Structure-activity studies on diphenylpyrazine derivatives: A novel class of prostacyclin receptor agonists

Article abstract of DOI:10.1016/j.bmc.2007.08.010

To develop nonprostanoid prostacyclin receptor agonists with a high degree of metabolic resistance and an extended duration of action, a novel series of diphenylpyrazine derivatives was synthesized and evaluated for their inhibition of ADP-induced human platelet aggregation. Structure-activity relationship studies on the side chain containing the carboxylic acid moiety of the lead compound 5c showed that the length of the linker and the presence of the concatenating nitrogen atom adjacent to the pyrazine ring are critical for the antiaggregatory activity. This study led to the discovery of 2-amino-5,6-diphenylpyrazine derivatives 8c, 15a, and 15b, which showed potent inhibition of platelet aggregation with IC₅₀ values of 0.2 μM. Among these compounds, 15b is an orally available and long-lasting prostacyclin receptor agonist which is promising for the treatment of various vascular diseases.

Full text of DOI:10.1016/j.bmc.2007.08.010

Bioorganic & Medicinal Chemistry 15 (2007) 6692–6704
Structure–activity studies on diphenylpyrazine derivatives: A
novel class of prostacyclin receptor agonists
Tetsuo Asaki,* Taisuke Hamamoto, Yukiteru Sugiyama,
Keiichi Kuwano and Kenji Kuwabara
Discovery Research Laboratories, Nippon Shinyaku Co., Ltd, 14 Nishinosho-Monguchi-Cho, Kisshoin,
Minami-ku, Kyoto 601-8550, Japan
Received 22 June 2007; revised 3 August 2007; accepted 4 August 2007
Available online 15 August 2007
Abstract—To develop nonprostanoid prostacyclin receptor agonists with a high degree of metabolic resistance and an extended
duration of action, a novel series of diphenylpyrazine derivatives was synthesized and evaluated for their inhibition of ADP-induced
human platelet aggregation. Structure–activity relationship studies on the side chain containing the carboxylic acid moiety of the
lead compound 5c showed that the length of the linker and the presence of the concatenating nitrogen atom adjacent to the pyrazine
ring are critical for the antiaggregatory activity. This study led to the discovery of 2-amino-5,6-diphenylpyrazine derivatives 8c, 15a,
and 15b, which showed potent inhibition of platelet aggregation with IC50 values of 0.2 lM. Among these compounds, 15b is an
orally available and long-lasting prostacyclin receptor agonist which is promising for the treatment of various vascular diseases.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
CO2H
CO2Na
Prostacyclin (PGI2) (1; Fig. 1) is an endogenous medi-
ator produced primarily in the vascular endothelial
cells. It plays an important role in vascular homeosta-
sis as an inhibitor of platelet aggregation and a potent
vasodilator.1 These physiological functions are medi-
ated by the activation of a specific G-protein-coupled
receptor, the PGI2 receptor (IP receptor).2 IP receptor
agonists are thought to be clinically useful agents for
the precise control of platelet and vascular function,
and the sodium salt of PGI2 itself is available as Flo-
lanꢁ (GlaxoSmithKline) for the treatment of arterial
pulmonary hypertension. However, its therapeutic
application has been limited because of its chemical
lability and complicated administration by continuous
infusion through a central venous catheter. To over-
come such drawbacks, much effort has been directed
toward developing chemically and metabolically stable
IP receptor agonists. Beraprost sodium (2),3 a repre-
sentative chemically stable PGI2 analogue, is now in
clinical use as an orally available drug for the treat-
ment of arteriosclerosis obliterans and idiopathic pul-
O
O
HO
HO
OH
1: Prostacyclin (PGI2)
OH
2: Beraprost sodium
Ph
Ph
Ph
Ph
N
N
N
O(CH2)7CO2H
O
O
Ph
CO2H
3: Octimibate
4: BMY42393
Ph
Ph
N
N
N
Me
CO2H
5c
Figure 1. Chemical structures of prostacyclin (PGI2) and prostacyclin
mimetics.
Keywords: Prostacyclin; IP receptor agonist; Diphenylpyrazine; Anti-
aggregatory activity.
monary arterial hypertension in the Asian area,
including Japan, but its duration of action is insuffi-
cient owing to its metabolic instability (t1/2 = 1 h in
*
Corresponding author. Tel.: +81 75 321 9168; fax: +81 75 321
9039; e-mail: t.asaki@po.nippon-shinyaku.co.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.010

T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
6693
humans).4 On the other hand, many nonprostanoid IP
2. Chemistry
receptor agonists,5–11 which do not have the prosta-
noid skeleton, have been reported. Octimibate 35 and
BMY42393 46 (Fig. 1), partial agonists for the IP
receptor, are structurally quite different from PGI2
and its analogues, except for the carboxylic acid moi-
ety. These observations demonstrate that the prosta-
noid skeleton is not essential for the activation of
the IP receptor. We similarly focused our attention
on nonprostanoid IP receptor agonists with a high de-
gree of metabolic resistance and an extended duration
of action.
The strategy for the synthesis of the pyrazine derivatives
related to 5a–d and 8a–c involved N-alkylation of the 2-
amino-5,6-diphenylpyrazines 7a–d with the appropriate
bromides as the key step (Scheme 1). Coupling of 2-
chloro-5,6-diphenylpyrazine 612 with various amines
provided the aminopyrazines 7a–d. N-Alkylation of
7a–d by deprotonation with sodium hydride followed
by treatment with the esters of x-bromoalkanoic acids
or the bromide 11 (prepared as described below) affor-
ded the corresponding esters, which were converted to
the desired compounds 5a–d and 8a–c by alkaline
hydrolysis. The alcohol 913 was O-alkylated with tert-
butyl bromoacetate under phase-transfer conditions14
to give the ester 10. Removal of the tetrahydropyranyl
(THP) protecting group on 10 by a standard protocol
followed by bromination afforded the bromide 11.
On the basis of the chemical structures of the known
nonprostanoid-type agonists, we performed extensive
chemical modification of these compounds. As part
of our drug-discovery effort to identify new IP recep-
tor agonists, we designed and synthesized the 2-amino-
5,6-diphenylpyrazine derivative 5c (Fig. 1), which has
a
pyrazine ring instead of the oxazole ring of
Alternative synthetic routes for the synthesis of the pyr-
azine derivatives related to 15a–d and 22a and 22b are
summarized in Schemes 2 and 3. N-Alkylation of 4-ami-
no-1-butanol 12 by formylation followed by LiAlH4
reduction (for 13a) or reductive alkylation (for 13b
and 13c) provided the amino alcohols 13a–c. The ben-
zylaminoalcohol 13d was prepared according to a litera-
ture procedure.15 The amino alcohols 13a–d were then
coupled with the chloropyrazine 6 to afford the corre-
sponding alcohols 14a–d, which were transformed into
the target compounds 15a–d according to the O-alkyl-
ation procedure described for 10 followed by alkaline
BMY42393. Our original lead compound 5c moder-
ately inhibited ADP-induced human platelet aggrega-
tion (IC50 = 1.5 lM). We next sought to increase the
potency of 5c by optimizing the side chain containing
the carboxylic acid moiety. In this report, we describe
the synthesis and structure–activity relationships
(SAR) of diphenylpyrazine derivatives as a novel class
of IP receptor agonists. Our goals were to investigate
the SAR based on the structure of 5c, to maximize
potency, and to identify an orally available agent with
a good pharmacokinetic profile.
Ph
Ph
N
Ph
N
Ph
Ph
N
c, d
5a: n = 5
a or b
5b: n = 6
(CH2)nCO2H
5c: n = 7
5d: n = 8
N
Cl
Ph
N
NHR
N
N
Me
6
7a: R = Me
7b:
7c:
7d:
R = Et
R = allyl
R = cyclopropyl
Ph
Ph
N
N
8a: R = Et
c, d
8b:
8c:
R = allyl
O
CO2H
R = cyclopropyl
N
R
e
OH
O
CO2t-Bu
THPO
R
9
10: R = OTHP
11: R = Br
f, g
Scheme 1. Reagents: (a) methylamine, MeOH for 7a; (b) amines, 1-butanol for 7b–d; (c) i—NaH, DMF; ii—Br(CH2)nCO2R for 5a–d, 11 for 8a–c;
(d) 1 N NaOH, MeOH; (e) BrCH2CO2t-Bu, n-Bu4NHSO4, aq KOH, benzene; (f) p-TsOH, MeOH; (g) Ph3P, CBr4, CH2Cl2.
a (for 13a)
d (for 14a)
e (for 14b)
OH
Ph
Ph
N
N
Ph
Ph
N
N
b (for 13b, 13c)
g
OH
H2N
HN
R
OH
O
CO2H
c (for 13d)
f (for 14c, 14d)
N
R
N
R
12
13a:R = Me
14a: R = Me
15a:R = Me
13b:
13c:
13d:
14b:
14c:
14d:
15b:
15c:
15d:
R = i-Pr
R = i-Pr
R = i-Pr
R = cyclopentyl
R = benzyl
R = cyclopentyl
R = benzyl
R = cyclopentyl
R = benzyl
Scheme 2. Reagents: (a) i—HCO2Et, EtOH, ii—LiAlH4, THF, for 13a; (b) acetone for 13b, cyclopentanone for 13c, H2, PtO2, EtOH; (c) i—PhCHO,
MeOH, CH2Cl2; ii—NaBH4, MeOH, CH2Cl2, for 13d (see Ref. 15); (d) 6, K2CO3, DMF; (e) 6, neat; (f) 6, triisopropanolamine, sulfolane; (g) i—
BrCH2CO2t-Bu, aq KOH, benzene; ii—1 N NaOH, MeOH.

