Development of 3,4-dihydro-2H-benzo[1,4]oxazine derivatives as dual thromboxane A2 receptor antagonists and prostacyclin receptor agonists

Article abstract of DOI:10.1016/j.bmc.2005.10.050

We discovered a novel series of 3,4-dihydro-2H-benzo[1,4]oxazin-8- yloxyacetic acid derivatives as potent dual-acting agents to block the TXA ₂ receptor and to activate the PGI₂ receptor. We report the synthesis, structure-activity r

Full text of DOI:10.1016/j.bmc.2005.10.050

Bioorganic & Medicinal Chemistry 14 (2006) 2005–2021
Development of 3,4-dihydro-2H-benzo[1,4]oxazine derivatives
as dual thromboxane A2 receptor antagonists and
prostacyclin receptor agonists
Michihiro Ohno,* Yoichiro Tanaka, Mitsuko Miyamoto, Takahiro Takeda,
Kazuhiro Hoshi, Naohiro Yamada and Atsushi Ohtake
Pharmaceutical Research Laboratories, Toray Industries, Inc. 1,111 Tebiro, Kamakura, Kanagawa 248-8555, Japan
Received 13 September 2005; revised 26 October 2005; accepted 27 October 2005
Available online 16 November 2005
Abstract—We discovered a novel series of 3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid derivatives as potent dual-acting
agents to block the TXA2 receptor and to activate the PGI2 receptor. We report the synthesis, structure–activity relationship,
and in vitro, ex vivo, and in vivo pharmacology of this series of compounds. 4-[2-(1,1-Diphenylethylsulfanyl)ethyl]-3,4-dihydro-
2H-benzo[1,4]oxazin-8-yloxyacetic acid N-methyl-D-glucamine salt (7) is a promising candidate for a novel treatment in the anti-
thrombotic and the cardiovascular fields avoiding hypotensive side effects.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
cardiovascular field, the therapeutic application of
PGI2 itself is limited by both chemical and metabolic
instability because of its labile enol–ether moiety. Thus,
the extensive efforts that have been focused on the
synthesis of PGI2 mimics were directed toward the
stabilization of the enol–ether moiety (i.e., 3).7,8 Recently,
non-prostanoids PGI2 mimetics with chemical and meta-
bolic instability have been reported (i.e., 4)9–14 (Chart 1).
Thromboxane A2 (TXA2) (1), discovered by Samuels-
son, is an unstable endogenous arachidonic acid metab-
olite that plays a pivotal role in platelet aggregation and
vasoconstriction,1 and has been implicated as a contrib-
utor to cardiovascular, renal, and pulmonary diseases.2,3
Because of the lack of clinical efficacy with these agents,4
a combined therapeutics using thromboxane receptor
antagonists (TRAs) and thromboxane synthase inhibi-
tors (TSIs) has been developed. This therapy has the
advantage that its TSI activity would prevent the
biosynthesis of TXA2, while the accumulated PGH2
would be redirected to produce beneficial prostaglandin
metabolites such as prostacyclin (PGI2), PGD2, and
PGE2. However, this conventional TRA/TSI therapy
exhibits unsatisfactory clinical effects.5
TXA2 and PGI2, both synthesized from arachidonic
acid, have opposite effects on platelet aggregation. Also
the balance between TXA2 and PGI2 greatly affects
maintenance of the homeostasis of the circulatory sys-
tem. In the case of ischemic disorders, the TXA2/PGI2
CO2H
CO2H
O
CO2Na
O
O
H
H
CO2H
O
O
Prostacyclin (2), discovered by Vane, is a powerful
endogenous inhibitor of platelet aggregation and also
plays an important role in biological homeostasis as
an endogenous autacoid distributed widely in various
tissues.6 Although these actions attracted notice in the
H
H
O N
Ph
OH
HO
HO
N
OH
OH
1thromboxane A2 (TXA2
)
2 prostacyclin (PGI2)
3 beraprost sodium
4 ONO-1301
CO2H
CO2
O
OH OH
O
CO2H
Me
OH
H
O
N
O
S
O
H2
OH OH
Ph
N
H
H
Ph
Ph
Me
N
HO
S
Keywords: Prostacyclin; Thromboxane A2 antagonist; Dual prosta-
noids; Anti-thrombosis.
OH
5 iloprost
6 sulotroban
7
*
Corresponding author. Tel.: +81 467 32 2111; fax: +81 467 32
4791; e-mail: michihiro_ono@nts.toray.co.jp
Chart 1.
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.050

2006
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
balance is shifted to the TXA2 side and phenomena such
as platelet activation, subsequent thrombogenesis ap-
pear. Thus, it is clinically important to achieve the prop-
er TXA2/PGI2 balance. A combination of an agent for
inhibiting TXA2 activity or an agent acting as a PGI2
receptor agonist is thought to be effective. Moreover,
Schering AG reported that PGI2 mimetic 5 (iloprost)
showed strong anti-thrombotic action without vasodila-
tion when it combined with TXA2/PGH2 receptor
antagonist 6 (sulotroban).15,16 Therefore, we are inter-
ested in developing agents that combine the TXA2
receptor antagonist activity with prostacyclin receptor
agonist activity within a single molecule. Such agents
would not only maximize the beneficial effects of each
agent but also address the potential clinical problem of
using two drugs with different pharmacokinetics. More-
over, one could expect a synergistic effect from the com-
bined therapeutic agent in a single chemical entity to
avoid the hypotensive effect of PGI2.
steps. We also preserved the terminal 1,1-diphenylethyl-
sulfide in the x-side chain, which was effective in trigger-
ing dual activity for the benzofuran scaffold,17 and wide
range of x-side chains were screened (10a–k) in the fol-
lowing optimization. The product in which the sulfide in
compound 10a was oxidized was also screened. Identifi-
cation of compounds was confirmed by H NMR, IR,
and low-resolution mass spectrometry (LRMS), and
the purity was demonstrated by elemental analysis.
1
The compounds in Tables 1 and 2 were prepared as de-
scribed in Schemes 1–7. The exploration of conventional
methods for thiol synthesis was the key objective of this
project. Nishio reported the single-step conversion from
secondary and tertiary alcohols to the corresponding
thiols by treatment with LawessonÕs reagent.18,19 We
also reported a conventional method for tertiary thiol
synthesis, which was improved in the b-elimination side
reaction of NishioÕs procedure and also enabled the
large-scale preparation.17 Our method became a power-
ful tool in synthesis of 9a–d and 10a–h.
In a previous publication,17 we reported dual-acting
benzofurans that possess TXA2 antagonism and PGI2
agonism. And we demonstrated that a synergistic effect
from the combined therapeutic agent in a single chemi-
cal entity enables us to avoid the hypotensive effect of
PGI2. In the present study, aimed at further investigat-
ing the dual prostanoids possessing TXA2 antagonism
and PGI2 agonism within a single molecule, we have
explored a new scaffold. We describe the design, synthe-
sis, and the biological evaluation of 3,4-dihydro-2H-
benzo[1,4]oxazine derivatives.
The preparation of indoline derivative 9a is outlined in
Scheme 1. We began the synthesis from commercially
available 4-methoxy-1H-indole (11). Compound 11
was reduced to indoline 12 with BH3 trimethylamine
complex.20 Indoline 12 was converted into N-(2-
hydroxyethyl)-indoline 13 in 11% yield in 5 steps. Thus,
compound 12 was coupled with 2-(2-bromoethoxy)tetr-
ahydropyran using phenyllithium as base, and tetrahy-
dropyranyl group was removed under mild acidic
condition. The methyl protection of the phenolic
hydroxyl group was removed using n-PrSK,21,22 and
then was treated with methyl bromoacetate to selectively
introduce the oxyacetic a-chain moiety at the 4-position
of indoline. After coupling compound 13 with 1,1-diphe-
nylethanethiol via the mesylate, compound 9a was
obtained upon hydrolysis of the methyl ester groups of 14.
2. Results and discussion
2.1. Chemistry
We designed our compounds from 3 (Chart 2). We pick-
ed out benzofuran scaffold, which is regarded as a char-
acteristic structure of 3 and reported a dual-acting
benzofuran 8. We expanded our research to replace
the scaffold since benzofuran derivatives are difficult to
synthesize. We designed compounds 9a–d and 10a
replacing the benzofuran ring to nitrogen containing
hetero-aromatics. To avoid enantiomeric problems, we
attached the x-side chain through a nitrogen atom.
We retained an oxyacetic acid group of compound 8
and its attached position for the following reasons. (1)
This derivatization at the 7-position of benzofuran is
known to maintain the PGI2 agonistic properties. (2)
This derivatization can avoid x-oxidation metabolism
of the a-chain. (3) It enables us to shorten synthetic
The preparation of tetrahydroquinoline derivative 9b is
outlined in Scheme 2. We began with the protection of
hydroxyl group of quinolin-5-ol 15 by t-butyldimethylsi-
lyl group. After hydrogenation of compound 16 using
Pd/C catalysis, tetrahydroquinoline 17 was sequentially
treated with 2-chloroacetyl chloride and sodium 1,1-
diphenylethanethiolate, which was accompanied by
removal of the t-butyldimethylsilyl group. The amide
group of compound 19 was reduced by BH3–tetrahydro-
H
N
OH
H
N
N
ii, iii, iv, v, vi
i
13
12
11
O
CO2Me
OMe
OMe
CO2H
Ph
Ph
Me
CO2Na
CO2H
Ph
Ph
Me
O
O
S
S
N
vii, viii
O
ix
N
H
O
X
(
)
n
Ph
Ph
Me
Ph
H
Y
N
O
CO2Me
14
S
S
Ph
Me
O
CO2H 9a
HO
OH
3
Scheme 1. Reagents: (i) BH3–Me3N, 87%; (ii) PhLi; (iii)
BrCH2CH2OTHP; (iv) HCl, MeOH; (v) n-PrSK, DMF; (vi)
BrCH2CO2Me, K2CO3, 11% for 5 steps; (vii) MsCl, Et3N; (viii)
Ph2MeCSNa, 99% for 2 steps; (ix) NaOH, 59%.
8
9a-d, 10a : n = 0, 1, 2
X = CH , O
Y = CH , CO
Chart 2.

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2007
O
H
H
CO2H
OMe
Cl
N
CO2Et
H
N
NH2
N
i
ii
iii
N
Cl
N
i
N
ii
iii
O
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
15
OTBS
17
OTBS
16
28
29
OTBS
30
31
18
OH
H
N
H
O
N
O
O
N
O
Ph
viii
Ph
Ph
Me
Ph
v
iv
vi, vii
O
S
Ph
Me
S
S
Ph
Me
O
O
iv
N
N
N
vi
v
OH
OH
OTHP
32
33
34
Ph
Ph
Me
OH
OH
O
CO2Me
Ph
Ph
Me
19
20
OH
S
21
S
Ph
Ph
Me
N
O
O
N
O
O
xi
N
O
ix, x
S
N
O
vii
O
CO2Me
O
CO2Me
O
CO2H
36
35
9d
O
CO2H
9b
Scheme 4. Reagents: (i) DPPA, Et3N, EtOH, 78%; (ii) NaOH, 93%;
(iii) (ClCH2CO)2O, 97%; (iv) BBr3, CH2Cl2 then K2CO3, DMF, 90%;
(v) DHP, PPTS, 80%; (vi) NaH, BrCH2CH2OTHP; (vii) p-TsOH,
MeOH, 94% for 2 steps; (viii) BrCH2CO2Me, K2CO3, DMF, 97%; (ix)
MsCl, Et3N; (x) Ph2MeCSNa, 42% for 2 steps; (xi) NaOH, 94%.
Scheme 2. Reagents: (i) TBDMS-Cl, imidazole, 100%; (ii) H2, Pd/C,
82%; (iii) ClCH2COCl, pyridine, 100%; (iv) Ph2MeCSNa, 98%; (v)
BH3, THF, 46%; (vi) BrCH2CO2Me, K2CO3, 72%; (vii) NaOH, 67%.
CO2Me
O
OH
O
H
N
O
N
N
outlined in Scheme 4. Compound 30 was synthesized
from 2,3-dimethoxybenzoic acid 28 using Schmidt
rearrangement. Thus, compound 28 was treated with
diphenylphosphorylazide and Et3N in ethanol, and the
resulting ethyl carbamate 29 was hydrolyzed to obtain
compound 30. After chloroacetylation of compound
30 followed by demethylation using BBr3, cyclization
of 3-oxo-3,4-dihydro-2H-1,4-benzoxazine was per-
formed by treating it with a base. The phenolic hydroxyl
group of compound 32 was protected by tetrahydropy-
ran. In the course of this synthesis, the compounds 32
and 33 were crystallized from ethyl acetate, respectively.
This enabled large-scale preparation and storage of the
compounds 32 and 33 as common intermediates. Com-
pound 33 was coupled with 2-(2-bromoethoxy)tetrahyd-
ropyran and tetrahydropyranyl groups were removed
under mild acidic condition. After selective introduction
of oxyacetic a-chain by treating with methyl bromoace-
tate, the resulting compound 35 was coupled with thiol
via mesylate. Compound 9d was obtained upon hydroly-
sis of the methyl ester groups of 36.
i, ii
iii
iv
OH
OAc
OAc
OH
22
23
24
25
Ph
Ph
Me
Ph
Ph
Me
S
S
OH
N
N
N
v
vi, vii
viii
O
O
O
CO2Me
CO2H
CO2Me
26
27
9c
Scheme 3. Reagents: (i) TFA, NaN3; (ii) Ac2O, pyridine, 61% for 2
steps; (iii) BrCH2CO2Me, K2CO3, 99%; (iv) LiAlH4, 81%; (v)
BrCH2CO2Me, K2CO3, 67%; (vi) CBr4, Ph3P; (vii) Ph2MeCSNa,
84% for 2 steps; (vii) NaOH, 62%.
furan complex, and the obtained compound 20 was
treated with methyl bromoacetate to introduce the oxy-
acetic a-chain moiety. Compound 9b was obtained upon
hydrolysis of the methyl ester groups of 21.
The preparation of tetrahydro-1H-benzazepine deriva-
tive 9c from 5-hydroxy-1-tetralone 22 is outlined in
Scheme 3. We performed the ring expansion using
Schmidt rearrangement.23 Thus, compound 22 was treat-
ed with sodium azide under acidic condition, and the phe-
nolic hydroxyl group was protected by the acetyl group.
N-Alkylation of the amide group of compound 23 was
performed by treating it with methyl bromoacetate and
potassium carbonate. The amide group and ester group
of 24 were simultaneously reduced to corresponding
amines and the primary alcohol using LiAlH4, which
was accompanied by reductive removal of acetyl group,
and the resulting compound 25 was treated with methyl
bromoacetate to selectively introduce the oxyacetic
a-chain moiety at the 6-position of tetrahydro-1H-ben-
zazepine. The alcohol 26 was coupled with 1,1-diphenyl-
ethanethiol via bromide. Compound 9c was obtained
upon hydrolysis of the methyl ester groups of 27.
The preparation of 3,4-dihydro-2H-1,4-benzoxazine
derivatives 10a–h is summarized in Scheme 5. Common
intermediate 33 was coupled with 2-(2-bromoethoxy)-
tetrahydropyran, and the amide group was reduced to
amine using the BH3–THF complex, then tetrahydro-
pyranyl groups were removed under mild acidic condi-
tion. Compound 38 was treated with methyl
bromoacetate to selectively introduce the oxyacetic a-
chain. The alcohol 39 was coupled with thiols via mesy-
late. Compound 10a–h was obtained upon hydrolysis of
the methyl ester group of 40a–h, respectively.
The preparation of compounds 7, 10k, and 41 is
described in Scheme 6. Compound 7 was obtained
in 95% yield by treating 10a with N-methyl-D-gluc-
amine and crystallizing from ethanol. The sulfoxide
analogue 10k was synthesized by direct oxidation of
10a with m-chloroperbenzoic acid. We also tried oxi-
dation of 10a or 40a using H2O2, m-chloroperbenzoic
acid, KMnO4, and OxoneÒ, but failed to obtain the
The preparation of 3-oxo-3,4-dihydro-2H-1,4-benzox-
azine derivative 9d from 2,3-dimethoxyaniline 30 is

