Development of prostaglandin D2 receptor antagonist: discovery of highly potent antagonists.

Article abstract of DOI:10.1016/j.bmc.2004.06.024

The process of discovery for highly potent prostaglandin D(2) (PGD(2)) receptor antagonists is reported. A series of N-(p-alkoxy)benzoyl-2-methylindole-4-acetic acids were synthesized and identified as a new class of selective PGD(2) receptor antagonists.

Full text of DOI:10.1016/j.bmc.2004.06.024

Bioorganic& Medicinal Chemistry 12 (2004) 4685–4700
Development of prostaglandin D2 receptor antagonist: discovery
of highly potent 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 4 June 2004; revised 21 June 2004; accepted 21 June 2004
Available online 24 July 2004
Abstract—The process of discovery for highly potent prostaglandin D2 (PGD2) receptor antagonists is reported. A series of N-(p-
alkoxy)benzoyl-2-methylindole-4-acetic acids were synthesized and identified as a new class of selective PGD2 receptor antagonists.
Most of them exhibited strong PGD2 receptor antagonism in binding studies and the cAMP formation assay. The structure–activity
relationships (SAR), including subtype selectivity of the synthesized compounds, are also discussed.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
of each receptor on various pathologies is still being
established by using potent but poorly selective ligands.
The situation is the same with regard to DP receptor
antagonists and only a few antagonists such as S-57513
are under going clinical trials for the treatment of aller-
gicrhinitis.
Prostanoids consist of prostaglandins (PGs) and throm-
boxanes (TX) and are products of cyclooxygenase de-
rived from arachidonic acid, which is the most
abundant precursor fatty acid in mammals (including
humans). Once prostanoids are produced by specific
processes, they act on the tissues where they are synthe-
sized via specific G-protein-coupled receptors. Each of
these receptors has been cloned, expressed, and charac-
terized.
In the preceding paper,4 we reported on the identifica-
tion of Indomethacin analog 2 as a new chemical lead
for a class of DP receptor antagonists. Optimization of
2 was continued to identify a superior mDP receptor
antagonist. As a result, we found that structural trans-
formation of 2 to 3a, which includes transformation of
N-benzoyl 2-methyl-5-methoxyindole-3-acetic acid to
N-benzoyl-2-methylindole-4-acetic acid, led to a marked
increase of binding affinity. Here we report on the dis-
covery of more optimized mDP receptor antagonists
3c, 3o–r, and 3t–v starting from our chemical lead 3a
(Fig. 1).
Coleman et al. proposed the existence of receptors spe-
cific for TX, PGI, PGE, PGF, and PGD that were
named the TP, IP, EP, FP, and DP receptors, respec-
tively.1 They further classified the EP receptor into four
subtypes (EP1, EP2, EP3, and EP4), all of which respond
to PGE2 in different ways.
Characterization of these receptors at the molecular
level has resulted in renewed interest in this field, but
few selective agonists and antagonists of human prosta-
noid receptors are available.2 As a result, the influence
2. Chemistry
The synthesis of 4–7 is outlined in Scheme 1. A key
intermediate 12 was prepared from 2-methyl-4-hydroxy-
indole 11 by the usual procedure.5 Palladium catalyzed
insertion of a carbon monoxide into 12 in the presence
of methanol provided 13,6 alkaline hydrolysis of which,
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.2004.06.024

4686
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
CO2H
MeO
Me
N
O
Cl
1 (Indomethacin)
CO2H
MeO
Me
N
Me
O
O
2
O
Me
Me
3r:
3a:
Me
CO2H
3c:
3o:
3p:
3q:
Me
O
O
Me
3t:
O
O
O
Me
Me
3u:
N
O
O
Me
Me
R
S
Me
O
3v:
3a, 3c, 3o-r and 3t-v
Figure 1. Discovery process of 3a, 3c, 3o–r, and 3t–v.
