Design, synthesis and biological evaluation of a sulfonylcyanoguanidine as thromboxane A2 receptor antagonist and thromboxane synthase inhibitor

Article abstract of DOI:10.1211/0022357011775992

The synthesis and the structure of N-isopropyl-N′-[2-(3′-methylphenylamino)-5-nitrobenzenesulfonyl] urea (14) was drawn from two thromboxane A₂ receptor antagonists structurally related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A₂ (TXA₂) receptor of human washed platelets. Compound 14 prevented platelet aggregation induced by arachidonic acid (0.6 mM) and U-46619 (1 μM) with an IC50 value of 0.45 and 0.15 μM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea precontracted by U-46619, a TXA₂ agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively. Finally, 14 (1 μM) completely inhibited TXA₂ synthase of human platelets. The pK_a value and the crystallographic data of 14 were determined and used to propose an interaction model between the TXA₂ antagonists related to torasemide and their receptor.

Full text of DOI:10.1211/0022357011775992

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 669–680
# 2001 The Authors
Received September 18, 2000
Accepted January 12, 2001
ISSN 0022-3573
Design, synthesis and biological evaluation of a
sulfonylcyanoguanidine as thromboxane A2 receptor
antagonist and thromboxane synthase inhibitor
Jean-Michel Dogne, Johan Wouters, Stephanie Rolin, Catherine Michaux,
Lionel Pochet, Francois Durant, Jacques Delarge and Bernard Masereel
Abstract
The synthesis and the structure of N-isopropyl-Nh-[2-(3h-methylphenylamino)-5-nitroben-
zenesulfonyl] urea (14) was drawn from two thromboxane A2 receptor antagonists structurally
related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A2
(TXA2) receptor of human washed platelets. Compound 14 prevented platelet aggregation
induced by arachidonic acid (0.6 mM) and U-46619 (1 µM) with an IC50 value of 0.45 and
0.15 µM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea
precontracted by U-46619, a TXA2 agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively.
Finally, 14 (1 µM) completely inhibited TXA2 synthase of human platelets. The pKa value and the
crystallographic data of 14 were determined and used to propose an interaction model
between the TXA2 antagonists related to torasemide and their receptor.
Department of Pharmacy,
University of Namur, 61 rue de
Bruxelles, 5000 Namur, Belgium
Stephanie Rolin, Lionel Pochet,
Bernard Masereel
Laboratory of Molecular
Structures, University of Namur,
61 rue de Bruxelles, 5000 Namur,
Belgium
Introduction
Thromboxane A2 (TXA2, 1) is an arachidonic acid metabolite playing a crucial role
in vasoconstriction, bronchoconstriction and platelet-aggregation. It is involved in
the aetiology of cardiovascular and pulmonary diseases such as myocardial
infarction and asthma (Ogletree 1987; Devillier & Bessard 1997). With the aim to
cure these pathologies, TXA2 synthase inhibitors, TXA2 receptor antagonists and
combined TXA2 synthase inhibitor\receptor antagonist compounds have been
developed (Dogne et al 2000). The lack of efficacy of TXA2 synthase inhibitors was
attributed to the accumulation of prostaglandin H2 (PGH2) which activated the
TXA2 receptor (Baghwat et al 1985; Fiddler & Lumley 1990). The efficacy of dual-
acting molecules as TXA2 synthase inhibitors\receptor antagonists would prevent
the biosynthesis of TXA2 and the action of accumulated PGH2. A wide variety of
TXA2 receptor antagonists have been synthesized. These antagonists (Figure 1) are
distributed into the sulfonamide derivatives from which sulotroban (2) is the lead
compound (Gresele et al 1984), into the potent ω-alkylcarboxylic compounds with
SQ-29548 (3) (Ogletree et al 1985) and seratrodast (4) (Kurokawa et al 1994) as
prototypes, and into a class of various tricyclic molecules (Hall et al 1987; Jessup
et al 1988; Ford-Hutchinson et al 1989; Miki & Ishii 1992; Theis et al 1992;
Romstedt et al 1993; Takeuchi et al 1998). In 1992, torasemide (5, Figure 2), a
Johan Wouters, Catherine
Michaux, Francois Durant
Laboratory of Medicinal
Chemistry, University of Liege,
4000 Liege, Belgium
Jean-Michel Dogne, Jacques
Delarge
Correspondence and X-ray
crystallographic data: B.
Masereel, Department of
Pharmacy, University of Namur,
FUNDP, 61 rue de Bruxelles,
5000 Namur, Belgium. E-mail:
bernard.masereel!fundp.ac.be
Funding: This study was
supported by grants obtained
from the National Fund for
Scientific Research (no.
3.4546.98, FNRS, Belgium) and
from the French Community of
Belgium. We are grateful to Ms
Ferauge of the Belgian Red
Cross for the PRP preparation.
+
+
−
diuretic sulfonylurea acting by inhibiting the Na K 2Cl co-transporter (Friedel
& Buckley 1991), was described as a poor TXA2 receptor antagonist able to relax
the canine coronary artery precontracted with TXA2 (Uchida et al 1992). A
669

670
Jean-Michel Dogne et al
O
O
COOH
SO2NH
O
COOH
OH
Thromboxane A2
1
Sulotroban 2
O
O
H3C
H3C
CH3
COOH
H
N
O
H
N
C
N
H
COOH
O
SQ-29548 3
Seratrodast 4
Figure 1 Structure of thromboxane A2 and some antagonists.
