Design, synthesis, and SAR study of a series of N-alkyl-N′-[2- (aryloxy)-5-nitrobenzenesulfonyl]ureas and -cyanoguanidine as selective antagonists of the TPα and TPβ isoforms of the human thromboxane A2 receptor

Article abstract of DOI:10.1021/jm070427h

The prostanoid thromboxane (TX)A₂ exerts its proaggregant and constrictive actions upon binding to the specific TXA₂ receptor (TP), a member of the G-protein coupled receptor superfamily. In humans, TXA ₂ signals through two distinct TP isoforms, TPα and TPβ. Herein, we describe the design, synthesis, and SAR study of a series of original N-alkyl-N′-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas and -cyanoguanidine. The SAR study was based on the results of a functional assay, TP-mediated intracellular calcium ([Ca²⁺]_i) mobilization performed on the two separate isoforms. Optimal nature and position of several structural moieties was defined for both activity and selectivity toward TPα and TPβ isoforms. Three compounds (9h, 9af, and 9ag), showing increased selectivity for TPβ relative to TPα (23.2:1, 18.1:1, 19.9:1, respectively), were selected for further experiments, and their activity was confirmed in a platelet aggregation assay. This study represents the first extended SAR study dealing with the identification of isoform selective antagonists for the human TXA₂ receptor.

Full text of DOI:10.1021/jm070427h

3928
J. Med. Chem. 2007, 50, 3928-3936
Design, Synthesis, and SAR Study of a Series of
N-Alkyl-N′-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas and -cyanoguanidine as Selective
Antagonists of the TPr and TPâ Isoforms of the Human Thromboxane A2 Receptor
Julien Hanson,*,† Jean-Michel Dogne´,†,§ Je´re´mie Ghiotto,† Anne-Lise Moray,† B. Therese Kinsella,‡ and Bernard Pirotte†
Drug Research Center, Laboratory of Medicinal Chemistry, UniVersity of Lie`ge, 1, AV. de l’Hoˆpital, B-4000 Lie`ge, Belgium, School of
Biomolecular & Biomedical Science, Conway Institute for Biomolecular and Biomedical Research, UniVersity College Dublin, Ireland, and
Department of Pharmacy, UniVersity of Namur, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium
ReceiVed April 11, 2007
The prostanoid thromboxane (TX)A2 exerts its proaggregant and constrictive actions upon binding to the
specific TXA2 receptor (TP), a member of the G-protein coupled receptor superfamily. In humans, TXA2
signals through two distinct TP isoforms, TPR and TPâ. Herein, we describe the design, synthesis, and
SAR study of a series of original N-alkyl-N′-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas and -cyanoguanidine.
The SAR study was based on the results of a functional assay, TP-mediated intracellular calcium ([Ca2+]i)
mobilization performed on the two separate isoforms. Optimal nature and position of several structural
moieties was defined for both activity and selectivity toward TPR and TPâ isoforms. Three compounds
(9h, 9af, and 9ag), showing increased selectivity for TPâ relative to TPR (23.2:1, 18.1:1, 19.9:1, respectively),
were selected for further experiments, and their activity was confirmed in a platelet aggregation assay. This
study represents the first extended SAR study dealing with the identification of isoform selective antagonists
for the human TXA2 receptor.
Introduction
On the other hand, because TPR appears to be the predominant
TP isoform expressed in platelets,11 specific inhibitor(s) of TPR
may be beneficial as more specific antiplatelet agent(s).
The lipid mediator thromboxane (TX)A2 plays a key role in
several physiologic processes including platelet aggregation and
vascular and bronchial smooth muscle constriction.1,2 An
Hence, the design and synthesis of selective isoform specific
TP receptor antagonists would be of great interest not only in
therapeutics but also as tools in pharmacological sciences to
decipher TP isoform specific roles. Despite great interest, the
identification of such TPR and/or TPâ isoform specific agonists/
antagonists has been poorly studied. Although early pharma-
cological reports have described selective TXA2 receptor (TP)
antagonists for separate tissues, the initial observations have
never been confirmed on isolated TP isoforms.12 Through
previous investigations, our group has addressed the influence
of the chemical structure of a series of nitrobenzenesulfonylureas
and -cyanoguanidines on selective antagonistic potency for TPR
or TPâ.13 In that study, we reported the synthesis and functional
characterization of a series of derivatives of the potent TP
receptor antagonist 1 (BM573, Figure 1),14 and from such
investigations a family of original N-alkyl-N′-[2-(4-methylphe-
noxy)-5-nitrobenzenesulfonyl]urea compounds was selected for
further detailed characterization through both chemical optimi-
zation and functional biologic/pharmacologic readouts. Three
of the most interesting previously reported compounds 2, 3, and
4 are illustrated in Figure 1. In the present study, we describe
the design, synthesis, and SAR study of a series of original
nitrobenzenesulfonylureas derived from the latter 2, 3, and 4
chemical leads. We have characterized the antagonistic potency
and TP-isoform selectivity of the new compounds using a
functional pharmacological test, namely inhibition of intracel-
lular calcium ([Ca2+]i) mobilization in model mammalian cell
lines that specifically overexpress the individual TPR or TPâ
isoforms. The antiplatelet activity of some of the most interesting
compounds was also determined in aggregation assays.
a
overproduction of TXA2 has been associated with many
pathological states such as myocardial infarction, thrombosis,
unstable angina, pulmonary embolism, septic shock, athero-
sclerosis, preeclampsia, and asthma.3 TXA2, a metabolite of
arachidonic acid (AA) released mainly by phospholipase (PL)-
A2 from membrane phospholipids, is primarily synthesized
through the sequential actions of cyclooxygenase (COX) and
TX synthase in platelets.3
The actions of TXA2 are mediated by its specific G-protein
coupled receptor (GPCR), referred to as the TXA2 receptor or
TP.4 In 1991, Hirata et al. reported that the human TP is encoded
by one gene,5 and in 1994 Raychowdhury et al. identified the
existence of a second isoform generated by alternative splicing.6
The two human TP isoforms, namely TPR and TPâ, share the
first 328 amino acids but differ within their carboxyl-terminal
tails (the last 15 amino acids of the R isoform being replaced
by 79 amino acids in the â isoform). To date, the exact role of
the TPR and TPâ isoforms is not fully understood although
several studies have established that they may have distinct
physiological functions.7-9 It has been proposed, for example,
that specific antagonism of TPâ would be useful to enhance
revascularization postmyocardial infarction since the stimulation
of TPâ seemed to be responsible for vascular endothelial growth
factor-induced endothelial cell differentiation and migration.10
* To whom correspondence should be addressed. Phone: +3243664382,
fax: +3243664362, e-mail: J.hanson@ulg.ac.be.
