Selexipag: An Oral and Selective IP Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.5b00698

Prostacyclin controls cardiovascular function via activation of the prostacyclin receptor. Decreased prostacyclin production occurs in several cardiovascular diseases. However, the clinical use of prostacyclin and its analogues is complicated by their chemical and metabolic instability. A medicinal chemistry program searched for novel nonprostanoid prostacyclin receptor agonists not subject to these limitations. A compound with a diphenylpyrazine structural core was synthesized. Metabolic stability and agonist potency were optimized through modification of the linear side chain. Compound 12b (MRE-269, ACT-333679) was identified as a potent and highly selective prostacyclin receptor agonist. Replacement of the terminal carboxyl group with an N-acylsulfonamide group yielded parent compound 26a (selexipag, NS-304, ACT-293987), which is orally active and provides sustained plasma exposure of 12b. Compound 26a was developed for the treatment of pulmonary arterial hypertension and shown to reduce the risk of the composite morbidity/mortality end point in a phase 3 event-driven clinical trial.

Full text of DOI:10.1021/acs.jmedchem.5b00698

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Drug Annotation
Selexipag: an oral and selective IP prostacyclin receptor
agonist for the treatment of pulmonary arterial hypertension
Tetsuo Asaki, Keiichi Kuwano, Keith Morrison, John Gatfield, Taisuke Hamamoto, and Martine Clozel
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00698 • Publication Date (Web): 20 Aug 2015
Downloaded from http://pubs.acs.org on August 22, 2015
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Selexipag:
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an oral and selective IP prostacyclin receptor agonist for the treatment of
pulmonary arterial hypertension
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Tetsuo Asaki1, Keiichi Kuwano1, Keith Morrison2, John Gatfield2, Taisuke Hamamoto1, Martine
Clozel2*
1Discovery Research Laboratories, Nippon Shinyaku Co., Ltd, 14 NishinoshoꢀMonguchiꢀCho,
Kisshoin, Minamiꢀku, Kyoto 601ꢀ8550, Japan
2Drug Discovery Department, Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil
CH4123, Switzerland
*Corresponding author
t.asaki@po.nipponꢀshinyaku.co.jp; k.kuwano@po.nipponꢀshinyaku.co.jp;
keith.morrison@actelion.com; john.gatfield@actelion.com; t.hamamoto@po.nipponꢀ
shinyaku.co.jp; martine.clozel@actelion.com
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ABSTRACT
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Prostacyclin controls cardiovascular function via activation of the prostacyclin
receptor. Decreased prostacyclin production occurs in several cardiovascular diseases. However,
the clinical use of prostacyclin and its analogs is complicated by their chemical and metabolic
instability. A medicinal chemistry program searched for novel nonꢀprostanoid prostacyclin
receptor agonists not subject to these limitations. A compound with a diphenylpyrazine structural
core was synthesized. Metabolic stability and agonist potency were optimized through
modification of the linear side chain. Compound 12b (MREꢀ269, ACTꢀ333679) was identified as
a potent and highly selective prostacyclin receptor agonist. Replacement of the terminal carboxyl
group with an Nꢀacylsulfonamide group yielded parent compound 26a (selexipag, NSꢀ304, ACTꢀ
293987), which is orally active and provides sustained plasma exposure of 12b. Compound 26a
was developed for the treatment of pulmonary arterial hypertension and recently shown to reduce
the risk of the composite morbidity/mortality endpoint in a phase 3 eventꢀdriven clinical trial.
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INTRODUCTION
Prostacyclin (PGI2)1, 2 is an endogenous prostanoid synthesized mainly by vascular endothelial
cells. PGI2 is a potent vasodilator and inhibitor of platelet aggregation, and plays an important
role in the maintenance of vascular homeostasis.1, 2 It also inhibits vascular smooth muscle cell
differentiation, proliferation, and migration.3 These physiological functions are mediated via
activation of the PGI2 receptor (IP receptor),4 a Gꢀproteinꢀcoupled receptor located on the surface
of vascular smooth muscle cells, platelets and other cells. Downꢀregulation of the PGI2 pathway
has been implicated in the pathogenesis of several vascular diseases,5, 6 supporting the use of
epoprostenol, the synthetic sodium salt of PGI2, and PGI2 analogs as therapeutic agents.
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However, epoprostenol is chemically and metabolically unstable at physiological temperature
and pH.2 As a consequence, its use requires continuous infusion through a central venous
catheter. PGI2 analogs such as iloprost and treprostinil have been developed in an effort to
improve chemical stability, but most of the approved compounds need to be administered by
subcutaneous/intravenous infusion or inhalation. The development of orally available PGI2
analogs with sufficient duration of action has proven challenging.7, 8 Beraprost sodium is an
orally available PGI2 analog, but its elimination halfꢀlife is very short and it has only transient
clinical benefits in pulmonary arterial hypertension (PAH).9 An oral formulation of treprostinil is
indicated in the US for the treatment of PAH to improve exercise capacity, but it did not show
clinical benefit in combination therapy.10
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We describe the development of 26a, a novel orally active IP receptor agonist with a high degree
of metabolic stability and an extended duration of action. Compound 26a was discovered by
Nippon Shinyaku,11, 12 and developed for the treatment of PAH by Nippon Shinyaku and
Actelion Pharmaceuticals Ltd.
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Figure 1. Chemical structures of prostacyclin (PGI2), PGI2 analogs and nonꢀprostanoid
prostacyclin (IP) receptor agonists including lead compound 4c.