CH3
HN
O
C
CH3
SO2NH
NH CH
CH3
CH3
CH3
N
Torasemide 5
CN
N
HN
HN
N
O
C
CH3
SO2NH
CH
NH
SO2NH
C
N
CH3
BM-144 6
BM-500 7
NO2
CH3
CN
N
C
HN
SO2NH
N
14
NO2
Figure 2 Design and origin of compound 14.
screening of molecules chemically related to torasemide of the isopropylsulfonylurea group with an unusual
led to the discovery of two TXA2 receptor antagonists, sulfonylcyanoguanidine side chain. BM-500 differed
BM-144 (6) and BM-500 (7) (Figure 2) (Masereel et al from torasemide by the presence of a nitrobenzene in-
1999). Their potency was higher than that of the parent stead of a pyridine ring. To reinforce the TXA2 antago-
compound and similar to that of sulotroban. Moreover nism, we have prepared the molecule 14 bearing the side
both torasemide derivatives lost the diuretic properties chain of BM-144 and the nitrobenzene ring of BM-500
of torasemide. As compared with the structure of tora- (Figure 2). To study the effect of the acidic proton
semide, the improvement of the TXA2 antagonism of the sulfonamide function, BM-500 was methylated
observed with BM-144 was due to the replacement (15).

671
A sulfonylcyanoguanidine as thromboxane A2 receptor antagonist
anol (60 mL) to give the title compound 11. Yield: 73%,
9.48 g; mp 153–155mC; IR (KBr) 1490 and 1338 (NO2),
Materials and Methods
1
1
−
1331 and 1148 (SO2) cm ; H NMR (DMSO-d6, 400
MHz). δ 8.59 (s, 1H, 6H-NO2phenyl), 8.20 (d, 1H, 4H-
NO2phenyl), 7.38 (d, 1H, 3H-NO2phenyl), 7.11–7.22
(m, 4H, phenyl), 2.36 (s, 3H, CH3). Anal. (C13H13N3O4S).
Expected (C, H, N, S in %) 50.81, 4.26, 13.67, 10.43;
found 51.17, 4.13, 13.58, 13.81.
Chemistry
Melting points were determined on a Buchi B-540
capillary apparatus. IR spectra were recorded as KBr
pellets on a Perkin-Elmer 1750 FT spectrophotometer.
1
The H NMR spectra were taken on a Jeol JNM-EX
400 (400 MHz) instrument in DMSO-d6 with hexa-
methyldisilane as an internal standard; chemical shifts
were reported in δ values (ppm) relative to internal
hexamethyldisilane. The abbreviations used are as
follows; s l singlet, d l doublet, t l triplet, m l
multiplet, and b l broad signal. Elemental analyses
(C, H, N, S) were performed on a Carlo-Erba NA 1500
elemental analyser and were withinp0.4% of the theo-
retical values. All reactions were routinely checked by
1-(1h-Hexamethyleneimino)-1-methylthio-2-cyanoimine
(13)
Hexamethyleneimine (3.1 mL, 27.5 mmol) was re-
fluxed with N-cyano-S,Sh-dimethyldithioiminocarb-
onate (Hantzsch & Wolvekamp 1904) (12; 3.6 g,
24.6 mmol). After 4-h of reaction, the medium was
evaporated under reduced pressure, the crude residue
added to 50 mL 0.1  HCl, and the bottom layer was
extracted three times with CHCl3. The organic phase
was dried with anhydrous MgSO4 and chloroform evap-
oratedunderreducedpressuretogivethetitlecompound
13. Yield: 64%, 3.1 g; mp 35–37mC; 1H NMR (DMSO-
d6, 400 MHz). δ 3.33 (m, 4H, CH2-N-CH2), 2.72 (s, 3H,
CH3S), 1.50–1.70 (2m, 8H, -(CH2)4-). Anal. (C9H15N3S).
Expected (C, H, N, S in %) 54.79, 7.66, 21.30, 16.25;
found 55.02, 7.48, 21.09, 16.17.
TLC on silica gel Merck 60F254
.