R1
OH
R
a
h
j
Me
Me
Me
N
N
H
N
H
R2
11
O
O
12 : R = OTf
b
c
d
20 : R1 = CO2Bn, R2= n-butyl
4 : R1 = CO2H, R 2= n-butyl
13 : R = CO2Me
14 : R = CO2H
15 : R = CO2Bn
16 : R =
e
22 : R1 =
7 : R1 =
5 : R1 =
21 : R1 =
6 : R1 =
, R2 = n-butyl
, R2 = n-butyl
CO2allyl
CO2H
i
j
CO2Me
CO2H
f
, R2 = n-butyl
, R2 = n-butyl
, R2 = n-butyl
CO2H
17 : R =
k,l,m
j
g
CO2allyl
18 : R =
CO2Bn
CO2H
ClOC
Me
O
19a
Scheme 1. Synthesis of 4–6 and 7a–g. Reagents: (a) Tf2O, 2,6-lutidine, CH2Cl2, ꢀ78ꢁC; (b) Pd(PPh3)4, CO, TEA, MeOH, DMF; (c) NaOH, MeOH,
1,4-dioxane; (d) benzyl bromide, K2CO3, DMF; (e) Pd(PPh3)2Cl2, methyl acrylate, DIPEA, DMF; (f) NaOH, MeOH, 1,4-dioxane; (g) allyl bromide,
K2CO3, DMF; (h) NaH, 19a, DMF (Method A), NaOH (powdered), 19a, TBACl, CH2Cl2 (Method B); (i) morpholine, Pd(PPh3)4, THF; (j) Pd–C,
H2, MeOH, EtOAc; (k) (COCl)2, DMF, toluene; (l) TMSCHN2, THF, CH3CN; (m) 2,4,6-collidine, benzyl alcohol.
followed by benzylation, led to the benzyl ester 15. Pal-
ladium catalyzed coupling of 12 with methyl acrylate af-
forded 16, alkaline hydrolysis of which, followed by
allylation, gave 18. N-Acylation of 15 with 19a resulted
in 20, catalytic hydrogenation of which produced 4. N-
Acylation of 18 with 19a led to 22, palladium catalyzed
deprotection of which produced the corresponding a,b-
unsaturated acid 7. Catalytichydrogenation of 7 af-
forded 5, which was converted to 21 by the conventional
C1 unit homologation procedure.7 Deprotection of 21
by catalytic hydrogenation gave 6.
The synthesis of various N-benzoyl-2-methylindole-4-
oxy acetic acids 8 is outlined in Scheme 2. O-Alkylation
of 2-methyl-4-hydroxyindole 11 with a methyl bromoac-
etate afforded 23. Alkaline hydrolysis of 23, followed by

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
CO2R1
Me
4687
O
O
CO2R1
a
d
11
Me
N
N
R2
H
O
O
23 : R1 = Me
24 : R1 = H
25 : R1 = allyl
b
c
26 : R1 = allyl, R2 = n-butyl
8 : R1 = H, R2 = n-butyl
e
ClOC
Me
O
19a
Scheme 2. Synthesis of 8. Reagents: (a) methyl bromoacetate, K2CO3, DMF; (b) NaOH, MeOH, THF; (c) allyl bromide, K2CO3, DMF; (d) NaH,
19a, DMF; (e) morpholine, Pd(Ph3)4, THF.
allylation with allyl bromide, resulted in 25. N-Acylation
of the ester 25 with benzoyl chloride 19a gave 26, depro-
tection of which produced 8.
by PGD2 in CHO cells in the presence of 0.1% BSA.9
Test compounds were also evaluated for binding to all
the mouse subtypes of PGE2 (mEP1–4) and to the hu-
man PGI2 receptor (hIP).
Compound 9 was prepared from 27 as described in
Scheme 3. Starting with 27, successive demethylation
with pyridinium hydrochloride, O-alkylation with ben-
zyl bromoacetate, and N-acylation with 19a afforded
28, deprotection of which produced 9.
