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

Article abstract of DOI:10.1021/jm060108a

Thromboxane A₂ (TXA₂) is an arachidonic acid metabolite involved in pathologies such as stroke, myocardial infarction, and atherosclerosis. Consequently, the design of TXA₂ receptor (TP) antagonists remains of great interest in cardiovascular medicine. The actions of TXA₂ are mediated by its specific G-protein coupled receptor of which two alternative spliced isoforms, TPα and TPβ, have been described in humans. In this study, we report the synthesis of a series of original N-alkyl-N′-[2-(cycloalkyl, alkylaryl)-5-nitrobenzenesulfonyl]urea and N-alkyl-N′-[2-(alkylaryl)-5-nitrobenzenesulfonyl]-N″- cyanoguanidines and outline their pharmacological evaluation using the individual TPα and TPβ isoforms. Among compounds analyzed, several of them exhibited greater affinity and/or functional activity for either TPα or TPβ. The most promising molecules were also found to be antiplatelet agents. From the present results, structural features involved in isoform selectivity can be proposed, and thereby several lead compounds have been identified for the further development of selective TP isoform antagonists.

Full text of DOI:10.1021/jm060108a

J. Med. Chem. 2006, 49, 3701-3709
3701
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
Julien Hanson,†,* Denis Reynaud,‡ Na Qiao,‡ Philippe Devel,† Anne-Lise Moray,† Jean-Franc¸ois Renard,† Leanne P. Kelley,§
Jean-Yves Winum, Jean-Louis Montero, B. Therese Kinsella,§ Bernard Pirotte,† Cecil R. Pace-Asciak,‡ and
Jean-Michel Dogne´†,|
Natural and Synthetic Drugs Research Centre, Department of Pharmacy, Laboratory of Medicinal Chemistry, UniVersity of Lie`ge, 1,
AV. de l’Hoˆpital, B-4000 Lie`ge, Belgium, Program in IntegratiVe Biology, Research Institute, The Hospital for Sick Children,
555 UniVersity AVenue, Toronto, Ontario, Canada M5G 1X8, School of Biomolecular & Biomedical Science, Conway Institute for
Biomolecular and Biomedical Research, UniVersity College Dublin, Ireland, Department of Pharmacy, UniVersity of Namur, FUNDP,
61 rue de Bruxelles, B-5000 Namur, Belgium, and UniVersite´ Montpellier II, Laboratoire de Chimie Biomoleculaire, UMR 5032,
Ecole Nationale Superieure de Chimie de Montpellier, 8 rue de l'Ecole Normale, 34296 Montpellier Cedex, France
ReceiVed February 1, 2006
Thromboxane A2 (TXA2) is an arachidonic acid metabolite involved in pathologies such as stroke, myocardial
infarction, and atherosclerosis. Consequently, the design of TXA2 receptor (TP) antagonists remains of great
interest in cardiovascular medicine. The actions of TXA2 are mediated by its specific G-protein coupled
receptor of which two alternative spliced isoforms, TPR and TPâ, have been described in humans. In this
study, we report the synthesis of a series of original N-alkyl-N′-[2-(cycloalkyl, alkylaryl)-5-nitrobenzene-
sulfonyl]urea and N-alkyl-N′-[2-(alkylaryl)-5-nitrobenzenesulfonyl]-N′′-cyanoguanidines and outline their
pharmacological evaluation using the individual TPR and TPâ isoforms. Among compounds analyzed, several
of them exhibited greater affinity and/or functional activity for either TPR or TPâ. The most promising
molecules were also found to be antiplatelet agents. From the present results, structural features involved in
isoform selectivity can be proposed, and thereby several lead compounds have been identified for the further
development of selective TP isoform antagonists.
Introduction
et al. identified a second isoform, generated by alternative
splicing.7 The two human TP isoforms, consequently named
TPR and TPâ, share the first 328 amino acids but differ
exclusively within their carboxy-terminal tails (15 amino acids
of the R isoform being replaced by 79 amino acids in the â
isoform).
In the 1980s, several compounds with differential activities
for TPs expressed in platelets or various types of smooth muscle
were characterized8 and none of them selectively bind to TPR
or TPâ.
Currently only one compound, 5,8-decadienoic acid, 10-
hydroxy-10-[2-(2Z)-2-octenylcyclopropyl]-, methyl ester, (5Z,8Z,-
10S)- (9CI) (PBT-3), a hepoxilin antagonist, has been described
as a preferential TPR antagonist.9 While the exact role of the
TPR and TPâ isoforms has not been fully elucidated to date, a
number of independent investigations highlight potentially
important physiological differences between them. For example,
Coyle et al. have recently demonstrated that the expression of
TPR and TPâ is under the control of two distinct promoters.10,11
Ashton et al. have also demonstrated that a specific inhibitor
of TPâ would be useful to enhance postmyocardial infarction
revascularization since the stimulation of TPâ seems to be
responsible for vascular endothelial growth factor-induced
endothelial cell differentiation and migration.12 On the other
hand, since TPR is the predominant TP isoform expressed in
platelets,13 specific inhibitor(s) of TPR may be beneficial as
antiplatelet agent(s). Moreover, it has been established that TPR,
but not TPâ, is subject to cross-desensitization of signaling by
prostacyclin leading to the proposal that TPR is the predominant
TP isoform involved in vascular hemeostasis.14 The potential
benefits of TP receptor/isoform selective antagonists in athero-
sclerosis have been recently highlighted where it was demon-
strated that compounds antagonizing TP receptors reduced
The lipid mediator thromboxane A2 (TXA2)a plays a key role
in several physiologic processes including platelet aggregation
as well as vascular and bronchial smooth muscle constriction.1,2
An 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
thromboxane synthase (TXS). COX exists as two main isoforms,
COX-1 and COX-2, which are encoded by two separate genes.
COX-1 is expressed constitutively in most tissues mediating
“housekeeping” functions, whereas COX-2 expression is mainly
induced at sites of inflammation by various stimuli.3 COX-2 is
also expressed in a constitutive manner in the brain, the vascular
endothelium, and the kidney.4 TXA2 synthesis mainly occurs
in platelets where both COX-1 and TXS are highly expressed.
The actions of TXA2 are mediated by its specific G-protein
coupled receptor (GPCR), referred to as the TXA2 receptor or
TP.5 In 1991, Hirata and colleagues reported that the human
TP was encoded by a single gene,6 and in 1994 Raychowdhury
* Corresponding author. Phone: +3243664382, Fax: +3243664362,
E-mail: J.hanson@ulg.ac.be.
† University of Lie`ge.
