e35.
[3] M.J. Wacker, L.M. Kosloski, W.J. Gilbert, C.D. Touchberry, D.S. Moore, J.K. Kelly,
M. Brotto, J.A. Orr, Inhibition of thromboxane A2-induced arrhythmias and
intracellular calcium changes in cardiac myocytes by blockade of the inositol
trisphosphate pathway, J. Pharmacol. Exp. Ther. 331 (2009) 917e924.
[4] S.S. Bhagwat, P.R. Hamann, W.C. Still, S. Bunting, F.A. Fitzpatrick, Synthesis and
structure of the platelet aggregation factor thromboxane A2, Nature (London)
315 (1985) 511e513.
with various concentrations of the test compound (10ꢀ5 to 10ꢀ9
M
final). In all cases, compound (1 mM) was diluted in dimethyl sulf-
oxide (DMSO)/PBS (30/70) prior to further dilution in PBS.
Fluorescence emission was read at 538 nm. At the end of each
experiment, fluorescence intensities were calibrated for determi-
nation of intracellular calcium concentration ([Ca2þ]i) values by
permeabilizing cells with 1% Triton X-100 to release all the dye
(Fmax) and subsequently chelating with 10 mM EGTA (Fmin). Cal-
cium concentrations were calculated using equation [Ca2þ]i) Kd
[5] G.I. Fiddler, P. Lumley, Preliminary clinical studies with thromboxane synthase
inhibitors and thromboxane receptor blockers.
A review, Circulation 81
(Suppl. 1) (1990) 169e178.

40
S.-M. Bambi-Nyanguile et al. / European Journal of Medicinal Chemistry 65 (2013) 32e40
[6] M. Hamberg, J. Svensson, B. Samuelsson, Thromboxanes: a new group of
[25] M.T. Walsh, J.F. Foley, B.T. Kinsella, Investigation of the role of the carboxyl-
terminal tails of the human thromboxane A2 receptor (TP) in mediating re-
ceptor: effector coupling, Biophys. Acta 1496 (2000) 164e182.
biologically active compounds derived from prostaglandin endoperoxides,
Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 2994e2998.
[7] J. Hanson, J.M. Dogne, J. Ghiotto, A.L. Moray, B.T. Kinsella, B. Pirotte, Design,
[26] T. Hirata, F. Ushikubi, A. Kakizuka, M. Okuma, S. Narumiya, Two thromboxane
A2 receptor isoforms in human platelets. Opposite coupling to adenylyl
cyclase with different sensitivity to Arg60 to Leu mutation, J. Clin. Invest. 97
(1996) 949e956.
synthesis, and SAR study of
nitrobenzenesulfonyl]ureas and e cyanoguanidine as selective antagonists
of the TP and TP ; isoforms of the human thromboxane A2 receptor, J. Med.
a
series of N-alkyl-N’-[2-(aryloxy)-5-
a
b
Chem. 50 (2007) 3928e3936.
[27] J.L. Parent, P. Labrecque, M.J. Orsini, J.L. Benovic, Internalization of the TXA2 re-
[8] J. Svensson, M. Hamberg, Thromboxane A2 and prostaglandin H2: potent
stimulators of the swine coronary artery, Prostaglandins 12 (1976) 943e950.
[9] S. Moncada, S.H. Ferreira, J.R. Vane, Bioassay of prostaglandins and biologically
active substances derived from arachidonic acid, Adv. Prostaglandin Throm-
boxane Res. 5 (1978) 211e236.
ceptor
agonist-promoted receptor internalization, J. Biol. Chem. 274 (1999) 8941e8948.
[28] H.M. Reid, B.T. Kinsella, Palmitoylation of the TP isoform of the human
thromboxane A2 receptor. Modulation of G protein: effector coupling and
modes of receptor internalization, Cell. Signalling 19 (2007) 1056e1070.
[29] A. Habib, G.A. FitzGerald, J. Maclouf, Phosphorylation of the thromboxane
a and b isoforms. Role of the differentially spliced COOH terminus in
b
[10] G. Davi, C. Patrono, Platelet activation and atherothrombosis, N. Engl. J. Med.
