Discovery of new orally active prostaglandin D2 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2011.08.065

To identify an orally available drug candidate, a series of 3-benzoylaminophenylacetic acids were synthesized and evaluated as prostaglandin D₂ (PGD₂) receptor antagonists. Some of the compounds tested were found to exhibit excellent

Full text of DOI:10.1016/j.bmc.2011.08.065

Bioorganic & Medicinal Chemistry 19 (2011) 6935–6948
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of new orally active prostaglandin D2 receptor antagonists
Maki Iwahashi a, , Atsushi Naganawa a, Atsushi Kinoshita a, Atsushi Shimabukuro a, Toshihiko Nishiyama a,
⇑
Seiji Ogawa a, Yoko Matsunaga a, Kohki Tsukamoto a, Yutaka Okada a, Ryoji Matsumoto b, Fumio Nambu a,
Rie Oumi a, Yoshihiko Odagaki a, Jun Katagi a, Koji Yano a, Kousuke Tani 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 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 an orally available drug candidate, a series of 3-benzoylaminophenylacetic acids were synthe-
sized and evaluated as prostaglandin D2 (PGD2) receptor antagonists. Some of the compounds tested
were found to exhibit excellent inhibitory activity against cAMP accumulation in human platelet rich
plasma (hPRP), which is one of the indexes of DP antagonism. The optimization process including
improvement of the physicochemical properties such as solubility, which may result in an improved
pharmacokinetic (PK) profile, is presented. Optimized compounds were studied for their pharmacokinet-
ics and in vivo potential. A structure–activity relationship study is also presented. Some of the test com-
pounds were found to have in vivo efficacy towards the inhibition of PGD2-induced and OVA-induced
vascular permeability in guinea pig conjunctiva.
Received 11 August 2011
Accepted 30 August 2011
Available online 2 September 2011
Keywords:
Prostaglandin
DP receptor
Antagonist
Phenylacetic acid
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Prostanoids are oxidative metabolites of arachidonic acid and
consist of prostaglandins (PGs) and thromboxane (TX). In vivo, they
act on the tissues where they were synthesized via specific G-pro-
tein coupled receptors named TP, IP, EP, FP and DP. Each of these
receptors has been cloned, expressed and characterized.
Test compounds listed in Tables 1–3 were synthesized as out-
lined in Schemes 1 and 2. Synthesis of methyl -disubstituted
phenylacetates, which can be used as the intermediates for the
a,a
synthesis of 10a–c and 11a–b, was carried out as described in
Scheme 1a. Conventional nitration of
a,a-dimethyl(4-chloro-
Characterization of these receptors at the molecular level has
resulted in renewed interest in this field, but few selective agonists
and antagonists of human prostanoid receptors are available.1 As a
result, the influence of each receptor on various pathologies is still
being established by using potent but poorly selective ligands. The
situation is similar with regard to DP receptor antagonists. Only a
few antagonists such as S-555739 are undergoing clinical trials for
the treatment of allergic rhinitis. Recently laropiprant (MK-0524)2
has proven to be effective in suppressing both subjective and
objective manifestations of niacin-induced vasodilatation.
In our previous papers,3 we reported on the discovery of a new
orally active indole-based structure 1b as a new class of DP recep-
tor antagonists starting from Indomethacin (Fig. 1). We also re-
ported on the discovery of another indole-based antagonist4a and
3-benzoylaminophenylacetic acids 2a–b4b as cost-effective DP
receptor antagonists. Here we report on the discovery of 3-ben-
zoylaminophenylacetic acids 5e and 10c possessing more im-
proved PK profiles.
phenyl)acetonitrile 13 and -dimethylene(4-chlorophenyl)ace-
a,a
tonitrile 14 with nitric acid-sulfuric acid afforded 15 and 18,
respectively. Acidic hydrolysis of the nitriles 15 and 18 provided
carboxylic acids 16 and 19, respectively. Esterification gave the
corresponding methyl esters 17 and 20, respectively. Catalytic
hydrogenation of their nitro groups afforded the corresponding
anilines 21 and 22, respectively.
Synthesis of ethyl
a,a-difluoro(3-amino-4-chlorophenyl)ace-
tate 29 is described in Scheme 1b. Treatment of 4-chloro-3-nitro-
benzaldehyde 23 with trimethylsilylcyanide in the presence of a
catalytic amount of zinc iodide afforded 24, acidic hydrolysis of
which followed by conventional esterification provided 26. Oxida-
tion of the
-ketoester 27, treatment of which with DAST gave ethyl
orophenylacetate 28. Reduction of 28 with iron metal resulted in
ethyl -difluoro(3-amino-4-chlorophenyl)acetate 29. Synthesis
a
-hydroxy ester 26 with sodium hypochlorite afforded
a
a,a-diflu-
a,a
of 2d–f, 3c–e, 5c–e, 10a–c, 11a–b and 12 is outlined in Scheme 2.
O-Alkylation of methyl 4-hydroxybenzoates 30 with the tosylate
31, preparation of which was reported previously,4a provided 32.
Alkaline hydrolysis of 32 followed by acid chloride formation affor-
ded 34a. Compounds 34b–d were previously reported.4a N-Acyla-
tion of methyl 3-aminophenylacetates 35a–c, 21, 22 and 29 with
⇑
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.2011.08.065

6936
M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
R
CO2H
CO2H
CO2H
R
A
R
N
NH
R
B
NH Me
Cl
O
O
O
O
O
O
O
O
Me
O
N
Me
N
Me
N
Me
R=H 1a
R=Me 1b
R=H 2a
R=Me 2b
R=H 5e
R=Me 10c
Figure 1. Molecular design of 3-benzoylaminophenylacetic acids 5e and 10c.
Table 1
Table 2
Optimization of A and B rings
Effects of chemical modifications of the N-methyl benzomorpholine moiety on
activity profiles
CO2H
2
CO2H
A
NH
NH Me
2
3
W
Cl
O
O
6
B
O
R
O
Y
X
Me
O
5
N
Me
a
Compd
R
IC50 (nM) mDP
Binding Ki (nM) hIP
O
a
Compd
W
X,Y
Binding Ki (nM) IC50 (nM) Binding Ki (nM) hIP
S
R
5e
7.6
>10,000
N
Me
O
mDP
1a
1b
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
4a
4b
5a
5b
5c
5d
5e
1700
16
310
18
NTb
2.4
220
3.3
20
2500
37
6400
3600
6
7
4.3
1.5
24
>10,000
>10,000
>10,000
>10,000
H
H
H
H
H
H
H
2-Me
2-Cl
O
X
5
16
2700
2,3-Me NTb
2,5-Me NTb
2,6-Me NTb
22
28
3.5
4.6
6.0
12
>10,000
>10,000
>10,000
2600
1400
5200
a X=H
R
S
b X=Me
Me 2-Me
Me 2-Cl
31
26
O
O
Me 2,3-Me NTb
Me 2,5-Me NTb
Me 2,6-Me NTb
8
9
14
5.7
4.8
1.8
7.4
NTb
4.3
54
2600
>10,000
>10,000
>10,000
1800
1600
3500
O
F
F
2-Me
2-Cl
2-Me
2-Cl
2,3-Me 28
2,5-Me NTb
2,6-Me 6.4
19
17
7.7
13
27
>10,000
O
Cl
Cl
Cl
Cl
Cl
a
IC50 (nM): mDP receptor antagonist activity.
5.1
7.6
1800
>10,000
a
IC50 (nM): mDP receptor antagonist activity.
NT: not tested.
b
with the above-described nosylates 39a–e afforded 6, 7a–b, 8
and 9, respectively. Synthesis of indol-4-yl acetic acid analog 1a
from 45 is outlined in Scheme 4. 2-(1H-Indol-4-yl)acetic acid 45
was converted to benzyl ester 46 by the conventional acidic ester-
ification condition. N-Acylation of 46 with acid chloride 474a using
the reported reaction conditions4a followed by deprotection by the
catalytic hydrogenolysis afforded 1a.
an optional acid chloride among 34a–d followed by alkaline hydro-
lysis resulted in 2d–f, 3c–e, 5c–e, 10a–c, 11a–b and 12. Synthesis
of building blocks for the chemical modification of the N-methyl
benzomorpholine moiety of 5e was carried out as shown in
Scheme 3a. O-Alkylation of 2-bromo-4-methylphenol with (S)-
(+)-glycidyl 3-nitrobenzenesulfonate afforded 37, which was then
treated with n-butyllithium followed by sulfonylation with 4-
nitrobenzenesulfonyl chloride afforded 39a. According to the same
procedure as described for the preparation of 39a from 38a, com-
pounds 39b–e were prepared from 38b–e.4a Synthesis of 6, 7a–b,
8 and 9 is described in Scheme 3b. O-Acetylation of 2,6-di-
methyl-4-hydroxybenzoic acid 40 by the conventional procedure
followed by the acid chloride formation with oxalyl chloride in
the presence of a catalytic amount of dimethyl formamide afforded
42. N-Acylation of methyl (3-amino-4-chlorophenyl)acetate 35c
with the acid chloride 42 afforded 43, methanolysis of which with
potassium carbonate in methanol afforded 44. O-Alkylation of 44
3. Results and discussion
The compounds listed in Tables 1–3 were all tested for inhibi-
tion of the specific binding of a radiolabeled ligand, [3H]PGD2, to
membrane fractions prepared from cells stably expressing the
mDP receptor. They were also evaluated for their potency to antag-
onize mDP receptors by measuring PGD2-stimulated changes in
intracellular second messenger cAMP (cyclic adenosine 30,50-
monophosphate) as an indicator of receptor function. The mDP
antagonism was measured in the presence of 0.1% of BSA for the
discovery of potent antagonists in the protein-rich in vivo animal
models. Because of their close homology to human receptors, all