† University of Lie`ge.
‡ University College Dublin.
§ University of Namur.
a Abbreviations: TXA2, thromboxane A2; TXS, thromboxane synthase;
PLA2, phospholipase A2; AA, arachidonic acid; GPCR, G-protein coupled
receptor; COX, cyclooxygenase; TP, thromboxane receptor; PLC, phos-
pholipase C; IP3, inositol 1,4,5-triphosphate.
Chemistry. The compounds evaluated in this work were
synthesized according to the synthesis pathway described in
Schemes 1-3. In the first step, which is common to all
10.1021/jm070427h CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/14/2007

SelectiVe Antagonists of the Human Thromboxane A2 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3929
Figure 1. Chemical structures of 1-4.
Scheme 1a
compounds based on a functional pharmacological evaluation.
To this end, the ability of the compounds to antagonize TPR-
and TPâ-mediated intracellular calcium ([Ca2+]i) mobilization
in response to the TXA2 mimetic U46619 (5-heptenoic acid,
7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo-
[2.2.1]hept-5-yl]-, (5i)- (9CI)) was evaluated in human embry-
onic kidney (HEK) 293 cell lines stably overexpressing either
TPR (HEK‚TPR cells) or TPâ (HEK‚TPâ cells). Indeed, both
TPR and TPâ are coupled to the Gq/phospholipase C effector
system8 and, hence, can readily induce a rise in [Ca2+]i upon
agonist stimulation of the TP receptor. HEK‚TPR and/or HEK‚
TPâ cells were preloaded with Ca2+ fluorescent dye Fluo-4,
and inhibition of [Ca2+]i mobilization in response to the well
characterized TXA2 mimetic U46619 (1 µM) stimulation was
determined in the absence or presence of increasing concentra-
tions of the test compounds, using concentrations ranging from
10-9 M to 10-5 M. Concentration-response curves were
determined, and the IC50, defined as the concentration (M) of
compound required to inhibit 50% of U46619-induced [Ca2+
]
i
a Reagents: (a) NaNO2, HCl; (b) SO2, HOAc, Cu2Cl2; (c) NH4OH, ∆;
mobilization, values of M were calculated for each compound
tested. A selectivity index, defined as ratio of the IC50 (M) of
TPR relative to the IC50 (M) of TPâ (i.e., IC50 TPR/IC50 TPâ)
was subsequently determined.
Furthermore, since TXA2 is a potent inducer of platelet
aggregation, the ability of the most interesting test compounds
to prevent aggregation of human platelets in response to the
TXA2 mimetic U46619 was also evaluated. In this test, the
compounds were incubated at concentrations ranging from 10-9
to 10-5 M with isolated human platelets, and aggregation was
triggered upon stimulation by U46619 (1 µM).
(d) appropriate phenolate, acetonitrile, K2CO3.
compounds described, commercially available 2-chloro-5-ni-
troaniline (5) reacted with sodium nitrite in acidic medium, and
the resulting diazonium salt solution was mixed with a solution
of sulfur dioxide in acetic acid in the presence of Cu(I) to
generate 2-chloro-5-nitrobenzenesulfonyl chloride (6) according
to the Meerwein variation of the Sandmeyer reaction.15 Com-
pound 6 was poured into a solution of ammonium hydroxide
and gently heated, resulting in the synthesis of 2-chloro-5-
nitrobenzenesulfonamide (7) which is the common intermediate
giving access to the compounds studied (Scheme 1).
The nucleophilic substitution of the chlorine atom in the para
position to the nitro group by various phenols led to the synthesis
of the intermediates 8a-p. Deprotonation of the sulfonamide
group of compounds 8a-p followed by reaction with the
appropriate isocyanate led to the synthesis of compounds 9a-
aj (Scheme 2).
For the synthesis of the sulfonylcyanoguanidine 12, a reactive
synthon was first prepared by direct reaction of diphenyl
N-cyanoiminocarbonate (10) with tert-butylamine. This synthon
(11) directly reacted with the sulfonamidate salt of 8a (9) to
generate compound 12 (Scheme 3).
Pharmacological Evaluation. Thereafter, we wished to
address the structure-activity relationships (SAR) of the latter
Results and Discussion
In a previous study, we demonstrated the activity of a family
of nitrobenzenesulfonylureas presenting an oxygen bond be-
tween the two aromatic rings as selective antagonists of the TPR
and TPâ isoforms of the human TXA2 receptor (TP).13 The three
most interesting leads identified in those previous investiga-
tions,13 namely compounds 2-4, are presented in Figure 1 and
their relative potency toward the TP receptor isoforms in Table
1. One single parameter was modulated in that preliminary
study, namely the side chain on the sulfonylurea group.
Consequently, herein, we designed and synthesized several series
of N-alkyl-N′-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas, derived
form 2-4 (Figure 1) in order to more precisely estimate the

3930 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hanson et al.
Scheme 2a
a Reagents: (a) NaOH 10%; (b) R2-NdCdO.
Scheme 3a
a Reagents: (a) NaOH 10%.
structure-activity relationships (SAR) for activity and selectivity
within this family of compounds toward the individual TP
receptor isoforms.