2-Chloro-5-nitrobenzenesulfonamide (10)
An aqueous solution of sodium nitrite (0.1 mol, 10 mL)
was added dropwise to a cooled solution (k5mC) of 2-
chloro-5-nitroaniline (8; 10 g, 58 mmol) in acetic acid
(100 mL) and 12  HCl (40 mL). Copper chloride (4 g,
30 mmol) dissolved in water (10 mL) was poured into
acetic acid (160 mL) previously saturated with SO2. This
last mixture was added to the diazonium salt and stirred
for 2 min. After the addition of crush-ice (200 g), the
precipitate 9 was collected by filtration and washed with
cold water. The resulting sulfonyl chloride (9) was
rapidly dissolved in 150 mL aqueous NH4OH (28–
32%). The solution was concentrated to 50 mL under
reduced pressure and acidified with 5  HCl to pre-
cipitate the title compound 10 which was collected,
washed with water and dried. Yield: 57%, 7.82 g; mp
Nhh-[2-(3h-methylphenylamino)-5-nitrobenzenesulfo-
nyl]-N-cyano-NhNh-hexamethylene guanidine (14)
The sodium salt of 2-(3h-methylphenylamino)-5-nitro-
benzene sulfonamide (11; 1.0 g, 3.3 mmol) and 1-(1h-
hexamethyleneimino)-1-methylthio-2-cyanoimine (13;
1.1 g, 5.6 mmol) were dissolved in a mixture of 1,4-
dioxane (3 mL) and N,N-dimethylformamide (2 mL),
and refluxed for 7 h. After evaporation of solvents under
reduced pressure the residue was dissolved in water
(50 mL) and 2.5  NaOH (2 mL). The solution was
extracted three times with diethyl ether (50 mL) and
adjusted to pH 1 with dilute HCl. The precipitate was
collected by filtration, washed with water, dried and
crystallized from boiling methanol (40 mL) to afford the
title compound 14. Yield: 30%, 0.44 g; mp 161–163mC;
IR (KBr) 1502 and 1306 (NO2), 1160 and 1329 (SO2),
177–179mC; IR (KBr) 1538 and 1354 (NO2), 1386 and
1
1166 (SO2) cm ; 1H NMR (DMSO-d6, 400 MHz).
−
δ 8.70 (s, 1H, 6H-NO2phenyl), 8.44 (d, 1H, 4H-
NO2phenyl), 7.96 (d, 1H, 3H-NO2phenyl). Anal.
(C6H5N2O4SCl). Expected (C, H, N, S in %) 30.45, 2.13,
11.84, 13.55; found 30.62, 2.23, 11.81, 13.92.
2-(3h-Methylphenylamino)-5-nitrobenzene sulfonamide
(11)
1
1
−
2-Chloro-5-nitro-benzenesulfonamide
(10;
10 g, 2182 CꢀN cm ; H NMR (DMSO-d6, 400 MHz).
42 mmol) was refluxed for 3 h with 3-methylaniline δ 8.80 (b, 1H, NH-phenyl), 8.49 (s, 1H, 6H-NO2phenyl),
(10 mL, 93 mmol) in 3-chlorotoluene (20 mL), the sol- 8.12 (d, 1H, 4H-NO2phenyl), 7.33 (d, 1H, 3H-
vent was evaporated under reduced pressure and the NO2phenyl), 7.02–7.20 (m, 4H, phenyl), 3.48 (m, 4H,
residue dissolved in 2.5  NaOH (100 mL). The solution CH2-N-CH2), 2.32 (s, 3H, CH3-phenyl), 1.41–1.57 (2m,
was extracted three times with cyclohexane (100 mL) 8H, -(CH2)4-). Anal. (C21H24N6O4S). Expected (C, H, N,
and adjusted to pH 1 with 5  HCl. The precipitate was S in %) 55.25, 5.30, 18.41, 7.02; found 55.36, 5.41,
collected, washed with water and crystallized in meth- 18.23, 7.14.

672
Jean-Michel Dogne et al
N-isopropyl-Nh-[2-(3h-methylphenylamino)-5-nitro-
benzenesulfonyl] urea (7)
NaH2PO4 0.4, NaHCO3 12, -glucose 5, HEPES pH
7.40. The reaction was terminated by addition of 4 mL
The sodium salt of 2-(3h-methylphenylamino)-5-nitro- ice-cold Tris-HCl buffer (10 m, pH 7.40), followed by
benzene sulfonamide (11; 1.0 g, 3.3 mmol) was dis- rapid filtration through Whatman GF\C glass filters.
solved in 20 mL methanol and tetrahydrofuran (50:50). Non-specific binding was defined as the amount of
Isopropyl isocyanate (0.65 mL, 6.8 mmol) was added radioactivity bound in the presence of a large molar
and the mixture refluxed for 30 min. The solvents were excess (10 µ) of SQ-29548. It is 5–7% of the total
evaporated under reduced pressure and the residue binding determined by the radioactivity in absence of
dissolved in water (50 mL) and 2.5  NaOH (2 mL). competing ligand. The IC50 was defined as the drug
The solution was extracted three times with diethyl concentration required to displace 50% [3H]SQ-29548
ether (50 mL) and adjusted to pH 1 with dilute HCl. bound to the TXA2 receptor. Values are the mean of
The precipitate was collected by filtration, washed with three concentration–response curves performed in trip-
water, dried and crystallized from boiling methano– licate and calculated by non-linear regression (Graph-
l–water (30 mL) to afford the title compound 7. Yield: Pad Prism software).
76%; IR (KBr) 1581 and 1307 (NO2), 1332 and 1158
1
1
−
(SO2) cm ; H NMR (DMSO-d6, 400 MHz). δ 10.88
(br s, 1H, -CO-NH-C!) 8.95 (br s, 1H, NH-phenyl),
8.77 (s, 1H, 6H-NO2phenyl), 8.36 (d, 1H, 4H-
NO2phenyl), 7.52–7.27 (m, 5H, 3H-NO2phenyl and
phenyl), 3.67 (m, 1H, -CH!), 2.50 (s, 3H, CH3-phenyl),
1.19 (2s, 6H, C(CH3)2. Anal. (C17H20N4O5S). Expected
(C, H, N, S in %) 52.03, 5.14, 14.28, 8.17; found 51.94,
5.23, 14.37, 8.28.
