Structure-activity relationship study on the 6-membered heteroaromatic ring system of diphenylpyrazine-type prostacyclin receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2007.09.066

A series of prostacyclin receptor agonists was prepared by modifying the central heteroaromatic ring of lead compound 2, and a docking study was performed to investigate their structure-activity relationships by using a homology-modeled structure of the p

Full text of DOI:10.1016/j.bmcl.2007.09.066

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6588–6592
Structure–activity relationship study on the 6-membered
heteroaromatic ring system of diphenylpyrazine-type
prostacyclin receptor agonists
Tetsuo Asaki,* Taisuke Hamamoto, Yukiteru Sugiyama, Keiichi Kuwano,
Kenji Kuwabara and Tomoko Niwa
Discovery Research Laboratories, Nippon Shinyaku Co., Ltd, 14, Nishinosho-Monguchi-Cho, Kisshoin, Minami-ku,
Kyoto 601-8550, Japan
Received 22 June 2007; revised 14 September 2007; accepted 19 September 2007
Available online 22 September 2007
Abstract—A series of prostacyclin receptor agonists was prepared by modifying the central heteroaromatic ring of lead compound 2,
and a docking study was performed to investigate their structure–activity relationships by using a homology-modeled structure of
the prostacyclin receptor. Compound 2 and its derivatives could be docked to the prostacyclin receptor in two ways depending on
the position of the nitrogen atom within the heteroaromatic ring. Furthermore, hydrogen bonding between the nitrogen atom in the
heteroaromatic ring and the hydroxyl group of Ser20 or Tyr75 of the receptor appears to be important for the potent expression of
biological activity.
Ó 2007 Published by Elsevier Ltd.
Prostacyclin (PGI2) (1; Fig. 1) is an endogenous media-
tor that contributes to the maintenance of homeostasis
in the circulatory system, where it plays an important
role in the regulation of blood flow as an inhibitor of
platelet aggregation and as a vasodilator.1,2 However,
therapeutic application of PGI2 has been limited by its
inherent chemical lability and complicated administra-
tion schedule.3 Prostacyclin receptor (IP receptor) ago-
nists with extended duration of action are expected to
be desirable therapeutic agents in the treatment of vari-
ous circulatory diseases, such as pulmonary arterial
hypertension and arteriosclerosis obliterans. Much ef-
fort has therefore been expended in the search for chem-
ically stable and orally available IP receptor agonists,
including PGI2 analogues4–6 and nonprostanoid PGI2
mimetics.7–10
length of the linker and the presence of the concatenat-
ing nitrogen atom adjacent to the pyrazine ring are crit-
ical for antiaggregatory activity. That investigation led
to the identification of several potent nonprostanoid-
type IP receptor agonists with superior biological activ-
ity. The representative compound 2 (Fig. 1) inhibits
ADP-induced platelet aggregation in human platelet-
rich plasma with an IC50 of 0.2 lM. Having optimized
the structural features of the carboxylic acid side chain,
we turned our attention to the pyrazine moiety of 2. In
the present study, we describe the chemical modification
of the pyrazine ring of 2 and a molecular modeling study
to investigate the importance of this region for the
expression of potent agonist activity.
In a recent previous study,11 we described structure–
activity relationships (SAR) associated with diphenyl-
pyrazine derivatives as a novel class of IP receptor ago-
nists. The SAR investigation demonstrated that the
CO2H
O
N
O
CO2H
N
N
HO
Me
OH
Keywords: Prostacyclin; IP receptor agonist; Antiaggregatory activity;
Molecular modeling; Structure–activity relationship.
1: Prostacyclin (PGI2)
2
*
Corresponding author. Tel.: +81 75 321 9168; fax: +81 75 321
9039; e-mail: t.asaki@po.nippon-shinyaku.co.jp
Figure 1. Chemical structures of prostacyclin (PGI2; 1) and 2.
