Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function.

Abstract

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.

Full text

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Pharmacological evaluation of the novel thromboxane modulator BM-567
(I/II). Effects of BM-567 on platelet function

J.-M. Dognéa, X. de Levala, P. Kolha, V. Sannaa, S. Rolinb, C. Michauxc, M. Mauerd, J.-L. Davide, B. Masereelb,
B. Pirottea

a Laboratory of Medicinal Chemistry, Department of Medicinal Chemistry, Natural and Synthetic Drugs Research Center, University of
Liège,1, avenue de l'Hôpital, Tour 4 (+5), Bât B36, B-4000 Liège (Sart-Tilman), Belgium
bDepartment of Pharmacy, University of Namur, B-5000 Namur, Belgium
cDepartment of Molecular and Structural Chemistry, University of Namur, B-5000 Namur, Belgium
dDepartment of Biostatistic, University Hospital, B-4000 Liège, Belgium
eDepartment of Thrombosis and Hemostasis, University Hospital, B-4000 Liège, Belgium
Abstract
The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-
nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A2 (TXA2) receptors (TP) and
thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been
determined. In this test, BM-567 showed a high affinity (IC50: 1.1±0.1 nM) for the TP receptors in comparison
with BM-531 (IC50: 7.8 ±0.7 nM) and sulotroban (IC50: 931±85 nM), two TXA2 antagonists. We also
demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 µM) (ED100:
0.20 ± 0.10 µM), U-46619, a stable TXA2 agonist (1 µM) (ED50: 0.30 ± 0.04 µM) and collagen (1 µg ml-1) (% of
inhibition: 44.3 ± 4.3% at 10 µM) and inhibited the second wave of ADP (2 µM). Moreover, when BM-567 was
incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer
(PFA-100®) was significantly prolonged (closure time: 215 ± 21 s) by using collagen/epinephrine cartridges.
Finally, at the concentration of 1 µM, BM-567 completely reduced the TXB2 production from human platelets
stimulated with AA (600 µM). These results indicate that BM-567 is a novel combined TXA2 receptor antagonist
and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.
1. INTRODUCTION
Platelet activation and vasoconstriction are intimately involved in thrombus formation. In particular,
thromboxane A2 (TXA2), which causes platelet aggregation and vasoconstriction, plays an important role in the
process of vascular thrombosis [1-6].
TXA2 is a biologically potent arachidonate metabolite derived from the cyclo-oxygenase pathway [7]. Indeed,
TXA2 is formed by the action of thromboxane synthase on the prostaglandin endoperoxide H2 (PGH2) [8] mainly
in activated platelets and macrophages [9,10]. Initially, there was a great interest in the potential use of TXA2
receptor (TP) antagonists such as sulotroban. However, these compounds were too selective to completely
inhibit platelet aggregation. In general, interest has switched to agents which are combined receptor antagonists
and thromboxane synthase inhibitors such as ridogrel . The rationale behind this is that the TP receptor
antagonist activity can block the aggregatory and vasoconstrictor actions of PGH2 accumulating after inhibition
of thromboxane synthesis and that this endoperoxide can be converted to either prostaglandin D2 (PGD2) by the
platelets or prostacyclin (PGI2) by the vessel wall, both of which increase platelet cyclic AMP levels to inhibit
platelet activation [5] (Fig. 1).
According to the observation that the pyridinic sulfonylurea torasemide, a high ceiling loop diuretic, was able to
induce a concentration-dependent relaxation of the canine coronary artery precontracted with the stable
carboxylic TXA2 [11], we designed and synthesized a series of its derivatives. Among these drugs, BM-500 (N-
isopropyl-N'-[(2-metatoluyl-amino-5-nitrobenzene)sulfonyl]urea) emerged as one of the most potent TXA2
receptor antagonists of this generation [12]. Later, we developed BM-531 (N-tert-butyl-N'-[(2-
cyclohexylamino-5-nitrobenzene) sulfonyl]urea) which was demonstrated to be a stronger TXA2 receptor

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antagonist than BM-500 and to combine a thromboxane synthase inhibitory activity [13,14]. These encouraging
results led us to design a new generation of derivatives chemically related to BM-531. Therefore, we selected
BM-567 and examined its effects on TP receptors and thromboxane synthase of human platelets (Fig. 2). Its
affinity for TXA2 receptors has been determined and its antiaggregatory potency has been evaluated by its ability
to prevent human platelet aggregation induced by arachidonic acid (AA), U-46619, a stable TXA2 agonist, ADP
and collagen. Moreover, we measured the activity of this drug on primary hemostasis measured by the Platelet
Function Analyzer (PFA-100®). Finally, we examined the effects of BM-567 on TXB2 production from platelets
stimulated with AA in order to evaluate the thromboxane synthase inhibitory potency.
Fig. 1. Prostaglandins and thromboxane biosynthesis.

