Design and synthesis of new prostaglandin D2 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2011.08.007

To identify new cost-effective prostaglandin D₂ (DP) receptor antagonists, a series of novel 3-benzoylaminophenylacetic acids were synthesized and biologically evaluated. Among those tested, some representative compounds were found to be orally

Full text of DOI:10.1016/j.bmc.2011.08.007

Bioorganic & Medicinal Chemistry 19 (2011) 5361–5371
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of new prostaglandin D2 receptor antagonists
Maki Iwahashi a, , Eiji Takahashi b, Motoyuki Tanaka a, Yoko Matsunaga a, Yutaka Okada a,
⇑
Ryoji Matsumoto c, Fumio Nambu a, Hisao Nakai a, Masaaki Toda a
a Minase Research Institute, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
b Quality Assurance Headquarters, Ono Pharmaceutical Co., Ltd, 17-2 Wadai Tsukuba, Ibaraki 300-4247, Japan
c Ono Pharmaceutical Co., Ltd, 2-1-5 Doshomachi, Chuoh, Osaka 541-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify new cost-effective prostaglandin D2 (DP) receptor antagonists, a series of novel 3-benzoylam-
inophenylacetic acids were synthesized and biologically evaluated. Among those tested, some represen-
tative compounds were found to be orally available. Receptor selectivity and rat PK profiles were also
evaluated. The structure–activity relationship (SAR) study is presented.
Received 13 June 2011
Revised 1 August 2011
Accepted 2 August 2011
Available online 8 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Prostaglandin
DP receptor
Antagonist
Phenylacetic acid
1. Introduction
affinity and the functional activity assays. The optimization process
of a new cost-effective chemical lead 2a is presented in this report.
Cyclooxygenase metabolizes arachidonic acid to five primary
prostanoids PGE2, PGF2 , PGI2, TXA2 and PGD2. Coleman et al.1 pro-
a
2. Materials and methods
posed the existence of specific receptors for TX, PGI, PGE, PGF, and
PGD named TP, IP, EP, FP, and DP receptors, respectively. Prosta-
glandin D2 (PGD2) is the major prostanoid released from mast cells
after challenge with IgE.2 PGD2 receptor antagonists might have
therapeutic potential for allergic disorders because PGD2 is consid-
ered to play an important role in various allergic diseases such as
allergic rhinitis,3 atopic asthma,2b allergic conjunctivitis,4 and
atopic dermatitis.5
Compounds listed in Tables 1–3 were synthesized as outlined
in Schemes 1–4. Methyl 3-aminophenylacetates 12a–c, 12f, 12h
and 12i were prepared as described in Scheme 1. Compounds
12d–e, 12g, 12j, and 13b were commercially available. Nucleo-
philic substitution of 3-nitrophenylmethyl bromide 7 with sodium
cyanide afforded 8, acidic hydrolysis of which followed by esteri-
fication with diazomethane provided 10a. Catalytic hydrogenation
of methyl esters 10a–c afforded the corresponding anilines 12f
and 12h–i, respectively. Other amino esters 12a–c were prepared
from the corresponding carboxylic acids 11a–c, respectively.
N-Acetylation of 12e with acetic anhydride in pyridine followed
by reduction with diborane-dimethyl sulfide complex afforded
methyl 3-ethylaminophenylacetate 13a. Synthesis of 9b is out-
lined in Scheme 1b. Sonogashira cross coupling reaction of 14
and 15 afforded 16, hydroboration of which followed by the treat-
ment with alkaline hydrogen peroxide afforded a phenylacetic
acid 9b. Synthesis of 2a, 3a–b, 4a–b, 5a–i, and 6a–g is described
in Scheme 2. N-Acylation of an optional aniline selected from
12a–j and 13a–b listed in Scheme 1a with 17–197 afforded 2a,
3a–b, 4a–b, 5a–i and 6a–g. Synthesis of a sulfonamide analog
2b was prepared from 12e as outlined in Scheme 3. N-Sulfonyla-
tion of 12e with 4-acetoxybenzenesulfonyl chloride 228 in the
presence of pyridine afforded 20, methanolysis of which with
potassium carbonate in methanol resulted in 21. O-Alkylation of
In our previous work,6 we reported on the discovery process of
N-benzoyl-2-methylindole-4-acetic acid analog 1 (Fig. 1) as an
optimized DP receptor antagonist starting with chemical modifica-
tion of indomethacin. Synthetic study of this new scaffold ‘2-meth-
ylindole-4-acetic acid’ strongly suggested the necessity of a more
cost-effective framework on which to build a drug candidate. As
well, the low hIP/DP receptor selectivity of 1 (Table 1) was consid-
ered a risk factor that might cause serious side effects such as
platelet aggregation. Thus, the molecular design of a new DP recep-
tor antagonist was expanded. As described in Figure 1, 3-amin-
ophenylacetic acid was selected as the new scaffold instead of 2-
methylindole-4-acetic acid because of the moderate potency of
3-benzoylaminophenylacetic acid 2a in terms of both the receptor
⇑
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: iwahashi@ono.co.jp (M. Iwahashi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.007

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M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
CO2H
CO2H
Me
N
NH
O
O
O
O
O
O
N
N
Me
Me
1
2a
Figure 1. Molecular design of a phenylacetic acid analog 2a.
Table 1
Effect of the linker X between rings A and B on activity profiles
CO2H
X
A
B
O
O
I
N
Me
a
Compd
X
Binding Ki (nM)
mEP4
IC50 (nM)
mDP
mEP1
6200
mEP2
99
mEP3
1700
hIP
37
mDP
16
1
>10,000
2.4
NH
2a
2100
520
>10,000
210
>10,000
6400
4600
310
220
NTb
NTb
O
O
NH
S
O
2b
2c
2700
2300
3500
140
430
O
>10,000
>10,000
>10,000
>10,000
5300
HN
a
IC50 (nM): mDP receptor antagonist activity.
NT: not tested.
b
Table 2
Effect of N-alkylation on the activity profiles
CO2H
R
N
O
O
O
N
Me
a
Compd
R
Binding Ki (nM)
mEP3
IC50 (nM)
mDP
mEP1
mEP2
mEP4
hIP
mDP
2a
3a
3b
H
Me
Et
2100
>10,000
1300
520
5300
>10,000
>10,000
1700
700
>10,000
>10,000
>10,000
6400
>10,000
2900
310
760
770
220
NTb
NTb
a
IC50 (nM): mDP receptor antagonist activity.
NT: not tested.
b
21 with the tosylate 237 in the presence of cesium carbonate
followed by alkaline hydrolysis produced 2b. Synthesis of 2c is
outlined in Scheme 4. O-Alkylation of the commercially available
p-nitrophenol 24 with the tosylate 237 in the presence of cesium

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
5363
Table 3
3. Results and discussion
Effect of substitution of rings A and B on the activity profiles
CO2H
The test compounds listed in Tables 1–3 were all tested for inhi-
bition of the specific binding of a radiolabeled ligand, [3H]PGD2, to
membrane fractions prepared from cells stably expressing mDP
receptor. They were also evaluated for their potency to antagonize
mDP receptors by measuring PGD2-stimulated changes in intracel-
lular second messenger cAMP (cyclic adenosine 3’, 5’-monophos-
phate) as an indicator of receptor function. The mDP antagonism
was measured in the presence of 0.1% BSA to mimic the protein-
rich environment of in vivo animal models. Because of their close
homology to human receptors, all the DP receptor affinities and
antagonist activities are assayed using the equivalent mouse
receptors unless otherwise noted.
2
6
A
NH
5
X
6
4
5
O
B
4
O
Y
O
2
II
3
N
Me
Compd
X
Y
mDP
a
Binding Ki (nM)
IC50 (nM)
Table 1 describes the SAR of the linkers between the rings A and
B of the general formula I. 3-Benzoylaminophenylacetic acid ana-
log 2a, which was designed based on the molecular design de-
scribed in Figure 1, exhibited moderate potency in both the
receptor affinity and the antagonist activity assays while the corre-
sponding retro-amide analog 2c showed 17-fold less potent affin-
ity in the binding assay. The corresponding sulfonamide analog
2b showed equipotent binding affinity while it did not show DP
receptor selectivity because of its increased affinity for both the
EP3 and EP4 receptors.
As shown in Table 2, N-alkyl amide analogs of 2a were prepared
and evaluated. N-Methylation and N-ethylation of 2a resulted in N-
methyl amide analog 3a and N-ethyl amide analog 3b, respectively.
Both the analogs 3a and 3b tended to have slightly less potent
receptor affinity for the DP receptor. Among the tested compounds,
3-benzoylaminophenylacetic acid analog 2a was found to have the
most potent antagonist activity. On the basis of the information de-
scribed above, 2a was used as a chemical lead for further optimiza-
tion. Accordingly, the following optimization was carried out using
3-benzoylaminophenylacetic acid as a scaffold.
2a
4a
4b
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
H
H
H
2-Me
4-Me
5-Me
6-Me
4-Cl
4-F
5-F
6-F
6-OMe
4-Me
5-Me
6-Me
4-Cl
H
Me
Cl
310
18
16
59
31
8.4
11
7.7
19
7.4
15
24
26
12
17
13
17
25
36
220
3.3
20
Me
Me
Me
Me
Me
Me
Me
Me
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
28
4.6
3.4
6.1
NTb
1.8
5.6
1.2
20
6.0
5.4
3.9
4.3
7.4
50
4-F
6-F
6-OMe
6g
41
a
IC50 (nM): mDP receptor antagonist activity.