‡ The Hospital for Sick Children.
§ University College Dublin.
| University of Namur.
Ecole Nationale Superieure de Chimie de Montpellier.
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.
10.1021/jm060108a CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/24/2006

3702 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Hanson et al.
Scheme 1a
solution of sulfur dioxide in acetic acid in the presence of Cu-
(I) to generate 2-chloro-5-nitrobenzenesulfonyl chloride (4)
according to the Meerwein variation of the Sandmeyer reac-
tion.17 Compound 4 was poured into a solution of ammonium
hydroxide and gently heated, resulting in the synthesis of
2-chloro-5-nitrobenzenesulfonamide (5) which is the common
intermediate for the compounds studied (Scheme 1).
a Reagents: (a) NaNO2, HCl; (b) SO2, HOAc, Cu2Cl2; (c) NH4OH, ∆.
The nucleophilic substitution of the chlorine atom in the
position para to the nitro group by either amines, phenols, or
thiols led to the synthesis of the intermediates 6a-c, 8a-c, and
10a-n, respectively. Deprotonation of the sulfonamide group
of compounds 6a-c followed by reaction with the appropriate
isocyanate led to the synthesis of compounds 7a-g (Scheme
2).
atherosclerotic plaque growth.15 Several groups have also
proposed that the adverse cardiovascular effects of the selective
COX-2 inhibitor rofecoxib were due to its reduction of
prostacylin synthesis without affecting TXA2 levels, thereby
altering the balance between the anti- and prothrombotic actions
of prostacyclin and TXA2 within the vasculature.16
In the current study, we describe the synthesis of a series of
novel nitrobenzenesulfonylureas and nitrobenzenesulfonylcy-
anoguanidines and outline their pharmacological evaluation as
TP receptor antagonists. For the first time, the structure-activity
relationships of this large series of compounds for both TPR
and/or TPâ isoforms are discussed. The antiplatelet activity of
the most pharmacologically interesting compounds is also
described.
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
compounds described, commercially available 2-chloro-5-ni-
troaniline (3) reacted with sodium nitrite in acidic medium, and
the resulting solution of diazonium salt was mixed with a
Intermediates 8a-c are characterized by a sulfur atom as a
bridge between the two aromatic rings. These compounds were
synthesized by reacting thiophenols, instead of phenols, with
compound 5 in the presence of K2CO3. As with the compounds
6a-c, intermediates 8a-c were deprotonated and reacted with
the appropriate isocyanates to afford the formation of com-
pounds 9a-e (Scheme 2).
Compounds 10a-m were obtained by reacting the appropriate
amines with compound 5. The sulfonamide group of compounds
10a-m was deprotonated and then submitted to different
reaction conditions. For the synthesis of sulfonylcyanoguanidines
13a-b, a reactive synthon was first prepared by direct reaction
of diphenyl N-cyanoiminocarbonate (11) with n-pentylamine.
This synthon (12) directly reacted with the sulfonamidate salts
of 10i and 10m to generate compounds 13a-b (Scheme 3).
Scheme 2a
a Reagents: (a) R1-ONa, K2CO3; (b) R1-SH, K2CO3; (c) R1-NH2, K2CO3; (d) NaOH; (e) R2-NdCdO; (f) NaOH; (g) R2-NdCdO.

Nitrobenzenic Thromboxane Modulators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3703
Scheme 3a
a Reagents: (a) R3-NH2, 2-propanol; (b) DMF; (c) NaOH; (d) R2-NdCdO; (e) ethyl chloroformate; (f) piperidine.
tion binding studies in COS-7 cell lines transiently transfected
with respective plasmids encoding TPR or TPâ. Experiments
were initially performed at a single concentration (1 nM) of
the 35 novel test compounds or reference compounds, 1 and 2.
Competition binding studies were performed with [3H]SQ29,-
548 {5-heptenoic acid, 7-[(1S,2R,3R,4R)-3-[[2-[(phenylamino)-
carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-, (5Z)-
(9CI)}, a selective TP antagonist, as the radioligand. It has been
previously confirmed that SQ29,548 had no apparent selectivity
for either the TPR or TPâ isoform.23 Thereafter, those test
compounds that displayed the highest affinities for one or both
TP isoforms as well as compounds characterized by the greatest
differences in affinity for either TPR or TPâ were selected for
a complete concentration-response curve determination with
concentrations ranging from 10-6 to 10-11 M. Values for the
inhibitory concentration (IC)50 were obtained from the concen-
tration-response curves and a “selectivity index”, defined as
the IC50 TPR/IC50 TPâ ratio, was calculated.
Because binding experiments do not reflect the specific
activity and pharmacological profile of a molecule/ligand at its
receptor, we also examined the effects of the compounds under
study on agonist-induced intracellular signaling by the individual
TPR and TPâ isoforms. To this end, the ability of the
compounds to antagonize TPR and TPâ-mediated intracellular
calcium mobilization ([Ca2+]i) in response to the TXA2 mimetic
Figure 1. Chemical structures of 1 and 2, two TP receptor antagonists
used as references in pharmacological tests.
Compounds 10a-m also reacted, after deprotonation, with
appropriate isocyanates to generate the sulfonylureas (16a-t).
Finally, one compound (15), bearing a sulfonyl group for which
the distal nitrogen atom was fully substituted, was obtained in
two steps: (i) reaction of 10i as the sodium salt with ethyl
chloroformate followed by (ii) reaction with piperidine that led
to formation of 15 (Scheme 3).
Pharmacological Evaluation. The overall aim of this study
was to evaluate a series of novel nitro-substituted benzene
sulfonamide derivatives as pharmacological antagonists of the
TPR and TPâ isoforms of the human TXA2 receptor. Conse-
quently, we synthesized several analogues of 1 (BM-573) and
2 (BM-613, both structures are presented in Figure 1), two potent
TP antagonists characterized by a nitrobenzenic ring bearing a
sulfonylurea moiety.18-22 The binding affinities of each TP
isoform for each test compound was assessed through competi-

3704 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Hanson et al.