2008
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
OH
OTHP
phenolic hydroxyl group of intermediate 32 was protect-
H
N
N
O
O
N
O
ed by acetyl group, and the resulting compound 42 was
coupled with 2-(1,1-diphenylethoxy)ethyl-p-toluenesulf-
onate or 4,4-diphenylpentyl-p-toluenesulfonate. The
amide group of 43a,b was reduced to the amine using
the BH3–tetrahydrofuran complex, which was accompa-
nied by removal of the acetyl group. The resulting com-
pound 44a,b was treated with methyl bromoacetate to
selectively introduce as oxyacetic a-chain moiety at the
6-position of 44. Compound 10i–j was obtained upon
hydrolysis of the methyl ester groups of 45a,b,
respectively.
iii
i, ii
iv
O
OTHP
OTHP
OH
33
37
38
OH
S
R
S R
N
O
N
O
N
O
vii
v, vi
O
CO2Me
O
CO2Me
O
CO2H
39
40a-h
10a-h
Ph
Ph
Me
Ph
Ph
CF3
Ph
Ph
Ph
Me e : R=
Me
Ph
a : R=
f : R=
b : R=
c : R=
d : R=
H
H
H
Ph
Ph g : R=
Me
Me
2.2. Biological activity
Me
S
F
h : R=
C
H2
All compounds synthesized were evaluated as their free
acid, sodium salt or N-methyl-D-glucamine salt. Com-
pounds synthesized were evaluated in terms of inhibition
of aggregation in human platelet-rich plasma (PRP)
induced by the P2Y receptor agonist adenosine diphos-
phate (ADP) or by a stable TXA2 agonist (U46619).
To confirm the mechanistic profile of these compounds,
we also performed receptor binding assay in human
platelet membrane fraction. These receptor binding
assays were carried out by using [3H]SQ-29548 (a
selective TXA2 receptor antagonist) and [3H]APS314d
sodium salt (a selective PGI2 receptor agonist) which
is one of the components of 3. Scatchard analysis of
the binding of [3H]SQ-29548 revealed a single binding
site (Kd = 10.2 0.51 nM, Bmax = 5.89 0.62 nM/mg
protein). [3H]APS314d sodium salt also had one binding
site (Kd = 14.3 0.51 nM, Bmax = 6.08 0.60 nM/mg
protein).
S
F
Scheme 5. Reagents: (i) NaH, BrCH2CH2OTHP; (ii) BH3, THF, 93%
for 2 steps; (iii) PPTS, MeOH, 97%; (iv) BrCH2CO2Me, K2CO3,
DMF, 74%; (v) MsCl, Et3N; (vi) NaH, thiols, 37–95%; (vii) NaOH,
61–91%.
Ph
O
S
Ph
Ph
S
Ph
Me
Ph
Me
S
Ph
Me
N
N
N
OH OH
i
ii
Me
OH
N
H2
O
O
O
OH OH
O
CO2
O
CO2
H
O
CO2H
iii
7
10a
10k
Ph
Ph
Me
S
N
O
O
CO2Na
41
Scheme 6. Reagents: (i) m-CPBA, AcOH, CH2Cl2, 81%; (ii) N-methyl-
D-glucamine, EtOH, 95%; (iii) NaOH, 100%.
We synthesized indoline analogue (9a), which had the
same 5–6-fused aromatic system as benzofuran. We
screened the scaffold using 1,1-diphenylethylsulfide
x-side chain, which was effective in triggering the PGI2
agonistic property for the benzofuran scaffold. Also, tet-
rahydroquinoline derivative (9b), having a 6–6-fused
aromatic system, and tetrahydro-1H-benzazepine deriv-
ative (9c), having a 6–7-fused aromatic system, were
tested. (Table 1) Compound 9a–c showed dual activity,
but it was not as potent as compound 8 in both proper-
ties. In the next step, we introduced oxygen atom on 9b,
which was the most potent among 9a–c, and designed
3,4-dihydro-2H-1,4-benzoxazine derivatives (9d and
10a). Compared to 9b, compound 10a was 2.4 times trig-
ger potent in the TXA2 antagonism. Compound 9d,
which is regarded as the oxidated analogue in the a-car-
bon of nitrogen of 10a, was less potent than 10a in anti-
aggregatory activity although it possessed similar dual
property as 10a. And compound 10a is the best dual
prostanoid in this series.
Ph
H
N
H
N
X
Ph
Me
O
O
i
N
O
O
iv
ii or iii
O
O
OH
OAc
OAc
42
32
43a (X=O)
43b (X=CH2)
Ph
Ph
Me
Ph
Ph
Me
Ph
Ph
Me
X
X
X
N
N
N
O
vi
v
O
O
O
CO2Me
O
CO2H
OH
44a (X=O)
44b (X=CH2)
10i(X=O)
10j(X=CH2)
45a (X=O)
45b (X=CH2)
Scheme 7. Reagents: (i) Ac2O, pyridine, 94%; (ii) NaH, p-TsOCH2-
CH2OCPh2Me, 57%; (iii) NaH, p-TsO(CH2)3CPh2Me, 61%; (iv) BH3,
THF, 99% for a, 99% for b; (v) BrCH2CO2Me, K2CO3, 80% for a, 92%
for b; (vi) NaOH, 65% for i, 79% for j.
sulfone analogue of 10a. Compound 41 was obtained
1 equivalent NaOH and
In the following optimization on 10a, we screened
through a wide range of x-side chains to enhance the
TXA2 antagonism and/or the PGI2 agonism (Table 2).
First, we investigated the influence of alkyl substitution
at the end of the x-side chain and 10b–f were synthe-
sized. Compound 10c was 10 times less potent than com-
pound 10a in TXA2 antagonistic property. And
compounds 10b and 10e completely lost TXA2 antago-
by treating 10a with
lyophilizing.
For comparison, we also attached the ether type and
carbohydrate type x-side chain at the 4-position of
3,4-dihydro-2H-1,4-benzoxazine. The preparation of
compound 10i and 10j is described in Scheme 7. The

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2009
Table 1. Inhibition of platelet aggregation and binding affinities of
nitrogen containing hetero-aromatic derivatives
Table 2. Inhibition of platelet aggregation and binding affinities of 3,4-
dihydro-2H-1,4-benzoxazine derivatives
Ph
Ph
Me
R
N
S
O
CO2H
A
O
O
CO2H
Compound
R
Anti-aggregatory
activity IC50
(lM)a
Receptor
affinity Ki
(lM)
Compound
A
Anti-aggregatory
activity IC50
(lM)a
Receptor
affinity Ki
(lM)
ADPb U46619c IP
TP
ADPb
U46619c
IP
TP
S
S
Ph
Ph
10a
10b
10c
10d
10e
10f
1.8
4.7
2.0
0.55
3.7
0.43
0.34
0.39
6.1
0.050
Me
8d
8.1
0.58
0.57
1.0
0.026
O
N
Ph
Ph
CF3
1.2
9a
9b
9c
4.2
1.3
1.8
15
1.6
0.14
S
S
S
Ph
Ph
1.3
0.54
0.046
2.6
N
N
H
0.59
1.3
0.52
1.3
0.12
Ph
Me
44
0.28
5.6
Me
0.19
Ph
H
45
1.5
N
O
O
H
9d
1.3
1.00
0.038
Ph
Ph
0.90
0.83
0.91
0.29
S
S
N
O
Me
10a
1.8
0.55
0.43
0.050
F
a IC50 represents the concentration that inhibits induced aggregation
by 50% in human platelet-rich plasma.
b Platelet aggregation was induced by ADP (5 lM).
c Platelet aggregation was induced by U46619 (2 lM).
d Ref. 17.
10g
23
0.54
1.9
0.13
Me
F
S
S
S
10h
10i
44
16
18
1.9
5.5
5.9
2.5
3.3
4.3
2.6
0.34
1.0
Me
O
Ph
Ph
Me
nistic property. Only compound 10d showed excellent
potency in TXA2 antagonistic property, but it complete-
ly lost PGI2 agonistic property. We checked the influ-
ence of carbon length of side chain. Compound 10f
showed moderate PGI2 agonistic property, but it is
about 6 times less than compound 10a in TXA2 antago-
nist activity. In second, we checked the influence of aro-
matic ring. The p-fluorophenyl analogue (10g) and the
thiophene analogue (10h) lost both TXA2 antagonistic
and PGI2 agonistic activities. We checked the influence
of the sulfide moiety in the side chain. The ether ana-
logue (10i) and the carbohydrate analogue (10j) lost
both TXA2 antagonistic property and PGI2 agonistic
property. The sulfide moiety in 10a is important to trig-
ger both TXA2 antagonistic and PGI2 agonistic activi-
ties. We also synthesized sulfoxide analogue (10k) and
investigated the oxidated stage of sulfide in compound
10a. Although compound 10k lost TXA2 antagonistic
property, its PGI2 agonistic property was dramatically
improved to be an excellent PGI2 agonist. The anti-
platelet activities of 10k, however, were less portent than
those expected from binding affinity because of its high
protein binding.
Ph
Ph
10j
2.6
Me
O
S
Ph
Ph
10k
7.9
0.023 0.97
Me
a IC50 represents the concentration that inhibits induced aggregation
by 50% in human platelet-rich plasma.
b Platelet aggregation was induced by ADP (5 lM).
c Platelet aggregation was induced by U46619 (2 lM).
In the next study, we have examined the pharmacologi-
cal profile of compound 10a in terms of its anti-platelet
effects. Compound 10a is a novel compound having a
potent TXA2 antagonistic activity together with a
moderate PGI2 agonistic activity. In fact, compound
10a showed an 8.6-fold higher affinity to thromboxane
A2 receptor (TP) than to prostacyclin receptor (IP), as
evidenced with the Ki values determined in binding
assays using human platelet membrane. PGI2 agonistic