During optimization of our new chemical lead 2, it was
found that 3a (in which the acetic acid residue is at-
tached to position 4) showed a marked increase of
mDP receptor affinity and antagonist activity (Fig. 1).4
Synthesis of 3a–v is outlined in Scheme 4. Protection of
the NH moiety of 12 with a tert-butoxy carbonyl pro-
duced 29, palladium catalyzed coupling reaction of
which with trimethylsilyl acetylene gave 30.8 Hydrobor-
ation of 30, followed by oxidation with alkaline hydro-
gen peroxide and deprotection, afforded 31.
Esterification of 31with allyl bromide and benzyl bro-
mide provided their corresponding esters 32 and 33,
respectively. N-Acylation of 32 and 33 with 19a–v re-
sulted in 34c,e–j,l–n, p–v and 34a,b,d,k,o, respectively,
deprotection of which produced 3a–v.
As described in Figure 2, the new chemical lead 3a was
thought to have a structural analogy to PGs regarding
the acidic part, which corresponds to a-chain, and the
lipophilic part, which corresponds to x-chain. Based
on many historical evidences, chemical modification of
the a- and x-chains of PGs has been known to be crucial
for the receptor affinity and/or subtype selectivity. In
such a reason, optimization of these acidic part and
the lipophilicpart of 3a was considered to be one of
the promising approaches for the increase of the recep-
tor affinity and subtype selectivity.
Compound 10 was prepared from 3v by the oxidation
using mCPBA as described in Scheme 5.
First, focus was placed on the optimization of 4-acetic
acid moiety. As described in Table 1, replacement of
the acetic acid residue of 3a with a carboxylic acid resi-
due afforded 4 with 3-fold lower affinity for the mDP re-
ceptor and 4-fold less potent antagonist activity. The
subtype selectivity of 4 was lower than that of 3a, mainly
because of a nearly 100-fold increase of its affinity for
the mEP3 receptor.
3. Results and discussion
The test compounds listed in Tables 1–3 were evaluated
for inhibition of the specific binding of a radiolabeled
ligand, [3H]PGD2, to membrane fractions prepared
from cells stably expressing each of the prostanoid re-
ceptors and for inhibition of cAMP formation evoked
Replacement of the acetic acid residue of 3a with a prop-
ionic acid residue and butanoic acid residue afforded 5
CO2H
O
CO2Bn
MeO
O
a, b, c
d
Me
Me
Me
N
N
H
N
Me
Me
27
O
O
O
O
9
28
Scheme 3. Synthesis of 9. Reagents: (a) HCl–pyridine; (b) benzyl bromoacetate, K2CO3, DMF; (c) NaOH (powdered), 19a, TBACl, CH2Cl2; (d) H2,
Pd–C, MeOH, EtOAc.

4688
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
TMS
OTf
a
b
12
Me
N
Me
Boc
29
N
Boc
30
CO2R1
CO2H
c, d
e
Me
Me
N
H
N
H
32 : R1 = allyl
33 : R1 = Bn
31
CO2R1
CO2H
f
g or h
Me
Me
N
N
O
R2
R2
O
O
O
34c, e-j, l-n, p-v : R1 = allyl
34a, b, d, k, o : R1 = Bn
3a-v
O
O
R2 :
q
r
i
a
b
c
Me
Me
Me
Me
Me
j
ClOC
Me
Me
O
Me
Me
s
t
R2
k
O
Me
d
e
f
Me
Me
O
O
19a-v
Me
O
Me
l
Me
Me
Me
u
v
O
O
m
n
Me
Me
S
Me
O
g
h
Me
o
p
O
Me
Scheme 4. Synthesis of 3a–v. Reagents: (a) Boc2O, DMAP (cat.), CH3CN; (b) PdCl2 (PPh3)2, trimethylsilylacetylene, CuI, TBAI, TEA, DMF, 70ꢁC
(c) (1) dicyclohexylborane, (2) NaOHaq, H2O2, THF; (d) NaOH aq, MeOH, 1,4-dioxane; (e) allyl bromide or benzyl bromide, K2CO3, DMF; (f)
NaOH (powdered), 19a–v, TBACl, CH2Cl2; (g) morpholine, Pd(PPh3)4, THF; (h) H2, Pd–C, i-PrOH, EtOAc.