Table 1. Displacement of Radiolabeled [3H]SQ29,548 from TPR and TPâ by Compounds at 1 nM
binding affinity (%)a
compound
R1
R2
Y
X
TPR
TPâ
1
2
4-methylphenyl
4-methylphenyl
cyclohexyl
cyclohexyl
cyclohexyl
tert-butylamino
n-pentylamino
n-hexylamino
n-heptylamino
n-octylamino
n-pentylamino
n-pentylamino
n-pentylamino
tert-butylamino
n-pentylamino
n-pentylamino
n-pentylamino
benzylamino
cyclohexylamino
n-pentylamino
n-pentylamino
n-pentylamino
sec-butylamino
tert-butylamino
sec-butylamino
n-pentylamino
n-pentylamino
tert-butylamino
n-pentylamino
n-hexylamino
cyclohexylamino
n-butylamino
n-pentylamino
n-pentylamino
tert-butylamino
n-pentylamino
n-pentylamino
tert-butylamino
n-pentylamino
n-pentylamino
n-pentylamino
piperidino
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
O
O
O
O
O
O
S
S
S
S
S
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
49.5 ( 1.6
39.6 ( 9.1
44.2 ( 16.5
29.5 ( 3.4
17.1 ( 3.7
27.9 ( 3.0
45.3 ( 8.9
48.5 ( 3.4
28.5 ( 5.4
28.6 ( 3.4
42.3 ( 4.0
36.5 ( 4.7
32.9 ( 4.5
31.1 ( 2.9
40.1 ( 8.8
49.7 ( 5.1
32.1 ( 5.2
48.4 ( 4.0
46.4 ( 2.2
40.7 ( 2.5
48.9 ( 7.7
44.6 ( 5.4
53.3 ( 5.7
35.5 ( 2.8
55.2 ( 2.1
49.8 ( 5.7
55.7 ( 1.7
20.1 ( 10.3
35.2 ( 1.6
47.0 ( 5.6
38.5 ( 2.7
33.4 ( 2.2
32.5 ( 1.8
24.7 ( 7.6
25.5 ( 1.8
48.0 ( 6.4
46.2 ( 9.2
53.6 ( 5.5
34.2 ( 2.9
42.5 ( 2.3
36.4 ( 11.1
24.1 ( 4.4
27.5 ( 4.4
45.3 ( 3.0
35.8 ( 9.2
25.0 ( 1.5
33.1 ( 4.9
39.9 ( 4.8
54.0 ( 2.1
49.3 ( 3.5
29.0 ( 3.3
48.7 ( 16.9
49.7 ( 11.0
49.9 ( 13.0
42.5 ( 6.0
46.3 ( 2.3
40.4 ( 6.5
52.7 ( 2.2
39.0 ( 7.8
44.7 ( 2.1
48.5 ( 3.8
46.1 ( 2.1
51.9 ( 3.8
56.0 ( 4.9
22.8 ( 3.3
36.8 ( 7.1
37.7 ( 6.3
43.0 ( 1.1
32.7 ( 7.7
31.7 ( 3.4
21.3 ( 5.7
27.2 ( 5.2
53.4 ( 3.2
54.2 ( 7.8
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16q
16r
16s
16t
7a
7b
7c
7d
7e
7f
7g
9a
9b
9c
9d
9e
13a
13b
15
2,4,6-trimethylphenyl
2-methylphenyl
3-methylphenyl
3-methyl-4-bromophenyl
2,5-dimethylphenyl
2,4-dimethylphenyl
2,6-dimethylphenyl
4-methylphenyl
4-methylphenyl
3,4-dimethylphenyl
3,5-dimethylphenyl
3-methyl-4-bromophenyl
2,6-dimethylphenyl
2,6-dimethylphenyl
4-methylphenyl
4-chlorophenyl
2,3-dimethylphenyl
4-methylphenyl
4-methylphenyl
4-methylphenyl
4-methylphenyl
4-methylphenyl
3-methylphenyl
2-methylphenyl
4-methylphenyl
4-methylphenyl
2-methylphenyl
4-chlorophenyl
4-chlorophenyl
4-methylphenyl
3,5-dimethylphenyl
4-methylphenyl
N-CN
N-CN
O
a Expressed as the percentage of displaced [3H]SQ29,548 by our compounds. Results are mean ( standard deviation of at least three determinations (n
g 3). Compounds were evaluated at a final concentration of 1 nM.
U46619 {5-Heptenoic acid, 7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hy-
droxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-, (5Z)- (9CI)}
was evaluated in HEK 293 cell lines stably overexpressing either
TPR (HEK.TPR cells) or TPâ (HEK.TPâ cells). HEK.TPR and/
or HEK.TPâ cells were preloaded with fluorescent dye Fluo-4
and inhibition of the TXA2 mimetic U46619 (1 µM) stimulation
of [Ca2+]i mobilization was determined in the absence or
presence of increasing concentrations of the compounds, using
concentrations ranging from 10-5 M to 10-8 M. As for the
competition binding studies, a selectivity index was determined.
Finally, since the TP receptor directly mediates human platelet
aggregation, the efficacy of test compounds in an ex vivo human
platelet aggregation model was evaluated.
were in the same range of order of previously published data.22
The bridging atom (O, S, or NH in the series 7a-g, 9a-e, or
13, 15, 16a-t, respectively) between the nitrobenzene and the
second ring did not affect the affinity of the molecules for the
TP receptors. Additionally, in the “NH” compounds family (13,
15, and 16a-t), a critical loss of affinity was not observed as
a result of the disubstitution of the distal nitrogen atom of the
urea group or as a result of the replacement of the urea function
with a cyanoguanidine isoster moiety. Thereafter, further
experiments focused on those compounds that either displayed
the highest affinity for the TP receptors or on those that showed
significant differences in their TPR/TPâ isoform selectivity.
For compounds 7a-e, characterized by the presence of an
oxygen atom as the bridge between two aromatic rings, several
concentration-response curves were determined and are sum-
marized in Table 2. Although the affinities of the compounds
for both TPR and TPâ lie within the same range, certain
compounds exhibited a significant ratio of selectivity between
the two receptor isoforms. For example, compound 7b exhibited
a significant ratio of 4.02 (p < 0.05), which was a result of a
greater affinity for TPâ. In this family of compounds, it was
apparent that the side chain (R2) had an impact on the affinity.