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
6937
Table 3
Effects of
6 resulted in the retention of the potent activities. For these rea-
sons, our focus was placed on further optimization of the ring B
of 2a. Results are summarized in Table 1. Introduction of 2-methyl,
2-chloro, 2,3-dimethyl, 2,5-dimethyl and 2,6-dimethyl residues
into the ring B of 2a afforded 2a–f, respectively. Among those
tested, 2b–f showed more potent binding affinities and/or antago-
nist activities relative to 2a. Above all, 2b and 2f exhibited more
potent antagonist activities than 2c–e. The same chemical modifi-
cation as described above in the ring B of the 3-benzoylamino-4-
methylphenylacetic acid framework afforded 3a–e, respectively.
All of these compounds exhibited nearly equipotent antagonist
activity as 2b and 2f. Introduction of 2-methyl and 2-chloro resi-
dues into the ring B of 3-benzoylamino-4-fluorophenylacetic acid
and 3-benzoylamino-4-chlorophenyl acetic acid frameworks affor-
ded 4a–b and 5a–b, respectively with tendency to retain strong
antagonist activity. Introduction of another methyl residue into
the ring B of 5a afforded 5c–e, respectively. Among the tested com-
pounds, 2,3-dimethylbenzoyl analog 5c showed nearly 10-fold less
potent antagonist activity relative to 5b and 5d–e. As described
above, 2-methylbenzoylaminophenylacetic acid analogs 2b, 3a
and 4a and 2,6-dimethylbenzoylaminophenylacetic acid analogs
2f, 3e and 5e tended to show more potent antagonist activity than
the others almost without exception. Especially, these 2,6-dim-
ethylbenzoyl analogs, which showed sufficient selectivity for hIP
receptor affinity, were expected to show good PK profiles due to
their presumed metabolic stability after oral dosing.
a
,
a
-substitution of the phenylacetic acid moiety on activity profiles
R
CO2H
R
NH
2
Cl
3
O
O
6
O
Y
X
5
N
Me
Compd
R
X,Y
IC50 (nM)
Binding Ki (nM) hIP
mDPa
hDPb
1b
5b
2e
5e
10a
10b
10c
11a
11b
12
2.4
4.3
28
7.6
7.0
43
0.7
26
1.0
14
57
37
1600
>10,000
>10,000
3300
3300
>10,000
1700
>10,000
520
H
H
H
Me
Me
Me
–CH2–CH2–
–CH2–CH2–
F
2-Cl
>50
NTc
4.4
280
32
2,5-Me
2,6-Me
2-Cl
2,5-Me
2,6-Me
2,5-Me
2,6-Me
2-Cl
8.1
19
6.5
NTc
a
mDP: mDP receptor antagonist activity.
hDP: hDP PRP assay, inhibition of the accumulation of cAMP in human platelet
b
The effects of the chemical modifications of the (2S)-N-meth-
ylbenzmorpholin-2-yl moiety on activity profiles were
investigated (Table 2). Replacement of the (2S)-N-meth-
ylbenzmorpholin-2-yl moiety of 5e with a (2R)-2,3-dihydro-1,4-
benzodioxin-2-yl, (3R)-dihydrobenzofuran-3-yl, (2S)-dihydroben-
zofuran-2-yl and 1,3-benzodioxol-2-yl moieties afforded 6–9,
respectively. Among the tested compounds, 6 and 7a exhibited
more potent antagonist activities than 7b, 8 and 9 with excellent
selectivity for the hIP receptor. Besides, the 2S-configuration of
5e, 2R-configuration of 6 and 3R-configuration of 7a were found
to be beneficial for the more potent activity relative to their corre-
sponding enantiomers.
rich plasma challenged with PGD2.
c
NT: not tested.
the DP receptor affinities and antagonist activities are assayed
using the mouse receptor unless otherwise noted.
In our previous paper,4b we reported on the discovery of a new
prostaglandin D2 (PGD2) receptor antagonist 2a. Introduction of a
substituent into the 2-position of the B ring was found to be ben-
eficial for increased activities. Introduction of a substituent into the
2-position of the ring A of 2a resulted in the reduction of the activ-
ities while introduction of a substituent into the positions 4, 5 and
R1
R1
R2
R1
R1
R1
R1
CN
CO2Me
NH2
d
a
NO2
Cl
Cl
Cl
15 : R1 = Me R2 = CN
16 : R1 = Me R2 = CO2H
17 : R1 = Me R2 = CO2Me
21 : R1 = Me
22 : R1 = CH2CH2
13 : R1 = Me
14 : R1 = CH2CH2
b
c
18 : R1 = CH2CH2 R2 = CN
19 : R1 = CH2CH2 R2 = CO2H
20 : R1 = CH2CH2 R2 = CO2Me
b
c
Scheme 1a. Synthesis of methyl 2, 2-disubstituted (3-amino-4-chlorophenyl)acetates. Reagents: (a) HNO3, H2SO4; (b) 50% H2SO4; (c) SOCl2, MeOH; (d) H2, Pd(OH)2–C, EtOAc.
O
O
O
TMSO
CN
CHO
F
F
HO
R1
O
O
OEt
NO2
OEt
R2
a
b
e
d
NO2
NO2
NO2
Cl
Cl
24
Cl
Cl
27
Cl
23
25 : R1 = H
26 : R1 = Et
28 : R2 = NO2
29 : R2 = NH2
c
f
Scheme 1b. Synthesis of ethyl 2,2-difluoro(3-amino-4-chlorophenyl)acetate. Reagents: (a) TMSCN, cat ZnI2, CH2Cl2; (b) concd HCl, AcOH; (c) EtOH, TsOH; (d) NaOCl, AcOH;
(e) DAST, CH2Cl2; (f) Fe, AcOH–H2O.

6938
M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
R
OMe
X
O
O
a
TsO
d, e
2d-f, 3c-e, 5c-e,
10a-c, 11a-b and 12
O
O
X
+
O
N
Me
Y
OH
Y
N
Me
30 : X,Y=2,6-Me
31
CO2Me
32 : R=OMe X,Y=2,6-Me
33 : R=OH X,Y=2,6-Me
b
c
34a : R=Cl
X,Y=2,6-Me
NH2
W
34b : R=Cl
34c : R=Cl
34d : R=Cl
X,Y=2,3-Me
X,Y=2,5-Me
X=H Y=2-Cl
35a : W=H
35b : W=Me
35c : W=Cl
Scheme 2. Synthesis of 2d–f, 3c–e, 5c–e, 10a–c, 11a–b and 12. Reagents: (a) Cs2CO3, DMF; (b) NaOH, 2-ethoxyethanol, dioxane; (c) (COCl)2, DMF, DME; (d) 21, 22, 29, and
35a–c, Py, CH2Cl2; (e) NaOH, MeOH, THF.
X
CO2R
Br
Me
Br
Me
a
b
Me
b, c
1a
O
HO
O
H
O
N
H
38a : X=OH
39a : X=ONs
36
37
c
45 : R = H
46 : R = Bn
a
O
O
O
HO
c
c
NsO
Cl
O
38b
O
39b
O
O
HO
NsO
N
Me
47
O
O
Scheme 4. Synthesis of 1a. Reagents: (a) BnOH, TsCl, pyridine, toluene; (b) 47,
NaOH, TBACl, CH2Cl2; (c) H2, Pd(OH)2–C, EtOAc.
38c
38d
39c
39d
HO
HO
NsO
NsO
O
O
c
c
Table 4
Effects of the substituents on the B ring on solubility
O
O
O
O
Compd
Solubilitya
(lg/mL)
5a
5d
129
17
38e
39e
5e
407
139
490
321
8.2
10a
10c
11b
1b
Scheme 3a. Synthesis of 39a–e. Reagents: (a) (S)-(+)-glycidyl 3-nitrobenzenesulf-
onate, Cs2CO3, DMF; (b) n-BuLi, THF; (c) 4-nitrobenzenesulfonyl chloride, Et3N,
DMAP, THF.
a
Solubility: kinetic solubility.
CO2Me
OH Me
X
Me
a
c
Table 5
O
O
Inhibitory effects of 5e and 10c on PGD2-induced vascular permeability in guinea pig
conjunctiva (n = 8)
NH Me
Me
OH
Me
OAc
Cl
O
Compd
Dose (mg/kg, po)
% Inhibition
41 : X = OH
42 : X = Cl
40
b
1b
5e
10c
0.1
0.1
0.1
67⁄
55⁄
58⁄
Me
OR
43 : R = Ac
44 : R = H
d
⁄⁄0.01 versus the control as measured with Dunnetts test.
Inhibition of increase in conjunctival vascular permeability caused by topical
application of PGD2 (0.01%, 20 L/eye) in guinea pigs. All antagonists were
e, f
6, 7a-b, 8 and 9
l
Scheme 3b. Synthesis of 6, 7a–b, 8 and 9. Reagents: (a) Ac2O, Py; (b) (COCl)2, DMF,
toluene; (c) 35c, Py, CH2Cl2; (d) K2CO3, MeOH; (e) 39a–e, Cs2CO3, DMF; (f) NaOH,
MeOH, THF.
administered po 1 h before the challenge.
antagonist, 10a–c, 11a–b and 12 were synthesized and evaluated
To avoid the predicted conjugation metabolism of the carbox-
as shown in Table 3.
ylic acid function, effect of the
tic acid moiety was investigated (Table 3). To look for
disubstituted compounds with an acceptable potency as a DP
a
,
a
-disubstitution of the phenylace-
Introduction of a a,a-dimethyl residue into the phenylacetic
acid moiety of 5b, 2e and 5e afforded 10a–c, respectively. Com-
pounds 10a and 10c showed more potent antagonist activity than
a,a
-

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
6939
Table 6
Table 7
Inhibitory effects of 5e on OVA-induced vascular permeability in guinea pig
conjunctiva (n = 7)
Rat PK study of 5e and 10c
Compd
Route
Dose
(mg/
kg)
AUCinf
g h/
mL)
Cmax
g/
mL)
CLtot
(mL/
min/kg)
T1/2
(h)
Vss
(mL/
kg)
BA
(%)
Compd
Dose (mg/kg, po)
% Inhibition
(l
(
l
1b
1
3
1
23
41*
46⁄
68
5e
iv
po
1
1
11.3
8.12
0.93
1.6
6.2
5.8
622
5e
72
87
Pyrilamine
1 (mg/kg, iv)
10c
iv
po
1
1
9.52
8.28
1.8
6.5
5.6
669
1.67
Inhibition of increase in conjunctival vascular permeability caused by topical
application of OVA (1%, 20 L/eye) in actively sensitized guinea pigs. All antagonists
l
PK study of 5e and 10c.
were administered po 1 h before the antigen challenge.
*
0.1 versus the control as measured by Dunnetts test.
an orally dosed molecule, solubility of phenylacetic acids 5a and
5d–e, -dimethylphenylacetic acids 10a and 10c, and -dime-
a,a
a,a
thylenephenylacetic acid 11b, which possess 2,6-disubstituted-
and 2-substituted-3-benzoylaminophenylacetic acid moieties,
were evaluated5 as shown in Table 4. Among the tested com-
pounds, 3-(2,6-dimethylbenzoylamino)phenylacetic acid analogs
5e, 10c and 11b showed remarkably improved solubility probably
because of the predicted change in their conformation based on the
complete loss of the planarity of the N-benzoylanilide moiety or
the loss of the conjugation of the two rings A and B via the anilide
moiety. The 2,6-disubstitution of the N-benzoyl moiety was proven
to be much more effective than the a,a-disubstitution of the phen-
ylacetic acid moiety as illustrated by the differences in solubility of
5e and 10c.
As shown in Table 5, the inhibitory effects of 5e and 10c, which
showed good antagonist activities and relatively better solubilities
among the tested compounds, on PGD2-induced vascular perme-
ability in guinea pig conjunctiva were investigated. Compound
1b was included for purposes of comparison. After oral dosing
(0.1 mg/kg, po), these three compounds exhibited nearly equipo-
tent efficacy 67%, 55% and 58%, respectively.
The inhibitory effects of 1b and 5e on OVA-induced vascular
permeability in guinea pig conjunctiva were further investigated
as shown in Table 6. The antihistaminic agent pyrilamine was in-
cluded as a positive control. Compound 5e demonstrated signifi-
cant efficacy after its 1 mg/kg of oral dosing while 3 mg/kg of
oral dosing of 1b was required to show equipotent efficacy.
Compounds 5e and 10c, which possess potent activities with
excellent solubilities, were evaluated for their PK profiles. A sin-
gle-dose rat PK study after oral dosing (1 mg/kg) and intravenous
dosing (1 mg/kg) of each of the compounds was conducted. As
shown in Table 7, both the compounds demonstrated excellent
Cmax, T1/2, tissue distribution (Vss), oral exposure (AUC) and bio-
availability (BA). Especially the value of clearance (CLtot) after iv
dosing was found to be much smaller than those of the chemical
leads 1b and 2a.4b As a result, the synthetic approach described
above was found to be effective to provide novel compounds with
improved PK profiles.
Figure 2. Conformational analysis of 1b and 5e using X-ray crystallography.
10b. Especially, 10c showed nearly 10-fold more potency than 5e.
Introduction of the
ded 11a–b, respectively. Again, 11b was found to be more potent
than 11a. Introduction of the -difluoro moiety into 5b afforded
a,a-dimethylene moiety into 2e and 5e affor-
a,a
12 with reduced antagonist activity. All the tested compounds
showed good to excellent selectivity for the hIP receptor. Espe-
cially, 10c and 11b showed very potent antagonist activities in
addition to their excellent selectivities for hIP receptor.
To estimate the potency of antagonism of these compounds
against the hDP receptor under conditions that are close to the
in vivo environment, PGD2-stimulated cAMP production in human
platelet rich plasma (hPRP) was analyzed as an indicator of recep-
tor function. Compounds 1b, 5b, 5e, 10a–c, 11a–b were evaluated
for their inhibition of cAMP accumulation in hPRP. Among the
tested, 5e, 10c and 11b were found to exhibit excellent inhibitory
activities while 10a and 10b showed less potency.
In an attempt to improve the PK profiles of the newly identified
DP antagonists, enhancement of their physicochemical properties
such as solubility is known to be effective. 2,6-Disubstitution and
2-substitution of the N-benzoyl moiety may provide effective ap-
proaches for such a purpose because of the predicted good solubil-
ity due to their non-planar conformations.
3.1. Conformational analysis of 1b and 5e based on the SAR
described above and X-ray crystallography (Fig. 2)
As shown in Table 1, compounds 2f exhibited nearly the same
in vitro potency as 3e and 5e while these three compounds showed
much more potent in vitro potency relative to 2a. Thus, the 2,6-di-
methyl substituent of the benzoyl moiety was found to be effective
for the increased in vitro activity while 4-chloro group of the ring A
of 5e was considered to be effective for the presumed improve-
ment of the PK profile based on the increased in vivo potency in Ta-
ble 6. On the basis of the data described above, the amide carbonyl
moieties of these three compounds were assumed not to be in the
same plane as the phenyl ring A. The 2-methyl group of the indole
nucleus of 1b was considered to play the same role as that of the
2,6-dimethyl group of 2f, 3e and 5e.
To confirm the improvement of the solubility, which is one of
the important factors which helps to improve the PK profiles of