2. MATERIALS AND METHODS
2.1. Drugs and chemicals
Drugs were synthesized according to general synthetic pathways previously described [12,15]. Drug mother
solutions were prepared in dimethylsulfoxide (DMSO). The final concentration of DMSO did not exceed 0.1%, a
concentration which did not affect the parameters measured. Stock solutions of sodium arachidonate (5mM;
Sigma, Belgium) were prepared in water. U-46619 (Cayman Chemical, Ann Arbor, MI) supplied in ethanolic
solution was diluted with the incubation buffer. SQ-29548 was purchased from RBI (Bioblock, Illkirch, France)
and sulotroban was synthesized according to a general procedure previously described [16]. The solution of
collagen was provided by Horm (France) and [5,6-3H]SQ-29548 (46 Ci mmol-1) was obtained from NEN
Products (Brussels, Belgium).

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Fig. 2. Chemical structures of torasemide, BM-500, BM-531 and BM-567.

2.2. Pharmacology
Platelet binding. The binding test realized on human washed platelets was performed according to a previously
described method [13,14]. Human platelet-rich plasma (PRP) was provided by the Belgian Red Cross. Fractions
(10 ml) of this plasma were centrifuged for 10 min at 1000 g (4°C). The supernatant was discarded, the pellet
resuspended in NaCl (0.2%, 5 ml), mixed for 20 s and then diluted with NaCl (1.6%, 5 ml). The suspension was
centrifuged again for 5min at 1000 g (4°C). The supernatant was removed and the pellet was suspended in
calcium- and magnesium-free Tyrode-Hepes buffer (mM: NaCl 137, KCl 2.7, NaH2PO4 0.4, NaHCO3 12, D-
glucose 5, Hepes q.s. ad pH 7.4) to a concentration of 2 x 108 cells ml-1. Freshly prepared samples of this
suspension (500 µl) were incubated with [5,6-3H]SQ-29548 (5nM final concentration, 100µl) for 60min at 25°C.
The displacement was initiated by addition of the studied ligand dissolved in the same buffer (400µl). After
incubation (30min, 25°C), ice-cold Tris-HCl buffer (10 mM, pH 7.4; 4ml) was added and the sample was rapidly
filtered through a glass-fiber filter (Whatman GF/C) and the tube was rinsed twice with ice-cold buffer (4 ml).
The filters were then placed in plastic scintillation vials containing an emulsion-type scintillation mixture (4 ml)
and the radioactivity was counted. The amount of [5,6-3H]SQ-29548 specifically bound to the human platelet TP
receptors (Bs, %) was calculated:
Bs = 100 x (B-NSB)/(Bt-NSB)
(1)
where Bt (total binding) and NSB (non-specific binding) are the radioactivity of [5,6-3H]SQ-29548 (5 nM) bound
to the platelets incubated in the absence of any competing ligand and in the presence of unlabelled SQ-29548 (50
µM), respectively. B is the radioactivity of the filtered platelets incubated with [5,6-3H]SQ-29548 (5 nM) and the
studied compound at a fixed concentration of 10-6M or at concentrations ranging from 10-5 to 10-10M. In each
experiment, NSB varied between 5% and 7% of Bt. For the most potent drugs, three concentration-response
curves were measured in triplicate using concentrations ranging from 10-5 to 10-10 M. The concentration of drug,
which reduced the amount of specifically bound [5,6-3H]SQ-29548 by 50% (IC50), was determined for each drug
by non-linear regression analysis (GraphPad Prism software). The results are expressed as mean ± SDM.
Platelet aggregation. The antiplatelet potency was determined according to a previously described method [13].
Briefly, blood was collected by venipuncture from volunteers reported to be free from medication for at least 10
days and diluted (9:1) with trisodium citrate (3.8% w/w) in a polypropylene tube. The PRP was obtained from
the supernatant fraction after centrifugation for 20min at 90 g (25°C). The remaining blood was centrifuged at
2000 g for 5min (25°C) and the supernatant gave the platelet-poor plasma (PPP). The platelet concentration of
PRP was adjusted to 3 x 108 cells ml-1 by dilution with PPP. Aggregation tests were performed according to
Born's turbidimetric method [19] by means of two-channel aggregometer (Chrono-log®). PPP was used to adjust