Results and Discussion
The results obtained with the initial competition-binding
studies are presented in Table 1. All compounds evaluated
displayed a high affinity for both TPR and TPâ receptor
isoforms. These affinities, expressed as the percentage displace-
ment of [3H]SQ29,548, were all in the nanomolar range and
were comparable to that of the reference compounds 1 and 2
(Figure 1) used throughout this study. The affinities of 1 and 2

Nitrobenzenic Thromboxane Modulators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3705
Table 3. Estimated IC50 Values for the Inhibition of [Ca2+
Mobilization Mediated by Either TPR or TPâ upon Stimulation by
U46619 (1 µM)
Table 2. Estimated IC50 Values for Displacement of [3H]SQ29,548
from TPR and TPâ
]
i
binding affinity IC50 (nM)a
ratioa
IC50 TPR/IC50 TPâ
U46619-mediated [Ca2+
mobilization (nM)a
]
i
compound
TPR
TPâ
ratioa
1
2
7a
7b
7c
7d
7e
9a
1.05 ( 0.43
2.65 ( 1.59
1.26 ( 0.74
2.63 ( 0.94
0.72 ( 0.08
1.52 ( 0.70
0.74 ( 0.02
0.77 ( 0.33
0.46 ( 0.11
0.71 ( 0.01
2.50 ( 1.21
1.01 ( 0.25
1.52 ( 0.31
1.90 ( 0.40
1.08 ( 0.33
1.82 ( 0.25
0.78 ( 0.15
0.78 ( 0.10
3.53 ( 1.38
1.77 ( 0.69
0.65 ( 0.15
1.58 ( 0.21
0.79 ( 0.15
0.70 ( 0.24
1.74 ( 0.28
0.68 ( 0.11
1.07 ( 0.26
2.68 ( 1.06
4.43 ( 0.47
1.48 ( 0.35
1.17 ( 0.12
0.70 ( 0.43
0.58 ( 0.27
0.56 ( 0.20
1.35
0.75
0.71
4.02
0.45
1.93
1.05
0.44
0.68
0.67
0.93
0.23
1.03
1.62
1.55
3.11
1.40
compound
TPR
TPâ
IC50 TPR/IC50 TPâ
1
2
7a
7b
7c
7d
7e
9a
318.89 ( 202.54
50.04 ( 0.73
53.10 ( 19.38
13.56 ( 1.08
57.63 ( 3.97
55.38 ( 4.85
58.48 ( 0.58
70.15 ( 5.90
52.89 ( 2.54
55.17 ( 3.45
38.52 ( 2.84
46.17 ( 1.32
55.55 ( 2.26
45.53 ( 1.50
47.10 ( 0.39
45.01 ( 2.59
76.50 ( 6.39
43.19 ( 9.98
30.78 ( 9.81
6.01
3.69
1.01
2.52
9.00
8.56
10.55
5.32
1.68
1.76
1.67
1.33
1.71
1.43
7.03
2.92
1.85
58.34 ( 43.01
139.32 ( 87.93
526.42 ( 29.61
600.71 ( 11.16
558.04 ( 33.91
293.54 ( 106.53
64.81 ( 3.54
15
16a
16b
16f
16j
16k
16n
16o
16q
15
16a
16b
16f
16j
16k
16n
16o
16q
81.22 ( 3.50
92.90 ( 9.03
60.39 ( 16.07
80.72 ( 20.39
64.47 ( 1.84
537.86 ( 8.18
126.28 ( 26.07
56.89 ( 2.35
a Results expressed as mean ( standard deviation of at least three
determinations (n g 3).
a Results expressed as mean ( standard deviation of at least three
determinations (n g 3).
For example in terms of TP isoform affinity, compound 7e with
R2 ) n-butyl was the most potent compound in this assay and
seems to bear the most interesting side chain, among those
tested, for these compounds. In terms of the calculated selectivity
ratio, the side chain was also found to be involved. On one
hand, compounds for which R2 corresponds to n-pentyl or
cyclohexyl (7b and 7d, respectively) expressed a preferential
affinity for TPâ (Table 2). On the other hand, when R2
corresponds to tert-butyl or n-hexyl (7a and 7c, respectively),
there was either a loss or no change in the selectivity ratio.
Additionally, Table 2 reports the results of the determination
of the IC50 value of compound 9a, characterized by a sulfur
atom as the intercycle bridge. This compound expressed a
nanomalor affinity for the TP receptors in our model. This
observation is consistent with our previous statement that the
nature of the intercycle bridge (NH, S, or O) did not markedly
influence the affinity of the compound for TPR/TPâ.
Compound 15 is characterized by having a NH as the bridge
between the two rings and having the distal N urea atom
incorporated into a ring. Although the affinity of compound 15
for TPR or TPâ was not markedly improved compared to the
reference compounds 1 and 2 (Table 2), the incorporation of
the distal N urea atom into an alicyclic ring appeared to be
favorable for the affinity of both isoforms, with no influences
on the selectivity.
in HEK293 cell lines stably overexpressing the individual TPR
or TPâ isoforms). Calcium was the obvious second messenger
to evaluate since TPR or TPâ each couple to Gq/phospholipase
(PL)C activation, leading to inositol 1,4,5-trisphosphate (IP3)-
mediated [Ca2+]i mobilization from intracellular stores.24,25 The
[Ca2+]i mobilization was measured with cells loaded with
fluorescent dye Fluo-4, whose fluorescence increases upon
binding with calcium ion released in response to the TXA2
mimetic U46619. Consequently, the ability of the compounds
to antagonize U46619-mediated [Ca2+]i mobilization was evalu-
ated.
The results obtained with the test compounds in this assay
(Table 3) do not fully correlate with those obtained through
competition binding studies (Table 2). This can be explained
by the fact that the affinity is not the same property as activity,
i.e., that affinity does not necessarily reflect the agonistic or
antagonistic properties of a compound at its receptor. Moreover,
the mechanism and intensity whereby a compound activates or
deactivates a receptor cannot be measured through competition
binding studies. Hence, when evaluating a receptor ligand
(agonist/antagonist), it is critical to evaluate that agent through
a number of independent mechanisms, such as through binding
studies and functional studies as outlined herein.
First, it is noteworthy that all compounds assayed exhibited
high activity as TPR and TPâ receptor antagonists. Compound
16q, one of the most potent compound in this assay, was
characterized by a 2,6-dimethylphenylamino group ortho to the
sulfonamide group in the aromatic ring. As a result, it is
suggestive that a pattern of substitution by methyl groups in
the 2- and 6- positions of the second ring may provide an
interesting lead for further experimentations. Moreover, in this
functional assay, each compound exhibited a better activity on
TPR compared with TPâ, except with compound 7a which
possessed activities in the same concentration range. These data
also confirmed the fact that the side chain of the molecules could
play a role in selectivity. For example, activities on TPâ of
compounds 7b-e were within the same range as compound 7a
characterized by a tert-butyl side chain. Nevertheless, their
activities on TPR were almost 10-fold less pronounced than that
of 7a. Additionally, it should also be noted that the rank order
of potency of 7b-e for TPR did not follow the pattern of the
size or steric hindrance of the side chain. Indeed, TPR potency
Finally, with compounds of the 16 family (16a-t) bearing a
NH bridge between the two rings, data on Table 2 indicates
that, when the compounds were characterized by a second ring
(R1), namely alicyclic, the length of the side chain influenced
the affinity. For example, compound 16a with R2 ) n-hexyl
was characterized by a ∼3-fold higher affinity than 16b with
R2 ) n-heptyl The results obtained with compound 16f for
which R2 ) n-pentyl and R1 ) m-methylphenyl were of
particular interest, since it is the most selective compound for
TPR isoform. Finally, it is noteworthy that PBT-3, a stable
analogue of hepoxilin which is known to be a selective TPR
antagonist, was reported to exhibit a selectivity ratio of ∼0.17
in the same assay.9
Thereafter, we sought to extend the characterization of the
selected high affinity test compounds through functional intra-
cellular signaling studies. Hence, we further examined the
properties of the various test compounds through assessment
of U46619-induced intracellular calcium ([Ca2+]i mobilization

3706 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Hanson et al.