In the 2-methylphenyl series (compounds 9b-e), the R2
n-butyl chain of analogue 9d was found to be the best substituent
and produced a 3- to10-fold increase in activity compared to
other compounds of the series (9c with an isopropyl chain as
R2 being the less active). Regarding the difference of activity
between the two isoforms, all compounds were characterized
by an isoform selectivity ratio (IC50 TPR/IC50 TPâ) of ∼16-
17 in this 2-substituted series except for analogue 9b with R2
) n-pentyl which had a lower ratio of 9. The higher ratio clearly
suggested increased selectivity for TPâ relative to TPR. Meta
substitution of the phenyl ring R1 (3-methylphenyl series)
seemed to have little influence on activity, except for compound
9h characterized by a ∼2- (compared to 9d) to 20-fold
(compared to 9c) increase in activity on both isoforms. More
interestingly, the compound 9h estimated selectivity ratio was
the highest in this table (23.2, Table 1) whereas other compounds
of this series displayed a ∼10 fold ratio. In previous work, the
compounds having a 4-methylphenyl moiety as the aromatic
ring R1 proved generally to be the most potent on TPR.13 The
two original R2 alkyl chains evaluated in this aryloxy series,
n-propyl (9j) and isopropyl (9i), produced an important (∼10
fold) decrease of activity on both isoforms. Collectively, the
results of Table 1 highlighted the following general trend for
most active R2 substituents on both isoforms: tert-butyl =
Several features of the structure were explored. Previous work
on nitrobenzene sulfonylurea suggested that the position and
the nature of the nitro group as well as the position of the
sulfonylurea moiety and the second aromatic ring were optimal,
so these parameters were kept constant (Dogne´ et al., unpub-
lished data). As shown in Table 1, we first varied the position
of the methyl group on the aromatic ring R1 (2- and 3-position),
and we addressed the influence of various alkyl side chains R2
for each position. We also completed the series of side chains
in the R1 ) 4-methylphenyl moiety series. All compounds
examined in the U46619-mediated [Ca2+]i mobilization assay
displayed potent micro- and submicromolar IC50 activities.
Consistent with previous observations, all compounds tested
were more potent on TPâ than on TPR13 in this assay.
Throughout the studies presented in Tables 1-4, 1 has been
included as the reference compound. Results obtained with
previously described compounds 9b and 9a (R1 ) 2- and
3-methyphenyl, respectively) have been included for comparison
with newly synthesized compounds. Parent compounds 2-4,
bearing a 4-methylphenyl moiety as R1, have also been included
into Table 1.

SelectiVe Antagonists of the Human Thromboxane A2 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3931
Table 1. Estimated IC50 Values for the Inhibition of [Ca2+]i Mobilization Mediated by Either TPR or TPâ upon Stimulation by U46619 (1 µM). First
Series: Influence of the Position of R1 Methyl Substituent and of the Side Chain
inhibition of U46619-mediated
[Ca2+]i mobilization (IC50, nM)a
compd
R1
R2
Y
TPR
TPâ
ratiob
1
4-methylphenyl
4-methylphenyl
4-methylphenyl
4-methylphenyl
3-methylphenyl
2-methylphenyl
2-methylphenyl
2-methylphenyl
2-methylphenyl
3-methylphenyl
3-methylphenyl
3-methylphenyl
4-methylphenyl
4-methylphenyl
tert-butyl
tert-butyl
n-pentyl
n-butyl
n-pentyl
n-pentyl
isopropyl
n-butyl
tert-butyl
isopropyl
n-butyl
tert-butyl
isopropyl
n-propyl
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
319 ( 203
58 ( 43
53 ( 19
58 ( 4
6.01
1.0
2.5
10.5
11.3
9.0
17.5
16.1
16.1
12.9
12.3
23.2
10.4
9.4
2c
3c
139 ( 88
55 ( 5
4c
558 ( 34
53 ( 2
9a
9b
9c
9d
9e
9f
9g
9h
9i
2530 ( 419
1630 ( 924
8750 ( 446
673 ( 339
3760 ( 637
2640 ( 492
1910 ( 689
398 ( 145
8760 ( 3780
4220 ( 2060
223 ( 68
181 ( 101
501 ( 63
42 ( 20
233 ( 88
205 ( 132
155 ( 30
17 ( 8
843 ( 124
448 ( 328
9j
a Results are expressed as mean ( SD of three separate experiments. b IC50TPR/IC50TPâ. c Data already published.13
Table 2. Estimated IC50 Values for the Inhibition of [Ca2+]i Mobilization Mediated by Either TPR or TPâ upon Stimulation by U46619 (1 µM).
Second Series: Influence of Nature and Position of R1 Phenyl Ring Substituents
inhibition of U46619-mediated
[Ca2+]i mobilization (IC50, nM)a
compd
R1
R2
Y
TPR
TPâ
ratiob
1
9k
9l
9m
9n
9o
9p
9q
9r
9s
4-methylphenyl
2-bromophenyl
2-bromophenyl
3-bromophenyl
3-bromophenyl
4-bromophenyl
4-bromophenyl
2-chlorophenyl
2-chlorophenyl
3-chlorophenyl
3-chlorophenyl
4-chlorophenyl
4-chlorophenyl
3-iodophenyl
tert-butyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
319 ( 203
2580 ( 1120
3130 ( 827
683 ( 189
5860 ( 2420
1660 ( 1290
1010 ( 257
2420 ( 1780
3060 ( 775
698 ( 226
53 ( 19
151 ( 73
423 ( 164
59 ( 30
413 ( 303
523 ( 145
120 ( 63
205 ( 156
387 ( 178
73 ( 9
6.01
17.0
7.4
11.6
14.2
3.2
8.4
11.8
7.9
9.6
11.9
7.6
8.9
4.9
9t
2390 ( 526
3050 ( 1670
871 ( 362
201 ( 42
400 ( 277
98 ( 45
9u
9v
9w
9x
9y
9z
470 ( 153
95 ( 35
3-iodophenyl
4-iodophenyl
4-iodophenyl
4520 ( 1800
2150 ( 307
2190 ( 1180
885 ( 389
401 ( 212
302 ( 153
5.1
5.4
7.2
a Results are expressed as mean ( SD of three separate experiments. b IC50TPR/IC50TPâ.
n-butyl > n-pentyl g n-propyl = isopropyl. For compounds
with tert-butyl and n-pentyl chains, the best substitution positions
for activity on both isoforms were 4 > 3 g 2. Regarding the
selectivity, the best alkyl side chain was R2 ) n-butyl or tert-
butyl, and the following trend was observed for these side chains
regarding the phenyl ring substitution: 3 g 2 . 4.
Furthermore, we designed compounds to address the specific
role in selectivity and activity of the nature of the substituent
on the phenyl ring R1. Former results showed that, in other
series, combination of a methyl or bromo substituent in the
4-position of the second ring with a n-pentyl side chain favored
the TPâ activity while some compounds with no substituents
in these positions showed a high affinity and activity on TPR.13
Consequently, we wished to compare the methyl group with
several other substituents. To this end, we designed and prepared
compounds which were characterized by a halogen atom instead
of the methyl group. Thus, monosubstituted derivatives with a
chlorine, bromine, and iodine atom in the 2-(except for iodine
series), 3-, and 4-position were synthesized (9k-z, Scheme 2).