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M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
Further conformational analysis of 1b and 5e by X-ray crystal-
mixture was poured into ice-water, and extracted with EtOAc
(ꢁ2). The combined organic layers were washed with water, brine,
dried over Na2SO4 and evaporated to yield 15 (4.6 g, 97% yield) as a
pale yellow solid; TLC Rf = 0.30 (n-hexane/EtOAc, 7:3); 1H NMR
(300 MHz, CDCl3) d 7.94 (dd, J = 2.4, 0.3 Hz, 1H), 7.68 (dd, J = 8.7,
2.4 Hz, 1H), 7.60 (dd, J = 8.7, 0.3 Hz, 1H), 1.77 (s, 6H).
lography was carried out. As presented in Figure 2, the benzoyl
moiety of 1b and the 2,6-dimethylbenzoyl moiety of 5e were found
not to be in the same plane as the indole nucleus of 1b and the phe-
nyl ring A (Table 1) of 5e, respectively. Both the N-benzoyl rings of
1b and 5e were found to occupy a nearly orthogonal orientation to
the core rings such as the indole nucleus and phenyl ring A, respec-
tively. Based on the SAR presented in Table 1, the above-described
conformation of 1b and 5e was strongly suggested to be beneficial
for the dramatic increase of their activities relative to those of 1a
and 2a, respectively. Experimental details were described in the
‘Section 5’ part.
Compound 18 was prepared as described above.
5.2.2. 1-(4-Chloro-3-nitrophenyl)cyclopropanecarbonitrile (18)
Yield 41%; Pale yellow powder; TLC Rf = 0.43 (n-hexane/EtOAc,
7:3); 1H NMR (300 MHz, CDCl3) d 7.71 (d, J = 2.4 Hz, 1H), 7.57 (d,
J = 8.1 Hz, 1H), 7.52 (dd, J = 8.1, 2.4 Hz, 1H), 1.91–1.84 (m, 2H),
1.52–1.45 (m, 2H).
4. Conclusion
5.3. General procedure for the preparation of 16 and 19
A series of 3-benzoylaminophenyl acetic acid analogs were syn-
thesized and evaluated for their mDP receptor affinity and/or
antagonist activities. Compounds 1b, 5b, 5e, 10a–c, 11a–b were
evaluated for their inhibition of cAMP accumulation in hPRP which
is one of the indexes of DP antagonism. Among the tested com-
pounds, 5e, 10c and 11b were found to exhibit excellent inhibitory
activities while 10a and 10b were less potent. The solubilities of
compounds 1b, 5a, 5d–e, 10a, 10c, 11b were also tested in an effort
to improve their PK profiles. As a result, we succeeded in the dis-
covery of some compounds possessing excellent activities, recep-
tor selectivities and PK profiles. These compounds showed
in vivo efficacy towards the inhibition of PGD2-induced and OVA-
induced vascular permeability in guinea pig conjunctiva.
5.3.1. 2-(4-Chloro-3-nitrophenyl)-2-methylpropanoic acid (16)
To a stirred suspension of 15 (3.0 g, 13.2 mmol) in water
(5.0 mL) was added concH2SO4 (5.0 mL) at room temperature. After
stirring for 5 h under reflux, the reaction mixture was poured into
ice-water, and extracted with EtOAc (ꢁ2). The combined organic
layers were washed with water, brine, dried over Na2SO4 and con-
centrated in vacuo. The resulting residue was purified by recrystal-
lization from EtOAc and n-hexane to yield 16 (2.26 g, 70% yield) as
a
white powder; TLC Rf = 0.47 (CHCl3/MeOH, 9:1); 1H NMR
(300 MHz, CDCl3) d 7.91 (d, J = 2.1 Hz, 1H), 7.58–7.50 (m, 2H),
1.64 (s, 6H).
Compound 19 was prepared as described above.
5.3.2. 1-(4-Chloro-3-nitrophenyl)cyclopropanecarboxylic acid
(19)
5. Experimental
Yield 66%; Ivory powder; TLC Rf = 0.43 (CHCl3/MeOH, 9:1); 1H
NMR (300 MHz, CDCl3) d 7.88–7.86 (m, 1H), 7.55–7.47 (m, 2H),
1.79–1.74 (m, 2H), 1.33–1.27 (m, 2H).
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, Varian GEM-
INI-200 or VXR-200s spectrometer using deuterated chloroform
(CDCl3) and deuterated methanol (CD3OD) as the solvent. Fast
atom bombardment (FABMS, HRMS) and electron ionization (EI)
mass spectra were obtained on a JEOL JMS-DX303HF spectrometer.
Atmospheric pressure chemical ionization (APCI) mass spectra
were determined on a HITACHI MI200H spectrometer. Infrared
spectra (IR) were measured in a Perkin-Elmer FT-IR 1760X spec-
trometer. Melting points and results of elemental analyses were
uncorrected. Column chromatography was carried out on silica
gel [Merck Silica Gel 60 (0.063–0.200 mm), Wako gel C-200, or Fuji
Silysia FL60D]. Thin layer chromatography was performed on silica
gel (Merck TLC or HPTLC plates, Silica Gel 60 F254). The following
abbreviations for solvents and reagents are used; diethylaminosul-
fur trifluoride (DAST), trimethylsilyl cyanide (TMSCN), t-butyl
methyl ether (MTBE), triethylamine (TEA), tetrabutylammonium
chloride (TBACl), acetic acid (AcOH), diethyl ether (Et2O), N,N-
dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol
(EtOH), ethyl acetate (EtOAc), methanol (MeOH), tetrahydrofuran
(THF), dichloromethane (CH2Cl2), chloroform (CHCl3), dimethoxy-
ethane (DME), acetonitrile (CH3CN), 4-(dimethylamino)pyridine
(DMAP).
5.4. General procedure for the preparation of 17 and 20
5.4.1. Methyl 2-(4-chloro-3-nitrophenyl)-2-methylpropanoate
(17)
To stirred MeOH (10 mL) were added dropwise thionyl chloride
(2.1 mL, 28.7 mmol) at ꢀ10 °C and
a solution of 16 (3.5 g,
14.4 mmol) in MeOH (10 mL) and DME (2 mL) at 0 °C. After stirring
for 1 h while gradually warmed up to room temperature and 3 h at
40 °C, the reaction mixture was cooled to room temperature and
concentrated in vacuo to yield 17 (3.8 g, quant), which was used
for the next reaction without further purification; TLC Rf = 0.42
(n-hexane/EtOAc, 4:1); 1H NMR (300 MHz, CDCl3) d 7.88–7.85
(m, 1H), 7.51–7.47 (m, 2H), 3.68 (s, 3H), 1.61 (s, 6H).
Compound 20 was prepared as described above.
5.4.2. Methyl 1-(4-chloro-3-
nitrophenyl)cyclopropanecarboxylate (20)
Pale yellow oil; TLC Rf = 0.62 (n-hexane/EtOAc, 2:1); 1H NMR
(300 MHz, CDCl3) d 7.87–7.83 (m, 1H), 7.54–7.46 (m, 2H), 3.65 (s,
3H), 1.73–1.66 (m, 2H), 1.25–1.19 (m, 2H).
5.5. General procedure for the preparation of 21 and 22
5.5.1. Methyl 2-(3-amino-4-chlorophenyl)-2-methylpropanoate
(21)
5.2. General procedure for the preparation of 15 and 18
To a stirred solution of 17 (1.2 g, 4.66 mmol) in EtOAc (10 mL)
and DMSO (16.5 lL, 0.233 mmol) was added Pd(OH)2–C (240 mg)
5.2.1. 2-(4-Chloro-3-nitrophenyl)-2-methylpropanenitrile (15)
To conc H2SO4 (5.7 mL, 107 mmol) were added successively
HNO3 (5.4 mL, 840 mmol) and then 13 (3.77 g, 210 mmol) at
ꢀ15 °C. After stirring for 2 h at room temperature, the reaction
at room temperature. The resulting suspension was vigorously stir-
red for 5 h at room temperature under hydrogen atmosphere.
Insoluble substance was removed by filtration. The filtrate was
washed with 0.1 M HCl, water, brine, dried over MgSO4 and