Table 4. Estimated IC50 Values for the Inhibition of Platelet
Aggregation Induced by U46619 (1 µM)
tion and confirmed their potent TP receptor antagonism and
their interest as antiplatelet agents.
inhibition of platelet aggregation
compound
induced by 1 µM U46619 IC50 (µM)a
Experimental Section
1
2
0.240 ( 0.013
0.278 ( 0.186
0.300 ( 0.003
0.670 ( 0.200
0.800 ( 0.090
0.900 ( 0.006
0.090 ( 0.007
Chemistry. All commercial chemicals (Sigma-Aldrich, Belgium)
and solvents are reagent grade and were used without further
purification unless otherwise stated. Compounds 5, 10d, 8a, and
8c were already described.26,27 6c was commercially available
(AmbinterStock Screening Collection). Nevertheless, these com-
pounds were synthesized in our lab according to the method of
preparation described below. All reactions were followed by thin-
layer chromatography (silica gel 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
either on a Bruker Avance 500 or on a Bruker DRX-400
spectrometer using DMSO-d6 as solvent and tetramethylsilane as
internal standard. For 1H NMR spectra, chemical shifts are
expressed in δ (ppm) downfield from tetramethylsilane. The
abbreviations d, doublet; t, 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.
General Procedure for the Reaction of 5 with Amines (10).
Compound 5 (0.01 mol) and the appropriate amine (0.05 mol) were
dissolved in 3-chlorotoluene (30 mL) and refluxed for 12-96 h.
When the reaction was finished (monitored by tcl), the mixture
was evaporated under reduced pressure. The residue was dissolved
in an aqueous NaOH solution (0.5 N, 10 mL/g of residue). This
mixture was extracted with diethyl ether and the aqueous layer was
separated and adjusted to pH 1 with 0.5 M hydrochloric acid. The
precipitate which appeared was collected by filtration, washed with
water and dried (yield: 35-95%).
General Procedure for the Reaction of 5 with Cresols (6).
Before starting the reaction, the cresolates were obtained from the
corresponding cresols (0.05 mol) after their neutralization by 0.06
mol NaOH (in aqueous solution, 10% w/v) in acetone. Evaporation
under reduced pressure provided crystals of the cresolates.
Compound 5 (0.01 mol) and the appropriate cresolate (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 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%).
7a
7b
9a
15
16q
a Results expressed as mean ( standard deviation of at least three
determinations (ng3).
was ranked 4b > 4c = 4e > 4d, indicating that n-pentyl >
n-butyl = n-hexyl > cyclohexyl. We could not conclude from
these results that the selectivity ratio is directly influenced by
the length of the side chain. Nevertheless, it is notable that in
this assay the n-pentyl side chain was responsible for the best
activity in this series compared to other linear side chains.
Finally, we sought to confirm the activity of our most
interesting molecules on human platelets by assessment of their
ability to inhibit platelet aggregation in response to the TXA2
mimetic U46619. Compounds 7a, 7b, 9a, 15, and 16q were
selected because of their ability to inhibit U46619-mediated
[Ca2+]i release, affinities, or apparent selectivity in different
models. The results obtained are summarized in Table 4. First,
compounds evaluated were indeed confirmed to act as TP
receptor antagonists inhibiting platelet aggregation in response
to 1 µM U46619 at sub-micromolar concentrations ranging from
0.09 to 0.9 µM.
It should be highlighted that all the compounds evaluated in
the three pharmacological assessment models exhibited promis-
ing results in terms of their affinities, as assessed through
competition of radioligand binding, measurements of inhibition
of [Ca2+]i mobilization, and inhibition of U46619-induced
platelet aggregation. Collective results highlighted compound
16q as a potential lead of major interest since its IC50 for
inhibition of platelet aggregation was 0.09 µM. Finally, it is
noteworthy that compound 16q is one of the most potent
compounds on TPR and is also the most potent inhibitor in
platelet aggregation studies. These data are consistent with
previous findings that TPR is the dominant isoform expressed
in human platelets,13 although this compound was equally active
on TPâ (Table 2).
Conclusions
General Procedure for the Reaction of 5 with Thiophenols
(8). Compound 5 (0.01 mol) and the appropriate thiophenol (0.05
mol) were dissolved in acetonitrile (30 mL). The mixture was
refluxed and potassium carbonate (0.007 mol) was added. When
the reaction was finished (0.1-1 h), the solution was acidified and
filtered and the filtrate was evaporated under reduced pressure. The
resulting crude oil was dissolved in methanol and ice was added.
The precipitate was recovered by filtration (yield: 50-75%).
General Procedure for the Preparation of Sulfonylureas with
Isocyanates (1, 2, 7, 9, and 16). 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 then was evaporated under reduced pressure. The
resulting solid was resuspended in acetone (30 mL) and gently
refluxed. The appropriate 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%).
We have synthesized and evaluated a series of some 40 novel
nitro-substituted benzenesulfonamide derivatives designed as
antagonists of the TP/TXA2 receptors. Since there are two TP
isoforms in humans, the development of selective compounds
for TPR and/or TPâ is clearly of great clinical interest for human
diseases. Hence, we have studied the affinity and activity of
our compounds on both TPR and TPâ. All compounds evaluated
exhibited very high affinity for both TP receptors, mainly acting
in the nanomolar range. Moreover, both receptor ligand binding
and inhibition of [Ca2+]i signaling concentration-response
curves have been determined with the most interesting com-
pounds. In these assays, all compounds confirmed their affinity
and activity for the TP receptors.
According to the biological data presented herein, we can
propose in our series of nitrobenzene-sulfonylureas and -sul-
fonylcyanoguanidines some structural factors involved in affinity
and activity on either TPR or TPâ, which could lead to the
development of selective TP receptor antagonists. Finally, the
most promising compounds were evaluated on platelet aggrega-

Nitrobenzenic Thromboxane Modulators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3707
N-tert-Butyl-N′-[2-(4-methylphenylamino)-5-nitrobenzene-
sulfonyl]urea (1). Mp: 126-127 °C. Anal. (C18H22N4O5S) C, H,
N, S.