For each compound, two alkyl side chains R2 were envisaged:
tert-butyl and n-pentyl, in order to collect information on the
influence of a long linear or short globular chain in this series.
Table 2 shows that the replacement of a methyl group by a
bromine atom had little or no positive effect, both in terms of
potency and selectivity. It is interesting to note that compound
9m (3-bromophenyl) had a ∼2 fold decrease of selectivity ratio

3932 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hanson et al.
Table 3. Estimated IC50 Values for the Inhibition of [Ca2+]i Mobilization Mediated by Either TPR or TPâ upon Stimulation by U46619 (1 µM). Third
Series: Influence of Large Substituent at Position 4 of R1 Aromatic Ring and Polysubstitution of R1
inhibition of U46619-mediated
[Ca2+]i mobilization (IC50, nM)a
compd
R1
R2
Y
TPR
TPâ
ratiob
1
4-methylphenyl
tert-butyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
n-pentyl
tert-butyl
tert-butyl
n-pentyl
tert-butyl
NH
O
O
O
O
O
O
O
O
O
O
319 ( 203
1940 ( 343
2670 ( 1970
10760 ( 5150
27230 ( 2650
19190 ( 11410
13560 ( 3440
1970 ( 622
1060 ( 207
3720 ( 769
528 ( 133
53 ( 19
152 ( 76
446 ( 416
875 ( 238
4840 ( 2520
1100 ( 401
749 ( 59
99 ( 61
6.01
12.7
6.0
12.3
5.6
17.5
18.1
19.9
9.6
9aa
9ab
9ac
9ad
9ae
9af
9ag
9ah
9ai
9aj
4-methoxyphenyl
4-methoxyphenyl
4-propoxyphenyl
4-propoxyphenyl
4-ethoxyphenyl
4-ethoxyphenyl
3-methoxyphenyl
3-methyl-4-chlorophenyl
3-methyl-4-chlorophenyl
2-methoxy-4-methylphenyl
111 ( 54
565 ( 200
52 ( 10
6.6
10.2
a Results are expressed as mean ( SD of three separate experiments. b IC50TPR/IC50TPâ.
compared with its potent methylated analogue 9h (11.6 and 23.2,
respectively). Consistent with our previous observation on the
activity trend, 9m, with a substituent in the 3-position and a
tert-butyl R2 alkyl chain, was the most potent compound of its
series. Compounds of the 4-bromophenyl series were less potent
than their methyl counterparts, but kept the same rank order of
selectivity ratio. In the chlorophenyl series, the same trends were
observed, with little differences compared to the bromophenyl
compounds series. Once again, the 3-position substitution of
phenyl ring combined with a tert-butyl side chain produced the
most potent compound, 9s, both on TPR and TPâ (Table 2).
Interestingly, in the iodophenyl series, although compound
potencies were in the same range as the other halo-substituted
compounds, the selectivity ratio was diminished. To this extent,
compound 9w was the most representative since its 3-position
substitution along with a tert-butyl side chain R2 increased its
TPR potency while decreasing its TPâ activity (Table 2). Thus,
9w was the most potent TPR halo-substituted antagonist in this
table with a potency close to that of 9h (TPR IC50 470 ( 153
nM and 398 ( 145 nM, respectively). From this table (Table
2), we can presume that the best combination for our derivatives
is a substituent at the 3-position of the phenyl ring R1 and a
tert-butyl side chain R2. The following trends have been
observed within these series: TPR activity: CH3 = I > Cl =
Br; TPâ activity: CH3 > Br = Cl > I; selectivity toward TPâ
isoform: CH3 > Br = Cl > I. Additionally, it is interesting to
point out that within bromo and chloro derivatives, the
combination of a 4-substituted phenyl moiety with a n-pentyl
side chain produced the most selective compounds whereas,
surprisingly, in the 4-iodo series, compounds were less selective.
In the series with a 3-substituted phenyl moiety (bromo, chloro,
and iodo), a tert-butyl side chain produced a ∼10 fold increase
in activity compared to compounds with n-pentyl side chain
(Table 2).
4-position of the phenyl ring R1 compared with the parent
methylated compounds (2 and 3). For both side chains (R2 )
tert-butyl or n-pentyl), the loss of activity depended on the length
of the alkoxy group. Interestingly, the activity on TPâ decreased
less markedly than activity on TPR, thus increasing the
selectivity ratio toward the TPâ isoform (4-ethoxyphenyl, 9ae
and 9af > 4-methoxyphenyl, 9aa = 4-propoxyphenyl, 9ac). The
most interesting compound of this table (9ag) was obtained with
the combination of a methoxy group in the 3-position of the
phenyl ring R1 and the R2 side chain being a tert-butyl moiety.
Fully consistent with observations in other series, this compound
displayed a potent antagonism of TPâ-mediated [Ca2+]i mobi-
lization combined with a good selectivity ratio (19.9). The
particular interest of this compound is that, although slightly
less potent than its methylated or halogenated analogues (9h,
9m, 9s, or 9w) on TPâ (IC50 ) ∼99 nM), it is characterized by
a much lower activity on TPR (IC50 ) ∼2 µM). Disubsituted
compounds 9ah and 9ai proved to have a combined profile of
monosubstituted 4-chlorophenyl and 3-methylphenyl analogues
(9u,v and 9a-h, respectively). Thus, the presence of a methyl
group at the 3-position of compound 9u increased ∼3-4 fold
its activity, without reaching the potent activity of the solely
3-methyl-substituted 9h (Tables 3 and 1, respectively). The
presence of a 2-methoxy group concomitant with a 4-methyl
group on the phenyl ring had little influence on TPâ activity
but decreased ∼10 fold the TPR activity compared to 2 (Tables
3 and 1, respectively). Since cyanoguanidines proved to be quite
active at both isoforms in other series,13 we synthesized an
oxygen-bonded cyanoguanidine, 10 (Scheme 3). This replace-
ment of sulfonylurea had a negative impact on the activity and
generated an inferior selectivity ratio (Table 4).