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
6941
concentrated in vacuo to yield 21 (3.21 g, quant) as a yellow liquid;
TLC Rf = 0.43 (n-hexane/EtOAc, 2:1); 1H NMR (300 MHz, CDCl3) d
7.18 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.66 (dd, J = 8.7,
2.4 Hz, 1H), 4.04 (br s, 2H), 3.65 (s, 3H), 1.53 (s, 6H).
sphere. After stirring for 2 h at room temperature and 4 h at
45 °C, the reaction mixture was poured into water and extracted
with EtOAc (ꢁ2). The combined organic layers were washed with
sat NaHCO3 aq, water, brine, dried over MgSO4 and concentrated
in vacuo to give a crude product, which was purified by column
chromatography on silica gel to yield 28 (278 mg, 62%) as a yellow
oil; TLC Rf = 0.57 (n-hexane/EtOAc, 4:1); 1H NMR (300 MHz, CDCl3)
d 8.13 (d, J = 3.0 Hz, 1H), 7.77 (dd, J = 8.1, 3.0 Hz, 1H), 7.68 (d,
J = 8.1 Hz, 1H), 4.34 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H).
Compound 22 was prepared as described above.
5.5.2. Methyl 1-(3-amino-4-
chlorophenyl)cyclopropanecarboxylate (22)
TLC Rf = 0.58 (n-hexane/EtOAc, 2:1); 1H NMR (300 MHz, CDCl3)
d 7.16 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 6.68–6.63 (m, 1H),
4.02 (br s, 2H), 1.60–1.52 (m, 2H), 1.17–1.10 (m, 2H).
5.11. Ethyl (3-amino-4-chlorophenyl)(difluoro)acetate (29)
5.6. (4-Chloro-3-nitrophenyl)[(trimethylsilyl)oxy]acetonitrile
(24)
To a stirred solution of 28 (253 mg, 0.907 mmol) in AcOH (3 mL)
and water (0.3 mL) was added Fe (253 mg, 4.54 mmol) at 80 °C.
After stirring for 30 min at 80 °C, the reaction mixture was cooled
to room temperature, poured into ice and extracted with EtOAc
(ꢁ2). The combined organic layers were washed with water, brine,
dried over MgSO4 and concentrated in vacuo to yield 29 (194 mg,
73%) as a dark brown oil, which was used for the next reaction
without further purification; TLC Rf = 0.48 (n-hexane/EtOAc, 4:1);
1H NMR (300 MHz, CDCl3) d 7.31 (d, J = 3.0 Hz, 1H), 6.99 (dd,
J = 8.1, 3.0 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 4.50–3.75 (br, 2H),
4.29 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H).
To stirred solution of 23 (3.71 g, 20.1 mmol) and Zn2I (128 mg,
0.402 mmol) in CH2Cl2 (50 mL) was added TMSCN (2.9 mL,
22.1 mmol) at 0 °C under argon atmosphere. After stirring for 1 h,
the reaction mixture was poured into 1 M HCl and extracted with
EtOAc (ꢁ2). The combined organic layers were washed with water,
brine, dried over MgSO4 and concentrated in vacuo to yield 24 as a
dark brown oil, which was used for the next reaction without fur-
ther purification; TLC Rf = 0.55 (n-hexane/EtOAc, 4:1).
5.7. (4-Chloro-3-nitrophenyl)(hydroxy)acetic acid (25)
5.12. Methyl 2,6-dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoate (32)
To a stirred solution of 24 (20.1 mmol) in AcOH (30 mL) was
added conc HCl (30 mL). After stirring overnight at 90 °C, the reac-
tion mixture was cooled to room temperature, poured into ice and
extracted with EtOAc (ꢁ2). The combined organic layers were
washed with water, brine, dried over MgSO4 and concentrated in
vacuo to yield 25 (4.48 g, 96% in 2 steps) as a yellow oil, which
was used for the next reaction without further purification; TLC
Rf = 0.14 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3) d 8.04
(d, J = 3.0 Hz, 1H), 7.67 (dd, J = 8.1, 3.0 Hz, 1H), 7.57 (d, J = 8.1 Hz,
1H), 6.00–4.50 (br, 2H).
To a stirred solution of 30 (2.05 g, 11.4 mmol) in DMF (40 mL)
were added Cs2CO3 (7.82 g, 24.0 mmol) and 31 (4.00 g, 12.0 mmol).
After stirring for 3 h at 65 °C, the reaction mixture was poured into
water and extracted with EtOAc (ꢁ2). The combined organic layers
were washed with water, brine, dried over MgSO4 and concen-
trated in vacuo. The resulting residue was purified by column chro-
matography on silica gel to yield 32 (3.85 g, 99%) as a colorless
viscous oil; TLC Rf = 0.70 (n-hexane/EtOAc, 7:3); 1H NMR
(300 MHz, CDCl3) d 6.95–6.76 (m, 2H), 6.75–6.63 (m, 2H), 6.60 (s,
2H), 4.73–4.54 (m, 1H), 4.31–4.15 (m, 1H), 4.17–4.03 (m, 1H),
3.88 (s, 3H), 3.37 (dd, J = 10.8, 2.1 Hz, 1H), 3.22 (dd, J = 11.5,
6.4 Hz, 1H), 2.89 (s, 3H), 2.30 (s, 6H).
5.8. Ethyl (4-chloro-3-nitrophenyl)(hydroxy)acetate (26)
To a stirred solution of 25 (4.48 g, 19.3 mmol) in EtOH (50 mL)
was added p-toluenesulfonic acid (367 mg, 1.93 mmol). After stir-
ring for 4 h at 70 °C, the reaction mixture was cooled to room tem-
perature, concentrated in vacuo, poured into water and extracted
with EtOAc (ꢁ2). The combined organic layers were washed with
water, brine, dried over MgSO4 and concentrated in vacuo to yield
26 (4.83 g, 96%) as a yellow oil, which was used for the next reac-
tion without further purification; TLC Rf = 0.37 (n-hexane/EtOAc,
2:1)
5.13. 2,6-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoic acid (33)
To a stirred solution of 32 (1 g, 2.93 mmol) in 2-ethoxyethanol
(10 mL) and dioxane (20 mL) was added 5 M NaOH aq (20 mL).
After stirring overnight at 120 °C, the reaction mixture was diluted
with water and extracted with MTBE. The aqueous layer was acid-
ified with 5 M HCl aq and extracted with EtOAc (ꢁ2). The com-
bined organic layers were washed with water, brine, dried over
MgSO4 and evaporated to yield 33 (5.90 g, 61%) as a white solid;
TLC Rf = 0.35 (CHCl3/MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d
6.94–6.77 (m, 2H), 6.75–6.67 (m, 2H), 6.64 (s, 2H), 4.73–4.55 (m,
1H), 4.23 (dd, J = 9.9, 5.1 Hz, 1H), 4.11 (dd, J = 9.7, 6.6 Hz, 1H),
3.38 (dd, J = 11.4, 2.7 Hz, 1H), 3.24 (dd, J = 11.7, 6.6 Hz, 1H), 2.91
(s, 3H), 2.42 (s, 6H).
5.9. Ethyl (4-chloro-3-nitrophenyl)(oxo)acetate (27)
To a stirred solution of 26 (1.59 g, 6.10 mmol) in AcOH (20 mL)
was added 10% NaClO aq (13.56 g, 18.32 mmol) at 0 °C. After stir-
ring for 4 h at 70 °C, the reaction mixture was poured into water
and extracted with EtOAc (ꢁ2). The combined organic layers were
washed with water, brine, dried over MgSO4 and concentrated in
vacuo to give a crude product, which was purified by column chro-
matography on silica gel to yield 27 (414 mg, 26%) as a yellow oil;
TLC Rf = 0.59 (n-hexane/EtOAc, 2:1); 1H NMR (300 MHz, CDCl3) d
8.61 (d, J = 3.0 Hz, 1H), 8.24 (dd, J = 8.1, 3.0 Hz, 1H), 7.72 (d,
J = 8.1 Hz, 1H), 4.48 (q, J = 7.5 Hz, 2H), 1.45 (t, J = 7.5 Hz, 3H).
5.14. 2,6-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]methoxy}benzoyl chloride (34a)
To a stirred solution of 33 (500 mg, 1.53 mmol) in DME (15 mL)
were added (COCl)2 (0.38 mL, 4.44 mmol) and DMF (3.4 lL,
0.044 mmol). After stirring for 1 h at 40 °C, the reaction mixture
was concentrated in vacuo to yield 34a (530 mg, 1.53 mmol) as a
green viscous oil, which was used for the next reaction without
further purification; 1H NMR (300 MHz, CDCl3) d 7.54–7.38 (m,
1H), 7.38–7.20 (m, 1H), 7.15–6.97 (m, 2H), 6.62 (s, 2H), 5.02–4.83
5.10. Ethyl (4-chloro-3-nitrophenyl)(difluoro)acetate (28)
To a stirred solution of 27 (414 mg, 1.61 mmol) in CH2Cl2 (5 mL)
was added DAST (311 mg, 1.93 mmol) at 0 °C under argon atmo-

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M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
(m, 1H), 4.34 (d, J = 4.0 Hz, 2H), 3.86–3.71 (m, 1H), 3.71–3.57 (m,
1H), 3.25 (d, J = 2.2 Hz, 3H), 2.41 (s, 6H).
7.54–7.46 (m, 1H), 7.46–7.38 (m, 1H), 7.37–7.22 (m, 2H), 7.10–
7.06 (m, 1H), 6.92–6.80 (m, 2H), 6.74–6.66 (m, 3H), 4.72–4.62
(m, 1H), 4.27 (dd, J = 9.9, 5.1 Hz, 1H), 4.16 (dd, J = 9.9, 6.6 Hz, 1H),
3.68 (s, 2H), 3.41 (dd, J = 11.7, 3.0 Hz, 1H), 3.28 (dd, J = 11.7,
6.3 Hz, 1H), 2.92 (s, 3H), 2.48 (s, 3H), 2.23 (s, 3H); MS (EI, Pos.)
m/z 460 (M+); IR (KBr) 3512, 3505, 3497, 3425, 3257, 3027,
2925, 1787, 1778, 1767, 1754, 1738, 1724, 1712, 1703, 1694,
1679, 1657, 1650, 1643, 1631, 1621, 1610, 1594, 1580, 1574,
1565, 1553, 1546, 1537, 1529, 1510, 1504, 1494, 1485, 1469,
1462, 1452, 1442, 1434, 1415, 1402, 1392, 1382, 1370, 1358,
1345, 1325, 1301, 1257, 1244, 1223, 1178, 1149, 1075, 1040,
998, 989, 955, 921, 910, 877, 858, 845, 828, 809, 796, 773,
5.15. General procedure for the preparation of 2d–f, 3c–e, 5c–e,
10a–c, 11a–b and 12
5.15.1. {4-Chloro-3-[(2,6-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}acetic acid (5e)
To a stirred solution of 35c (781 mg, 3.9 mmol) and pyridine
(0.95 mL, 11.7 mmol) in CH2Cl2 (10 mL) was added a solution of
34a (1.3 g, 3.9 mmol) in CH2Cl2 (10 mL). After stirring overnight
at room temperature, the reaction mixture was quenched with
water and extracted with EtOAc (ꢁ2). The combined organic layers
were washed with water, brine, dried over MgSO4 and concen-
trated in vacuo. The resulting residue was purified by column chro-
matography on silica gel to yield methyl ester (1.3 g, 63% in 2
steps) as a pale yellow amorphous powder; TLC Rf = 0.54 (n-hex-
ane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3) d 8.49 (d, J = 2.2 Hz,
1H), 7.72 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.04 (dd, J = 8.2, 2.2 Hz,
1H), 6.96–6.76 (m, 2H), 6.75–6.58 (m, 4H), 4.72–4.56 (m, 1H),
4.24 (dd, J = 9.6, 4.5 Hz, 1H), 4.18–4.04 (m, 1H), 3.73 (s, 3H), 3.68
(s, 2H), 3.39 (dd, J = 11.4, 2.4 Hz, 1H), 3.24 (dd, J = 11.7, 6.4 Hz,
1H), 2.91 (s, 3H), 2.39 (s, 6H).
741 cmꢀ1; Optical rotation ½a 2D3
ꢂ
+13.03 (c 0.75, DMSO); mp
200.0–202.0 °C.
5.15.4. {3-[(2,6-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (2f)
Yield 56% in 2 steps; White powder; TLC Rf = 0.32 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 7.61 (m, 1H), 7.52 (m, 1H), 7.34
(t, J = 7.8 Hz, 1H), 7.30 (s, 1H), 7.10 (d, J = 7.5 Hz, 1H), 6.86 (m, 2H),
6.69 (m, 2H), 6.64 (s, 2H), 4.62 (m, 1H), 4.22 (dd, J = 9.9, 5.1 Hz, 1H),
4.10 (dd, J = 9.9, 6.6 Hz, 1H), 3.69 (s, 2H), 3.38 (dd, J = 11.7, 2.7 Hz,
1H), 3.24 (dd, J = 11.7, 6.6 Hz, 1H), 2.90 (s, 3H), 2.36 (s, 6H); MS
(APCI, Neg, 20 V) m/z 459 (MꢀH)ꢀ; IR (KBr) 3276, 3039, 2934,
2919, 2882, 2865, 2659, 2555, 1692, 1655, 1628, 1604, 1594,
1523, 1505, 1467, 1439, 1414, 1375, 1362, 1319, 1293, 1243,
1231, 1173, 1162, 1133, 1109, 1086, 1058, 1039, 988, 971, 935,
908, 866, 860, 829, 781, 739, 708, 682, 625 cmꢀ1; Optical rotation
To a stirred solution of methyl ester (1.3 g, 2.6 mmol) described
above in MeOH (15 mL) and dioxane (15 mL) was added 5 M NaOH
aq (10 mL). After stirring for 2 h at room temperature, the reaction
mixture was quenched with 2 M HCl aq and extracted with EtOAc
(ꢁ2). The combined organic layers were washed with water, brine,
dried over MgSO4 and concentrated in vacuo. The resultant residue
was recrystallized from EtOH to yield 5e (1.1 g, 87%) as a white
powder; TLC Rf = 0.28 (CHCl3/MeOH, 9:1); 1H NMR (300 MHz,
CDCl3) d 8.51 (d, J = 2.2 Hz, 1H), 7.73 (s, 1H), 7.37 (d, J = 8.2 Hz,
1H), 7.05 (dd, J = 8.2, 2.2 Hz, 1H), 6.95–6.79 (m, 2H), 6.75–6.58
(m, 4H), 4.71–4.55 (m, 1H), 4.24 (dd, J = 9.6, 4.8 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),
3.24 (dd, J = 11.7, 6.6 Hz, 1H), 2.91 (s, 3H), 2.38 (s, 6H); MS (APCI,
Neg, 20 V) m/z 493 (MꢀH)ꢀ; HRMS (FAB) calcd for C27H28ClN2O5:
495.1687, Found: 495.1671; IR (KBr) 3268, 2936, 1692, 1660, 1604,
½
a 2D3
+11.89 (c 0.75, DMSO); mp 138.0–140.0 °C.
ꢂ
5.15.5. {3-[(2,3-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]-4-
methylphenyl}acetic acid (3c)
Yield 85% in 2 steps; White powder; TLC Rf = 0.47 (CHCl3/
CH3OH/AcOH, 9:1:0.1); 1H NMR (300 MHz, DMSO-d6) d 12.28 (s,
1H), 9.59 (s, 1H), 7.36–7.25 (m, 2H), 7.17 (d, J = 7.5 Hz, 1H),
7.06–6.90 (m, 2H), 6.85–6.68 (m, 3H), 6.65–6.54 (m, 1H), 4.67–
4.53 (m, 1H), 4.21 (d, J = 5.1 Hz, 2H), 3.52 (s, 2H), 3.45–3.36 (m,
1H), 3.22 (dd, J = 11.6, 7.4 Hz, 1H), 2.85 (s, 3H), 2.32 (s, 3H),
2.22 (s, 3H), 2.16 (s, 3H); MS (APCI, Neg 20 V) m/z 473 (M-H)-;
IR (neat) 3430, 3267, 3040, 2917, 2640, 2360, 1856, 1688, 1646,
1609, 1581, 1530, 1505, 1480, 1457, 1451, 1288, 1266, 1256,
1224, 1190, 1137, 1109, 1041, 976, 912, 870, 823, 741, 682,
1584, 1505, 1465, 1426, 1316, 1293, 1245, 1228, 1168, 1058 cmꢀ1
;
Optical rotation ½a D27
ꢂ
+14.98 (c 1.00, DMF); mp 183.5–185.0 °C.
Compounds 2d–f, 3c–e, 5c–d, 10a–c, 11a–b and 12 were pre-
pared as described above.
5.15.2. {3-[(2,3-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (2d)
628, 565, 553, 483, 459 cmꢀ1, Optical rotation ½a 2D4
ꢂ
+13.73 (c
0.75, DMSO).
Yield 75% in 2 steps; Pale yellow powder; TLC Rf = 0.42 (CHCl3/
MeOH/AcOH, 9:1:0.1); 1H NMR (300 MHz, DMSO-d6) d 12.30 (s,
1H), 10.15 (s, 1H), 7.68 (s, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.25 (m,
2H), 6.95 (m, 2H), 6.76 (m, 3H), 6.59 (m, 1H), 4.59 (m, 1H), 4.22
(d, J = 4.8 Hz, 2H), 3.52 (s, 2H), 3.40 (dd, J = 11.7, 2.7 Hz, 1H), 3.22
(dd, J = 11.7, 7.5 Hz, 1H), 2.84 (s, 3H), 2.26 (s, 3H), 2.16 (s, 3H);
MS (APCI, Neg, 20 V) m/z 459 (MꢀH)ꢀ; IR (KBr) 3430, 3287,
3045, 2930, 2878, 2363, 1715, 1701, 1649, 1609, 1594, 1579,
1528, 1506, 1490, 1459, 1449, 1439, 1432, 1319, 1300, 1260,
1226, 1193, 1139, 1111, 1041, 991, 910, 892, 827, 811, 776, 759,
740, 705, 670, 607, 558, 529, 521, 503, 494, 485, 453 cmꢀ1; Optical
5.15.6. {3-[(2,5-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]-4-
methylphenyl}acetic acid (3d)
Yield 76% in 2 steps; White powder; TLC Rf = 0.62 (CHCl3/
CH3OH, 9:1); 1H NMR (300 MHz, DMSO-d6) d 9.55 (s, 1H), 7.35 (s,
1H), 7.25 (d, J = 1.7 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.01 (dd,
J = 7.8, 1.7 Hz, 1H), 6.91 (s, 1H), 6.84–6.75 (m, 1H), 6.75–6.68 (m,
2H), 6.64–6.54 (m, 1H), 4.67–4.51 (m, 1H), 4.23 (d, J = 5.1 Hz,
2H), 3.51 (s, 2H), 3.43–3.36 (m, 1H), 3.21 (dd, J = 11.7, 7.2 Hz,
1H), 2.84 (s, 3H), 2.40 (s, 3H), 2.21 (s, 3H), 2.18 (s, 3H); MS (APCI,
Neg 20 V) m/z 473 (M-H)-; IR (KBr) 3589, 3569, 3554, 3547,
3538, 3423, 3266, 3027, 2925, 2367, 2345, 1911, 1871, 1847,
1832, 1794, 1774, 1734, 1719, 1709, 1690, 1647, 1630, 1609,
1579, 1561, 1527, 1506, 1476, 1459, 1450, 1389, 1321, 1300,
1259, 1224, 1172, 1153, 1138, 1073, 1040, 995, 894, 816, 781,
742, 698, 680, 670, 638, 568, 535, 526, 503, 467, 456 cmꢀ1; Optical
rotation ½a 2D3
ꢂ
+14.70 (c 0.75, DMSO).
5.15.3. {3-[(2,5-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]phenyl}acetic
acid (2e)
Yield 47% in 2 steps; Pale blue powder; TLC Rf = 0.60 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.62–7.54 (m, 1H),
rotation ½a 2D4
ꢂ
+14.52 (c 0.75, DMSO); mp 192.0–193.0 °C.