6694
T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
a
b
H
N
OHC
OMOM
OBn
Me
Me
OMOM
c
Ph
Ph
N
N
Ph
Ph
N
N
16
18
17
19
f, g
OR
OCH2CO2H
n
n
N
Me
N
Me
c
20a: n = 1, R = MOM
21a: n = 1, R = H
20b: n = 2, R = Bn
H2N
N
H
OBn
22a: n = 1
22b: n = 2
d
e
21b: n = 2, R = H
Scheme 3. Reagents: (a) MeNH2, H2, 5% Pd–C, MeOH; (b) i—HCO2Et, EtOH; ii—LiAlH4, THF; (c) 6, K2CO3, DMF; (d) HCl, MeOH; (e) c-HCl,
EtOH; (f) BrCH2CO2Me, K2CO3, cat KI, MeCN; (g) 1 N NaOH, MeOH.
hydrolysis. The transformation from 13c–d to 14c–d was
carried out by our own original method. To overcome
the steric hindrance of the amines, the reaction was car-
ried out at high temperature in the presence of high-boil-
ing triisopropanolamine base with high-boiling
sulfolane as the solvent. Under these conditions, the re-
quired number of equivalents of the expensive amino
alcohols could be reduced. The phenoxyacetic acids
22a and 22b were prepared from the methylamines 17
and 19, which were easily derived from the benzaldehyde
1616 and the phenethylamine 18,17 respectively. Cou-
pling of 17 with the chloropyrazine 6 followed by
O-deprotection yielded the phenol 21a. Subsequent O-
alkylation of 21a with methyl bromoacetate followed
by alkaline hydrolysis gave the desired compound 22a.
A similar sequence of reactions starting with 19 led to
the acid 22b.
The pyrazine derivatives 28a–d were prepared according
to Scheme 5. Coupling of the hydroxypyrazine 26a12
and the mercaptopyrazine 26b18 with the bromide 11
afforded the esters 27a and 27b, respectively. The desired
acids 28a and 28b were synthesized by alkaline hydroly-
sis of 27a and 27b. The sulfoxide 28c and the sulfone 28d
were prepared by oxidation of the sulfide 27b by m-
chloroperbenzoic acid (mCPBA) followed by acid
hydrolysis.
The synthetic routes to the tetrazole 30 and the N-meth-
ylsulfonamide 31 are shown in Schemes 6 and 7, respec-
tively. O-Alkylation of the alcohol 14b with
bromoacetonitrile gave the nitrile 29, which was con-
verted to the tetrazole 30 by heating with sodium azide.
The carboxylic acid 15b was easily converted to the sul-
fonamide 31 through condensation with methanesulf-
onamide in the presence of 1,10-carbonyldiimidazole
(CDI).11
The synthetic route to the alkylated pyrazine 25 is
shown in Scheme 4. Sonogashira reaction of the chloro-
pyrazine 6 with 4-pentyn-1-ol gave the coupling product
23, which was converted to the alcohol 24 by catalytic
hydrogenation. The alcohol 24 underwent a two-step
reaction sequence as described above that involved
introduction of the ester moiety followed by alkaline
hydrolysis to give the desired compound 25.
3. Results and discussion
The test compounds prepared were evaluated for their
potency to inhibit ADP-induced platelet aggregation in
human platelet-rich plasma. The concentration of test
Ph
Ph
N
Ph
Ph
N
N
Ph
Ph
N
N
Ph
Ph
N
N
OH
b
c
OH
O
CO2H
a
N
Cl
OH
6
23
24
25
Scheme 4. Reagents: (a) PdCl2(PPh3)2, CuI, Et3N; (b) H2, 5% Pd–C, EtOH; (c) i—BrCH2CO2 t-Bu, n-Bu4NHSO4, aq KOH, benzene; ii—1 N
NaOH, MeOH.
Ph
Ph
N
N
Ph
Ph
N
N
Ph
Ph
N
N
a (for 27a)
b (for 27b)
c
O
CO2t-Bu
O
CO2H
CO2H
X
X
X
d, e
26a: X = OH
26b: X = SH
27a: X = O
27b: X = S
28a: X = O
28b: X = S
Ph
Ph
N
N
O
S
(O)n
28c: n = 1
28d: n = 2
Scheme 5. Reagents: (a) i—NaH, DMF; ii—11; (b) 11, Na2CO3, acetone; (c) 1 N NaOH, MeOH; (d) mCPBA (1 eq for 28c, 2 eq for 28d), CHCl3;
(e) 4 N HCl, 1,4-dioxane.

T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
6695
Ph
Ph
N
N
Ph
Ph
N
N
Ph
Ph
N
N
N
N
a
b
N
OH
O
CN
O
N
N
N
N
H
14b
29
30
Scheme 6. Reagents: (a) BrCH2CN, n-Bu4NHSO4, aq KOH, benzene; (b) NaN3, NH4Cl, DMF.
Ph
Ph
N
N
Ph
Ph
N
N
a
O
CO2H
O
CONHSO2Me
N
N
15b
31
Scheme 7. Reagents: (a) i—CDI, THF; ii—MeSO2NH2, DBU.
Table 2. Effect of concatenating atoms between the pyrazine and the
linker on inhibition of ADP-induced human platelet aggregation
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 Tables 1–4.
Ph
Ph
N
N
O
CO2H
X
Compound
X
Inhibition of human
platelet aggregation
IC50a (lM)
Previous studies on nonprostanoid PGI2 mimetics such
as the oxazole6 and pyrazole series11 provide key infor-
mation on the structural requirements for the design
of potent IP receptor agonists. These studies demon-
strate that the agonist activity strongly depends on the
topographical relationship between the diphenylated
heterocycle and the carboxylic acid functionality, which
comprise the nonprostanoid PGI2 mimetic pharmaco-
phore. With this information in hand, our first efforts to-
ward optimization of the lead compound 5c
(IC50 = 1.5 lM) were focused on altering the conforma-
tionally flexible alkylene chain while keeping the other
features intact. We investigated the effects of the length
and rigidity of the methylene linker (Table 1). Structure–
activity studies on 5a–d clearly demonstrated that a hex-
amethylene linker was of the optimal length. Compound
5b (hexamethylene linker), which is one carbon shorter
15ab
28bb
25
NMe
S
0.2
2
CH2
O
17
34
28a
28c
28d
SO
SO2
37
>100
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.
Table 3. Effect of substituents at the N-atom adjacent to the pyrazine
ring on inhibition of ADP-induced human platelet aggregation
Ph
Ph
N
N
O
CO2H
N
R
Table 1. Effect of linker variation on inhibition of ADP-induced
human platelet aggregation
Compound
R
Inhibition of human
platelet aggregation
IC50a (lM)
Ph
Ph
N
N
8ab
8b
Et
Allyl
0.5
1
Linker
N
CO2H
Me
8c
Cyclopropyl
Me
i-Pr
0.2
0.2
0.2
0.8
50
15ab
15b
15cb
15d
Compound
Linker
Inhibition of human
platelet aggregation
a
Cyclopentyl
Benzyl
IC50 (lM)
5a
5b
–(CH2)5–
–(CH2)6–
–(CH2)7–
–(CH2)8–
8
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.
0.3
1.5
1.5
0.2
5c
5d
15a
–(CH2)4–OCH2–
than the lead 5c (heptamethylene), showed fivefold-
more-potent inhibition than 5c (IC50 = 0.3 lM). Reduc-
ing the length of 5c by two carbons, however, resulted in
a significant loss of potency (5a (pentamethylene),
IC50 = 8 lM), while extension of the linker by one car-
bon to give 5d (octamethylene) had no effect on potency.
Replacement of the methylene unit in the b-position
22a
22b
5
2
-CH2
OCH2-
-(CH2)2
OCH2-
a Inhibition of platelet aggregation induced by ADP (10 lM) in human
platelet-rich plasma.

6696
T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
Table 4. Effect of alteration of terminal carboxyl group on inhibition
of ADP-induced human platelet aggregation
15d (R = benzyl). These results suggest that the N-sub-
stituent may interact with a sterically restricted pocket
of the receptor ligand-binding domain which can accept
only a small hydrophobic group.
Ph
Ph
N
N
O
Y
N
Finally, modification of the terminal carboxyl group of
15b was investigated (Table 4). Tetrazole 30 showed
only a marginally reduced potency (IC50 = 0.4 lM)
compared to 15b, as observed in early studies of nonpro-
stanoid IP receptor agonists.6,11 The tetrazole ring there-
fore functioned as an effective isostere of the carboxyl
group in 15b. Although N-acylsulfonamides are well
known as carboxylic acid mimics,20,21 the N-methylsulf-
onamide 31 showed significantly reduced inhibitory
activity (IC50 = 6.4 lM), suggesting that the terminal
carboxyl group is important for biological activity.
Compound
Y
Inhibition of human
platelet aggregation
IC50a (lM)
15b
CO2H
0.2
N
N
30
31
0.4
6.4
N
N
H
CONHSO2Me
a Inhibition of platelet aggregation induced by ADP (10 lM) in human
platelet-rich plasma.
When a therapeutic agent targeting the IP receptor is
being developed for long-term administration, a critical
issue to be considered is the risk of unpredictable side ef-
fects mediated by other prostanoid receptors. To avoid
such side effects, it is desirable to develop an agent that
is selective for the IP receptor. On the basis of its perfor-
mance in in vitro functional assays and other character-
istics, including favorable pharmacokinetic and low
toxicity in animal studies, we selected 15b as a promising
candidate and investigated its binding affinity for the IP
and other prostanoid receptors. In another paper,22 we
demonstrate that 15b shows a high binding affinity for
the human IP receptor with a Ki value of 20 nM in
our receptor binding assay, displaying more than 130-
fold selectivity over the other human prostanoid recep-
tors (EP1–4, DP, FP, and TP). In addition, no significant
off-target activity in binding or enzyme assays was ob-
served for 15b (data not shown).
with respect to the carboxyl group by an oxygen atom
was explored in expectation of increasing metabolic
resistance to b-oxidative degradation in vivo.19 Intro-
duction of an oxygen atom at this position gave 15a
(IC50 = 0.2 lM), which was compatible with mainte-
nance of in vitro activity. Next, to reduce the flexibility
of the linear alkyl chain, we incorporated a phenyl ring
within the methylene linker to obtain 22a and 22b and
compared their activities with those of 5b and 5c, which
have a simple methylene linker. We used a meta-substi-
tution pattern because the length of the resulting linker
is approximately equal to that of the methylene linker in
5b and 5c in the extended form. This was to try to ensure
that the terminal carboxyl group would be correctly
positioned. Compounds 22a and 22b showed weaker
activity than 5c (22a, IC50 = 5 lM; 22b, IC50 = 2 lM).
The marked enhancement in potency observed for 15a
prompted us to explore the SAR of compounds incorpo-
rating an oxygen atom in the methylene linker. Keeping
this oxygen atom in the linker, we then investigated the
necessity of the nitrogen atom which joins the pyrazine
ring and the alkyl side chain (Table 2). Replacement
of the N-methylamino group by methylene or oxygen
led to a two-order-of-magnitude reduction in potency
(25, IC50 = 17 lM; 28a, IC50 = 34 lM). A sulfur atom
at this site produced a 10-fold reduction in activity
(28b, IC50 = 2 lM), and the sulfoxide 28c and the sul-
fone 28d also showed substantially lower potency. These
results show that the N-methylamino group played an
important role in the biological activity of 15a.
After intravenous and oral administration of 15b to rats,
dogs, and monkeys (Table 5), 15b displayed good oral
bioavailability (BA rat, 102%; dog, 80%) and appropri-
ately extended duration (t1/2 rat, 3.6 h; dog, 6.2 h; mon-
key, 5.6 h).
4. Conclusions
To develop nonprostanoid IP receptor agonists with a
high degree of metabolic resistance and a long duration
Table 5. Pharmacokinetic parameters of 15b after intravenous or oral
administration to rats, dogs, and monkeys
To explore the steric requirements for the substituent at
the nitrogen atom adjacent to the pyrazine ring, the ef-
fects of replacing the N-methyl group were also evalu-
ated (Table 3). Some sensitivity to variation in this
region was observed. Compound 8a (R = Et) had a
slightly lower antiaggregatory activity against human
platelets (IC50 = 0.5 lM), and compounds 8c
(R = cyclopropyl) and 15b (R = i-Pr) showed activity
equal to that of 15a. Thus, small-sized alkyl groups were
well tolerated. In contrast, replacement of the N-methyl
group by larger substituents gave compounds 8b
(R = allyl) and 15c (R = cyclopentyl), with 4- to 5-fold
lower potency, and the relatively inactive compound
Route Parameter
Ratsa
Dogsa
Monkeysa
b
—
iv
Dose (mg/kg)
t1/2 (h)
3
4.9 1.4
1
5.9 0.2
72.5 9.1
—
—
AUC (lg · h/mL) 5.2 0.7
po
Dose (mg/kg)
Tmax (h)
10
3
0.8 0.4
14.9 3.6 0.11 0.04
1
2.3 1.5
0.8 0.3
1.9 0.5
3.6 0.7
Cmax (lg/mL)
t1/2 (h)
6.2 1.0
5.6 1.5
0.65 0.12
—
AUC (lg · h/mL) 17.7 1.9 176 41
BA (%) 102 80
a Each value is the mean SD (n = 3).
b Not tested.