MEDICINAL CHEMISTRY STRATEGY
The discovery strategy for a longꢀacting IP receptor agonist was based on the observation that
the prototypical prostanoid structure is not necessary for activation of the IP receptor.11
Octimibate13 and 1 (BMYꢀ42393),14, 15 both partial agonists at the IP receptor, bear no structural
resemblance to PGI2 and its analogs, apart from a carboxylic acid moiety (Figure 1).
Compound 1, which has a long duration of action in animal models of thrombosis,16 was
chemically modified by replacing the oxazole ring with a pyrazine ring to obtain the lead
compound, the 2ꢀaminoꢀ5,6ꢀdiphenylpyrazine derivative 4c (Figure 1). The potency of
compounds at the human IP receptor was measured by their inhibition of adenosine diphosphate
(ADP)ꢀinduced platelet aggregation. Compound 4c inhibited platelet aggregation with a half
maximal inhibitory concentration (IC50) value of 1.5 ꢀM. Based on the observation that the
topological relationship between the diphenyl heterocycle and the carboxylic acid moiety in the
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and pyrazole17 series determines agonist activity, the effects of the length and
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oxazole
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rigidity of the alkylene linker were investigated (Table 1). Structureꢀactivity relationship studies
on 4a–d demonstrated that the presence of a hexamethylene linker in 4b provided the optimal
length, with fiveꢀfold more potent inhibition of platelet aggregation (IC50 = 0.3 ꢀM) than was
shown by 4c.
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Replacing the methylene unit in the βꢀposition with respect to the carboxyl group with an oxygen
atom18 was envisaged to increase metabolic stability in vivo. This approach generated 12a, which
maintained high in vitro potency (IC50 = 0.2 ꢀM).
Replacing the flexible alkylene linker in 4b and 4c with a more rigid metaꢀdisubstituted phenyl
linker as shown in 19a and 19b led to weaker activity in the platelet aggregation assay.
Table 1. Effect of linker variation on inhibition of ADPꢀinduced human platelet aggregation
a Inhibition of platelet aggregation induced by ADP (10 ꢀM) in human platelet rich plasma.
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Reproduced with permission from reference 11.
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Next, the impact of the concatenating nitrogen atom between the pyrazine ring and the linker was
investigated (Table 2). Replacing the Nꢀmethylamino group in 12a with methylene, oxygen,
sulfur, sulfoxide, or sulfone reduced potency in this assay, suggesting that the nitrogen atom
adjacent to the pyrazine ring is important for the pharmacological action of 12a.
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Table 2. Effect of concatenating atoms between the pyrazine and the linker on inhibition of
ADPꢀinduced human platelet aggregation
a Inhibition of platelet aggregation induced by ADP (10 ꢀM) in human platelet rich plasma.
b The biological activity of the sodium salt was evaluated.
Reproduced with permission from reference 11.
Further optimization of 12a focused on exploring the substituent attached to the exocyclic
nitrogen. As illustrated by the examples in Table 3, small groups such as an ethyl, cyclopropyl or
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isopropyl group were generally preferred (5a, IC50 = 0.5 ꢀM; 5c, IC50 = 0.2 ꢀM; 12b, IC50
0.2 ꢀM), while a benzyl group led to a substantial loss of activity (12d, IC50 = 50 ꢀM).
=
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Table 3. Effect of substituents at the Nꢀatom adjacent to the pyrazine ring on inhibition of ADPꢀ
induced human platelet aggregation
a, b See footnotes to Table 2.
Reproduced with permission from reference 11.
The prostanoid receptor binding profile of 12b was characterized because this compound, in
addition to being orally bioavailable with a long plasma halfꢀlife in animals (Table 4), displayed
low potential for causing offꢀtarget sideꢀeffects (low toxicity in animal studies).19 Compound
12b bound to the human IP receptor with high affinity (binding affinity (Ki) of 20 nM) and with
more than 130ꢀfold selectivity over other prostanoid receptors19 (prostaglandin E receptors, EP1ꢀ
4; prostaglandin D receptor, DP1; prostaglandin F2 receptor, FP; and thromboxane A2 receptor,
TP).
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Table 4. Pharmacokinetic parameters of 12b after intravenous or oral administration to rats,
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dogs, and monkeys
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Route
iv
Parameter
Dose (mg/kg)
t1/2 (h)
Ratsa
3
Dogsa
1
Monkeysa
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ꢀb
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4.9 ±1.4
5.2 ± 0.7
5.9 ±0.2
72.5 ± 9.1
ꢀ
AUC (ꢀg.h/mL)
Dose (mg/kg)
Tmax (h)
po
10
3
1
0.8 ± 0.3
1.9 ± 0.5
0.8 ± 0.4
14.9 ± 3.6
2.3 ± 1.5
0.11 ± 0.04
Cmax (ꢀg /mL)
t1/2 (h)
3.6 ± 0.7
6.2 ± 1.0
176 ±4 1
5.6 ± 1.5
17.7 ± 1.9
0.65 ± 0.12
AUC (ꢀg.h/mL)
Bioavailability (%)
102
80
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a Each value is the mean ± SD (n = 3).
b Not tested.
iv, intravenous; AUC, area under the curve; Cmax, maximum observed plasma concentration; t1/2,
apparent terminal halfꢀlife; po, per os; Tmax, time to reach maximum observed plasma
concentration.
Reproduced with permission from reference 11.