357 (2007) 2482e2494.
receptor
a, the predominant isoform expressed in human platelets, J. Biol.
[11] J.W. Eikelboom, J. Hirsh, J.I. Weitz, M. Johnston, Q. Yi, S. Yusuf, Aspirin resistant
thromboxane biosynthesis and the risk of myocardial infarction, stroke, or
cardiovascular death in patients at high risk for cardiovascular events, Cir-
culation 105 (2002) 1650e1655.
Chem. 274 (1999) 2645e2651.
[30] O. Moussa, A.W. Ashton, M. Fraig, E. Garrett-Mayer, M.A. Ghoneim,
P.V. Halushka, D.K. Watson, Novel role of thromboxane receptors
bladder cancer pathogenesis, Cancer Res. 68 (2008) 4097e4104.
b isoform in
[12] G. Niccoli, S. Giubilato, E. Russo, C. Spaziani, A. Leo, I. Porto, M. Leone,
F. Burzotta, S. Riondino, F. Pulcinelli, L.M. Biasucci, F. Crea, Plasma levels of
thromboxane A2 on admission are associated with no-reflow after primary
percutaneous coronary intervention, Eur. Heart J. 29 (2008) 1843e1850.
[13] S. Offermanns, Activation of platelet function through G-protein-coupled re-
ceptors, Circ. Res. 99 (2006) 1293e1304.
[31] L. Navarro-Nunez, J. Castillo, M.L. Lozano, C. Martinez, O. Benavente-Garcia,
V. Vicente, J. Rivera, Thromboxane A2 receptor antagonism by flavonoids:
structureeactivity relationships, J. Agric. Food Chem. 57 (2009) 1589e1594.
[32] J. Hanson, D. Reynaud, N. Qiao, P. Devel, A.L. Moray, J.F. Renard, L.P. Kelley,
J.Y. Winum, J.L. Montero, B.T. Kinsella, B. Pirotte, C.R. Pace-Asciak, J.M. Dogné,
Synthesis and pharmacological evaluation of novel nitrobenzenic thromboxane
modulators as antiplatelet agents acting on both the alpha and beta isoforms of
the human thromboxane receptor, J. Med. Chem. 49 (2006) 3701e3709.
[33] H. Meerwein, G. Dittmar, R. Gollner, K. Hafner, F. Mensch, O. Steinfort, Aro-
matic diazo compounds. II. Preparation of aromatic sulfonyl chlorides, a new
modification of the Sandmeyer reaction, Chem. Ber. 90 (1957) 841e852.
[34] T. Cyrus, L.X. Tang, J. Rokach, G.A. FitzGerald, D. Praticò, Lipid peroxidation and
platelet activation in murine atherosclerosis, Circulation 104 (2001) 1940e1945.
[35] D. Praticò, T. Cyrus, H. Li, G.A. FitzGerald, Endogenous biosynthesis of
thromboxane and prostacyclin in two distinct murine models of atheroscle-
rosis, Blood 96 (2000) 3823e3826.
[14] J.I. Osende, D. Shimbo, V. Fuster, M. Dubar, J.J. Badimon, Antithrombotic effects
of S18886,
a novel orally active thromboxane A2 receptor antagonist,
J. Thromb. Haemostasis 2 (2004) 492e498.
[15] C.W. Hamm, R.L. Lorenz, W. Bleifeld, W. Kupper, W. Wober, P.C. Weber,
Biochemical evidence of platelet activation in patients with persistent un-
stable angina, J. Am. Coll. Cardiol. 10 (1987) 998e1006.
[16] J.M. Dogné, C.T. Supuran, D. Pratico, Adverse cardiovascular effects of the
coxibs, J. Med. Chem. 48 (2005) 2251e2257.
[17] S. Koba, R. Pakala, T. Watanabe, T. Katagiri, C.R. Benedict, Synergistic inter-
action between thromboxane A2 and mildly oxidized low density lipoproteins
on vascular smooth muscle cell proliferation, prostaglandins leukotrienes
essent, Fatty Acids 63 (2000) 329e335.
