Discovery of a new class of potent, selective, and orally active prostaglandin D2 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2004.07.048

N-(p-Alkoxy)benzoyl-2-methylindole-4-acetic acids 2r and 2s were identified as a new class of orally active prostaglandin D₂ (PGD₂) receptor antagonists. The process of discovering a series of N-(p-alkoxy)benzoyl-2-methylindole-4-ace

Full text of DOI:10.1016/j.bmc.2004.07.048

Bioorganic& Medicinal Chemistry 12 (2004) 5361–5378
Discovery of a new class of potent, selective, and orally
active prostaglandin D2 receptor antagonists
a
a
a
Kazuhiko Torisu,a, Kaoru Kobayashi, Maki Iwahashi, Yoshihiko Nakai,
*
Takahiro Onoda,b Toshihiko Nagase,a Isamu Sugimoto,a Yutaka Okada,a
Ryoji Matsumoto,a Fumio Nanbu,a Shuichi Ohuchida,b Hisao Nakaia and Masaaki Todaa
aMinase Research Institute, Ono Pharmaceutical Co., Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
bFukui Research Institute, Ono Pharmaceutical Co., Ltd, Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
Received 28 June 2004; revised 23 July 2004; accepted 23 July 2004
Available online 23 August 2004
Abstract—The process of discovering a series of N-(p-alkoxy)benzoyl-2-methylindole-4-acetic acid analogs is presented since these
compounds represent a new class of potent, selective, and orally active prostaglandin D2 (PGD2) receptor antagonists. Most of these
compounds exhibit strong PGD2 receptor binding and PGD2 receptor antagonism in cAMP formation assays. When given orally,
these new antagonists dramatically suppress allergic inflammatory responses, such as the PGD2-induced or OVA-induced increase
of vascular permeability. Structure–activity relationship (SAR) data are also discussed.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
In preceding papers,7 we have reported on the discovery
of a new class of selective DP receptor antagonists start-
ing from the chemical modification of Indomethacin
analogs. Here we report on the identification of hDP
receptor antagonists 2r–s, which exhibited efficacy in
animal models, starting from the chemical modification
of 1 and 4 (Fig. 1).
Prostanoids (prostaglandins and thromboxanes) are
products of cyclooxygenase that are derived from ara-
chidonic acid. It has been proposed that specific prosta-
noid receptors exist for each of the primary prostanoids,
including PGD2, PGE2, PGF2 , PGI2, and TXA2, which
a
have been designated as the DP, EP, FP, IP, and TP
receptors, respectively.1 In addition, the EP receptor is
classified into four subtypes (EP1, EP2, EP3, and EP4),
all of which respond to PGE2 in different ways.
2. Chemistry
The synthesis of 5a–i is outlined in Scheme 1. N-Acyl-
ation of 2-methylindole-4-acetic acid esters 6 and 7 with
acid chlorides 8a–i afforded 9a–i, respectively, after
which deprotection by conventional methods gave 5a–i,
respectively.
Prostaglandin D2 (PGD2) receptor antagonists might
have potential clinical uses because PGD2 is considered
to play an important role in various allergicdiseases,
3
including allergic rhinitis,2 atopicasthma, allergiccon-
junctivitis,4 and atopicdermatitis. 5 However, there have
been few reports on the efficacy of PGD2 receptor antag-
onists in animal models of allergy or human allergicdis-
eases.6 A selective DP receptor antagonist would be
expected to show potential therapeutic value for various
allergicdisorders.
Synthesis of 2a–s according to a polymer-supported
solid phase technique is described in Scheme 2.8 N-Acyl-
ation of 7 with p-acetyloxybenzoyl chloride provided
10, catalytic hydrogenation of which led to 11. The
carboxylic acid 11 was supported with a resin to
produce 12, the acetyl moiety of which was removed
by aminolysis to give a phenol 13. Mitsunobu reaction9
of the polymer-supported phenol 13 with alcohols 15a–s
resulted in 14a–s, respectively. Removal of the resin
from 14a–s was achieved by a reported method8 to give
2a–s, respectively.
Keywords: Prostaglandin; DP receptor; Antagonist.
*
Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-
9314; e-mail: torisu@ono.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.048

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K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
CO2H
CO2H
Me
Me
N
N
O
O
X
X
Y
O
O
O
1 : X = CH2O
4 : X = CH2
N
Me
2r : X = F, Y = H
2s : X = H, Y = Me
Figure 1.
CO2R
CO2H
CO2R
X
X
a
b or c
Me
Me
Me
N
O
N
N
H
O
O
O
6 : R = allyl
7 : R = Bn
9a-i
5a-i
a: R = allyl, X = o-OMe
b: R = allyl, X = m-OMe
c: R = benzyl, X = p-OMe
f: R = allyl, X = p-Me
g: R = allyl, X = o-F
h: R = allyl, X = m-F
i: R = allyl, X = p-F
a: X = o-OMe
b: X = m-OMe
c: X = p-OMe
f: X = p-Me
g: X = o-F
h: X = m-F
i: X = p-F
d: R = allyl, X = o-Me
e: R = allyl, X = m-Me
d: X = o-Me
e: X = m-Me
X
a: X = o-OMe
b: X = m-OMe
c: X = p-OMe
f: X = p-Me
ClOC
g: X = o-F
h: X = m-F
i: X = p-F
O
d: X = o-Me
e: X = m-Me
8a-i
Scheme 1. Synthesis of 5a–i. Reagents: (a) NaOH (powdered), 8a–i, TBACl, H2Cl2; (b) morpholine, Pd(PPh3)4, THF; (c) H2, Pd–C, MeOH, EtOAc.
Preparation of 15c, 15k, and 15m–s is described in
Scheme 3. O-Alkylation of a catechol 16 with methyl
dichloroacetate in the presence of sodium hydride affor-
ded 17, after which lithium aluminum hydride reduction
led to 15c (Scheme 3a). N-Methylation of 18,10 followed
by lithium aluminum–hydride reduction, produced 15k11
(Scheme 3b). The optically active N-methyl benzoxazine
15m was prepared by N-methylation of 19,12 followed by
catalytic hydrogenolysis (Scheme 3c). The N-ethyl ben-
zoxazine 15n was prepared by N-ethylation of 15j
(Scheme 3d). Sodium borohydride reduction of a qui-
noxaline 2013 led to the tetrahydroquinoxaline 21,14 after
which N,N0-dimethylation provided 15o (Scheme 3e).
N-Methylation of indoline-2-carboxylic acid 22, fol-
lowed by lithium aluminum hydride reduction, produced
15p (Scheme 3f). Lithium aluminum hydride reduction of
N-tbutoxycarbonylindoline 2415 resulted in a N-methyl-
indoline 15q (Scheme 3g). Compounds 15r–s were pre-
pared from 25a–b, respectively as outlined in Scheme
3h. N-Tosylation of 25a–b with tosyl chloride gave
26a–b, respectively. N-Alkylation of 26a–b with (S)-
(À)-glycidyl triphenylmethyl ether provided 27a–b,
respectively. Cyclization reaction of 27a–b, followed by
removal of the N-tosyl moiety, produced 28a–b, respec-
tively. N-Methylation of 28a–b followed by acidic depro-
tection produced 15r–s, respectively.
3. Results and discussion
The test compounds listed in Tables 1–4 were biolog-
ically evaluated for inhibition of the specific binding of
a radiolabeled ligand, [3H]PGD2, to membrane fractions
prepared from cells stably expressing each prostanoid
receptor and for inhibition of cAMP formation evoked
by PGD2 in CHO cells in the presence of bovine serum
albumin (BSA). Test compounds were also evaluated for
binding to all the subtypes of the mouse PGE2 receptor
(mEP1, mEP2, mEP3, and mEP4) and to the human
PGI2 receptor (hIP).
In one of the earlier papers,7b we reported on the high
mDP receptor affinity and antagonist activity of N-
benzoyl-2-methylindole-4-acetic acids 3, 4, and 1.
However, the hDP receptor affinity and antagonist
activity of these compounds was found to be relatively
lower than their mDP receptor affinity and mDP
antagonist activity, respectively, as described in
Table 1.
Based on the results reported in the preceding paper,7b
the optimum position of the acetic acid residue, which
corresponds to the acidic part of PGs, was identified
as the position-4 of the N-benzoyl-2-methylindole tem-
plate. Thus, our next focus was placed on further opti-
mization of the p-alkoxy moiety, which corresponds to
the lipophilicpart of PGs.
The synthesis of 15a, 15b, 15d and 15e is reported in
Refs. [16–19].

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5363
COOBn
CO2H
a
b
c
7
Me
Me
N
N
O
O
OAc
O
OAc
O
11
10
O
resin
Me
resin
Me
resin
Me
O
N
O
O
N
d
e
N
R
O
OAc
O
OH
O
O
12
14a-s
13
CO2H
Cl
f
Me
resin :
polymer
N
R
O
O
2a-s
b)20
b)16
b)17
c)
c)
O
O
N
OH
OH
OH
OH
OH
OH
15a-s :
h
a
n
O
O
O
b)21
b)11
Et
Me
i
b
N
O
O
O
O
o
p
c)
OH
OH
OH
N
Me
j
c
d
e
O
O
N
H
O
c)
OH
b)18
b)19
c)
OH
N
Me
k
N
Me
O
OH
OH
b)22
c)
q
OH
OH
O
O
l
N
Me
O
a)
a)
OH
N
Me
f
F
c)
OH
OH
O
O
c)
r
O
N
Me
O
m
OH
g
N
Me
O
c)
s
Me
N
Me
Scheme 2. Synthesis of 2a–s. Reagents: (a) NaOH (powdered), p-acetoxybenzoyl chloride, CH2Cl2; (b) Pd(OH)2, H2, EtOAc; (c) resin,
diisopropylethylamine; (d) piperidine, CH2Cl2; (e) 15a–s, DEAD, PPh3; (f) AcOH, CF3CH2OH, CH2Cl2. (a) Commercially available; (b) reported;
(c) described in Scheme 3a–h.
Because more diverse chemical modification of the ter-
minal phenyl moiety relative to 3 was expected to be
possible, compounds 4 and 1 were selected as the chem-
ical leads for further optimizations.
receptor affinity, nearly equal hDP receptor affinity,
and slightly lower hDP antagonist activity relative to 4.
The m-methoxy derivative 5b showed more than 25-fold
weaker mDP receptor affinity, nearly 2-fold weaker hDP
receptor affinity, and 10-fold higher hDP antagonist
activity compared with 4.
Introduction of a substituent into the terminal phenyl
moiety of 4 was followed by biological evaluation, as
shown in Table 2. Introduction of a methoxy moiety
at the terminal phenyl moiety of 4 afforded 5a–c. The
o-methoxy derivative 5a showed slightly weaker mDP
The p-methoxy derivative 5c demonstrated nearly 3-fold
weaker mDP receptor affinity, slightly weaker hDP

5364
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
OH
OH
O
OH
a
O
b
a)
b)
CO2Me
O
O
16
17
15c
CO2Et
O
O
a, b
OH
N
N
H
Me
18
15k
O
O
OH
a, b
OBn
c)
d)
N
N
H
Me
19
15m
O
O
OH
a
OH
N
H
N
Me
15n
15j
Me
N
H
N
N
CO2Et
a
OH
b
OH
e)
f)
N
N
H
N
Me
20
21
15o
OH
a
b
CO2H
OH
CO2Me
OH
N
N
N
Me
15p
H
Me
22
23
a
g)
h)
N
N
Me
15q
Boc
24
X
Y
F
X
Y
F
X
F
b
a
NHTs
N
OTr
Y
NH2
Ts OH
25a: X = F, Y = H
25b: X = H, Y = Me
26a: X = F, Y = H
27a: X = F, Y = H
26b:X = H, Y = Me
27b: X = H, Y = Me
O
X
Y
O
X
Y
c, d
OH
OTr
e, f
N
N
H
Me
28a: X = F, Y = H
28b:X = H, Y = Me
15r: X = F, Y = H
15s:X = H, Y = Me
Scheme 3. Synthesis of 15c, 15k, and 15m–s. (a) Reagents: (a) NaH, methyl dichloroacetate, DMF; (b) LiAlH4, THF. (b) Reagents: (a) K2CO3, MeI,
acetone; (b) LiAlH4, THF. (c) Reagents: (a) K2CO3, MeI, acetone; (b) H2, Pd–C, HCl, MeOH. (d) Reagents: (a) K2CO3, EtI, DMF. (e) Reagents: (a)
NaBH4, MeOH, THF; (b) K2CO3, MeI, acetone. (f) Reagents: (a) K2CO3, MeI, acetone; (b) LiAlH4, THF. (g) Reagents: (a) LiAlH4, ether. (h)
Reagents: (a) TsCl, Pyridine, CH2Cl2; (b) (S)-(À)-glycidyl triphenylmethyl ether, K2CO3, BnEt3NCl, 1,4-dioxane; (c) tBuOK, THF; (d) Na,
naphthalene, DME; (e) MeI, K2CO3, acetone; (f) AcOH, THF, H2O.
receptor affinity, and nearly 3-fold lower hDP receptor
antagonist activity compared with 4. Introduction of a
methyl moiety at the terminal phenyl moiety of 4
afforded 5d–f. The o-methyl derivative 5d showed more
than 10-fold weaker mDP receptor affinity, nearly equal
hDP receptor affinity, and 5-fold higher hDP antagonist