N-n-Pentyl-N′-[2-(3, 5-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16n). Mp: 122-125 °C. Anal. (C20H26N4O5S)
C, H, N, S.
N-n-Pentyl-N′-[2-(4-methylphenylamino)-5-nitrobenzenesulfo-
nyl]urea (2). Mp: 145-147 °C. Anal. (C19H24N4O5S) C, H, N, S.
N-tert-Butyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (7a). Mp: 162-165 °C. Anal. (C18H21N3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (7b). Mp: 148-150 °C. Anal. (C19H23N3O6S) C, H, N, S.
N-n-Hexyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (7c). Mp: 128-130 °C. Anal. (C20H25N3O6S) C, H, N, S.
N-Cyclohexyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfo-
nyl]urea (7d). Mp: 170-173 °C. Anal. (C20H23N3O6S) C, H, N,
S.
N-n-Butyl-N′-[2-(4-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (7e). Mp: 207-210 °C. Anal. (C18H21N3O6S) C, H, N, S.
N-n-Pentyl-N′-[2-(3-methylphenoxy)-5-nitrobenzenesulfonyl]-
urea (7f). Mp: 157-158 °C. Anal. (C19H23N3O6S) C, H, N, S.
N-n-Pentyl -N′-[2-(2-methylphenoxy)-5-nitrobenzenesulfo-
nyl]urea (7g). Mp: 150-153 °C. Anal. (C19H23N3O6S) C, H, N,
S.
N-tert-Butyl-N′-[2-(4-methylphenylthio)-5-nitrobenzenesulfo-
nyl]urea (9a). Mp: 181-183 °C. Anal. (C18H21N3O5S2) C, H, N,
S.
N-n-Pentyl-N′-[2-(4-methylphenylthio)-5-nitrobenzenesulfo-
nyl]urea (9b). Mp: 149-151 °C. Anal. (C19H23N3O5S2) C, H, N,
S.
N-n-Pentyl-N′-[2-(2-methylphenylthio)-5-nitrobenzenesulfo-
nyl]urea (9c). Mp: 156-158 °C. Anal. (C19H23N3O5S2) C, H, N,
S.
N-tert-Butyl-N′-[2-(4-chlorophenylthio)-5-nitrobenzenesulfo-
nyl]urea (9d). Mp: 139-142 °C. Anal. (C17H18ClN3O5S2) C, H,
N, S.
N-n-Pentyl-N′-[2-(3-methyl-4-bromophenylamino)-5-nitroben-
zenesulfonyl]urea (16o). Mp: 130-132 °C. Anal. (C19H23-
BrN4O5S) C, H, N, S.
N-sec-Butyl-N′-[2-(2,6-dimethylphenylamino)-5-nitrobenzene-
sulfonyl]urea (16p). Mp: 97-99 °C. Anal. (C19H24N4O5S) C, H,
N, S.
N-tert-Butyl-N′-[2-(2,6-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16q). Mp: 95-97 °C. Anal. (C19H24N4O5S) C,
H, N, S.
N-sec-Butyl-N′-[2-(4-methylphenylamino)-5-nitrobenzene-
sulfonyl]urea (16r). Mp: 104-106 °C. Anal. (C19H24N4O5S) C,
H, N, S.
N-n-Pentyl-N′-[2-(4-Chlorophenylamino)-5-nitrobenzenesulfo-
nyl]urea (16s). Mp: 124-126 °C. Anal. (C18H21ClN4O5S) C, H,
N, S.
N-n-Pentyl-N′-[2-(2, 3-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16t). Mp: 139-142 °C. Anal. (C20H26N4O5S2)
C, H, N, S.
Procedure for the preparation of N-n-Pentyl-N′-cyano-O-
phenylisourea (12). A mixture of 11 (0.01 mol) and n-pentylamine
(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 ) 7 Hz, NH-CH2-CH2-CH2-CH2-
CH3); 1.1-1.35 (m, 6H, NH-CH2-CH2-CH2-CH2-CH3); 3.26
(q, 2H, J ) 7 Hz, NH-CH2-CH2-CH2-CH2-CH3); 7.06-7.41
(m, 5H, Haro).
General Procedure for the Preparation of Nitrobenzenesulfo-
nylcyanoguanidines (13). 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.
N-n-Pentyl-N′-[2-(4-methylphenylamino)-5-nitrobenzenesulfo-
nyl]-N′′-cyanoguanidine(13a).Mp: 162-166°C.Anal.(C20H24N6O4S)
C, H, N, S.
N-n-Pentyl-N′-[2-(3,5-dimethylphenylamino)-5-nitrobenzene-
sulfonyl]-N′′-cyanoguanidine (13b). Mp: 149-151 °C. Anal.
(C21H26N6O4S) C, H, N, S.
N-n-Pentyl -N′-[2-(4-chlorophenylthio)-5-nitrobenzenesulfo-
nyl]urea (9e). Mp: 131-135 °C. Anal. (C18H20ClN3O5S2) C, H,
N, S.
N-n-Hexyl-N′-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]-
urea (16a). Mp: 115-116 °C. Anal. (C19H30N4O5S) C, H, N, S.
N-n-Heptyl-N′-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]-
urea (16b). Mp: 117-118 °C. Anal. (C20H32N4O5S) C, H, N, S.
N-n-Octyl-N′-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]-
urea (16c). Mp: 93-94 °C. Anal. (C21H34N4O5S) C, H, N, S.
N-n-Pentyl-N′-[2-(2,4,6-trimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16d). Mp: 143-145 °C. Anal. (C21H28N4O5S)
C, H, N, S.
N-n-Pentyl-N′-[2-(2-methylphenylamino)-5-nitrobenzenesulfo-
nyl]urea (16e). Mp: 127-128 °C. Anal. (C19H24N4O5S) C, H, N,
S.
N-n-Pentyl-N′-[2-(3-methylphenylamino)-5-nitrobenzenesulfo-
nyl]urea (16f). Mp: 128-130 °C. Anal. (C19H24N4O5S) C, H, N,
S.
N-tert-Butyl-N′-[2-(3-methyl-4-bromophenylamino)-5-nitroben-
zenesulfonyl]urea (16g). Mp: 141-143 °C. Anal. (C18H21-
BrN4O5S) C, H, N, S.