T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
6697
of action, we performed a SAR study on the side chain
containing the carboxylic acid moiety of diphenylpyr-
azine derivative 5c. This study revealed that the inhibi-
tion of ADP-induced human platelet aggregation was
strongly influenced by the length of the linker and the
presence of the concatenating nitrogen atom. In addi-
tion, we investigated the effects of altering the N-substi-
tuent and the terminal carboxyl group and identified the
structural requirements for effective PGI2 mimicry in
this series. The optimized compounds 8c, 15a, and 15b
inhibited platelet aggregation equally potently, with
IC50 values of 0.2 lM. Compound 15b displayed good
oral bioavailability and fairly long plasma duration in
rats, dogs, and monkeys. Further studies on this series
of derivatives are in progress to develop clinical candi-
dates for orally available and long-lasting IP receptor
agonists.
over MgSO4 and evaporated in vacuo. The residual so-
lid was washed with isopropyl ether and filtered to give
7b (1.56 g, 76%) as colorless crystals. Mp 124–126 ꢂC.
FAB-MS m/z 276 [MH]+. 1H NMR (CDCl3) d: 1.31
(3H, t, J = 7.2 Hz), 3.47 (2H, qd, J = 7.2, 5.4 Hz), 4.67
(1H, br), 7.10–7.50 (10H, m), 7.93 (1H, s). Anal. Calcd
for C18H17N3: C, 78.52; H, 6.22; N, 15.26. Found: C,
78.46; H, 6.23; N, 15.22.
5.1.3. N-(5,6-Diphenylpyrazin-2-yl)allylamine (7c). Com-
pound 7c was prepared from 6 and allylamine in a man-
ner similar to that described for 7b except that the crude
7c was purified by silica gel column chromatography
with hexane–EtOAc (4:1) as the eluent Yield 48%, pale
yellow crystals. Mp 102–103 ꢂC. FAB-MS m/z 288
1
[MH]+. H NMR (CDCl3) d: 4.03–4.14 (2H, m), 4.88
(1H, t, J = 5.6 Hz), 5.21 (1H, dt, J = 10.2, 2.2 Hz),
5.32 (1H, dt, J = 17.0, 1.6 Hz), 5.98 (1H, ddt, J = 17.0,
10.2, 5.6 Hz), 7.19–7.50 (10H, m), 7.94 (1H, s). Anal.
Calcd for C19H17N3: C, 79.41; H, 5.96; N, 14.62. Found:
C, 79.42; H, 5.96; N, 14.57.
5. Experimental
Reagents and solvents were used as obtained from the
supplier without further purification. Melting points
were determined on a Shibata melting-point apparatus
and are uncorrected. Column chromatography was car-
ried out on a silica gel column (Wako Wakogelꢁ C-200
or Fuji Silysia PSQ-100B). TLC was carried out on
Merck TLC aluminum sheets silica gel 60 F254, and
detection was by UV quenching at 254 nm or spraying
with phosphomolybdic acid. Yields were not optimized.
1H NMR spectra were recorded on a Varian Gemini
2000 (200 MHz) spectrometer. Chemical shifts (d) are gi-
ven in ppm relative to the internal standard, tetrameth-
ylsilane, and coupling constants are given in Hertz (Hz).
Mass spectra were recorded on a JEOL JMS-700 mass
spectrometer and IR spectra on a Shimadzu FT IR-
8100 spectrometer.
5.1.4. N-(5,6-Diphenylpyrazin-2-yl)cyclopropylamine (7d).
Compound 7d was prepared from 6 and cyclopropyl-
amine in a manner similar to that described for 7b. Yield
61%, pale yellow crystals. Mp 136–138 ꢂC. FAB-MS m/z
1
288 [MH]+. H NMR (CDCl3) d: 0.60–0.70 (2H, m),
0.82–0.95 (2H, m), 2.60–2.75 (1H, m), 5.20 (1H, s),
7.15–7.45 (10H, m), 8.30 (1H, s). Anal. Calcd for
C19H17N3: C, 79.41; H, 5.96; N,14.62. Found: C,
79.49; H, 6.08; N, 14.49.
5.1.5. tert-Butyl[4-(2-tetrahydropyranyloxy)butoxy]ace-
tate (10). Compound 10 was prepared from the alcohol
913 essentially according to a previously described meth-
od.14 A mixture of 9 (35.98 g, 0.207 mol), tetra-n-butyl-
ammonium hydrogensulfate (33.95 g, 0.100 mol),
benzene (300 mL), and 40% aqueous KOH (300 mL)
was stirred vigorously and then cooled to 5 ꢂC. tert-Bu-
tyl bromoacetate (48.45 g, 0.248 mol) was added drop-
wise as rapidly as possible while keeping the
temperature below 10 ꢂC. After stirring had been contin-
ued for 45 min, the mixture was stirred at room temper-
ature for a further 1 h. The reaction mixture was diluted
with ice water and extracted with Et2O. The extract was
washed with water and brine and dried over MgSO4.
After the solvent was concentrated in vacuo, the residual
oil was subjected to chromatography on silica gel with
hexane–EtOAc (4:1) as the eluent to give 10 (36.21 g,
61%) as a colorless oil. FAB-MS m/z 289 [MH]+.1H
NMR (CDCl3) d: 1.40–1.95 (10H, m), 1.48 (9H, s),
3.30–3.65 (4H, m), 3.69–3.93 (2H, m), 3.96 (2H, s),
4.59 (1H, t, J = 3.3 Hz). Anal. Calcd for C15H28O5Æ0.2-
H2OÆ0.3C4H8O2: C, 60.76; H, 9.72. Found: C, 60.52;
H, 9.30.
5.1. Preparation of 5a–d and 8a–c
5.1.1. N-(5,6-Diphenylpyrazin-2-yl)methylamine (7a). A
mixture of the chloropyrazine 612 (14.00 g, 52 mmol)
and a 40% solution of methylamine in methanol
(57 mL) was heated at 80 ꢂC for 12 h in a sealed tube.
The mixture was cooled to room temperature and con-
centrated. The residue was then diluted with water and
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated in vacuo,
after which the residual solid was recrystallized from
EtOAc–hexane to give 7a (7.82 g, 57%) as pale yellow
crystals. Mp 137–138 ꢂC [lit. 138–139 ꢂC23]. FAB-MS
m/z 262 [MH]+. 1H NMR (CDCl3) d: 3.04 (3H, d,
J = 5.0 Hz), 4.78 (1H, br), 7.10–7.50 (10H, m), 7.94
(1H, s). Anal. Calcd for C17H15N3: C, 78.13; H, 5.79;
N, 16.08. Found: C, 78.31; H, 5.97; N, 16.05.
5.1.2. N-(5,6-Diphenylpyrazin-2-yl)ethylamine (7b). A
mixture of 6 (2.00 g, 7.5 mmol) and a 70% solution of
ethylamine in water (9.66 g, 150 mmol) in 1-butanol
(10 mL) was heated at 150 ꢂC for 24 h in a sealed tube.
The mixture was cooled to room temperature and con-
centrated. The residue was diluted with water and ex-
tracted with CHCl3, after which the extract was dried
5.1.6. tert-Butyl(4-bromobutoxy)acetate (11). To a solu-
tion of the ester 10 (36.21 g, 0.126 mol) in MeOH
(360 mL) was added p-toluenesulfonic acid monohy-
drate (47.77 g, 0.251 mol). The mixture was stirred at
room temperature for 30 min. The reaction mixture
was poured into saturated aqueous NaHCO3 and ex-
tracted twice with Et2O. The extracts were dried over