2010
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
Table 3. Solubility of 10a with N-methyl-D-glucamine salt and sodium
salt
This is also supported by the evidence that these results,
together with the results of binding assays, indicate that
the PGI2 receptor agonistic activity of compound 7 is
relatively less potent than its TXA2 receptor antagonis-
tic activity.
Salt form of 10a
dist H2O Saline 5% xylitol
(mg/mL) (mg/mL) (mg/mL)
N-Methyl-D-glucamine salt (7) >30
Sodium salt (41) <0.1
<0.1a
<0.1
>30
<0.1
To confirm the anti-thrombotic character of compound
7, we tried ex vivo experiment. The inhibitory effects ob-
served with cynomolgus monkeyÕs PRP were
IC50 = 2.7 0.7 lM (induced by 10 lM ADP) and
IC50 = 0.56 0.02 lM (induced by 600 lM arachidonic
acid). Since these data were quite similar to those
observed with human PRP, our further experiments
were carried out in cynomolgus monkeys (Table 5).
Infusion of PGI2 receptor agonist 4 showed dose-depen-
dent inhibition of ADP-induced platelet aggregation,
but severe vasodilation together with increase of heart
rate was observed at the dose of 3 lg/kg/min. The
anti-platelet activity of compound 4 is linked to its po-
tent vasodilation. In a similar manner, compound 7
caused dose-dependent inhibitions of the ADP-induced
platelet aggregation, which was completely inhibited at
the dose of 30 lg/kg/min. And the arachidonic acid-in-
duced aggregation was completely inhibited at the dose
of more than 3 lg/kg/min. Furthermore, compound 7
did not show any significant changes in blood pressure
and heart rate at the dose less than 10 lg/kg/min. At
the dose 30 lg/kg/min, compound 7 showed an increase
of heart rate, but no significant change in blood pressure
was observed.
a The compound was precipitated as a sodium salt.
Table 4. Effects of 7, SQ-29548, 3 (beraprost sodium), and 4 (ONO-
1301) on in vitro platelet aggregation in human PRP
Aggregating agent
IC50 (nM)
SQ-29548
640 110 21
1800 540 > 10000
7
3
4
U-46619
ADP
3
7.6 0.7 170 11
5.7 1.0 170
3
Platelet aggregation was induced by U-46619 (4 lM) or by ADP
(5 lM). Values are means SE of three to four determinations.
property of compound 10a was also confirmed by
monitoring intracellular c-AMP concentration using
platelet.24
2.3. Solubility improvement
To eliminate the effect of dimethyl sulfoxide in pharma-
cological experiments, we synthesized sodium salt. Com-
pound 10a possessing two aromatic rings at the end of
the side chain, however, is highly lipophilic, so sodium
salt does not dissolve even in distilled water. Then we
performed a wide range of screening of conjugate
amines and found out the N-methyl-D-glucamine salt
of 10a (7). Compound 7 showed excellent solubility in
the 5% xylitol. In saline, compound 7 did not dissolve
more than 0.1 mg/mL, since the N-methyl-D-glucamine
salt turned into sodium salt. In a previous report,17 we
succeeded in obtaining a diethanolamine salt to improve
the compoundÕs solubility, but diethanolamine salt of
10a did not crystallize, which is important for the com-
poundÕs stability. From these results, we found out that
compound 7 with 5% xylitol is the practical formula for
pharmacological experiments (Table 3).
The neuroprotective effect of compound 7 on arterial
thrombosis was studied in cynomolgus monkey middle
cerebral artery (MCA) occlusion–reperfusion model.26
Via a transorbital approach,27,28 cynomolgus monkeys
underwent a 3-h occlusion of the right middle cerebral
artery (MCA), followed by reperfusion and observation
for 4 days. Starting 2 h after MCA occlusion, compound
7 was administered. The control animals received drug
vehicle bolus and infusion administration after MCA
occlusion. Steady-state 15O continuous inhalation was
used for assessment of local cerebral blood flow
(CBF), cerebral metabolic rate of oxygen (CMRO2),
and oxygen extraction fraction (OEF) using high-resolu-
tion PET. Five consecutive PET scans (before occlusion,
at 2 h after occlusion, and at 2 h, 24 h, and 4 days after
reperfusion) were performed on each monkey. The size
of the cerebral damage due to ischemia was measured
histologically at 4 days after reperfusion. Treatment
with compound 7 (15 lg/kg/min infusion for 0.5 h, fol-
lowed by 7.5 lg/kg/min infusion for 23.5 h), reduces
cerebral damage detected by histological observation
at 4 days after MCA occlusion. Compound 7 significant-
ly suppressed the CMRO2, which is observed from 2 h
to 4 days after the MCA occlusion, but compound 7
did not affect CBF during the experiments. Compound
7 inhibits platelet aggregation induced by ADP by
90% without showing any significant changes in blood
pressure and heart rate in this stroke model. These
results suggest that the anti-thrombotic effect and the
neuronal protective effect of compound 7 are separable
from its potent vasodilation.
2.4. Pharmacological activity
The TXA2 antagonistic and PGI2 agonistic activities of
compound 7 were examined in in vitro platelet aggrega-
tion (Table 4).24,25 Compound 7 exhibited inhibitory ef-
fects on the ADP- and U-46619-induced aggregation.
The IC50 value of inhibitory effects on the ADP-induced
aggregation was about 3-fold less potent than that on
the U-46619-induced aggregation. Similar tendency
was observed with the TXA2 receptor antagonist, SQ-
29548. On the other hand, the IC50 value of inhibitory
effects by the selective PGI2 receptor agonists 3 (bera-
prost sodium) and 4 (ONO-1301) were almost the same
on both ADP- and U-46619-induced platelet aggrega-
tion. These results are consistent with the fact that com-
pound 7 has a PGI2 receptor agonistic activity in
addition to the TXA2 receptor antagonistic activity.

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2011
Table 5. Effects of 7 and 4 (ONO-1301) on blood pressure, heart rate, and ex vivo platelet aggregation in monkey
7
4
Dose (lg/kg/min)a
10
Dose (lg/kg/min)a
0.3
Pre
1
3
30
86
191
Pre
1
3
Mean blood pressure (mmHg)b
Heart rate (beats/min)b
92
165
4
5
92 17
8
98
171
7
4
96
172
8
6
8
3
98
151
9
6
96
155
8
7
87
165
8
8
71
176
5
4
170
Percentage of platelet aggregation
ADPc
Arachidonic acidc
67
83
3
2
62
5
61
7
5
1*
36 11*
1*
12 8*
1*
66
77
6
1
57
74
8
1
32 13
54 22
21 19
52 22
28 20*
7
8
a Drugs were infused for 30 min at each of the doses in a dose-escalation manner.
b Data are expressed as means SE of three to seven determinations.
c Platelet aggregation was induced by ADP (10 lM) or by arachidonic acid (600 lM). Values are means SE of three to seven determinations.
* Significantly different from the vehicle group (p < 0.01).
3. Conclusion
Chemical (MI, USA), ADP from Sigma (MO, USA),
3.8% sodium citrate from Yamanouchi Pharmaceutical
(Tokyo, Japan), and a low molecular weight heparin sodi-
um dalteparin from Kissei Pharmaceutical (Nagano,
Japan).
In a previous study, we demonstrated the ex vivo exper-
iment of benzofuran-7-oxyacetic acid analogue and
illustrated the beneficial properties of PGI2 stable
mimetics in terms of avoiding hypotensive side effects.
In this paper, we have expanded the range of structures
suitable as dual prostanoids. A variety of 3,4-dihydro-
2H-benzo[1,4]oxazin-8-yloxyacetic acid analogues were
prepared by versatile synthetic route, which allows
large-scale preparation. Among the analogues synthe-
sized, we found the dual-acting prostanoid 7 possessing
a potent TXA2 antagonism and a moderate PGI2
agonism.
4.2. Chemistry
All melting points were obtained with a Yanagimoto
melting point apparatus and are uncorrected. Infrared
(IR) spectrawere measured on aJASCO FT/IR-410 infra-
red spectrophotometer. 1H NMR spectra were recorded
with a Varian Gemini-2000 spectrometer (300 MHz) with
tetramethylsilane as an internal standard. Low mass spec-
tra (MS) or high-resolution mass spectra (HR-MS) were
obtained with a JEOL JMS-DX303 mass spectrometer.
The fast atom bombardment mass spectra (FAB-MS)
were obtained by using glycerol as the matrix. Elemental
analysis was performed by Toray Research Center. Thin-
layer chromatography was performed on pre-coated TLC
plates (silica gel 60 F-254, layer thickness 0.25 mm or
DIOL F-254s) manufactured by E. Merck. Silica gel col-
umn chromatography was performed on Silica gel 60
(0.063–0.200 mm) manufactured by E. Merck. In general,
reactions were carried out in dry solvents under an argon
atmosphere unless otherwise mentioned. All reactions
that required anhydrous condition were performed under
argon or nitrogen, and all glassware was either oven-dried
or flame-dried before use.
In conclusion, our results reported in this paper clearly
illustrate the advantage of compound 7 having both
PGI2 receptor agonistic and TXA2 receptor antagonistic
activities. The TXA2 receptor antagonistic and PGI2
receptor agonistic activities of compound 7 are demon-
strated in in vitro platelet aggregation. The ex vivo
and in vivo experiments of compound 7 illustrated the
beneficial anti-thrombotic properties in terms of avoid-
ing vasodilation side effect. Remarkably, compound 7
was found to be a promising candidate for a novel med-
icine in anti-thrombotic and cardiovascular fields. Fur-
ther experimental evaluations using monkey cerebral
artery occlusion–reperfusion model are now in progress
on pharmacological properties.
4.3. (4-(2-(1,1-Diphenylethylthio)ethyl)-3,4-dihydro-2H-
1,4-benzoxazin-8-yloxy)acetic acid N-methyl-D-glucamine
salt (7)
4. Experimental
4.1. Materials
To a solution of (4-(2-(1,1-diphenylethylthio)ethyl)-3,4-
dihydro-2H-1,4-benzoxazin-8-yloxy)acetic acid 10a
(1.50 g) in ethanol (150 mL) was added a solution of
N-methyl-D-glucamine (0.74 g) in ethanol (74 mL) on
heating, and the resulting mixture stood at room tem-
perature. The precipitated solid was filtered off to obtain
the object compound 7 (2.03 g, 95%). Colorless needles,
Synthetic reagents were purchased from Aldrich (Mil-
waukee, WI), Kanto Kagaku Co. (Tokyo, Japan), TCI
(Tokyo, Japan), and Sigma Chemical Co. (St. Louis,
MO). Anhydrous tetrahydrofuran, methanol, dichloro-
methane, DMF, and pyridine were purchased from
Kanto Kagaku Co. (Tokyo, Japan). All of the reagents
and solvents used were of analytical grade from standard
commercial source or were purified by standard methods
before use. The active isomer of beraprost sodium,
[3H]APS-314d sodium, and [3H]SQ-29548 were synthe-
sized at Daiichi Pure Chemicals (Tokyo, Japan).
SQ-29548 and U-46619 were purchased from Cayman
1
mp 137.0–141.5 °C; H NMR (500 MHz, DMSO-d6) d
2.04 (3H, s), 2.45 (3H, s), 2.42–2.52 (2H, m), 2.85 (1H,
dd, J = 8.5, 12.5 Hz), 2.94 (1H, dd, J = 3.5, 12.5 Hz),
3.13 (2H, t, J = 7.8 Hz), 3.16 (2H, t, J = 4.3 Hz), 3.36–
3.44 (2H, m), 3.46–3.52 (1H, m), 3.58 (1H, dd, J = 3.5,
11.0 Hz), 3.65 (1H, dd, J = 1.5, 5.0 Hz), 3.81–3.87 (1H,

2012
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
m), 4.05 (2H, t, J = 4.0 Hz), 4.16 (2H, s), 5.88 (1H, d,
J = 7.5 Hz), 6.09 (1H, dd, J = 1.0, 8.5 Hz), 6.50 (1H, t,
J = 8.3 Hz), 7.22–7.28 (2H, m), 7.30–7.42 (8H, m); H
(4H, m); IR (liquid film method) 1734 cmÀ1 (COOH);
Mass (EI, m/e) 461 (M+); Calcd. for C28H31NO3S: C,
72.85; H, 6.77; N, 3.03; S, 6.95. Found: C, 73.08; H,
6.81; N, 3.00; S, 7.03.
1
NMR (300 MHz, CD3OD) d 2.13 (3H, s), 2.60 (2H, t,
J = 7.7 Hz), 2.77 (3H, s), 3.16–3.32 (6H, m), 3.67–3.93
(5H, m), 4.03–4.17 (1H, m), 4.22 (2H, t, J = 4.4 Hz),
4.42 (2H, s), 6.05 (1H, d, J = 8.2 Hz), 6.32 (1H, d,
J = 8.2 Hz), 6.64 (1H, t, J = 8.2 Hz), 7.26–7.75 (10H,
m); Mass (FAB, m/e) 448 (MÀH)+; Elemental analysis
Calcd. for C33H44N2O9S: C, 61.47; H, 6.88; N, 4.34; S,
4.97. Found: C, 61.22; H, 6.80; N, 4.36; S, 4.99.
4.7. (4-(2-(1,1-Diphenylethylthio)ethyl)-3-oxo-3,4-dihy-
dro-2H-1,4-benzoxazin-8-yloxy)acetic acid (9d)
By the procedure used in 9a, compound 9d (155 mg,
94%) was prepared from 170 mg of 36. White pow-
der, mp 106 °C (recrystallized from dichloromethane/
n-hexane); 1H NMR (300 MHz, CDCl3) d 2.08 (3H,
s), 2.57 (2H, m), 3.81 (2H, m), 4.60 (2H, s), 4.70
(2H, s), 6.16 (1H, dd, J = 1, 8 Hz), 6.61 (1H, dd,
J = 1, 8 Hz), 6.84 (1H, t, J = 8 Hz), 7.21–7.34 (6H,
m), 7.40–7.45 (4H, m); IR (KBr) 1734 (COOH),
1665 (amide) cmÀ1; Mass (EI, m/e) 463 (M+); Ele-
mental analysis Calcd. for C26H25NO5S: C, 67.37;
H, 5.44; N, 3.02; S, 6.92. Found: C, 67.02; H, 5.46;
N, 2.76; S, 6.94.
4.4. General procedure of methyl ester hydrolysis; {1-[2-
(1,1-Diphenylethylsulfanyl)ethyl]-2,3-dihydro-1H-indol-4-
yloxy}acetic acid (9a)
To a solution of {1-[2-(1,1-diphenylethylsulfanyl)ethyl]-
2,3-dihydro-1H-indol-4-yloxy}acetic acid methyl ester
(14) (333 mg) in methanol (5 mL) and THF (2 mL)
was added 1 N sodium hydroxide aqueous solution
(1.00 mL), and the mixture was stirred at room temper-
ature for 20 min. The solvent was distilled off under re-
duced pressure, and the residue was poured into a 5%
citric acid aqueous solution, and then extracted with
ethyl acetate. The organic layer was washed with water
and brine, dried over sodium sulfate, and concentrated.
The residue was recrystallized from ethyl acetate/n-hex-
ane to obtain compound 9a (191 mg, 59%). Colorless
prisms, mp 155.5–157.5 °C; 1H NMR (300 MHz,
CDCl3) d 2.08 (3H, s), 2.47–2.54 (2H, m), 2.93 (2H, t,
J = 8.7 Hz), 2.97–3.04 (2H, m), 3.27 (2H, t,
J = 8.7 Hz), 4.64 (2H, s), 5.96 (1H, d, J = 8.1 Hz), 6.10
(1H, d, J = 8.1 Hz), 6.96 (1H, t, J = 8.1 Hz), 7.20–7.34
(6H, m), 7.40–7.47 (4H, m); IR (KBr) 1744 cmÀ1
(COOH); Mass (FAB, m/e) 433 (M+); Elemental analy-
sis Calcd. for C26H27NO3S: C, 72.03; H, 6.28; N, 3.23; S,
7.40. Found: C, 71.98; H, 6.32; N, 3.37; S, 7.41.
4.8. (4-(2-(1,1-Diphenylethylthio)ethyl)-3,4-dihydro-2H-
1,4-benzoxazin-8-yloxy)acetic acid (10a)
By the procedure used in 9a, compound 10a (15.61 g,
77%) was prepared from 17.29 g of 40a. Colorless nee-
dles, mp 161–161.5 °C (recrystallized from ethyl ace-
tate/n-hexane); 1H NMR (300 MHz, CDCl3) d 2.06
(3H, s), 2.52 (2H, t, J = 7.5 Hz), 3.14 (2H, t,
J = 7.5 Hz), 3.22 (2H, t, J = 4.5 Hz), 4.23 (2H, t,
J = 4.5 Hz), 4.61 (2H, s), 6.06 (1H, dd, J = 1, 8 Hz),
6.31 (1H, dd, J = 1, 8 Hz), 6.68 (1H, t, J = 8 Hz),
7.20–7.35 (6H, m), 7.39–7.44 (4H, m); 13C NMR
(125 MHz, CDCl3) d 26.4, 31.0, 46.8, 50.8, 56.7, 64.7,
68.8, 105.8, 107.4, 121.3, 126.8, 128.0, 128.1, 133.9,
135.5, 146.0, 146.7, 171.1; IR (KBr) 1744 cmÀ1
(COOH); Mass (EI, FAB, m/e) 449 (M+); HR-MS
(FAB, m/e) Calcd. for C26H27NO4S is 449.1631, Found
449.1661; Elemental analysis Calcd. for C26H27NO4S: C,
69.46; H, 6.05; N, 3.12; S, 7.13. Found: C, 69.20; H,
6.05; N, 3.21; S, 7.12.
4.5. (1-(2-(1,1-Diphenylethylthio)ethyl)-1,2,3,4-tetra-
hydroquinolin-5-yloxy)acetic acid (9b)
By the procedure used in 9a, compound 9b (173 mg,
67%) was prepared from 268 mg of 21. Pale yellow nee-
dles; mp 150.5–152.0 °C (recrystallized from methylene
4.9. (4-(2-(1,1-Diphenyl-2,2,2-trifluoroethylthio)ethyl)-
3,4-dihydro-2H-1,4-benzoxazin-8-yloxy)acetic acid (10b)
1
chloride/hexane); H NMR (300 MHz, CDCl3) d 1.80–
190 (2H, m), 2.06 (3H, s), 2.47–2.55 (2H, m), 2.67
(2H, t, J = 6.6 Hz), 3.04–3.19 (4H, m), 4.61 (2H, s),
5.93 (1H, 6, J = 8.1 Hz), 6.05 (1H, d, J = 8.1 Hz), 6.88
(1H, t, J = 8.1 Hz), 7.20–7.34 (6H, m), 7.39–7.46 (4H,
m); IR (KBr) 1745 cmÀ1 (COOH); Mass (FAB, m/e)
448 (M+H)+; Calcd. for C27H29NO3S: C, 72.45; H,
6.53; N, 3.13; S, 7.16. Found: C, 72.75; H, 6.35; N,
3.26; S, 7.32.
By the procedure used in 9a, compound 10b (472 mg,
83%) was prepared from 585 mg of 40b. Colorless plates,
mp 140–140.5 °C (recrystallized from ethyl acetate/n-hex-
ane); 1H NMR (300 MHz, CDCl3) d 2.56 (2H, t,
J = 7.5 Hz), 3.19 (4H, m), 4.23 (2H m), 4.61 (2H, s),
5.98 (1H, dd, J = 1, 8 Hz), 6.30 (1H, dd, J = 1, 8 Hz),
6.65 (1H, t, 1 = 8 Hz), 7.32–7.36 (6H, m), 7.42–7.46 (4H,
m); IR (KBr) 1748 cmÀ1 (COOH); Mass (EI, m/e) 503
(M+); Elemental analysis Calcd. for C26H24F3NO4S: C,
62.02; H, 4.80; N, 2.78; S, 6.37; F, 11.32. Found: C,
61.78; H, 4.85; N, 2.87; S, 6.54; F, 11.37.
4.6. (1-(2-(1,1-Diphenylethylthio)ethyl)-2,3,4,5-tetrahy-
dro-1H-benzazepin-6-yloxy)acetic acid (9c)
By the procedure used in 9a, compound 9c (171 mg,
62%) was prepared from 283 mg of 27. Colorless solid;
1H NMR (300 MHz, CDCl3) d 1.68–1.52 (4H, m), 2.05
(3H, s), 2.45–2.53 (2H, m), 2.80–2.91 (4H, m), 3.06–
3.14 (2H, m), 4.63 (2H, s), 6.39 (2H, d, J = 8.1 Hz),
6.97 (1H, t, J = 8.1 Hz), 7.16–7.32 (6H, m), 7.36–7.44
4.10. (4-(2-(Diphenylmethylthio)ethyl)-3,4-dihydro-2H-
1,4-benzoxazin-8-yloxy)acetic acid (10c)
By the procedure used in 9a, compound 10c (381 mg,
90%) was prepared from 439 mg of 40c. Colorless nee-
dles, mp 172.5 °C (recrystallized from ethyl acetate/n-