Efforts were then directed towards extending the in vivo exposure of 12b. Replacement of the
carboxyl group with acidic surrogates such as Nꢀacylsulfonamide is a recognized structural
modification to improve pharmacokinetic properties of carboxylic acid drugs.20 Compound 26a,
the Nꢀmethylsulfonamide derivative of 12b (Figure 2), bound to the human IP receptor with 13ꢀ
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fold lower affinity than 12b, and was 26ꢀfold less potent than 12b in platelet aggregation
assays.19 However, in vitro, 26a was slowly hydrolyzed to the active form 12b by animal and
human hepatic microsomes (Table 5), suggesting that 26a is converted to the carboxylic acid in
vivo. The serine hydrolase inhibitor phenylmethylsulfonyl fluoride completely inhibited
hydrolysis of 26a to 12b in monkey hepatic microsomes, suggesting that conversion is mediated
by a carboxylesterase. In vivo hydrolysis to 12b following oral administration of 26a was
confirmed in monkeys and humans.12
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Figure 2. Chemical structures of sulfonamide and sulfonylurea analogs
Table 5. Hydrolysis of 26a to 12b in plasma or by hepatic microsomes from animals and humans
Hydrolysis rate (nmol/min/mL or nmol/min/mg protein)
Rats
0.500
0.090
Dogs
ꢀa
Monkeys
Humans
ꢀ
Plasma
ꢀ
Hepatic microsomes
0.030
0.057
0.019
26a was incubated with plasma or hepatic microsomes at 37°C for 10 min.
a Below the limits of detection of 12b.
Reproduced with permission from reference 12.
These results prompted investigation of the pharmacokinetic properties of additional sulfonamide
and
sulfonylurea
analogs
of
26a:
the
Nꢀisopropylsulfonamide
26b,
the
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trifluoromethanesulfonamide 26c, the Nꢀarylsulfonamides 26d and 26e, and the sulfonylureas
26f and 26g (Figure 2)12. Compounds 26b, 26c, and 26f displayed low rates of in vitro
hydrolysis, similar to that of 26a in hepatic microsomes from monkeys and humans. In vivo
conversion of all three compounds to 12b was confirmed after oral administration in monkeys
(Table 6). Two important differences in the measured pharmacokinetic properties of 12b were
observed following administration of parent compounds compared to administration of 12b: 10%
to 30% lower maximum plasma concentration (Cmax) values; and a more than 1.5ꢀfold increase in
apparent terminal halfꢀlife (t1/2). These compounds are therefore potential parent drugs with longꢀ
lasting agonistic activity due to sustained formation and prolonged exposure of 12b. It is
expected that slower hydrolysis of the parent compound prolongs its pharmacological activity
while potentially reducing adverse effects.
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Table 6. Pharmacokinetic parameters for 12b after oral administration of 12b, the sulfonamides
26a–c, or the sulfonylurea 26f (1 mg/kg) to monkeys
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Compound
Compound
n
Tmax
(h)
2.3
14.0
6.7
10
Cmax
(ng/mL)
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AUC0–24h
(ng.h/mL)
652
t1/2
(h)
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administered measured
12ba
12b
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3
5.6
10.7
4.9
14.5
2.3
8.5
ꢀc
859
26a
12b
26a
12b
26b
12b
12b
47
384
13
170
26b
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17
128
26cb
26fb
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308
6
20
374
Each value is the mean of 2 or 3 animals.
a See reference 11.
b
The plasma concentrations were not determined because the compound was insufficiently
ionized in liquidꢀchromatographyꢀmass spectrometry.
c
Not calculated because the plasma concentrations of 12b after administration of 26f continued
to increase until the end of the sampling period.
AUC0–24h, area under the curve from 0 to 24 h after dosing; Cmax, maximum observed plasma
concentration; t1/2, apparent terminal halfꢀlife; Tmax, time to reach maximum observed plasma
concentration.
Reproduced with permission from reference 12.
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This medicinal chemistry program culminated in the discovery of 26a, a novel IP receptor
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agonist with properties that differentiate it from PGI2 and its analogs:
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chemical and metabolic stability are provided by the nonꢀprostanoid structure
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potency is provided by the optimized linker length, heteroꢀatom and Nꢀsubstituent
prolonged exposure to the potent active metabolite (carboxylic acid) is provided by the
Nꢀacylsulfonamide parent drug, which is slowly converted to the carboxylic acid in vivo
TARGET DISEASE: PULMONARY ARTERIAL HYPERTENSION
Pulmonary arterial hypertension is a progressive disease that is characterized by increased
resistance in the pulmonary circulation caused by pulmonary artery constriction and gradual
pulmonary artery remodeling. Pulmonary arterial hypertension ultimately leads to right heart
failure and premature death.21 Alterations of several signaling pathways contribute to the
development and progression of PAH. Current therapeutic management of PAH targets pathways
mediated by PGI2, endothelin, and nitric oxide. The PGI2 pathway is activated when PGI2
stimulates the IP receptor, leading to increased cyclic adenosine monophosphate (cAMP) levels
and subsequent vasodilatory and antiproliferative effects in pulmonary arterial smooth muscle
cells.3 Dysregulation of the PGI2 pathway in PAH5, 22, 23 results in a decrease in PGI2 production
and an imbalance between relaxant PGI2 and contractile mediators such as endothelinꢀ1 and
thromboxane A2. These studies support the use of IP receptor agonists in management of PAH.