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5365
Table 1. New chemical leads for a DP receptor antagonist
hDP receptor affinity, and nearly 2-fold lower hDP
receptor antagonist activity relative to 4. The p-fluoro
derivative 5i demonstrated nearly 4.5-fold weaker
mDP receptor affinity, nearly equal hDP receptor affin-
ity, and nearly 3-fold lower hDP receptor antagonist
activity relative to 4. The subtype selectivity of the com-
pounds listed in Table 2 was not improved relative to
that of 4. hDP Receptor antagonist activity was in-
creased for the m-methoxy and m-methyl derivatives
5b and 5e relative to their o-and p-isomers, while it
was not always improved for the m-fluoro derivative 5h.
CO2H
Me
N
R
O
O
Compd
R
Binding Ki
(lM)
IC50 (lM)a
mDP hDP
mDP
hDP
0.18
As a result, it was concluded that the increase of hDP
receptor affinity was not so great as that of mDP recep-
tor affinity in the above-described chemical modification
of 4. Thus structural transformation of the terminal
phenoxy moiety of 1 to a fused bicyclic structures as de-
scribed in Figure 1 instead of simply introducing a sub-
stituent was carried out as another approach for the
optimization.
O
O
3
4
0.020
0.074
0.12
0.20
0.15
0.0018
0.087
0.29
1
0.017
0.053
0.25
a IC50 (lM): hDP receptor antagonist activity.
activity compared with 4. The m-methyl derivative 5e
showed nearly 7-fold weaker mDP receptor affinity,
nearly equal hDP receptor affinity and nearly 10-fold
higher hDP receptor antagonist activity relative to 4.
The p-methyl derivative demonstrated nearly 10-fold
weaker mDP receptor affinity, nearly 3-fold weaker
hDP receptor affinity, and nearly 2-fold lower hDP
receptor antagonist activity relative to 4. Introduction
of a fluoro residue at the terminal phenyl moiety of 4
provided 5g–i.
Synthesis and evaluation of the dihydrobenzofuran, 1,2-
methylenedioxybenzene, dihydrobenzopyran, and ben-
zodioxane derivatives listed in Table 3 was carried out.
As shown in Table 3, dihydrobenzofuran derivative 2a
demonstrated slightly weaker mDP receptor affinity
and 10-fold stronger hDP receptor affinity relative to
1, while it showed nearly 7-fold higher hDP receptor
antagonist activity. The regio isomer 2b exhibited higher
hDP receptor antagonist activity, although its mDP and
hDP receptor affinity was weaker than that of 2a.
The o-fluoro derivative 5g showed nearly equal mDP
receptor affinity, nearly 2-fold stronger hDP receptor
affinity, and equal hDP receptor antagonist activity rel-
ative to 4. The m-fluoro derivative 5h showed nearly 10-
fold weaker mDP receptor affinity, nearly 3-fold weaker
Introduction of another oxygen into the fused
dihydrofuran of 2a afforded 2c, which showed nearly
2-fold stronger mDP receptor affinity, slightly weaker
hDP receptor affinity, and slightly lower hDP receptor
Table 2. Effect of the terminal phenyl moiety on the activity profiles
CO2H
Me
R
N
O
O
Compd
R
Binding Ki (lM)
IC50 (lM)b
mEP1
mEP2
mEP3
mEP4
hIP
mDP
hDP
hDP
4
H
0.40
2.4
1.3
2.2
4.1
1.9
1.9
2.9
2.6
1.8
1.8
3.9
1.9
1.6
3.5
0.14
0.0018
0.0039
0.045
0.087
0.10
0.190
0.14
0.11
0.13
0.30
0.046
0.20
0.13
0.15
0.25
0.015
0.40
0.032
0.016
0.28
0.15
0.37
0.47
5a
5b
5c
5d
5e
5f
5g
5h
5i
o-OMe
m-OMe
p-OMe
o-Me
m-Me
p-Me
o-F
0.42
0.55
0.68
0.23
0.79
1.7
0.099
NTa
0.95
>10
0.69
0.65
1.4
>10
>10
2.3
0.0064
0.026
0.013
0.050
0.083
0.29
>10
>10
1.4
1.1
0.025
0.27
1.2
0.24
0.73
0.71
0.026
NTa
0.29
0.0024
0.019
0.0093
m-F
p-F
2.0
2.8
0.20
a NT: not tested.
b IC50 (lM): hDP receptor antagonist activity.

5366
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
Table 3. Activity profiles of benzofuran, benzopyran, and benzodioxane derivatives
CO2H
Me
N
R
O
O
Compd
R
Binding Ki (lM)
IC50 (lM)b
mEP1
5.6
mEP2
0.54
mEP3
>10
mEP4
hIP
>10
mDP
0.017
hDP
0.29
hDP
O
O
>10
>10
0.25
1
2a
7.8
0.45
0.98
0.27
0.022
0.029
0.036
2b
2c
2d
2e
2f
3.1
0.78
3.8
0.78
0.25
0.092
0.28
2.9
1.4
0.75
>10
>10
>10
>10
>10
>10
2.9
>10
3.1
0.33
0.33
0.16
0.068
0.0094
0.061
0.18
0.042
0.039
0.052
0.39
0.013
0.056
0.030
NTa
O
O
O
O
>10
7.0
3.5
NTa
O
O
O
O
>10
>10
>10
1.3
NTa
0.18
NTa
0.24
2g
2h
>10
2.0
0.24
0.064
1.3
0.27
0.012
0.016
0.12
0.032
0.018
0.13
0.024
0.031
0.27
O
O
R
S
0.22
1.6
O
O
2i
2.3
O
a NT: not tested.
b IC50 (lM): hDP receptor antagonist activity.
antagonist activity. Subtype selectivity of 2a–c was re-
duced relative to that of 4, mainly because of the in-
creased mEP3 and hIP receptor affinity.
with a benzodioxane-2-yl ethyl moiety afforded 2f, which
showed nearly 10-fold weaker mDP receptor affinity and
10-fold lower hDP receptor antagonist activity.
Benzopyran derivative 2d showed 3.5-fold weaker mDP
receptor affinity, and 5.5-fold stronger hDP receptor
affinity relative to 1, while it showed nearly 8-fold higher
hDP receptor antagonist activity. Another dihydrobenz-
opyran 2e showed weaker affinity for the mDP and hDP
receptors compared with 2d. Introduction of another
phenolicoxygen at the fused dihydropyran of 2d afforded
2g, which showed nearly 5-fold stronger mDP receptor
affinity and 2-fold stronger hDP receptor affinity. As
illustrated in the transformation from 2d to 2g, introduc-
tion of another oxygen was effective for improving sub-
type selectivity and maintaining strong hDP antagonist
activity. Replacement of the benzodioxane-2-yl methyl
Synthesis and biological evaluation of the optically ac-
tive forms 2h and 2i was also conducted. The R-isomer
2h demonstrated nearly equal mDP receptor affinity,
nearly 2-fold stronger hDP receptor affinity, and nearly
equal hDP receptor antagonist activity relative to its
racemic form 2g, while the S-isomer 2i showed more
than 10-fold weaker affinity for the mDP and hDP recep-
tors and nearly 10-fold lower hDP receptor antagonist
activity. The subtype selectivity of the dihydrobenzofu-
ran analogs 2a–c and the benzodioxane analog 2g was
lower than that of chemical lead 1, while the subtype
selectivity of the optically active R-isomer 2h was higher
than that of the S-isomer 2i.

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5367
Table 4. Activity profiles of benzomorpholine, benzopiperazine, and indoline derivatives
CO2H
Me
N
R
O
O
Compd
R
Binding Ki (lM)
IC50 (lM)b
mEP1
2.0
mEP2
0.45
mEP3
>10
mEP4
hIP
mDP
0.16
hDP
hDP
O
2j
2.8
4.7
NTa
0.087
0.021
N
H
O
2k
>10
0.11
0.10
0.41
1.7
>10
0.13
0.037
NTa
0.94
0.027
0.016
0.23
0.0038
0.0035
0.17
0.0039
0.0012
0.019
N
Me
O
S
R
2l
6.2
1.7
>10
>10
3.3
N
Me
O
2m
2n
>10
>10
>10
>10
N
Me
O
0.024
0.018
0.013
N
Et
Me
N
2o
>10
2.7
>10
>10
NTa
0.19
0.21
0.022
N
Me
2p
2q
8.1
3.9
>10
1.4
>10
>10
NTa
0.19
0.22
0.30
0.22
0.16
N
Me
>10
0.74
0.066
0.011
N
Me
O
F
S
S
7
2r
>10
>10
2.1
2.4
1.7
>10
>10
0.26
1.2
0.023
0.021
0.0053
0.018
0.00081
0.0021
N
Me
O
2s
>10
N
Me
6
Me
a NT: not tested.
b IC50 (lM): hDP receptor antagonist activity.
Further optimization of 2a–b and 2g was successfully
carried out by introducing a nitrogen instead of an oxy-
gen at the same position, as illustrated in Table 4.
Replacing the 4-oxygen of the benzodioxane moiety of
2g with NH led to 2j, which showed reduced mDP
and hDP receptor affinity, while 2j showed equal hDP
receptor antagonist activity relative to 2g. N-Methyl-
ation of the nitrogen of the benzomorpholine of 2j gave
2k, with improved mDP and hDP receptor affinity as
well as a marked increase of hDP receptor antagonist
activity. N-Ethylation of the benzomorpholine of 2j
afforded 2n, which showed increased mDP and hDP
receptor affinity as well as nearly 2-fold higher hDP
receptor antagonist activity, although the increase of
antagonist activity was not as great as that of the N-
methylated compound. Replacement of the oxygen of
the benzomorpholine moiety of 2k with a N-methyl moi-
ety afforded 2o, which demonstrated a marked decrease