Finally, within this “oxygen-bridged” family of chemical
compounds, we wished to confirm the antiplatelet activity of
the most potent compounds. Selection relied on their activity
on TPR and TPâ or the importance of their selectivity ratio. 9h
was the first to be evaluated because it displayed the optimal
structure for both activity and selectivity among the compounds
presented herein. The IC50 of compound 9h for inhibition of
U46619-induced platelet aggregation was found to be similar
to the one for the inhibition of [Ca2+]i mobilization triggered
by TPR isoform (513 ( 28 nM and 398 ( 145 nM, respec-
tively). The concentration-response curve obtained with this
We subsequently aimed to explore the influence of longer
substituents at the 4-position of the second aromatic ring R1.
Consequently, several derivatives with a 4-alkoxyphenyl moiety
as R1 were prepared (9aa-af, Scheme 2). Moreover, some
disubstituted compounds were synthesized (9ag-aj). The R2
alkyl chain in this series was either a tert-butyl or a n-pentyl
moiety. We observed a systematically important loss of activity
on both isoforms for compounds with alkoxy groups at the

SelectiVe Antagonists of the Human Thromboxane A2 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3933
Table 4. Estimated IC50 Values for the Inhibition of [Ca2+]i Mobilization Mediated by Either TPR or TPâ upon Stimulation by U46619 (1 µM). Fourth
Series: Impact of the Sulfonylurea Function
inhibition of U46619-mediated
[Ca2+]i mobilization (IC50, nM)a
compd
R1
R2
Y
X
TPR
TPâ
ratiob
1
13c
4-methylphenyl
4-methylphenyl
tert-butyl
tert-butyl
NH
O
O
N-C N
319 ( 203
2080 ( 179
53 ( 19
579 ( 53
6.01
3.6
a Results are expressed as mean ( SD of three separate experiments. b IC50TPR/IC50TPâ.
Figure 2. (A) Concentration-response curves for inhibition of [Ca2+]i mobilization by 9 h on separate isoforms. (B) Concentration response curve
for inhibition of platelet aggregation by 9 h.
Figure 3. (A) Concentration-response curves for inhibition of [Ca2+]i mobilization by 9af on separate isoforms. (B) Concentration response curve
for inhibition of platelet aggregation by 9af.
compound on platelets can overlay the TPR curve (Figure 2,
panels A and B). These results confirmed the theory postulating
that platelet aggregation is solely mediated by TPR isoform. It
is noteworthy that 9h is inactive or almost inactive on TPR
(expressed in platelets and in HEK‚TPR cells) when tested at
0.1 µM, although is has complete antagonistic activity on TPâ
(expressed in HEK‚TPâ cells) at this concentration (Figure 2,
panel A). Thus, this compound is theoretically selective for TPâ
at this concentration.
We have conducted similar experiments on platelets with
other potentially selective compounds. Thus, 9af displayed a
similar profile with comparable IC50s for inhibition of U46619-
induced platelet aggregation and [Ca2+]i mobilization. This
compound had nevertheless its curves strongly shifted to the
right in inhibition of [Ca2+]i mobilization (Figure 3, panel A)
and was thus less active on platelets (IC50: 40 ( 9 µM, Figure
3, panel B). Consequently, with a selectivity ratio within the
same rank order, it could be more interesting since it is almost

3934 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hanson et al.
Figure 4. (A) Concentration-response curves for inhibition of [Ca2+]i mobilization by 9ag on separate isoforms. (B) Concentration response
curve for inhibition of platelet aggregation by 9ag.
triplet; m, multiplet; br, broad were used throughout. Infrared
spectra were recorded using a Perkin-Elmer FT-IR 1750. All
compounds described were recrystallized from hot methanol
(40 °C)/H2O mixture (60/40; 10 mL/100 mg of product) unless
otherwise stated.
inactive on platelets at concentrations below 10 µM but keeps
a good activity on TPâ isoform expressed in HEK293 cell lines.
Similarly, compound 9ag was one of the most interesting
compounds, since it displayed one of the best activity on TPâ,
while being poorly active on platelets (IC50: 985 ( 49 nM)
(Figure 4).
General Procedure for the Reaction of 7 with Phenols. Before
the reaction was started, the phenolates were obtained from the
corresponding cresols (0.05 mol) after their neutralization by 0.06
M NaOH (in aqueous solution, 10% w/v) in acetone. Evaporation
under reduced pressure provided crystals of the phenolates.
Compound 7 (0.01 mol) and the appropriate phenolate (0.05 mol)
were dissolved in acetonitrile. The mixture was refluxed, and
potassium carbonate (0.007 mol) was added. After completion of
the reaction monitored by TLC (12-36 h), the solution was
acidified by means of hydrochloric acid solution (10 M) and filtered,
and 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: 60-70%).
2-(4-Chlorophenoxy)-5-nitrobenzenesulfonamide (8d). Mp:
180-181 °C. Anal. (C12H9ClN2O5S) C, H, N, S.
2-(3-Chlorophenoxy)-5-nitrobenzenesulfonamide (8e). Mp:
128-130 °C. Anal. (C12H9ClN2O5S) C, H, N, S.
2-(2-Chlorophenoxy)-5-nitrobenzenesulfonamide (8f). Mp:
170-171 °C. Anal. (C12H9ClN2O5S) C, H, N, S.
2-(4-Bromophenoxy)-5-nitrobenzenesulfonamide (8g). Mp:
170-171 °C. Anal. (C12H9BrN2O5S) C, H, N, S.
Conclusions
TXA2 exerts its proaggregatory and constrictive actions upon
binding to specific GPCR named TP. In humans, two isoforms
(TPR and TPâ) of this receptor have been described that differ
exclusively in their C-carboxyl terminal tail domains. Herein,
we present the design, synthesis and SAR study of a series of
35 original N-alkyl-N′-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas
and -cyanoguanidines. These compounds were designed based
on a previous report by our group that highlighted this family
as potentially interesting in terms of antagonistic activity as well
as selectivity toward the individual TP isoforms. Results
obtained in an inhibition of [Ca2+]i mobilization test performed
on both TPR and TPâ isoforms allowed us to define the optimal
nature and position of several structural moieties for both activity
and selectivity. The study represents the first extended SAR
study dealing with selectivity among TP receptor isoforms.
Three compounds were selected for their selectivity and activity,
and their TPR antagonistic potency was confirmed in a human
platelet aggregation test.