NT: not tested.
b
On the basis of the SAR study described in Tables 1 and 2, the
sulfonamide and retro-amide moieties were found to show no
selectivity for the DP receptor as illustrated by the results for 2b
and 2c, respectively. The secondary anilide moiety was found to
be more beneficial than the tertiary amide moiety as a linker be-
tween the rings A and B as illustrated by the results of 2a and
3a–b. N-Methyl-N-phenylbenzamides have been reported to
carbonate afforded 25, catalytic hydrogenation of which afforded
the corresponding aniline 26. N-Acylation of 26 with an acid
chloride 28, which was prepared from 3-methoxycarbonylmethyl-
benzoic acid 27 and oxalyl chloride followed by alkaline hydroly-
sis resulted in 2c.
CO2Me
R
R
d
e, f
Et
N
H
NO2
NH2
X
X
11a : R = CO2H
12a : R = CO2Me
11b : R = CO2H
12b : R = CO2Me
11c : R = CO2H
12c : R = CO2Me
X = 2-Me
13a
7
8
: R = Br
: R = CN
X = 5-Me
X = 5-Me
X = 5-Me
a
b
c
c
c
c
X = 2-Me
X = 4-Me
X = 4-Me
X = 6-F
9a : R = CO2H
10a : R = CO2Me
X = 5-Me
CO2Me
9b : R = CO2H X = 5-F
10b : R = CO2Me
c
X = 5-F
X = 6-F
12d : R = CO2Me X = 4-Cl
12e : R = CO2Me X = H
12f : R = CO2Me X = 5-Me
Me
10c : R = CO2Me
X = 4-F
N
H
13b
X = 6-Me
12g : R = CO2Me
12h : R = CO2Me
X = 5-F
12i : R = CO2Me X = 4-F
X = 6-OMe
12j : R = CO2Me
Scheme 1a. Synthesis of methyl (3-aminophenyl)acetates 12a–j and 13a–b. Reagents: (a) NaCN, DMSO; (b) H2SO4; (c) CH2N2, EtOAc; (d) H2, 10%Pd–C, EtOAc, MeOH; (e)
Ac2O, Py, CH2Cl2; (f) BH3ꢀSMe, THF.

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M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
TMS
CO2H
I
TMS
b
a
+
F
NO2
Cl
F
NO2
F
NO2
15
14
9b
16
Scheme 1b. Synthesis of (3-fluoro-5-nitrophenyl)acetic acid 9b. Reagents: (a) PdCl2(PPh3)2, CuI, n-Bu4NI, Et3N, DMF; (b) cyclohexene, BH3–THF, THF and then 2 M NaOH aq,
H2O2.
that both the analogs 4a and 4b exhibited increased binding affin-
ity and antagonist activity relative to the unsubstituted analog 2a.
Effect of the substitution of ring A on the activity profiles was fur-
ther investigated for both the series 5a–i and 6a–g. Introduction of
2-methyl, 4-methyl, 5-methyl and 6-methyl residues into ring A of
4a afforded 5a–d, respectively. 2-Methyl analog 5a showed re-
duced potency in both the binding affinity and antagonist activity
assays relative to 4a while the 4-methyl analog 5b exhibited
slightly reduced binding affinity and retained antagonist activity.
5-Methyl analog 5c and 6-methyl analog 5d showed slightly in-
creased binding affinity and nearly equipotent antagonist activity.
As a result, 4-methyl, 5-methyl and 6-methyl analogs 5b–d tended
to show equipotent antagonist activity relative to 4a. Introduction
of a halogen residue into the ring A of 4a afforded 4-chloro, 4-flu-
oro, 5-fluoro and 6-fluorophenyl analogs 5e–h, respectively, also
with retention of the potency relative to 4a in binding affinity
and antagonist activity assays. Introduction of a 6-methoxy residue
as a hydrophilic substituent into the ring A of 4a afforded 5i with a
tendency for retained receptor affinity and reduced antagonist
activity with a decrease in potency compared with 5e–h.
Cl
Y
O
a, b
2a, 3a–b, 4a–b, 5a–i
and 6a–g
O
O
N
17 :Y = H
18 :Y = Me
19 :Y = Cl
Me
Scheme 2. Synthesis of 2a, 3a–b, 4a–b, 5a–i and 6a–g. Reagents: (a) 12a–j and
13a–b, Py, CH2Cl2; (b) 5 M NaOH aq, MeOH, THF.
CO2Me
a
c, d
12e
2b
NH
O S
O
OR
Second, the corresponding SAR was investigated for a series of
3-(2-chlorophenyl)aminoacetic acid analogs. Introduction of a
methyl residue into positions 4, 5 and 6 of the ring A of 4b resulted
in 6a–c, respectively, a tendency for retained binding affinity and
an increase in the antagonist activity. Introduction of a halogen
such as 4-chloro, 4-fluoro and 6-fluoro residues into ring A of 4b
afforded 6d–f, respectively. Compounds 6d–e showed retained
binding affinity potency and increased antagonist activity while
6f had reduced antagonist activity although it retained strong
receptor affinity. Introduction of a methoxy residue into ring A of
4b afforded 6g with a reduction in the activities of both binding
affinity and antagonist activity.
Based on the SAR study described in Table 3, the 2-methyl res-
idue of ring A of 5a was estimated to cause a deleterious effect on
the conformation of both the acetic acid and 3-N-benzoyl moieties.
Introduction of a substituent into the 6-position of ring A of 4a
and/or 4b, which affords 5d, 5h, 5i, 6c, 6f and 6g, resulted in com-
plicated results. 3-(2-Methylbenzoyl)aminophenylacetic acid ana-
logs 5d and 5h showed retained potency for both the binding
affinity and antagonist activity relative to their parent compound
4a while the corresponding 3-(2-chlorobenzoylamino)phenylace-
tic acid analogs 6c and 6f tended to show increased potency and
slightly less potency, respectively, in their antagonist activity with
retained receptor affinity. Substitution with the 6-methoxy residue
on ring A was found not to be beneficial for further optimization as
illustrated by the results of 5i and 6g.
20 : R = Ac
21 : R = H
b
Cl
S
O
O
O
TsO
N
OAc
Me
22
23
Scheme 3. Synthesis of sulfonamide analogs 2b. Reagents: (a) 22, Py, CH2Cl2; (b)
K2CO3, MeOH, DME; (c) 23, Cs2CO3, DMF; (d) 5 M NaOH aq, MeOH, THF.
occupy s-cis conformation with remarkable higher field shifts of
the aromatic protons while N-phenylbenzamides have been re-
ported to occupy s-trans conformation.9 Based on the information
mentioned above, 3a was considered to occupy s-cis conformation
with the observed higher shifts of the aromatic protons (see
Section 5.9.3) while 2a was considered to occupy s-trans conforma-
tion. More potency of 2a relative to 3a strongly suggests that
s-trans amide conformation is more preferred to the corresponding
s-cis conformation for DP receptor. More detailed SAR study of our
designed and synthesized compounds regarding conformational
analysis using X-ray analysis will be reported in the following
paper.
Table 3 describes the effect of the substituents on rings A and B
of the general formula II on the activity profiles. It has been fre-
quently observed that 3-benzoylaminophenylacetic acid analogs
such as 2a are hydrolyzed by metabolic enzymes. Since our objec-
tive is to identify an orally active drug candidate, the targeted DP
antagonist has to possess tolerable metabolic stability to be sys-
temically effective. To identify a metabolically stable antagonist,
we focused on the synthesis and evaluation of 3-(2-meth-
ylbenzoyl)aminophenylacetic acid and 3-(2-chlorobenzoyl)amin-
ophenylacetic acid analogs such as 4a and 4b. It was fortunate
Table 4 describes the selectivity profiles of 4a, 5b–c and 6d–e
including those for the mEP, mFP, hTP, hIP and mDP receptors.
As shown in Table 4, all the tested compounds were proven to
show better selectivity for the human IP receptor compared with
the 2-methylindole analog 1 because of their reduced hIP receptor
affinities and their retained potent mDP receptor affinities. They
showed nearly equipotent mDP antagonist activities with the
chemical lead 1.

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
5365
X
O2N
O
c, d
a
O
2c
OH
N
Me
24
25 : X = NO
26 : X = NH2
2
b
CO2Me
CO2Me
e
O
O
OH
Cl
27
28
Scheme 4. Synthesis of 2c. Reagents: (a) 23, Cs2CO3, DMF; (b) H2, 10%Pd–C, MeOH, EtOAc; (c) 28, Py, CH2Cl2; (d) 5 M NaOH aq, MeOH, THF; (e) (COCl)2, DMF, DME.
Table 4
Activity profiles of 1, 4a, 5b, 5c, 6d and 6e
a
Compd
Binding Ki (nM)
IC50 (nM)
mDP
mEP1
mEP2
mEP3
mEP4
mFP
hTP
hIP
mDP
1
6200
99
1700
>10,000
>10,000
>10,000
670
>10,000
>10,000
3100
260
37
16
18
31
8.4
13
17
2.4
3.3
4.6
3.4
4.3
7.4
4a
5b
5c
6d
6e
>10,000
>10,000
>10,000
6600
NTb
170
62
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3600
2600
3500
1600
>10,000
44
22
>10,000
a
IC50 (nM): mDP receptor antagonist activity.
NT: not tested.
b
Table 5
Effect of the pharmacokinetic profiles of the representative compounds in rats
Compd
Route
Dose (mg/kg)
AUC (
lg h/mL)
Cmax
(l
g/mL)
CLtot (mL/min/kg)
2.4
T1/2 (h)
Vss (mL/kg)
BA (%)
1
iv
po
iv
po
iv
po
1
10
1
10
1
7.2a
9.8
5.4
0.27
1.8
3.1
5.2
1800
24.3a
3.5
4.9
8.7
34
119
62
2a
5b
0.67a
8.0a
25
220
610
4.6b
3.3
10
28.8b
a
AUCinf
AUClast
.
b
.