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2013
1
hexane); H NMR (300 MHz, CDCl3) d 2.60 (2H, m),
dles, mp 142–143 °C (recrystallized from ethyl acetate/
1
3.27 (2H, t, J = 4 Hz), 3.37 (2H, m), 4.24 (2H, t,
J = 4 Hz), 4.61 (2H, s), 5.23 (1H, s), 6.10 (1H, dd,
J = 1, 8 Hz), 6.31 (1H, dd, J = 1, 8 Hz), 6.66 (1H, t,
J = 8 Hz), 7.22–7.37 (6H, m), 7.42–7.46 (4H, m); IR
(KBr) 1743 cmÀ1 (COOH); Mass (EI, m/e) 435 (M+);
Elemental analysis Calcd. for C25H25NO4S: C, 68.94;
H, 5.79; N, 3.22; S, 7.36. Found: C, 68.85; H, 5.77; N,
3.24; S, 7.25.
n-hexane); H NMR (300 MHz, CDCl3) d 2.02 (3H, s),
2.50 (2H, t, J = 7.6 Hz), 3.21 (2H, t, J = 7.6 Hz), 3.26
(2H, t, J = 7.6 Hz), 4.25 (2H, t, J = 4.4 Hz), 4.62 (2H,
s), 6.06 (1H, dd, J = 8.2, 1.1 Hz), 6.31 (1H, dd, J = 8.2,
1.1 Hz), 6.69 (1H, t, J = 8.2 Hz), 6.95–7.03 (4H, m),
7.33–7.40 (4H, m); IR (KBr) 1744 cmÀ1 (COOH); Mass
(EI, m/e) 485 (M+); Elemental analysis Calcd. for
C26H25F2NO4S: C, 64.32; H, 5.19; N, 2.88; S, 6.60.
Found: C, 64.22; H, 5.13; N, 2.99; S, 6.61.
4.11. (4-(2-(1-Methyl-1-phenylethylthio)ethyl)-3,4-dihy-
dro-2H-1,4-benzoxazin-8-yloxy)acetic acid (10d)
4.15. (4-(2-(1,1-bis-(3-Thienyl)ethylthio)ethyl)-3,4-dihy-
dro-2H-1,4-benzoxazin-8-yloxy)acetic acid (10h)
By the procedure used in 9a, compound 10d (271 mg,
78%) was prepared from 358 mg of 40d. Colorless nee-
dles, mp 114 °C (recrystallized from ethyl acetate/n-hex-
By the procedure used in 9a, compound 10h (161 mg,
91%) was prepared from 183 mg of 40h. Colorless gran-
ular, mp 149 °C (recrystallized from ethyl acetate/n-hex-
1
ane); H NMR (300 MHz, CDCl3) d 1.72 (6H, s), 2.42
1
(2H, m), 3.10 (2H, m), 3.19 (2H, t, J = 4.5 Hz), 4.22
(2H, t, J = 4.5 Hz), 4.60 (2H, s), 5.98 (1H, dd, J = 1,
8 Hz), 6.29 (1H, dd, J = 1, 8 Hz), 6.66 (1H, t,
J = 8 Hz), 7.24 (1H, m), 7.35 (2H, m), 7.56 (2H, m);
IR (KBr) 1744 cmÀ1 (COOH); Mass (EI, m/e) 387
(M+); Elemental analysis Calcd. for C21H25NO4S: C,
65.09; H, 6.50; N, 3.61; S, 8.27. Found: C, 64.72; H,
6.51; N, 3.57; S, 8.03.
ane); H NMR (300 MHz, CDCl3) d 2.05 (3H, s), 2.54
(2H, t, J = 7.4 Hz), 3.17 (2H, t, J = 7.4 Hz), 3.25 (2H,
t, J = 4.4 Hz), 4.25 (2H, t, J = 4.4 Hz), 4.62 (2H, s),
6.11 (1H, dd, J = 8.2, 1.1 Hz), 6.31 (1H, dd, J = 8.2,
1.1 Hz), 6.72 (1H, t, J = 8.2 Hz), 7.12 (2H, dd, J = 3.0,
1.4 Hz), 7.14 (2H, dd, J = 5.2, 1.4 Hz), 7.29 (2H, dd,
J = 5.2, 3.0 Hz); IR (KBr) 1744 cmÀ1 (COOH); Mass
(EI, m/e) 461 (M+); Elemental analysis Calcd. for
C22H23NO4S3: C, 57.24; H, 5.02; N, 3.03; S, 20.84.
Found: C, 56.84; H, 5.04; N, 3.03; S, 20.45.
4.12. (4-(2-(Benzylthio)ethyl)-3,4-dihydro-2H-1,4-benz-
oxazin-8-yloxy)acetic acid (10e)
4.16. (4-(2-(1,1-Diphenylethoxy)ethyl)-3,4-dihydro-2H-
1,4-benzoxazin-8-yloxy)acetic acid (10i)
By the procedure used in 40a, compound 39 (242 mg)
was coupled with phenylmethanethiol and the methyl es-
ter of the obtained sulfide was hydrolyzed by the proce-
dure used in 9a, to obtain compound 10e (199 mg, 61%).
Colorless needles, mp 127–131 °C (recrystallized from
By the procedure used in 9a, compound 10i (193 mg,
65%) was prepared from 308 mg of 45a. Colorless
granular, mp 108 °C (recrystallized from ethyl acetate/
1
1
ethyl acetate/n-hexane); H NMR (300 MHz, CDCl3) d
n-hexane); H NMR (300 MHz, CDCl3) d 1.83 (3H, s),
2.61 (2H, t, J = 7.6 Hz), 3.30–3.38 (4H, m), 3.76 (2H,
s), 4.27 (2H, t, J = 4.4 Hz), 4.63 (2H, s), 6.25 (1H, dd,
J = 8.2, 1.1 Hz), 6.32 (1H, dd, J = 8.2, 1.1 Hz), 6.72
(1H, t, J = 8.2 Hz), 7.25–7.35 (5H, m); IR (KBr)
1742 cmÀ1 (COOH); Mass (EI, m/e) 359 (M+); Elemen-
tal analysis Calcd. for C19H21NO4S: C, 63.49; H, 5.89;
N, 3.90; S, 8.92. Found: C, 63.20; H, 5.90; N, 3.97; S,
8.78.
3.40–3.51 (6H, m), 4.27 (2H, t, J = 4.4 Hz), 4.63 (2H, s),
6.30 (1H, dd, J = 8.2, 1.1 Hz), 6.33 (1H, dd, J = 8.2,
1.1 Hz), 6.69 (1H, t, J = 8.2 Hz), 7.17–7.33 (10H, m);
IR (KBr) 1746 cmÀ1 (COOH); Mass (EI, m/e) 433
(M+); Elemental analysis Calcd. for C26H27NO5: C,
72.04; H, 6.28; N, 3.23. Found: C, 72.22; H, 5.98; N, 3.40.
4.17. (4-(4,4-Diphenylpentyl)-8-hydroxy-3-oxo-3,4-dihy-
dro-2H-1,4-benzoxazin-8-yloxy)acetic acid (10j)
4.13. (4-(2-(2,2-Diphenylpropylthio)ethyl)-3,4-dihydro-
2H-1,4-benzoxazin-8-yloxy)acetic acid (10f)
By the procedure used in 9a, compound 10j (249 mg,
79%) was prepared from 308 mg of 45b. Colorless gran-
ular, mp 133 °C (recrystallized from ethyl acetate/n-hex-
ane); 1H NMR (300 MHz, CDCl3) d 1.36–1.48 (2H, m),
1.63 (3H, s), 2.08–2.17 (2H, m), 3.16 (2H, t, J = 8.4 Hz),
3.21 (2H, t, J = 4.4 Hz), 4.25 (2H, t, J = 4.4 Hz), 4.62
(2H, s), 6.26 (1H, d, J = 8.5 Hz), 6.30 (1H, d,
J = 8.0 Hz), 6.71 (1H, t, J = 8.2 Hz), 7.16–7.22 (6H,
m), 7.23–7.31 (4H, m); IR (KBr) 1744 cmÀ1 (COOH);
Mass (EI, m/e) 431 (M+); Elemental analysis Calcd.
for C27H29NO4: C, 75.15; H, 6.77; N, 3.25. Found: C,
75.36; H, 6.81; N, 3.21.
By the procedure used in 9a, compound 10f (85 mg, 74%)
was prepared from 119 mg of 40f. Colorless needles, mp
138–139 °C (recrystallized from ethyl acetate/n-hexane);
1H NMR (300 MHz, CDCl3) d 1.78 (3H, s), 2.35 (2H,
t, J = 7.1 Hz), 3.24–3.33 (6H, m), 4.23 (2H, t,
J = 4.4 Hz), 4.62 (2H, s), 6.28–6.33 (2H, m), 6.76 (1H,
t, J = 8.2 Hz), 7.17–7.31 (10H, m); IR (KBr) 1746 cmÀ1
(COOH); Mass (EI, m/e) 463 (M+); Elemental analysis
Calcd. for C27H29NO4S: C, 69.95; H, 6.31; N, 3.02; S,
6.92. Found: C, 69.80; H, 6.22; N, 3.07; S, 6.78.
4.14. (4-(2-(1,1-bis-(4-Fluorophenyl)ethylthio)ethyl)-3,4-
dihydro-2H-1,4-benzoxazin-8-yloxy)acetic acid (10g)
4.18. (4-(2-(1,1-Diphenylethylsulfinyl)ethyl)-3,4-dihydro-
2H-1,4-benzoxazin-8-yloxy)acetic acid (10k)
By the procedure used in 9a, compound 10g (166 mg,
67%) was prepared from 254 mg of 40g. Colorless nee-
To a solution of (4-(2-(1,1-diphenylethylthio)ethyl)-3,4-
dihydro-2H-1,4-benzoxazin-8-yloxy)acetic acid 10a