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
6943
5.15.7. {3-[(2,6-Dimethyl-4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)amino]-4-
methylphenyl}acetic acid (3e)
1222, 1137, 1106, 1044, 965, 917, 819, 743, 661, 611, 571,
453 cmꢀ1
;
Optical rotation ½a D22
ꢂ
+12.37 (c 0.75, DMSO); mp
151.0–152.0 °C.
Yield 40% in 2 steps; White powder; TLC Rf = 0.46 (CHCl3/
CH3OH/AcOH, 9:1:0.1); 1H NMR (300 MHz, DMSO-d6) d 9.71 (s,
1H), 7.28 (d, J = 1.5 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.02 (dd,
J = 7.8, 1.5 Hz, 1H), 6.84–6.68 (m, 5H), 6.64–6.55 (m, 1H), 4.64–
4.49 (m, 1H), 4.18 (d, J = 5.1 Hz, 2H), 3.52 (s, 2H), 3.41–3.32 (m,
1H), 3.16 (dd, J = 11.7, 7.2 Hz, 1H), 2.84 (s, 3H), 2.32 (s, 6H), 2.24
(s, 3H); MS (APCI, Neg 20 V) m/z 473 (MꢀH)ꢀ; IR (KBr) 3589,
3569, 3434, 3266, 3038, 2925, 2865, 2651, 2548, 2368, 1899,
1871, 1693, 1651, 1605, 1561, 1505, 1468, 1458, 1452, 1407,
1361, 1317, 1293, 1262, 1244, 1229, 1173, 1159, 1133, 1101,
1059, 1040, 990, 959, 913, 860, 828, 791, 777, 740, 683, 626,
5.15.11. 2-{4-Chloro-3-[(2,5-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}-2-methylpropanoic acid
(10b)
Yield 66% in 2 steps; White amorphous powder; TLC Rf = 0.18
(Hex/AcOEt, 2:1); 1H NMR (300 MHz, DMSO-d6) d 9.69 (s, 1H),
7.57 (d, J = 2.1 Hz, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.22
(dd, J = 8.7, 2.1 Hz, 1H), 6.91 (s, 1H), 6.86–6.75 (m, 1H), 6.75–6.69
(m, 2H), 6.65–6.54 (m, 1H), 4.67–4.55 (m, 1H), 4.24 (d, J = 5.1 Hz,
2H), 3.46–3.35 (m, 1H), 3.21 (dd, J = 11.7, 7.2 Hz, 1H), 2.84 (s,
3H), 2.42 (s, 3H), 2.18 (s, 3H), 1.47 (s, 6H); MS (APCI, Neg 20 V)
m/z 521 (MꢀH)ꢀ; IR (KBr); 3415, 3041, 2975, 2929, 2872, 2819,
2633, 1732, 1703, 1609, 1581, 1505, 1455, 1414, 1323, 1297,
1259, 1224, 1147, 1105, 1073, 1041, 944, 915, 818, 743, 661,
571 cmꢀ1
;
Optical rotation ½a D21
ꢂ
+12.58 (c 0.75, DMSO); mp
200.0–201.0 °C.
5.15.8. {4-Chloro-3-[(2,3-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}acetic acid (5c)
566, 453 cmꢀ1; Optical rotation ½a 2D1
ꢂ
+13.58 (c 0.75, DMSO).
Yield 58% in 2 steps; Beige powder; TLC Rf = 0.40 (Hex/EtOAc/
AcOH, 9:1:0.1); 1H NMR (300 MHz, CDCl3) d 8.52 (s, 1H), 7.94 (s,
1H), 7.46–7.32 (m, 2H), 7.02 (dd, J = 8.1, 2.1 Hz, 1H), 6.93–6.76
(m, 3H), 6.75–6.66 (m, 2H), 4.73–4.60 (m, 1H), 4.34–4.23 (m,
1H), 4.16 (dd, J = 9.9, 6.6 Hz, 1H), 3.70 (s, 2H), 3.55–3.37 (m, 1H),
3.29 (dd, J = 11.4, 6.9 Hz, 1H), 2.92 (s, 3H), 2.43 (s, 3H), 2.23 (s,
3H); MS (APCI, Neg, 20 V) m/z 493 (MꢀH)ꢀ; IR (KBr) 3589, 3569,
3414, 3274, 3028, 2925, 2621, 2347, 2278, 1961, 1870, 1794,
1709, 1686, 1655, 1607, 1584, 1561, 1519, 1506, 1485, 1458,
1427, 1298, 1268, 1244, 1225, 1188, 1159, 1138, 1108, 1056,
992, 936, 927, 916, 906, 881, 871, 861, 849, 825, 816, 795, 781,
768, 741, 715, 706, 696, 673, 651, 641, 607, 596, 587, 572, 562,
553, 544, 516, 506, 499, 489, 481, 465, 454 cmꢀ1; Optical rotation
5.15.12. 2-{4-Chloro-3-[(2,6-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}-2-methylpropanoic acid
(10c)
Yield 58% in 2 steps; White powder; TLC Rf = 0.51 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 8.66 (d, J = 2.4 Hz, 1H), 7.72 (s,
1H), 7.35 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.4, 2.4 Hz, 1H), 6.93–
6.79 (m, 2H), 6.74–6.68 (m, 2H), 6.66 (s, 2H), 4.69–4.58 (m, 1H),
4.24 (dd, J = 9.6, 4.8 Hz, 1H), 4.12 (dd, J = 9.6, 6.6 Hz, 1H), 3.39
(dd, J = 11.4, 2.7 Hz, 1H), 3.24 (dd, J = 11.4, 6.6 Hz, 1H), 2.91 (s,
3H), 2.39 (s, 6H), 1.66 (s, 6H); MS (APCI, Neg, 20 V) m/z 521
(MꢀH)ꢀ; IR (KBr) 3409, 3198, 2993, 2984, 2925, 2870, 2644,
2551, 1691, 1639, 1605, 1579, 1504, 1474, 1457, 1412, 1402,
1380, 1356, 1321, 1298, 1277, 1266, 1248, 1225, 1171, 1136,
1110, 1078, 1065, 1052, 1041, 940, 912, 893, 853, 823, 745, 723,
½
a 2D4
+13.34 (c 0.75, DMSO).
ꢂ
5.15.9. {4-Chloro-3-[(2,5-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
672, 558, 522, 459 cmꢀ1; Optical rotation ½a 2D6
ꢂ
+17.54 (c 1.00,
CH3CN); mp 131.0–133.0 °C.
yl]methoxy}benzoyl)amino]phenyl}acetic acid (5d)
Yield 76% in 2 steps; Pale brown powder; TLC Rf = 0.29 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, DMSO-d6) d 12.36 (s, 1H), 9.61 (s,
1H), 7.54 (d, J = 2.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H),
7.12 (dd, J = 8.1, 2.1 Hz, 1H), 6.87 (s, 1H), 6.82–6.73 (m, 1H),
6.73–6.66 (m, 2H), 6.62–6.53 (m, 1H), 4.67–4.52 (m, 1H), 4.28–
4.16 (m, 2H), 3.58 (s, 2H), 3.38 (dd, J = 11.7, 2.7 Hz, 1H), 3.22 (dd,
J = 11.7, 7.2 Hz, 1H), 2.85 (s, 3H), 2.42 (s, 3H), 2.18 (s, 3H); MS
(APCI, Neg, 20 V) m/z 493 (MꢀH)ꢀ; IR (KBr) 3447, 3029, 2940,
2884, 2775, 2625, 2539, 1917, 1877, 1714, 1688, 1607, 1584,
1572, 1530, 1503, 1469, 1454, 1426, 1376, 1317, 1288, 1264,
1245, 1191, 1168, 1138, 1075, 1064, 1044, 984, 911, 879, 849,
831, 807, 786, 765, 750, 743, 732, 663, 651, 606, 574, 556, 513,
5.15.13. 1-{4-Chloro-3-[(2,5-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}cyclopropanecarboxylic
acid (11a)
Yield 48% in 2 steps; White amorphous powder; TLC Rf = 0.59
(CHCl3/MeOH, 9:1); 1H NMR (300 MHz, DMSO-d6) d 9.67 (s, 1H),
7.56 (d, J = 2.1 Hz, 1H), 7.46–7.38 (m, 2H), 7.20 (dd, J = 8.1, 2.1 Hz,
1H), 6.91 (s, 1H), 6.83–6.75 (m, 1H), 6.75–6.68 (m, 2H), 6.64–
6.54 (m, 1H), 4.66–4.54 (m, 1H), 4.24 (d, J = 5.1 Hz, 2H), 3.43–
3.35 (m, 1H), 3.21 (dd, J = 11.7, 7.2 Hz, 1H), 2.84 (s, 3H), 2.42 (s,
3H), 2.18 (s, 3H), 1.52–1.42 (m, 2H), 1.16–1.10 (m, 2H); MS (APCI,
Neg, 20 V) m/z 519 (MꢀH)ꢀ; IR (KBr) 3409, 3198, 2993, 2984,
2925, 2870, 2644, 2551, 1691, 1639, 1605, 1579, 1504, 1474,
1457, 1412, 1402, 1380, 1356, 1321, 1298, 1277, 1266, 1248,
1225, 1171, 1136, 1110, 1078, 1065, 1052, 1041, 940, 912, 893,
853, 823, 745, 723, 672, 558, 522, 459 cmꢀ1; Optical rotation
468, 456 cmꢀ1; Optical rotation ½a 2D1
ꢂ
+13.60 (c 0.75, DMSO).
5.15.10. 2-{4-Chloro-3-[(2-chloro-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}-2-methylpropanoic acid
(10a)
½
a 2D2
+11.61 (c 0.75, DMSO).
ꢂ
Yield 53% in 2 steps; White amorphous powder; TLC Rf = 0.26
(Hex/AcOEt, 1:1); 1H NMR (300 MHz, CDCl3) d 8.80–8.62 (m, 2H),
7.88 (d, J = 9.3 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.11 (dd, J = 8.4,
2.4 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.96 (dd, J = 8.7, 2.4 Hz, 1H),
6.92–6.81 (m, 2H), 6.74–6.67 (m, 2H), 4.70–4.62 (m, 1H), 4.27
(dd, J = 9.9, 5.4 Hz, 1H), 4.18 (dd, J = 9.9, 6.3 Hz, 1H), 3.38 (dd,
J = 11.4, 3.0 Hz, 1H), 3.25 (dd, J = 11.4, 6.3 Hz, 1H), 2.91 (s, 3H),
1.64 (s, 6H); MS (APCI, Neg 40 V) m/z 527 (MꢀH)ꢀ; IR (KBr);
3383, 3068, 2977, 2937, 2871, 2821, 2639, 2548, 1733, 1703,
1672, 1602, 1583, 1526, 1504, 1459, 1414, 1358, 1297, 1242,
5.15.14. 1-{4-Chloro-3-[(2,6-dimethyl-4-{[(2S)-4-methyl-3,4-
dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}cyclopropanecarboxylic
acid (11b)
Yield 48% in 2 steps; White amorphous powder; TLC Rf = 0.59
(CHCl3/MeOH, 9:1); 1H NMR (300 MHz, DMSO-d6) d 9.93 (s, 1H),
7.54 (d, J = 2.1 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.4,
2.1 Hz, 1H), 6.84–6.67 (m, 5H), 6.64–6.55 (m, 1H), 4.62–4.50 (m,
1H), 4.18 (d, J = 5.1 Hz, 2H), 3.41–3.33 (m, 1H), 3.22–3.11
(m, 1H), 2.84 (s, 3H), 2.32 (s, 6H), 1.50–1.43 (m, 2H), 1.16–1.10