CO2H
CO2H
a
Me
Me
Me
N
O
S
Me
N
O
S
O
O
O
O
3v
Scheme 5. Synthesis of 10. Reagents: (a) mCPBA, CH2Cl2.
10
and 6, respectively, which showed lower affinity for the
mDP receptor and less potent antagonist activity, while
5 and 6 showed lower subtype selectivity.
antagonist activity, while the subtype selectivity of 7
was lower relative to that of 5 mainly because of a nearly
10-fold increase of mEP2 receptor affinity. Replacement
of the acetic acid residue of 3a with a glycolic acid resi-
due afforded 8 with nearly 4-fold lower affinity for the
mDP receptor and a more than 2-fold decrease of antag-
onist activity, while 8 showed much lower subtype selec-
Introduction of a 2,3-trans double bond into the propi-
onicacid residue of 5 gave 7 with nearly 3-fold lower
affinity to mDP receptor and more than 2-fold weaker

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
Table 1. Effect of the 4-acidic moieties on the activity profiles
4689
R
Me
N
Me
O
O
Compd
R
Binding Ki (lM)
IC50 (lM)
mEP1
>10
mEP2
mEP3
0.029
mEP4
>10
mDP
0.033
mDP
1.2
CO2H
4
5.1
CO2H
3a
>10
2.0
3.3
>10
0.010
0.30
CO2H
CO2H
CO2H
5
6
>10
>10
3.5
>10
>10
3.0
0.013
0.033
0.43
0.57
0.046
>10
7
>10
>10
>10
0.32
4.3
>10
0.50
>10
>10
2.6
0.032
0.047
1.2
1.1
O
CO2H
8
0.69
NTa
HO2C
O
Me
9
>10
>10
N
a NT: not tested.
tivity mainly because of its increased affinity for the
mEP3 and mEP4 receptors. Transfer of the glycolic acid
residue of 8 from position-4 to position-5 led to 9, which
showed a marked decrease of mDP receptor affinity.
Accordingly, the optimization of N-benzoyl-2-methylin-
dole-4-acetic acid was pursued.
3a. Replacement of the butoxy moiety of 3a with a 3-
butenyloxy moiety provided 3i, which showed 4.6-fold
lower mDP affinity.
Replacement of the p-butoxy moiety of 3a with various
phenylalkoxy moieties, such as p-phenylmethoxy, p-
phenylethoxy and p-phenylpropyloxy moieties, resulted
in 3j–l, respectively. Among these compounds, 3j and
3l showed lower mDP receptor affinity while 3k showed
nearly 5-fold higher mDP receptor affinity and more
than 2-fold stronger antagonist activity. The subtype
selectivity of these three compounds 3j–l seemed to be
lower than that of 3a. Thus, 3c was found to show the
highest antagonist activity, although its subtype selectiv-
ity was not satisfactory.
Our second focus was placed on the optimization of the
p-alkoxy moiety of 3a (Tables 2 and 3). As described in
Table 2, replacement of the n-butoxy moiety of 3a with
longer alkoxy moieties, such as n-pentyloxy and n-hexyl-
oxy, gave 3b and 3c, which had slightly higher and more
than 10-fold higher antagonist activity relative to 3a,
respectively, but subtype selectivity was not improved
compared with 3a. Replacement of the p-butoxy moiety
of 3a with branched p-alkoxy moieties provided 3d–f.