0960-894X/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.09.066

T. Asaki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6588–6592
6589
Ph
Ph
X
Z
Ph
Ph
X
Z
Ph
Ph
X
Z
Y
Y
3-5a
3-5b
3-5c
3-5d
a
: X = CH, Y = CH, Z = N
: X = CH, Y = N, Z = CH
: X = N, Y = N, Z = CH
: X = N, Y = N, Z = N
b, c
Y
O
CO2H
OH
Cl (or Br)
N
N
Me
Me
3a-d
4a-d
5a-d
Scheme 1. Reagents and conditions for synthesis of 5a–d: (a) 4-(methylamino)-1-butanol (10 equiv for 3a,b, and c, 1.5 equiv for 3d), 3a, neat, 100 °C;
3b, neat, 190 °C; 3c, neat, 170 °C; 3d, K2CO3, DMF, rt; (b) BrCH2CO2t-Bu, n-Bu4NHSO4, aq KOH, benzene, 0 °C to rt; (c) 1 N NaOH, MeOH,
reflux.
The strategy for the synthesis of diphenylated heterocy-
cles 5a–d, which is similar to that described for the cor-
responding pyrazine derivatives,11 is summarized in
Scheme 1. Coupling of 3a–d with 4-(methylamino)-1-
butanol afforded the corresponding alcohols 4a–d.
O-Alkylation of the alcohols 4a–d with tert-butyl bro-
moacetate under phase-transfer conditions12 followed
by saponification afforded the desired compounds 5a–
d. The starting material 3a was synthesized by N-oxida-
tion of 2,3-diphenylpyridine 613 and subsequent reaction
of the resulting N-oxide 7 with phosphorus oxychloride
as shown in Scheme 2. Other starting materials 3b,14 c15,
and d16 were prepared according to literature
procedures.
1119 gave the 2-(methylamino)pyrimidine 12, which
was N-alkylated with tert-butyl (4-bromobutyloxy)ace-
tate in the presence of sodium hydride as a base, fol-
lowed by saponification to give the target compound 13.
The test compounds prepared were evaluated for their
potency to inhibit ADP-induced platelet aggregation in
human platelet-rich plasma. The concentration of test
compound giving 50% inhibition of aggregation (IC50)
was determined from dose–response curves. In this as-
say, iloprost, beraprost sodium, and BMY42393 exhib-
ited IC50 values of 5 nM, 17 nM, and 1.5 lM,
respectively. The biological activity of the compounds
synthesized is summarized in Table 1.
The synthetic route to the diphenylpyridine 10 is shown
in Scheme 3. The requisite pyridine 9 was prepared by
catalytic hydrogenation of 817 followed by N-methyla-
tion of the resulting amine by reductive alkylation.18
N-Alkylation of 9 with tert-butyl (4-bromobutyl-
oxy)acetate11 in the presence of K2CO3 and NaI gave
the corresponding ester, which was converted to the tar-
get compound 10 by alkaline hydrolysis.
Using compound 2 as a template, we investigated the ef-
fect on biological potency of replacing the diphenylpyr-
azine ring by various diphenylated 6-membered
heteroaromatic rings. Platelet inhibitory activity was
considerably affected by modification of the pyrazine
ring (Table 1). To gain an understanding of the struc-
tural elements required for potency, three pyridine ana-
logues 5a,b and 10 were prepared. Replacement of the
2-amino-5,6-diphenylpyrazine ring in 2 with a 2-ami-
no-5,6-diphenylpyridine ring gave 5a, which retained
the potency of 2. The regioisomeric pyridines 5b and
10 showed severalfold-reduced potency compared to 2
and 5a. This result indicates that the nitrogen atom at
the 1-position (numbering according to the structure
shown in Table 1) has some impact on biological activ-
ity. We next synthesized the pyrimidine 13, the pyrida-
zine 5c, and the triazine 5d, which have two or three
nitrogen atoms in the 6-membered heteroaromatic ring.