N-n-Pentyl-N′-[2-(2, 5-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16h). Mp: 126-128 °C. Anal. (C20H24N6O4S)
C, H, N, S.
N-n-Pentyl-N′-[2-(2, 4-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16i). Mp: 127-129 °C. Anal. (C20H26N4O5S) C,
H, N, S.
N-n-Pentyl-N′-[2-(2, 6-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16j). Mp: 129-131 °C. Anal. (C20H26N4O5S)
C, H, N, S.
Procedure for the Synthesis of N-[2-(4-Methylphenylamino)-
5-nitrobenzenesulfonyl]-piperidine-1-carboxamide (15). Com-
pound 10i (0.01 mol) was dissolved in acetone. NaOH (0.01 mol)
(10% sol. w/v) was added. The mixture was gently mixed during
10 min and was evaporated under reduced pressure. The solid was
resuspended in acetone (10 mL). A large excess of ethyl chloro-
formate (0.03 mol) was added dropwise under vigorous stirring.
The mixture was stirred for 15 min, and the solution was evaporated
under reduced pressure. The ethyl carbamate obtained was purified
by crystallization in methanol. Ethyl 2-(4-methylphenylamino)-5-
nitrobenzenesulfonylcarbamate (14, 0.01 mol) was dissolved in a
mixture of anhydrous toluene (40 mL) and piperidine (0.02 mol).
The resulting solution was gently refluxed overnight. At the end
of the reaction, the solution was evaporated under reduced pressure,
and the residue was dissolved into an aqueous NaOH solution (0.5
N, 20 mL). This mixture was extracted with diethyl ether. The
aqueous layer was recovered and adjusted to pH 1 with hydrochloric
acid (0.5 N). The precipitate which appeared was recovered by
N-Benzyl-N′-[2-(4-methylphenylamino)-5-nitrobenzenesulfo-
nyl]urea (16k). Mp: 148-150 °C. Anal. (C21H20N4O5S) C, H,
N, S.. N-Cyclohexyl-N′-[2-(4-methylphenylamino)-5-nitroben-
zenesulfonyl]urea (16l). Mp: 166-168 °C. Anal. (C20H24N4O5S)
C, H, N, S.
1
filtration (yield: 65%). Mp: 139-141 °C. H NMR (CDCl3) δ:
N-n-Pentyl-N′-[2-(3, 4-dimethylphenylamino)-5-nitrobenze-
nesulfonyl]urea (16m). Mp: 88-90 °C. Anal. (C20H26N4O5S) C,
H, N, S.
1.60 (b, 6H, piperidine); 2.38 (s, 3H, CH3-4′); 3.39 (s, 4H,
piperidine); 7.03 (d, 1H, J ) 9 Hz, H-3); 7.17-7.29 (m, 4H, Haro′);
8.07 (dd, 1H, J ) 9 Hz, J ) 2.5 Hz, H-4); 8.75 (d, 1H, J ) 2.5

3708 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Hanson et al.
Hz, H-6); 8.85 (s, 1H, Ph-NH-Ph); 11.14 (s, 1H, S02-NH-CO-).
Human in Vitro Platelet Aggregation. The antiaggregant
potency has been determined according to the turbidimetric Born’s
method.30 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.26,31 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) connected to a linear recorder as
previously described.32 PRP (294 µL) was added in a silanised
cuvette and stirred (1000 rpm). Each compound was diluted (1 mM)
in dimethyl sulfoxide (DMSO)/PBS (30/70) and preincubated in
PRP for three minutes 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
substance concentration preventing 50% of platelet aggregation
(IC50) induced by U46619 was calculated by nonlinear regression
analysis (GraphPad Prism software) from at least three dose-
response curves.
Anal. (C19H22N4O5S) C, H, N, S.
Radioligand Binding Assay. COS-7 cells were maintained in
Dulbecco’s modified Eagle’s medium (Hyclone Laboratories,
Logan, UT) supplemented with 10% fetal bovine serum and 1%
of solution containing 10 000 units/mL penicillin G, 10 000 µg/
mL streptomycin, and 25 µg/mL amphotericin (Cellgro; Mediatech,
Herndon, VA). Cells were grown at 37 °C in a humidified
atmosphere of 95% O2 and 5% CO2. cDNAs (the cDNA was
generously provided by Prof. Perry Halushka, Medical University
of South Carolina) for the TPR and TPâ were subcloned into
pcDNA3, the resultant plasmid, pcDNA3:TPR and pcDNA3:TPâ
were introduced into COS-7 cells by the DEAE-dextran/chloroquine
method. Forty eight hours posttransfection, cells were harvested
by centrifugation at 500g for 5 min and washed three times in ice-
cold phosphate-buffered saline. Cells were resuspended in buffer
containing 25 mM HEPES/125 mM NaCl/10 µM indomethacin,
pH 7.4, and kept on ice for the binding study. Binding reactions
were carried out on 5 × 105 cells in a total volume of 0.2 mL in
the above buffer with 10 nM [3H]SQ29,548 (Perkin-Elmer Life
and analytical services, Boston, MA) added to all tubes in triplicate,
containing various concentrations of studied compounds (10-9 for
screening assay or 10-6 to 10-11 M for competition binding curves)
in 1 µL of ethanol. Additional tubes containing excess unlabeled
SQ29,548 (10 µM) (Cayman Chemical Co, Ann Arbor, MI) were
included to assess the extent of nonspecific binding. Binding was
allowed to take place for 30 min at 37 °C; free radioligand was
removed by rapid vacuum filtration through Whatman (Maidstone,
UK) GF/B glass fiber filters prewashed with the cell suspension
buffer. The tubes and the filters were rapidly washed with ice-cold
10 mM Tris buffer, pH 7.4 (three times with 3 mL). The
radioactivity on the filters containing the ligand-receptor complexes
was counted in 10 mL of Ecolite scintillation fluid (ICN, St.
Laurent, QC, Canada) in a Beckman (model LS 3800) liquid
scintillation counter. The binding experiments were performed on
whole cells.
Statistical Analysis. Results are expressed as the mean (
standard deviation from at least three determinations (n g 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.
Acknowledgment. Julien Hanson is funded by the “Fonds
pour la Formation a` la Recherche dans l’Industrie et dans
l’Agriculture” (FRIA., from Belgium). This work was supported
by a grant from the Fonds National de la Recherche Scientifique
(FNRS) from Belgium. The authors are grateful to Pierre Close
for his scientific advice and wish to thank MM. Philippe Neven
and Didier Botty for excellent technical assistance. B.T.K.
acknowledges the support of the Wellcome Trust.