Platelet aggregation
Blood was obtained from the antecubital vein of volun-
teers who had not taken any medication within the
previous 10 days. Platelet-rich plasma (PRP) and plate-
let-poor plasma (PPP) were prepared as described by
Masereel et al (1999). The PRP was diluted to 3i108
1
−
platelets mL , and the PPP was used to adjust the
photometric measurement to the minimum optical den-
sity. The drug (or vehicle) was added to the PRP and
N-isopropyl-Nh-methyl-Nh-[2-(3h-methylphenylamino)-
5-nitrobenzenesulfonyl] urea (15)
1
−
stirred at 1100 rev min for 3 min at 25mC (aggre-
gometer Chronolog Corporation). The aggregation was
induced by addition of a freshly prepared solution of the
sodium salt of arachidonic acid (0.6 m final) or U-
46619 (1 µ). The aggregation curve was recorded for
5 min. The drug concentration (IC50) reducing platelet
aggregation by 50% was calculated by non-linear re-
gression analysis (GraphPad Prism software) from at
least four dose–response curves.
N-isopropyl-Nh-[2-(3h-methylphenylamino)-5-nitro-
benzenesulfonyl] urea (14; 1.0 g, 2.5 mmol) was
dissolved in methanol (20 mL) containing KOH
(2.5 mmol). After cooling (4mC), methyl iodide
(0.19 mL, 3 mmol) was added and the mixture stirred
for 1 h. Methanol was evaporated under reduced press-
ure, the crude residue was crystallized from water-
methanol to afford the title compound. Yield: 68%; mp
143–145mC; IR (KBr) 1576 and 1308 (NO2), 1335 and
1
1
−
1161 (SO2) cm ; H NMR (DMSO-d6, 400 MHz). δ
10.90 (br s, 1H, -CO-NH-C!) 8.93 (br s, 1H, NH-
Rat aorta and guinea-pig trachea contraction
phenyl), 8.76 (s, 1H, 6H-NO2phenyl), 8.38 (d, 1H, 4H- Rat aortic (male Wistar rats) and guinea-pig tracheal
NO2phenyl), 7.50–7.27 (m, 5H, 3H-NO2phenyl and phe- rings were taken from anaesthetised animals (80 mg
1
−
nyl), 3.70 (m, 1H, -CH!), 3.01 (s, 3H, SO2-N-CH3) 2.51 kg nembutal). The rings were suspended under 1g
(s, 3H, CH3-phenyl), 1.21 (2s, 6H, C(CH3)2. Anal. tension, equilibrated for 1 h in an organ bath containing
(C18H22N4O5S). Expected (C, H, N, S in %) 53.19, 5.46, 20 mL Krebs solution (in m: NaCl 118, KCl 4.7,
13.78, 7.89; found 52.94, 5.62, 13.89, 8.11.
KH2PO4 1.2, MgSO4 1.2; CaCl2 2.5, NaHCO3 25, -
glucose 5, indomethacin 1) kept at 37mC and bubbled
continuously with a mixture of O2\CO2 (95\5). The
aortic and tracheal rings were then exposed to 20 and
Receptor binding assay
Washed human platelets were prepared as described by 10 n U-46619, respectively. When the tension was
Masereel et al (1999). Incubation (1 mL) containing stable, the TXA2 receptor antagonists (50 µL) were
8
1
−
500 µL platelet suspension (2i10 cells mL ), 100 µL added to the bath at cumulatively increasing concen-
[3H]SQ-29548 (5 n final) and 400 µL drug at a fixed trations until relaxation had reached maximum. The
concentration was performed at 25mC for 60 min. The tension was measured with an isometric transducer and
incubation medium was (in m): NaCl 137, KCl 2.7, recorded by the Iox acquisition data software (Emka

673
A sulfonylcyanoguanidine as thromboxane A2 receptor antagonist
Technologies, Paris, France). The IC50 value of each 13.85(2), α l 94.30(1), β l 97.56(1), γ l 103.22(1),
drug was assessed for six concentration–response curves V l 1593.8(3) A3) were refined from 25 well-centred re-
and expressed as the concentration evoking 50% in- flections. A total of 5985 reflections were measured.
hibition of the plateau induced by U-46619. The IC50 Data were corrected for background, Lorentz-polar-
values and their standard error were calculated by non- ization, and absorption (Tmin l 0.42, Tmax l 0.63)
linear regression analysis (GraphPad Prism software).
effects. The structure was solved by direct methods and
refined (full matrix least squares on intensities) with
Shelxl97 (Sheldrick & Schneider 1997). Molecule 14
crystallized with one molecule of chloroform and one
Thromboxane synthase inhibition
8
1
−
PRP (3i10 platelets mL ) and PPP were prepared as molecule of toluene. Final statistics: C21H24N6O4S-
described by Masereel et al (1999). Six minutes after the CHCl3-C7H8, Mr l 668.0, P-1, Dx l 1.392, Z l 2,
addition of drug (or vehicle) to the PRP, the throm- F(000) l 696, µ l 3.59, R1 l 0.0745 for 4215 I " 2σ(I)
boxane synthesis was stimulated by addition of arachi- and wR l 0.198, S l 1.037, =ρ max l 1.05 (close to
donic acid (0.6 m). The incubation (25mC) was stopped the methyl group of the toluene solvent molecule).
after 4 min by addition of indomethacin (1 m), the
PRP was centrifuged (16000 g for 10 s) and two samples
(400 µL) of the supernatant assayed for TXB2. Serum
TXB2, the stable metabolite of TXA2, was measured by
Molecular modelling
enzyme immunoassay (TXB2 EIA kit, Cayman Chemi- Docking simulations of 6, 7, and 14 in the human TXA2
cal). Results were expressed as the percentage of TXB2 receptor were performed. A model of the trans-
production compared with that of PRP incubated with membrane spanning helices of the receptor was built
arachidonic acid in absence of any drug (100%). The using the GPCR mode of SwissModel and bacteriorho-
basal production of TXB2 by unstimulated platelets was dopsin (1bac.pdb) as the template (Guex & Peitsch
3.2%. Results are the meanps.d., n l 3.