2014
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
(51 mg) in CH2Cl2 (5 mL) was added m-CPBA (20 mg),
and the mixture was stirred at room temperature for 4 h.
The reaction mixture was added to water (20 mL) and
then extracted with ethyl acetate. The organic layer
was washed with brine, dried over sodium sulfate, and
concentrated. The residue was purified by column chro-
matography (DIOL type silica gel, solvent: n-hexane/
ethyl acetate = 1/2) to obtain compound 10k (43 mg,
81%). 1H NMR (300 MHz, CDCl3) d 1.97 (3H, s),
2.26–2.46 (2H, m), 3.20–3.37 (2H, m), 3.52–3.60 (2H,
m), 4.22–4.27 (2H, m), 4.64 (2H, s), 6.15 (1H, dd,
J = 1.2, 8.4 Hz), 6.30 (1H, dd, J = 1.2, 8.4 Hz), 6.65
(1H, t, J = 8.4 Hz), 7.27–7.46 (10H, m); Mass (FAB,
m/e) 466 (M+H)+; Elemental analysis Calcd. for
C26H27NO5S: C, 67.08; H, 5.85; N, 3.01; S, 6.89. Found:
C, 67.35; H, 5.72; N, 3.07; S, 6.95.
solved in DMF (5 mL) and treated with K2CO3
(1.00 g) and methyl bromoacetate (0.30 mL) at room
temperature. To the reaction mixture was added a satu-
rated ammonium chloride aqueous solution (30 mL)
and extracted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, and con-
centrated. The residue was purified by column chroma-
tography (silica gel: n-hexane/ethyl acetate = 1/1) to
obtain compound 13 (187 mg, 11%). 1H NMR
(300 MHz, CDCl3) d 2.05–2.16 (1H, m), 3.03 (2H, t,
J = 8.6 Hz), 3.20–3.27 (2H, m), 3.43 (2H, t,
J = 8.6 Hz), 3.79 (3H, s), 3.75–3.83 (2H, m), 4.65 (2H,
s), 6.16 (1H, d, J = 8.4 Hz), 6.27 (1H, d, J = 8.1 Hz),
6.99–7.06 (1H, m); Mass (EI, m/e) 251 (M+).
4.21. {1-[2-(1,1-Diphenylethylsulfanyl)-ethyl]-2,3-dihy-
dro-1H-indol-4-yloxy}acetic acid methyl ester (14)
4.19. 4-Methoxy-2,3-dihydro-1H-indole (12)
To a solution of [1-(2-hydroxyethyl)-2,3-dihydro-1H-in-
dol-4-yloxy]acetic acid methyl ester (13) (187 mg) in
CH2Cl2 (5.0 mL) were added Et3N (0.18 mL) and meth-
anesulfonyl chloride (0.08 mL), and the mixture was
stirred at 0 °C for 1 h. The reaction solution was poured
into a 5% citric acid aqueous solution and then extracted
with ethyl acetate. The organic layer was washed with
water and brine, dried over magnesium sulfate, and then
concentrated to obtain the mesylate of 13. Sodium
hydride (50 mg) was washed with n-hexane and dried
under reduced pressure. A solution of 1,1-diphenyletha-
nethiol (327 mg) in DMF (3.0 mL) was added to the
sodium hydride at 0 °C, and the mixture was stirred at
room temperature for 10 min. A solution of the above
mesylate in DMF (5.0 mL) was added to the mixture,
and the mixture was stirred at room temperature for
3 h. The solvent was distilled off under reduced pressure,
and the residue was poured into a 5% citric acid aqueous
solution, and then extracted with ethyl acetate. The
organic layer was washed with water and brine, dried
over magnesium sulfate, and concentrated. The residue
was purified by column chromatography (neutral alumi-
na: n-hexane/ethyl acetate = 7/1) to obtain compound 14
(333 mg, 99%). 1H NMR (300 MHz, CDCl3) d 2.08 (3H,
s), 2.47–2.54 (2H, m), 2.93 (2H, t, J = 8.4 Hz), 2.96–3.04
(2H, m), 3.25 (2H, t, J = 8.4 Hz), 3.78 (3H, s), 4.61 (2H,
s), 5.94 (1H, d, J = 8.1 Hz), 6.07 (1H, d, J = 8.1 Hz),
6.93 (1H, t, J = 8.1 Hz), 7.20–7.34 (6H, m), 7.40–7.47
(4H, m); IR (neat) 1765 cmÀ1 (COOMe); Mass (EI,
m/e) 447 (M+).
To a solution of 4-methoxy-1H-indole (11) (3.95 g) in
dioxane (50 mL) were added BH3–Me3N complex
(6.37 g) and 10.5 N HCl (8.0 mL), and the resulting
solution was refluxed for 70 min. The reaction mixture
was cooled to room temperature and concentrated un-
der reduced pressure. The residue was diluted with water
(100 mL), neutralized with KOH solution, and then
extracted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, and con-
centrated. The residue was purified by column chroma-
tography (silica gel: n-hexane/ethyl acetate = 5/1) to
obtain compound 12 (3.48 g, 87%). 1H NMR
(300 MHz, CDCl3) d 2.80–3.20 (2H, m), 3.60–3.80
(2H, m), 3.80 (3H, s), 6.28 (1H, d, J = 7.9 Hz), 6.30
(1H, d, J = 7.9 Hz), 6.99 (1H, t, J = 7.9 Hz); Mass (EI,
m/e) 149 (M+).
4.20. [1-(2-Hydroxyethyl)-2,3-dihydro-1H-indol-4-
yloxy]acetic acid methyl ester (13)
To a solution of 4-methoxy-2,3-dihydro-1H-indole (12)
(996 mg) in Et2O (15 mL) was added phenyllithium
Et2O solution (1.08 M, 9.3 mL), and the resulting solu-
tion was stirred at room temperature for 15 min. 2-(2-
Bromoethoxy)tetrahydropyran (2039 mg) was added to
the reaction mixture, and the resulting solution was stir-
red overnight at room temperature. The reaction mix-
ture was poured into saturated ammonium chloride
aqueous solution (30 mL) and then extracted with ethyl
acetate. The organic layer was washed with brine, dried
over sodium sulfate, and concentrated. The residue was
dissolved in methanol (20 mL) and treated with 1 N HCl
(1.5 mL) at room temperature. The reaction mixture was
concentrated and the residue was purified by column
chromatography (silica gel: n-hexane/ethyl acetate =
2/1), and the obtained compound was dissolved in
DMF (15 mL). n-PrSH (3.0 mL) and t-BuOK (2.10 g)
were added to the solution, and the resulting mixture
was refluxed for 3 h. The reaction mixture was poured
into water (40 mL) and extracted with ethyl acetate.
The organic layer was washed with brine, dried over
sodium sulfate, and concentrated. The residue was puri-
fied by column chromatography (silica gel: n-hexane/
ethyl acetate = 1/1). The obtained compound was dis-
4.22. 5-(t-Butyldimethylsiloxy)quinoline (16)
To a solution of 5-hydroxyquinoline (15) (101 mg) in
DMF (5 mL) were added imidazole (113 mg) and
t-butyldimethylsilyl chloride (162 mg) with stirring over-
night at room temperature under argon atmosphere.
Water (5 mL) was added to the reaction solution, and
the mixture was then extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium
sulfate, and then concentrated. The residue was purified
by column chromatography (silica gel: hexane/ethyl ace-
1
tate = 9/1) to obtain compound 16 (197 mg, 100%). H
NMR (300 MHz, CDCl3) d 0.30 (6H, s), 1.09 (9H, s),
6.92 (1H, dd, J = 7.8, 0.9 Hz), 7.38 (1H, dd, J = 8.7,

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2015
4.5 Hz), 7.56 (1H, dd, J = 8.4, 7.5 Hz), 7.73 (1H, dt,
J = 8.4, 0.9 Hz), 8.50 (1H, ddd, J = 8.7, 2.1, 0.9 Hz),
4.26. 1-(2-(1,1-Diphenylethylthio)ethyl)-5-hydroxy-
1,2,3,4-tetrahydroquinoline (20)
8.89 (1H, dd, J = 4.5, 1.8 Hz); Mass (EI, m/e) 259 (M+).
To a solution of 1-(1,1-diphenylethylthioacetyl)-5-hy-
droxy-1,2,3,4-tetrahydroquinoline (19) (136 mg) in
THF (5 mL) was added BH3–THF complex (1.0 M,
2.0 mL) at 0 °C, and the resulting solution was stirred
at room temperature for 5 h. The reaction solution
was added to a saturated ammonium chloride aqueous
solution (20 mL) and extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium
sulfate, and concentrated. The residue was purified by
column chromatography (silica gel: n-hexane/ethyl ace-
tate = 5/1) to obtain compound 20 (60 mg, 46%). 1H
NMR (300 MHz, CDCl3) d 1.82–1.92 (2H, m), 2.07
(3H, s), 2.48–2.64 (4H, m), 3.04–3.19 (4H, m), 4.54
(1H, br s), 5.83 (1H, d, J = 8.1 Hz), 6.07 (1H, dd,
J = 8.1, 0.9 Hz), 6.81 (1H, t, J = 8.1 Hz), 7.20–7.35
(6H, m), 7.39–7.45 (4H, m); Mass (EI, m/e) 389 (M+).
4.23. 5-(t-Butyldimethylsiloxy)-1,2,3,4-tetrahydro-quino-
line (17)
To a solution of 5-(t-butyldimethylsiloxy)quinoline (16)
(1715 mg) in EtOH (50 mL) was added 10% Pd–C
(100 mg), and the mixture was stirred under hydrogen
atmosphere at room temperature. After disappearance
of the starting material, the solid catalyst was filtered
off, and the filtrate was concentrated. The residue was
purified by column chromatography (silica gel: n-hex-
ane/ethyl acetate = 10/1) to obtain compound 17
1
(1426 mg, 82%). H NMR (300 MHz, CDCl3) d 0.22
(6H, d, J = 0.6 Hz), 1.00 (9H, d, J = 0.6 Hz), 1.86–1.97
(2H, m), 2.65 (2H, t, J = 6.6 Hz), 3.21–3.56 (2H, m),
3.80 (1H, br s), 6.13 (1H, d, J = 8.1 Hz), 6.14 (1H, d,
J = 8.1 Hz), 6.82 (1H, td, J = 7.8, 0.6 Hz); Mass (EI,
m/e) 263 (M+).
4.27. Methyl(1-(2-(1,1-diphenylethylthio)ethyl)-1,2,3,4-
tetrahydroquinolin-5-yloxy)acetate (21)
4.24. 5-(t-Butyldimethylsiloxy)-1-chloroacetyl-1,2,3,4-
tetrahydroquinoline (18)
To a solution of 1-(2-(1,1-diphenylethylthio)ethyl)-5-hy-
droxy-1,2,3,4-tetrahydroquinoline 20 (60 mg) in DMF
(5 mL) were added K2CO3 (100 mg) and methyl bro-
moacetate (0.050 mL), and the resulting mixture was
stirred at room temperature for 4 h. The reaction solu-
tion was added to a saturated ammonium chloride aque-
ous solution (15 mL) and then extracted with ethyl
acetate. The organic layer was washed with brine, dried
over sodium sulfate, and concentrated. The residue was
purified by column chromatography (silica gel: n-hex-
ane/ethyl acetate = 9/1) to obtain compound 21
(51 mg, 72%). 1H NMR (300 MHz, CDCl3) d 1.78–
1.89 (2H, m), 2.06 (3H, s), 2.47–2.55 (2H, m), 2.69
(2H, t, J = 6.6 Hz), 3.04–3.18 (4H, m), 3.78 (3H, s),
4.58 (2H, s), 5.91 (1H, d, J = 8.4 Hz), 6.02 (1H, d,
J = 8.4 Hz), 6.86 (1H, t, J = 1, 8.4 Hz), 7.20–7.35 (6H,
m), 7.38–7.47 (4H, m); IR (liquid film method)
1763 cmÀ1 (COOMe); Mass (EI, m/e) 461 (M+).
To a solution of 5-(t-butyldimethylsiloxy)-1,2,3,4-tetra-
hydroquinoline (17) (1426 mg) in CH2Cl2 (50 mL) were
added pyridine (1.00 mL) and chloroacetyl chloride
(0.70 mL), and the resulting solution was stirred for
5 h at 0 °C under argon atmosphere. The reaction solu-
tion was added to water (50 mL) and extracted with eth-
yl acetate. The organic layer was washed with brine,
dried over sodium sulfate, and concentrated. The resi-
due was purified by column chromatography (silica
gel: n-hexane/ethyl acetate = 5/1) to obtain compound
18 (1839 mg, 100%). 1H NMR (300 MHz, CDCl3) d
0.24 (6H, s), 1.02 (9H, s), 1.98 (1H, quint, J = 6.6 Hz),
2.70 (2H, t, J = 6.6 Hz), 3.81 (2H, t, J = 6.6 Hz), 4.25
(2H, s), 6.68 (1H, d, J = 8.4 Hz), 6.75–7.00 (1H, m),
7.08 (1H, t, J = 8.0 Hz); IR (liquid film method)
1667 cmÀ1 (amide); Mass (EI, m/e) 339 (M+).
4.25. 1-(1,1-Diphenylethylthioacetyl)-5-hydroxy-1,2,3,4-
tetrahydroquinoline (19)
4.28. 6-Acetoxy-2-oxo-2,3,4,5-tetrahydro-1H-benzaze-
pine (23)
To a solution of 1,1-diphenylethanethiol (203 mg) in
DMF (3 mL) was added sodium hydride (65 mg), and
the mixture was stirred at 0 °C for 10 min under argon
atmosphere. A solution of 5-(t-butyldimethylsiloxy)-
1-chloroacetyl-1,2,3,4-tetrahydroquinoline (18) (252 mg)
in DMF (3 mL) was then added, and the mixture was
stirred at room temperature for 3 h. The reaction solu-
tion was added to water (30 mL) and then extracted with
ethyl acetate. The organic layer was washed with brine,
dried over sodium sulfate, and concentrated. The
residue was purified by column chromatography (silica
gel: n-hexane/ethyl acetate = 2/1) to obtain compound
19 (294 mg, 98%). 1H NMR (300 MHz, CDCl3) d
1.88–1.98 (2H, m), 2.05 (3H, s), 2.65 (2H, t,
J = 7.1 Hz), 3.25 (2H, s), 3.50–3.80 (2H, m), 4.96 (1H,
br s), 6.50–6.80 (1H, m), 6.60 (1H, d, J = 8.7 Hz), 6.94
(1H, t, J = 8.1 Hz), 7.14–7.40 (10H, m); IR (KBr)
1622 cmÀ1 (amide); Mass (EI, m/e) 403 (M+).
To a solution of 5-hydroxy-1-tetralone (22) (506 mg) in
trifluoroacetic acid (10 mL) was added sodium azide
(254 mg), and the mixture was heated under reflux.
After disappearance of the starting material, the reac-
tion solution was poured into water (20 mL), neutralized
with a sodium bicarbonate aqueous solution, and then
extracted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, and con-
centrated to obtain a residue, which was dissolved in
CH2Cl2 (5 mL) and pyridine (2 mL). Ac2O (0.45 mL)
was added to the solution, and the mixture was stirred
at room temperature for 6 h. The reaction solution
was added to saturated ammonium chloride aqueous
solution (30 mL) and extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium
sulfate, and then concentrated. The residue was purified
by column chromatography (silica gel: n-hexane/ethyl
acetate = 1/1) to obtain compound 23 (416 mg, 61%).