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K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
Table 5. Inhibitory effects of 4, 2r, and 2s on PGD2-induced vascular
permeability in guinea pig conjunctiva (n = 8). ** 0.01 versus Control
with DunnettÕs test
of mDP and hDP receptor affinity as well as 5-fold lower
antagonist activity. Accordingly, synthesis and biologi-
cal evaluation of the optically active forms of the most
potent analog 2k was conducted.
Compd
Dose (mg/kg, po)
% inhibition
4
1
46
51
The 2S-isomer 2l, which showed an increase of receptor
affinity and antagonist activity compared with the race-
mate 2k, demonstrated markedly higher DP receptor
affinity and antagonist activity than the 2R-isomer 2m.
Replacement of the oxygen atom of the benzofuran
derivative 2a with an N-methyl moiety afforded 2p,
which showed nearly 10-fold weaker affinity for mDP
and hDP receptors and nearly 4-fold lower hDP antag-
onist activity. The regioisomer 2q showed nearly 14-fold
higher hDP antagonist activity than 2p, while it showed
nearly equal affinity and nearly 3-fold stronger affinity
for the mDP and hDP receptors, respectively.
3
2r
2s
0.3
0.3
60**
61**
Inhibition of increase in conjunctival vascular permeability caused by
topical application of PGD2 (0.01%, 20lL/eye) in guinea pigs. All
antagonists were administered po 1h before the challenge.
Table 6. Inhibitory effects of 2r and 2s on OVA-induced vascular
permeability in guinea pig conjunctiva (n = 7) *0.1 versus Control with
DunnettÕs test
Compd
Dose (mg/kg, po)
% inhibition
2r
10
10
21
Introduction of a 7-fluoro moiety and a 6-methyl moiety
at the N-methylbenzomorpholine of the more active
enantiomer 2l afforded 2r and 2s, respectively, with
nearly equal mDP receptor affinity. Compound 2r exhib-
ited nearly 1.5-fold lower hDP receptor affinity relative
to 2l, while 2s showed 5-fold weaker hDP receptor affin-
ity. With regard to hDP receptor antagonist activity, that
of 2r was 1.5-fold higher relative to 2l, while that of 2s
was nearly 1.8-fold lower. As a result, 2r exhibited the
most potent hDP antagonist activity among the test com-
pounds. Thus, 2r and 2s were identified as more opti-
mized subtype selective hDP antagonist because of the
increased affinity of 2l for the hIP receptor.
2s
Pyrilamine
40*
68
1 (mg/kg, iv)
Inhibition of increase in conjunctival vascular permeability caused by
topical application of OVA (1%, 20lL/eye) in actively sensitized gui-
nea pigs. All antagonists were administered po 1h before the antigen
challenge.
Based on these findings, the newly discovered DP antag-
onists such as 2r and 2s were expected to have therapeu-
ticpotential for allergicdiseases.
The results of pharmacokinetic (PK) study of compound
2s was shown in Table 7 as a representive example.
When 2s was administered orally to male rats at a dose
of 10mg/kg under fasting condition. Compound 2s had
a good pharmacokinetic profile in rats (Table 7).
Administration of this compound to rats (1mg/kg, iv;
10mg/kg, po; n = 3) led to detectable plasma levels, with
a T1/2 of 9.2h (iv) and 7.8h (po). The volume of distri-
bution (Vss) was calculated to be 1408mL/kg, indicating
distribution to the tissues. Systemic clearance (CL)
was 2.10mL/min/kg. The peak plasma level (Cmax) was
4371lg/mL at 0.7h, and the oral bioavailability was
48% at a dose of 10mg/kg. The AUC was 8.69lgh/
mL (iv) and 42.1lgh/mL (po).
Pharmacological evaluation of the antagonist activity of
the optimized compounds was carried out as follows.
Some of the selected compounds were evaluated for
inhibition of the increase of vascular permeability in
the guinea pig conjunctiva after oral administration.
As shown in Table 5, when chemical lead 4 was given
orally (po), it caused 46% inhibition and 51% inhibition
at a dose of 1 and 3mg/kg, respectively. The optically
active isomer 2r had significantly greater potency than
4, and achieved 60% inhibition at 0.3mg/kg, po. The
optically active N-methyl benzomorpholine analog 2s
was equipotent to 2r, but exhibited more potency than 4.
4. Summary
These compounds were also evaluated for their ability to
inhibit the increase of vascular permeability caused by
OVA (1%, 20lL) in the guinea pig conjunctiva.
Further optimization of the new chemical leads 1 and 4
was successfully achieved. Modification of these chemi-
cal leads was concentrated on their terminal phenyl moi-
ety, which was still not optimized. The N-methyl
benzomorpholine derivative 2k showed the most potent
hDP receptor antagonist activity among the racemic
compounds tested. Optically active 2r and 2s were iden-
tified as more optimum hDP receptor selective
As shown in Table 6, when compounds 2r and 2s were
given orally, they caused 21% inhibition and 40% inhibi-
tion at 10mg/kg, respectively, while a positive control
(Pyrilamine) caused 68% inhibition when a dose of
1mg/kg was given intravenously.
Table 7. Pharmacokinetic data for 2s in rats
Dose (mg/kg)
AUCinf (lgh/mL)
Tmax (h)
Cmax (lg/mL)
CLtot (mL/min/kg)
T1/2 (h)
Vss (mL/kg)
1408
BA (%)
48
iv
po
1
10
8.69
42.1
2.1
9.2
7.8
0.7
4371

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5369
antagonists relative to 2l because both showed weaker
hIP receptor affinity. Some of the selected compounds
were evaluated in animal models, and compound 2s
exhibited good biological and PK profiles.
5.2.2. Allyl (1-{4-[2-(2-methoxyphenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetate (9a). 65% yield; TLCC
1
Rf = 0.35 (n-hexane/EtOAc, 3/1); H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.22 (d, J = 7.2Hz,
2H), 7.04–6.90 (m, 7H), 6.50 (s, 1H), 6.02–5.80 (m,
1H), 5.32–5.17 (m, 2H), 4.63–4.60 (m, 2H), 4.24 (t,
J = 7.4Hz, 2H), 3.85 (s, 2H), 3.84 (s, 3H), 3.12 (t, J
7.4Hz, 2H), 2.45 (s, 3H).
5. Experimental
5.1. General directions
5.2.3. Allyl (1-{4-[2-(3-Methoxyphenyl)ethoxy]benzoyl}-
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 GEMINI-200 or
VXR-200s spectrometer using deuterated chloroform
(CDCl3) or deuterated methanol (CD3OD) or deuter-
ated dimethylsulfoxide (DMSO-d6) as the solvent. Fast
atom bombardment mass spectra (FAB-MS) and elect-
ron 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. Column chromatography
was carried out on silica gel [Merck silica gel 60
(0.063ꢀ0.200mm), Wako gel C200 or Fuji Silysia
BW235]. 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; tetrahydrofuran (THF), ethyl acetate
(EtOAc), dimethylformamide (DMF), dichloromethane
(CH2Cl2), chloroform (CHCl3), methanol (MeOH), ace-
tic acid (AcOH), triethylamine (TEA) and diisopropyl-
ethylamine (DIPEA).
2-methyl-1H-indol-4-yl)acetate
(9b).
70%
yield;
TLC Rf = 0.37 (n-hexane/EtOAc, 3/1); 1H NMR
(300MHz, CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.44–
7.36 (m, 1H), 7.27–7.20 (m, 2H), 7.06–6.90 (m, 6H),
6.50 (s, 1H), 6.02–5.79 (m, 1H), 5.32–5.18 (m, 2H),
4.63–4.60 (m, 2H), 4.24 (t, J = 7.0Hz, 2H), 3.88 (s,
5H), 3.15 (t, J = 7.0Hz, 2H), 2.45 (s, 3H).
5.2.4. Allyl (2-methyl-1-{4-[2-(3-methylphenyl)ethoxy]-
benzoyl}-1H-indol-4-yl)acetate (9e). 81% yield; TLC
1
Rf = 0.40 (n-hexane/EtOAc, 1/1); H NMR (200MHz,
CDCl3) d 7.69 (d, J = 9.0Hz, 2H), 7.22–6.92 (m, 9H),
6.50 (s, 1H), 6.01–5.81 (m, 1H), 5.32–5.18 (m, 2H),
4.61 (dt, J = 5.4, 1.2Hz, 2H), 4.25 (t, J = 7.0Hz, 2H),
3.86 (s, 2H), 3.10 (t, J = 7.0Hz, 2H), 2.44 (d,
J = 1.0Hz, 3H), 2.40 (s, 3H); MS (APCI, Pos, 20V.)
m/z 468 (M+H)+.
5.2.5. Allyl (2-methyl-1-{4-[2-(4-methylphenyl)ethoxy]-
benzoyl}-1H-indol-4-yl)acetate (9f). 34% yield; TLC
1
Rf = 0.25 (n-hexane/EtOAc, 4/1); H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.6Hz, 2H), 7.18–7.16 (m, 4H),
7.02–6.92 (m, 5H), 6.50 (s, 1H), 6.01–5.81 (m, 1H),
5.32–5.18 (m, 2H), 4.61 (dt, J = 5.6, 1.4Hz, 2H), 4.23
(t, J = 7.2Hz, 2H), 3.86 (s, 2H), 3.10 (t, J = 7.2Hz,
2H), 2.44 (d, J = 0.8Hz, 3H), 2.34 (s, 3H); MS (APCI,
Pos, 20V.) m/z 468 (M+H)+.
5.2.6. Allyl (1-{4-[2-(2-fluorophenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetate (9g). 87% yield; TLC
1
Rf = 0.47 (n-hexane/EtOAc, 3/1); H NMR (200MHz,
5.2. General procedure for the preparation of
N-(p-(2-arylethyloxy)benzoyl)-2-methyl-indole-4-acetic
acids (9a–i)
CDCl3) d 7.68 (d, J = 8.8Hz, 2H), 7.40–7.10 (m, 4H),
7.06–6.89 (m, 5H), 6.49 (s, 1H), 6.01–5.80 (m, 1H),
5.32–5.18 (m, 2H), 4.63–4.60 (m, 2H), 4.25 (t, J 6.9Hz,
2H), 3.86 (s, 2H), 3.18 (t, J = 6.9Hz, 2H), 2.42 (s, 3H).
5.2.1. Allyl (2-methyl-1-{4-[2-(2-methylphenyl)ethoxy]-
benzoyl}-1H-indol-4-yl)acetate (9d). To a stirred mixture
of 6 (100mg, 0.436mmol) and NaOH (powdered,
87.2mg, 2.18mmol) in CH2Cl2 (5mL) were added
TBACl (6mg, 0.02mmol) and p-((2-(o-methylphe-
nyl)ethyloxy))benzoyl chloride (180mg, 0.655mmol) at
room temperature. After stirring for 30min at room
temperature, the reaction mixture was quenched with
2M HCl aq and extracted with EtOAc. The combined
organiclayers were washed with water, brine, dried over
Na2SO4 and evaporated to afford the residue, which was
purified by column chromatography on silica gel to yield
9d (107mg, 53%); TLC Rf = 0.38 (n-hexane/EtOAc, 3/1);
1H NMR (300MHz, CDCl3) d 7.69 (d, J = 8.8Hz, 2H),
7.22–7.17 (m, 4H), 7.02–6.92 (m, 5H), 6.50 (s, 1H), 6.01–
5.81 (m, 1H), 5.31–5.18 (m, 2H), 4.61 (dt, J = 5.6,
1.4Hz, 2H), 4.23 (t, J = 7.2Hz, 2H), 3.86 (s, 2H), 3.16
(t, J = 7.2Hz, 2H), 2.44 (s, 3H), 2.40 (s, 3H); MS (APCI,
Pos, 20V.) m/z 468 (M+H)+.
5.2.7. Allyl (1-{4-[2-(3-fluorophenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetate (9h). 80% yield; TLC
1
Rf = 0.45 (n-hexane/EtOAc, 3/1); H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.38–7.20 (m, 2H),
7.09–6.85 (m, 7H), 6.49 (s, 1H), 6.01–5.81 (m, 1H),
5.33–5.16 (m, 2H), 4.63–4.60 (m, 2H), 4.25 (t,
J = 6.6Hz, 2H), 3.86 (s, 2H), 3.13 (t, J = 6.6Hz, 2H),
2.42 (s, 3H).
5.2.8. Allyl (1-{4-[2-(4-fluorophenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetate (9i). 90% yield; TLC
1
Rf = 0.50 (n-hexane/EtOAc, 3/1); H NMR (200MHz,
CDCl3) d 7.72–7.63 (m, 2H), 7.23–7.17 (m, 2H), 7.06–
6.82 (m, 7H), 6.49 (s, 1H), 6.02–5.80 (m, 1H), 5.35–
5.17 (m, 2H), 4.63–4.59 (m, 2H), 4.21 (t, J =
7.0Hz, 2H), 3.88 (s, 2H), 3.08 (t, J = 7.0Hz, 2H), 2.42
(s, 3H).