There are currently three PGI2 analogs, in addition to epoprostenol, approved for the treatment of
PAH: treprostinil (intravenous, subcutaneous, and inhaled; oral in US only), iloprost (inhaled)
and beraprost (oral, in Japan and South Korea only).24ꢀ26 Among these, only epoprostenol has
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demonstrated improved patient survival in a randomized clinical trial in treatmentꢀnaïve New
York Heart Association functional class III and IV patients.27 However, the use of epoprostenol
is limited by its short halfꢀlife requiring continuous intravenous infusion and by the risk of lifeꢀ
threatening infections.28 Inhaled and subcutaneously administered PGI2 analogs have greater
chemical stability compared to epoprostenol but require frequent dosing for inhaled iloprost (six
to nine times daily) and may induce pain at the injection site for subcutaneous treprostinil, in
addition to the usual prostanoidꢀassociated side effects such as headache, flushing, diarrhea and
jaw pain.7 An IP receptor agonist with oral activity, such as 26a, could provide an important
advance in the pharmacological treatment of PAH.
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An oral IP receptor agonist could also have potential applications in other clinical diseases, such
as chronic thromboembolic pulmonary hypertension, peripheral artery disease, or Raynaud's
syndrome. In part because of their pharmacokinetic properties, orally available PGI2 analogs,
such as treprostinil and beraprost, have shown inconsistent clinical benefits in PAH9, 25 and other
diseases.29ꢀ31
PRECLINICAL EVALUATION OF 26a
Compounds 26a and 12b are agonists at the human IP receptor
Compounds 26a and 12b are both agonists at the human IP receptor. Subsequent cAMP
accumulation assays using recombinant Chinese hamster ovary (CHO) cells, demonstrated that
12b is 16ꢀfold more potent than 26a at the human IP receptor (EC50 = 11 nM for 12b vs 177 nM
for 26a). These results combined with the observation that 12b is present at 3ꢀ to 4ꢀfold higher
levels than the parent drug at steadyꢀstate in humans32 demonstrate that the active metabolite 12b
is the major contributor to the efficacy of 26a in man.
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Compound 12b is a functional agonist in human pulmonary arterial smooth muscle cells
Pulmonary arterial smooth muscle cells (PASMCs) display increased contractile and
proliferative activity and contribute to the pathology of PAH. The functional effects of 12b
mediated through activation of the IP receptor were therefore measured in human PASMCs.
Compound 12b potently induces accumulation of intracellular cAMP leading to longꢀlasting
(> 10 h) membrane hyperpolarization (EC50 = 32 nM), which contributes to cell relaxation.33
Compound 12b also causes longꢀlasting and pronounced relaxation of PASMCs as measured by
changes in cell shape (EC50 = 4.3 nM). In addition, 12b potently inhibits plateletꢀderived growth
factorꢀBBꢀstimulated proliferation of human PASMCs (IC50 = 2.9 nM). These in vitro results
demonstrate that 12b possesses characteristics that may be beneficial in the treatment of PAH.
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Compound 26a is effective in experimental pulmonary hypertension
The effects of repeated oral administration of 26a (1 mg/kg administered twice daily) on disease
progression were measured in a rat model of pulmonary hypertension induced by monocrotaline
(MCT). Compound 26a administered for 19 days reduces the increase in right ventricular
systolic pressure evoked by MCT without affecting the heart rate.34 Compound 26a also restores
vascular endothelial function (as assessed by improvement of endotheliumꢀdependent relaxation
responses to acetylcholine), decreases pulmonary artery wall thickness, and reduces right
ventricular hypertrophy. The survival of MCT rats at 45 days is significantly increased by 26a
(Figure 3). These results indicate that 26a suppresses various pathological processes associated
with pulmonary hypertension in the rat.
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P＝0.0307
MCT + vehicle
MCT + selexipag
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Time after MCT injection (days)
Figure 3. Effect of 26a on the survival of MCT rats. Compound 26a at 1 mg/kg or vehicle was
administered twice daily for 45 days starting on day 0. Survival curves were calculated by the
KaplanꢀMeier method and compared by the logꢀrank test (n = 15).
Reproduced with permission from reference 34.
Compound 26a does not cause tachyphylaxis
Receptor desensitization followed by internalization has been observed following chronic
exposure of the IP receptor to PGI2 analogs in vitro.35 Furthermore, dose escalation may be
required to maintain the efficacy of PGI2 analogs in patients with PAH, although disease
progression may also contribute to this apparent loss of efficacy (tachyphylaxis).36 The potential
of 26a to cause tachyphylaxis was evaluated in normotensive and hypertensive rats following
repeated oral administration.19, 37 In rats that have received prior repeated administration of 26a
for up to 4 weeks, vasodilation of the femoral artery in response to 26a is not significantly
diminished. Additional experiments measured the hemodynamic effects of repeated
administration of 26a in spontaneously hypertensive rats. Compound 26a (10 to 300 mg/kg/day)
decreases mean arterial pressure (MAP) in a doseꢀdependent manner, and the effect at each dose
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is sustained for 4 days without any loss of response.37 MAP rapidly returns to preꢀdrug levels
after cessation of administration of 26a and no rebound effect is observed. These findings
indicate that repeated oral administration of 26a does not cause tachyphylaxis.