Compound 3d (p-isoamyloxy moiety) demonstrated
slightly higher affinity for the mDP receptor and stron-
ger antagonist activity relative to 3a, while its subtype
selectivity was not improved.
Insertion of a hetero atom, such as oxygen or sulfur,
into the p-alkoxy substituent of the N-benzoyl moiety
produced the compounds listed in Table 3 with better
mDP receptor antagonist activity and subtype selectiv-
ity. Replacement of the p-butoxy moiety of 3a with a
methoxyethoxy moiety afforded 3m, which showed more
than 17-fold lower affinity for the mDP receptor. Meth-
oxypropyloxy derivative 3n showed 3-fold stronger
antagonist activity and 3-fold lower affinity for the
mDP receptor. Replacement of the p-butoxy moiety of
3a with a ethoxyethoxy moiety gave 3o, which showed
more potent antagonist activity and much improved
subtype selectivity, while 3o showed 2-fold lower mDP
receptor affinity relative to 3a. Ethoxypropyloxy deriva-
tive 3p demonstrated nearly 3-fold stronger antagonist
activity and nearly 5-fold higher mDP receptor affinity
than 3o. The n-propyloxyethoxy derivative 3q showed
more potent antagonist activity and slightly higher
Two other branched alkoxy derivatives, 3e–f, showed
slightly lowert and 2-fold higher antagonist activity,
respectively, while their mDP receptor affinity was more
than 10-fold lower than that of 3a. Introduction of p-(2-
cycloalkyl)ethoxy moieties as the alkoxy moiety created
3g and 3h. Compound 3g, with a p-(2-cyclohexyl)ethoxy
benzoyl moiety, showed slightly stronger antagonist
activity relative to 3a, while its mDP receptor affinity
was nearly 20-fold lower. Compound 3h, with a p-(2-cy-
clopropyl)ethoxy benzoyl moiety, showed stronger
antagonist activity relative to 3a, while its mDP receptor
affinity was slightly lower. The subtype selectivity of
these two compounds was estimated to be inferior to

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K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
Table 2. Effect of the p-alkoxy moieties on the activity profiles
CO2H
Me
N
O
R
O
Compd
R
Binding Ki (lM)
IC50 (lM)
mEP1
3.1
mEP2
mEP3
2.8
mEP4
3.1
hIP
1.9
mDP
mDP
0.25
3b
3c
0.80
1.0
0.0043
Me
Me
5.3
3.0
>10
>10
3.1
2.3
0.74
>10
0.0085
0.0092
0.020
0.20
Me
Me
Me
3d
3e
0.92
2.8
NTa
0.34
0.46
Me
Me
Me
>10
>10
>10
3f
>10
4.3
2.6
2.9
>10
>10
>10
4.0
NTa
NTa
0.19
0.22
0.14
0.20
Me
3g
3h
3i
3j
4.3
2.7
1.2
1.3
>10
>10
1.2
4.9
0.85
>10
2.5
0.016
0.046
0.068
0.080
NTa
1.3
1.8
>10
>10
0.33
3k
0.40
7.7
1.3
2.2
3.3
1.9
2.1
0.14
NTa
0.0018
0.17
0.12
NTa
3l
NTa
a NT: not tested.
mDP receptor affinity than 3o, but its subtype selectivity
was lower because of increased affinity for mEP1,
mEP2, and mEP3 relative to 3o. The i-propyloxyethoxy
derivative 3r demonstrated 1.5-fold higher antagonist
activity than 3o, while its subtype selectivity was lower
mainly because of its increased affinity for the mEP1 re-
ceptor relative to 3o. The tert-butoxyethoxy derivative
3s unexpectedly showed lower antagonist activity com-
pared with its high affinity for the mDP receptor. Meth-
oxyethoxyethoxy derivative 3t showed slightly stronger
antagonist activity and nearly 2-fold higher affinity for
the mDP receptor than 3q. The subtype selectivity of
compounds 3s and 3t was excellent. The unexpectedly
weaker antagonist activity of 3s relative to its high
mDP receptor affinity was considered to be attributable
to relatively stronger protein binding because of its more
lipophilic tert-butoxy moiety. Phenoxyethoxy derivative
3u showed slightly stronger antagonist activity and high-
er mDP receptor affinity relative to 3o, while subtype
selectivity was lower because of increased affinity for
the mEP1 and mEP2 receptors. The subtype selectivity
of compounds 3n and 3p was lower than that of 3o,
mainly because of increased mEP2 receptor affinity.