5370
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5.2.9. Benzyl (1-{4-[2-(4-methoxyphenyl)ethoxy]benzoyl}-
2-methyl-1H-indol-4-yl)acetate (9c). 28% yield; TLC
1683, 1604, 1574, 1510, 1434, 1370, 1299, 1256, 1221,
1169, 1025, 912, 779, 760, 701, 642, 542cmÀ1
.
1
Rf = 0.68 (n-hexane/EtOAc, 5/1); H NMR (200MHz,
CDCl3) d 7.73–7.65 (m, 2H), 7.40–7.16 (m, 7H), 7.06–
6.83 (m, 7H), 6.45 (s, 1H), 5.15 (s, 2H), 4.20 (t,
J = 7.0Hz, 2H), 3.86 (s, 2H), 3.80 (s, 3H), 3.08 (t,
J = 7.0Hz, 2H), 2.42 (s, 3H).
5.3.5. (2-Methyl-1-{4-[2-(4-methylphenyl)ethoxy]benzo-
yl}-1H-indol-4-yl)acetic acid (5f). 34% yield; TLC
Rf = 0.50 (CH3Cl/MeOH, 9/1); 1H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.18–6.92 (m, 9H),
6.49 (s, 1H), 4.23 (t, J = 6.6Hz, 2H), 3.87 (s, 2H), 3.10
(t, J = 6.6Hz, 2H), 2.44 (s, 3H), 2.34 (s, 3H); MS (APCI,
Neg, 20V.) m/z 426 (MÀH)À; HRMS (EI, Pos.) calcd for
C27H25NO4: 427.1784; found: 427.1783; IR (KBr) 3419,
2923, 1696, 1680, 1606, 1511, 1435, 1371, 1318, 1255,
5.3. General procedure for the preparation of
N-(p-(2-arylethyloxy)benzoyl)-2-methyl-indole-4-
acetic acids (5a, b, d–i)
5.3.1.
(2-Methyl-1-{4-[2-(2-methylphenyl)ethoxy]ben-
1222, 1172, 1021, 912, 811, 761, 639, 613, 554cmÀ1
.
zoyl}-1H-indol-4-yl)acetic acid (5d). To a stirred solution
of 9d (107mg, 0.229mmol) in THF (5mL) was added
Pd(PPh3)4 (26mg, 0.022mmol) at room temperature
under argon atmosphere. After stirring for 15min, mor-
pholine (0.10mL, 1.2mmol) was added. After 1h, 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 a residue, which was purified
by column chromatography on silica gel to yield 5d
5.3.6. (1-{4-[2-(2-Fluorophenyl)ethoxy]benzoyl}-2-meth-
yl-1H-indol-4-yl)acetic acid (5g). 85% yield; TLC
Rf = 0.45 (CH3Cl/MeOH, 9/1); 1H NMR (200MHz,
CDCl3) d 7.68 (d, J = 8.8Hz, 2H), 7.40–7.08 (m, 4H),
7.05–6.90 (m, 5H), 6.47 (s, 1H), 4.26 (t, J = 6.8Hz,
2H), 3.84 (s, 2H), 3.18 (t, J = 6.8Hz, 2H), 2.43 (s, 3H);
MS (APCI, Neg, 20V.) m/z 430 (MÀH)À; HRMS (EI,
Pos.) calcd for C26H22FNO4: 431.1533; found:
431.1539; IR (KBr) 2927, 1685, 1605, 1494, 1434,
1
(45mg, 48%); TLC Rf = 0.50 (CHCl3/CH3OH, 9/1); H
1301, 1256, 1170cmÀ1
.
NMR (200MHz, CDCl3) d 7.69 (d, J = 9.0Hz, 2H),
7.23–6.91 (m, 9H), 6.48 (s, 1H), 4.23 (t, J = 7.2Hz,
2H), 3.86 (s, 2H), 3.16 (t, J = 7.2Hz, 2H), 2.44 (s, 3H),
2.39 (s, 3H); MS (APCI, Neg, 20V.) m/z 426 (MÀH)À;
HRMS (EI, Pos.) calcd for C27H25NO4: 427.1784;
found: 427.1765; IR (KBr) 3419, 2926, 1684, 1605,
1574, 1510, 1434, 1370, 1300, 1256, 1221, 1169, 1019,
5.3.7. (1-{4-[2-(3-Fluorophenyl)ethoxy]benzoyl}-2-meth-
yl-1H-indol-4-yl)acetic acid (5h). 86% yield; TLC
Rf = 0.45 (CH3Cl/MeOH, 10/1); 1H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.35–7.20 (m, 2H),
7.09–6.85 (m, 7H), 6.47 (s, 1H), 4.25 (t, J = 6.6Hz,
2H), 3.84 (s, 2H), 3.12 (t, J = 6.6Hz, 2H), 2.42 (s, 3H);
MS (APCI, Neg, 20V.) m/z 430 (MÀH)À; HRMS (EI,
Pos.) calcd for C26H22FNO4: 431.1533; found:
431.1539; IR (KBr) 2928, 1686, 1605, 1510, 1434,
912, 778, 760, 642, 615cmÀ1
.
5.3.2. (1-{4-[2-(2-Methoxyphenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetic acid (5a). 42% yield; TLC
1256, 1170, 1142, 1027cmÀ1
.
1
Rf = 0.45 (CHCl3/CH3OH, 10/1); H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.22 (d, J = 7.2Hz,
2H), 7.04–6.93 (m, 7H), 6.47 (s, 1H), 4.24 (t,
J = 7.4Hz, 2H), 3.85 (s, 2H), 3.85 (s, 3H), 3.14 (t,
J = 7.4Hz, 2H), 2.43 (s, 3H); MS (APCI, Neg, 20V.)
m/z 442 (MÀH)À; HRMS (EI, Pos.) calcd for C27H25
NO5: 443.1733; found:443.1732; IR (KBr) 2926, 1684,
5.3.8. (1-{4-[2-(4-Fluorophenyl)ethoxy]benzoyl}-2-meth-
yl-1H-indol-4-yl)acetic acid (5i). 76% yield; TLC
Rf = 0.45 (CH3Cl/MeOH, 10/1); 1H NMR (200MHz,
CDCl3) d 7.72–7.65 (m, 2H), 7.23–7.17 (m, 2H), 7.06–
6.84 (m, 7H), 6.47 (s, 1H), 4.21 (t, J = 7.0Hz, 2H),
3.85 (s, 2H), 3.80 (s, 3H), 3.08 (t, J = 7.0Hz, 2H), 2.43
(s, 3H); MS (APCI, Neg, 20V.) m/z 430 (MÀH)À;
HRMS (EI, Pos.) calcd for C26H22FNO4: 431.1533;
found: 431.1532; IR (neat) 2926, 1684, 1605, 1510,
1604, 1435, 1299, 1255, 756cmÀ1
.
5.3.3. (1-{4-[2-(3-Methoxyphenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetic acid (5b). 50% yield; TLC
Rf = 0.35 (EtOAc); 1H NMR (300MHz, CDCl3) d
7.72–7.68 (m, 2H), 7.44–7.36 (m, 1H), 7.28–7.22 (m,
2H), 7.06–6.88 (m, 6H), 6.48 (s, 1H), 4.24 (t,
J = 7.0Hz, 2H), 3.86 (s, 5H), 3.15 (t, J = 7.0Hz, 2H),
2.44 (s, 3H); MS (APCI, Neg, 20V.) m/z 442 (MÀH)À;
HRMS (EI, Pos.) calcd for C27H25 NO5: 443.1733;
found: 443.1734; IR (neat) 2936, 1682, 1602, 1494,
1434, 1370, 1300, 1256, 1222, 1170, 760cmÀ1
.
5.4. (1-{4-[2-(4-Methoxyphenyl)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetic acid (5c)
To a stirred solution of the compound 9c (110mg,
206mmol) in EtOAc(1mL) and MeOH (2mL) was
added 10wt% Pd–C (10mg) at room temperature. The
resulting suspension was stirred for 30min at room tem-
perature 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 5c (25.2mg, 28%); TLC Rf = 0.50 (CHCl3/
CH3OH, 10/1); 1H NMR (200MHz, CDCl3) d 7.72–
7.65 (m, 2H), 7.23–7.17 (m, 2H), 7.06–6.84 (m, 7H),
6.47 (s, 1H), 4.21 (t, J = 7.0Hz, 2H), 3.85 (s, 2H), 3.80
(s, 3H), 3.08 (t, J = 7.0Hz, 2H), 2.43 (s, 3H); MS (APCI,
1433, 1369, 1253, 1168, 1019, 910, 731, 642, 520cmÀ1
.
5.3.4. (2-Methyl-1-{4-[2-(3-methylphenyl)ethoxy]benz-
oyl}-1H-indol-4-yl)acetic acid (5e). 42% yield; TLC
Rf = 0.50 (CH3Cl/MeOH, 9/1); 1H NMR (200MHz,
CDCl3) d 7.69 (d, J = 8.8Hz, 2H), 7.19–6.92 (m, 9H),
6.48 (s, 1H), 4.24 (t, J = 7.2Hz, 2H), 3.86 (s, 2H), 3.10
(t, J = 7.2Hz, 2H), 2.44 (s, 3H), 2.36 (s, 3H); MS (APCI,
Neg, 20V.) m/z 426 (MÀH)À; HRMS (EI, Pos.) calcd for
C27H25NO4: 427.1784; found: 427.1774; IR (KBr) 2924,