6944
M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
(m, 2H); MS (APCI, Neg, 20 V) m/z 519 (MꢀH)ꢀ; IR (KBr) 3565,
3414, 3203, 3037, 2957, 2927, 2863, 2686, 2604, 2360, 2342,
1681, 1639, 1605, 1579, 1557, 1505, 1471, 1456, 1413, 1313,
1298, 1257, 1219, 1206, 1163, 1138, 1105, 1101, 1048, 976, 926,
858, 843, 822, 745, 726, 708, 668, 654, 628, 609, 598, 587, 558,
547, 538, 526, 513, 499, 485, 472, 456 cmꢀ1; Optical rotation
1H), 4.28 (dd, J = 9.0, 5.4 Hz, 1H), 4.14 (t, J = 9.6 Hz, 1H), 3.83–
3.73 (m, 1H), 2.23 (s, 3H).
Compounds 39b–e were prepared as described above.
5.18.2. (2S)-2,3-Dihydro-1,4-benzodioxin-2-ylmethyl 4-
nitrobenzenesulfonate (39b)
½
a 2D4
ꢂ
+11.74 (c 0.75, DMSO); mp 171.8–172.7 °C.
TLC Rf = 0.47 (n-hexane/EtOAc, 7:3); 1H NMR (300 MHz, CDCl3)
d 8.38 (d, J = 9.0 Hz, 2H), 8.11 (d, J = 9.0 Hz, 2H), 6.84–6.77 (m, 3H),
6.72–6.66 (m, 1H), 4.48–4.41 (m, 1H), 4.37–4.33 (m, 2H), 4.24 (dd,
J = 11.7, 2.4 Hz, 1H), 4.07 (dd, J = 11.7, 6.0 Hz, 1H).
5.15.15. {4-Chloro-3-[(2-chloro-4-{[(2S)-4-methyl-3,4-dihydro-
2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)amino]phenyl}(difluoro)acetic acid (12)
Yield 35% in 2 steps; Green powder; TLC Rf = 0.50 (CHCl3/MeOH/
H2O, 80:20:1); 1H NMR (300 MHz, DMSO-d6) d 10.12 (br s, 1H),
7.80 (br s, 1H), 7.58 (m, 2H), 7.40 (m, 1H), 7.21 (d, J = 2.1 Hz, 1H),
7.09 (dd, J = 8.4, 2.1 Hz, 1H), 6.76 (m, 4H), 6.59 (m, 1H), 4.57 (m,
1H), 4.28 (m, 2H), 3.37 (dd, J = 11.7, 2.4 Hz, 1H), 3.17 (dd, J = 11.7,
7.5 Hz, 1H), 2.84 (s, 3H); MS (FAB, Pos.) m/z 537 (M + H)+; IR
(KBr) 3381, 1652, 1602, 1589, 1532, 1504, 1458, 1420, 1300,
5.18.3. (3S)-2,3-Dihydro-1-benzofuran-3-ylmethyl 4-
nitrobenzenesulfonate (39c)
TLC Rf = 0.72 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3)
d 8.36 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 7.20–7.09 (m, 2H),
6.86–6.75 (m, 2H), 4.58 (t, J = 9.6 Hz, 1H), 4.37 (dd, J = 9.6, 4.5 Hz,
1H), 4.29 (dd, J = 9.6, 5.7 Hz, 1H), 4.17 (dd, J = 9.6, 8.1 Hz, 1H),
3.86–3.76 (m, 1H).
1243, 1223, 1133, 1099, 1048, 824, 758, 744, 612, 567 cmꢀ1
.
5.18.4. (2S)-2,3-Dihydro-1-benzofuran-2-ylmethyl 4-
nitrobenzenesulfonate (39d)
5.16. (2S)-2-[(2-Bromo-4-methylphenoxy)methyl]oxirane (37)
TLC Rf = 0.78 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3)
d 8.35 (d, J = 9.3 Hz, 2H), 8.06 (d, J = 9.3 Hz, 2H), 7.17–7.04 (m, 2H),
6.84 (td, J = 7.5, 0.9 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.02–4.92 (m,
1H), 4.40 (dd, J = 10.8, 3.3 Hz, 1H), 4.30 (dd, J = 10.8, 5.7 Hz, 1H),
3.32 (dd, J = 15.9, 9.9 Hz, 1H), 3.01 (dd, J = 15.9, 6.9 Hz, 1H).
To a stirred solution of 2-bromo-4-methylphenol 36 (6.8 g,
26.2 mmol) in DMF (50 mL) were added Cs2CO3 (12.8 g,
39.3 mmol) and (S)-(+)-glycidyl 3-nitrobenzenesulfonate (6.8 g,
26.2 mmol) at room temperature under argon atmosphere, and
stirring was continued for 1 h. 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 con-
centrated in vacuo to give 37 (6.1 g, 96%) as a pale yellow oil, which
was used for the next reaction without further purification; TLC
Rf = 0.50 (n-hexane/EtOAc, 2:1).
5.18.5. 1,3-Benzodioxol-2-ylmethyl 4-nitrobenzenesulfonate
(39e)
TLC Rf = 0.80 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3)
d 8.35 (d, J = 9.0 Hz, 2H), 8.06 (d, J = 9.0 Hz, 2H), 6.80 (dd, J = 5.7,
3.3 Hz, 2H), 6.68 (dd, J = 5.7, 3.3 Hz, 2H), 6.25 (t, J = 3.3 Hz, 1H),
4.44 (d, J = 3.3 Hz, 2H).
5.17. [(3R)-5-Methyl-2,3-dihydro-1-benzofuran-3-yl]methanol
(38a)
5.19. 4-(Acetyloxy)-2,6-dimethylbenzoic acid (41)
To a stirred solution of the above-described crude product 37
(6.0 g, 24.5 mmol) in THF (35 mL) was added dropwise a 1.56 M
solution of n-butyl lithium in hexane (15.7 mL, 24.5 mmol) at
ꢀ78 °C. After stirring for 1 h at ꢀ78 °C and 1 h while gradually
warmed up to room temperature, the reaction mixture was
quenched with 1 M HCl aq and extracted with EtOAc (ꢁ2). The
combined organic layers were washed with brine, dried over
Na2SO4 and concentrated in vacuo to give a crude product, which
was purified by column chromatography on silica gel to yield
38a (2.2 g, 55%) as a pale yellow oil; TLC Rf = 0.32 (n-hexane/EtOAc,
2:1); 1H NMR (300 MHz, CDCl3) d 7.03 (s, 1H), 6.96 (m, 1H), 6.70 (d,
J = 8.4 Hz, 1H), 4.62 (t, J = 9.0 Hz, 1H), 4.45 (dd, J = 9.0, 5.1 Hz, 1H),
3.86–3.73 (m, 2H), 3.67–3.53 (m, 1H), 2.29 (s, 3H).
To a stirred solution of 4-hydroxy-2,6-dimethylbenzoic acid 40
(20 g, 121 mmol) in pyridine (50 mL) was added Ac2O (50 mL).
After stirring for 2 h at room temperature, the reaction mixture
was diluted with EtOAc and added water at 0 °C. After stirring
for 2 min at 0 °C, the reaction mixture was extracted with EtOAc
(ꢁ2). The combined organic layers were washed with 2 M HCl
aq, water and brine, dried over MgSO4 and concentrated in vacuo
to give a crude product, which was washed with EtOAc/n-hexane
to yield 41 (15 g, 60%) as an ivory powder; TLC Rf = 0.33 (CHCl3/
MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 6.82 (s, 2H), 2.44 (s,
6H), 2.30 (s, 3H).
5.20. 4-(Chlorocarbonyl)-3,5-dimethylphenyl acetate (42)
5.18. General procedure for the preparation of 39a–e
To a stirred solution of 41 (2.0 g, 9.6 mmol) in toluene (15 mL)
were added (COCl)2 (1.7 mL, 19.2 mmol) and DMF (4 lL,
5.18.1. [(3S)-5-Methyl-2,3-dihydro-1-benzofuran-3-yl]methyl 4-
nitrobenzenesulfonate (39a)
0.05 mmol). After stirring for 1 h at 40 °C, the reaction mixture
was concentrated in vacuo to yield 42 (2.2 g, 9.6 mmol), which
was used for the next reaction without further purification; 1H
NMR (300 MHz, CDCl3) d 6.82 (s, 2H), 2.41 (s, 6H), 2.30 (s, 3H).
To a stirred solution of 38a (164 mg, 1.0 mmol) in THF (5 mL)
were added TEA (0.33 mL, 2.4 mmol), DMAP (12 mg, 0.1 mmol)
and 4-nitrobenzenesulfonyl chloride (266 mg, 1.2 mmol) at room
temperature. After stirring overnight at room temperature, the
reaction mixture was quenched with water and extracted with
EtOAc (ꢁ2). The combined organic layers were washed with 1 M
HCl aq, water, brine, dried over Na2SO4 and concentrated in vacuo
to give 39a (349 mg, quant), which was used for the next reaction
without further purification; TLC Rf = 0.81 (n-hexane/EtOAc, 1:1);
1H NMR (300 MHz, CDCl3) d 8.36 (d, J = 8.4 Hz, 2H), 8.03 (d,
J = 8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H), 6.89 (s, 1H), 6.66 (d,
J = 8.4 Hz, 1H), 4.55 (t, J = 9.0 Hz, 1H), 4.35 (dd, J = 9.0, 4.5 Hz,
5.21. Methyl (3-{[4-(acetyloxy)-2,6-dimethylbenzoyl]amino}-4-
chlorophenyl)acetate (43)
To a stirred solution of 35c (958 mg, 4.8 mmol) and pyridine
(1.16 mL, 14.4 mmol) in CH2Cl2 (10 mL) was added a solution of
42 (1.1 g, 4.8 mmol) in CH2Cl2 (10 mL). After stirring overnight at
40 °C, the reaction mixture was quenched with water and ex-
tracted with EtOAc (ꢁ2). The combined organic layers were
washed with 2 M HCl aq, water and brine, dried over MgSO4 and