2-(3-Bromophenoxy)-5-nitrobenzenesulfonamide (8h). Mp:
149-150 °C. Anal. (C12H9BrN2O5S) C, H, N, S.
2-(2-Bromophenoxy)-5-nitrobenzenesulfonamide(8i).Mp: 190-192
°C. Anal. (C12H9BrN2O5S) C, H, N, S.
2-(4-Iodophenoxy)-5-nitrobenzenesulfonamide (8j). Mp: 178-
179 °C. Anal. (C12H9IN2O5S) C, H, N, S.
Experimental Section
2-(3-Iodophenoxy)-5-nitrobenzenesulfonamide (8k). Mp: 153-
Chemistry. All commercial chemicals (Sigma-Aldrich, Belgium)
and solvents are reagent grade and were used without further
purification unless otherwise stated. Compounds 1, 7,16 2, 3, 4, 8a,b,
and 9a,b13 were synthesized as previously described. 2-(2-Meth-
ylphenoxy)-5-nitrobenzenesulfonamide (8c) was commercially avail-
able (AmbinterStock Screening Collection). Nevertheless, this
compound was synthesized in our lab according to the method of
preparation described below. All reactions were followed by thin-
layer chromatography (Silicagel 60F254 Merck), and visualization
was accomplished with UV light (254 nm). Elemental analyses (C,
H, N, S) were determined on a Carbo Erba EA 1108 and were
within (0.4% of the theoretical values. NMR spectra were recorded
on a Bruker Avance 500 spectrometer using DMSO-d6 or CDCl3
as solvent and tetramethylsilane as internal standard. For 1H
NMR spectra, chemical shifts are expressed in δ (ppm) downfield
from tetramethylsilane. The abbreviations s, singlet; d, doublet; t,
154 °C. Anal. (C12H9IN2O5S) C, H, N,S.
2-(4-Methoxyphenoxy)-5-nitrobenzenesulfonamide (8l). Mp:
161-166 °C. Anal. (C13H12N2O6S) C, H, N, S.
2-(4-Propoxyphenoxy)-5-nitrobenzenesulfonamide
(8m).
Mp: 135-138 °C. Anal. (C15H16N2O6S) C, H, N, S.
2-(4-Ethoxyphenoxy)-5-nitrobenzenesulfonamide (8n). Mp:
158-162 °C. Anal. (C14H14N2O6S) C, H, N, S.
2-(3-Methyl-4-chlorophenoxy)-5-nitrobenzenesulfonamide (8o).
Mp: 182-184 °C. Anal. (C13H11ClN2O5S) C, H, N, S.
2-(3-Methoxyphenoxy)-5-nitrobenzenesulfonamide
(8p).
Mp: 136-137 °C. Anal. (C13H12N2O6S) C, H, N, S.
General Procedure for the Preparation of Sulfonylureas with
Isocyanates. The appropriate sulfonamide (0.01 mol) was dissolved
in acetone (30 mL), and 0.01 mol NaOH (10% aqueous sol. w/v)
was added. The mixture was gently mixed during 10 min and then

SelectiVe Antagonists of the Human Thromboxane A2 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3935
was evaporated under reduced pressure. The resulting solid was
resuspended in acetone (30 mL) and gently refluxed. The appropri-
ate isocyanate (0.02 mol) was added to the mixture. At the end of
the reaction (0.1-1 h), the mixture was evaporated under reduced
pressure, and the crude product was washed with AcOEt. The solid
was collected by filtration and dissolved in an aqueous NaOH
solution (0.5 N; 20 mL). The resulting solution was adjusted to
pH ) 1 with hydrochloric acid (12 N), and the solid which
precipitated was isolated by filtration (Yield: 40-60%).
N-Isopropyl-N′-[2-(2-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9c). Mp: 196-198 °C. Anal. (C19c19N3O6S) C, H, N, S.
N-n-Butyl-N′-[2-(2-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9d). Mp: 179-181 °C. Anal. (C18H21N3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(2-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9e). Mp: 150-153 °C. Anal. (C18H21N3O6S) C, H, N, S.
N-Isopropyl-N′-[2-(3-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9f). Mp: 173-174 °C. Anal. (C17H19N3O6S) C, H, N, S.
N-n-Butyl-N′-[2-(3-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9g). Mp: 183-184 °C. Anal. (C18H21N3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(3-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9h). Mp: 150-152 °C. Anal. (C18H21N3O6S) C, H, N, S.
N-Isopropyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9i). Mp: 183-184 °C. Anal. (C17H19N3O6S) C, H, N, S.
N-n-Propyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (9j). Mp: 206-208 °C. Anal. (C17H19N3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(2-bromophenoxy)-5-nitrobenzenesulfonyl]-
urea (9k). Mp: 169-173 °C. Anal. (C17H18BrN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(2-bromophenoxy)-5-nitrobenzenesulfonyl]-
urea (9l). Mp: 130-135 °C. Anal. (C18H20BrN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(3-bromophenoxy)-5-nitrobenzenesulfonyl]-
urea (9m). Mp: 148-149 °C. Anal. (C17H18BrN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(3-bromophenoxy)-5-nitrobenzenesulfonyl]-
urea (9n). Mp: 136-138 °C. Anal. (C18H20BrN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-bromophenoxy)-5-nitrobenzenesulfonyl]-
urea (9o). Mp: 173-174 °C. Anal. (C17H18BrN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(4-bromophenoxy)-5-nitrobenzenesulfonyl]-
urea (9p). Mp: 166-168 °C. Anal. (C18H20BrN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(2-chlorophenoxy)-5-nitrobenzenesulfonyl]-
urea (9q). Mp: 186-187 °C. Anal. (C17H18ClN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(2-chlorophenoxy)-5-nitrobenzenesulfonyl]-
urea (9r). Mp: 132-133 °C. Anal. (C18H20ClN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(3-chlorophenoxy)-5-nitrobenzenesulfonyl]-
urea (9s). Mp: 154-156 °C. Anal. (C17H18ClN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(3-chlorophenoxy)-5-nitrobenzenesulfonyl]-
urea (9t). Mp: 158-159 °C. Anal. (C18H20ClN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-chlorophenoxy)-5-nitrobenzenesulfonyl]-
urea (9u). Mp: 161-163 °C. Anal. (C17H18ClN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(4-chloroxyphenoxy)-5-nitrobenzenesulfony-
l]urea (9v). Mp: 162-164 °C. Anal. (C18H20ClN3O6S) C, H, N,
S.