As shown in Table 5, pharmacokinetic (PK) profiles of the repre-
5. Experimental
sentative compounds 1, 2a and 5b were investigated. The newly
synthesized antagonist 5b tended to have improved PK profiles rel-
ative to the chemical lead 2a especially in terms of oral exposure
(AUC) and clearance (CL) with good bioavailability (BA). As shown
by the PK data of 5b, concurrent introduction of a 4-methyl residue
into the A ring and a 2-methyl residue into the B ring was consid-
ered effective to block the enzymatic degradation of 5b. The tissue
availability (Vss) of 5b was 610 ml/kg, which was found to be be-
tween the Vss values of 2a (220 ml/kg) and 1 (1800 ml/kg).
5.1. General directions
Analytical samples were homogeneous as confirmed by TLC,
and afforded spectroscopic results consistent with the assigned
structures. Proton nuclear magnetic resonance spectra (1H NMR)
were taken on a Varian Mercury 300 spectrometer or Varian GEM-
INI-200 or VXR-200s spectrometer using deuterated chloroform
(CDCl3) or deuterated methanol (CD3OD) or deuterated dimethyl-
sulfoxide (DMSO-d6) as the solvent. Fast atom bombardment mass
spectra (FAB-MS) and electron ionization (EI) were obtained on a
JEOL JMS-DX303HF spectrometer. The matrix assisted laser
desorption ionization-time of flight high-resolution mass spectra
(MALDI-TOF, HRMS) were obtained on a PerSeptive Voyager Elite
spectrometer. Atmospheric pressure chemical ionization (APCI)
was determined on a HITACHI M1200H spectrometer. Infrared
spectra (IR) were measured on a Perkin-Elmer FT-IR 1760X
spectrometer. Melting points and results of elemental analyses
were uncorrected. Optical rotations were measured in a JASCO
4. Conclusion
Design and synthesis of new prostaglandin D2 receptor antago-
nists starting from 3-benzoylaminophenylacetic acid 2a, which
was designed based on the structural simplification of our original
2-methylindole antagonist 1 (Fig. 1), as a chemical lead led us to
discover a new cost-effective antagonist 5b. Further effort to iden-
tify an orally available subtype selective DP antagonist as a drug
candidate will be disclosed in due course.

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M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
DIP-1000 digital polarimeter. Column chromatography was carried
out on silica gel [Merck Silica Gel 60 (0.063–0.200 mm), Wako gel
C200 or Fuji Silysia BW235]. Thin layer chromatography was per-
formed on silica gel (Merck TLC or HPTLC plates, Silica Gel 60 F254).
The following abbreviations for solvents and reagents are used; tet-
rahydrofuran (THF), ethyl acetate (EtOAc), dimethylformamide
(DMF), dichloromethane (CH2Cl2), chloroform (CHCl3), methanol
(MeOH), triethylamine (TEA), 1,2-dimethoxyethane (DME), dimeth-
ylsulfoxide (DMSO), acetic anhydride (Ac2O), pyridine (Py).
5.5. General procedure for the preparation of methyl
aminophenyl acetates 12f and 12h–i
5.5.1. Methyl (3-amino-5-methylphenyl)acetate (12f)
To a stirred solution of 10a (146 mg, 0.7 mmol) in EtOAc (3 mL)
and MeOH (3 mL) was added 10% Pd–C (30 mg) at room tempera-
ture. The resulting suspension was vigorously stirred for 30 min at
room temperature under hydrogen atmosphere. Insoluble sub-
stance was removed by filtration. The filtrate was concentrated
in vacuo to give a crude product, which was purified by column
chromatography on silica gel to yield 12f (77 mg, 65%) as a pale
yellow oil; TLC Rf = 0.26 (n-hexane/EtOAc, 7:3); 1H NMR
(300 MHz, CDCl3) d 6.49 (s, 1H), 6.43 (s, 2H), 3.68 (s, 3H), 3.70–
3.50 (br, 2H), 3.49 (s, 2H), 2.24 (s, 3H).
5.2. (3-Methyl-5-nitrophenyl)acetonitrile (8)
To a stirred solution of 7 (782 mg, 3.4 mmol) in DMSO (5 mL)
was added NaCN (183 mg, 3.74 mmol) at room temperature. After
stirring overnight, the reaction mixture was quenched with water
and extracted with EtOAc (ꢁ2). The combined organic layers were
washed with water, brine, dried over Na2SO4 and concentrated in
vacuo to yield 8 as a dark brown oil, which was used for the next
reaction without further purification; TLC Rf = 0.14 (n-hexane/
EtOAc, 9:1).
Compounds 12h–i were prepared as described above.
5.5.2. Methyl (3-amino-5-fluorophenyl)acetate (12h)
Yield quant; Pale yellow oil; TLC Rf = 0.26 (n-hexane/EtOAc,
7:3); 1H NMR (300 MHz, CDCl3) d 6.42–6.33 (m, 2H), 6.32–6.24
(m, 1H), 3.85–3.70 (br, 2H), 3.70 (s, 3H), 3.50 (s, 2H).
5.3. (3-Methyl-5-nitrophenyl)acetic acid (9a)
5.5.3. Methyl (3-amino-4-fluorophenyl)acetate (12i)
Yield quant; Pale yellow powder; TLC Rf = 0.63 (n-hexane/
EtOAc, 7:3); 1H NMR (300 MHz, CDCl3) d 6.91 (dd, J = 11.1, 8.4 Hz,
1H), 6.71 (dd, J = 8.4, 2.1 Hz, 1H), 6.62–6.54 (m, 1H), 3.80–3.60
(br, 2H), 3.68 (s, 3H), 3.50 (s, 2H).
To a stirred suspension of 8 (3.4 mmol) in water (2.5 mL) was
added concd H2SO4 (2.5 mL). After stirring for 1.5 h under reflux,
the reaction mixture was cooled to room temperature, poured into
water and extracted with EtOAc (ꢁ2). The combined organic layers
were washed with water, brine, dried over Na2SO4 and concen-
trated in vacuo to yield 9a (130 mg, 20% in 2 steps) as a pale orange
solid, which was used for the next reaction without further purifi-
cation; TLC Rf = 0.50 (CHCl3/CH3OH, 9:1); 1H NMR (300 MHz,
CDCl3) d 7.98 (s, 2H), 7.44 (s, 1H), 3.74 (s, 2H), 2.47 (s, 3H).
5.6. Methyl [3-(ethylamino)phenyl]acetate (13a)
To a stirred solution of 12e (820 mg, 4.97 mmol) in CH2Cl2
(5 mL) were added pyridine (0.82 mL, 10.1 mmol) and Ac2O
(0.52 mL, 5.50 mmol). After stirring for 30 min at room tempera-
ture, the reaction mixture was poured into water and extracted
with EtOAc (ꢁ2). The combined organic layers were washed with
1 M HCl aq, brine, dried over Na2SO4 and concentrated in vacuo
to yield the corresponding N-acetate as a pale yellow oil, which
was used for the next reaction without further purification; TLC
Rf = 0.22 (n-hexane/EtOAc, 1:1).
5.4. General procedure for the preparation of methyl
nitrophenyl acetates 10a–b and methyl aminophenyl
acetates 12a–c
5.4.1. Methyl (3-methyl-5-nitrophenyl)acetate (10a)
To a stirred solution of 9a (130 mg, 0.7 mmol) in EtOAc (10 mL)
was added dropwise a solution of CH2N2 in ether until bubbling
ceased. The reaction mixture was concentrated in vacuo to yield
10a (146 mg, quant) as a pale yellow oil; TLC Rf = 0.51 (n-hexane/
EtOAc, 7:3); 1H NMR (300 MHz, CDCl3) d 7.96 (s, 2H), 7.43 (s,
1H), 3.73 (s, 3H), 3.70 (s, 2H), 2.46 (s, 3H).
To a stirred solution of the N-acetate (4.97 mmol) in THF (3 mL)
was added a 2 M solution of BH3SMe2 in THF (1.9 mL, 3.79 mmol)
at 0 °C. After stirring for 1 h without cooling bath, the reaction mix-
ture was additionally stirred for 15 h at 60 °C and then poured into
sat NaHCO3 aq and extracted with EtOAc (ꢁ2). The combined or-
ganic layers were washed with water, brine, dried over Na2SO4
and concentrated in vacuo. The resulting residue was purified by
column chromatography on silica gel to yield 13a (320 mg, 33%
in 2 steps) as a colorless oil; TLC Rf = 0.49 (n-hexane/EtOAc, 2:1);
1H NMR (300 MHz, CDCl3) d 7.12 (dd, J = 7.5, 7.5 Hz, 1H), 6.59 (d,
J = 7.5 Hz, 1H), 6.55–6.48 (m, 2H), 3.68 (s, 3H), 3.54 (s, 2H), 3.15
(q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H).
Compounds 10b and 12a–c were prepared as described above.
5.4.2. Methyl (3-fluoro-5-nitrophenyl)acetate (10b)
Yield quant; Pale yellow oil; TLC Rf = 0.56 (n-hexane/EtOAc,
7:3); 1H NMR (300 MHz, CDCl3) d 7.99 (s, 1H), 7.86 (dt, J = 8.4,
2.4 Hz, 1H), 7.39 (dt, J = 8.4, 1.8 Hz, 1H), 3.75 (s, 5H).
5.4.3. Methyl (3-amino-2-methylphenyl)acetate (12a)
Yield quant; Pale yellow powder; TLC Rf = 0.26 (n-hexane/
EtOAc, 7:3); 1H NMR (300 MHz, CDCl3) d 6.97 (t, J = 7.5 Hz, 1H),
6.69–6.61 (m, 2H), 2.68 (s, 3H) 3.64 (s, 2H), 3.70–3.50 (br, 2H),
2.10 (s, 3H).