1997, 1999; Guex et al 1999). The alignment of the
target and template sequences was taken from the work
of Yamamoto et al (1993). The crystal geometry of 14
was used as input for the co-ordinates of the ligands.
pKa determination
Compound 7 was dissolved at a concentration of 20 µ The geometry for 6 and 7 were obtained by replacing the
in a mixture containing 10, 20 or 30% CH3OH and the nitrobenzene or the N-cyano-NhNh-hexamethylene-
appropriate buffer prepared with sodium citrate\HCl guanidinemoietyof14byapyridineorNh-isopropylurea
(pH 1–4.5) or Na2HPO4\KH2PO4 (pH 4.5–8.0). The pH group, respectively. Starting geometry for the complexes
of each solution was measured, and its optical density were obtained using a Monte-Carlo procedure available
(230–500 nm) was recorded against blank with a double- from the Affinity program of MSI (San Diego, CA)
beam UV-visible spectrophotometer (Perkin-Elmer using default parameters. Parameters of the extensible
Lambda 20). The wavelength of the maximum absorp- and sytematic force field were used along the entire
tion was plotted against the pH value (Figure 4). For procedure. According to previous studies (Yamamoto
each percentage of CH3OH, the inflection point of the et al 1993; Wouters et al 1999) a binding site was defined
sigmoid curve calculated by non-linear regression ana- in the receptor that comprised residues: Met-112, Ile-
lysis (GraphPad Prism software) gave the pKa value. 113, Phe-115, Gly-116, Leu-120, Leu-161, Leu-163, Gly-
Finally, the pKa values obtained at 10, 20 and 30% 164, Leu-165, Leu-166, Pro-167, Leu-168, Leu-169 Val-
(v\v) CH3OH were plotted against the percentage of co- 171, Ser-201, Met-202, Gly-204, Gly-205, Leu-206,
solvent used, and the linear regression led to extrapolate Val-208, Leu-210, Phe-212, Leu-262, Arg-295. The best
the pKa value of 7 at 0% of CH3OH (Figure 4): pKa l structures of the complex of the human TXA2 receptor
(0.0176i%CH3OH)j3.905; r l 0.9999; P ! 0.001 with 6, 7, and 14 were optimized further with the
(GraphPad Prism software).
Discover program (MSI, San Diego, CA) using a first
molecular dynamics run (300 K, 1000 fs) followed by
energy minimization (steepest descent and conjugated
X-ray crystallography
1
−
gradient) to a final convergence of 1 kcal mol . The
The crystal used to collect X-ray diffraction data of 14 conformation of the ligands and the lateral chains of all
was obtained by slow evaporation of a concentrated amino acid residues of the binding site were allowed to
solution in a chloroform\toluene mixture. Cell para- move. The solvent effect was approached by using a
meters (triclinic, a l 9.980(1), b l 12.020(10), c l distance dependent dielectric constant (1*r).

674
Jean-Michel Dogne et al
enesulfonyl]-N-cyano-NhNh-hexamethyleneguanidine
(14) was prepared by reaction of 13 with the sodium salt
of the sulfonamide 11. The sulfonylurea 7 was pre-
pared by the reaction of the sodium salt of 11 with
isopropylisocyanate, then the potassium salt of 7 was
methylated by CH3I to give 15.
Results and Discussion
Chemistry
The diazonium salt prepared from 2-chloro-5-nitroanil-
ine (8) reacted with sulfur dioxide to afford the cor-
responding sulfonyl chloride 9 which reacted immedi-
ately with NH4OH to give 2-chloro-5-nitrobenzene-
sulfonamide (10; Figure 3). The sulfonamide
10 refluxed with 3-methylaniline to give 2-(3h-
Biological evaluation
methylphenylamino)-5-nitro-benzenesulfonamide (11). The affinity for the TXA2 receptor of human washed
The N-cyano-S,Sh-dimethyldithioiminocarbonate 12 platelets was evaluated as the drug concentration (IC50)
(Hantzsch & Wolvekamp 1904) refluxed with an excess required to displace 50% [3H]SQ-29548, a potent com-
(1.1 equiv.) of hexamethyleneimine to afford 1-(1h- petitive and specific ligand of this site (Table 1) (Ogletree
hexamethyleneimino)-1-methylthio-2-cyanoimine (13). et al 1985). Compound 14 was found as active as SQ-
Finally, Nh’-[2-(3h-methylphenylamino)-5-nitro-benz- 29548 and more active than the parent compounds (6
Cl
Cl
Cl
(i)
NH2
(ii)
SO2Cl
SO2NH2
8
9
10
NO2
NO2
NO2
(iii)
CN
N
C
H3C
S
S
CH3
CH3
CH3
12
(iv)
HN
HN
O
C
CH3
CH3
(vi)
CN
N
SO2NH2
SO2NH
NH CH
H3C
S
C
N
11
7
NO2
13
NO2
(vii)
(v)
CH3
CH3
CN
N
HN
HN
O
C
CH3
CH3
SO2N
CH3
15
NH CH
SO2NH
C
N
14
NO2
NO2
Figure 3 Synthesis of TXA2 receptor antagonists related to torasemide. i, CH3COOH, HCl, NaNO2 ; CH3COOH, SO2, CuCl2 ; ii, NH4OH;
iii, 3-CH3-C6H4-NH2 ; iv, hexamethyleneimine; v, 1 equiv. NaOH, dimethylformamide, dioxane; vi, NaOH, (CH3)2CH-NlClO; vii, KOH,
CH3OH, CH3I.