6698
T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
MgSO4. After evaporation of the solvent, the residue
was purified by chromatography on silica gel with hex-
ane–EtOAc (2:1) as the eluent to give tert-butyl(4-
hydroxybutoxy)acetate (17.02 g, 66%) as a colorless
oil. 1H NMR (CDCl3) d: 1.48 (9H, s), 1.70 (2H, t,
J = 7.0 Hz), 1.73 (2H, t, J = 7.0 Hz), 3.57 (2H, t,
J = 5.8 Hz), 3.68 (2H, t, J = 6.0 Hz), 3.97 (2H, s). To a
solution of this alcohol (5.48 g, 27 mmol) in CH2Cl2
(150 mL) were added triphenylphosphine (7.74 g,
30 mmol) and carbon tetrabromide (10.22 g, 30 mmol).
The mixture was stirred at room temperature for 1 h.
After evaporation of the solvent, the residue was puri-
fied by chromatography on silica gel with CHCl3 as
the eluent to give 11 (5.18 g, 72%) as a colorless oil.
FAB-MS m/z 269 [M+2]+, 267 [MH]+. 1H NMR
(CDCl3) d: 1.48 (9H, s), 1.70–1.83 (2H, m), 1.93–2.07
(2H, m), 3.47 (2H, t, J = 6.6 Hz), 3.55 (2H, t,
J = 6.2 Hz), 3.95 (2H, s). Anal. Calcd for
C10H19BrO3Æ0.6CHCl3: C, 37.58; H, 5.83. Found: C,
37.31; H, 5.66.
698 cmꢀ1 1H NMR (CDCl3) d: 1.30–1.50 (2H, m),
.
1.60–1.80 (4H, m), 2.36 (2H, t, J = 7.3 Hz), 3.17 (3H,
s), 3.63 (2H, t, J = 7.4 Hz), 7.15–7.50 (10H, m), 8.03
(1H, s). Anal. Calcd for C23H25N3O2Æ0.1H2O: C,
73.22; H, 6.73; N, 11.14. Found: C, 73.16; H, 6.82; N,
11.01.
5.1.9. 7-[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]hepta-
noic acid (5b). Compound 5b was prepared from 7a and
ethyl 7-bromoheptanoate. Yield 8% (in two steps), col-
orless crystals. Mp 114–118 ꢂC. FAB-MS m/z 390
[MH]+. IR (KBr): 2950, 1717, 1583, 1568, 1208,
698 cmꢀ1 1H NMR (CDCl3) d: 1.30–1.80 (8H, m),
.
2.32 (2H, t, J = 7.3 Hz), 3.17 (3H, s), 3.61 (2H, t,
J = 7.1 Hz), 4.30 (1H, br), 7.20–7.50 (10H, m), 8.02
(1H, s). Anal. Calcd for C24H27N3O2Æ0.2H2O: C,
73.33; H, 7.03; N, 10.69. Found: C, 73.32; H, 7.06; N,
10.41.
5.1.10. 8-[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]octa-
noic acid (5c). Compound 5c was prepared from 7a
and methyl 8-bromooctanoate. Yield 14% (in two
steps), colorless crystals. Mp 116–117 ꢂC. FAB-MS
m/z 404 [MH]+. IR (KBr): 2934, 1714, 1584, 1566,
5.1.7. General procedure for the synthesis of 5a–d and 8a–c:
9-[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]nonanoic acid
(5d). To a suspension of NaH (60% dispersion in oil,
42 mg, 1.05 mmol) in DMF (2 mL) was added the
aminopyrazine 7a (233 mg, 0.89 mmol) at room temper-
ature, and the mixture was stirred at 80 ꢂC for 30 min.
The mixture was ice cooled and a solution of methyl
9-bromononanoate (209 mg, 0.87 mmol) in DMF
(0.5 mL) was added dropwise. The mixture was stirred
at room temperature for 2 h, diluted with ice water,
and extracted with EtOAc. The extract was washed with
brine and dried over MgSO4. After the solvent was
evaporated, the crude product was purified by silica
gel column chromatography with hexane–EtOAc (9:1)
as the eluent to give the corresponding ester (95 mg,
1395, 1184, 698 cmꢀ1 1H NMR (CDCl3) d: 1.30–
.
1.80 (10H, m), 2.33 (2H, t, J = 7.4 Hz), 3.17 (3H, s),
3.60 (2H, t, J = 7.5 Hz), 4.00 (1H, br), 7.20–7.50
(10H, m), 8.03 (1H, s). Anal. Calcd for C25H29N3O2:
C, 74.41; H, 7.24; N, 10.41. Found: C, 74.16; H,
7.25; N, 10.29.
5.1.11. {4-[(5,6-Diphenylpyrazin-2-yl)(ethyl)amino]but-
oxy}acetic acid (8a). Compound 8a was prepared from
the aminopyrazine 7b and the bromide 11. Yield 26%
1
(in two steps), colorless oil. H NMR (CDCl3) d: 1.25
(3H, t, J = 7.1 Hz), 1.60–1.90 (4H, m), 3.45–3.73 (6H,
m), 4.05 (2H, s), 5.43 (1H, br), 7.15–7.50 (10H, m),
8.07 (1H, s). The analytical sample was obtained as
the sodium salt as follows. To a solution of 8a in MeOH
was added 1 N NaOH (1.0 equiv), and the reaction mix-
ture was evaporated in vacuo. The residual water was re-
moved by co-distillation with EtOH in vacuo. The
sodium salt of 8a was collected by filtration, and after
an Et2O rinse it was obtained as a colorless amorphous
powder. FAB-MS m/z 450 [M+Na]+. 1H NMR (CDCl3)
d: 1.05 (3H, t), 1.53 (4H, br), 2.93 (2H, br), 3.35 (4H, br),
3.79 (2H, s), 6.90–7.50 (10H, m), 7.89 (1H, s). Anal.
Calcd for C24H26N3O3NaÆH2O: C, 64.71; H, 6.34; N,
9.43. Found: C, 64.88; H, 6.25; N, 9.16.
1
25%) as a pale yellow oil. H NMR (CDCl3) d: 1.20–
1.40 (8H, m), 1.50–1.75 (4H, m), 2.29 (2H, t,
J = 7.5 Hz), 3.17 (3H, s), 3.60 (2H, t, J = 7.3 Hz), 3.66
(3H, s), 7.20–7.50 (10H, m), 8.01 (1H, s). To a stirred
solution of this ester (95 mg, 0.22 mmol) in MeOH
(2 mL) was added 1 N NaOH (1 mL), and the mixture
was heated at reflux for 1 h. After evaporation of the
solvent, the residue was dissolved in water and washed
with Et2O. The aqueous layer was separated, neutralized
with 1 N HCl (1 mL), and extracted with EtOAc. The
organic extract was dried over MgSO4 and evaporated
in vacuo. The residual solid was washed with a mixture
of isopropyl ether and hexane (1:1) and filtered to give
5d (79 mg, 86%) as colorless crystals. Mp 103–105 ꢂC.
FAB-MS m/z 418 [MH]+. IR (KBr): 2930, 2855, 1713,
1586, 1202, 695 cmꢀ1.1H NMR (CDCl3) d: 1.20–1.50
(8H, m), 1.50–1.80 (4H, m), 2.33 (2H, t, J = 7.3 Hz),
3.18 (3H, s), 3.60 (2H, t, J = 7.3 Hz), 7.20–7.50 (10H,
m), 8.03 (1H, s). Anal. Calcd for C26H31N3O2Æ0.25H2O:
C, 73.99; H, 7.52; N, 9.96. Found: C, 73.80; H, 7.55; N,
9.65.
5.1.12. {4-[(Allyl)(5,6-diphenylpyrazin-2-yl)amino]butoxy}
acetic acid (8b). Compound 8b was prepared from the
aminopyrazine 7c and 11. Yield 26% (in two steps), col-
1
orless oil. H NMR (CDCl3) d: 1.60–1.90 (4H, m), 3.61
(2H, t, J = 5.8 Hz), 3.64 (2H, t, J = 7.4 Hz), 4.06 (2H, s),
4.21 (2H, d, J = 5.0 Hz), 5.21 (1H, dd, J = 10.6, 1.4 Hz),
5.22 (1H, dd, J = 17.1, 1.4 Hz), 5.90 (1H, ddt, J = 17.1,
10.6, 5.0 Hz), 7.15–7.50 (10H, m), 8.06 (1H, s). The ana-
lytical sample was obtained as the sodium salt in the
form of a colorless amorphous powder in a manner sim-
ilar to that described for 8a. FAB-MS m/z 462 [M+Na]+.
1H NMR (DMSO-d6) d: 1.47 (9H, s), 1.40–1.90 (4H, m),
3.00–3.90 (6H, overlapping with solvent signal), 4.23
5.1.8. 6-[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]hexa-
noic acid (5a). Compound 5a was prepared from 7a
and ethyl 6-bromohexanoate. Yield 19% (in two steps),
pale yellow crystals. Mp 159–160 ꢂC. FAB-MS m/z 376
[MH]+. IR (KBr): 2950, 1715, 1586, 1572, 1186,