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In vivo tachyphylaxis on continued exposure to G proteinꢀcoupled receptor agonists can be
caused by recruitment of βꢀarrestin to the activated and phosphorylated receptor leading to
uncoupling of receptor signaling and subsequent internalization and removal of the receptor from
the cell surface. To estimate their receptor uncoupling potential, the potency and efficacy of 26a
and 12b at the human IP receptor were measured with respect to Gꢀprotein signaling (adenylate
cyclase activation) and βꢀarrestin recruitment / receptor internalization (uncoupling) in CHO
cells expressing the human IP receptor. Comparison was made with PGI2 analogs (iloprost,
treprostinil and beraprost).38 All compounds induce comparable maximal responses for increased
cAMP production. However, the compounds differ markedly in their effects on βꢀarrestin
recruitment. Compounds 26a and 12b have a low efficacy for recruitment of βꢀarrestin, in
contrast to the PGI2 analogs. This difference between PGI2 analogs on the one hand and 26a and
12b on the other is substantiated by receptor internalization assays. Compounds 26a and 12b –
even at high concentrations – induce only minimal internalization of human IP receptors from
the cell surface in recombinant cells, whereas PGI2 analogs induce pronounced receptor
internalization. These in vitro studies demonstrate that 26a and 12b induce limited βꢀarrestin
recruitment and minimal receptor internalization. Together, these findings might explain why
26a does not cause tachyphylaxis in animal models.
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Compounds 26a and 12b are selective agonists at the IP receptor
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Compounds 26a and 12b, contrary to PGI2 analogs, are highly selective for the IP receptor.19, 39
The binding affinity of 26a for the human IP receptor is 260 nM, whereas Ki values > 10,000 nM
are measured at the other human Gꢀproteinꢀcoupled prostanoid receptors (EP1ꢀ4, DP, FP and TP)
(Table 7). Compound 12b binds the human IP receptor with 13ꢀfold higher affinity than 26a
(Ki = 20 nM), and binds other prostanoid receptors with at least 130ꢀfold lower affinity. In
contrast, the PGI2 analogs beraprost, treprostinil and iloprost bind much less selectively to
prostanoid receptors.19, 39
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Table 7 Binding affinity and receptor selectivity of 26a, 12b and PGI2 analogs for human
prostanoid receptors
Binding affinity (Ki, nM)
IP
260
20
EP1
EP2
EP3
EP4
DP
FP
TP
26aa
>10000 >10000 >10000 >10000 >10000 >10000 >10000
12ba
>10000
5800
>10000
0.68
4900
7.2
2600
>10000 >10000
Beraprosta
Treprostinilb
Iloprostb
39
>10000 >10000
>10000 >10000 >10000
32
212
1.1
3.6
2505
208
826
212
4.4
4680
131
>10000
3778
3.9
1172
1016
a See reference 19.
b See reference 39.
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EP1ꢀ4, prostaglandin E receptors; DP, prostaglandin D receptor; FP, prostaglandin F2 receptor;
TP, thromboxane A2 receptor.
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The seven nonꢀIP prostanoid receptors fall into two categories: i) contractile receptors (EP1, EP3,
TP, and FP), which contract smooth muscle in the gastrointestinal tract and blood vessels, and ii)
vasodilating receptors (EP2, EP4, and DP), which are not functional in human pulmonary arteries,
and have immunosuppressive effects by inhibiting functions of macrophages, natural killer cells,
T cells and other immune cell types.40
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The high degree of selectivity of 26a and 12b for the IP receptor may provide benefits over PGI2
analogs in terms of both agonist efficacy and patient tolerability. Vasorelaxation in response to
12b is comparable in pulmonary arteries isolated from MCT pulmonary hypertensive rats and in
vessels isolated from normal rats, whereas vasorelaxation induced by the nonꢀselective PGI2
analogs beraprost34 and treprostinil41 is significantly lower in MCT pulmonary hypertensive rats
than in normal rats, because of coactivation of the contractile EP3 receptor in pathological
conditions.41 The mRNA expression and pharmacological activity of the EP3 receptor is upꢀ
regulated in experimental pulmonary hypertension models, as it is in human PAH lungs.42 The
effective pulmonary vasodilatation evoked by 12b in pathological conditions such as PAH
supports the therapeutic potential of 26a.
Oral administration of 26a does not significantly alter gastric function, and neither 26a nor 12b
contract rat gastric fundus ex vivo. These findings suggest that the high selectivity of 26a and
12b for the IP receptor along with the lower potency of 26a, the main molecular entity before
absorption, may help minimize the severity of gastric side effects such as nausea and vomiting,
which are mediated through activation of EP3 receptors.43, 44 In contrast, the PGI2 analogs
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iloprost, beraprost, and treprostinil contract gastric smooth muscle via the stimulation of EP3
receptors.44 Iloprost and beraprost also activate contractile EP1 receptors, and orally administered
beraprost slows gastric emptying and gastrointestinal transport.44
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A role for EP3 receptors has also been suggested in the development of peripheral pain45, 46, a
side effect reported in patients treated with PGI2 analogs.7 Treprostinil exacerbates whereas 12b
inhibits vasoconstriction of rat peripheral arteries in response to sympathetic nerve stimulation.47
The contractile effect of treprostinil is EP3ꢀreceptorꢀdependent and may accentuate ischemic
muscle pain under conditions of heightened adrenergic activity, as observed in patients with
PAH.48 Compound 26a is not expected to evoke the side effects associated with concomitant
stimulation of other prostanoid receptors and may demonstrate improved tolerability in patients
with PAH.
CLINICAL EVALUATION OF 26a
A microdose study of oral 26a (100 ꢁg) conducted in five healthy volunteers19 showed that it is
metabolized to 12b in the human body with an elimination halfꢀlife of 7.9 h, permitting twiceꢀ
daily dosing.
The safety, tolerability, pharmacokinetics, and pharmacodynamics of 26a were subsequently
evaluated in healthy male subjects in a doubleꢀblind, placeboꢀcontrolled, randomized, multipleꢀ
ascendingꢀdose, upꢀtitration study (400 to 1,800 ꢁg twice daily for 3 days each, in steps of 200
ꢁg; n = 12) or placebo (n = 4).32 Compound 26a was well tolerated at doses up to 1,600 ꢁg.