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5371
Neg 20V) m/z 442 (MÀH)À; HRMS (EI, Pos.) calcd for
C27H25NO5: 443.1733; found: 443.1714; IR (neat) 2931,
1708, 1683, 1604, 1513, 1434, 1370, 1301, 1250, 1169,
30min at room temperature, the reaction mixture was fil-
tered, washed with CH2Cl2 (·5), dried in vacuo to afford
13 (24.7g, 100%); IR (KBr) 3410, 3059, 3025, 2923, 2849,
1738, 1684, 1604, 1492, 1432, 1367, 1285, 1219, 1163,
1040, 981, 957, 909, 826, 754, 735, 698, 634, 607, 589,
1025, 912, 829, 759cmÀ1
.
5.5. Benzyl {1-[4-(acetyloxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetate (10)
461cmÀ1
.
5.9. General procedure for the preparation of
N-(p-(alkyloxy)benzoyl)-2-methylindole-4-
acetic acids (2a–q)
To a stirred solution of 7 (15g, 53.7mmol) and NaOH
(powdered, 10.74g, 269mmol) in CH2Cl2 (400mL) were
added TBACl (746mg, 2.69mmol) and p-acetoxy ben-
zoyl chloride (20.4g, 102mmol) at room temperature.
After stirring for 30min at room temperature, the reac-
tion mixture was filtered through a pad of Celite, and
the filtrate was concentrated in vacuo. The residue was
purified by column chromatography on silica gel to yield
10 (12.2g, 51%); TLC Rf = 0.34 (n-hexane/EtOAc, 7/3);
1H NMR (300MHz, CDCl3) d 7.76 (d, J = 8.7Hz,
2H), 7.40–7.20 (m, 7H), 7.08–6.92 (m, 3H), 6.47 (s,
1H), 5.15 (s, 2H), 3.88 (s, 2H), 2.40 (s, 3H), 2.35 (s, 3H).
5.9.1. {1-[4-(2,3-Dihydro-1-benzofuran-2-ylmethoxy)ben-
zoyl]-2-methyl-1H-indol-4-yl}acetic acid (2a). To an agi-
tated mixture of 13 (500mg, 0.470mmol) in CH2Cl2
(5mL) were added a solution of 15a (423mg, 2.82mmol)
in CH2Cl2 (1mL), Ph3P (740mg, 2.82mmol) and DEAD
(40% in toluene) (1.29mL, 2.82mmol) at room tempera-
ture. After agitating for 2h at room temperature, the
reaction mixture was filtered to collect insoluble sub-
stance, which was washed with CH2Cl2 and dried in
vacuo to afford 14a.
5.6. {1-[4-(Acetyloxy)benzoyl]-2-methyl-1H-indol-4-
yl}acetic acid (11)
To a stirred suspension of 14a described above in
CH2Cl2 (2mL) and CF3CH2OH (0.6mL) was added
acetic acid (0.3mL) at room temperature. After agitat-
ing for 2h at room temperature, 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 2a
(91.4mg, 44%); TLC Rf = 0.34 (CHCl3/CH3OH, 10/1);
1H NMR (300MHz, CDCl3) d 7.73–7.69 (m, 2H),
7.23–7.12 (m, 2H), 7.06–6.82 (m, 7H), 6.49 (d,
J = 1.2Hz, 1H), 5.20 (m, 1H), 4.29 (dd, J = 9.9, 6.3Hz,
1H), 4.20 (dd, J = 9.9, 4.2Hz, 1H), 3.86 (s, 2H), 3.42
(dd, J = 15.9, 9.6Hz, 1H), 3.17 (dd, J = 15.9, 8.4Hz,
1H), 2.44 (d, J = 1.2Hz, 3H); MS (EI, Pos) m/z 441
(M+); HRMS (EI, Pos.) calcd for C27H23NO5:
441.1576; found: 441.1573; IR (KBr) 2925, 1684,
1604, 1509, 1481, 1434, 1369, 1257, 1169, 1041, 911,
To a stirred solution of 10 (12.2g, 27.6mmol) in EtOAc
(250mL) was added Pd(OH)2 (1.2g) at room tempera-
ture. The resulting suspension was stirred for 2h at
room temperature under hydrogen atmosphere. Insolu-
ble substance was removed by filtration. The filtrate
was concentrated in vacuo to give a crude product,
which was purified by recrystallization from EtOAc to
yield 11 (7.39g, 80%); TLC Rf = 0.52 (CHCl3/CH3OH,
9/1); 1H NMR (300MHz, CDCl3)
d
7.77 (d,
J = 8.7Hz, 2H), 7.24 (d, J = 8.7Hz, 2H), 7.10–6.94 (m,
3H), 6.51 (s, 1H), 3.87 (s, 2H), 2.43 (s, 3H), 2.35 (s, 3H).
5.7. Polymer supported 2-chlorotriphenylmethyl{1-[4-
(acetyloxy)benzoyl]-2-methyl-1H-indol-4-yl}acetate (12)
To an agitated solution of 11 (7.77g, 23.2mmol) and
DIPEA (16.0mL, 92.7mmol) in CH2Cl2 (200mL) were
added polymer supported 2-chlorotriphenylmethyl chlo-
ride (1.2mmol/g, 100–200 mesh, 1% DVB) (19.3g,
23.2mmol) at room temperature. After agitating for
2.5h at room temperature, the reaction mixture was fil-
tered to collect a insoluble substance, which was washed
with CH2Cl2 (·5).
753cmÀ1
.
5.9.2. {1-[4-(2,3-Dihydro-1-benzofuran-3-ylmethoxy)ben-
zoyl]-2-methyl-1H-indol-4-yl}acetic acid (2b). 21% yield;
TLC Rf = 0.50 (CH3Cl/MeOH, 10/1); 1H NMR
(200MHz, CDCl3) d 7.70 (m, 2H), 7.31 (d, J = 7.4Hz,
1H), 7.21 (m, 1H), 7.10–6.82 (m, 7H), 6.49 (s, 1H),
4.73 (t, J = 9.6Hz, 1H), 4.55 (dd, J = 9.6, 4.6Hz, 1H),
4.29–3.88 (m, 3H), 3.86 (s, 2H), 2.44 (s, 3H); MS
(FAB, Pos.) m/z 442 (M+H)+; HRMS (EI, Pos.) calcd
for C27H23NO5: 441.1576; found: 441.1566; IR (neat)
3348, 1708, 1682, 1603, 1574, 1509, 1483, 1461, 1434,
1369, 1300, 1254, 1221, 1169, 1097, 1021, 960, 911,
To a suspension of this insoluble substance in CH2Cl2
(150mL) were added a solution of AcOH (13.4mL,
232mmol), DIPEA (160mL, 927mmol). After agitating
for 1h at room temperature, the reaction mixture was
filtered, washed with CH2Cl2 (·5) to afford the 12
(26.1g, 100%); IR (KBr) 3569, 3026, 2923, 1736, 1686,
1601, 1492, 1432, 1367, 1304, 1191, 1042, 908, 826,
837, 753, 614cmÀ1
.
5.9.3. {1-[4-(1,3-Benzodioxol-2-ylmethoxy)benzoyl]-2-
740, 697, 457cmÀ1
.
methyl-1H-indol-4-yl}acetic acid (2c). 43% yield; TLC
Rf = 0.33 (CH3Cl/MeOH, 10/1); 1H NMR (300MHz,
CDCl3) d 7.74–7.69 (m, 2H), 7.06–6.85 (m, 9H), 6.50–
6.47 (m, 2H), 4.35 (d, J = 4.2Hz, 2H), 3.85 (s, 2H),
2.43 (d, J = 0.9Hz, 3H); MS (FAB, Pos.) m/z 444
(M+H)+; HRMS (EI, Pos.) calcd for C26H21NO6:
443.1369; found: 443.1360; IR (KBr) 2925, 1685, 1604,
5.8. Polymer supported 2-chlorotriphenylmethyl [1-(4-
hydroxybenzoyl)-2-methyl-1H-indol-4-yl]acetate (13)
To an agitated mixture of 12 (26.1g, 23.2mmol) in
CH2Cl2 (240mL) was added piperidine (12.5mL,
127mmol) at room temperature. After agitating for
1509, 1484, 1434, 1231, 1043, 911, 743, 612cmÀ1
.

5372
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5.9.4. {1-[4-(3,4-Dihydro-2H-chromen-2-ylmethoxy)ben-
zoyl]-2-methyl-1H-indol-4-yl}acetic acid (2d). 48% yield;
TLC Rf = 0.49 (CH3Cl/MeOH, 10/1); 1H NMR
(200MHz, CDCl3) d 7.74–7.67 (m, 2H), 7.06–6.87 (m,
9H), 6.49 (s, 1H), 4.45 (m, 1H), 4.38–4.15 (m, 2H),
3.86 (s, 2H), 3.00–2.80 (m, 2H), 2.44 (s, 3H), 2.30–1.90
(m, 2H); MS (APCI, Neg. 20V) m/z 454 (MÀH)À;
HRMS (EI, Pos.) calcd for C28H25NO5: 455.1733;
found: 455.1716; IR (KBr) 2926, 1684, 1604, 1575,
5.9.9. (1-{4-[(2S)-2,3-Dihydro-1,4-benzodioxin-2-ylmeth-
oxy]benzoyl}-2-methyl-1H-indol-4-yl)acetic acid (2i).
24% yield; 98.6% ee; TLC Rf = 0.30 (CH3Cl/MeOH,
10/1); 1H NMR (300MHz, CDCl3) d 7.75–7.70 (m,
2H), 7.07–6.86 (m, 9H), 6.49 (m, 1H), 4.61 (m, 1H),
4.42 (dd, J = 11.4, 2.1Hz, 1H), 4.33 (dd, J = 10.2,
5.1Hz, 1H), 4.26 (dd, J = 11.4, 6.3Hz, 1H), 4.25 (dd,
J = 10.2, 6.0Hz, 1H), 3.87 (s, 2H), 2.44 (d, J = 1.2Hz,
3H); MS (FAB, Pos.) m/z 458 (M+H)+; HRMS (EI,
Pos.) calcd for C27H23NO6: 457.1525; found: 457.1540;
IR (KBr) 2925, 1685, 1604, 1495, 1434, 1369, 1248,
1509, 1434, 1369, 1301, 1257, 1170, 1039cmÀ1
.
1171, 1045, 911, 841, 752, 615cmÀ1
.
5.9.5. {1-[4-(3,4-Dihydro-2H-chromen-3-ylmethoxy)ben-
zoyl]-2-methyl-1H-indol-4-yl}acetic acid (2e). 41% yield;
TLC Rf = 0.34 (CH3Cl/MeOH, 10/1); 1H NMR
(300MHz, CDCl3) d 7.72–7.43 (m, 5H), 7.15–6.82 (m,
6H), 6.49 (s, 1H), 4.36 (m, 1H), 4.17 (m, 1H), 4.08 (d,
J = 6.9Hz, 2H), 3.87 (s, 2H), 3.04 (dd, J = 16.5,
6.0Hz, 1H), 2.78 (dd, J = 16.5, 7.2Hz, 1H), 2.63 (m,
1H), 2.44 (d, J = 1.2Hz, 3H); MS (APCI, Neg. 20V)
m/z 454 (MÀH)À; HRMS (EI, Pos.) calcd for
C28H25NO5: 455.1733; found: 455.1740; IR (KBr)
2924, 1683, 1604, 1509, 1490, 1435, 1369, 1255, 1169,
5.9.10. {1-[4-(3,4-Dihydro-2H-1,4-benzoxazin-2-ylmeth-
oxy)benzoyl]-2-methyl-1H-indol-4-yl}acetic acid (2j).
28% yield; TLC Rf = 0.25 (CH3Cl/MeOH, 10/1); 1H
NMR (300MHz, CDCl3) d 7.76–7.40 (m, 3H), 7.10–
6.89 (m, 4H), 6.89–6.60 (m, 4H), 6.49 (s, 1H), 4.60 (m,
1H), 4.31 (dd, J = 9.8, 5.0Hz, 1H), 4.24 (dd, J = 9.8,
6.2Hz, 1H), 3.86 (s, 2H), 3.58 (dd, J = 11.8, 3.0Hz,
1H), 3.42 (dd, J = 11.8, 6.6Hz, 1H), 2.44 (s, 3H); MS
(APCI, Neg. 20V) m/z 455 (MÀH)À; HRMS (EI, Pos.)
calcd for C27H24N2O5: 456.1685; found: 456.1687; IR
(neat) 3384, 2924, 1681, 1603, 1574, 1502, 1433, 1369,
1301, 1256, 1215, 1169, 1120, 1038, 958, 910, 836, 779,
911, 837, 757, 723, 694, 614, 542cmÀ1
.
5.9.6. (1-{4-[2-(2,3-Dihydro-1,4-benzodioxin-2-yl)eth-
729, 695, 639, 616, 541cmÀ1
.
oxy]benzoyl}-2-methyl-1H-indol-4-yl)acetic acid (2f).
12% yield; TLC Rf = 0.44 (CH3Cl/MeOH, 10/1); 1H
NMR (300MHz, CDCl3) d 7.71 (d, J = 8.7Hz, 2H),
7.10–6.80 (m, 9H), 6.49 (s, 1H), 4.50–4.40 (m, 1H),
4.38–4.17 (m, 3H), 4.01 (dd, J = 11.4, 7.2Hz, 1H), 3.86
(s, 2H), 2.44 (s, 3H), 2.17 (q, J = 6.0Hz, 2H); MS
(APCI, Neg. 20V) m/z 470 (MÀH)À; HRMS (EI, Pos.)
calcd for C28H25NO6: 471.1682; found: 471.1669; IR
(neat) 2926, 1709, 1683, 1604, 1509, 1494, 1434,
1369, 1303, 1253, 1221, 1169, 1044, 910, 837, 749,
5.9.11. (2-Methyl-1-{4-[(4-methyl-3,4-dihydro-2H-1,4-
benzoxazin-2-yl)methoxy]benzoyl}-1H-indol-4-yl)acetic
acid (2k). 28% yield; TLC Rf = 0.26 (CH3Cl/MeOH, 10/
1
1); H NMR (200MHz, CDCl3) d 7.78–7.40 (m, 3H),
7.10–6.78 (m, 6H), 6.73 (d, J = 8.0Hz, 2H), 6.49 (s,
1H), 4.68 (m, 1H), 4.31 (dd, J = 10.0, 5.2Hz, 1H), 4.20
(dd, J = 10.0, 6.4Hz, 1H), 3.86 (s, 2H), 3.41 (dd,
J = 11.6, 2.8Hz, 1H), 3.27 (dd, J = 11.6, 6.6Hz, 1H),
2.92 (s, 3H), 2.44 (s, 3H); MS (APCI, Neg. 20V) m/z
469 (MÀH)À; HRMS (EI, Pos.) calcd for
C28H26N2O5: 470.1842; found: 470.1836; IR (neat)
2924, 2359, 1683, 1604, 1505, 1434, 1368, 1299, 1254,
641cmÀ1
.
5.9.7. {1-[4-(2,3-Dihydro-1,4-benzodioxin-2-ylmethoxy)-
benzoyl]-2-methyl-1H-indol-4-yl}acetic acid (2g). 35%
1221, 1170, 1041, 959, 911, 837, 727, 542cmÀ1
.
1
yield; TLC Rf = 0.34 (CH3Cl/MeOH, 10/1); H NMR
(200MHz, CDCl3) d 7.71 (d, J = 8.8Hz, 2H), 7.09–
6.82 (m, 9H), 6.48 (s, 1H), 4.61 (m, 2H), 4.42 (dd,
J = 11.8, 2.6Hz, 1H), 4.38–4.18 (m, 3H), 3.84 (s, 2H),
2.43 (s, 3H); MS (APCI, Neg. 20V) m/z 456 (MÀH)À;
HRMS (MALDI-TOF, Pos.) calcd for C27H23NO6+H+:
458.1604; found: 458.1064; IR (KBr) 3046, 2925, 1734,
1708, 1684, 1603, 1574, 1509, 1494, 1433, 1369, 1302,
1246, 1220, 1170, 1044, 958, 911, 842, 780, 751, 637,
5.9.12. [2-Methyl-1-(4-{[(2S)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)-1H-indol-4-yl]ace-
tic acid (2l). 30% yield; TLC Rf = 0.26 (CH3Cl/MeOH,
10/1); 1H NMR (300MHz, CDCl3)
d 7.71 (d,
J = 9.0Hz, 2H), 7.20–6.78 (m, 7H), 6.78–6.64 (m, 2H),
6.49 (s, 1H), 4.68 (m, 1H), 4.30 (dd, J = 9.9, 5.4Hz,
1H), 4.21 (dd, J = 9.9, 6.0Hz, 1H), 3.86 (s, 2H), 3.40
(dd, J = 11.4, 2.7Hz, 1H), 3.27 (dd, J = 11.4, 6.6Hz,
1H), 2.92 (s, 3H), 2.44 (s, 3H); MS (APCI, Neg. 20V)
m/z 469 (MÀH)À; HRMS (MALDI-TOF, Pos.) calcd
for C28H26N2O5+H+: 471.1920; found: 470.1914; IR
(KBr) 2923, 1699, 1604, 1506, 1434, 1368, 1240, 1172,
614cmÀ1
.
5.9.8. (1-{4-[(2R)-2,3-Dihydro-1,4-benzodioxin-2-ylmeth-
oxy]benzoyl}-2-methyl-1H-indol-4-yl)acetic acid (2h).
38% yield; 98.6% ee; TLC Rf = 0.34 (CH3Cl/MeOH,
10/1); 1H NMR (300MHz, CDCl3) d 7.75–7.70 (m,
2H), 7.07–6.86 (m, 9H), 6.49 (m, 1H), 4.61 (m, 1H),
4.42 (dd, J = 11.4, 2.1Hz, 1H), 4.33 (dd, J = 10.2,
5.1Hz, 1H), 4.26 (dd, J = 11.4, 6.3Hz, 1H), 4.25 (dd,
J = 10.2, 6.0Hz, 1H), 3.87 (s, 2H), 2.44 (d, J = 1.2Hz,
3H); MS (APCI, Neg. 20V) m/z 456 (MÀH)À; HRMS
(EI, Pos.) calcd for C27H23NO6: 457.1525; found:
457.1523; IR (KBr) 2927, 1685, 1604, 1494, 1434,
1043, 962, 844, 745cmÀ1
.
5.9.13. [2-Methyl-1-(4-{[(2R)-4-methyl-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]methoxy}benzoyl)-1H-indol-4-yl]ace-
tic acid (2m). 48% yield; TLC Rf = 0.26 (CH3Cl/MeOH,
10/1); 1H NMR (200MHz, CDCl3) d 7.72 (d, J = 8.8Hz,
2H), 7.12–6.79 (m, 7H), 6.72 (d, J = 7.4Hz, 2H), 6.49 (s,
1H), 4.68 (m, 1H), 4.31 (dd, J = 9.6, 5.2Hz, 1H), 4.20
(dd, J = 9.6, 6.2Hz, 1H), 3.87 (s, 2H), 3.40 (dd,
J = 11.6, 2.8Hz, 1H), 3.28 (dd, J = 11.6, 6.2Hz, 1H),
1369, 1248, 1170, 1044, 910, 751, 542cmÀ1
.