2016
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
1H NMR (300 MHz, CDCl3) d 1.99–2.07 (2H, m), 2.14
(2H, t, J = 6.9 Hz), 2.32 (3 H, s), 2.56 (2H, t,
J = 7.2 Hz), 6.89 (2H, d, J = 8.1 Hz), 7.24 (1H, t,
J = 8.1 Hz), 9.66 (1H, s); IR (KBr) 1758 (OAc), 1676
(CONH) cmÀ1; Mass (EI, m/e) 219 (M+).
(10 mL) was added sodium hydride (65 mg), and the
mixture was stirred at 0 °C for 5 min. A solution of
the bromide of 24 in DMF (3 mL) was added, and
the reaction mixture was stirred at 0 °C for 3 h. The
reaction solution was added to a saturated ammonium
chloride aqueous solution (30 mL) and then extracted
with ethyl acetate. The organic layer was washed with
brine, dried over sodium sulfate, and concentrated.
The residue was purified by column chromatography
(silica gel: n-hexane/ethyl acetate = 8/1) to obtain com-
4.29. Methyl(6-acetoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-
benzazepin-1-yl)acetate (24)
By the procedure used in 21, compound 24 (333 mg,
99%) was prepared from 252 mg of 23. 1H NMR
(300 MHz, CDCl3) d 1.95–2.30 (2H, m), 2.30–2.41
(2H, m), 2.35 (3H, s), 2.76–2.90 (2H, m), 3.75 (3H, s),
4.51 (2H, s), 6.96 (1H, dd, J = 8.1, 0.9 Hz), 7.06 (1H,
dd, J = 8.1, 0.9 Hz), 7.27 (1H, t, J = 8.1 Hz); IR (liquid
1
pound 27 (532 mg, 84%). H NMR (300 MHz, CDCl3)
d 1.50–1.68 (4H, m), 2.05 (3H, s), 2.45–2.53 (2H, m),
2.78–2.95 (4H, m), 3.00–3.15 (2H, m), 3.78 (3H, s),
4.59 (2H, s), 6.35 (2H, d, J = 8.1 Hz), 6.95(1H, t,
J = 8.1 Hz), 7.17–7.36 (6H, m), 7.38–7.44 (4H, m);
IR (liquid film method) 1765 cmÀ1 (COOMe); Mass
(EI, m/e) 475 (M+).
film method) 1750 (COOMe, OAc), 1661(amide) cmÀ1
;
Mass (EI, m/e) 291 (M+).
4.30. 1-(2-Hydroxyethyl)-6-hydroxy-2,3,4,5-tetrahydro-
1H-1-benzazepine (25)
4.33. 2,3-Dimethoxyphenylcarbamic acid ethyl ester (29)
To a solution of 2,3-dimethoxybenzoic acid (28) (1.11 g)
in dioxane (25 mL) and Et3N (10 mL) was added diph-
enylphosphorylazide (1.70 g), and the mixture was stir-
red at 100 °C for 7.5 h. The solvent was removed
under reduced pressure. The residue was added to 5%
citric acid aqueous solution and extracted with ethyl
acetate. The organic layer was washed with brine, dried
over sodium sulfate, and concentrated. The residue was
purified by column chromatography (silica gel: n-hex-
ane/ethyl acetate = 3/1) to obtain compound 29
(1.08 g, 78%). Colorless plates, mp 45 °C; 1H NMR
(300 MHz, CDCl3) d 1.33 (3H, t, J = 7.0 Hz), 3.86
(3H, s), 3.87 (3H, s), 4.23 (2H, q, J = 7.0 Hz),
6.62 (1H, dd, J = 1.5, 8.0 Hz), 7.03 (1H, t, J = 8.0 Hz),
7.28 (1H, br s), 7.74 (1H, dd, J = 1.5, 8.5 Hz); Mass
(EI, m/e) 225 (M+).
To a solution of methyl(6-acetoxy-2-oxo-2,3,4,5-tetra-
hydro-1H-1-benzazepin-1-yl)acetate (24) (1080 mg) in
THF (15 mL) was added LiAlH4 (355 mg), and the mix-
ture was stirred at 0 °C for 3 h. The reaction solution
was added to water (100 mL) and then extracted with
ethyl acetate. The organic layer was washed with brine,
dried over sodium sulfate, and then concentrated. The
residue was purified by column chromatography (silica
gel: n-hexane/ethyl acetate = 2/1) to obtain compound
25 (624 mg, 81%). 1H NMR (300 MHz, CDCl3) d
1.55–1.66 (2H, m), 1.68–1.81 (2H, m), 2.63 (1H, br s),
2.82–2.90 (2H, m), 2.90–2.97 (2H, m), 3.31 (2H, t,
J = 5.3 Hz), 3.71 (2H, t, J = 5.3 Hz), 5.31 (1H, br s),
6.47 (1H, dd, J = 8.1, 1.2 Hz), 6.57 (1H, d, J = 8.1 Hz),
6.98 (1H, t, J = 8.1 Hz); Mass (EI, m/e) 207 (M+).
4.31. Methyl(1-(2-hydroxyethyl)-2,3,4,5-tetrahydro-1H-
1-benzazepin-6-yloxy)acetate (26)
4.34. 2,3-Dimethoxyaniline (30)
To a solution of 2,3-dimethoxyphenylcarbamic acid eth-
yl ester (29) (385 mg) in EtOH (10 mL) was added 5.48 N
NaOH (4.0 mL), and the mixture was stirred at 100 °C
for 9 h. The solvent was removed under reduced pres-
sure. The residue was added to 5% citric acid aqueous
solution and then extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium
sulfate, and concentrated. The residue was purified by
column chromatography (silica gel: n-hexane/ethyl
acetate = 3/1) to obtain compound 30 (244 mg, 93%).
By the procedure used in 21, compound 26 (47 mg, 67%)
was prepared from 52 mg of 25. H NMR (300 MHz,
1
CDCl3) d 1.55–1.67 (2H, m), 1.68–1.80 (2H, m), 2.51
(1H, br s), 2.93–2.98 (4H, m), 3.31 (2H, t, J = 5.1 Hz),
3.69 (2H, t, J = 5.1 Hz), 3.80 (3H, s), 4.62 (2H, s), 6.46
(1H, dd, J = 8.1, 0.9 Hz), 6.67 (1H, dd, J = 8.1,
0.9 Hz), 7.06 (1H, t, J = 8.1 Hz); IR (liquid film method)
1760 cmÀ1 (COOMe); Mass (EI, m/e) 279 (M+).
1
4.32. Methyl(1-(2-(1,1-diphenylethylthio)ethyl)-2,3,4,5-
tetrahydro-1H-1-benzazepin-6-yloxy)acetate (27)
Colorless oil; H NMR (300 MHz, CDCl3) d 3.82 (3H,
s), 3.84 (3H, s), 6.34 (1H, dd, J = 1.0, 8.0 Hz), 6.39
(1H, dd, J = 1.0, 8.0 Hz), 6.854 (1H, t, J = 8.0 Hz); Mass
(EI, m/e) 153 (M+).
To a solution of methyl(1-(2-hydroxyethyl)-2,3,4,5-tet-
rahydro-1H-1-benzazepin-6-yloxy)acetate (26) (370 mg)
in CH2Cl2 (15 mL) were added triphenylphosphine
(1085 mg) and carbon tetrabromide (922 mg), and
the mixture was stirred at 0 °C for 3.5 h. A saturated
sodium bicarbonate aqueous solution (15 mL) was
added to the reaction solution, and the mixture was
extracted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, and
concentrated to obtain the bromide of 24. To a solu-
tion of 1,1-diphenylethanethiol (428 mg) in DMF
4.35. 2,3-Dimethoxy-a-chloroacetoanilide (31)
To a solution of 2,3-dimethoxyaniline (30) (1.00 g) in
THF (5 mL) was added chloroacetic anhydride
(1.26 g), and the resulting solution was stirred at room
temperature for 4 h. The solvent was removed under re-
duced pressure, and the reaction solution was poured
into a saturated sodium bicarbonate solution, and then
extracted with ethyl acetate. The organic layer was

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2017
washed with brine, dried over magnesium sulfate, and
concentrated. The residue was purified by column chro-
matography (silica gel; ethyl acetate/n-hexane = 1/2) to
obtain compound 31 (1.64 g, 97%). Colorless flakes,
mp 58–59 °C (recrystallized from dichloromethane/n-
(15 mL) was added to sodium hydride (127 mg),
which was washed with n-hexane and dried under
reduced pressure, and the mixture was stirred at
room temperature for 10 min. A solution of 2-(2-
bromoethoxy)tetrahydropyran (935 mg) in DMF
(3.0 mL) was added, and the mixture was stirred at
room temperature for 5.5 h. The solvent was distilled
off under reduced pressure, and the residue was
poured into a 5% citric acid aqueous solution, and
then extracted with ethyl acetate. The organic layer
was washed with water and brine, dried over magne-
sium sulfate, and then concentrated. The residue was
dissolved in MeOH (50 mL), and p-toluenesulfonic
acid hydrate (160 mg) was added. The mixture was
stirred at room temperature for 2 h and the solvent
was distilled off under reduced pressure. The residue
was poured into 5% citric acid and then extracted
with ethyl acetate. The organic layer was washed
with water and brine, dried over magnesium sulfate,
and then concentrated. The residue was recrystallized
from ethyl acetate/n-hexane to obtain compound 34
(415 mg, 94%). Colorless plates, mp 166 °C; 1H
NMR (300 MHz, DMSO-d6) d 3.54 (2H, m), 3.90
(2H, t, J = 6 Hz), 4.55 (2H, s), 4.85 (1H, br), 6.55
(1H, dd, J = 1, 8 Hz), 6.70 (1H, dd, J = 1, 8 Hz),
6.70 (1H, dd, J = 1, 8 Hz), 6.82 (1H, t, J = 8 Hz),
9.42 (1H, br s); IR (KBr) 1669 cmÀ1 (amide); Mass
(EI, m/e) 209 (M+).
1
hexane); H NMR (300 MHz, CDCl3) d 3.88 (3H, s),
3.91 (3H, s), 4.20 (2H, s), 6.73 (1H, dd, J = 1.5,
8.5 Hz), 7.06 (1H, t, J = 8.5 Hz), 7.96 (1H, dd, J = 1.5,
8.5 Hz), 9.05 (1H, br s); Mass (EI, m/e) 229, 231 (M+)
(peak height is 3:1).
4.36. 8-Hydroxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazine
(32)
To a solution of 2,3-dimethoxy-a-chloroacetoanilide
(31) (1.43 g) in dichloromethane (30 mL) cooled to
À78 °C was added BBr3 in CH2Cl2 (1.0 M, 13.0 mL).
The mixture was stirred at 0 °C for 2 h, poured into
water, and then extracted with ethyl acetate. The organ-
ic layer was washed with water and brine, dried over
magnesium sulfate, and then concentrated. The residue
was dissolved in DMF (30 mL), and potassium carbon-
ate (1.03 g) was added. The mixture was stirred for 2.5 h
and the solvent was removed under reduced pressure.
The residue was poured into 5% citric acid and then
extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over magnesium sul-
fate, and then concentrated. The residue was recrystal-
lized from ethyl acetate to obtain compound 32
1
(922 mg, 90%). Colorless plates, mp 226 °C; H NMR
4.39. Methyl(4-(2-hydroxyethyl)-3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-8-yloxy)acetate (35)
(300 MHz, DMSO-d6) d 4.49 (2H, s), 6.34 (1H, dd,
J = 1, 8 Hz), 6.45 (1H, dd, J = 1, 8 Hz), 6.71 (1H, t,
J = 8 Hz), 9.37 (1H, br s), 10.57 (1H, br s); IR (KBr)
1682 cmÀ1 (CONH); Mass (EI, m/e) 165 (M+).
By the procedure used in 21, the compound 35 (280 mg,
97%) was prepared from 216 mg of 34. Colorless plates,
mp 106 °C (recrystallized from ethyl acetate/n-hexane);
1H NMR (300 MHz, CDCl3) d 2.13 (1H, br s), 3.80
(3H, s), 3.94 (2H, bt, J = 5.5 Hz), 4.13 (2H, t,
J = 5.5 Hz), 4.70 (2H, s), 4.72 (2H, s), 6.62 (1H, dd,
J = 1, 8 Hz), 6.80 (1H, dd, J = 1, 8 Hz), 6.96 (1H, t,
J = 8 Hz); IR (KBr) 1763 (COOMe), 1663 (amide)
cmÀ1; Mass (EI, m/e) 281 (M+).
4.37. 3-Oxo-8-(tetrahydropyran-2-yloxy)-3,4-dihydro-
2H-1,4-benzoxazine (33)
To a solution of 8-hydroxy-3-oxo-3,4-dihydro-2H-1,4-
benzoxazine (32) (740 mg) in DMF (1.5 mL) were
added dihydropyran (1.0 g) and pyridinium p-toluene-
sulfonate (360 mg), and the mixture was stirred at
room temperature for 18 h. The solvent was removed
under reduced pressure, and the residue was poured
into saturated sodium bicarbonate aqueous solution,
and then extracted with ethyl acetate. The organic
layer was washed with water, 5% citric acid, water,
and brine, dried over magnesium sulfate, and then
concentrated. The residue was recrystallized from eth-
yl acetate to obtain compound 33 (890 mg, 80%).
Colorless plates, mp 186 °C; 1H NMR (300 MHz,
DMSO-d6) d 1.54 (3H, m), 1.76 (3H, m), 3.52 (1H,
m), 3.79 (1H, m), 4.54 (2H s), 5.40 (1H, t,
J = 4 Hz), 6.54 (1H, dd, J = 1.5, 8 Hz), 6.76 (1H,
dd, J = 1.5, 8 Hz), 6.83 (1H, t, J = 8 Hz), 10.67
(1H, br s); IR (KBr) 1684 cmÀ1 (CONH); Mass
(EI, m/e) 249 (M+).
4.40. Methyl(4-(2-(1,1-diphenylethylthio)ethyl)-3-oxo-
3,4-dihydro-2H-1,4-benzoxazin-8-yloxy)acetate (36)
By the procedure used in 14, compound 36 (172.5 mg,
42%) was prepared from 187 mg of 35. Colorless oil;
1H NMR (300 MHz, CDCl3) d 2.08 (3H, s), 2.57
(2H, m), 3.79 (3H, s), 3.81 (2H, m), 4.59 (2H, s),
4.69 (2H, s), 6.13(1H, dd, J = 1, 8 Hz), 6.56 (1H, dd,
J = 1, 8 Hz), 6.81 (1H, t, J = 8 Hz), 7.21–7.34 (6H,
m), 7.40–7.45 (4H, m); IR (liquid film method) 1763
(COOMe), 1686 (amide) cmÀ1; Mass (EI, m/e) 477
(M+).
4.41. 4-(2-(Tetrahydropyran-2-yloxy)ethyl)-8-(tetrahy-
dro-pyran-2-yloxy)-3,4-dihydro-2H-1,4-benzoxazine (37)
4.38. 4-(2-Hydroxyethyl)-8-hydroxy-3-oxo-3,4-dihydro-
2H-1,4-benzoxazine (34)
To a solution of 3-oxo-8-tetrahydropyranyloxy-3,4-
dihydro-2H-1,4-benzoxazine (33) (6.04 g) in anhydrous
DMF (100 mL) was added sodium hydride (1.02 g),
and the mixture was stirred at room temperature for
1 h. 2-(2-Bromoethoxy)tetrahydropyrane (7.60 g) was
A solution of 3-oxo-(8-tetrahydropyrane-2-yloxy)-3,4-
dihydro-2H-1,4-benzoxazine (33) (529 mg) in DMF