T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
6699
(2H, br), 5.00–5.40 (2H, m), 5.70–6.10 (1H, m), 7.10–
7.65 (10H, m), 8.13 (1H, s). Anal. Calcd for
C25H26N3O3NaÆ0.7H2O: C, 66.42; H, 6.11; N, 9.29.
Found: C, 66.31; H, 5.97; N, 8.98.
J = 5.1 Hz), 2.90–4.80 (2H, br), 3.07 (1H, qn, J = 6.5
Hz), 3.57 (2H, t, J = 4.8 Hz).
5.2.4. 4-[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]-1-buta-
nol (14a). A mixture of the chloropyrazine 6 (6.00 g,
22 mmol), 13a (2.78 g, 27 mmol), and K2CO3 (4.67 g,
34 mmol) in DMF (22 mL) was stirred at 80 ꢂC for
40 h. The resulting mixture was diluted with ice water
and extracted with Et2O. The extract was washed with
water and brine and dried over MgSO4. After the sol-
vent was concentrated in vacuo, the residual oil was sub-
jected to chromatography on silica gel with hexane–
EtOAc (1:2) as the eluent to give 14a (6.13 g, 82%) as
colorless crystals. An analytical sample was obtained
by recrystallization from isopropyl ether. Mp 97–
5.1.13. {4-[(Cyclopropyl)(5,6-diphenylpyrazin-2-yl)amino]-
butoxy}acetic acid (8c). Compound 8c was prepared from
the aminopyrazine 7d and 11. Yield 24% (in two steps),
pale green crystals. Mp 108–109 ꢂC. FAB-MS m/z 418
[MH]+. IR (KBr): 2942, 1725, 1565, 1364, 1221, 1144,
698 cmꢀ1 1H NMR (CDCl3) d: 0.70–0.82 (2H, m),
.
0.94–1.05 (2H, m), 1.58–1.88 (4H, m), 2.60–2.72 (1H,
m), 3.57 (2H, t, J = 6.2 Hz), 3.75 (2H, t, J = 6.9 Hz),
4.01 (2H, s), 5.96 (1H, br), 7.10–7.50 (10H, m), 8.48
(1H, s). Anal. Calcd for C25H27N3O3: C, 71.92; H, 6.52;
N, 10.06. Found: C, 71.77; H, 6.56; N, 10.04.
1
98.5 ꢂC. FAB-MS m/z 334 [MH]+. H NMR (CDCl3)
d: 1.50–1.85 (5H, m), 3.18 (3H, s), 3.68 (4H, t,
J = 7.1 Hz), 7.15–7.50 (10H, m), 8.03 (1H, s). Anal.
Calcd for C21H23N3O: C, 75.65; H, 6.95; N, 12.60.
Found: C, 75.52; H, 6.94; N, 12.56.
5.2. Preparation of 15a–d
5.2.1. 4-(Methylamino)-1-butanol (13a). To a stirred
solution of 4-amino-1-butanol 12 (10.00 g, 112 mmol)
in EtOH (100 mL) was added ethyl formate
(13.6 mL, 168 mmol), and the mixture was heated at
reflux for 18 h. The volatiles were evaporated in vacuo
and the resulting crude N-(4-hydroxybutyl)formamide
was used for the next step without further purifica-
tion. To a suspension of LiAlH4 (6.36 g, 168 mmol)
in THF (100 mL) was added a solution of the crude
product (13.29 g) in THF (50 mL) at a rate compatible
with slow reflux. After the mixture was refluxed for
1.5 h, the reaction was quenched with water
(6.3 mL), aqueous 15% NaOH (6.3 mL), and water
(18.9 mL), in that order, with ice cooling. The mixture
was stirred at room temperature for 30 min, the insol-
uble precipitate was filtered off, and the filtrate was
evaporated in vacuo. The residue was dissolved in
CHCl3 and the solution was dried over MgSO4 and
concentrated in vacuo. The residue was purified by
distillation to afford 13a (6.73 g, 58% in two steps)
as a colorless oil. Bp 84–85 ꢂC (16 mmHg). FAB-MS
5.2.5. 4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]-1-
butanol (14b). A mixture of 6 (30.00 g, 0.11 mol) and
13b (131.22 g, 1.00 mol) was heated at 190 ꢂC for 10 h.
The resulting mixture was diluted with ice water and ex-
tracted with Et2O. The extract was washed with water
and brine and dried over MgSO4. After the solvent
was concentrated in vacuo, the residual oil was subjected
to chromatography on silica gel with hexane–EtOAc
(1:1) as the eluent to give 14b (22.96 g, 56%) as colorless
1
crystals. Mp 102–103 ꢂC. FAB-MS m/z 362 [MH]+. H
NMR (CDCl3) d: 1.28 (6H, d, J = 6.6 Hz), 1.40–1.88
(5H, m), 3.45 (2H, t, J = 7.7 Hz), 3.62–3.76 (2H, m),
4.78 (1H, qn, J = 6.6 Hz), 7.19–7.50 (10H, m), 8.02
(1H, s). Anal. Calcd for C23H27N3OÆ0.5H2O: C, 74.56;
H, 7.62; N, 11.34. Found: C, 74.39; H, 7.87; N, 11.32.
5.2.6. 4-[(Cyclopentyl)(5,6-diphenylpyrazin-2-yl)amino]-
1-butanol (14c). A mixture of 6 (4.00 g, 15 mmol), 13c
(7.08 g, 45 mmol) and triisopropanolamine (8.61 g,
45 mmol) in sulfolane (16 mL) was heated at 190 ꢂC
for 22 h. The resulting mixture was diluted with ice
water and extracted with Et2O. The extract was washed
with water and brine and dried over MgSO4. After the
solvent was concentrated in vacuo, the residual oil was
subjected to chromatography on silica gel with hex-
ane–EtOAc (1:1) as the eluent to give 14c (3.47 g,
60%) as pale yellow crystals. The analytical sample
was obtained as pale yellow crystals after washing with
isopropyl ether. Mp 94–96 ꢂC. FAB-MS m/z 388
1
m/z 104 [MH]+. H NMR (CDCl3) d: 1.50–1.75 (4H,
m), 2.43 (3H, s), 2.62 (2H, t, J = 5.5 Hz), 3.57 (2H,
t, J = 5.1 Hz), 3.74 (2H, br).
5.2.2. 4-(Isopropylamino)-1-butanol (13b). A mixture of
12 (100.40 g, 1.13 mol), platinum (IV) oxide (2.10 g,
9 mmol), and acetone (108 mL, 1.47 mol) in EtOH
(160 mL) was stirred under an atmosphere of hydrogen
(2–3 atm) at room temperature for 48 h. The catalyst
was filtered through a pad of Celite and the cake was
washed with EtOH. The filtrate was concentrated in va-
cuo to give 13b (147.64 g, quant.) as a colorless oil,
which was used for the next step without further purifi-
1
[MH]+. H NMR (CDCl3) d: 1.50–2.20 (12H, m), 1.54
(1H, t, J = 5.7 Hz) 3.48 (2H, t, J = 7.7 Hz), 3.69 (2H,
q, J = 5.7 Hz), 4.56–4.80 (1H, m), 7.10–7.50 (10H, m),
8.04 (1H, s). Anal. Calcd for C25H29N3OÆ0.5H2O: C,
77.13; H, 7.56; N, 10.79. Found: C, 76.97; H, 7.66; N,
10.80.
cation. FAB-MS m/z 132 [MH]+. H NMR (CDCl3) d:
1.09 (6H, d, J = 6.2 Hz), 1.52–1.76 (4H, m), 2.64 (2H,
t, J = 5.5 Hz), 2.80 (1H, qn, J = 6.2 Hz), 2.90–4.40
(2H, br), 3.57 (2H, t, J = 4.9 Hz).
1
5.2.7. 4-[(Benzyl)(5,6-diphenylpyrazin-2-yl)amino]-1-buta-
nol (14d). Compound 14d was prepared from 6 and 4-
(benzylamino)-1-butanol15 in a manner similar to that
described for 14c. Yield 69%, pale yellow crystals. The
analytical sample was obtained as colorless crystals after
washing with isopropyl ether. Mp 96–98 ꢂC. FAB-MS
5.2.3. 4-(Cyclopentylamino)-1-butanol (13c). Compound
13c was prepared from cyclopentanone (1 equiv) in a
manner similar to that described for 13b. Yield quant.,
pale yellow oil. FAB-MS m/z 158 [MH]+. 1H NMR
(CDCl3) d: 1.25–1.90 (12H, m), 2.63 (2H, t,

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T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
m/z 410 [MH]+. 1H NMR (CDCl3) d: 1.48 (1H, br),
1.52–1.93 (4H, m), 3.59–3.80 (4H, m), 4.85 (2H, s),
7.15–7.55 (15H, m), 7.99 (1H, s). Anal. Calcd for
C27H27N3O: C, 79.19; H, 6.65; N, 10.26. Found: C,
79.03; H, 6.62; N, 10.27.
6.94 (1H, br), 7.10–7.50 (10H, m), 8.10 (1H, s). Anal.
Calcd for C27H31N3O3Æ0.5H2O: C, 72.20; H, 7.05; N,
9.36. Found: C, 72.06; H, 7.02; N, 9.19.
5.2.11. {4-[(Benzyl)(5,6-diphenylpyrazin-2-yl)amino]but-
oxy}acetic acid (15d). Compound 15d was prepared
from 14d in a manner similar to that described for
15a. Yield 57% (in two steps), pale yellow amorphous
5.2.8. General procedure for the synthesis of 15a–d: {4-
[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]butoxy}acetic
acid (15a). A mixture of 14a (18.00 g, 54 mmol), tetra-n-
butylammonium hydrogensulfate (9.16 g, 27 mmol),
benzene (40 mL) and 40% aqueous KOH (40 mL) was
stirred vigorously and then cooled to 5 ꢂC. tert-Butyl
bromoacetate (10.5 mL, 65 mmol) was added dropwise
as rapidly as possible while keeping the temperature be-
low 10 ꢂC. After stirring had been continued for 45 min,
the reaction mixture was stirred at room temperature for
a further 1 h. The reaction mixture was diluted with ice
water and extracted with Et2O. The extract was then
washed with water and brine and dried over MgSO4.
After the solvent had been concentrated in vacuo, the
residual oil was subjected to chromatography on silica
gel with hexane–EtOAc (2:1) as the eluent to give tert-
butyl {4-[(5,6-diphenylpyrazin-2-yl)(methyl)amino]but-
oxy}acetate (16.35 g, 68%) as a pale yellow solid. The
analytical sample was obtained as colorless crystals after
washing with hexane. Mp 71–74 ꢂC. FAB-MS m/z 448
[MH]+. 1H NMR (CDCl3) d: 1.47 (9H, s), 1.55–1.85
(4H, m), 3.18 (3H, s), 3.56 (2H, t, J = 6.0 Hz), 3.68
(2H, t, J = 6.9 Hz), 3.93 (2H, s), 7.20–7.50 (10H, m),
8.03 (1H, s). Anal. Calcd for C27H33N3O3: C, 72.46;
H, 7.43; N, 9.39. Found: C, 72.44; H, 7.32; N, 9.41.
Saponification of this ester according to the procedure
described for the preparation of 5d gave 15a as pale yel-
low crystals (85% yield). Mp 102–103 ꢂC. FAB-MS m/z
392 [MH]+. IR (KBr): 2984, 1700, 1584, 1566, 1393,
1
powder. H NMR (CDCl3) d: 1.58–1.90 (4H, m), 3.58
(2H, t, J = 5.9 Hz), 3.67 (2H, t, J = 7.3 Hz), 4.04 (2H,
s), 4.86 (2H, s), 5.29 (1H, br), 7.13–7.48 (15H, m), 8.08
(1H, s). The analytical sample was obtained as the so-
dium salt in the form of colorless crystals in a manner
similar to that described for 8a. Mp 133–137 ꢂC. FAB-
MS m/z 512 [M+Na]+. IR (KBr): 3440, 1601, 1557,
1
1482, 1210, 696 cmꢀ1. H NMR (DMSO-d6) d: 1.44–
1.80 (4H, m), 3.47 (2H, t, J = 5.4 Hz), 3.51 (2H, s),
3.63 (2H, t, J = 7.4 Hz), 4.86 (2H, s), 7.17–7.45 (15H,
m), 8.16 (1H, s). Anal. Calcd for C29H28N3O3NaÆ1.7-
H2O: C, 66.96; H, 6.08; N, 8.08. Found: C, 67.17; H,
6.48; N, 8.21.
5.3. Preparation of 22a and 22b
5.3.1. 3-(Methoxymethoxy)-N-methylbenzylamine (17).
A mixture of 3-(methoxymethoxy)benzaldehyde 1616
(3.00 g, 18 mmol), 5% platinum on carbon (0.10 g), a
40% solution of methylamine in MeOH (2.1 mL), and
MeOH (10 mL) was stirred under an atmosphere of
hydrogen (2 atm) at room temperature for 7 h. The reac-
tion mixture was allowed to warm to 70 ꢂC and was con-
tinuously stirred for 15 h. The catalyst was removed by
filtration of the reaction mixture through a pad of Celite
and the cake was washed with MeOH. The filtrate was
concentrated in vacuo and the residue was purified by
silica gel column chromatography with CHCl3–MeOH
(20:1–10:1) as the eluent to give 17 (2.51 g, 77%) as a
pale yellow oil. FAB-MS m/z 182 [MH]+. 1H NMR
(CDCl3) d: 1.39 (1H, s), 2.46 (3H, s), 3.48 (3H, s), 3.73
(2H, s), 5.18 (2H, s), 6.90–7.00 (3H, m), 7.20–7.30
(1H, m). Anal. Calcd for C10H15NO2Æ0.35CHCl3: C,
55.74; H, 6.94; N, 6.28. Found: C, 55.62; H, 6.94; N,
6.41.
1127, 770, 698 cmꢀ1 1H NMR (CDCl3) d: 1.60–1.90
.
(4H, m), 3.18 (3H, s), 3.60 (2H, t, J = 6.0 Hz), 3.68
(2H, t, J = 6.9 Hz), 4.04 (2H, s), 4.69 (1H, br), 7.20–
7.50 (10H, m), 8.09 (1H, s). Anal. Calcd for
C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found: C,
70.44; H, 6.42; N, 10.64.
5.2.9. {4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]but-
oxy}acetic acid (15b). Compound 15b was prepared
from 14b in a manner similar to that described for
15a. Yield 62% (in two steps), pale yellow crystals. Mp
114–116 ꢂC. FAB-MS m/z 420 [MH]+. IR (KBr): 2984,
5.3.2. 2-[3-(Benzyloxy)phenyl]-N-methylethylamine (19).
To a stirred solution of 2-[3-(benzyloxy)phenyl]ethyla-
mine 1817 (8.63 g, 38 mmol) in EtOH (50 mL) was added
ethyl formate (4.6 mL, 57 mmol), and the mixture was
heated at reflux for 22 h. The volatiles were evaporated
in vacuo and the residue was purified by silica gel col-
umn chromatography with hexane–EtOAc (1:2) as the
eluent to give N-[3-(benzyloxy)phenethyl]formamide
(4.04 g, 42%) as a pale brown oil. FAB-MS m/z 256
1
1719, 1561, 1482, 1221, 1142, 772, 698 cmꢀ1. H NMR
(CDCl3) d: 1.27 (6H, d, J = 6.8 Hz), 1.60–1.90 (4H,
m), 3.44 (2H, t, J = 7.5 Hz), 3.62 (2H, t, J = 5.9 Hz),
4.07 (2H, s), 4.84 (1H, qn, J = 6.8 Hz), 7.01 (1H, br),
7.18–7.49 (10H, m), 8.09 (1H, s). Anal. Calcd for
C25H29N3O3: C, 71.58; H, 6.97; N, 10.02. Found: C,
71.64; H, 6.96; N, 9.97.
1
[MH]+. H NMR (CDCl3) d: 2.81 (2H, t, J = 6.6 Hz),
3.50–3.61 (2H, m), 5.06 (2H, s), 5.45 (1H, br), 6.70–
6.90 (3H, m), 7.20–7.50 (6H, m), 8.10 (1H, s). Anal.
Calcd for C16H17NO2Æ0.2H2O: C, 74.22; H, 6.77; N,
5.41. Found: C, 74.20; H, 6.65; N, 5.44. To a suspension
of LiAlH4 (0.88 g, 23 mmol) in THF (20 mL) was added
dropwise a solution of this formamide (3.96 g, 16 mmol)
in THF (10 mL) at room temperature. After the mixture
had been refluxed for 2 h, the reaction was quenched
with water (0.9 mL), aqueous 15% NaOH (0.9 mL),
5.2.10. {4-[(Cyclopentyl)(5,6-diphenylpyrazin-2-yl)amino]but-
oxy}acetic acid (15c). Compound 15c was prepared from
14c in a manner similar to that described for 15a. Yield
36% (in two steps), pale yellow crystals. Mp 104–106 ꢂC.
FAB-MS m/z 446 [MH]+. IR (KBr): 2953, 1732, 1563,
1
1470, 1221, 1134, 772, 698 cmꢀ1. H NMR (CDCl3) d:
1.50–2.15 (12H, m), 3.47 (2H, t, J = 7.5 Hz), 3.61 (2H,
t, J = 6.0 Hz), 4.07 (2H, s), 4.74 (1H, qn, J = 7.7 Hz),