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the photometric measurement to the minimum optical density. PRP (225 µl) was added in a silanized cuvette and
stirred (1100 rev min-1). Drug solution (20 µl) was then added and the mixture was incubated at 37°C for 3min.
Platelet aggregation was initiated by addition of (5 µl) AA (600 µM final), U-46619 (l µM final) ADP (2 µM
final) and collagen (l µg ml-1 final). To evaluate platelet aggregation, the maximum increase in light transmission
was determined from the aggregation curve 6min after addition of the inducer. The drug concentration
preventing 100% of the platelet aggregation induced by AA (ED100) was measured and expressed as mean ±
SDM (n = 3). The drug concentration reducing 50% of the platelet aggregation induced by U-46619 (ED50) was
calculated by non-linear regression analysis from at least three dose-response curves. The results are expressed
as mean + SDM.
PFA-100. The PFA-100 system® (Dade Behring International®) has been described elsewhere in detail [17].
The system consists of an instrument and a disposable test cartridge where primary hemostasis is stimulated.
Briefly, the system monitors platelet aggregation on a collagen-epinephrine-coated membrane as whole blood
sample is aspirated under controlled flow conditions through a microscopic aperture cut into the membrane. The
time required for the platelet plug to occlude the aperture is indicative of the platelet function in the sample.
Blood samples collected by venipuncture from four healthy volunteers reported to be free from medication for at
least 10 days and diluted (9:1) with trisodium citrate (3.8% w/w) in a polypropylene tube were incubated with or
without the drug (10 µM) prior to test at room temperature typically for 5min with occasional mixing by gentle
inversion. Then, the blood sample is added to the sample reservoir, and the test cartridge is added into the PFA-
100® and incubated at 37°C. The sample is aspirated through a central aperture (150 µM diam.) cut into the
membrane under steady vacuum created by displacement of the piston of a syringe pump connected to the
vacuum chuck. Platelet activation and aggregation occurs at the area surrounding the aperture due to the
presence of agonists and local shear conditions, eventually leading to complete occlusion of the aperture. The
instrument monitors blood flow through the aperture and reports the time required for occlusion of the aperture
by the platelet plug, thus defining the closure time (CT). The CT measured are expressed as mean + SDM (n =
4).
Thromboxane synthase activity. PRP preparation is identical to that described for the platelet aggregation
experiments. Each drug was dissolved in dimethylsulfoxide (DMSO) and diluted with a Tyrode-Hepes buffer
(mM: NaCl 137, KC1 2.7, NaH2PO4·-H2O 0.4, D-glucose 5, NaHCO3 12, Hepes; pH 7.4). To 900 ml of PRP, 50
ml NaCl, 0.9% and 10 ml of drug solution were added. After 6min incubation at 37°C under stirring (600 rpm),
aggregation was induced by 40 ml sodium arachidonate (0.6 mM). After 4min, the reaction was stopped by
adding 50 ml of indomethacin (0.02 M in ethanol). The sample was immediately centrifuged (17 500 g for 10 s)
and the supernatant was removed and frozen (-78°C) until assayed for TXB2. Basal and maximal production of
TXB2 was estimated in the absence and in the presence of AA, respectively. Thromboxane synthase activity was
expressed as the TXB2 production, which was measured by using a competitive enzyme immunoassay (TXB2
EIA Kit, Cayman Chemical Company).
3. RESULTS
In a first experimentation, we evaluated the affinity of BM-567 for human platelet TP receptors. Thereby, we
measured the capacity of our drug to displace [5,6-3H]SQ-29548, a potent competitive ligand of TP receptors,
from its binding site on human washed platelets. We realized concentration-response curves and calculated IC50
values, which represent the drug concentration, needed to displace 50% of [5,6-3H]SQ-29548 from TP receptors.
Table 1 shows that BM-567 (IC50: 1.1±0.1 nM) is characterized by a higher affinity than sulotroban (IC50: 931 ±
85nM), BM-500 (IC50: 79 ± 7.9nM) and BM-531 (IC50: 7.8 ± 0.7nM).
In a second step, we determined the property of BM-567 to prevent human platelet aggregation induced by AA
(600 µM), U-46619 (1 µM), ADP (2 µM) and collagen (lµgml-1). When AA was used as inducer, BM-567
totally prevented the aggregation at the lower dose of 0.20 ± 0.10 µM (ED100)· Compared to the other TXA2
receptor antagonists referenced in Table 1, BM-567 was much more active than both sulotroban (ED100
2.1 µM) and BM-500 (ED100: 14.0 ± 2.3µM) and no significant difference was found in comparison with BM-
531 (ED100: 0.125 ± 0.015 µM). When the TXA2 stable agonist U-46619 was used as platelet aggregating agent,
BM-567 (ED50: 0.30 ± 0.04 µM) was slightly more active than BM-531 in terms of ED50, which correspond to
the concentration required preventing 50% of the aggregation. It was nevertheless much more potent than BM-
500 and sulotroban (ED50: 9.5 ± 2.4 and 11.3 ± 4.1 µM, respectively). The same ranking can be made when
collagen was used as platelet aggregating agent. Indeed, at the concentration of 10 µM, BM-567 and BM-531,
respectively, prevented 44.3 ± 4.3% and 42.9 ± 49% of platelet aggregation while BM-500 and sulotroban were
not active. Finally, when ADP was used as inducer, none of the tested drugs prevented the initial phase of
aggregation. On the other hand, BM-500 and sulotroban (10 µM) weakly prevented the second wave of platelet
: 10.2 ±