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5373
2.92 (s, 3H), 2.45 (s, 3H); MS (APCI, Neg. 20V) m/z 469
(MÀH)À; HRMS (EI, Pos.) calcd for C28H26N2O5:
470.1842; found: 470.1846; IR (neat) 2925, 1708, 1682,
1604, 1504, 1433, 1368, 1299, 1255, 1220, 1170, 1136,
J = 8.7Hz, 2H), 7.08–6.85 (m, 5H), 6.60 (d, J = 8.7Hz,
3H), 6.49 (s, 1H), 4.73–4.64 (m, 1H), 4.30 (dd, J = 9.9,
5.1Hz, 1H), 4.21 (dd, J = 9.9, 6.3Hz, 1H), 3.87 (s,
2H), 3.36 (dd, J = 11.7, 2.7Hz, 1H), 3.22 (dd, J = 11.7,
6.6Hz, 1H), 2.88 (s, 3H), 2.45 (s, 3H); MS (APCI,
Neg. 20V) m/z 487 (MÀH)À; HRMS (EI, Pos.) calcd
for C28H25FN2O5: 488.1748; found: 488.1741; IR
(KBr) 3438, 2925, 1684, 1603, 1574, 1512, 1434, 1369,
1299, 1254, 1170, 1151, 1039, 984, 959, 911, 846, 780,
1041, 958, 910, 836, 735, 616cmÀ1
.
5.9.14. (1-{4-[(4-Ethyl-3,4-dihydro-2H-1,4-benzoxazin-2-
yl)methoxy]benzoyl}-2-methyl-1H-indol-4-yl)acetic acid
(2n). 35% yield; TLC Rf = 0.28 (CH3Cl/MeOH, 10/1);
1H NMR (200MHz, CDCl3) d 7.80–7.57 (m, 2H),
7.10–6.79 (m, 7H), 6.79–6.56 (m, 2H), 6.49 (s, 1H),
4.68–4.50 (m, 1H), 4.31 (dd, J = 9.6, 5.2Hz, 1H), 4.21
(dd, J = 9.6, 6.2Hz, 1H), 3.86 (s, 2H), 3.57–3.20 (m,
4H), 2.44 (s, 3H), 1.17 (t, J = 7.4Hz, 3H); MS (APCI,
Neg. 20V) m/z 483 (MÀH)À; HRMS (EI, Pos.) calcd
for C29H28N2O5: 484.1998; found: 484.2000; IR (neat)
2970, 2925, 1708, 1684, 1604, 1576, 1503, 1434, 1368,
1300, 1255, 1219, 1169, 1043, 958, 910, 836, 779, 741,
760, 703, 615cmÀ1
.
5.9.19. [2-Methyl-1-(4-{[(2S)-4, 6-dimethyl-3,4-dihydro-
2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)-1H-indol-4-yl]-
acetic acid (2s). 32% yield; TLC Rf = 0.37 (CH3Cl/
MeOH, 10/1); 1H NMR (300MHz, CDCl3) d 7.74–
7.69 (m, 2H), 7.06–6.82 (m, 5H), 6.73 (d, J = 8.1Hz,
1H), 6.54–6.47 (m, 3H), 4.64 (m, 1H), 4.30 (dd,
J = 9.9, 5.1Hz, 1H), 4.20 (dd, J = 9.9, 6.3Hz, 1H),
3.86 (s, 2H), 3.39 (dd, J = 11.4, 2.7Hz, 1H), 3.26 (dd,
J = 11.4, 6.6Hz, 1H), 2.90 (s, 3H), 2.45 (d, J = 0.9Hz,
3H), 2.28 (s, 3H); MS (EI, Pos.) m/z 484 (M+); Anal.
Calcd for C29H28N2O5: C, 71.88; H, 5.82; N, 5.78.
Found: C, 71.95; H, 5.91; N, 5.85; IR (KBr) 3430,
2921, 1699, 1666, 1604, 1576, 1510, 1478, 1435, 1419,
1368, 1338, 1288, 1240, 1189, 1171, 1136, 1065, 1039,
1015, 963, 912, 845, 807, 777, 758, 744, 706, 677, 641,
614, 542cmÀ1
.
5.9.15. (1-{4-[(1,4-Dimethyl-1,2,3,4-tetrahydroquinoxa-
lin-2-yl)methoxy]benzoyl}-2-methyl-1H-indol-4-yl)acetic
acid (2o). 10% yield; TLC Rf = 0.48 (CH3Cl/MeOH, 9/
1
1); H NMR (300MHz, CDCl3) d 7.70 (d, J = 9.0Hz,
2H), 7.08–6.92 (m, 5H), 6.80–6.66 (m, 2H), 6.62–6.48
(m, 3H), 4.27–4.07 (m, 2H), 3.87 (s, 2H), 3.83–3.73 (m,
1H), 3.36–3.20 (m, 2H), 3.05 (s, 3H), 2.88 (s, 3H), 2.45
(s, 3H); MS (APCI, Neg. 20V) m/z 482 (MÀH)À;
HRMS (EI, Pos.) calcd for C29H29N3O4: 483.2158;
found: 483.2161; IR (neat) 2925, 1682, 1602,
1508, 1433, 1369, 1298, 1254, 1221, 1169, 1022, 911,
607, 591, 554, 521, 461cmÀ1
.
5.10. Methyl 1,3-benzodioxole-2-carboxylate (17)
737cmÀ1
.
To a stirred suspension of sodium hydride (60% oil dis-
persion, 8.0g, 210mmol) were added 16 (11.0g,
100mmol) in DMF (400mL) and methyl dichloro-
acetate (52mL, 500mmol) at room temperature under
argon atmosphere. After stirring for 3h at 95ꢁC, the
reaction mixture was quenched with 2M HCl aq and ex-
tracted with EtOAc (·2). The combined organic layers
were washed with water, then brine, dried over Na2SO4
and concentrated in vacuo, to give a crude product,
which was purified by column chromatography on silica
gel to yield 17 (4.86g, 27%); TLC Rf = 0.43 (n-hexane/
5.9.16. (2-Methyl-1-{4-[(1-methyl-2,3-dihydro-1H-indol-
2-yl)methoxy]benzoyl}-1H-indol-4-yl)acetic acid (2p).
45% yield; TLC Rf = 0.49 (CH3Cl/MeOH, 9/1); H NMR
1
(300MHz, CDCl3) d 7.76–7.66 (m, 2H), 7.20–6.46
(m, 10H), 5.00–2.80 (m, 5H), 3.87 (s, 2H), 2.94 and
2.91 (s · 2, total 3H), 2.45 (s, 3H); MS (APCI, Neg.
20V) m/z 453 (MÀH)À; HRMS (EI, Pos.) calcd for
C28H26N2O4: 454.1893; found: 454.1893; IR (neat)
2925, 2360, 1708, 1683, 1603, 1573, 1507, 1433, 1368,
1300, 1254, 1221, 1168, 1036, 958, 910, 837, 779, 733,
1
EtOAc, 3/1; H NMR (300MHz, CDCl3) d 6.90–6.87
(m, 4H), 6.33 (s, 1H), 3.86 (s, 3H).
668, 614cmÀ1
.
5.9.17. (2-Methyl-1-{4-[(1-methyl-2,3-dihydro-1H-indol-
3-yl)methoxy]benzoyl}-1H-indol-4-yl)acetic acid (2q).
43% yield; TLC Rf = 0.47 (CH3Cl/MeOH, 9/1); 1H
NMR (300MHz, CDCl3) d 7.74–7.68 (m, 3H), 7.22–
7.10 (m, 2H), 7.08–6.90 (m, 4H), 6.72 (t, J = 6.6Hz,
1H), 6.53 (d, J = 8.1Hz, 1H), 6.49 (s, 1H), 4.24–4.06
(m, 2H), 3.87 (s, 2H), 3.78–3.70 (m, 1H), 3.49 (t,
J = 8.1Hz, 1H), 3.38 (dd, J = 9.0, 5.1Hz, 1H), 2.79 (s,
3H), 2.45 (s, 3H); MS (APCI, Neg. 20V) m/z 453
(MÀH)À; HRMS (EI, Pos.) calcd for C28H26N2O4:
454.1893; found: 454.1896; IR (neat) 3376, 1682, 1604,
1493, 1434, 1369, 1300, 1254, 1168, 1022, 910, 727,
5.11. 1,3-Benzodioxol-2-ylmethanol (15c)
To a stirred suspension of lithium aluminium hydride
(760mg, 20.0mmol) in THF (10mL) was added a solu-
tion of 17 (3.0g, 16.7mmol) in THF (20mL) at 0ꢁC.
After stirring for 20min at room temperature, the reac-
tion mixture was diluted with ether (50mL) and
quenched with saturated aqueous sodium sulfate under
cooling. The resulting mixture was stirred for additional
1h at room temperature. Insoluble substances were re-
moved by filtration and the filtrate was evaporated to
give a crude product, which was purified by column
chromatography on silica gel to yield 15c (602mg, 8%
542cmÀ1
.
1
5.9.18. [2-Methyl-1-(4-{[(2S)-7-fluoro-4-methyl-3,4-dihy-
dro-2H-1,4-benzoxazin-2-yl]methoxy}benzoyl)-1H-indol-
4-yl]acetic acid (2r). 33% yield; TLC Rf = 0.49 (CH3Cl/
MeOH, 9/1); 1H NMR (MHz, CDCl3) d 7.72 (d,
for 2 steps); TLC Rf = 0.39 (n-hexane/EtOAc, 2/1); H
NMR (300MHz, CDCl3) d 6.83–6.81 (m, 4H), 6.18 (t,
J = 3.6Hz, 1H), 3.93 (dd, J = 6.6, 3.6Hz, 2H), 1.88 (t,
J = 6.6Hz, 1H).