675
A sulfonylcyanoguanidine as thromboxane A2 receptor antagonist
Table 1 Affinity for the TXA2 receptor, anti-aggregating efficacy on human platelets, rat aorta and guinea-pig trachea relaxation induced by
reference molecules, torasemide and its related compounds.
Drug
Affinity for the TXA2
receptor (IC50 µ)a
Platelet aggregation
Aorta IC50 (n)c
Trachea IC50 (n)c
Arachidonic acid
(IC50 µ)b
U-46619 (IC50 µ)b
Sulotroban (2)
0.93p0.15
0.021p0.001
nd
12.3p2.1
0.034p0.002
35.0p4.3
" 100
9.4p0.8
14.0p0.3
0.45p0.04
nd
10.1p1.7
0.035p0.002
5.1p0.7
" 100
12.9p1.0
9.5p0.7
0.15p0.02
nd
1620p182
21.1p0.8
48.5p4.5
3608p260
120p10.3
190p16.6
20.4p1.8
nd
465p39
3.75p0.56
5.68p0.96
4020p380
110p13.4
112p7.5
5.47p0.95
nd
SQ-29548 (3)
Seratrodast (4)
Torasemide (5)
2.69p0.07
0.28p0.02
0.080p0.08
0.022p0.002
" 100
6
7
14
15
aConcentration displacing 50% of [3H]SQ-29548 specifically bound to the TXA2 receptor of human washed platelets. bConcentration required
c
to reduce by 50% platelet aggregation induced by arachidonic acid (0.6 m) or U-46619 (1 µ). Concentration required to reduce by 50%
the rat aortic tonus and the guinea-pig tracheal tonus induced by 20 and 10 n U-46619, respectively ; nd, not determined. Values are
meanps.d., n l 3–5.
Table2 InhibitionoftheTXA2 synthaseisexpressedasthereduction
of the production of TXB2 induced by 0.6 m arachidonic acid
(control l 100%) on human platelets. The TXB2 production of
unstimulated platelets was 2.7p0.4%.
and 7). Its IC50 value was 122- and 42-times lower than
that of torasemide and sulotroban, respectively. The
Hill coefficient of 14 (1.035p0.08) was similar to that of
SQ-29548 (1.023p0.03). This suggested a competitive
binding to the TXA2 receptor. The N-methylsulfonyl-
urea 15 had no affinity.
1
−
Drug
Concn (µmol L
)
% of TXB2
production
TXA2 antagonism was evaluated by the ability of the
compounds to inhibit human platelet aggregation in-
duced by arachidonic acid, the natural precursor of
TXA2, or by U-46619, a TXA2 agonist (Coleman et al
1981; Liel et al 1987). When using arachidonic acid, the
anti-aggregating potency of 14 was higher than that of
6, 7, sulotroban and seratrodast (Table 1). SQ-29548
remained the most powerful antagonist studied. Similar
results were obtained when U-46619 was used as the
aggregating agent (Table 1). Indeed, SQ-29548 was still
four-times more active than 14. The molecules related to
torasemide showed a discrepancy between the IC50
values calculated from the binding experiment on
Control
100p5.9
105p6.8
32.5p4.7***
98.8p8.6
99.3p1.3
104p4.1
Furegrelate
1
10
10
10
0.1
1
Furegrelate
6
7
14
14
14
4.4p1.1***
3.7p0.1***
10
Values are meanps.d., nl3. ***P ! 0.001 compared with the control
value.
washed platelets and from the aggregating tests. This isolated trachea contracted by U-46619 (10 n). Com-
could be assigned to the high affinity of sulfonamide pound 14 was 20-times more active than its precursors
compounds for plasma proteins present in the aggre- (6,7). Thepotencyof14wassimilartothatofseratrodast
gating experiment (Cozzi et al 1994; Soyka et al 1994). and SQ-29548 (Table 1).