T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
6701
and water (2.7 mL), in that order, with ice cooling. The
mixture was stirred at room temperature for 30 min. The
insoluble precipitate was filtered off and the filtrate was
evaporated in vacuo. The residue was then purified by
silica gel column chromatography with EtOAc as the
eluent to give 19 (3.00 g, 80%) as a pale yellow oil.
FAB-MS m/z 242 [MH]+. 1H NMR (CDCl3) d: 1.12
(1H, br), 2.42 (3H, s), 2.70–2.90 (4H, m), 5.06 (2H, s),
6.75–6.90 (3H, m), 7.17–7.47 (6H, m). Anal. Calcd for
C16H19NOÆ0.5H2O: C, 76.77; H, 8.05; N, 5.60. Found:
C, 76.57; H, 7.78; N, 5.51.
ture was neutralized with saturated aqueous NaHCO3
and extracted with EtOAc. The extract was washed with
water and dried over MgSO4. After the solvent was con-
centrated in vacuo, the crude solid was washed with iso-
propyl ether and dried in vacuo to give 21b (0.87 g, 92%)
as pale yellow crystals. Mp 158–161 ꢂC. FAB-MS m/z
382 [MH]+. 1H NMR (CDCl3) d: 2.89 (2H, t,
J = 7.6 Hz), 3.12 (3H, s), 3.83 (2H, t, J = 7.6 Hz), 5.40
(1H, br), 6.62–6.67 (2H, m), 6.78 (1H, d, J = 7.6 Hz),
7.10–7.50 (11H, m), 8.00 (1H, s). Anal. Calcd for
C25H23N3O: C, 78.71; H, 6.08; N, 11.02. Found: C,
78.51; H, 6.10; N, 10.73.
5.3.3. N-(5,6-Diphenylpyrazin-2-yl)-3-(methoxymethoxy)-
N-methylbenzylamine (20a). A mixture of the chloropyr-
azine 6 (1.00 g, 3.7 mmol), the methylamine 17 (0.82 g,
4.5 mmol) and K2CO3 (0.77 g, 5.6 mmol) in DMF
(4 mL) was stirred at 80 ꢂC for 40 h. The resulting mix-
ture was diluted with ice water and extracted with
EtOAc. The extract was washed with water and brine
and dried over MgSO4. After the solvent was concen-
trated in vacuo, the resulting solid was washed with iso-
propyl ether–Et2O (2:1) and dried in vacuo to give 20a
(0.93 g, 60%) as colorless crystals. Mp 109–111 ꢂC.
FAB-MS m/z 412 [MH]+. 1H NMR (CDCl3) d: 3.22
(3H, s), 3.46 (3H, s), 4.87 (2H, s), 5.15 (2H, s), 6.90–
7.00 (3H, m), 7.20–7.50 (11H, m), 8.05 (1H, s). Anal.
Calcd for C26H25N3O2: C, 75.89; H, 6.12; N, 10.21.
Found: C, 75.81; H, 6.11; N, 10.18.
5.3.7. General procedure for the synthesis of 22a and 22b:
[3-{[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]methyl}-
phenoxy]acetic acid (22a). A mixture of 21a (605 mg,
1.65 mmol), methyl bromoacetate (278 mg, 1.82 mmol),
K2CO3 (273 mg, 1.98 mmol), and KI (catalytic amount)
in MeCN (10 mL) was heated at reflux for 3 h. The
insoluble materials were filtered off and the filtrate was
concentrated in vacuo. The residue was purified by silica
gel column chromatography with CHCl3–MeOH (40:1)
as the eluent, and the resulting solid was washed with
Et2O and dried in vacuo to give methyl [3-{[(5,6-diphe-
nylpyrazin-2-yl)(methyl)amino]methyl}phenoxy]acetate
(632 mg, 87%) as colorless crystals. Mp 132–134 ꢂC.
FAB-MS m/z 440 [MH]+. 1H NMR (CDCl3) d: 3.20
(3H, s), 3.77 (3H, s), 4.58 (2H, s), 4.87 (2H, s), 6.79
(1H, dd, J = 1.8, 8.0 Hz), 6.88 (1H, m), 6.94 (1H, d,
J = 8.0 Hz), 7.20–7.50 (11H, m), 8.05 (1H, s). Anal.
Calcd for C27H25N3O3: C, 73.78; H, 5.73; N, 9.56.
Found: C, 73.76; H, 5.68; N, 9.60. Saponification of this
ester according to the procedure described for the prep-
aration of 5d gave 22a as pale yellow crystals (79%
yield). Mp 182–187 ꢂC. FAB-MS m/z 426 [MH]+. IR
(KBr): 3050, 1754, 1586, 1560, 1395, 1229, 770,
5.3.4. 2-[3-(Benzyloxy)phenyl]-N-(5,6-diphenylpyrazin-2-
yl)-N-methylethylamine (20b). Compound 20b was pre-
pared from 6 and the methylamine 19 in a manner sim-
ilar to that described for 14a. Yield 71%, pale yellow
1
crystals. Mp 78–78.5 ꢂC. FAB-MS m/z 472 [MH]+. H
NMR (CDCl3) d: 2.94 (2H, t, J = 7.5 Hz), 3.12 (3H,
s), 3.85 (2H, t, J = 7.5 Hz), 5.02 (2H, s), 6.81–6.87
(3H, m), 7.17–7.51 (16H, m), 8.02 (1H, s). Anal. Calcd
for C32H29N3O: C, 81.50; H, 6.20; N, 8.91. Found: C,
81.39; H, 6.22; N, 8.95.
1
700 cmꢀ1. H NMR (DMSO-d6) d: 3.19 (3H, s), 4.62
(2H, s), 4.85 (2H, s), 6.70–7.90 (3H, m), 7.15–7.40
(11H, m), 8.21 (1H, s). Anal. Calcd for C26H23N3O3:
C, 73.39; H, 5.45; N, 9.88. Found: C, 73.26; H, 5.47;
N, 9.83.
5.3.5. N-(5,6-Diphenylpyrazin-2-yl)-3-hydroxy-N-meth-
ylbenzylamine (21a). To a suspension of 20a (0.91 g,
2.2 mmol) in MeOH (10 mL) was added an 18% solu-
tion of HCl in MeOH (2 mL) at room temperature.
After the suspension had been stirred for 2 h, a further
portion of 18% HCl in MeOH (1 mL) was added and
the mixture was stirred continuously at the same tem-
perature for 1 h. After evaporation of the volatiles, the
residue was diluted with saturated aqueous NaHCO3
and extracted with EtOAc. The organic layer was dried
over MgSO4 and concentrated in vacuo. The resulting
solid was washed with Et2O and dried in vacuo to give
21a (0.66 g, 81%) as colorless crystals. Mp 156–157 ꢂC.
FAB-MS m/z 368 [MH]+. 1H NMR (CDCl3) d: 3.17
(3H, s), 4.81 (2H, s), 5.92 (1H, br), 6.65–6.75 (2H, m),
6.82 (1H, d, J = 7.8 Hz), 7.10–7.50 (11H, m), 8.00 (1H,
s). Anal. Calcd for C24H21N3O: C, 78.45; H, 5.76; N,
11.44. Found: C, 78.22; H, 5.87; N, 11.34.
5.3.8. [3-{[(5,6-Diphenylpyrazin-2-yl)(methyl)amino]ethyl}
phenoxy]acetic acid (22b). Compound 22b was prepared
from 21b in a manner similar to that described for 22a.
Yield 51% (in two steps), pale yellow crystals. Mp 174–
176 ꢂC. FAB-MS m/z 440 [MH]+. IR (KBr): 2900, 1709,
1586, 1570, 1262, 1248, 1177, 696 cmꢀ1 1H NMR
.
(CDCl3) d: 2.94 (2H, t, J = 7.6 Hz), 3.13 (3H, s), 3.85
(2H, t, J = 7.6 Hz), 4.57 (2H, s), 6.77–6.89 (3H, m),
7.17–7.45 (11H, m), 8.03 (1H, s). Anal. Calcd for
C27H25N3O3: C, 73.78; H, 5.73; N, 9.56. Found: C,
73.43; H, 5.79; N, 9.32.
5.4. Preparation of 25
5.4.1. 5-(5,6-Diphenylpyrazin-2-yl)-4-pentyn-1-ol (23). A
mixture of 6 (1.58 g, 5.9 mmol), 4-pentyn-1-ol (0.60 g,
7.1 mmol), dichlorobis(triphenylphosphine)palladium
(II) (210 mg, 0.3 mmol), and copper (I) iodide (57 mg,
0.3 mmol) in triethylamine (10 mL) was stirred under
an argon atmosphere at 80 ꢂC for 7 h. The resulting mix-
ture was diluted with Et2O and the insoluble materials
5.3.6. N-(5,6-Diphenylpyrazin-2-yl)-2-(3-hydroxyphenyl)-
N-methylethylamine (21b). A mixture of 20b (1.17 g,
2.5 mmol), EtOH (12 mL) and 35% aqueous HCl
(6 mL) was heated at 80 ꢂC for 17 h. The reaction mix-