5.7. [(3-Fluoro-5-nitrophenyl)ethynyl](trimethyl)silane (16)
To a stirred solution of 1-fluoro-3-iodo-5-nitrobenzene 14 (2 g,
7.5 mmol) in DMF (30 mL) under argon atmosphere were added
Pd(PPh3)2Cl2 (263 mg, 0.4 mmol), CuI (143 mg, 0.75 mmol), n-
Bu4NI (553 mg, 1.5 mmol), triethylamine (1.57 mL, 11.2 mmol)
and (trimethylsilyl)acetylene 15 (1.8 mL, 12.7 mmol). After stirring
overnight at 70 °C, the reaction mixture was cooled to room tem-
perature, poured into sat NH4Cl aq and extracted with EtOAc
(ꢁ2). The combined organic layers were washed with water, brine,
dried over Na2SO4 and concentrated in vacuo. The resulting residue
was purified by column chromatography on silica gel to yield 16
(1.31 g, 74%) as a brown oil; TLC Rf = 0.82 (n-hexane/EtOAc, 9:1);
1H NMR (300 MHz, CDCl3) d 8.11 (s, 1H), 7.91–7.84 (m, 1H),
7.50–7.44 (m, 1H), 0.27 (s, 9H).
5.4.4. Methyl (3-amino-4-methylphenyl)acetate (12b)
Yield quant; Pale pink oil; TLC Rf = 0.29 (n-hexane/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3) d 6.99 (d, J = 7.8 Hz, 1H), 6.65–6.59 (m,
2H), 3.68 (s, 3H), 3.70–3.50 (br, 2H), 3.52 (s, 2H), 2.14 (s, 3H).
5.4.5. Methyl (5-amino-2-fluorophenyl)acetate (12c)
Yield quant; Pale yellow oil; TLC Rf = 0.47 (n-hexane/EtOAc,
1:1); 1H NMR (300 MHz, CDCl3) d 6.85 (t, J = 9.3 Hz, 1H), 6.60–
6.51 (m, 2H), 3.71 (s, 3H), 3.58 (s, 2H), 3.60–3.45 (br, 2H).

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
5367
5.8. (3-Fluoro-5-nitrophenyl)acetic acid (9b)
20 V) m/z 431 (MꢂH)ꢂ; IR (neat) 2925, 1712, 1649, 1606,
1544, 1505, 1441, 1304, 1245, 1179, 1042, 909, 844, 735 cmꢂ1
;
To a stirred solution of cyclohexene (1.81 g, 22.1 mmol) in THF
(15 mL) were added a 1 M solution of BH3 in THF (11 mL, 11 mmol)
and then a solution of 16 (1.31 g, 5.5 mmol) in THF (9 mL) at 0 °C.
The reaction mixture was stirred overnight without cooling bath.
To the above-described reaction mixture were added slowly 2 M
NaOH aq (18 mL) and then 30% H2O2 (6.0 mL). After stirring for
15 min, the reaction mixture was diluted with water and extracted
with n-hexane. The aqueous layer was acidified with concd HCl
and extracted with EtOAc (ꢁ2). The combined organic layers were
washed with water, brine, dried over Na2SO4 and evaporated to
yield 9b (650 mg, 59%), which was used for the next reaction with-
out further purification; TLC Rf = 0.38 (CHCl3/MeOH, 9:1); 1H NMR
(300 MHz, CDCl3) d 8.00 (s, 1H), 7.88 (dt, J = 8.4, 2.4 Hz, 1H), 7.40
(dt, J = 8.4, 1.8 Hz, 1H), 3.79 (s, 2H).
Optical rotation ½a D24
ꢃ
+8.86 (c 0.75, DMSO).
5.9.3. {3-[Methyl(4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic acid
(3a)
Yield 38% in two steps; Green amorphous powder; TLC Rf = 0.63
(AcOEt/MeOH, 19:1); 1H NMR (300 MHz, CDCl3) d 7.29–7.20 (m,
4H), 7.10–7.02 (m, 2H), 6.91–6.69 (m, 6H), 4.55 (m, 1H), 4.28
(dd, J = 10.8, 4.8 Hz, 1H), 4.04 (dd, J = 10.8, 7.2 Hz, 1H), 3.49 (s,
3H), 3.43 (s, 2H), 3.38 (dd, J = 11.4, 2.4 Hz, 1H), 3.09 (dd, J = 11.4,
7.2 Hz, 1H), 2.86 (s, 3H); MS (APCI, Neg, 20 V) m/z 445 (MꢂH)ꢂ;
IR (neat) 2939, 1721, 1605, 1504, 1451, 1376, 1301, 1223, 1177,
1042, 906, 841, 738, 703, 600 cmꢂ1; Optical rotation ½a 2D4
ꢃ
+6.65
(c 0.75, DMSO).
5.9. General procedure for the preparation of 2a, 3a–b, 4a–b,
5a–i, and 6a–g
5.9.4. {3-[Ethyl(4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (3b)
5.9.1. {3-[(2-Methyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-benz-
oxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic acid (4a)
Yield 49% in two steps; Blue amorphous powder; TLC Rf = 0.49
(AcOEt/MeOH, 19:1); 1H NMR (300 MHz, CDCl3) d 7.30–7.18 (m,
3H), 7.12–7.02 (m, 2H), 6.92–6.66 (m, 7H), 4.54 (m, 1H), 4.30
(dd, J = 10.8, 4.8 Hz, 1H), 4.03 (dd, J = 10.8, 7.5 Hz, 1H), 3.99 (dq,
J = 2.4, 7.2 Hz, 2H), 3.41 (s, 2H), 3.39 (dd, J = 11.7, 2.4 Hz, 1H),
3.07 (dd, J = 11.7, 7.8 Hz, 1H), 2.86 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H);
MS (APCI, Neg, 20 V) m/z 459 (MꢂH)ꢂ; IR (neat) 2932, 1726,
To a stirred solution of 12e (62 mg, 0.4 mmol) in CH2Cl2 (5 mL)
were added a solution of 18 (133 mg, 0.4 mmol) in CH2Cl2 (3 mL)
and then pyridine (0.067 mL, 0.8 mmol). After stirring overnight
at room temperature, the reaction mixture was diluted with water
and extracted with EtOAc (ꢁ2). The combined organic layers were
washed with 1 M NaOH aq, water, brine, dried over Na2SO4 and
concentrated in vacuo. The resulting residue was purified by col-
umn chromatography on silica gel to yield methyl ester (120 mg,
63%); TLC Rf = 0.14 (n-hexane/EtOAc, 7:3); 1H NMR (300 MHz,
CDCl3) d 7.60–7.38 (m, 4H), 7.32 (t, J = 7.8 Hz, 1H), 7.06 (d,
J = 7.5 Hz, 1H), 6.92–6.77 (m, 4H), 6.74–6.66 (m, 2H), 4.70–4.60
(m, 1H), 4.26 (dd, J = 9.6, 5.4 Hz, 1H), 4.14 (dd, J = 11.4, 6.6 Hz,
1H), 3.71 (s, 3H), 3.65 (s, 2H), 3.39 (dd, J = 11.4, 3.0 Hz, 1H), 3.26
(dd, J = 11.4, 7.8 Hz, 1H), 2.91 (s, 3H), 2.51 (s, 3H).
1605, 1505, 1450, 1396, 1301, 1245, 1177, 1043, 839, 743 cmꢂ1
;
Optical rotation ½a D24
ꢃ
+4.05 (c 0.75, DMSO).
5.9.5. {3-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (4b)
Yield 40% in two steps; Ivory powder; TLC Rf = 0.29 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.06 (s, 1H), 7.82 (d,
J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.34 (t,
J = 7.5 Hz, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.01 (d, J = 1.8 Hz, 1H),
6.98–6.80 (m, 3H), 6.76–6.67 (m, 2H), 4.71–4.61 (m, 1H), 4.26
(dd, J = 9.9, 5.1 Hz, 1H), 4.17 (dd, J = 9.9, 6.0 Hz, 1H), 3.68 (s, 2H),
3.38 (dd, J = 11.7, 3.0 Hz, 1H), 3.25 (dd, J = 11.7, 6.0 Hz, 1H), 2.91
(s, 3H); MS (APCI, Neg, 20 V) m/z 465 (MꢂH)ꢂ; IR (KBr) 3255,
2944, 1699, 1655, 1604, 1531, 1494, 1443, 1295, 1261, 1223,
1174, 1135, 1047, 968, 915, 860, 779, 741, 708, 607 cmꢂ1; Optical
To a stirred solution of methyl ester (120 mg, 0.25 mmol) de-
scribed above in MeOH (4 mL) and THF (4 mL) was added 5 M
NaOH aq (2 mL). After stirring for 1 h at room temperature, the
reaction mixture was diluted with 2 M HCl aq and extracted with
EtOAc (ꢁ2). The combined organic layers were washed with water,
brine, dried over Na2SO4 and concentrated in vacuo to give a crude
product, which was washed with EtOAc/n-hexane to yield 4a
(85 mg, 73%) as a pale blue powder; TLC Rf = 0.40 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 7.62–7.40 (m, 4H), 7.33 (t,
J = 8.1 Hz, 1H), 7.10–7.04 (m, 1H), 6.92–6.76 (m, 4H), 6.74–6.66
(m, 2H), 4.70–4.60 (m, 1H), 4.26 (dd, J = 9.6, 4.8 Hz, 1H), 4.14 (dd,
J = 9.6, 6.6 Hz, 1H), 3.68 (s, 2H), 3.39 (dd, J = 11.4, 2.7 Hz, 1H),
3.25 (dd, J = 11.4, 6.6 Hz, 1H), 2.91 (s, 3H), 2.50 (s, 3H); MS (APCI,
Neg, 20 V) m/z 445 (MꢂH)ꢂ; IR (KBr) 3256, 3040, 2925, 1698,
1650, 1607, 1572, 1532, 1503, 1442, 1355, 1318, 1299, 1244,
1225, 1176, 1136, 1098, 1041, 994, 975, 912, 868, 825, 773, 739,
rotation ½a 2D7
ꢃ
+13.19 (c 0.75, DMSO).