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 de-
scribed.28 HEK 293 cells or their stable cell line equivalents were
routinely grown in Dulbecco’s modified Eagle’s medium (DMEM)
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.
containing 10% fetal bovine serum (FBS). Measurement of [Ca2+
]
i
mobilization 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.29 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 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 IC50s
were calculated by nonlinear regression analysis (GraphPad Prism
software) from at least three concentration-response curves.
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) Cheng, Y.; Austin, S. C.; Rocca, B.; Koller, B. H.; Coffman, T. M.
et al. Role of prostacyclin in the cardiovascular response to
thromboxane A2. Science 2002, 296, 539-541.
(5) 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.
(6) 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.
(7) 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.
(8) Mais, D. E.; Saussy, D. L., Jr.; Chaikhouni, A.; Kochel, P. J.; Knapp,
D. R. et al. Pharmacologic characterization of human and canine
thromboxane A2/prostaglandin H2 receptors in platelets and blood
vessels: evidence for different receptors. J. Pharmacol. Exp. Ther.
1985, 233, 418-424.

Nitrobenzenic Thromboxane Modulators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3709
(9) Qiao, N.; Reynaud, D.; Demin, P.; Halushka, P. V.; Pace-Asciak, C.
R. The thromboxane receptor antagonist PBT-3, a hepoxilin stable
analogue, selectively antagonizes the TPalpha isoform in transfected
COS-7 cells. J. Pharmacol. Exp. Ther. 2003, 307, 1142-1147.
(10) Coyle, A. T.; Kinsella, B. T. Characterization of promoter 3 of the
human thromboxane A receptor gene. A functional AP-1 and octamer
motif are required for basal promoter activity. FEBS J. 2005, 272,
1036-1053.
(11) Coyle, A. T.; O’Keeffe, M. B.; Kinsella, B. T. 15-deoxy Delta12,
14-prostaglandin J2 suppresses transcription by promoter 3 of the
human thromboxane A2 receptor gene through peroxisome prolif-
erator-activated receptor gamma in human erythroleukemia cells.
FEBS J. 2005, 272, 4754-4773.
(12) 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.
(13) 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.
(14) 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.
(15) Egan, K. M.; Wang, M.; Fries, S.; Lucitt, M. B.; Zukas, A. M. et al.
Cyclooxygenases, thromboxane, and atherosclerosis: plaque desta-
bilization by cyclooxygenase-2 inhibition combined with thrombox-
ane receptor antagonism. Circulation 2005, 111, 334-342.
(16) Dogne, J. M.; Supuran, C. T.; Pratico, D. Adverse cardiovascular
effects of the coxibs. J. Med. Chem. 2005, 48, 2251-2257.
(17) 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.
(18) Dogne, J. M.; Hanson, J.; de Leval, X.; Kolh, P.; Tchana-Sato, V. et
al. Pharmacological characterization of N-tert-butyl-N′-[2-(4′-meth-
ylphenylamino)-5-nitrobenzenesulfonyl]urea (BM-573), a novel throm-
boxane 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.
(21) Tchana-Sato, V.; Dogne, J. M.; Lambermont, B.; Ghuysen, A.; Magis,
D. et al. Effects of BM-573, a thromboxane A2 modulator on
systemic hemodynamics perturbations induced by U-46619 in the
pig. Prostaglandins Other Lipid Mediat. 2005, 78, 82-95.
(22) Hanson, J.; Rolin, S.; Reynaud, D.; Qiao, N.; Kelley, L. P. et al. In
vitro and in vivo pharmacological characterization of BM-613 [N-n-
pentyl-N′-[2-(4′-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a
novel dual thromboxane synthase inhibitor and thromboxane receptor
antagonist. J. Pharmacol. Exp. Ther. 2005, 313, 293-301.
(23) Parent, J. L.; Labrecque, P.; Orsini, M. J.; Benovic, J. L. Internaliza-
tion of the TXA2 receptor alpha and beta isoforms. Role of the
differentially spliced cooh terminus in agonist-promoted receptor
internalization. J. Biol. Chem. 1999, 274, 8941-8948.
(24) Kinsella, B. T.; O’Mahony, D. J.; Fitzgerald, G. A. The human
thromboxane A2 receptor 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.
1997, 281, 957-964.
(25) Walsh, M. T.; Foley, J. F.; Kinsella, B. T. Characterization of the
role of N-linked glycosylation on the cell signaling and expression
of the human thromboxane A2 receptor alpha and beta isoforms. J.
Pharmacol. Exp. Ther. 1998, 286, 1026-1036.
(26) 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.
(27) Wagner, A. W.; Banholzer, R. Heterocycles with endocyclic sulfur-
nitrogen double bond. I. 1-Substituted 5-nitro-1H-1,3,2-benzodithia-
zole 3,3-dioxides. Berichte 1963, 96, 1177-1186.
(28) 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.
(29) Lin, K.; Sadee, W.; Quillan, J. M. Rapid measurements of intracellular
calcium using a fluorescence plate reader. Biotechniques 1999, 26,
318-322, 324-316.
(30) Born, G. V.; Cross, M. J. The Aggregation of Blood Platelets. J.
Physiol. 1963, 168, 178-195.
(31) Dogne, J. M.; de Leval, X.; Neven, P.; Rolin, S.; Wauters, J. et al.
Effects of a novel non-carboxylic thromboxane A2 receptor antagonist
(BM-531) derived from torasemide on platelet function. Prostag-
landins, Leukotrienes Essent. Fatty Acids 2000, 62, 311-317.
(32) Harris, D. N.; Asaad, M. M.; Phillips, M. B.; Goldenberg, H. J.;
Antonaccio, M. J. Inhibition of adenylate cyclase in human blood
platelets by 9-substituted adenine derivatives. J. Cyclic Nucleotide
Res. 1979, 5, 125-134.
(19) Ghuysen, A.; Lambermont, B.; Dogne, J. M.; Kolh, P.; Tchana-Sato,
V. et al. Effect of BM-573 [N-terbutyl-N′-[2-(4′-methylphenylamino)-
5-nitro-benzenesulfonyl]urea], a dual thromboxane synthase inhibitor
and thromboxane receptor antagonist, in a porcine model of acute
pulmonary embolism. J. Pharmacol. Exp. Ther. 2004, 310, 964-
972.
(20) Rolin, S.; Petein, M.; Tchana-Sato, V.; Dogne, J. M.; Benoit, P. et
al. BM-573, a dual thromboxane synthase inhibitor and thromboxane
receptor antagonist, prevents pig myocardial infarction induced by
coronary thrombosis. J. Pharmacol. Exp. Ther. 2003, 306, 59-65.
JM060108A