N-n-Pentyl-N′-[2-(4-ethoxyphenoxy)-5-nitrobenzenesulfonyl]-
urea (9af). Mp: 182-185 °C. Anal. (C20H25N3O7S) C, H, N, S.
N-tert-Butyl-N′-[2-(3-methoxyphenoxy)-5-nitrobenzenesulfo-
nyl]urea (9ag). Mp: 128-131 °C. Anal. (C18H21N3O7S) C, H, N,
S.
N-tert-Butyl-N′-[2-(3-methyl-4-chlorophenoxy)-5-nitrobenze-
nesulfonyl]urea (9ah). Mp: 174-176 °C. Anal. (C18H20ClN3O6S)
C, H, N, S.
N-n-Pentyl-N′-[2-(3-methyl-4-chlorophenoxy)-5-nitrobenze-
nesulfonyl]urea (9ai). Mp: 174-176 °C. Anal. (C19H22ClN3O6S)
C, H, N, S.
N-tert-Butyl-N′-[2-(2-methoxy-4-methylphenoxy)-5-nitroben-
zenesulfonyl]u rea (9aj). Mp: 160-162 °C. Anal. (C19H23N3O7S)
C, H, N, S.
Procedure for the Preparation of N-tert-Butyl-N′-cyano-O-
phenylisourea (11). A mixture of 10 (0.01 mol) and tert-butylamine
(0.015 mol) in 2-propanol (30 mL) was stirred at room temperature
for 10-15 min. The solution was evaporated, and the crude oil
1
was crystallized in cold methanol. Mp: 134-136 °C. H NMR
(DMSO) δ: 0.86 (t, 2H, J ) 9 Hz, NHCH2CH2CH2CH2CH3); 1.1-
1.35 (m, 6H, NHCH2CH2CH2CH2CH3); 3.26 (q, 2H, J ) 9 Hz,
NHCH2CH2CH2CH2CH3); 7.06-7.41 (m, 5H, Haro).
Procedure for the Preparation of N-tert-Butyl-N′-[2-(4-me-
thylphenoxy)-5-nitrobenzenesulfonyl]-N′′-cyanoguanidine (12).
The appropriate sulfonamide (0.01 mol) was dissolved in acetone
(30 mL). NaOH (0.01 mol, 10% aqueous sol. w/v) was added. The
mixture was gently mixed during 10 min and was evaporated under
reduced pressure. The solid was resuspended in dimethylformamide
(20 mL) at room temperature, and the appropriate N-alkyl-N′-cyano-
O-phenylisourea was added. The mixture was stirred at RT for 20-
24 h. At the end of the reaction, the mixture was evaporated under
reduced pressure and the crude oil was suspended in a mixture of
methanol and hydrochloric acid (5 N aqueous solution) from which
crystals of the desired product appeared. Mp: 161-163 °C. Anal.
(C19H21N5O5S) C, H, N, S.
Calcium Measurements. HEK‚TPR and HEK‚TPâ cell lines,
stably overexpressing HA-tagged forms of TPR and TPâ in human
embryonic kidney (HEK) 293 cells have been previously described.8
HEK 293 cells or their stable cell line equivalents were routinely
grown in Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS). Measurement of [Ca2+]i mobiliza-
tion either in HEK‚TPR or HEK‚TPâ cells was carried out using
fluorescent microplate reader Fluoroskan Ascent FL equipped with
two dispenser (Thermo Electron Corporation, Finland) according
to modified method of Lin et al.17 Briefly, cells were trypsinized
and 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 D-glucose, 1.3 mM CaCl2, 10 mM HEPES, pH 7.4) and
incubated for 1 h with fluorescent dye Fluo-4/AM (5 µg/mL;
Molecular Probes, Invitrogen, Merelbeke, Belgium). Cells were then
rinsed three times with Krebs-HEPES buffer, and 150 µL of a
suspension 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 with various concentrations of the test compound (10-5 to
10-8 M final; 10 µL) prior to stimulation with U46619 (1 µM final,
50 µL). In all cases, compound (1 mM) was diluted in dimethyl
sulfoxide (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 ([Ca2+]i) values by
permeabilizing cells with 1% Triton X-100 to release all the dye
(Fmax) and subsequently chelating with 10 mM EGTA (Fmin).
Calcium concentrations were calculated using equation [Ca2+]I )
Kd(F - Fmin)/(Fmax - F), assuming a Kd of 385 nM for Fluo-4.
The results (IC50) presented are the concentration required to inhibit
50% of the normal rise of [Ca2+]i upon stimulation with 1 µM
U46619, determined in the absence of any compounds. The IC50
were calculated by nonlinear regression analysis (GraphPad Prism
software) from at least three concentration-response curves.
Human in Vitro Platelet Aggregation. The antiaggregatory
potency has been determined according to the turbidimetric Born’s
N-tert-Butyl-N′-[2-(3-iodophenoxy)-5-nitrobenzenesulfonyl]-
urea (9w). Mp: 154-157 °C. Anal. (C17H18IN3O6S) C, H, N,S.
N-n-Pentyl-N′-[2-(3-iodophenoxy)-5-nitrobenzenesulfonyl]-
urea (9x). Mp: 123-126 °C. Anal. (C18H20IN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-iodophenoxy)-5-nitrobenzenesulfonyl]-
urea (9y). Mp: 176-179 °C. Anal. (C17H18IN3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(4-iodophenoxy)-5-nitrobenzenesulfonyl]-
urea (9z). Mp: 151-154 °C. Anal. (C18H20IN3O6S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-methoxyphenoxy)-5-nitrobenzenesulfo-
nyl]urea (9aa). Mp: 176-177 °C. Anal. (C18H21N3O7S) C, H, N,
S.
N-n-Pentyl-N′-[2-(4-methoxyphenoxy)-5-nitrobenzenesulfony-
l]urea (9ab). Mp: 170-172 °C. Anal. (C19H23N3O7S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-propoxyphenoxy)-5-nitrobenzenesulfo-
nyl]urea (9ac). Mp: 145-149 °C. Anal. (C20H25N3O7S) C, H, N,
S.