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aggregation while at the same concentration both BM-567 and BM-531 completely inhibited this secondary
phase of ADP-induced aggregation. In this aggregometry study, torasemide (100 µM) was inactive, irrespective
of the inducer used (Table 1).
The antiplatelet potency of BM-567 has been confirmed on human whole blood by measuring with the platelet
function analyzer (PFA-100®). In this study, we examined the effects of BM-567 on primary hemostasis.
Compared to reference values (CT: 110±10 s), torasemide, BM-500 and sulotroban did not modify closure time
of the central aperture of a membrane coated with a mixture of collagen and epinephrine (CT: 108 ± 9; 114 ± 8;
112±5 s, respectively) while both BM-531 and BM-567 significantly prolonged CT (CT: 175 ± 24 and 215 ± 21
s, respectively).
Finally, we examined the effects of BM-567 on the thromboxane synthase activity from human platelets. At the
concentrations of 10 and 1 µM, BM-567 totally prevented the production of TXB2 by human platelets activated
by AA (600 µM). At the same dosage, the thromboxane synthase inhibitor furegrelate reduced the production of
TXB2 from 67.52% while torasemide and BM-500 showed no inhibitory potency. At lower concentration (0.1
µM), BM-567 did not inhibit the production of TXB2 anymore. In this test, BM-531 showed the same inhibitory
profile as BM-567 (Fig. 3).
4. DISCUSSION AND CONCLUSIONS
In 1992, Uchida et al. [11] demonstrated that torasemide elicited a dose-dependent vasodilating action in the
isolated canine coronary arteries contracted by the carbocyclic thromboxane A2 (CTA2), a stable analogue of the
potent coronary vasoconstrictor TXA2, whereas indapamide or furosemide had little effect on this preparation.
Nevertheless, these effects were observed at non-therapeutic doses. In a previous work, we confirmed that
torasemide possessed a weak affinity for TXA2 receptor of human washed platelets and that this potency was not
strong enough to prevent platelet aggregation induced by AA, U-46619, collagen and ADP, even at the dose of
100 µM. Thereby, we synthesized and developed a series of its derivatives.
Table 1
Aggregometry
U-46619c
(ED50, µM)
11.3 ± 4.1
>100
9.5 ± 2.4
Drugs


Bindinga (IC50, nM)