5.9.6. {2-Methyl-3-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5a)
Yield 69% in two steps; Yellow powder; TLC Rf = 0.38 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.82–7.68 (m, 1H),
7.54–7.44 (m, 1H), 7.38–7.18 (m, 2H), 7.10 (d, J = 8.1 Hz, 1H),
6.92–6.77 (m, 4H), 6.75–6.66 (m, 2H), 4.70–4.60 (m, 1H), 4.26
(dd, J = 9.9, 5.4 Hz, 1H), 4.14 (dd, J = 9.9, 6.3 Hz, 1H), 3.73 (s,
2H), 3.39 (dd, J = 11.4, 2.7 Hz, 1H), 3.25 (dd, J = 11.4, 6.6 Hz,
1H), 2.91 (s, 3H), 2.52 (s, 3H), 2.24 (s, 3H); MS (APCI, Neg,
20 V) m/z 459 (MꢂH)ꢂ; IR (KBr) 3250, 2925, 1695, 1649, 1605,
1503, 1472, 1449, 1278, 1242, 1223, 1174, 1136, 1042, 939,
707, 607 cmꢂ1, Optical rotation ½a 2D4
ꢃ
+14.49 (c 0.60, DMSO); mp
173–175 °C.
Compounds 2a, 3a–b, 4b, 5a–i and 6a–g were prepared as de-
scribed above.
5.9.2. {3-[(4-{[(2S)-4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}acetic acid (2a)
822, 785, 737, 692, 607, 479 cmꢂ1; Optical rotation ½a 2D7
ꢃ
+10.14
(c 0.75, DMSO).
Yield 57% in two steps; Green amorphous powder; TLC
Rf = 0.52 (AcOEt/MeOH, 19:1); 1H NMR (300 MHz, CDCl3)
d
5.9.7. {4-Methyl-3-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5b)
Yield 68% in two steps; Ivory powder; TLC Rf = 0.35 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.04–7.86 (br, 1H),
7.53–7.42 (m, 1H), 7.34–7.22 (m, 1H), 7.19 (d, J = 7.8 Hz, 1H),
7.90–7.78 (m, 3H), 7.57 (s, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.29
(m, 1H), 7.06–6.95 (m, 3H), 6.91–6.82 (m, 2H), 6.75–6.66 (m,
2H), 4.65 (m, 1H), 4.27 (dd, J = 9.6, 4.8 Hz, 1H), 4.16 (dd,
J = 9.6, 6.6 Hz, 1H), 3.63 (s, 2H), 3.38 (dd, J = 11.4, 2.7 Hz, 1H),
3.25 (dd, J = 11.4, 6.6 Hz, 1H), 2.90 (s, 3H); MS (APCI, Neg,

5368
M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
7.07–6.99 (m, 1H), 6.92–6.76 (m, 4H), 6.75–6.66 (m, 2H), 4.70–
4.60 (m, 1H), 4.27 (dd, J = 9.9, 4.8 Hz, 1H), 4.15 (dd, J = 9.9,
6.6 Hz, 1H), 3.66 (s, 2H), 3.39 (dd, J = 11.7, 2.7 Hz, 1H), 3.25
(dd, J = 11.7, 6.6 Hz, 1H), 2.91 (s, 3H), 2.52 (s, 3H), 2.28 (s, 3H);
MS (APCI, Neg, 20 V) m/z 459 (MꢂH)ꢂ; IR (KBr) 3274, 3033,
2926, 2871, 2824, 2643, 2350, 2304, 2242, 1690, 1650, 1608,
1579, 1529, 1504, 1481, 1449, 1402, 1357, 1311, 1298, 1288,
1270, 1244, 1221, 1176, 1149, 1138, 1101, 1041, 1010, 976,
935, 913, 867, 853, 842, 823, 810, 780, 743, 735, 700, 690,
681, 638, 628, 609, 595, 586, 577, 564, 555, 546, 532, 522,
2.53 (s, 3H); MS (APCI, Neg, 20 V) m/z 463 (MꢂH)ꢂ; IR (KBr)
3254, 3141, 3034, 2927, 2866, 2649, 2619, 2345, 1699, 1655,
1608, 1572, 1536, 1503, 1460, 1435, 1408, 1320, 1289, 1264,
1240, 1223, 1177, 1135, 1119, 1051, 995, 977, 911, 865, 824,
785, 771, 734, 679, 665, 613, 566, 523, 499, 484, 454 cmꢂ1; Optical
rotation ½a 2D7
ꢃ
+9.03 (c 0.75, DMSO).
5.9.12. {3-Fluoro-5-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5g)
514, 493, 472, 460 cmꢂ1; Optical rotation ½a 2D4
ꢃ
+12.57 (c 0.75,
Yield 59% in two steps; Pale blue powder; TLC Rf = 0.41 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.56–7.40 (m, 3H), 7.20
(br s, 1H), 6.93–6.75 (m, 5H), 6.76–6.66 (m, 2H), 4.70–4.60 (m,
1H), 4.26 (dd, J = 9.6, 5.1 Hz, 1H), 4.14 (dd, J = 9.6, 6.6 Hz, 1H),
3.64 (s, 2H), 3.39 (dd, J = 11.7, 2.4 Hz, 1H), 3.25 (dd, J = 11.7,
6.6 Hz, 1H), 2.91 (s, 3H), 2.49 (s, 3H); MS (APCI, Neg, 20 V) m/z
463 (MꢂH)ꢂ; IR (KBr) 3268, 3067, 2927, 1702, 1654, 1623, 1604,
1573, 1535, 1503, 1474, 1430, 1278, 1243, 1225, 1175, 1137,
1096, 1042, 1001, 940, 910, 850, 824, 769, 741, 678, 607, 567,
DMSO); mp 176–177 °C.
5.9.8. {3-Methyl-5-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5c)
Yield 51% in two steps; Ivory powder; TLC Rf = 0.35 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.48–7.28 (m, 4H), 6.92–
6.76 (m, 5H), 6.74–6.66 (m, 2H), 4.70–4.60 (m, 1H), 4.26 (dd,
J = 9.9, 5.1 Hz, 1H), 4.14 (dd, J = 9.9, 6.6 Hz, 1H), 3.63 (s, 2H), 3.39
(dd, J = 11.4, 2.7 Hz, 1H), 3.25 (dd, J = 11.4, 6.6 Hz, 1H), 2.91 (s,
3H), 2.50 (s, 3H), 2.35 (s, 3H); MS (APCI, Neg, 20 V) m/z 459 (MꢂH)ꢂ;
IR (KBr) 3262, 3150, 3035, 2924, 2871, 1693, 1650, 1609, 1573,
1534, 1504, 1455, 1414, 1290, 1245, 1223, 1178, 1137, 1041, 947,
909, 867, 847, 824, 735, 689, 654, 609, 569, 522 cmꢂ1; Optical rota-
524 cmꢂ1; Optical rotation ½a 2D7
ꢃ
+12.49 (c 0.75, DMSO).
5.9.13. {2-Fluoro-5-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5h)
Yield 53% in two steps; Ivory powder; TLC Rf = 0.31 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.61 (br s, 1H), 7.52–
7.38 (m, 3H), 7.07 (t, J = 8.7 Hz, 1H), 6.92–6.76 (m, 4H), 6.76–6.66
(m, 2H), 4.70–4.60 (m, 1H), 4.26 (dd, J = 9.3, 5.1 Hz, 1H), 4.14 (dd,
J = 9.3, 6.3 Hz, 1H), 3.73 (s, 2H), 3.39 (dd, J = 11.7, 2.7 Hz, 1H),
3.25 (dd, J = 11.7, 6.6 Hz, 1H), 2.91 (s, 3H), 2.50 (s, 3H); MS (APCI,
Neg, 20 V) m/z 463 (MꢂH)ꢂ; IR (KBr) 3275, 3041, 2929, 2619,
2547, 1700, 1649, 1609, 1573, 1528, 1502, 1455, 1419, 1350,
1321, 1303, 1289, 1233, 1214, 1178, 1136, 1113, 1103, 1065,
1041, 975, 909, 869, 826, 775, 742, 578, 545, 453 cmꢂ1; Optical
tion ½a 2D4
ꢃ
+13.59 (c 0.75, DMSO).
5.9.9. {2-Methyl-5-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5d)
Yield 45% in two steps; Ivory powder; TLC Rf = 0.32 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.56–7.34 (m, 4H), 7.18
(d, J = 8.4 Hz, 1H), 6.92–6.76 (m, 4H), 6.75–6.66 (m, 2H), 4.70–
4.60 (m, 1H), 4.26 (dd, J = 9.6, 4.5 Hz, 1H), 4.14 (dd, J = 9.6, 6.3 Hz,
1H), 3.69 (s, 2H), 3.39 (dd, J = 11.4, 2.1 Hz, 1H), 3.25 (dd, J = 11.4,
6.6 Hz, 1H), 2.91 (s, 3H), 2.50 (s, 3H), 2.30 (s, 3H); MS (APCI, Neg,
20 V) m/z 459 (MꢂH)ꢂ; IR (KBr) 3256, 3042, 2926, 1699, 1647,
1608, 1572, 1505, 1451, 1411, 1356, 1320, 1298, 1244, 1223,
1177, 1148, 1137, 1092, 1052, 997, 975, 913, 864, 825, 790, 775,
rotation ½a 2D7
ꢃ
+14.12 (c 0.75, DMSO).