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K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5.12. (4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-yl)-
methanol (15k)
hexane. The resulting precipitates were removed by fil-
tration. The filtrate was concentrated in vacuo, to give
a crude product, which was purified by column chroma-
tography on silica gel to yield 15n (1.32g, 68%); TLC
Rf = 0.30 (n-hexane/EtOAc); 1H NMR (200MHz,
CDCl3) d 7.00–6.50 (m, 4H), 4.35 (m, 1H), 4.05–3.70
(m, 2H), 3.50–3.00 (m, 4H), 2.20–1.90 (m, 1H), 1.13 (t,
J = 7.5Hz, 3H).
To a stirred solution of 18 (4.47g, 21.6mmol) in acetone
(50mL) were added K2CO3 (5.96g, 43.1mmol) and
methyl iodide (2.7mL, 43.1mmol) at room temperature
under argon atmosphere, and stirring was continued for
16h at 40ꢁC. The reaction mixture was diluted with
EtOAc/n-hexane. The resulting precipitates were re-
moved by filtration. The filtrate was concentrated in
vacuo, to give a residue, which was used for the next
reaction without further purification.
5.15. 1,2,3,4-Tetrahydroquinoxalin-2-ylmethanol (2l)
To a stirred solution of 20 (500mg, 2.47mmol) in THF
(3mL) and MeOH (6mL) were added sodium borohy-
dride (196mg, 5.19mmol) portionwise at 0ꢁC. After stir-
ring for 1h, the reaction mixture was quenched with
saturated aqueous ammonium chloride, and extracted
with EtOAc( ·2). The combined organic layers were
washed with water, brine, dried over Na2SO4 and con-
centrated in vacuo to give a residue, which was purified
by column chromatography on silica gel to yield 21
To a suspension of lithium aluminium hydride (1.59g,
42mmol) in THF (20mL) was added a solution of
above-described ethyl ester (4.78g, 21.6mmol) in THF
(100mL) at room temperature. After stirring for 1h at
room temperature, the reaction mixture was diluted with
ether and quenched with saturated aqueous sodium sul-
fate at room temperature. The resulting mixture was
stirred for additional 1h at room temperature. Insoluble
substance was removed by filtration and the filtrate was
evaporated to give a crude product, which was purified
by column chromatography on silica gel to yield 15k
(2.80g, 75%); TLC Rf = 0.10 (n-hexane/EtOAc, 3/1);
1H NMR (200MHz, CDCl3) 7.00–6.60 (m, 4H), 4.35
(m, 1H), 4.00–3.70 (m, 2H), 3.33–3.10 (m, 2H), 2.88 (s,
3H), 2.01 (m, 1H).
1
(180mg, 44%); TLC Rf = 0.24 (CHCl3/MeOH, 9/1); H
NMR (300MHz, CDCl3) d 6.66–6.48 (m, 4H), 3.73
(dd, J = 10.5, 4.8Hz, 1H), 3.68–3.52 (m, 2H), 3.34 (dd,
J = 10.5, 3.0Hz, 1H), 3.24 (dd, J = 10.5, 6.0Hz, 1H),
3.10–2.60 (br, 2H).
5.16. (1,4-Dimethyl-1,2,3,4-tetrahydroquinoxalin-2-
yl)methanol (15o)
5.13. [(2R)-4-Methyl-3,4-dihydro-2H-1,4-benzoxazin-2-
yl]methanol (15m)
To a stirred solution of 21 (180mg, 1.10mmol) in ace-
tone (10mL) were added K2CO3 (455mg, 3.29mmol)
and methyl iodide (0.40mL, 6.58mmol) at room temper-
ature under argon atmosphere, and stirring was contin-
ued for 16h at 40ꢁC. 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 give a residue, which was purified by column chroma-
tography on silica gel to yield 15o (60mg, 28%); TLC
To a stirred solution of 19 (3.60g, 14.1mmol) in acetone
(50mL) were added K2CO3 (5.90g, 42.3mmol) and
methyl iodide (2.7mL, 42.3mmol) at room temperature
under argon atmosphere, and stirring was continued for
3h at 40ꢁC. The reaction mixture was diluted with
EtOAc/n-hexane. The resulting precipitates were re-
moved by filtration. The filtrate was concentrated in va-
cuo, to give a residue, which was used for the next
reaction without further purification.
1
Rf = 0.20 (n-hexane/EtOAc, 7/3); H NMR (200MHz,
CDCl3) d 6.85–6.73 (m, 1H), 6.72–6.53 (m, 3H), 3.86
(dd, J = 10.8, 6.0Hz, 1H), 3.75 (dd, J = 10.8, 3.9Hz,
1H), 3.46–3.36 (m, 1H), 3.27–3.18 (m, 1H), 3.16–3.07
(m, 1H), 3.00 (s, 3H), 2.88 (s, 3H).
To a stirred solution of above-described benzyl ether in
MeOH (30mL) were added 10wt% Pd–C (0.30g) and
10% HCl–MeOH (10mL) at room temperature. The
resulting suspension was refluxed for 2h under hydrogen
atmosphere. Insoluble substance was removed by filtra-
tion. The filtrate was concentrated in vacuo, to give a
crude product, which was purified by column chroma-
tography on silica gel to yield 15m (0.590g, 23%);
TLC Rf = 0.10 (n-hexane/EtOAc, 3/1); 1H NMR
(200MHz, CDCl3) d 7.00–6.60 (m, 4H), 4.35 (m, 1H),
4.00–3.70 (m, 2H), 3.33–3.10 (m, 2H), 2.88 (s, 3H),
2.01 (m, 1H).
5.17. Methyl 1-methylindoline-2-carboxylate (23)
To a stirred solution of 22 (2.0g, 12.26mmol) in acetone
(20mL) were added K2CO3 (6.78g, 49.0mmol) and
methyl iodide (3.1mL, 49.0mmol) at room temperature
under argon atmosphere. After stirring for 16h at room
temperature, the reaction mixture was diluted with
EtOAc/n-hexane. The resulting precipitates were re-
moved by filtration. The filtrate was concentrated in
vacuo, to give a residue, which was purified by column
chromatography on silica gel to yield 23 (1.70g, 73%);
TLC Rf = 0.70 (n-hexane/EtOAc, 7/3); 1H NMR
(300MHz, CDCl3) d 7.11 (t, J = 7.5Hz, 1H), 7.05 (d,
J = 7.5Hz, 1H), 6.70 (t, J = 7.5Hz, 1H), 6.51 (d,
J = 7.5Hz, 1H), 4.05 (t, J = 9.6Hz, 1H), 3.80 (s, 3H),
3.34 (dd, J = 15.6, 9.6Hz, 1H), 3.13 (dd, J = 15.6, 9.6
Hz, 1H), 2.84 (s, 3H).
5.14. (4-Ethyl-3,4-dihydro-2H-1,4-benzoxazin-2-yl)-
methanol (15n)
To a stirred solution of 15j (1.65g, 10mmol) in DMF
(10mL) were added K2CO3 (2.7 g, 20mmol) and ethyl
iodide (1.6mL, 20mmol) at room temperature under ar-
gon atmosphere, and stirring was continued for 16h at
50ꢁC. The reaction mixture was diluted with EtOAc/n-

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5375
5.18. (1-Methyl-2,3-dihydro-1H-indol-2-yl)methanol
(15p)
Rf = 0.50 (CH2Cl2); 1H NMR (300MHz, CDCl3) d
7.67–7.63 (m, 2H), 7.39 (m, 1H), 7.24–7.20 (m, 2H),
6.86–6.81 (m, 2H), 6.63 (brs, 1H), 2.38 (s, 3H), 2.30 (s,
3H).
To a stirred suspension of lithium aluminium hydride
(675mg, 17.8mmol) in THF (20mL) was added a solu-
tion of 23 (1.70g, 8.89mmol) in THF (10mL) at 0ꢁC.
After stirring for 1h at room temperature, the reaction
mixture was diluted with ether and quenched with satu-
rated aqueous sodium sulfate under cooling. The result-
ing mixture was stirred for additional 1h at room
temperature. Insoluble substance was removed by filtra-
tion and the filtrate was evaporated to give a crude
product, which was purified by column chromatography
on silica gel to yield 15p (1.30 g, 90%); TLC Rf = 0.30
(n-hexane/EtOAc, 7/3); 1H NMR (300MHz, CDCl3)
d 7.16–7.05 (m, 2H), 6.73 (t, J = 7.5Hz, 1H), 6.55 (d,
J = 7.5Hz, 1H), 3.95 (dd, J = 11.7, 3.6Hz, 1H), 3.74–
3.60 (m, 1H), 3.56–3.44 (m, 1H), 3.16–2.96 (m, 2H),
2.77 (s, 3H), 1.94–1.76 (m, 1H).
5.21. General procedure for the preparation of 27a and
27b
5.21.1. N-(2,4-Difluorophenyl)-N-[(2S)-3-triphenylmethyl-
oxy-2-hydroxypropyl]-4-methylbenzenesulfonamide (27a).
To a stirred solution of 26a (46.2g, 160mmol) and (S)-
(À)-glycidyl triphenylmethyl ether (55.7g, 180mmol)
in 1,4-dioxane (90mL) were added K2CO3 (2.21g,
16mmol) and benzyltriethylammonium chloride
(3.64g, 16mmol) at room temperature under argon
atmosphere, and stirring was continued for 5h at
120ꢁC. The reaction mixture was diluted with water
and extracted with EtOAc (·2). The combined organic
layers were washed with water, brine, dried over Na2SO4
and concentrated in vacuo to give 27a (115.6g, 100%),
which was used for the next reaction without further
purification; TLC Rf = 0.29 (n-hexane/EtOAc, 3/1);
MS (EI, Pos.) m/z 599 (M+).
5.19. (1-Methyl-2,3-dihydro-1H-indol-3-yl)methanol
(15q)
To a stirred suspension of lithium aluminium hydride
(276mg, 7.20mmol) in ether (7mL) was added a solu-
tion of 23 (600mg, 2.40mmol) in ether (3mL) at 0ꢁC.
After stirring for 2h at room temperature, the reaction
mixture was diluted with ether and quenched with satu-
rated aqueous sodium sulfate under cooling. The result-
ing mixture was stirred for additional 1h at room
temperature. Insoluble substance was removed by filtra-
tion and the filtrate was evaporated to give a crude
product, which was purified by column chromatography
on silica gel to yield 15q (350mg, 89%); TLC
5.21.2. N-[(2S)-3-Trityloxy-2-hydroxypropyl]-N-(2-flu-
oro-5-methylphenyl)-4-methylbenzenesulfonamide (27b).
The title compound 27b was prepared from 26b accord-
ing to the same procedure as described for the prepara-
tion of 27a from 26a: 100% yield; TLC Rf = 0.46 (n-
hexane/EtOAc, 2/1).
5.22. General procedure for the preparation of 28a and
28b
1
5.22.1. (2S)-2-[(Trityloxy)methyl]-7-fluoro-3,4-dihydro-
2H-1,4-benzoxazine (28a). To a stirred solution of 27a
(109g, 150mmol) in THF (150mL) was added BuOK
Rf = 0.23 (n-hexane/EtOAc, 7/3); H NMR (300MHz,
CDCl3) d 7.16–7.08 (m, 3H), 6.68 (dt, J = 0.9, 7.5Hz,
1H), 6.49 (d, J = 7.5Hz, 1H), 3.85–3.72 (m, 2H), 3.52–
3.34 (m, 2H), 3.32–3.22 (m, 1H), 2.75 (s, 3H); MS
(APCI, Pos, 20V.) m/z 164 (M+H)+.
t
(20.2g, 180mmol) at room temperature under argon
atmosphere, and then refluxed for 1h. The reaction mix-
ture was diluted with saturated NH4Cl 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 used for the
next reaction without further purification; TLC
Rf = 0.47 (n-hexane/EtOAc, 3/1); MS (EI, Pos.) m/z
579 (M+).
5.20. General procedure for the preparation of 26a and
26b
5.20.1.
N-(2,4-Difluorophenyl)-4-methylbenzenesulfon-
amide (26a). To a stirred solution of 25a (20.8g,
160mmol) and pyridine (15.6mL, 190mmol) in CH2Cl2
(160mL) were added p-toluenesulfonyl chloride (32.2g,
170mmol) at room temperature. After stirring for 16h
at room temperature, the reaction mixture was
quenched with 1 M HCl aq and extracted with CH2Cl2
(x2). The combined organic layers were washed with
brine, dried over Na2SO4 and concentrated in vacuo
to give 26a (46.2g, 100%); TLC Rf = 0.46 (CH2Cl2);
1H NMR (200MHz, CDCl3) d 7.65–7.50 (m, 2H),
7.35–7.15 (m, 3H), 6.92–6.80 (m, 1H), 6.78–6.65 (m,
1H), 6.52–6.42 (m, 1H), 2.39 (s, 3H); MS (APCI, Pos,
20V.) m/z 284 (M+H)+.
To a stirred solution of the above-described crude prod-
uct in THF (800mL) was added dropwise a solution of
sodium napthalenide at À78ꢁC, which was prepared
from Na (22.2g, 0.96mol) and naphthalene (74.4g,
0.6mol) in DME (600mL) at room temperature. After
stirring for 3h at À78ꢁC, the reaction mixture was
quenched with saturated NH4Claq 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 28a
(44.1g, 69%) as a pale yellow oil; TLC Rf = 0.46
1
5.20.2.
N-(2-Fluoro-5-methylphenyl)-4-methylbenzene-
(n-hexane/EtOAc, 3/1); H NMR (300MHz, CDCl3) d
sulfonamide (26b). The title compound 26b was prepared
from 25b according to the same procedure as described
for the preparation of 26a from 25a: 88% yield; TLC
7.35–7.18 (m, 15H), 6.58–6.42 (m, 3H), 4.38–4.28 (m,
1H), 3.48–3.39 (m, 2H), 3.28–3.18 (m, 2H); MS (EI,
Pos.) m/z 425 (M+).