The pyrimidine 13 showed a fourfold loss of potency
compared with 2, much the same as 5b and 10. A similar
The pyrimidine 13 was synthesized as shown in Scheme
4. Cyclization of 1-methylguanidine and the enamine
Ph
Ph
Ph
Ph
Ph
Ph
a
b
N
N
O
N
Cl
6
7
3a
Scheme 2. Reagents and conditions for synthesis of 3a: (a) mCPBA,
CHCl3, rt; (b) POCl3, 100 °C.
Ph
Ph
N
Ph
Ph
N
Ph
Ph
N
a, b
c, d
O
CO2H
N
NO2
NH
Me
Me
8
9
10
Scheme 3. Reagents and conditions for synthesis of 10: (a) HCO2NH4, 10% Pd–C, MeOH, rt; (b) i—HCO2Et, EtOH, reflux; ii—LiAlH4, THF, rt; (c)
Br(CH2)4OCH2CO2t-Bu, K2CO3, NaI, DMF, 90 °C; (d) 1 N NaOH, MeOH, reflux.
Ph
Ph
Ph
Ph
CHNMe2
O
Ph
Ph
N
N
a
b, c
O
CO2H
N
NH
Me
N
N
Me
11
12
13
Scheme 4. Reagents and conditions for synthesis of 13: (a) 1-methylguanidine hydrochloride, K2CO3, xylenes, reflux; (b) i—NaH, DMF, 80 °C; ii—
Br(CH2)4OCH2CO2t-Bu, 0 °C to rt; (c) 1 N NaOH, MeOH, reflux.

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T. Asaki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6588–6592
Stitham et al.20 showed that mutation of Tyr75, Phe95,
Phe278, or Arg279 of the IP receptor to Ala significantly
reduces the binding affinity for iloprost, providing evi-
dence that these amino acids are important for binding.
Taking this result into account, we manually docked 5a,
which is as active as 2, into the binding site of the IP
receptor by rotating around the single bonds of 5a and
the side chains of the ligand-contacting amino acids in
the binding site. We then performed energy minimiza-
tion with the MMFF94x force field21 implemented in
Table 1. Effect of alteration of the pyrazine ring of 2 on inhibition of
ADP-induced human platelet aggregation
4
3
Het
O
CO2H
N
Me
2
1
Compound
Het
Inhibition of human platelet
aggregation IC50 (lM)a
˚
MOE. Compound 5a and the amino acids within 7 A
of 5a were energy-minimized until the root-mean-square
N
N
2
0.2
0.2
1.1
8.6
1.3
0.8
0.8
gradient of the potential energy was less than 0.1 kcal
˚
molÀ1
A
À1. An automatic docking study with GLIDE
5ab
5b
5c
version 3.0 (Schro¨dinger, Inc.) produced similar binding
models. The entire docked model, in which the IP recep-
tor is shown as seven transmembrane (TM) helices, is
shown in Figure 2a. Compound 5a interacts mainly with
helices TM1, TM2, TM3, and TM7. Figure 2b shows a
close-up view of the binding site of the complex. The
carboxyl group of 5a is located close to the guanidino
group of Arg279, so that it is well placed for electrostatic
interaction with this amino acid. The nitrogen atom at
the 1-position of the pyridine ring is well placed to form
a hydrogen bond with the hydroxyl group of Tyr75. The
hydroxyl group of Ser20 is likely to form a hydrogen
bond with the hydroxyl group of Tyr75, thereby stabiliz-
ing the interaction between the 1-position nitrogen atom
of 5a and the hydroxyl group of Tyr75. The amino acids
Met23, Leu67, Val71, Phe95, Met99, Phe102, Phe278,
Ala282, and Pro285 form a large hydrophobic pocket
which accommodates well the two phenyl groups of
5a, and van der Waals interactions between the hydro-
phobic side chains of the amino acids and the phenyl
groups of 5a are facilitated. Similar modes of interaction
were observed in the docking model for prostacyclin
bound to the IP receptor20: the carboxyl group of pros-
tacyclin interacts with the guanidino group of Arg279
and the hydroxyl group at C11 interacts with the hydro-
xyl group of Tyr75, while the hydrophobic methylene
chain is located close to the hydrophobic amino acids
mentioned above.