6702
T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
were filtered off. The filtrate was concentrated in vacuo.
The residue was purified by silica gel column chroma-
tography with hexane–EtOAc (1:1) as the eluent, and
the resulting solid was washed with isopropyl ether
and dried in vacuo to give 23 (1.12 g, 60%) as pale yel-
low crystals. Mp 97–99 ꢂC. FAB-MS m/z 315 [MH]+.
1H NMR (CDCl3) d: 1.62 (1H, t, J = 5.2 Hz), 1.92
(2H, qn, J = 6.6 Hz), 2.64 (2H, t, J = 7.2 Hz), 3.83
(2H, t, J = 5.7 Hz), 7.20–7.60 (10H, m), 8.62 (1H, s).
Anal. Calcd for C21H18N2O: C, 80.23; H, 5.77; N,
8.91. Found: C, 80.27; H, 5.73; N, 8.88.
J = 6.0 Hz), 7.20–7.50 (10H, m), 8.23 (1H, s). Anal.
Calcd for C26H30N2O4Æ0.2H2O: C, 71.28; H, 6.99; N,
6.39. Found: C, 71.00; H, 6.86; N, 6.26.
5.5.2. tert-Butyl[4-(5,6-diphenylpyrazin-2-ylsulfanyl)but-
oxy]acetate (27b). To an ice-cooled mixture of 5,6-diphe-
nyl-2-pyrazinethiol 26b18 (500 mg, 1.9 mmol) and
Na2CO3 (321 mg, 3.0 mmol) in acetone (20 mL) was
added dropwise a solution of 11 (556 mg, 2.1 mmol) in
acetone (2 mL). The solution was allowed to warm to
room temperature and stirred for 24 h. After evapora-
tion of the solvent, the residue was diluted with water
and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by chromatography on silica
gel with hexane–EtOAc (7:3) as the eluent to give 27b
5.4.2. 5-(5,6-Diphenylpyrazin-2-yl)-1-pentanol (24). A
mixture of 23 (409 mg, 1.3 mmol) and 5% palladium
on carbon (40 mg) in MeOH (20 mL) was stirred under
an atmosphere of hydrogen (3 atm) at room temperature
for 4 h. The catalyst was filtered through a pad of Celite
and the cake was washed with MeOH. After the filtrate
was concentrated in vacuo, the residual oil was subjected
to chromatography on silica gel with hexane–EtOAc
(1:1) as the eluent to give 24 (409 mg, 99%) as a pale yel-
1
(701 mg, 82%) as a pale yellow oil. H NMR (CDCl3)
d: 1.47 (9H, s), 1.70–2.00 (4H, m), 3.31 (2H, t,
J = 7.0 Hz), 3.55 (2H, t, J = 6.0 Hz), 3.92 (2H, s),
7.20–7.50 (10H, m), 8.44 (1H, s).
1
low oil. FAB-MS m/z 319 [MH]+. H NMR (CDCl3) d:
5.5.3. [4-(5,6-Diphenylpyrazin-2-yloxy)butoxy]acetic acid
(28a). Saponification of 27a according to the procedure
described for the preparation of 5d gave 28a as colorless
crystals. Yield 62%. Mp 65–66 ꢂC. FAB-MS m/z 379
[MH]+. IR (KBr): 2959, 1715, 1483, 1327, 1148, 696
1.30–1.80 (5H, m), 1.80–2.00 (2H, m), 2.93 (2H, t,
J = 7.7 Hz), 3.68 (2H, t, J = 6.0 Hz), 7.20–7.50 (10H,
m), 8.47 (1H, s). Anal. Calcd for C21H22N2OÆ0.3-
C4H8O2: C, 77.32; H, 7.13; N, 8.12. Found: C, 77.30;
H, 6.75; N, 8.48.
cmꢀ1 1H NMR (CDCl3) d: 1.75–2.05 (4H, m), 3.66
.
(2H, t, J = 5.9 Hz), 4.13 (2H, s), 4.48 (2H, t,
J = 5.9 Hz), 7.15–7.55 (10H, m), 8.25 (1H, s). Anal.
Calcd for C22H22N2O4: C, 69.83; H, 5.86; N, 7.40.
Found: C, 69.84; H, 5.90; N, 7.34.
5.4.3. [5-(5,6-Diphenylpyrazin-2-yl)pentyloxy]acetic acid
(25). Compound 25 was prepared from 24 in a manner
similar to that described for 15a. Yield 25% (in two
1
steps), pale yellow oil. H NMR (CDCl3) d: 1.40–2.00
(6H, m), 2.93 (2H, t, J = 7.7 Hz), 3.58 (2H, t,
J = 6.4 Hz), 4.09 (2H, s), 7.20–7.50 (10H, m), 8.50
(1H, s), 9.05 (1H, br). The analytical sample was ob-
tained as the sodium salt in the form of a pale brown
amorphous powder in a manner similar to that de-
5.5.4. [4-(5,6-Diphenylpyrazin-2-ylsulfanyl)butoxy]acetic
acid (28b). Saponification of 27b according to the proce-
dure described for the preparation of 5d gave 28b as pale
yellow crystals. Yield 61%. Mp 86–88 ꢂC. FAB-MS m/z
395 [MH]+. IR (KBr): 2936, 1748, 1734, 1416, 1138,
1
scribed for 8a. FAB-MS m/z 421 [M+Na]+. H NMR
696 cmꢀ1 1H NMR (CDCl3) d: 1.70–2.00 (4H, m),
.
(CDCl3) d: 1.00–2.00 (6H, m), 2.74 (2H, br), 3.40 (2H,
br), 3.80 (2H, s), 6.90–7.50 (10H, m), 8.32 (1H, s). Anal.
Calcd for C23H23N2O3NaÆ0.5H2O: C, 67.80; H, 5.94; N,
6.88. Found: C, 68.03; H, 6.22; N, 6.48.
3.31 (2H, t, J = 6.9 Hz), 3.59 (2H, t, J = 6.1 Hz), 4.05
(2H, s), 6.00 (1H, br), 7.20–7.50 (10H, m), 8.46 (1H,
s). Anal. Calcd for C22H22N2O3S: C, 66.98; H, 5.62;
N, 7.10. Found: C, 66.77; H, 5.65; N, 7.45.
5.5. Preparation of 28a–d
5.5.5. [4-(5,6-Diphenylpyrazin-2-ylsulfinyl)butoxy]acetic
acid (28c). To an ice-cooled solution of 27b (350 mg,
0.78 mmol) in CHCl3 (5 mL) was added 3-chloroperoxy-
benzoic acid (purity ca. 70%; 191 mg, 0.77 mmol). The
mixture was stirred at 0 ꢂC for 2 h, diluted with 0.2 N
NaOH (20 mL), and extracted with CHCl3. The organic
layer was dried over MgSO4 and concentrated in vacuo.
The residue was purified by chromatography on silica
gel with hexane–EtOAc (4:1–1:1) as the eluent to give
5.5.1. tert-Butyl[4-(5,6-diphenylpyrazin-2-yloxy)butoxy]ace-
tate (27a). To a suspension of NaH (60% dispersion in
oil, 96 mg, 2.4 mmol) in DMF (4 mL) was added 26a1
(500 mg, 2.0 mmol) by portions at room temperature.
The mixture was stirred for 30 min and then ice cooled.
A solution of the bromide 11 (641 mg, 2.4 mmol) in
DMF (1 mL) was added dropwise, and the resulting
mixture was allowed to warm to room temperature
and stirred for 18 h. The mixture was diluted with ice
water and extracted with EtOAc. The extract was
washed with water and dried over MgSO4. After the sol-
vent was evaporated, the crude product was purified by
silica gel column chromatography with hexane–EtOAc
(6:1) as the eluent to give 27a (474 mg, 54%) as a color-
less oil. The alternative N-linked regioisomer was almost
undetectable by TLC. FAB-MS m/z 435 [MH]+. 1H
NMR (CDCl3) d: 1.48 (9H, s), 1.72–2.05 (4H, m), 3.61
(2H, t, J = 6.0 Hz), 3.97 (2H, s), 4.48 (2H, t,
tert-butyl
[4-(5,6-diphenylpyrazin-2-ylsulfinyl)but-
oxy]acetate (145 mg, 40%) as a pale yellow oil. 1H
NMR (CDCl3) d: 1.46 (9H, s), 1.70–2.20 (4H, m),
3.06–3.39 (2H, m), 3.56 (2H, t, J = 5.8 Hz), 3.92 (2H,
s), 7.27–7.53 (10H, m), 9.16 (1H, s). To a solution of this
oil (145 mg, 0.31 mmol) in 1,4-dioxane (2 mL) was
added 4 N HCl (1 mL). The mixture was stirred at
80 ꢂC for 30 min, diluted with ice water, neutralized with
2 N NaOH, and extracted with EtOAc. The extract was
washed with brine and dried over MgSO4. Evaporation
of the solvent gave 28c (125 mg, 98%) as a pale yellow