Steady state was reached after 3 days with no evidence of accumulation of 26a. At the highest
dose (1,800 ꢁg), the geometric mean terminal halfꢀlives of 26a and 12b were 1.4 and 8.7 h,
respectively.
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Compound 26a was investigated in a randomized, doubleꢀblind, placeboꢀcontrolled, phase 2,
proofꢀofꢀconcept trial in 43 patients with PAH49 receiving an endothelin receptor antagonist, a
phosphodiesterase typeꢀ5 inhibitor, or both, as background therapy. Compound 26a was upꢀ
titrated in 200 ꢁg twiceꢀdaily increments to the maximum tolerated dose or to the maximal dose
of 800 ꢁg. More than 60% of patients reached a final dose of 26a of 600–800 ꢁg twice daily
(12%, n = 4: 200 ꢁg bid; 18%, n = 6: 400 ꢁg bid; 21%, n = 7: 600 ꢁg bid; 42%, n = 14: 800 ꢁg
bid; 2 patients discontinued prematurely). At week 17, 26a significantly lowered pulmonary
vascular resistance (treatment effect: −30.3%; p = 0.0045) and increased cardiac index (treatment
effect: +0.5 L.minꢀ1.mꢀ2; p = 0.01). In addition, the sixꢀminute walk distance improved by 24.7 m
in the 26a treatment group (95% confidence limits: −23.7 to 72.2). Compound 26a was well
tolerated and the majority of adverse events were mild or moderate in severity. The high
selectivity of 26a for the IP receptor, the pharmacokinetic properties that reduce peakꢀtrough
fluctuations, and the upꢀtitration regimen used are collectively considered to minimize the
adverse effects. A decrease over time in the prevalence of adverse events frequently associated
with PGI2 analog treatment, such as headache, pain in an extremity, pain in the jaw, nausea and
diarrhea, was observed in patients treated with 26a.
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The longꢀterm effect of 26a on the composite endpoint of morbidity/mortality was investigated
in the doubleꢀblind, randomized, placeboꢀcontrolled, phase 3 GRIPHON (Prostaglandin (PGI2)
Receptor Agonist in Pulmonary Arterial Hypertension) study, which enrolled 1,156 patients with
PAH.50 The dose was initiated at 200 ꢁg twice daily and increased up to a maximum of 1,600 ꢁg
twice daily. A large majority of patients (80.5%) were on stable background therapy composed
of an endothelin receptor antagonist and/or a phosphodiesterase typeꢀ5 inhibitor. In the
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GRIPHON study, 26a demonstrated a statistically significant effect on morbidity/mortality, the
primary endpoint.
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Based on these longꢀterm efficacy and safety studies, the oral selective IP receptor agonist 26a
has the potential to overcome the limitations associated with PGI2 analogs and, if approved, may
represent an important new drug for the treatment of PAH as it would offer longꢀterm outcome
benefits with an oral therapy targeting the prostacyclin pathway.
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CHEMISTRY
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Syntheses of diphenylpyrazine derivatives leading to identification of 12b as a long-lasting
IP receptor agonist
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The detailed chemistry has been described previously.11
NꢀAlkylation of 2ꢀaminoꢀ5,6ꢀdiphenylpyrazines 3a–d with the appropriate bromides was the key
step in the synthesis of the pyrazine derivatives related to 4a–d and 5a–c as described in
Scheme 1.
Scheme 1: (a) methylamine, MeOH, 80°C for 12 h, for 3a (57%); (b) amines, 1ꢀbutanol, 150°C
for 24 h, for 3b–d (48ꢀ76%); (c) i—NaH, DMF, 80°C for 30 min; ii—Br(CH2)nCO2R, room
temperature (rt) for 2 h, for 4a–d (8ꢀ21% in 2 steps), 8 for 5a–c (24ꢀ26% in 2 steps); (d) 1 N
NaOH, MeOH, heated at reflux for 1 h; (e) BrCH2CO2tꢀBu, nꢀBu4NHSO4, aq KOH, benzene,
<10°C for 45 min, rt for 1 h (61%); (f) pꢀTsOH, MeOH, rt for 30 min (66%); (g) Ph3P, CBr4,
CH2Cl2, rt for 1 h (72%).
Reproduced with permission from reference 11.
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Alternative synthetic routes for the synthesis of the pyrazine derivatives related to 12a–d and 19a
and 19b are summarized in Schemes 2 and 3.
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Scheme 2: (a) i—HCO2Et, EtOH, heated at reflux for 18 h, ii—LiAlH4, THF, slow reflux for 1.5
h (58%); (b) acetone for 10b, cyclopentanone for 10c, H2, rt for 48 h, PtO2, EtOH (quantitative);
(c) i—PhCHO, MeOH, CH2Cl2, H2, rt overnight; ii—NaBH4, MeOH, CH2Cl2, rt overnight; (d) 2,
K2CO3, DMF, 80°C for 40 h (82%); (e) 2, neat, 190°C for 10 h (56%); (f) 2,
triisopropanolamine, sulfolane, 190°C for 22 h (60% for 11c, 69% for 11d); (g) i— BrCH2CO2tꢀ
Bu, aq KOH, benzene, <10°C for 45 min, rt for 1 h; ii—1 N NaOH, MeOH, heated at reflux for
1 h (36ꢀ62% in 2 steps).
Reproduced with permission from reference 11.