N
N
N
N
N
N
5d
10a
13
N
N
N
N
a Inhibition of platelet aggregation induced by ADP (10 lM) in human
platelet rich-plasma.
b The biological activity of the sodium salt was evaluated.
result was obtained for the triazine 5d. The pyridazine 5c
was substantially less potent than the other analogues, a
result that provides evidence for the importance of the
nitrogen atom at the 1-position.
To further elucidate their SAR, we performed docking
studies for the representative agonists shown in Table
1. Stitham et al.20 have constructed a 3D model of
the IP receptor with the Internet-based protein struc-
ture homology-modeling server Swiss Model (http://
swissmodel.expasy.org/) by using the X-ray crystallo-
graphic structure of the bovine rhodopsin receptor
(Protein Data Bank code 1HZX) as a template. They
have also proposed a docking model for prostacyclin
bound to the IP receptor. Our 3D model of the IP
receptor was prepared according to their method by
using MOE version 2003.01 (Chemical Computing
Group, Inc.). The sequences of the IP receptor
(Swiss-Prot accession number P43119) and the bovine
rhodopsin receptor were aligned as described by Sti-
tham et al.20 A set of 10 intermediate homology models
was generated with the Homology module, and each
The pyridine derivatives 5b and 10 do not have nitrogen
atoms at the 1-position, yet they were fairly active. To
investigate the reason for this, we docked 5b into the
binding site of the IP receptor and found that it bound
well when it was flipped so that the nitrogen atom at
the 3-position could interact with the hydroxyl group
of Tyr75 (Fig. 2c). When compound 10 was similarly
docked (Fig. 2d), the nitrogen atom at the 4-position
could interact with the hydroxyl group of Ser20 instead
of that of Tyr75, and the hydroxyl group of Tyr75
apparently forms a hydrogen bond with the hydroxyl
group of Ser20. This flipping of 5b and 10 does not
greatly affect the mode of interaction of the substituents
at the 2-, 5-, and 6-positions with the IP receptor. In 5a,
the carboxyl group is located close to the guanidino
group of Arg279, and the two phenyl groups at the 5-
and 6-positions interact with the hydrophobic pocket
formed by Met23, Leu67, Val71, Phe95, Met99,
Phe102, Phe278, Ala282, and Pro285. The flexible meth-
ylene chain of the substituent at the 2-position can easily
intermediate was minimized to an energy gradient of
˚
0.01 kcal molÀ1
A
À1. The quality of the models gener-
ated was validated with the Protein Report module,
and the model with the lowest energy was selected
for further study.

T. Asaki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6588–6592
6591
Van der Waals interactions
between hydrophobic moieties
a
b
Phe278
Arg279
TM4
Met99
TM3
Electrostatic
interaction
Phe102
TM5
Ala282
5a
TM2
Phe95
Leu67
Pro285
Val71
Tyr75
Met23
Ser20
Van der Waals interactions
TM6
TM1
Hydrogen-bonding
interaction
TM7
5a
c
Van der Waals interactions
between hydrophobic moieties
d
between hydrophobic moieties
Phe278
Arg279
Phe278
Met99
Met99
Electrostatic
interaction
Electrostatic
Arg279
Phe102
Phe102
interaction
Ala282
Ala282
5b
10
Phe95
Leu67
Phe95
Leu67
Val71
Pro285
Val71
Pro285
Tyr75
Tyr75
Met23
Ser20
Met23
Ser20
Hydrogen-bonding
interaction
Hydrogen-bonding
interaction
Figure 2. Docking models of the homology-modeled IP receptor in complex with 5a, 5b, or 10. Hydrophobic amino acids are shown in white, and
hydrogen-bonding interactions are shown as broken lines. The figure was prepared with PyMOL version 0.99 (DeLano Scientific).