AAb
(ED100, µM)
10.2 ± 2.1
> 100
14 ± 2.3
0.125 ± 0.015 0.48 ± 0.09
0.20 ± 0.10
Collagend
(%)
NI
NI
NI
42.9 ± 4.9 175 ± 24
44.3 ± 4.3 215 ± 21
PFA-100e


Sulotroban
Torasemide
BM-500
BM-531
BM-567
a Drug affinity for TXA2 receptor of human platelet: IC50 represents the drug concentrations needed to displace 50% of [5,6-3H]SQ-29548
from TXA2 receptor of human washed platelets.
b Drug concentrations required preventing 100% of the platelet aggregation induced by 600 µM of AA.
c Drug concentrations required preventing 50% of the platelet aggregation induced by 1 µM of U-46619.
d Inhibition percentage of platelet aggregation induced by collagen with a 10 µM drug prevention (NI = no inhibition).
e Closure times measured by PFA-100® (with drugs used at 10 µM) compared to reference value (110 ± 10 s). Results are expressed as mean
± SDM (n≥3).

931 ± 85
2691± 72
79 ± 7.9
7.8 ± 0.7
1.1 ± 0.1
112 ± 5
108 ± 9
114 ± 8
0.30 ± 0.04

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Fig. 3. Effects of furegrelate and BM-567 on thromboxane B2 production induced by human platelets which
expresses the thromboxane synthase inhibitory activity.

This work led to a first generation of TXA2 receptor antagonist of which BM-500 was the most active. These
encouraging results led us to design and synthesize a series of torasemide derivatives of second generation
represented by BM-531, which was demonstrated as the first combined thromboxane receptor antagonist and
thromboxane synthase inhibitor belonging to the sulfonylurea class. Finally, with the aim of improving the
pharmacological profile of BM-531, we designed different other derivatives of which BM-567 emerged. Indeed,
we demonstrated that BM-567 strongly displaced [5,6-3H]SQ-29548 from TXA2 receptor of human washed
platelets with a higher potency than BM-500, BM-531 and sulotroban. We also confirmed its powerful TXA2
antagonism and antiplatelet potency using standard aggregometric techniques and the recently developed
platelet function analyzer (PFA-100®). In these tests, BM-567 was at least as active as BM-531, while BM-500
and sulotroban were less potent. Finally, we confirmed that BM-567 also reduced the production of TXB2 by
human platelets. This effect cannot be attributed to a cyclo-oxygenase inhibition since we demonstrated that BM-
567 did not prevent the prostaglandin E2 (PGE2) production induced by both ovine COX-1 and COX-2
separately incubated with AA. This result confirms the thromboxane synthase inhibitory activity of BM-567.
This has the advantage that other AA metabolites can still be produced. Thus, in the setting of platelet activation,
as would occur locally at a vascular injury site, the platelet-produced endoperoxides can be taken up by other
cells (smooth muscle cells, endothelial cells and white blood cells) which can then produce PGI2 and other
antithrombotic prostanoids [18,19]. Therefore, the antithrombotic effect due to the presence of locally produced
prostacyclin could exceed that expected by only blocking TP receptors. Additionally, the conversion of the
endoperoxides to E-type prostaglandins could help to reduce thrombus formation due to their vasodilatory
action. A variety of compounds with both activities have been reported. Ridogrel was the first putative dual
inhibitor of TXA2 to be tested clinically, but it yielded disappointing clinical results because its receptor affinity
(IC50: 1.7 µM) was demonstrated too weak in regards to its thromboxane inhibitory (IC50: 0.004 µM) effects to
adequately qualify as a compound with a dual mode of action [20]. This is not the case with BM-567 which
exhibits a combined activity on both TP receptors and thromboxane synthase at a similar concentration range.
BM-567 was found slightly more active than BM-531 in preventing platelet aggregation induced by U-46619
and prolonging closure time with collagen-epinephrine cartridges from PFA-100®. From a structural point of
view, both drugs feature an acidic sulfonylurea moiety which appears to mimic the carboxylic acid function
found in all prostanoids and the majority of their pharmacological modulators [21]. In conclusion, this work led
to the discovery of BM-567, an original, well-balanced non-carboxylic TXA2 receptor antagonist and
thromboxane synthase inhibitor. This compound has been selected for further in vivo experiments as
antithrombotic agent.
Acknowledgements
This work was support by grants of the "La Communauté Française de Belgique".

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