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
6945
concentrated in vacuo to yield 43 as a ivory powder, which was
used for the next reaction without further purification; TLC
Rf = 0.54 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3) d 8.47
(d, J = 2.1 Hz, 1H), 7.76 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.06 (dd,
J = 8.1, 2.1 Hz, 1H), 6.83 (s, 2H), 3.73 (s, 3H), 3.69 (s, 2H), 2.41 (s,
6H), 2.31 (s, 3H).
5.1 Hz, 1H), 4.16 (dd, J = 9, 5.1 Hz, 1H), 4.02 (t, J = 8.4 Hz, 1H),
3.92 (m, 1H), 3.72 (s, 2H) 2.38 (s, 6H); MS (APCI, Neg, 40 V) m/z
464 (MꢀH)ꢀ; IR (KBr) 3569, 3547, 3504, 3496, 3449, 3413, 3239,
3033, 2958, 2926, 2737, 1734, 1703, 1656, 1625, 1601, 1583,
1521, 1483, 1460, 1426, 1381, 1319, 1292, 1242, 1227, 1173,
1161, 1108, 1100, 1052, 963, 954, 940, 868, 859, 849, 837, 748,
457 cmꢀ1; mp 147.0–149.0 °C; Optical rotation ½a 2D5
ꢂ
+15.48 (c
5.22. Methyl {4-chloro-3-[(4-hydroxy-2,6-
dimethylbenzoyl)amino]phenyl}acetate (44)
0.57, CHCl3).
5.23.3. {4-Chloro-3-[(2,6-dimethyl-4-{[(3R)-5-methyl-2,3-
dihydro-1-benzofuran-3-
yl]methoxy}benzoyl)amino]phenyl}acetic acid (7b)
To a stirred solution of the above-described crude product 43
(4.8 mmol) in MeOH (20 mL) and THF (20 mL) was added K2CO3
(2.0 g, 14.4 mmol) at room temperature. After stirring for 2 h at
room temperature, insoluble substance was removed by filtration.
The filtrate was diluted with EtOAc, washed with 2 M HCl aq, water
and brine, dried over MgSO4 and concentrated in vacuo to give a
crude product, which was washed with EtOAc/n-hexane to yield
44 (800 mg, 48% in 3 steps) as a beige powder; TLC Rf = 0.40 (n-
Yield 24% in 2 steps; White powder; TLC Rf = 0.39 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 8.52 (d, J = 2.1 Hz, 1H), 7.73 (s,
1H), 7.37 (d, J = 8.1 Hz, 1H), 7.09 (m, 1H), 7.05 (dd, J = 8.1, 2.1 Hz,
1H), 6.98 (m, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.63 (s, 2H), 4.69 (t,
J = 9.00 Hz, 1H), 4.50 (dd, J = 9.00, 5.1 Hz, 1H), 4.16 (dd, J = 8.4,
5.1 Hz, 1H), 3.99 (t, J = 9.00 Hz, 1H), 3.88 (m, 1H), 3.73 (s, 2H)
2.38 (s, 6H), 2.30 (s, 3H); MS (APCI, Neg, 40 V) m/z 478 (MꢀH)ꢀ;
IR (KBr) 3570, 3562, 3547, 3413, 3255, 3013, 2923, 2863, 2737,
1734, 1692, 1658, 1606, 1583, 1519, 1491, 1464, 1426, 1406,
1376, 1316, 1283, 1245, 1226, 1215, 1175, 1162, 1140, 1104,
1064, 1054, 966, 807 cmꢀ1; mp 168.0–169.0 °C; Optical rotation
hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3)
d 8.47 (d,
J = 2.1 Hz, 1H), 7.74 (s, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.04 (dd,
J = 8.1, 2.1 Hz, 1H), 6.53 (s, 2H), 5.16 (s, 1H), 3.73 (s, 3H), 3.68 (s,
2H), 2.35 (s, 6H).
5.23. General procedure for the preparation of 6, 7a–b, 8 and 9
½
a 2D5
ꢂ
ꢀ18.92 (c 0.76, CHCl3).
5.23.1. [4-Chloro-3-({4-[(2R)-2,3-dihydro-1,4-benzodioxin-2-
ylmethoxy]-2,6-dimethylbenzoyl}amino)phenyl]acetic acid (6)
To stirred solution of 44 (194 mg, 0.56 mmol) and 39b (197 mg,
0.56 mmol) in DMF (3 mL) was added Cs2CO3 (365 mg, 1.12 mmol).
After stirring for 1 h at rt, the reaction mixture was 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. The resulting residue was purified by column chro-
matography on silica gel to yield methyl ester (120 mg, 43%) as a
pale yellow viscous oil; TLC Rf = 0.68 (n-hexane/EtOAc, 1:1); 1H
NMR (300 MHz, CDCl3) d 8.50–8.47 (m, 1H), 7.72 (s, 1H), 7.36 (d,
J = 8.1 Hz, 1H), 7.07–7.02 (m, 1H), 6.95–6.80 (m, 4H), 6.66 (s, 2H),
4.60–4.54 (m, 1H), 4.44–4.36 (m, 1H), 4.33–4.10 (m, 3H), 3.73 (s,
3H) 3.68 (s, 2H), 2.39 (s, 6H).
5.23.4. [4-Chloro-3-({4-[(2S)-2,3-dihydro-1-benzofuran-2-
ylmethoxy]-2,6-dimethylbenzoyl}amino)phenyl]acetic acid (8)
Yield 31% in 2 steps; White powder; TLC Rf = 0.40 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 8.51 (d, J = 2.1 Hz, 1H), 7.73 (s,
1H), 7.37 (d, J = 8.1 Hz, 1H), 7.17 (m, 2H), 7.05 (dd, J = 8.1, 2.1 Hz,
1H), 6.85 (m, 2H), 6.65 (s, 2H), 5.15 (m, 1H), 4.22 (dd, J = 9.9,
6.3 Hz, 1H), 4.10 (dd, J = 9.9, 4.8 Hz, 1H), 3.73 (s, 2H) 3.40 (dd,
J = 15.3, 9.3 Hz, 1H), 3.13 (dd, J = 15.3, 6.3 Hz, 1H), 2.38 (s, 6H);
MS (APCI, Neg, 40 V.) m/z 464 (MꢀH)ꢀ; IR (KBr) 3407, 3238,
3030, 2930, 1693, 1656, 1603, 1584, 1520, 1482, 1464, 1425,
1409, 1378, 1321, 1288, 1233, 1176, 1163, 1109, 1054, 1017,
984, 954, 914, 865, 827, 789, 744, 689, 661 cmꢀ1; mp 160.0–
161.0 °C; Optical rotation ½a D25
ꢂ
+41.89 (c 0.68, CHCl3).
To a stirred solution of methyl ester (120 mg, 0.24 mmol) de-
scribed above in MeOH (3 mL) and THF (3 mL) was added 5 M
NaOH aq (2 mL). After stirring for 2 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 6
(60 mg, 51%) as a white powder; TLC Rf = 0.46 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 8.51 (d, J = 2.1 Hz, 1H), 7.73 (s,
1H), 7.37 (d, J = 8.1 Hz, 1H), 7.06 (dd, J = 8.1, 2.1 Hz, 1H), 6.88 (m,
4H), 6.66 (s, 2H), 4.57 (m, 1H), 4.40 (dd, J = 11.4, 2.4 Hz, 1H), 4.21
(m, 3H), 3.73 (s, 2H) 2.39 (s, 6H); MS (APCI, Neg, 40 V) m/z 480
(MꢀH)ꢀ; IR (KBr) 3266, 3045, 3018, 2955, 2911, 2883, 1735,
1692, 1659, 1630, 1602, 1593, 1585, 1519, 1493, 1467, 1425,
1406, 1317, 1305, 1283, 1269, 1249, 1226, 1176, 1166, 1144,
1108, 1062, 1052, 1046, 926, 913, 887, 867, 850, 828, 789,
5.23.5. (3-{[4-(1,3-Benzodioxol-2-ylmethoxy)-2,6-
dimethylbenzoyl]amino}-4-chlorophenyl)acetic acid (9)
Yield 34% in 2 steps; White powder; TLC Rf = 0.39 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 8.50 (d, J = 1.8 Hz, 1H), 7.73 (s,
1H), 7.37 (d, J = 8.1 Hz, 1H), 7.05 (dd, J = 8.1, 2.1 Hz, 1H), 6.85 (m,
4H), 6.66 (s, 2H), 6.44 (t, J = 4.2 Hz, 1H), 4.27 (d, J = 4.2 Hz, 2H),
3.72 (s, 2H) 2.38 (s, 6H); MS (APCI, Neg, 40 V) m/z 466 (MꢀH)ꢀ;
IR (KBr) 3405, 3246, 3064, 2956, 2920, 2870, 2765, 1693, 1658,
1631, 1605, 1584, 1520, 1484, 1467, 1454, 1426, 1407, 1378,
1353, 1319, 1305, 1284, 1235, 1177, 1169, 1109, 1101, 1089,
1066, 1054, 1005, 983, 956, 926, 913, 897, 869, 861, 827, 800,
789, 766, 736, 689, 662, 654, 560, cmꢀ1; mp 165.0–168.0 °C.
5.24. Benzyl 1H-indol-4-ylacetate (46)
750 cmꢀ1; mp 174.0–176.0 °C; Optical rotation ½a 2D5
ꢂ
+4.85 (c 0.84,
To a stirred solution of 2-(1H-indol-4-yl)acetic acid 45 (11.0 g,
62.9 mmol) in pyridine (45 mL) were added benzyl alcohol
(6.9 mL, 66.0 mmol) and a solution of p-toluenesulfonyl chloride
(13.2 g, 69.2 mmol) in toluene (38 mL) at 0 °C. After stirring vigor-
ously for 2 h at room temperature, the reaction mixture was di-
luted with water and extracted with MTBE (ꢁ2). The combined
organic layers were washed with NaHCO3 aq, water and brine,
dried over Na2SO4 and concentrated in vacuo to give 46 as a dark
brown oil, which was used for the next reaction without further
purification; TLC Rf = 0.45 (n-hexane/EtOAc, 2:1).
CHCl3).
Compounds 7a–b, 8 and 9 were prepared as described above.
5.23.2. [4-Chloro-3-({4-[(3R)-2,3-dihydro-1-benzofuran-3-
ylmethoxy]-2,6-dimethylbenzoyl}amino)phenyl]acetic acid (7a)
Yield 6% in 2 steps; Ivory powder; TLC Rf = 0.39 (CHCl3/MeOH,
9:1); 1H NMR (300 MHz, CDCl3) d 8.51 (d, J = 1.8 Hz, 1H), 7.73 (s,
1H), 7.37 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 6.6 Hz, 1H), 7.18 (m, 1H),
7.05 (dd, J = 8.4, 2.1 Hz, 1H), 6.89 (t, J = 7.5 Hz, 1H), 6.84 (d,
J = 8.1 Hz, 1H), 6.63 (s, 2H), 4.71 (t, J = 9 Hz, 1H), 4.51 (dd, J = 9.3,