2018
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
added to the reaction mixture, and the resulting mixture
was stirred at room temperature for 17.5 h. The solvent
was removed under reduced pressure, and the residue
was poured into a 5% citric acid aqueous solution, and
then extracted with ethyl acetate. The organic layer
was washed with water and brine, dried over magnesium
sulfate, and then concentrated. The residue was dis-
solved in THF (150 mL), and a 1.0 M BH3–THF solu-
tion (60 mL) was added, and the mixture was stirred
at room temperature for 2 h. The reaction mixture was
added to saturated sodium bicarbonate aqueous solu-
tion and extracted with ethyl acetate. The organic layer
was washed with water and brine, dried over magnesium
sulfate, and then concentrated. The residue was purified
by column chromatography (silica gel; ethyl acetate/n-
hexane = 1/2) to obtain compound 37 (7.01 g, 93%).
4.44. General procedure for coupling with thiols; meth-
yl(4-(2-(1,1-diphenylethylthio)ethyl)-3,4-dihydro-2H-1,4-
benzoxazin-8-yloxy)acetate (40a)
To a solution of methyl(4-(2-hydroxyethyl)-3,4-dihydro-
2H-1,4-benzoxazin-8-yloxy)acetate (39) (15.7 g) in
CH2Cl2 (360 mL) were added Et3N (29.5 mL) and meth-
anesulfonyl chloride (14.6 mL), and the mixture was
stirred at 0 °C for 1 h. The reaction mixture was poured
into a 5% citric acid aqueous solution and then extracted
with ethyl acetate. The organic layer was washed with
water, saturated sodium bicarbonate water, and water
and brine, dried over magnesium sulfate, and then con-
centrated to obtain mesylate of 39 (17.8 g). To sodium
hydride (2.82 g), which was washed with n-hexane, was
added a solution of 1,1-diphenylethanethiol (15.1 g) in
DMF (1000 mL) and the mixture was stirred at room
temperature for 1 h. A solution of the mesylate of 39
above (17.8 g) in DMF (100 mL) was added to the reac-
tion mixture, and the resulting mixture was stirred at
room temperature for 18 h. The solvent was removed
under reduced pressure, and the residue was poured into
a 5% citric acid aqueous solution, and then extracted
with ethyl acetate. The organic layer was washed with
water and brine, dried over magnesium sulfate, and con-
centrated. The residue was purified by column chroma-
tography (neutral alumina: methyl acetate/n-hexane = 1/
3) to obtain compound 40a (19.7 g, 83%). Colorless
prisms, mp 101 °C (recrystallized from ethyl acetate/n-
1
Colorless oil; H NMR (300 MHz, CDCl3) d 1.50–2.10
(12H, m), 3.47 (5H, m), 3.62 (2H, m), 3.82 (1H, m),
3.91 (1H, m), 4.03 (1H, m), 4.25 (2H, t, J = 4.5 Hz),
4.59 (1H, t, J = 3 Hz), 5.36 (1H, t, J = 3 Hz), 6.41 (1H,
dd, J = 1, 8 Hz), 6.52 (1H, dd, J = 1, 8 Hz), 6.72 (1H,
t, J = 8 Hz); Mass (EI, m/e) 363 (M+).
4.42. 4-(2-Hydroxyethyl)-8-hydroxy-3,4-dihydro-2H-1,4-
benzoxazine (38)
To a solution of 4-(2-(tetrahydropyran-2-yloxy)ethyl)-8-
(tetrahydropyran-2-yloxy)-3,4-dihydro-2H-1,4-benzox-
azine (37) (3.89 g) in methanol (80 mL) was added
pyridinium p-toluenesulfonate (520 mg), and the mix-
ture was stirred at room temperature for 1.5 h. The sol-
vent was removed under reduced pressure, and the
residue was poured into 5% citric acid, and then extract-
ed with ethyl acetate. The organic layer was washed with
water and brine, dried over magnesium sulfate, and then
concentrated. The residue was recrystallized from ethyl
acetate/n-hexane to obtain compound 38 (2.00 g, 97%).
Colorless plates, mp 105.5 °C; 1H NMR (300 MHz,
DMSO-d6) d 3.26 (1H, t, J = 5.5 Hz), 3.36 (2H, t,
J = 4 Hz), 3.54 (2H, q, J = 5.5 Hz), 4.10 (2H, t,
J = 4 Hz), 4.65 (1H, t, J = 5.5 Hz), 6.07 (1H, dd, J = 1,
8 Hz), 6.15 (1H, dd, J = 1, 8 Hz), 6.49 (1H, t,
J = 8 Hz), 8.49 (1H, s); Mass (EI, m/e) 195 (M+).
1
hexane); H NMR (300 MHz, CDCl3) d 2.06 (3H, s),
2.51 (2H, t, J = 8.0 Hz), 3.13 (2H, t, J = 8.0 Hz), 3.19
(2H, t, J = 4.5 Hz), 3.78 (3H, s), 4.22 (2H, t,
J = 4.5 Hz), 4.65 (2H, s), 6.01 (1H, dd, J = 1.0,
8.0 Hz), 6.19 (1H, dd, J = 1.0, 8.0 Hz), 6.62 (1H, t,
J = 8.0 Hz), 7.21–7.44 (10H, m); IR (KBr) 1765 cmÀ1
(COOMe); Mass (EI, m/e) 463 (M+).
4.45. Methyl(4-(2-(1,1-diphenyl-2,2,2-trifluoroethylthio)-
ethyl)-3,4-dihydro-2H-1,4-benzoxazin-8-yloxy)acetate
(40b)
By the procedure used in 40a, compound 40b (614 mg,
85%) was prepared from 375 mg of 39. Pale yellow oil;
1H NMR (300 MHz, CDCl3) d 2.55 (2H, t, J = 7 Hz),
3.17 (4H, m), 3.78 (3H, s), 4.21 (2H), 4.64 (2H, s),
5.94 (1H, dd, J = 1, 8 Hz), 6.19 (1H, dd, J = 1, 8 Hz),
6.60 (1H, t, J = 8 Hz), 7.31–7.36 (6H, m), 7.42–7.46
(4H, m); IR (liquid film method) 1765 cmÀ1 (COOMe);
Mass (EI, m/e) 517 (M+).
4.43. Methyl(4-(2-hydroxyethyl)-3,4-dihydro-2H-1,4-
benzoxazin-8-yloxy)acetate (39)
To a solution of 4-(2-hydroxyethyl)-8-hydroxy-3,4-dihy-
dro-2H-1,4-benzoxazine (38) (17.3 g) in DMF (200 mL)
were added anhydrous potassium carbonate (30.6 g) and
methyl bromoacetate (24.1 g), and the mixture was stir-
red at room temperature for 5.5 h. The solvent was dis-
tilled off, and the residue was poured into 5% citric acid,
and then extracted with ethyl acetate. The organic layer
was washed with water and brine, dried over magnesium
sulfate, and then concentrated. The residue was recrys-
tallized from ethyl acetate/n-hexane to obtain compound
39 (17.6 g, 74%). Colorless plates, mp 64–66 °C; 1H
NMR (300 MHz, CDCl3) d 1.78 (1H, t, J = 5.5 Hz),
3.42 (4H, m), 3.79 (3H, s), 3.83 (2H, q, J = 5.5 Hz),
4.31 (2H, m), 4.68 (2H, s), 6.26 (1H, dd, J = 1, 8 Hz),
6.47 (1H, dd, J = 1, 8 Hz), 6.73 (1H, t, J = 8 Hz); IR
(KBr) 1719 cmÀ1 (COOMe); Mass (EI, m/e) 267 (M+).
4.46. Methyl(4-(2-(diphenylmethylthio)ethyl)-3,4-dihy-
dro-2H-1,4-benzoxazin-8-yloxy)acetate (40c)
By the procedure used in 40a, compound 40c (476 mg,
81%) was prepared from 347 mg of 39. Colorless
plates, mp 94–95 °C (recrystallized from ethyl ace-
tate/n-hexane); 1H NMR (300 MHz, CDCl3) d 2.61
(2H, m), 3.26 (2H, m), 3.36 (2H, m), 3.78 (3H, s),
4.24 (2H, m), 4.65 (2H, s), 5.24 (1H, s), 6.06 (1H,
dd, J = 1, 8 Hz), 6.20 (1H, dd, J = 1, 8 Hz), 6.61
(1H, t, J = 8 Hz), 7.26–7.31 (6H, m), 7.41–7.46 (4H,
m); IR (KBr) 1763 cmÀ1 (COOMe); Mass (EI, m/e)
449 (M+).