Replacement of the ether oxygen of 3o with a sulfur af-
forded 3v, which showed slightly more potent antagonist
activity and nearly 2-fold higher mDP receptor affinity
relative to 3o, while its subtype selectivity was much
lower because of increased affinity for the mEP1,
mEP2, and mEP3 receptor. Oxidation of the sulfur atom
of 3v to a sulfone moiety provided 10, which showed
markedly lower affinity for the mDP receptor than 3v.
As a result, the optimal subtype-selective mDP antago-
nists were 3o and 3t. The subtype selectivity of 3s was
excellent, although its antagonist activity was reduced
compared with its relatively high affinity for the mDP
receptor because of its predicted high level of protein
binding.
4. Conclusion
In summary, structural optimization of 3a was contin-
ued to identify a more potent mDP receptor antagonist.
Among the compounds tested, 3c, 3o–r, and 3t–v dem-
onstrated higher mDP receptor affinity and antagonist
activity relative to 3a. Compound 3c also showed affin-
ity for the hIP receptor at 0.74lM (Ki), while 3o–r and
3t–v did not show hIP receptor affinity at 10lM.
The acetic acid residue at position-4 of the indole moiety
of 3a was optimal among the residues investigated, as

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
Table 3. Effect of the hetero atom-containing p-alkoxy moieties on the activity profiles
4691
CO2H
Me
N
O
R
O
Compd
R
Binding Ki (lM)
IC50 (lM)
mEP1
>10
mEP2
mEP3
NTa
mEP4
hIP
mDP
0.17
mDP
NTa
NTa
O
O
3m
>10
>10
Me
O
Me
Me
3n
3o
>10
>10
0.42
>10
>10
>10
>10
>10
>10
>10
0.031
0.020
0.10
0.074
3p
3q
>10
4.7
6.6
>10
2.0
>10
>10
>10
>10
0.0043
0.014
0.023
0.027
O
Me
Me
Me
Me
Me
O
O
0.57
3r
5.3
>10
>10
>10
>10
0.075
0.048
O
3s
3t
3u
>10
>10
5.6
>10
>10
0.54
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.067
0.0078
0.017
1.0
Me
Me
O
O
Me
0.063
0.053
O
3v
S
S
Me
Me
8.8
2.7
3.6
>10
>10
>10
0.012
1.2
0.066
NTa
NTa
O
O
10
>10
>10
>10
a NT: not tested.
acidic part
CO2H
CO2H
Me
-chain)
-chain)
PGs
Me
N
OH
O
O
Me
3a
PGs
lipophilic part
Figure 2. Structural analogy of PGs and 3a.
illustrated in Table 1. Position-4 was also found to be
better than position-5 for substitution by an acetic acid
residue, as illustrated by the increased mDP affinity of 8
relative to that of 9. Further optimization and in vivo
evaluation of the optimum compound will be reported
in due course.
nance spectra (1H NMR) were taken on a Varian Mer-
cury 300 spectrometer or Varian GEMINI-200 or VXR-
200s spectrometer using deuterated chloroform (CDCl3)
or deuterated methanol (CD3OD) or deuterated dimeth-
ylsulfoxide (DMSO-d6) as the solvent. Fast atom bom-
bardment mass spectra (FAB-MS) and electron
ionization (EI) were obtained on a JEOL JMS-
DX303HF spectrometer. The matrix assisted laser de-
sorption 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
5. Experimental
5.1. General directions
Analytical samples were homogeneous as confirmed by
TLC, and afforded spectroscopic results consistent with
the assigned structures. Proton nuclear magnetic reso-

4692
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
analyses were uncorrected. Column chromatography
was carried out on silica gel [Merck silica gel 60
(0.063ꢁ0.200mm), 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-
ticacid (AcOH), triethylamine (TEA) and diisopropyl-
ethylamine (DIPEA).
zyl bromide (0.7mL, 5.91mmol) at room temperature.