adapt its conformation so that the terminal carboxyl
group can interact with the guanidino group of
Arg279. Because the substituents at the 5- and 6-posi-
tions are the same, the flipping of 5b or 10 does not sig-
nificantly affect the interactions of these substituents
with the IP receptor.
computation times, we replaced the phenyl groups and
the carboxylate side chain with simple methyl groups,
which are unlikely to greatly influence the electronic
environment of the heteroaromatic rings. In the electro-
static-potential maps (Fig. 3), the colors range from red
Lead compound 2 can be docked into the IP receptor in
the same manner as 5a or 10 (Fig. 2b and d). Though it
is difficult to determine which nitrogen atom of the pyr-
azine ring interacts with the hydroxyl group of Tyr75 or
Ser20, it is reasonable to assume that a 5a-type interac-
tion (Fig. 2b) is more probable than a 10-type interac-
tion (Fig. 2d) because 5a is more potent than 10.
2
5a
5b
5c
N
Me
Me
Me
Me
N
Me
Me
Me
Me
N
N
Me
Me
Me
Me
N
N
N
N
N
N
Me
Me
Me
Similarly, 13 can be docked into the receptor in the same
manner as 5a or 5b (Fig. 2b and c). In 13, a 5a-type
interaction is more plausible than a 5b-type interaction,
because 5a is more potent than 5b.
Me
The result for 5c is less clear, because this compound has
lower activity than 5b or 10. Electrostatic-potential
maps are widely used in drug design to evaluate elec-
tronic properties.22 We therefore calculated electro-
5d
10
13
Me
Me
N
Me
Me
Me
Me
N
N
N
Me
Me
Me
N
N
N
N
Me
N
static-potential
maps
of
the
6-membered
Me
Me
heteroaromatic rings by using a Hartree–Fock quantum
mechanical model with a 6-31G(*) basis set with Trident
software (Wavefunction, Inc.). To avoid excessively long
Figure 3. Electrostatic-potential maps of the central heteroaromatic
rings of test compounds calculated with a 6-31G(*) basis set.

6592
T. Asaki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6588–6592
(high electronegativity) through green to blue (high elec-
tropositivity). The electrostatic-potential map of 5c
clearly differs from those of 2, 5a, 5b, 10, and 13, in that
the adjacent location of the two nitrogen atoms greatly
increases the red area around the nitrogen atoms. In
the IP receptor, the hydroxyl groups of Ser20 and
Tyr75 are close together and form an electronegative
environment. The electronegativity of the two adjacent
nitrogen atoms in 5c may lead to unfavorable electronic
interactions between the nitrogen atoms and the electro-
negative hydroxyl groups of Ser20 and Tyr75, thereby
weakening the hydrogen-bonding interaction between
them. Even though 5d also has two adjacent nitrogen
atoms, it is more active than 5c. This is reasonable be-
cause 5d also has a nitrogen atom at the 1-position, so
that a 5a-type interaction is possible.
References and notes
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Our results can be summarized as follows. (1) The
important interactions with the IP are the same for pros-
tacyclin and the diphenylpyrazine derivatives: electro-
static interactions between the terminal carboxyl group
and the guanidino group of Arg279, hydrogen-bonding
interactions between a nitrogen atom of the heteroaro-
matic ring and the hydroxyl group of Ser20 or Tyr75,
and van der Waals interactions with the hydrophobic
side chains of Met23, Leu67, Val71, Phe95, Met99,
Phe102, Phe278, Ala282, and Pro285. (2) The flexibility
of the substituent at the 2-position and the presence of
the same group at the 5- and 6-positions allow two
modes of binding depending on the position of the nitro-
gen atom in the heteroaromatic ring. (3) A favorable
hydrogen-bonding interaction between a nitrogen atom
of the heteroaromatic ring and Ser20 or Tyr75 greatly
contributes to the agonistic activity.
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We thank Dr. Gerald E. Smyth for helpful suggestions
during the preparation of the manuscript.