Scheme 3: (a) MeNH2, H2, 5% Pd–C, MeOH, rt for 7 h, warmed to 70°C and stirred for 15 h
(77%); (b) i—HCO2Et, EtOH, heated at reflux for 22 h (42%); ii—LiAlH4, THF, rt, reflux for 2
h (80%); (c) 2, K2CO3, DMF, 80°C for 40 h (60% for 17a, 71% for 17b); (d) HCl, MeOH, rt for
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3 h (81%); (e) cꢀHCl, EtOH, 80°C for 17 h (92%); (f) BrCH2CO2Me, K2CO3, cat KI, MeCN,
heated at reflux for 3 h; (g) 1 N NaOH, MeOH, heated at reflux for 1 h (69% for 19a, 51% for
19b in 2 steps).
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Reproduced with permission from reference 11.
The synthetic route to the alkylated pyrazine 22 is shown in Scheme 4.
Scheme 4: (a) PdCl2(PPh3)2, CuI, Et3N, 80°C for 7 h (60%); (b) H2, 5% Pd–C, EtOH, rt for 4 h
(99%); (c) i—BrCH2CO2 tꢀBu, nꢀBu4NHSO4, aq KOH, benzene, <10°C for 45 min, rt for 1 h;
ii—1 N NaOH, MeOH, heated at reflux for 1 h (25% in 2 steps).
Reproduced with permission from reference 11.
Pyrazine derivatives 25a–d were prepared according to Scheme 5.
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Scheme 5: (a) i—NaH, DMF, rt for 0.5 h ii—8, rt for 18 h (54%); (b) 8, Na2CO3, acetone, rt for
24 h (82%); (c) 1 N NaOH, MeOH, heated at reflux for 1 h (62% for 25a, 61% for 25b); (d)
mCPBA (1 eq for 25c, 2 eq for 25d), CHCl3, 0°C for 2 h for 25c, 19 h for 25d; (e) 4 N HCl, 1,4ꢀ
dioxane, 80°C for 0.5 h (39% for 25c, 66% for 25d, in 2 steps).
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Reproduced with permission from reference 11.
Synthesis of sulfonamide and sulfonylurea derivatives of 12b leading to identification of
26a as a parent drug of 12b
The detailed chemistry has been described previously.12
The synthetic route to 26a–g is shown in Scheme 6. The carboxylic acid 12b was used to prepare
Nꢀacylsulfonamides 26a–e and Nꢀacylsulfonylureas 26f and 26g.
Scheme 6: (a) i—CDI, THF, rt for 0.5 h, then reflux 0.5 h; ii—RSO2NH2, rt for 10 min, DBU, rt
for 12 h for 26a (77%) and 36 h for 26b-g (43ꢀ65 %).
Reproduced with permission from reference 12.
ANCILLARY INFORMATION
Supporting Information Available:
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SMILES for all compounds and compound numbers used in the original publications are
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
Martine Clozel
Phone: + 41 61 565 65 13, email: martine.clozel@actelion.com
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript
Notes
The only funding source for the authors was Actelion Pharmaceuticals Ltd and Nippon Shinyaku
Co.
ACKNOWLEDGEMENTS
The authors thank Sylvie I. Ertel, PhD (Sundgau Medical Writers, Habsheim, France) for
editorial assistance and Dr. Gerald E. Smyth, Nippon Shinyaku Co., Ltd, for helpful suggestions
on the manuscript.
ABBREVIATIONS USED
ADP, adenosine diphosphate; AUC0–24h, area under the curve from 0 to 24 h after dosing; cAMP,
cyclic adenosine monophosphate; CHO, Chinese hamster ovary; Cmax, maximum observed
plasma concentration; DP, prostaglandin D; EC50, half maximal effective concentration; EP1ꢀ4,
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prostaglandin E; FP, prostaglandin F2; IC50, half maximal inhibitory concentration; IP/PGI2,
prostacyclin; iv, intravenous; Ki, binding affinity; MAP, mean arterial pressure; MCT,
monocrotaline; PAH, pulmonary arterial hypertension; PASMC, pulmonary arterial smooth
muscle cells; po, per os; rt, room temperature; t1/2, apparent terminal halfꢀlife; Tmax, time to reach
maximum observed plasma concentration; TP, thromboxane A2
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REFERENCES
(1)
Moncada, S.; Gryglewski, R.; Bunting, S.; Vane, J. R. An enzyme isolated from
arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet
aggregation. Nature 1976, 263, 663ꢀ665.
(2)
endoperoxides, thromboxane A2, and prostacyclin. Pharmacol. Rev. 1978, 30, 293ꢀ331.
(3) Fetalvero, K. M.; Martin, K. A.; Hwa, J. Cardioprotective prostacyclin signaling
in vascular smooth muscle. Prostaglandins Other Lipid Mediat. 2007, 82, 109ꢀ118.
(4) Coleman, R. A.; Smith, W. L.; Narumiya, S. International Union of
Moncada, S.; Vane, J. R. Pharmacology and endogenous roles of prostaglandin
Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the
receptors and their subtypes. Pharmacol. Rev. 1994, 46, 205ꢀ229.
(5)
Tuder, R. M.; Cool, C. D.; Geraci, M. W.; Wang, J.; Abman, S. H.; Wright, L.;
Badesch, D.; Voelkel, N. F. Prostacyclin synthase expression is decreased in lungs from patients
with severe pulmonary hypertension. Am. J. Respir. Crit. Care Med. 1999, 159, 1925ꢀ1932.