5.9.14. {2-Methoxy-5-[(2-methyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phe-
nyl}acetic acid (5i)
737, 704, 664, 633, 600, 570, 545 cmꢂ1; Optical rotation ½a 2D4
ꢃ
Yield 52% in two steps; Gray powder; TLC Rf = 0.36 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.58–7.39 (m, 3H), 7.34
(br s, 1H), 6.93–6.76 (m, 5H), 6.74–6.66 (m, 2H), 4.70–4.60 (m,
1H), 4.26 (dd, J = 9.3, 4.8 Hz, 1H), 4.14 (dd, J = 9.3, 6.3 Hz, 1H),
3.85 (s, 3H), 3.69 (s, 2H), 3.39 (dd, J = 11.4, 2.7 Hz, 1H), 3.25 (dd,
J = 11.4, 6.6 Hz, 1H), 2.91 (s, 3H), 2.50 (s, 3H); MS (APCI, Neg,
20 V) m/z 475 (MꢂH)ꢂ; IR (KBr) 3262, 3131, 3044, 2929, 2838,
2646, 2553, 1699, 1645, 1607, 1573, 1504, 1459, 1428, 1300,
1287, 1260, 1239, 1227, 1176, 1147, 1135, 1103, 1092, 1063,
1050, 1026, 975, 939, 865, 817, 807, 775, 744, 633, 598, 574,
+12.31 (c 0.60, DMSO).
5.9.10. {4-Chloro-3-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5e)
Yield 54% in two steps; Yellow powder; TLC Rf = 0.41 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.49 (br s, 1H), 7.99 (s,
1H), 7.53 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.01 (dd, J = 7.8,
1.8 Hz, 1H), 6.93–6.78 (m, 4H), 6.76–6.66 (m, 2H), 4.70–4.60 (m,
1H), 4.27 (dd, J = 9.9, 4.8 Hz, 1H), 4.16 (dd, J = 9.9, 6.6 Hz, 1H), 3.70
(s, 2H), 3.40 (dd, J = 11.7, 2.7 Hz, 1H), 3.25 (dd, J = 11.7, 6.6 Hz, 1H),
2.91 (s, 3H), 2.55 (s, 3H); MS (APCI, Neg, 20 V) m/z 479 (MꢂH)ꢂ; IR
(KBr) 3275, 3036, 2926, 1687, 1656, 1607, 1583, 1524, 1502, 1459,
1426, 1357, 1290, 1245, 1221, 1177, 1136, 1092, 1051, 978, 914,
545, 455 cmꢂ1; Optical rotation ½a 2D7
ꢃ
+9.44 (c 0.75, DMSO).
5.9.15. {3-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]-4-methylphenyl}
acetic acid (6a)
864, 822, 782, 741, 655, 504, 479, 469 cmꢂ1; Optical rotation ½a 2D7
ꢃ
Yield 50% in two steps; Ivory powder; TLC Rf = 0.42 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.02 (s, 2H), 7.90 (d,
J = 8.7 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.09–7.00 (m, 2H), 7.00–
6.93 (m, 1H), 6.93–6.81 (m, 2H), 6.76–6.66 (m, 2H), 4.70–4.60
(m, 1H), 4.27 (dd, J = 9.6, 5.1 Hz, 1H), 4.18 (dd, J = 9.6, 6.3 Hz, 1H),
3.68 (s, 2H), 3.39 (dd, J = 11.7, 2.7 Hz, 1H), 3.25 (dd, J = 11.7,
6.3 Hz, 1H), 2.91 (s, 3H), 2.32 (s, 3H); MS (APCI, Neg, 20 V) m/z
479 (MꢂH)ꢂ; IR (KBr) 3257, 3147, 3040, 2928, 2823, 2557, 1698,
1657, 1639, 1604, 1581, 1534, 1503, 1450, 1426, 1407, 1358,
1291, 1264, 1245, 1225, 1138, 1046, 993, 962, 918, 846, 827,
785, 740, 718, 688, 615, 586, 566, 557, 536, 526, 506, 495, 472,
+7.96 (c 0.75, DMSO).
5.9.11. {4-Fluoro-3-[(2-methyl-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (5f)
Yield 22% in two steps; Pale orange powder; TLC Rf = 0.31
(CHCl3/MeOH, 9:1); 1H NMR (300 MHz, CDCl3)
d 7.67 (d,
J = 2.7 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.14–6.76 (m,
6H), 6.76–6.68 (m, 2H), 4.70–4.60 (m, 1H), 4.27 (dd, J = 9.3,
5.1 Hz, 1H), 4.15 (dd, J = 9.3, 6.6 Hz, 1H), 3.69 (s, 2H), 3.40 (dd,
J = 11.7, 2.7 Hz, 1H), 3.26 (dd, J = 11.7, 6.9 Hz, 1H), 2.92 (s, 3H),
462 cmꢂ1; Optical rotation ½a 2D4
ꢃ
+17.39 (c 0.75, DMSO).

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
5369
5.9.16. {3-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-ben-
zoxazin-2-yl]methoxy}benzoyl)amino]-5-methylphenyl}acetic
acid (6b)
5.9.20. {5-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]-2-fluorophenyl}
acetic acid (6f)
Yield 57% in two steps; Ivory powder; TLC Rf = 0.39 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.00 (s, 1H), 7.81 (d,
J = 8.7 Hz, 1H), 7.41 (s, 2H), 7.01 (d, J = 2.4 Hz, 1H), 6.98–6.81 (m,
4H), 6.75–6.67 (m, 2H), 4.70–4.60 (m, 1H), 4.26 (dd, J = 9.9,
5.1 Hz, 1H), 4.17 (dd, J = 9.9, 6.3 Hz, 1H), 3.64 (s, 2H), 3.38 (dd,
J = 11.4, 2.7 Hz, 1H), 3.25 (dd, J = 11.4, 6.3 Hz, 1H), 2.91 (s, 3H),
2.36 (s, 3H); MS (APCI, Neg, 20 V) m/z 479 (MꢂH)ꢂ; IR (KBr)
3236, 3066, 2927, 2868, 2821, 2664, 2593, 1701, 1648, 1608,
1602, 1545, 1505, 1494, 1456, 1355, 1302, 1276, 1257, 1220,
1177, 1134, 1047, 967, 919, 851, 790, 740, 732, 688, 637, 614,
Yield 50% in two steps; Pale gray powder; TLC Rf = 0.41 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.06 (s, 1H), 7.82 (d,
J = 8.7 Hz, 1H), 7.67–7.60 (m, 1H), 7.56–7.47 (m, 1H), 7.08 (t,
J = 9.0 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H), 6.99–6.81 (m, 3H), 6.76–
6.67 (m, 2H), 4.70–4.60 (m, 1H), 4.26 (dd, J = 9.9, 5.4 Hz, 1H),
4.17 (dd, J = 9.9, 6.0 Hz, 1H), 3.74 (s, 2H), 3.38 (dd, J = 11.7,
2.7 Hz, 1H), 3.25 (dd, J = 11.7, 6.3 Hz, 1H), 2.91 (s, 3H); MS (APCI,
Neg, 20 V) m/z 483 (MꢂH)ꢂ; IR (KBr) 3266, 3068, 2930, 2826,
2653, 2545, 2319, 1701, 1655, 1625, 1605, 1561, 1530, 1502,
1420, 1321, 1300, 1272, 1255, 1212, 1107, 1064, 1041, 969, 943,
565, 522 cmꢂ1; Optical rotation ½a 2D4
ꢃ
+11.90 (c 0.75, DMSO).
920, 860, 838, 825, 790, 768, 742, 620, 574, 534, 489 cmꢂ1; Optical
21
rotation [
a
]
+11.99 (c 0.75, DMSO).
D
5.9.17. {5-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]-2-methylphenyl}
acetic acid (6c)
5.9.21. {5-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl)amino]-2-methoxyphenyl}
acetic acid (6g)
Yield 36% in two steps; Ivory powder; TLC Rf = 0.47 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.00 (s, 1H), 7.81 (d,
J = 9.0 Hz, 1H), 7.57–7.52 (m, 1H), 7.49–7.42 (m, 1H), 7.19 (d,
J = 8.4 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 6.98–6.81 (m, 3H), 6.76–
6.66 (m, 2H), 4.70–4.60 (m, 1H), 4.26 (dd, J = 9.9, 5.1 Hz, 1H),
4.17 (dd, J = 9.9, 6.3 Hz, 1H), 3.69 (s, 2H), 3.38 (dd, J = 11.4,
2.7 Hz, 1H), 3.25 (dd, J = 11.4, 6.6 Hz, 1H), 2.91 (s, 3H), 2.31 (s,
3H); MS (APCI, Neg, 20 V) m/z 479 (MꢂH)ꢂ; IR (KBr) 3238, 3045,
2942, 2865, 2639, 1699, 1652, 1604, 1561, 1525, 1505, 1474,
1452, 1411, 1320, 1294, 1275, 1259, 1217, 1173, 1161, 1134,
1102, 1064, 1050, 997, 968, 912, 859, 824, 819, 790, 749, 735,
Yield 44% in two steps; White powder; TLC Rf = 0.41 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.96 (s, 1H), 7.82 (d,
J = 8.4 Hz, 1H), 7.57 (dd, J = 8.4, 2.7 Hz, 1H), 7.48 (d, J = 2.7 Hz,
1H), 7.01 (d, J = 2.7 Hz, 1H), 6.97–6.81 (m, 4H), 6.75–6.66 (m,
2H), 4.70–4.60 (m, 1H), 4.26 (dd, J = 9.9, 5.4 Hz, 1H), 4.16 (dd,
J = 9.9, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 2H), 3.38 (dd, J = 11.7,
3.0 Hz, 1H), 3.25 (dd, J = 11.7, 6.6 Hz, 1H), 2.91 (s, 3H); MS (APCI,
Neg, 20 V) m/z 495 (MꢂH)ꢂ; IR (KBr) 3404, 3247, 3132, 3068,
2931, 2838, 2644, 2558, 1701, 1651, 1604, 1560, 1529, 1504,
1459, 1418, 1279, 1221, 1136, 1119, 1040, 968, 917, 863, 810,
694, 632, 568, 546 cmꢂ1; Optical rotation ½a 2D4
ꢃ
+13.67 (c 0.75,
740, 572 cmꢂ1; Optical rotation ½a 2D4
ꢃ
+8.66 (c 0.75, DMSO).
DMSO).