[36] J.M. Dogné, J. Hanson, X. de Leval, P. Kolh, V. Tchana-Sato, L. de Leval, S. Rolin,
A. Ghuysen, P. Segers, B. Lambermont, B. Masereel, B. Pirotte, Pharmacological
[18] R. Pakala, C.R. Benedict, Effect of serotonin and thromboxane A2 on endo-
thelial cell proliferation: effect of specific receptor antagonists, J. Lab. Clin.
Med. 131 (1998) 527e537.
[19] D.F. Woodward, R.L. Jones, S. Narumiya, International union of basic and
clinical pharmacology. LXXXIII: classification of prostanoid receptors, updat-
ing 15 years of progress, Pharmacol. Rev. 63 (2011) 471e538.
[20] M. Hirata, Y. Hayashi, F. Ushikubi, Y. Yokota, R. Kageyama, S. Nakanishi,
S. Narumiya, Cloning and expression of cDNA for a human thromboxane A2
receptor, Nature 349 (1991) 617e620.
characterization
of
N-tert-butyl-N’-[2-(4’-methylphenylamino)-5-
nitrobenzenesulfonyl]urea (BM-573), a novel thromboxane A2 receptor antag-
onist and thromboxane synthase inhibitor in a rat model of arterial thrombosis
and its effects on bleeding time, J. Pharmacol. Exp. Ther. 309 (2004) 498e505.
[37] M.T. Walsh, J.F. Foley, B.T. Kinsella, The alpha, but not the beta, isoform of the
human thromboxane A2 receptor is
a target for prostacyclin-mediated
desensitization, J. Biol. Chem. 275 (2000) 20412e20423.
[38] K. Lin, W. Sadee, J.M. Quillan, Rapid measurements of intracellular calcium
using a fluorescence plate reader, Biotechniques 26 (1999) 318e322.
[39] G.V. Born, M.J. Cross, The aggregation of blood platelets, J. Physiol. 168 (1963)
178e195.
[21] M.K. Raychowdhury, M. Yukawa, L.J. Collins, S.H. McGrail, K.C. Kent, J.A. Ware,
Alternative splicing produces
a divergent cytoplasmic tail in the human
endothelial thromboxane A2 receptors, J. Biol. Chem. 269 (1994) 19256e19261.
[22] R.M. Nusing, M. Hirata, A. Kakizuka, T. Eki, K. Ozawa, S. Narumiya, Charac-
terization and chromosomal mapping of the human thromboxane A2 receptor
gene, J. Biol. Chem. 268 (1993) 25253e25259.
[40] J.M. Dogne, X. de Leval, P. Neven, S. Rolin, J. Wauters, J.L. David, J. Delarge,
B. Masereel, Effects of
a novel non-carboxylic thromboxane A2 receptor
antagonist (BM-531) derived from torasemide on platelet function prosta-
glandins, leukotrienes essent, Fatty Acids 62 (2000) 311e317.
[23] B.T. Kinsella, D.J. O’Mahony, G.A. Fitzgerald, The human thromboxane A2 re-
ceptor alpha isoform (TP alpha) functionally couples to the G proteins Gq and
G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2 alpha,
J. Pharmacol. Exp. Ther. 281 (1997) 957e964.
[41] J.M. Dogné, J. Wouters, S. Rolin, C. Michaux, L. Pochet, F. Durant, J. Delarge,
B. Masereel, Design, synthesis and biological evaluation of a sulfonylcyano-
guanidine as thromboxane A2 receptor antagonist and thromboxane synthase
inhibitor, J. Pharm. Pharmacol. 53 (2001) 669e680.
[24] M.T. Walsh, J.F. Foley, B.T. Kinsella, Characterization of the role of N-linked glyco-
sylation on the cell signaling and expression of the human thromboxane A2 re-
ceptor alpha and beta isoforms, J. Pharmacol. Exp. Ther. 286 (1998) 1026e1036.
[42] D.N. Harris, M.M. Asaad, M.B. Phillips, H.J. Goldenberg, M.J. Antonaccio, Inhi-
bition of adenylate cyclase in human blood platelets by 9-substituted adenine
derivatives, J. Cyclic Nucleotide Res. 5 (1979) 125e134.