(6)
Hirsh, P. D.; Campbell, W. B.; Willerson, J. T.; Hillis, L. D. Prostaglandins and
ischemic heart disease. Am. J. Med. 1981, 71, 1009ꢀ1026.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 35
1
2
3
4
(7)
Badesch, D. B.; McLaughlin, V. V.; Delcroix, M.; Vizza, C. D.; Olschewski, H.;
5
6
7
8
Sitbon, O.; Barst, R. J. Prostanoid therapy for pulmonary arterial hypertension. J. Am. Coll.
Cardiol. 2004, 43, 56Sꢀ61S.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(8)
Database Syst Rev [Online] 2013, 4, DOI: 10.1002/14651858.CD000986.pub3.
(9) Barst, R. J.; McGoon, M.; McLaughlin, V.; Tapson, V.; Oudiz, R.; Shapiro, S.;
Robertson, L.; Andras, A. Prostanoids for intermittent claudication. Cochrane
Robbins, I. M.; Channick, R.; Badesch, D.; Rayburn, B. K.; Flinchbaugh, R.; Sigman, J.;
Arneson, C.; Jeffs, R. Beraprost therapy for pulmonary arterial hypertension. J. Am. Coll.
Cardiol. 2003, 41, 2119ꢀ2125.
(10) Tapson, V. F.; Jing, Z. C.; Xu, K. F.; Pan, L.; Feldman, J.; Kiely, D. G.; Kotlyar,
E.; McSwain, C. S.; Laliberte, K.; Arneson, C.; Rubin, L. J.; Team, F.ꢀC. S. Oral treprostinil for
the treatment of pulmonary arterial hypertension in patients receiving background endothelin
receptor antagonist and phosphodiesterase type 5 inhibitor therapy (the FREEDOMꢀC2 study): a
randomized controlled trial. Chest 2013, 144, 952ꢀ958.
(11) Asaki, T.; Hamamoto, T.; Sugiyama, Y.; Kuwano, K.; Kuwabara, K. Structureꢀ
activity studies on diphenylpyrazine derivatives: a novel class of prostacyclin receptor agonists.
Bioorg. Med. Chem. 2007, 15, 6692ꢀ6704.
(12) Nakamura, A.; Yamada, T.; Asaki, T. Synthesis and evaluation of Nꢀ
acylsulfonamide and Nꢀacylsulfonylurea prodrugs of a prostacyclin receptor agonist. Bioorg.
Med. Chem. 2007, 15, 7720ꢀ7725.
(13) Seiler, S.; Brassard, C. L.; Arnold, A. J.; Meanwell, N. A.; Fleming, J. S.; Keely,
S. L., Jr. Octimibate inhibition of platelet aggregation: stimulation of adenylate cyclase through
prostacyclin receptor activation. J. Pharmacol. Exp. Ther. 1990, 255, 1021ꢀ1026.
28
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Page 29 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(14) Meanwell, N. A.; Rosenfeld, M. J.; Trehan, A. K.; Wright, J. J.; Brassard, C. L.;
Buchanan, J. O.; Federici, M. E.; Fleming, J. S.; Gamberdella, M.; Zavoico, G. B.; Seiler, S. M.
Nonprostanoid prostacyclin mimetics. 2. 4,5ꢀDiphenyloxazole derivatives. J. Med. Chem. 1992,
35, 3483ꢀ3497.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(15) Meanwell, N. A.; Rosenfeld, M. J.; Trehan, A. K.; Romine, J. L.; Wright, J. J.;
Brassard, C. L.; Buchanan, J. O.; Federici, M. E.; Fleming, J. S.; Gamberdella, M.; Zavoica, G.
B.; Seiler, S. M. Nonprostanoid prostacyclin mimetics. 3. Structural variations of the diphenyl
heterocycle moiety. J. Med. Chem. 1992, 35, 3498ꢀ3512.
(16) Seiler, S. M.; Meanwell, N. A. BMY 42393, An orally active prostacyclin partial
agonist of novel Structure. Cardiovasc. Drug Rev. 1995, 13, 289ꢀ304.
(17) Meanwell, N. A.; Rosenfeld, M. J.; Wright, J. J.; Brassard, C. L.; Buchanan, J. O.;
Federici, M. E.; Fleming, J. S.; Seiler, S. M. Structureꢀactivity relationships associated with
3,4,5ꢀtriphenylꢀ1Hꢀpyrazoleꢀ1ꢀnonanoic acid, a nonprostanoid prostacyclin mimetic. J. Med.
Chem. 1992, 35, 389ꢀ397.
(18) Skuballa, W.; Schillinger, E.; Sturzebecher, C. S.; Vorbruggen, H. Synthesis of a
new chemically and metabolically stable prostacyclin analogue with high and longꢀlasting oral
activity. J. Med. Chem. 1986, 29, 313ꢀ315.
(19) Kuwano, K.; Hashino, A.; Asaki, T.; Hamamoto, T.; Yamada, T.; Okubo, K.;
Kuwabara,
K.
2ꢀ[4ꢀ[(5,6ꢀdiphenylpyrazinꢀ2ꢀyl)(isopropyl)amino]butoxy]ꢀNꢀ
(methylsulfonyl)acetam ide (NSꢀ304), an orally available and longꢀacting prostacyclin receptor
agonist prodrug. J. Pharmacol. Exp. Ther. 2007, 322, 1181ꢀ1188.
(20) Gallant, M.; Carriere, M. C.; Chateauneuf, A.; Denis, D.; Gareau, Y.; Godbout,
C.; Greig, G.; Juteau, H.; Lachance, N.; Lacombe, P.; Lamontagne, S.; Metters, K. M.; Rochette,
29
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