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K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5.22.2. (2S)-2-[(Trityloxy)methyl]-6-methyl-3,4-dihydro-
2H-1,4-benzoxazine (28b). The title compound 28b was
prepared from 27b according to the same procedure as
described for the preparation of 28a from 27a: 94%
mDP and hDP or 5.0nM of [3H]Iloprost for IP) and
the test compounds at various concentrations in assay
buffer (10mM KH2PO4–KOH buffer containing
100mM NaCl, pH6.0 for mEP1-4, 25 mM HEPES-
NaOH buffer containing 1mM EDTA, 5mM MgCl2
and 10mM MnCl2, pH7.4 for mDP and hDP, 50mM
Tris–HCl buffer containing 1mM EDTA and 10mM
MgCl2, pH7.5, for IP-receptor). Incubation was carried
out at room temperature for 60min except for mEP1,
mDP, and hDP (20min), IP (30min). The incubation
was terminated by filtration through Whatman GF/B fil-
ters. The filters were washed with ice-cold buffer (10mM
KH2PO4–KOH buffer containing 100mM NaCl, pH6.0
for mEP1-4, 10mM Tris–HCl buffer containing 100mM
NaCl and 0.01 w/v% BSA, pH7.4 for mDP and hDP,
10mM Tris–HCl buffer containing 100mM NaCl,
pH7.5, for IP receptor), dried for 60min at 60ꢁC and
the radioactivity on the filter was measured in 6mL of
liquid scintillation (ACSII) mixture with a liquid scintil-
lation counter. Nonspecific binding was achieved by
adding excess amounts of unlabeled PGE2 (for mEP1-
4) or unlabeled PGD2 (for mDP and hDP) or unlabeled
Iloprost (for IP) with 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:
1
yield; TLC Rf = 0.36 (n-hexane/EtOAc, 3/1); H NMR
(300MHz, CDCl3) d 7.48–7.20 (m, 15H), 6.67 (d,
J = 8.1Hz, 1H), 6.45 (dd, J = 8.1, 1.8Hz, 1H), 6.40 (d,
J = 1.8Hz, 1H), 4.29 (m, 1H), 3.49–3.42 (m, 2H),
3.27–3.20 (m, 2H), 2.19 (s, 3H).
5.23. General procedure for the preparation of 15c and
15s
5.23.1. [(2S)-7-Fluoro-4-methyl-3,4-dihydro-2H-1,4-benz-
oxazin-2-yl]methanol (15c). To a stirred solution of 28a
(1.89g, 4.4mmol) in acetone (18mL) were added
K2CO3 (1.84g, 13mmol) and methyl iodide (0.83mL,
13mmol) at room temperature under argon atmosphere.
After stirring for 9h at 35ꢁC, the reaction mixture was
quenched with saturated NaHCO3 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 used for the
next reaction without further purification; TLC
Rf = 0.52 (n-hexane/EtOAc, 3/1); MS (EI, Pos.) m/z
439 (M+).
A solution of the above-described crude product in THF
(5mL), acetic acid (13mL) and water (2mL) was heated
at 100ꢁC. After stirring for 40min at 100ꢁC, the reaction
mixture was concentrated in vacuo to give an oily resi-
due, which was purified by column chromatography
on silica gel to yield 15r (0.368mg, 42%) as a pale brown
oil; TLC Rf = 0.09 (n-hexane/EtOAc, 3/1); 1H NMR
(300MHz, CDCl3) d 6.62–6.55 (m, 3H), 4.40–4.31 (m,
1H), 3.92–3.78 (m, 2H), 3.22–3.08 (m, 2H), 2.84 (s,
3H), 2.10–1.98 (m, 1H); MS (APCI, Pos. 20V) m/z 439
(M+).
Ki ¼ IC50=ð1 þ ½LŠ=KdÞ
[L]: concentration of radioligand, Kd: dissociation con-
stant of radiolabeled ligand towards the prostanoid
receptors.
6.2. Measurement of the mDP and hDP receptor
antagonist activity
To confirm that test compounds antagonize the mDP
and hDP receptors and to estimate potencies of antago-
nism for the mDP and hDP receptors, a functional assay
was performed by measuring PGD2-stimulated changes
in intracellular second messenger cAMP (cyclic adeno-
sine 30, 50-monophosphate) as an indicator of receptor
function.
5.23.2. [(2S)-4,6-Dimethyl-3,4-dihydro-2H-1,4-benzoxa-
zin-2-yl]methanol (15s). The title compound 15s was pre-
pared from 28b according to the same procedure as
described for the preparation of 28a from 15r: 45%
1
yield; TLC Rf = 0.44 (n-hexane/EtOAc, 2/1); H NMR
(300MHz, CDCl3) d 6.70 (d, J = 7.8Hz, 1H), 6.50–
6.46 (m, 2H), 4.30 (m, 1H), 3.90–3.75 (m, 2H), 3.23–
3.13 (m, 2H), 2.87 (s, 3H), 2.26 (s, 3H), 2.03 (brs, 1H).
For the assessment of the antagonist activity of test
compounds, a suspension of CHO cells expressing
mDP or hDP receptor was seeded at a cell density of
1 · 105 cells/well and cultivated for 2days. The cells in
each well were rinsed with minimum essential medium
(MEM), and MEM containing 2lM of Diclofenac was
added to each well. The cells were incubated for approx-
imately 10min at 37ꢁC and the culture medium was re-
moved. The assay medium (MEM containing 0.1or1%
BSA, 1mM IBMX and 2lM Diclofenac) was added
to each well and the cells were incubated for approxi-
mately 10min at 37ꢁC. The assay medium containing
100nM of PGD2 for mDP or 10nM of PGD2 for
hDP, or assay medium containing various concentra-
tions of test compounds and 100nM of PGD2 for
mDP or 10nM of PGD2 for hDP was added to each
well zand the cells were further incubated for 10min at
37ꢁC. The reaction was terminated by the addition of
ice-cold trichloroacetic acid (TCA; 10w/v%) and the
6. Biological assay method
6.1. Prostanoid mEP, mDP, hDP, and hIP receptor
binding assay
Competitive binding studies were conducted using radio-
ligand and membrane fractions prepared from Chinese
hamster ovary (CHO) cells stably expressing the respec-
tive prostanoid receptors, mEP1, mEP2, mEP3a, mEP4,
mDP, hDP, and hIP.
Membranes from CHO cells expressing prostanoid
receptors were incubated with radioligand (2.5nM of
[3H]PGE2 for mEP1-4 or 2.5nM of [3H]PGD2 for

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 5361–5378
5377
incubation mixture was frozen at À80ꢁC until the assay
using 30% HP-b-CD containing 5% DMSO (1mg/ml/
kg). Formulation for oral dosing was prepared using
0.5% MC (10mg/5ml/kg). Test compounds (1mg/kg)
were dosed intravenously to the fasted male rats
(n = 3). Test compounds (10mg/kg) were dosed orally
to the fasted male rats (n = 3). After dosing, blood sam-
ples (250lL) were collected from the jugular vein using a
heparinized syringe at the selected time points (iv: pre-
dosing, 2, 5, 15 and 30min, 1, 2, 4, 6 and 8h; po: pre-
dosing, 5, 15 and 30min 1, 2, 4, 6 and 8h, respectively).
The blood samples were ice-chilled and then centrifuged
at 12,000 rpm for 2min at 4ꢁC to obtain plasma, which
for cAMP.
The frozen incubation mixture was thawed, and the cells
were detached with a cell scraper. After centrifugation of
the reaction mixture, TCA was extracted by adding a
mixture of 0.5M tri-n-octylamine and chloroform (53/
239, v/v) to the resultant supernatant, mixing and re-
centrifugation. The cAMP level in the resultant aqueous
layer (upper layer) was determined by radioimmuno-
assay using a cAMP assay kit (Amersham). The relative
responsiveness (%) of cAMP production was calculated
relative to the maximum increase in cAMP that
occurred in the absence of test compound (100%) to
estimate the IC50 values.
was preserved at À80ꢁC in a freezer. The AUC, Cmax
,
Tmax, T1/2, Vss and CL were obtained by measuring
the time course of the plasma concentration of the test
compounds. Bioavailability (BA) was calculated accord-
ing to the following equation:
6.3. Inhibitory effects of selected compounds on PGD2-
induced vascular permeability in guinea pig conjunctiva
BA ð%Þ ¼ ðAUCpo=DpoÞ=AUCiv=DivÞ ꢁ 100
Topical application of PGD2 to the eye of guinea pigs is
known to cause plasma exudation in the conjunctiva via
DP receptor. We assessed the PGD2 receptor antago-
nism of test compounds in conjunctival vascular perme-
ability 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%, 20 lL/eye) to the eye, and then 5% Evans blue
dye (1mL/kg, iv) was immediately injected as a marker
of plasma exudation. All antagonists were orally admin-
istered 1h before the challenge. After 30min, the guinea
pigs were exsanguinated, and the eye tissue including
conjunctiva were extracted. Isolated conjunctiva were
incubated in DMF (1mL) at 37ꢁC to extract the extrav-
asated dye, and the incubation mixture was centrifuged.
The absorption of the supernatant at 620 nm was deter-
mined, and the amount of Evans blue dye leaked into
the tissues was quantified by interpolation on the stan-
dard curve.
AUCpo: AUC after oral dosing; AUCiv: AUC after
intravenous dosing; Dpo: Dosage of oral administration;
Div: Dosage of intravenous administration.
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