N-n-Pentyl-N′-[2-(4-propoxyphenoxy)-5-nitrobenzenesulfony-
l]urea (9ad). Mp: 148-150 °C. Anal. (C21H27N3O7S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-ethoxyphenoxy)-5-nitrobenzenesulfonyl]-
urea (9ae). Mp: 178-179 °C. Anal. (C19H23N3O7S) C, H, N, S.

3936 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hanson et al.
method.18 The blood was drawn from 10 healthy donors of both
genders, aged 20-30. 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.13 Platelet concentration of PRP was adjusted
to 3 × 108 cells/mL by dilution with PPP. Platelet aggregation of
PRP was studied using a double channel aggregometer (Chronolog
Corporation, Chicago, IL). PRP (294 µL) was added in a silanized
cuvette and stirred (1000 rpm). Each drug was diluted (1 mM) in
dimethyl sulfoxide (DMSO)/PBS (30/70) and preincubated in PRP
for 3 min at 37 °C before the aggregating agent was added. Platelet
aggregation was initiated by addition of a fresh solution of U46619
(1 µM 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 drug concentration
preventing 50% of platelet aggregation (IC50) induced by arachi-
donic acid and U46619 was calculated by nonlinear regression
analysis (GraphPad Prism software) from at least three dose-
response curves.
(5) Hirata, M.; Hayashi, Y.; Ushikubi, F.; Yokota, Y.; Kageyama, R., et
al. Cloning and expression of cDNA for a human thromboxane A2
receptor. Nature 1991, 349, 617-620.
(6) Raychowdhury, M. K.; Yukawa, M.; Collins, L. J.; McGrail, S. H.;
Kent, K. C., et al. Alternative splicing produces a divergent
cytoplasmic tail in the human endothelial thromboxane A2 receptor.
J. Biol. Chem. 1994, 269, 19256-19261.
(7) Reid, H. M.; Kinsella, B. T. The alpha, but not the beta, isoform of
the human thromboxane A2 receptor is a target for nitric oxide-
mediated desensitization. Independent modulation of Tp alpha
signaling by nitric oxide and prostacyclin. J. Biol. Chem. 2003, 278,
51190-51202.
(8) Walsh, M. T.; Foley, J. F.; Kinsella, B. T. The alpha, but not the
beta, isoform of the human thromboxane A2 receptor is a target for
prostacyclin-mediated desensitization. J. Biol. Chem. 2000, 275,
20412-20423.
(9) Kinsella, B. T. Thromboxane A2 signalling in humans: a ‘Tail’ of
two receptors. Biochem. Soc. Trans. 2001, 29, 641-654.
(10) Ashton, A. W.; Ware, J. A. Thromboxane A2 receptor signaling
inhibits vascular endothelial growth factor-induced endothelial cell
differentiation and migration. Circ. Res. 2004, 95, 372-379.
(11) Habib, A.; FitzGerald, G. A.; Maclouf, J. Phosphorylation of the
thromboxane receptor alpha, the predominant isoform expressed in
human platelets. J. Biol. Chem. 1999, 274, 2645-2651.
(12) Halushka, P. V. Thromboxane A(2) receptors: where have you gone?
Prostaglandins Other Lipid Mediat. 2000, 60, 175-189.
(13) Hanson, J.; Reynaud, D.; Qiao, N.; Devel, P.; Moray, A. L., et al.
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. 2006, 49, 3701-3709.
Acknowledgment. Julien Hanson is funded by the “Fonds
pour la Formation a` la Recherche dans l’Industrie et dans
l’Agriculture” (F.R.I.A., from Belgium). This work was sup-
ported by a grant from the “Fonds National de la Recherche
Scientifique” (FNRS) from Belgium. The authors wish to thank
M. Philippe Neven for excellent technical assistance. B.T.
Kinsella acknowledges the financial support of the Wellcome
Trust and Science Foundation Ireland.
(14) Dogne, J. M.; Hanson, J.; de Leval, X.; Kolh, P.; Tchana-Sato, V.,
et al. Pharmacological characterization of N-tert-butyl-N′-[2-(4′-
methylphenylamino)-5-nitrobenzenesulfonyl]urea (BM-573), a novel
thromboxane A2 receptor antagonist and thromboxane synthase
inhibitor in a rat model of arterial thrombosis and its effects on
bleeding time. J. Pharmacol. Exp. Ther. 2004, 309, 498-505.
(15) Meerwein, H.; Dittmar, G.; Gollner, R.; Hafner, K.; Mensch, F., et
al. Aromatic diazo compounds. II. Preparation of aromatic sulfonyl
chlorides, a new modification of the Sandmeyer reaction. Chem. Ber.
1957, 90, 841-852.
(16) Dogne, J. M.; Wouters, J.; Rolin, S.; Michaux, C.; Pochet, L., et al.
Design, synthesis and biological evaluation of a sulfonylcyanoguani-
dine as thromboxane A2 receptor antagonist and thromboxane
synthase inhibitor. J. Pharm. Pharmacol. 2001, 53, 669-680.
(17) Lin, K.; Sadee, W.; Quillan, J. M. Rapid measurements of intracellular
calcium using a fluorescence plate reader. Biotechniques 1999, 26,
318-322, 324-316.
Supporting Information Available: NMR, elemental analysis,
melting points, and IR peaks for all compounds presented. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Hamberg, M.; Svensson, J.; Samuelsson, B. Thromboxanes: a new
group of biologically active compounds derived from prostaglandin
endoperoxides. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 2994-2998.
(2) Moncada, S.; Ferreira, S. H.; Vane, J. R. Bioassay of prostaglandins
and biologically active substances derived from arachidonic acid. AdV
Prostaglandin Thromboxane Res. 1978, 5, 211-236.
(3) Dogne, J. M.; de Leval, X.; Hanson, J.; Frederich, M.; Lambermont,
B., et al. New developments on thromboxane and prostacyclin
modulators part I: thromboxane modulators. Curr. Med. Chem. 2004,
11, 1223-1241.
(4) Coleman, R. A.; Smith, W. L.; Narumiya, S. International Union of
Pharmacology classification of prostanoid receptors: properties,
distribution, and structure of the receptors and their subtypes.
Pharmacol. ReV. 1994, 46, 205-229.
(18) Born, G. V.; Cross, M. J. The Aggregation of Blood Platelets. J.
Physiol. 1963, 168, 178-195.
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