5.10. Methyl [3-({[4-(acetyloxy)phenyl]sulfonyl}amino)phenyl]
acetate (20)
5.9.18. {4-Chloro-3-[(2-chloro-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (6d)
To a stirred solution of 12e (300 mg, 1.8 mmol) and pyridine
(0.29 mL, 3.6 mmol) in CH2Cl2 (5.0 mL) was added dropwise a solu-
tion of 22 (426 mg, 1.8 mmol) in CH2Cl2 (3.0 mL) dropwise. After
stirring for 1 h at room temperature, the reaction mixture was di-
luted with 2 M HCl aq and extracted with EtOAc (ꢁ2). The com-
bined organic layers were washed with water, brine, dried over
Na2SO4 and concentrated in vacuo to yield 20, which was used
for the next reaction without further purification; TLC Rf = 0.11
(n-hexane/EtOAc, 7:3);1H NMR (300 MHz, CDCl3) d 7.82–7.74 (m,
2H), 7.24–7.15 (m, 3H), 7.08–6.94 (m, 3H), 6.56 (s, 1H), 3.68 (s,
3H), 3.56 (s, 2H), 2.30 (s, 3H).
Yield 44% in two steps; Ivory powder; TLC Rf = 0.41 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.70 (br s, 1H), 8.56 (br
s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.07–7.00
(m, 2H), 6.96 (dd, J = 9.0, 2.4 Hz, 1H), 6.93–6.80 (m, 2H), 6.76–
6.66 (m, 2H), 4.70–4.60 (m, 1H), 4.28 (dd, J = 9.9, 5.4 Hz, 1H),
4.18 (dd, J = 9.9, 6.3 Hz, 1H), 3.71 (s, 2H), 3.39 (dd, J = 11.7,
2.7 Hz, 1H), 3.25 (dd, J = 11.7, 6.3 Hz, 1H), 2.91 (s, 3H); MS (APCI,
Neg, 20 V) m/z 499 (MꢂH)ꢂ; IR (KBr) 3370, 3228, 3042, 2943,
2888, 2821, 2657, 2558, 1931, 1891, 1711, 1664, 1600, 1586,
1540, 1497, 1467, 1425, 1412, 1347, 1291, 1242, 1223, 1199,
1172, 1150, 1134, 1085, 1054, 984, 939, 915, 856, 825, 815, 775,
752, 728, 709, 693, 659, 606, 600, 577, 558, 475 cmꢂ1; Optical rota-
5.11. Methyl (3-{[(4-hydroxyphenyl)sulfonyl]amino}phenyl)
acetate (21)
tion ½a 2D4
ꢃ
+13.25 (c 0.75, DMSO).
5.9.19. {3-[(2-Chloro-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-ben-
zoxazin-2-yl]methoxy}benzoyl)amino]-4-fluorophenyl}acetic
acid (6e)
To a stirred solution of 20 (1.8 mmol) in MeOH (10 mL) and
DME (5 mL) was added K2CO3 (354 mg, 2.6 mmol). After stirring
for 30 min at room temperature, the resulting insoluble substance
was removed by filtration. The filtrate was concentrated in vacuo
to give a crude product, which was purified by column chromatog-
raphy on silica gel to yield 21 (370 mg, 68% in 2 steps); TLC
Rf = 0.22 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3) d
7.68–7.58 (m, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.06–6.92 (m, 3H),
6.86–6.74 (m, 2H), 6.70–6.40 (br, 1H), 3.68 (s, 3H), 3.56 (s, 2H).
Yield 10% in two steps; White powder; TLC Rf = 0.43 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 8.51–8.42 (m, 1H), 7.88
(d, J = 9.6 Hz, 1H), 7.15–6.80 (m, 7H), 6.76–6.66 (m, 2H), 4.71–
4.61 (m, 1H), 4.26 (dd, J = 9.6, 5.4 Hz, 1H), 4.18 (dd, J = 9.6, 6.3 Hz,
1H), 3.70 (s, 2H), 3.40 (dd, J = 12.0, 3.3 Hz, 1H), 3.25 (dd, J = 12.0,
6.6 Hz, 1H), 2.91 (s, 3H); MS (APCI, Neg, 20 V) m/z 483 (MꢂH)ꢂ;
IR (KBr) 3397, 3244, 3039, 2942, 2888, 2817, 2644, 2552, 2379,
2345, 2279, 2251, 2219, 2174, 2041, 1994, 1922, 1870, 1847,
1803, 1793, 1773, 1717, 1668, 1649, 1638, 1615, 1599, 1548,
1498, 1483, 1468, 1436, 1406, 1375, 1348, 1317, 1290, 1258,
1241, 1224, 1187, 1138, 1098, 1084, 1052, 1010, 969, 938, 916,
908, 897, 876, 859, 845, 824, 815, 792, 776, 752, 707, 681, 647,
616, 602, 589, 579, 542, 532, 515, 503, 486, 474 cmꢂ1; Optical rota-
5.12. (3-{[(4-{[(2S)-4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}phenyl)sulfonyl]amino}phenyl)acetic acid (2b)
To a stirred solution of 21 (370 mg, 1.1 mmol) in DMF (15 mL)
were added Cs2CO3 (750 mg, 2.3 mmol) and 23 (384 mg,
1.1 mmol). After stirring for 2 h at 60 °C, the reaction mixture
was poured into water and extracted with EtOAc (ꢁ2). The com-
tion ½a 2D4
ꢃ
+14.73 (c 0.75, DMSO).

5370
M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
bined organic layers were washed with water, brine, dried over
Na2SO4 and concentrated in vacuo. The resulting residue was puri-
fied by column chromatography on silica gel to yield methyl ester
(282 mg, 51%); TLC Rf = 0.46 (n-hexane/EtOAc, 1:1); 1H NMR
(300 MHz, CDCl3) d 7.69 (d, J = 9.3 Hz, 2H), 7.19 (t, J = 7.8 Hz, 1H),
7.06–6.78 (m, 7H), 6.74–6.65 (m, 2H), 6.36 (s, 1H), 4.67–4.57 (m,
1H), 4.23 (dd, J = 9.6, 5.4 Hz, 1H), 4.13 (dd, J = 9.6, 6.0 Hz, 1H),
3.67 (s, 3H), 3.55 (s, 2H), 3.36 (dd, J = 11.7, 2.7 Hz, 1H), 3.22 (dd,
J = 11.7, 6.6 Hz, 1H), 2.89 (s, 3H).
5.16. (3-{[(4-{[(2S)-4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}phenyl)amino]carbonyl}phenyl)acetic acid (2c)
To a stirred solution of 26 (365 mg, 1.4 mmol) in CH2Cl2 (7 mL)
were added a solution of the above-described crude product 28
(1.5 mmol) in CH2Cl2 (3 mL) and then pyridine (0.24 mL,
3.0 mmol). After stirring for 2 h at room temperature, the reaction
mixture was diluted with water and extracted with EtOAc (ꢁ2).
The combined organic layers were washed with 1 M NaOH aq,
water, brine, dried over Na2SO4 and concentrated in vacuo to give
a crude product, which was used for the next reaction without fur-
ther purification; TLC Rf = 0.49 (n-hexane/EtOAc, 1:1); 1H NMR
(300 MHz, CDCl3) d 7.80–7.72 (m, 3H), 7.55 (d, J = 9.0 Hz, 2H),
7.50–7.44 (m, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.91–6.81 (m, 2H),
6.75–6.65 (m, 2H), 4.70–4.60 (m, 1H), 4.24 (dd, J = 9.6, 5.1 Hz,
1H), 4.14 (dd, J = 9.6, 6.6 Hz, 1H), 3.72 (s, 2H), 3.40 (dd, J = 11.4,
2.7 Hz, 1H), 3.25 (dd, J = 11.4, 6.6 Hz, 1H), 2.91 (s, 3H), 2.05 (s, 3H).
To a stirred solution of the above-described crude product
(1.4 mmol) in MeOH (5 mL) and THF (5 mL) was added 5 M NaOH
aq (3 mL). After stirring for 3 h at room temperature, the reaction
mixture was diluted with 2 M HCl aq and extracted with EtOAc
(ꢁ2). The combined organic layers were washed with water, brine,
dried over Na2SO4 and concentrated in vacuo to give a crude prod-
uct, which was washed with EtOAc/n-hexane to yield 2c (330 mg,
51% in 2 steps) as a pale blue amorphous powder; TLC Rf = 0.34
(CHCl3/MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.86–7.70 (m,
3H), 7.58–7.48 (m, 2H), 7.48–7.40 (m, 2H), 6.98–6.88 (m, 2H),
6.88–6.80 (m, 2H), 6.75–6.64 (m, 2H), 4.68–4.58 (m, 1H), 4.23
(dd, J = 9.6, 4.5 Hz, 1H), 4.11 (dd, J = 9.6, 6.6 Hz, 1H), 3.72 (s, 2H),
3.39 (dd, J = 11.7, 2.7 Hz, 1H), 3.25 (dd, J = 11.7, 6.9 Hz, 1H), 2.90
(s, 3H); MS (APCI, Neg, 20 V) m/z 431 (MꢂH)ꢂ; IR (KBr) 3277,
3042, 2923, 1699, 1648, 1608, 1584, 1510, 1456, 1411, 1301,
1221, 1176, 1136, 1042, 990, 957, 915, 823, 740, 711, 607, 520,
To a stirred solution of methyl ester described above (282 mg,
0.56 mmol) in MeOH (3 mL) and THF (3 mL) was added 5 M
NaOH aq (1.5 mL). After stirring for 1 h at room temperature,
the reaction mixture was poured into 1 M HCl aq and extracted
with EtOAc (ꢁ2). The combined organic layers were washed
with water, brine, dried over Na2SO4 and concentrated in vacuo.