T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
6703
1
oil. H NMR (CDCl3) d: 1.70–2.22 (4H, m), 3.11–3.41
C25H29N7OÆ0.25CHCl3: C, 64.06; H, 6.23; N, 20.71.
Found: C, 64.23; H, 6.31; N, 20.74.
(2H, m), 3.61 (2H, t, J = 5.8 Hz), 4.08 (2H, s), 5.60
(1H, br), 7.20–7.60 (10H, m), 9.17 (1H, s). The analytical
sample was obtained as the sodium salt in the form of a
pale yellow amorphous powder in a manner similar to
5.7. Preparation of 31
that described for 8a. FAB-MS m/z 455 [M+Na]+. H
5.7.1. 2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]but-
oxy}-N-(methylsulfonyl)acetamide (31). Compound 31
was prepared from the carboxylic acid 15b by a method
similar to one described in the literature.11 A mixture of
15b (300 mg, 0.72 mmol) and 1,10-carbonyldiimidazole
(128 mg, 0.79 mmol) in THF (5 mL) was stirred at room
temperature under an argon atmosphere for 30 min and
at reflux for a further 30 min. After cooling, methane-
sulfonamide (69 mg, 0.73 mmol) was added and the mix-
ture was stirred at room temperature for 10 min. 1,8-
1
NMR (CDCl3) d: 1.50–2.10 (4H, m), 2.90–3.25 (2H,
m), 3.43 (2H, br), 3.82 (2H, s), 7.10–7.50 (10H, m),
9.07 (1H, s). Anal. Calcd for C22H21N2O4SNaÆ0.2-
C4H10OÆ1.4H2O: C, 57.96; H, 5.50; N, 5.93. Found: C,
58.30; H, 5.10; N, 5.45.
5.5.6. [4-(5,6-Diphenylpyrazin-2-ylsulfonyl)butoxy]acetic
acid (28d). tert-Butyl[4-(5,6-diphenylpyrazin-2-ylsulfo-
nyl)butoxy]acetate was prepared from 27b in a manner
similar to that described for 28c except that the reaction
was carried out for 19 h with 2 mol equivalents of 3-
chloroperoxybenzoic acid, and it was obtained as a col-
Diazabicyclo[5.4.0]-7-undecene
(DBU)
(0.11 mL,
0.74 mmol) was then added dropwise to the mixture.
After being stirred at room temperature for 12 h, the
reaction mixture was poured into 1 N HCl and extracted
with Et2O. The extract was washed with water and dried
over MgSO4. After the solvent was concentrated in va-
cuo, the residue was subjected to chromatography on
silica gel to give 31 (272 mg, 77%) as a pale yellow solid.
FAB-MS m/z 497 [MH]+. 1H NMR (CDCl3) d: 1.29
(6H, d, J = 6.6 Hz), 1.60–1.90 (4H, m), 3.29 (3H, s),
3.46 (2H, t, J = 7.3 Hz), 3.59 (2H, t, J = 6.0 Hz), 3.97
(2H, s), 4.72 (1H, qn, J = 6.6 Hz), 7.15–7.50 (10H, m),
8.02 (1H, s). Anal. Calcd for C26H32N4O4S: C, 62.88;
H, 6.49; N, 11.28. Found: C, 63.06; H, 6.53; N, 10.98.
1
orless oil (yield quant.). H NMR (CDCl3) d: 1.45 (9H,
s), 1.71–1.85 (2H, m), 1.90–2.09 (2H, m), 3.53 (2H, t,
J = 6.0 Hz), 3.56 (2H, t, J = 7.6 Hz), 3.89 (2H, s),
7.27–7.53 (10H, m), 9.22 (1H, s). Compound 28d was
prepared by saponification of this oil in a manner simi-
lar to that described for 28c except that it was purified
by recrystallization from EtOAc–hexane. Yield 66%,
colorless crystals. Mp 123–125 ꢂC. FAB-MS m/z 427
[MH]+. IR (KBr): 2950, 1738, 1323, 1121, 698 cmꢀ1
.
1H NMR (CDCl3) d: 1.72–1.90 (2H, m), 1.90–2.13
(2H, m), 3.55 (2H, t, J = 7.8 Hz), 3.58 (2H, t, J = 5.8
Hz), 4.04 (2H, s), 7.20–7.60 (10H, m), 9.23 (1H, s). Anal.
Calcd for C22H22N2O5S: C, 61.96; H, 5.20; N, 6.57.
Found: C, 61.95; H, 5.25; N, 6.41.
5.8. Inhibition of platelet aggregation
Blood samples freshly collected from volunteers were
mixed with 1/10th volume of 3.8% trisodium citrate
solution. Platelet-rich plasma was prepared by centrifu-
gation of blood samples at 150g for 10 min at room tem-
perature. Platelet aggregation induced by ADP (10 lM)
was monitored at 37 ꢂC with an aggregometer (model
PAM-8C, Mebanix, Tokyo, Japan) in the presence or
absence of the test compound by a literature method.24
5.6. Preparation of 30
5.6.1. 2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]but-
oxy}acetonitrile (29). Compound 29 was prepared from
the alcohol 14b and bromoacetonitrile in a manner sim-
ilar to that described for 10. Yield 28%, yellow oil. FAB-
1
MS m/z 401 [MH]+. H NMR (CDCl3) d: 1.28 (6H, d,
J = 6.6 Hz), 1.60–1.90 (4H, m), 3.44 (2H, t,
J = 7.7 Hz), 3.64 (2H, t, J = 5.8 Hz), 4.19 (2H, s), 4.75
(1H, qn, J = 6.6 Hz), 7.10–7.50 (10H, m), 8.01 (1H, s).
Anal. Calcd for C25H28N4OÆ0.2C4H8O2: C, 74.11; H,
7.14; N, 13.40. Found: C, 73.87; H, 6.86; N, 13.57.
5.9. Pharmacokinetic studies in animals
Male Sprague–Dawley rats (7 weeks old; Japan SLC,
Shizuoka, Japan), male beagle dogs weighing 10–12 kg
(Nosan Corporation, Tokyo), and male cynomolgus
monkeys weighing 4–6 kg (Japan SLC) were used. The
animals were acclimatized to the laboratory conditions
for more than 1 week (rats) or more than 3 weeks (dogs
and monkeys) before dosing at a room temperature of
21–25 ꢂC and 45–65% humidity. The rats, dogs, and
monkeys were fed diet F-2 (Funabashi Farms, Funa-
bashi, Japan), diet TC-1 (Aixia, Tokyo), and diet PS
(Oriental Yeast Co., Tokyo), respectively. None of the
animals were fasted before the experiments. Compound
15b was intravenously or orally administered to groups
of three animals. Blood samples were collected at vari-
ous times (before administration and 5, 15, and 30 min
and 1, 2, 4, 6, 8, 10, and 24 h after administration) by
venous withdrawal. Each sample was taken into a hepa-
rinized tube and plasma was prepared by centrifugation
(3000 rpm, 15 min, 4 ꢂC). The plasma concentrations of
test compounds after administration were measured by
5.6.2. 5-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]but-
oxymethyl}-1H-tetrazole (30). A mixture of 29 (0.80 g,
2.0 mmol), sodium azide (1.04 g, 16.0 mmol), and
ammonium chloride (0.75 g, 14 mmol) in DMF (7 mL)
was stirred at 120 ꢂC under an argon atmosphere for
1.5 h. The reaction mixture was poured into ice water
and extracted with EtOAc. The extract was washed with
water and dried over MgSO4. After the solvent was con-
centrated in vacuo, the residue was purified by chroma-
tography on silica gel with CHCl3–MeOH (50:1) as the
eluent to give 30 (0.55 g, 62%) as a pale yellow amor-
phous powder. FAB-MS m/z 444 [MH]+. IR (KBr):
1
2973, 1557, 1447, 1372, 1235, 1125, 772, 698 cmꢀ1. H
NMR (CDCl3) d: 1.25 (6H, d, J = 6.6 Hz), 1.67–2.00
(4H, m), 3.39 (2H, t, J = 7.9 Hz), 3.71 (2H, t,
J = 5.1 Hz), 4.90 (2H, s), 5.08 (1H, qn, J = 6.6 Hz),
7.20–7.53 (10H, m), 8.33 (1H, s). Anal. Calcd for

6704
T. Asaki et al. / Bioorg. Med. Chem. 15 (2007) 6692–6704
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liquid chromatography mass spectrometry (LC/MS) or
liquid chromatography tandem mass spectrometry
(LC/MS/MS). Briefly, plasma was mixed with an equal
volume of 0.2 N HCl and a small amount of MeOH
containing the internal standard. The compound was ex-
tracted by liquid–liquid extraction or solid-phase extrac-
tion. The extracted sample was then assayed by LC/
MS(/MS). For LC/MS(/MS), a Series 1050 (Agilent,
Palo Alto, CA, USA) or Alliance 2795 (Waters, Milford,
MA, USA) liquid chromatograph was used with a Quat-
tro micro API tandem quadrupole mass spectrometer
(Waters) equipped with a field-desorption apparatus
(M-2500; Hitachi, Tokyo). The ionization mode was
atmospheric pressure chemical ionization positive or
electrospray ionization positive. The peak intensities of
characteristic ions (m/z) were measured in the selected-
ion-monitoring mode or the selected-reaction-monitoring
mode. HPLC was carried out on a SymmetryShield RP8
(4.6 · 20 mm, Waters) or Xterra MS18IS (3.5 lm,
2.1 · 20 mm, Waters) column. The mobile phase was
MeCN/0.1% formic acid (80/20) and the column was
operated at a flow rate of 1 or 0.3 mL/min and a temper-
ature of 40 ꢂC.
Acknowledgments
15. Wang, C.; Delcros, J.-G.; Cannon, L.; Konate, F.; Carias,
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We thank Dr. Akio Nakamura and Mr. Tetsuhiro
Yamada for performing the pharmacokinetic studies,
and Dr. Gerald E. Smyth, Mr. Syouji Yasufuku, and
Mr. Yoshiaki Shirouchi for helpful suggestions during
the preparation of the manuscript.
18. Ohta, A.; Okimura, K.; Tonomura, Y.; Ohta, M.;
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