6946
M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
5.25. [1-(4-{[(2S)-4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-
yl]methoxy}benzoyl)-1H-indol-4-yl]acetic acid (1a)
6.2. Measurement of the mDP receptor antagonist activity
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.
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
To a stirred solution of 46 (1 g, 3.69 mmol) and 47 (1.76 g,
5.54 mmol) in CH2Cl2 (17 mL) were added 20 M NaOH aq
(0.92 mL, 18.5 mmol) and TBACl (83 mg, 0.30 mmol). After stirring
for 30 min at room temperature, the reaction mixture was
quenched with water and extracted with EtOAc (ꢁ2). The com-
bined organic layers were washed with water and brine, dried over
Na2SO4 and concentrated in vacuo. The resulting residue was puri-
fied by column chromatography on silica gel to yield benzyl ester
(1.47 g, 73%) as a pale yellow oil; TLC Rf = 0.22 (n-hexane /EtOAc,
3:1).
(MEM), and MEM containing 2 lM of Diclofenac was added to each
well. The cells were incubated for approximately 10 min at 37 °C
To a stirred solution of benzyl ester (0.80 g, 1.46 mmol) in
EtOAc (10 mL) was added 20% Pd(OH)2/C (100 mg) at room tem-
perature. The resulting suspension was vigorously stirred for
30 min at room temperature under hydrogen atmosphere. The
reaction mixture was filtered through a pad of Celite, and the fil-
trate was concentrated in vacuo to give a crude product, which
was washed with EtOAc/n-hexane to yield 1a (0.39 g, 59%) as a
white powder; TLC Rf = 0.33 (EtOAc/n-hexane, 2:1); 1H NMR
(300 MHz, CDCl3) d 8.27 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H),
7.37 (d, J = 3.9 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 6.6 Hz,
1H), 7.05 (d, J = 8.7 Hz, 2H), 6.93–6.80 (m, 2H), 6.77–6.63 (m,
3H), 4.73–4.63 (m, 1H), 4.31 (dd, J = 9.9, 5.1 Hz, 1H), 4.21 (dd,
J = 9.9, 6.0 Hz, 1H), 3.90 (s, 2H) 3.40 (dd, J = 11.7, 2.7 Hz, 1H), 3.28
(dd, J = 11.7, 6.6 Hz, 1H), 2.92 (s, 3H); MS (EI, Pos.) m/z 456 (M+);
IR (KBr) 3422, 2935, 1702, 1669, 1606, 1509, 1489, 1461,1431,
1385, 1338, 1306, 1253, 1227, 1209, 1178, 1160, 1138, 1042,
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 enzyme immunoassay using a cAMP assay kit (GE
Healthcare UK Ltd). The relative responsiveness (%) of cAMP pro-
duction was calculated relative to the maximum increase in cAMP
that occurred in the absence of test compound (100%) to estimate
of the IC50 values.
994, 955, 891, 848, 814, 757, 683 cmꢀ1
.
6. Biological assay method
6.3. Measurement of DP receptor antagonistic activity using
human platelet-rich plasma (PRP)
6.1. Prostanoid mDP, and hIP receptor binding assay
Competitive binding studies were conducted using radiolabeled
ligands and membrane fractions prepared from Chinese hamster
ovary (CHO) cells stably expressing the respective prostanoid
receptors, mDP, and hIP.
To estimate the potency of antagonism of test compounds
against the human prostanoid DP receptor in the conditions that
may be close to the in vivo environment, a functional assay was
performed by using PGD2-stimulated cAMP production in human
platelet rich plasma (PRP) as an indicator of receptor function.
For the assessment of the antagonist activity of test compounds,
blood was collected from the cubital vein of a healthy adult who
gave written informed consent, using a syringe filled with a 3.8
percent sodium citrate solution of about one ninth of predeter-
mined volume. The collected blood was subjected to centrifugal
separation at 100 G, at room temperature for 15 minutes to obtain
PRP in the upper layer. EDTA was added to the obtained PRP so that
the final concentration thereof to be about 10 mmol/L. The PRP was
subjected to centrifugal separation at 1500 G, at room temperature
for 15 min to obtain supernatant platelet poor plasma (PPP). After
the obtained platelet pellets were suspended, the suspension was
diluted with PPP so that the platelet density to be adjusted to
Membranes from CHO cells expressing prostanoid receptors
were incubated with radioligands (2.5 nM of [3H]PGD2 for mDP;
5.0 nM of [3H]Iloprost for hIP) and the test compounds at various
concentrations in assay buffer (25 mM HEPES–NaOH buffer con-
taining 1 mM EDTA, 5 mM MgCl2 and 10 mM MnCl2, pH 7.4 for
mDP; 50 mM Tris–HCl buffer containing 1 mM EDTA and 10 mM
MgCl2, pH 7.5, for hIP). Incubation was carried out at room temper-
ature for 20 min for mDP and 30 min for hIP. The incubation was
terminated by filtration through Whatman GF/B filters. The filters
were washed with ice-cold buffer (10 mM Tris–HCl buffer contain-
ing 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 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 scintil-
lation counter. Nonspecific binding was achieved by adding excess
amounts of unlabeled PGD2 (for mDP) or unlabeled Iloprost (for IP)
with assay buffer. The concentrations of the test substance re-
quired 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.
5.0 ꢁ 105/
lL. To the obtained platelet suspension, 3-isobutyl-1-
methylxanthine and prostanoid EP3 receptor antagonist were
added so that the final concentrations thereof to be 8 mmol/L
and 1
pensed to each test tube, followed by subjecting to incubation at
37 °C for 5 min. After adding 1.5 L of DMSO or a variety of concen-
trations of the compound of the present invention, 10 min of incu-
bation was carried out at 37 °C. 1.5 L of DMSO or PGD2 (final
concentration: 3 mol/L) was added thereto to initiate the reac-
tion. After 15 min of incubation was carried out at 37 °C,
300 mol/L of ice-cooled 10 percent trichloroacetic acid (TCA)
lmol/L, respectively. 297 lL of the prepared PRP was dis-
l
l
l
Ki ¼ IC50=ð1 þ ½Lꢂ=KdÞ
l
[L]: Concentration of radioligand; Kd: Dissociation constant of
was added thereto to terminate the reaction. The TCA-treated sam-
radiolabeled ligand towards the prostanoid receptors.
ple was subjected to centrifugal separation at 15,000g for 3 min at

M. Iwahashi et al. / Bioorg. Med. Chem. 19 (2011) 6935–6948
6947
4 °C. The concentration of thus obtained supernatant cAMP was
measured by enzyme immunoassay using cAMP EIA system
administered 1 h before the antigen challenge. After 30 min, the gui-
nea pigs were exsanguinated, and the eye tissue including conjunc-
tiva were extracted. Isolated conjunctiva was incubated in DMF
(1 mL) at 37 °C to extract the extravasated dye, and the incubation
mixture was centrifuged. The absorption of the supernatant at
620 nm was determined, and the amount of Evans blue dye leaked
into the tissues was quantified by interpolation on the standard
curve.
(Amersham plc). 300
lL of the supernatant obtained above was
mixed with 600 L of a solution of 0.5 mol/L tri-n-octylamine in
l
chloroform. After extracting TCA in the organic layer, the cAMP
content in the water layer sample was measured according to
the method described in cAMP assay kit. The strength of DP recep-
tor antagonistic activity of the test compounds was represented by
IC50 (concentration of the test compounds required to inhibit by
50% the production of cAMP in the absence of the test compounds)
calculated from an inhibition ratio against a cAMP production
10. X-ray crystallography of 1bꢃNa salt and 5e
amount which increases by 3 lmol/L PGD2 stimulation.
The crystals of 1bꢃNa salt were obtained from MeCN/H2O (95:5)
solution. The crystals of approximately 0.4 ꢁ 0.4 ꢁ 0.01 mm were
selected for the data collection and mounted in a sealed capillary
tube with a small amount of the mother liquid to prevent decom-
position in air.
7. Single dose rat pharmacokinetic study of 5e and 10c
Single dose pharmacokinetics of 5e and 10c were studied in rats.
Formulation for intravenousinjection was prepared using 30% HP-b-
CD containing 5% DMSO (1 mg/mL/kg) and NaHCO3 (1eq). Formula-
tionfororaldosingwaspreparedusing0.5%MC(1 mg/5 mL/kg). Test
compounds (1 mg/kg) were dosed intravenously to the fasted male
rats (n = 3). Test compounds (1 mg/kg) were dosed orally to the
All measurements were made on a RIGAKU AFC5R diffractome-
ter with graphite-monochromated Cu-K
a radiation and RU-200
rotating anode X-ray generator. Of 4615 reflections that were col-
lected, 3930 were unique. Equivalent reflections were merged. An
empirical absorption correction was applied.
The program package of teXan (1) was used for analysis. The po-
sition of all the non-H atoms were determined by the program
SHELXL86 (2). Hydrogen atoms were placed at calculated positions
and confirmed by the difference Fourier synthesis.
Crystallographic data for 1b: monoclinic P21(#4), a = 8.98(1),
b = 7.74(2), c = 38.53(4) Å, b = 93.8(1)°, V = 2671(6) Å3, Z = 4,
k = 1.54178 Å, R; Rw = 0.106, 0.114.
fasted male rats (n = 3). After dosing, blood samples (250 lL) were
collected from the jugular vein using a heparinized syringe at the se-
lected time points (iv: pre-dosing, 5, 15 and 30 min, 1, 2, 4, 6, 8 and
24 h; po: pre-dosing, 15 and 30 min 1, 2, 4, 6, 8 and 24 h, respec-
tively). The blood samples were ice-chilled and then centrifuged at
12,000 rpm for 2 min at 4 °C to obtain plasma, which was preserved
at -80 °C in a freezer. The AUC, Cmax, Tmax, T1/2, Vss and CL were ob-
tained by measuring the time course of the plasma concentration
of the test compounds. Bioavailability (BA) was calculated according
to the following equation:
(1) teXan:Molecular Structure Corporation; 1993.
(2) Sheldrick, G. M.; Kruger, C.; Goddard R., Eds.; Oxford Univer-
sity Press, 1985; pp 175–189.
BAð%Þ ¼ ðAUCpo=DpoÞ=AUCiv=DivÞ ꢁ 100
AUCpo: AUC after oral dosing; AUCiv: AUC after intravenous dos-
ing; Dpo: dosage of oral administration; Div: dosage of intravenous
administration.
The crystals of 5e were obtained by recrystallization from EtOH/
H2O solution.
A
pale yellow platelet crystal (0.34 ꢁ 0.40 ꢁ
0.04 mm) was mounted in a cryoloop and frozen at 93 K in a liquid
nitrogen stream.
8. Inhibitory effects of selected compounds on PGD2-induced
vascular permeability in guinea pig conjunctiva
Data was collected on Rigaku R-AXIS RAPID with graphite-
monochromated Cu-Ka radiation at 93 K. A total of 25894
reflections were collected, of which 8541 were unique. Equivalent
reflections were merged.
Topical application of PGD2 to the eye of guinea pigs is known to
cause plasma exudation in the conjunctiva via DP receptor. We as-
sessed the PGD2 receptor antagonism of test compounds in con-
junctival vascular permeability induced by PGD2. For the
assessment of the antagonist activity of the test compounds, male
Hartley guinea pigs were challenged by instillation of PGD2 (0.01%,
The program package of Crystal Structure (1) was used for anal-
ysis. The position of all the non-H atoms were determined by the
program SHELXL 97 (2) and DIRDIF (3), and refined anisotropically.
Hydrogen atoms were placed at calculated positions and refined
using a riding model. Crystallographic data for 5e: monoclinic
20 lL/eye) to the eye, and then 5% Evans blue dye (1 mL/kg, iv) was
P21(#4),
b = 95.485(7)°, V = 2356.4(1) Å3, Z = 4, k = 1.54178 Å, R; Rw
(I > 3.00 (I)) = 0.0938, 0.0973.
a = 13.986(2),
b = 4.8040(5),
c = 35.232(4)
Å,
immediately injected as a marker of plasma exudation. All antago-
nists were orally administered 1 h before the challenge. After
30 min, the guinea pigs were exsanguinated, and the eye tissue
including conjunctiva were extracted. Isolated conjunctiva were
incubated in DMF (1 mL) at 37 °C to extract the extravasated dye,
and the incubation mixture was centrifuged. The absorption of
the supernatant at 620 nm was determined, and the amount of
Evans blue dye leaked into the tissues was quantified by interpola-
tion on the standard curve.
r
These data have submitted to the Cambridge Structure Data-
base with reference number CCDC 843498.
(3) Crystal Structure, version 3.6.0, 2000–2004, Crystal Struc-
ture Analysis Package, Rigaku and Rigaku /MSC.
(4) Sheldrick, G. M.; SHELX97; University of Göttingen, Ger-
many 1997.
(5) Beurskens, P. T.; Admiraal, G.; Beurskens, G.; Bosman, W. P.;
Garcia-Granda, S.; Gould, R. O.; Smits, J. M. M.; Smykalla, C.
Technical Report of the Crystallography Laboratory, Univer-
sity of Nijmegen, The Netherlands. 1996.
9. Inhibitory effects of 5e on ovalbumin-induced vascular
permeability in guinea pig conjunctiva
Male Hartley guinea pigs were sensitized by intraperitoneal
injection of a mixture of ovalbumin (OVA) (1 mg) and inactivated
Bordetella pertussis (5 ꢁ 109). Two weeks later, animals were chal-
References and notes
lenged by topical application of OVA (1%, 20 lL/eye) to the eye,
and then Evans blue dye (20 mg/kg, iv) was immediately injected
as a marker of plasma exudation. All antagonists were orally
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