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2019
4.47. Methyl(4-(2-(1-methyl-1-phenylethylthio)ethyl)-3,4-
dihydro-2H-1,4-benzoxazin-8-yloxy)acetate (40d)
s), 5.66 (1H, br), 6.08 (1H, br), 6.37 (1H, br), 7.00
(6H, m), 7.23 (4H, m); Mass (FAB-Pos, m/e) 449
(M+H)+, 472 (M+H+Na)+; Elemental analysis Calcd.
for C26H26NNaO4S + 1.5H2O: C, 62.64; H, 5.86; N,
2.81; S, 6.43. Found: C, 62.35; H, 5.77; N, 3.02; S, 6.59.
By the procedure used in 40a, compound 40d (483 mg,
94%) was prepared from 343 mg of 39. Pale yellow oil;
1H NMR (300 MHz, CDCl3) d 1.72 (6H, s), 2.42 (2H,
t, J = 8 Hz), 3.09 (2H, t, J = 8 Hz), 3.17 (2H, t,
J = 4.5 Hz), 3.78 (3H, s), 4.20 (2H, t, J = 4.5 Hz), 4.64
(2H, s), 5.94(1H, dd, J = 1, 8 Hz), 6.17 (1H, dd, J = 1,
8 Hz), 6.60 (1H, t, J = 8 Hz), 7.24 (1H, m), 7.34 (2H,
m), 7.54 (2H, m); IR (liquid film method) 1765 cmÀ1
(COOMe); Mass (EI, m/e) 401 (M+).
4.52. 8-Acetoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazine
(42)
To a solution of 8-hydroxy-3-oxo-3,4-dihydro-2H-1,4-
benzoxazine (32) (10.56 g) in toluene (30 mL) and pyri-
dine (6 mL) was added acetic anhydride (7.09 mL),
and the mixture was stirred for 24 h at room tempera-
ture. The reaction solution was poured into a 1 N
hydrochloric acid aqueous solution and then extracted
with ethyl acetate. The organic layer was washed with
water and brine, dried over sodium sulfate, and then
concentrated. The residue was recrystallized from ethyl
acetate/n-hexane to obtain compound 42 (12.5 g, 94%).
Colorless needles, mp 227 °C; 1H NMR (300 MHz,
CDCl3) d 2.33 (3H, s), 4.62 (2H, s), 6.70 (1H, dd,
J = 8.0, 1.5 Hz), 6.77 (1H, dd, J = 8.0, 1.5 Hz), 6.96
(1H, t, J = 8.0 Hz), 7.89 (1H, br s); IR (KBr) 1767
(OAc), 1693 (CONH) cmÀ1; Mass (EI, m/e) 207 (M+).
4.48. Methyl(4-(2-(2,2-diphenylpropylthio)ethyl)-3,4-
dihydro-2H-1,4-benzoxazin-8-yloxy)acetate (40f)
By the procedure used in 40a, compound 40f (134 mg,
37%) was prepared from 200 mg of 39. Colorless oil;
1H NMR (300 MHz, CDCl3) d 1.78 (3H, s), 2.36 (2H,
t, J = 7.6 Hz), 3.24–3.35 (6H, m), 3.79 (3H, s), 4.24
(2H, t, J = 4.4 Hz), 4.66 (2H, s), 6.22(1H, d,
J = 8.5 Hz), 6.27 (1H, d, J = 8.5 Hz), 6.70 (1H, t,
J = 8.5 Hz), 7.17–7.32 (10H, m); IR (liquid film method)
1765 cmÀ1 (COOMe); Mass (EI, m/e) 477 (M+).
4.49. Methyl(4-(2-(1,1-bis-(4-fluorophenyl)ethylthio)eth-
yl)-3,4-dihydro-2H-1,4-benzoxazin-8-yloxy)acetate (40g)
4.53. 8-Acetoxy-4-(2-(1,1-diphenylethoxy)ethyl)-3-oxo-
3,4-dihydro-2H-1,4-benzoxazine (43a)
By the procedure used in 40a, compound 40g (311 mg,
83%) was prepared from 200 mg of 39. Colorless oil;
1H NMR (300 MHz, CDCl3) d 2.02 (3H, s), 2.50 (2H,
t, J = 7.6 Hz), 3.21–3.35 (6H, m), 3.79 (3H, s), 4.24
(2H, t, J = 4.4 Hz), 4.66 (2H, s), 6.22 (1H, d,
J = 8.5 Hz), 6.27 (1H, d, J = 8.5 Hz), 6.70 (1H, t,
J = 8.5 Hz), 7.17–7.32 (10H, m); IR (liquid film method)
1763 cmÀ1 (COOMe); Mass (EI, m/e) 499 (M+).
To a solution of 2-(1,1-diphenylethoxy)ethanol (606 mg)
in THF (10 mL) was added n-butyllithium hexane solu-
tion (1.47 M, 2.6 mL), and the mixture was stirred for
10 min at À40 °C. p-Toluenesulfonyl chloride (715 mg)
was added to the solution, and the mixture was stirred
for 1.5 h at À40 °C. The reaction mixture was poured into
water and then extracted with ethyl acetate. The organic
layer was washed with water and brine, dried over sodium
sulfate, and concentrated to obtain tosylate of 2-(1,1-di-
phenyl-ethoxy)ethanol. To a suspension of sodium hy-
dride (84 mg) in DMF (5 mL) was added a solution of
8-acetoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazine (42)
(363 mg) in DMF (5 mL), and the mixture was stirred at
room temperature for 30 min. The tosylate above was
added, and the mixture was stirred overnight at room tem-
perature. The reaction solution was poured into a 5% cit-
ric acid aqueous solution and then extracted with ethyl
acetate. The organic layer was washed with water and
brine, dried over sodium sulfate, and then concentrated.
The residue was purified by column chromatography (sol-
vent: ethyl acetate/cyclohexane = 1/4) to obtain com-
pound 43a (429 mg, 57%). Colorless oil; 1H NMR
(300 MHz, CDCl3) d 1.81 (3H, s), 2.32 (3H, s), 3.52 (2H,
t, J = 5.8 Hz), 4.12 (2H, t, J = 5.8 Hz), 4.53 (2H, s), 6.77
(1H, dd, J = 8.2, 1.4 Hz), 6.94 (1H, t, J = 8.2 Hz), 7.04
(1H, dd, J = 8.2, 1.4 Hz), 7.18–7.29 (10H, m); IR (KBr)
1773 (OAc), 1688 (amide) cmÀ1; Mass (EI, m/e) 431 (M+).
4.50. Methyl(4-(2-(1,1-bis-(3-thienyl)ethylthio)ethyl)-3,4-
dihydro-2H-1,4-benzoxazin-8-yloxy)acetate (40h)
By the procedure used in 40a, compound 40g (253 mg,
95%) was prepared from 150 mg of 39. Colorless oil;
1H NMR (300 MHz, CDCl3) d 2.05 (3H, s), 2.51–2.58
(2H, m), 3.16 (2H, t, J = 7.7 Hz), 3.23 (2H, t,
J = 4.4 Hz), 3.79 (3H, s), 4.23 (2H, t, J = 4.4 Hz), 4.65
(2H, s), 6.07(1H, dd, J = 8.2, 1.1 Hz), 6.20 (1H, dd,
J = 8.2, 1.1 Hz), 6.66 (1H, t, J = 8.2 Hz), 7.11 (2H, dd,
J = 3.0, 1.4 Hz), 7.14 (2H, dd, J = 5.2, 1.4 Hz), 7.29
(2H, dd, J = 5.2, 3.0 Hz); IR (liquid film method)
1767 cmÀ1 (COOMe); Mass (EI, m/e) 475 (M+).
4.51. (4-(2-(1,1-Diphenylethylthio)ethyl)-3,4-dihydro-2H-
1,4-benzoxazin-8-yloxy)acetic acid sodium salt (41)
(4-(2-(1,1-Diphenylethylthio)ethyl)-3,4-dihydro-2H-1,4-
benzoxazin-8-yloxy)acetic acid 10a (510 mg) was dis-
solved in distilled water (10 mL) and sodium hydroxide
aqueous solution (0.0978 N, 11.54 mL) with heating,
and the solution was filtered with a membrane filter.
The filtrate was lyophilized to obtain the object com-
pound (535 mg, 100%). Colorless amorphous, mp
4.54. 8-Acetoxy-4-(4,4-diphenylpentyl)-3-oxo-3,4-dihy-
dro-2H-1,4-benzoxazine (43b)
By the procedure used in 43a, compound 43b (428 mg,
61%) was prepared from 391 mg of 4,4-diphenylpen-
tane-1-ol and 507 mg of 42. Colorless oil; 1H NMR
(300 MHz, CDCl3) d 1.42–1.55 (4H, m), 1.61 (3H, s),
1
110 °C; H NMR (300 MHz, D2O, 30 °C) d 1.79 (3H,
s), 2.24 (2H, m), 2.75 (4H, m), 3.86 (2H, m), 4.23 (2H,

2020
M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2.14–2.24 (2H, m), 2.31 (3H, s), 3.83 (2H, t, J = 7.7 Hz),
4.55 (2H, s), 6.46 (1H, dd, J = 8.2, 1.4 Hz), 6.74 (1H, dd,
J = 8.2, 1.4 Hz), 6.87 (1H, t, J = 8.2 Hz), 7.13–7.21 (6H,
m), 7.21–7.30 (4H, m); Mass (EI, m/e) 429 (M+).
(3H, s), 2.08–2.16 (2H, m), 3.15 (2H, t, J = 7.4 Hz),
3.19 (2H, t, J = 4.4 Hz), 3.79 (3H, a), 4.24 (2H, t,
J = 4.4 Hz), 4.66 (2H, s), 6.19 (1H, dd, J = 8.2,
1.4 Hz), 6.24 (1H, dd, J = 8.2, 1.4 Hz), 6.66 (1H, t,
J = 8.2 Hz), 7.14–7.21 (6H, m), 7.23–7.30 (4H, m); IR
(liquid film method) 1763 cmÀ1 (COOMe); Mass (EI,
m/e) 445 (M+).
4.55. 4-(2-(1,1-Diphenylethoxy)ethyl)-8-hydroxy-3,4-
dihyro-2H-1,4-benzoxazine (44a)
To a solution of 8-acetoxy-3,4-(2-(1,1-diphenylethoxy)-
ethyl)-3-oxo-3,4-dihydro-2H-1,4-benzoxazine
4.59. Blood samples
(43a)
(386 mg) in THF (10 mL) was added BH3–THF solution
(1.0 M, 3 mL), and the resulting mixture was stirred at
room temperature for 4 h. The reaction solution was
poured into water and extracted with ethyl acetate.
The organic layer was washed with water, sodium bicar-
bonate water, and water and brine, dried over sodium
sulfate, and then concentrated. The obtained oily resi-
due was dissolved in THF (1 mL) and MeOH (10 mL),
and anhydrous potassium carbonate (170 mg) was add-
ed. The mixture was stirred at room temperature for 1 h
and the solvent was removed under reduced pressure.
The residue was poured into 5% citric acid and then
extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over sodium sulfate,
and concentrated. The residue was purified by column
chromatography (solvent: ethyl acetate/cyclohex-
ane = 1/5) to obtain compound 44a (339 mg, 99%). Col-
Blood samples were collected from healthy human vol-
unteers under the approval by the Institutional Ethics
Committee of the Pharmaceutical Research Laborato-
ries, Toray Industries, Inc. Written informed consent
was obtained from each of the volunteers. The volun-
teers did not take any drugs at least within 2 weeks be-
fore their participation in this study. Blood samples were
also collected from male cynomolgus monkeys (Japan
SLC, Shizuoka, Japan) in accordance with the Guide-
lines for the Animal Care and Use established at the
Pharmaceutical Research Laboratories, Toray Indus-
tries, Inc.
4.60. Platelet aggregation in PRP
Nine volumes of blood collected from human volun-
teers were mixed with one volume of 3.8% sodium cit-
rate in a tube. The citrated blood samples were
immediately centrifuged at 90–140g for 10 min at room
temperature. The resulting supernatant was used as the
PRP fraction. The remaining blood was further centri-
fuged at 1400g for 10 min. The resulting supernatant
was used as the platelet-poor plasma fraction. Human
PRP were pretreated with compound at various con-
centrations for 1 min before the addition of U-46619
(2 lM), arachidonic acid (600 lM), collagen (1 lg/
mL) or ADP (5 lM). The platelet stimulation with
ADP was carried out in the presence or absence of
SQ-29548 (10 lM). Platelet aggregation was monitored
by recording transmittance on a four-channel light
transmission aggregometer (NBS HematracerÒ 601,
MC Medical, Japan) for 5 min after the addition of a
platelet-stimulating agent. For evaluating the effect of
the test drugs, percent inhibitions of platelet aggrega-
tion were calculated from the increases in transmittance
observed with the test drugs (N = 3), on the assump-
tion that no inhibition was observed in the control
incubations of PRP with vehicle alone. And the optical
density of platelet-poor plasma was taken to represent
100% aggregation.
1
orless oil; H NMR (300 MHz, CDCl3) d 1.83 (3H, s),
3.40–3.52 (6H, m), 4.25 (2H, t, J = 4.5 Hz), 5.38 (1H,
s), 6.11 (1H, dd, J = 8.1, 1.4 Hz), 6.30 (1H, dd, J = 8.1,
1.4 Hz), 6.63 (1H, t, J = 8.1 Hz), 7.17–7.36 (10H, m);
Mass (EI, m/e) 375 (M+).
4.56. 4-(4,4-Diphenylpentyl)-8-hydroxy-3-oxo-3,4-dihy-
dro-2H-1,4-benzoxazine (44b)
By the procedure used in 44a, compound 44b (391 mg,
99%) was prepared from 424 mg of 43b. Colorless oil;
1H NMR (300 MHz, CDCl3) d 1.36–1.48 (2H, m), 1.63
(3H, s), 2.08–2.16 (2H, m), 3.15 (2H, t, J = 7.3 Hz),
3.19 (2H, t, J = 4.4 Hz), 4.22 (2H, t, J = 4.4 Hz), 5.36
(1H, s), 6.10 (1H, dd, J = 8.1, 1.1 Hz), 6.31 (1H, dd,
J = 8.1, 1.1 Hz), 6.66 (1H, t, J = 8.1 Hz), 7.14–7.21
(6H, m), 7.23–7.29 (4H, m); Mass (EI, m/e) 373 (M+).
4.57. Methyl(4-(2-(1,1-diphenylethoxy)ethyl)-3,4-dihy-
dro-2H-1,4-benzoxazin-8-yloxy)acetate (45a)
By the procedure used in 39, compound 45a (229 mg,
80%) was prepared from 239 mg of 44a. Colorless oil;
1H NMR (300 MHz, CDCl3) d 1.83 (3H, s), 3.40–3.51
(6H, m), 3.79 (3H, s), 4.27 (2H, t, J = 4.4 Hz), 4.67
(2H, s), 6.19 (1H, dd, J = 8.2, 1.1 Hz), 6.27 (1H, dd,
J = 8.2, 1.1 Hz), 6.64 (1H, t, J = 8.2 Hz), 7.17–7.34
(10H, m); IR (liquid film method) 1763 cmÀ1 (COOMe);
Mass (EI, m/e) 447 (M+).
4.61. Binding assay for TP and IP receptor in human
platelet membrane
Platelet-rich plasma (PRP), prepared above, was washed
twice in washing buffer, pH 6.5, containing 115 mM
NaCl, 4.3 mM K2HPO4, 24.4 mM Na2HPO4, 5 mM glu-
cose, 1 mM EDTA-2Na, and 0.01 mM indomethacin,
and resuspended in 10 mM Tris buffer, pH 7.4, contain-
ing 5 mM MgCl2 and 2 mM EDTA-2Na. The platelets
were alternately frozen and thawed three times and then
centrifuged at 40,000g for 20 min at 4 °C. The mem-
brane preparation was resuspended at 4 °C in assay buff-
4.58. Methyl(4-(4,4-diphenylpentyl)-8-hydroxy-3-oxo-
3,4-dihydro-2H-1,4-benzoxazin-8-yloxy)acetate (45b)
By the procedure used in 39, compound 45b (386 mg,
92%) was prepared from 353 mg of 44b. Colorless oil;
1H NMR (300 MHz, CDCl3) d 1.35–1.47 (2H, m), 1.63

M. Ohno et al. / Bioorg. Med. Chem. 14 (2006) 2005–2021
2021
er, pH 7.4, containing 50 mM Tris and 5 mM MgCl2,
and stored at À80 °C. For TP receptor binding assay,
human platelet membrane (10 lg protein) was incubated
in assay buffer in the presence of the selective TP recep-
tor antagonist, [3H]SQ-29548, and 7, for 30 min at
25 °C. For IP receptor binding assay, human platelet
membrane (10 lg protein) was incubated in assay buffer
in the presence of the selective IP receptor agonist,
[3H]APS-314d sodium, and 7, for 60 min at 4 °C. The
reaction mixture was separated bound and free radiola-
beled ligand by rapid filtration through GF/C filters
presoaked in 10 mM Tris–HCl buffer. Filters were
washed and the residual [3H]SQ-29548 or [3H]APS-
314d sodium bound to the filter was determined by li-
quid scintillation counting. Specific binding was defined
as the difference between total binding and non-specific
binding, which was determined in the presence of 10 lM
SQ-29548 or 10 lM APS-314d sodium. Ki was calculat-
ed using the equation Ki = 1C50/(1 + L/Kd), where L is
the concentration of ligand.
We thank Dr. K. A. Jacobson (NIDDK, NIH, MD) for
helpful discussion and proofreading.
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Cynomolgus monkeys were anesthetized with sodium
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4.63. Statistics
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We dedicate this publication to Dr. Kiyotaka Ohno
(Toray, Kamakura). We thank Dr. Katsuhiko Iseki
(Toray, Kamakura) for helpful discussion. We thank
Prof. K. Umemura (Hamamatsu University School of
Medicine, Hamamatsu) and Dr. H. Tsukada (Hamama-
tsu Photnics K. K., Hamakita) for providing us with the
pharmacological data employing positron emission
tomography. We thank Dr. Masafumi Isogaya and
Mika Nukaya for providing us with the biological data.