The resulting residue was purified by column chromatography
on silica gel to yield 2b (90 mg, 84%) as a pale blue amorphous
powder; TLC Rf = 0.33 (CHCl3/MeOH, 9:1); 1H NMR (300 MHz,
CDCl3) d 7.63 (d, J = 9.3 Hz, 2H), 7.24–7.17 (m, 1H), 7.13–7.06
(m, 1H), 7.04–6.97 (m, 1H), 6.94–6.70 (m, 8H), 4.67–4.57 (m,
1H), 4.27 (dd, J = 10.2, 5.1 Hz, 1H), 4.14 (dd, J = 10.2, 5.7 Hz,
1H), 3.53 (s, 2H), 3.37 (dd, J = 11.4, 2.4 Hz, 1H), 3.17 (dd,
J = 11.4, 7.2 Hz, 1H), 2.88 (s, 3H); MS (APCI, Neg, 20 V) m/z 467
(MꢂH)ꢂ; IR (KBr) 3254, 2944, 1711, 1594, 1501, 1460, 1414,
1334, 1303, 1248, 1223, 1153, 1093, 1041, 981, 913, 834, 746,
703, 646, 584, 554 cmꢂ1; Optical rotation ½a 2D5
ꢃ
+9.27 (c 0.75,
DMSO).
5.13. (2S)-4-Methyl-2-[(4-nitrophenoxy)methyl]-3,4-dihydro-
2H-1,4-benzoxazin (25)
To a stirred solution of 4-nitrophenol 24 (1 g, 7.2 mmol) in DMF
(20 mL) were added Cs2CO3 (4.73 g, 14.4 mmol) and 23 (2.4 g,
7.2 mmol). After stirring overnight at 60 °C, the reaction mixture
was diluted with water and extracted with EtOAc (ꢁ2). The com-
bined organic layers were washed with water, brine, dried over
Na2SO4 and concentrated in vacuo to yield 25, which was used
for the next reaction without further purification; TLC Rf = 0.52
(n-hexane/EtOAc, 7:3).
459 cmꢂ1; Optical rotation ½a 2D5
ꢃ
+6.23 (c 0.75, DMSO).
6. Biological assay method
6.1. Prostanoid mEP1–4, mDP, hTP, mFP and hIP receptor
binding assay
5.14. 4-{(2S)-4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-yl]
methoxy}aniline (26)
Competitive binding studies were conducted using radiolabeled
ligands and membrane fractions prepared from Chinese hamster
ovary (CHO) cells stably expressing the respective prostanoid
To a stirred solution of 25 (7.2 mmol) in EtOAc (15 mL) and
MeOH (15 mL) was added 10% Pd–C (500 mg) at room temperature
under argon atmosphere. The resulting suspension was vigorously
stirred for 3.5 h at room temperature under hydrogen atmosphere.
Insoluble substance was removed by filtration. The filtrate was
concentrated in vacuo to give a crude product, which was purified
by column chromatography on silica gel to yield 26 (1.2 g, 62% in 2
steps); TLC Rf = 0.12 (toluene/EtOAc, 9:1); 1H NMR (300 MHz,
CDCl3) d 6.90–6.75 (m, 4H), 6.72–6.61 (m, 4H), 4.64–4.54 (m,
1H), 4.16 (dd, J = 9.6, 5.1 Hz, 1H), 4.02 (dd, J = 9.6, 6.9 Hz, 1H),
3.60–3.30 (br, 2H), 3.38 (dd, J = 11.4, 2.4 Hz, 1H), 3.22 (dd,
J = 11.4, 6.6 Hz, 1H), 2.89 (s, 3H).
receptors, mEP1, mEP2, mEP3a, mEP4, mDP, hTP mFP, and hIP.
Membranes from CHO cells expressing prostanoid receptors
were incubated with radioligands (2.5 nM of [3H]PGE2 for mEP1–
4; 2.5 nM of [3H]PGD2 for mDP; 5.0 nM [3H]-SQ29548 for
a
hTP;2.5 nM [3H]PGF2 for mFP; 5.0 nM of [3H]Iloprost for hIP)
and the test compounds at various concentrations in assay buffer
(10 mM KH2PO4–KOH buffer containing 1 mM EDTA, 10 mM
MgCl2, and 100 mM NaCl, pH 6.0 for mEP1–4 and mFP; 25 mM
HEPES–NaOH buffer containing 1 mM EDTA, 5 mM MgCl2 and
10 mM MnCl2, pH 7.4 for mDP; 10 mM Tris–HCl buffer containing
100 mM NaCl, pH 7.5, for hTP; 50 mM Tris–HCl buffer containing
1 mM EDTA and 10 mM MgCl2, pH 7.5, for hIP). Incubation was
carried out at room temperature for 60 min except for mEP1, and
mDP (20 min), and hTP and hIP (30 min). The incubation was ter-
minated by filtration through Whatman GF/B filters. The filters
were washed with ice-cold buffer (10 mM KH2PO4–KOH buffer
containing 100 mM NaCl, pH 6.0 for mEP1–4 and mFP; 10 mM
Tris–HCl buffer containing 100 mM NaCl and 0.01w/v% BSA, pH
7.4 for mDP; 10 mM Tris–HCl buffer containing 100 mM NaCl, pH
7.5, for hTP and hIP), dried for 60 min at 60 °C and the radioactivity
on the filter was measured in 6 mL of liquid scintillation (ACSII)
mixture with a liquid scintillation counter. Nonspecific binding
5.15. Methyl [3-(chlorocarbonyl)phenyl]acetate (28)
To a stirred solution of 3-(2-methoxy-2-oxoethyl)benzoic acid
27 (300 mg, 1.5 mmol) in DME (10 mL) were added oxalyl chlo-
ride (0.34 mL, 3.9 mmol) and DMF (1.2 L, 0.015 mmol). After stir-
ring for 30 min at 40 °C, the reaction mixture was concentrated
in vacuo to give a crude product, which was used for the next
reaction without further purification; 1H NMR (300 MHz, CDCl3)
d 8.10–8.00 (m, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.49 (t, J = 7.5 Hz,
1H), 3.73 (s, 5H).

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 5361–5371
5371
was achieved by adding excess amounts of unlabeled PGE2 (for
mEP1–4), unlabeled PGD2 (for mDP), unlabeled SQ29548 (for
hTP), unlabeled PGF2a (for mFP) or unlabeled Iloprost (for IP) with
oral dosing was prepared using 0.5% MC (10 mg/5 ml/kg). Test
compounds (1 mg/kg) were dosed intravenously to the fasted male
rats (n = 3). Test compounds (10 mg/kg) were dosed orally to the
assay buffer. The concentrations of the test substance required to
inhibit the amounts of the specific binding in the vehicle group
by 50% (IC50 value) were estimated from the regression curve.
The Ki value (M) was calculated according to the following
equation.
fasted male rats (n = 3). After dosing, blood samples (250 ll) were
collected from the jugular vein using a heparinized syringe at the
selected time points (iv: pre-dosing, 2, 5, 15 and 30 min, 1, 2, 4,
6 and 8 h; po: pre-dosing, 5, 15 and 30 min 1, 2, 4, 6 and 8 h,
respectively). The blood samples were ice-chilled and then centri-
fuged at 12,000 rpm for 2 min at 4 °C to obtain plasma, which was
preserved at ꢂ80 °C in a freezer. The AUC, Cmax, Tmax, T1/2, Vss and
CL were obtained by measuring the time course of the plasma con-
centration of the test compounds. Bioavailability (BA) was calcu-
lated according to the following equation:
Ki ¼ IC50=ð1 þ ½Lꢃ=KdÞ
where [L]: concentration of radioligand and Kd: dissociation con-
stant of radiolabeled ligand towards the prostanoid receptors.
6.2. Measurement of the mDP receptor antagonist activity
BAð%Þ ¼ ðAUCpo=DpoÞ=AUCiv=DivÞ ꢁ 100
To confirm that test compounds antagonize the mDP receptor
and to estimate potencies of antagonism for the mDP receptor, a
functional assay was performed by measuring PGD2-stimulated
changes in intracellular second messenger cAMP as an indicator
of receptor function.
where AUCpo: AUC after oral dosing; AUCiv: AUC after intravenous
dosing; Dpo: dosage of oral administration; Div: dosage of intrave-
nous administration.
References and notes
For the assessment of the antagonist activity of test compounds,
a suspension of CHO cells expressing mDP receptor was seeded at a
cell density of 1 ꢁ 105 cells per well and cultivated for 2 days. The
cells in each well were rinsed with minimum essential medium
(MEM), and MEM containing 2 lM of Diclofenac was added to each
well. The cells were incubated for approximately 10 min at 37 °C
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and the culture medium was removed. The assay medium (MEM
containing 0.1% BSA, 1 mM IBMX and 2 lM Diclofenac) was added
to each well and the cells were incubated for approximately 10 min
at 37 °C. The assay medium, assay medium containing 10 nM of
PGD2, or assay medium containing various concentrations of test
compounds and 10 nM of PGD2 was added to each well and the
cells were further incubated for 10 min at 37 °C. The reaction
was terminated by the addition of ice-cold trichloroacetic acid
(TCA; 10 w/v%).
After centrifugation of the reaction mixture, TCA was extracted
by adding a mixture of tri-n-octylamine and chloroform (5:18 v/v)
to the resultant supernatant, mixing and re-centrifugation. The
cAMP level in the resultant aqueous layer (upper layer) was deter-
mined by enzymeimmunoassay using a cAMP assay kit (GE Health-
care UK Ltd). The relative responsiveness (%) of cAMP production
was calculated relative to the maximum increase in cAMP that oc-
curred in the absence of test compound (100%) to estimate of the
IC50 values.
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7. Single dose rat pharmacokinetic study of 2a, 5b and 1
Single dose pharmacokinetics of 2a, 5b and 1 were studied in
rats. Formulation for intravenous injection was prepared using
30% HP-b-CD containing 5% DMSO (1 mg/ml/kg). Formulation for