The TXA2 antagonism of 14 was confirmed also by its
According to the interest of a combined TXA2 re-
ability to relax the rat aortic ring precontracted by U- ceptor antagonist\synthase inhibitor therapy, their in-
46619 (20 n). Its potency was similar to that of SQ- hibitory potency on thromboxane synthase of human
29548. Once more, 14 was six- and ten-times more platelets was evaluated. The inhibition of thromboxane
potent than 6 and 7, respectively. As seratrodast, the synthase was estimated by the reduction of the platelet
only TXA2 receptor antagonist launched, is indicated in production of TXB2, the stable metabolite of TXA2,
the treatment of asthma, we evaluated the ability of induced by 0.6 m arachidonic acid (Table 2). At 1 µ,
torasemide and its derivatives to relax the guinea-pig 14 was found more active than furegrelate, a TXA2

676
Jean-Michel Dogne et al
Crystallography
synthase inhibitor chosen as reference (Johnson et al
1986), while 6 and 7 were inactive at 10 µ.
Molecule 14 crystallized with one molecule of chloro-
form and one molecule of toluene. An Oak Ridge
Thermal Ellipsoid Plot diagram of the crystal structure
is given in Figure 5. In the crystal structure, 14 adopted
a folded conformation (Figure 5) with the cyano group
facing the nitrophenyl ring to favour van der Waals
interactions. Stacking between the toluene solvent
molecule and the toluyl ring of 14 also stabilized the
molecules in the solid state. Crystal cohesion was further
reinforced by intra (N8-H8 … O20 : 2.786(5) A, 138.6m)
and inter (N21-H21 … N25 (1-x, 1-y, 1-z): 2.880(5) A,
177.6m) molecular hydrogen bonds.
Bond lengths and valence angles corresponded to the
standard values measured for torasemide and its related
compounds. Based on the values of the torsion angles
φ1, φ2, φ3 and φ4 measured in the crystal, four theor-
etical conformations (α, β, γ, δ) were defined for these
molecules (Table 3) (Masereel et al 1995). Nevertheless,
the conformation of 14 differed from the δ one observed
with the pyridylsulfonylcyanoguanidine (Dupont et al
1995) and from those adopted by torasemide and its
related pyridylsulfonylureas (Dupont et al 1978, 1991).
Indeed, the conformation deduced from the crystal
structure of 14 did not corresponded to any one de-
scribed among our series (Table 3). More structure
determinations of other analogues are required to see if
this is specific to the nitrobenzene series.
Dissociation constants (pKa)
Except for 6 and 7, all the TXA2 receptor antagonists
described bear a carboxylic function, which is claimed
to interact with the TXA2 receptor (Yamamoto et al
1993; Masereel et al 1999). For this reason, the pKa
value of the sulfonylurea moiety of 7 was determined
by UV-spectrophotometry. As observed for many pH
indicators, 7 revealed a bathochromic effect associated
to the decrease of pH (Figure 4). For 7, the maximum
optical densities (λmax) of the molecular and anionic
forms were 375 and 391 nm, respectively. Due to the
poor solubility of 7 in aqueous buffers, methanol (10, 20
and 30%) was used as co-solvent. As shown, the pH
value corresponding to the inflection point was the pKa
value of 7, where the concentration of both forms was
equal. Extrapolated to 0% of methanol, the pKa value
of 7 was 3.90p0.01 (Figure 4). Compared with tora-
semide (5; pKa l 6.68) (Masereel et al 1994), the re-
placement of the pyridine by a nitrobenzene ring
strongly dropped the pKa value of the sulfonylurea
function. These results indicated clearly that 7 was under
its anionic form at physiological pH (7.4) and was able
to establish an ionic bond with the TXA2 receptor.
O
O
C
Molecular modelling and receptor–ligand
interactions
pKa = 3.90
SO2NH C NH
SO2N
NH
Compounds 6, 7, and 14 have been docked into a model
of the human TXA2 receptor to compare their inter-
actions with the protein (Figure 6). Starting geometry
for the complexes were obtained using a Monte-Carlo
approach (Affinity, MSI, San Diego, CA) and optimized
further by a molecular dynamics run followed by energy
minimization (Discover, MSI, San Diego, CA).
390
385
380
375
370
4.5
4.4
4.3
4.2
Although limited e.g. no mutational data were re-
ported for the receptor that could identify or check the
influence of amino acids in the binding of ligands, the
present approach attempted to rationalize the existing
pharmacological data and could be useful for designing
original molecules. The total non-bonded interaction
energy calculated between the binding site of the re-
4.1
4.0
3.9
3.8
0
10
20
30
40
% CH3OH
1
−
ceptor and 14 was k24.2 kcal mol . This value was
1
2
3
4
5
6
7
8
1
pH
−
only k23.2 and k18.3 kcal mol for 6 and 7, re-
spectively. Although those energy differences should
not be over interpreted, they were in agreement with the
experimental affinities. The most common molecular
Figure 4 Variation of the maximum of the optical density of 7
related to the pH in presence of methanol (30%, v\v), and deter-
mination of its pKa value by extrapolation to 0% of methanol.

677
A sulfonylcyanoguanidine as thromboxane A2 receptor antagonist
C(7)
C(1)
C(6)
C(5)
C(2)
C(3)
C(4)
C(28)
C(27)
C(10)
N(8)
C(29)
C(9)
C(11)
N(23)
N(25)
C(24)
O(16)
C(12)
C(13)
N(26)
C(32)
C(14)
C(22)
C(30)
O(20)
N(15)
C(31)
N(21)
S(18)
O(17)
O(19)
Figure 5 ORTEP diagram of the crystal structure of 14.
model describing the interactions between the TXA2 al 1995). According to these considerations we postu-
receptor antagonists and their receptor was based on lated that the pKa of 14 was still lower than that of 7.
three main interactions (Nicolaı et al 1993; Yamamoto Thus, in physiological conditions (pH 7.4), the sulphon-
et al 1993; Wouters et al 1999). Firstly, all TXA2 receptor amide nitrogen of compounds 6, 7 and 14 was negatively
antagonists such as SQ-29548, seratrodast, sulotroban charged and able to establish an ionic interaction with
or tricycle derivatives were postulated to establish an Arg295ofsegmentII. Thelackofactivityof15, deprived
ionic bond between their carboxylate function and the of acid function, reinforced this theory.
positivechargeofArg295. Secondly, asecondanchoring
In our docked structures (Figure 6), all three ligands
point to the receptor involved a hydrophobic binding were placed in such a way that the sulfonamide group
pocket that comprised the side chain of Ser 201 involved could interact with Arg 295 of segment II. Interestingly,
in H-bond formation. Thirdly, the TXA2 receptor an- the oxygen involved in the interaction was different for
tagonists were thought to fill a second hydrophobic the pyridinic analogue (O20 … NH2IArg 295 l 2.71 A
pocket. Those two hydrophobic pockets appeared large for 6) than for the nitrobenzenic analogues (O19 …
enough to accommodate different lipophilic substituents NH2IArg 295 l 2.61 A for 7 and O19 … NH1IArg 295
of antagonists\ligands (Wouters et al 1999).
l 2.65 A for 14). This could be related to the different
conformational preferences observed in the crystal
structures of ligands.
Interaction with Arg 295
Due to the nitrobenzene ring, the pKa value of the
sulfonylurea 7 (3.90) was lower than that of torasemide First hydrophobic pocket
(6.68), its pyridine counterpart (Masereel et al 1994). In The m-toluyl moiety and aromatic (nitrophenyl or
the pyridine series, we demonstrated that the pKa of pyridinyl) ring of 6, 7 or 14 occupied a hydrophobic
torasemide was higher than that of its sulfonyl- pocket formed by residues Leu 198, Ser 201, Met 202 on
cyanoguanidine counterpart (pKa l 6.00) (Masereel et helice V and Leu 262 on helice VI. The absence of the

678
Jean-Michel Dogne et al
Table 3 Main torsion angles observed in the crystal structure of 14
and description of the four conformations observed with torasemide
and its derivatives.
a
V
VI
1
CH3
2
II
3
4
7
6
25
N
24
8
C
20
O
NH
5
23
31
28
32
N
30
29
9
14
21
N
H
10
11
S
N26
C
16
13
22
27
O
12
N
15
O
16
O
17
IV
III
φ1 = C9–C14–S18–N21
φ2 = C14–S18–N21–C22
φ3 = S18–N21–C22–N26
φ4 = C21–C22–N26–C27
φ4′ = N21–C22–N26–C32
φ2
φ4
φ4′
(°)
Conformer φ1
(°)
φ3
(°)
(°)
(°)
b
crystal
–80.9
–90
+90
+90
+90
+69.2
+90
+90
+90
+90
+78.8
180
180
180
0
–170.6
180
0
180
180
+13.7
…
…
…
…
α
β
γ
δ
nitro function in 6 placed this compound deeper in the
hydrophobic pocket. The hydroxyl group of Ser 255 on
helice VI pointed into this hydrophobic pocket but was
not within binding distance of the nitro (7 or 14) nor the
pyridinic nitrogen of 6. In contrast to the model pro-
posed for other TXA2 receptor antagonists (Yamamoto
et al 1993) Ser 201 did not H-bond with any of the
antagonists investigated. It is suggested that introduc-
tion of an appropriate substituent on the toluyl ring able
to interact with this Ser 201 could increase the affinity
for the receptor.
c
Second hydrophobic pocket
A second hydrophobic pocket was formed by residues
Leu 161 and Leu 168 of helice IV and Ile 113 of helice
III. It accommodated the lateral NhNh-hexamethyl-
eneguanidine or Nh-isopropylurea chains of the TXA2
receptor antagonists under study. The folded confor-
mation of 6 and 14, stabilized by the intra-molecular
stacking of the cyano and aromatic (nitrophenyl or
pyridinyl) ring, properly oriented the lateral hexa-
methylene moiety into the second hydrophobic pocket.
In conclusion, the combination of the pharmaco-
phores of the torasemide derivatives 6 and 7 led to the
Figure 6 Final models of the complexes between the human TXA2
receptor and compounds 6 (a), 7 (b) and 14 (c).

679
A sulfonylcyanoguanidine as thromboxane A2 receptor antagonist
Guex, N., Diemand, A., Peitsch, M. C. (1999) Protein modelling for
all. Trends Biochem. Sci. 24: 364–367
design of 14, a very potent TXA2 receptor antagonist
bearing a TXA2 synthase inhibitory potency. Its docking
in the TXA2 receptor-binding site led us to propose an
interaction model suggesting a modulation to improve
the original structure of BM-519 (14).
Hall, R. A., Gillard, J., Guindon, Y., Letts, J., Champion, E., Ethier,
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135: 193–201
Hantzsch, A., Wolvekamp, M. (1904) Die constitution der sogenann-
ten dithiocyansaure und persulfocyansaure. Justus Liebigs Ann.
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