After stirring for 2h at 80ꢁC, the reaction mixture was
poured into ice-water and extracted with EtOAc (·3).
The combined organic layers were washed with water,
brine, dried over Na2SO4. The solvent was removed by
evaporation and the residue was purified by column chro-
matography on silica gel to afford 15 (610mg, 58%). TLC
Rf =0.44 (n-hexane/EtOAc, 8/3); 1H NMR (300MHz,
CDCl3) d 8.05 (br s, 1H), 7.91 (d, J=7.2Hz, 1H), 7.54–
7.24 (m, 7H), 6.88 (m, 1H), 5.44 (s, 2H), 2.48 (s, 3H).
5.6. Methyl (2E)-3-(2-methyl-1H-indol-4-yl)acrylate (16)
5.2. 2-Methyl-1H-indol-4-yl trifluoromethanesulfonate
(12)
To a stirred solution of 12 (3.16g, 11.3mmol) in DMF
(200mL) were added methyl acrylate (2.24mL,
24.9mmol), DIPEA (5.9mL, 33.9mmol) and
Pd(PPh3)2Cl2 (238mg, 0.339mmol) at room temperature.
After stirring for 16h at 95ꢁC, the reaction mixture was
poured into water and extracted with EtOAc (·2). The
combined organic layers were washed with water, brine,
and dried over Na2SO4 and evaporated to afford a resi-
due, which was purified by column chromatography on
silica gel to yield 16 (950mg, 39% yield); TLC Rf =0.50
To a stirred solution of 2-methyl-1H-indol-4-ol (11)
(10.0g, 67.9mmol) in CH2Cl2 (100mL) were added luti-
dine (10.3mL, 88.3mmol) and Tf2O (13.72mL,
81.54mmol) at 0ꢁC. After stirring for 1h, the reaction
mixture was poured into water and extracted with EtO-
Ac( ·2). The combined organic layers were washed with
water, brine, dried over Na2SO4 and evaporated to af-
ford a residue, which was used for the next reaction
without further purification; TLC Rf =0.57 (n-hexane/
EtOAc, 7/3).
1
(n-hexane/EtOAc, 8/2); H NMR (200MHz, CDCl3) d
7.97 (d, J=16.2Hz, 1H), 7.96 (d, J=8.1Hz, 1H), 7.56
(d, J=8.1Hz, 1H), 7.33 (d, J=8.1Hz, 1H), 6.82 (s, 1H),
6.54 (d, J=16.2Hz, 1H), 3.84 (s, 3H), 2.61 (s, 3H).
5.3. Methyl 2-methyl-1H-indole-4-carboxylate (13)
To a stirred solution of 12 (6.34g, 22.7mmol) in MeOH
(33mL) and DMF (200mL) were added TEA (6.3mL,
45.29mmol) and Pd(PPh3)4 (2.6g, 2.26mmol) at room
temperature. After stirring over night at 60ꢁC under
an atmosphere of carbon monoxide, the reaction mix-
ture was filtered through a pad of Celite to remove the
catalyst. The filtrate was poured into water and ex-
tracted with EtOAc (·2). The combined organic layers
were washed with water, brine, and dried over Na2SO4
and evaporated to afford a residue, which was purified
by column chromatography on silica gel to yield 13
(100% yield); TLC Rf =0.18 (toluene); 1H NMR
(200MHz, CDCl3) d 8.14–8.00 (br s, 1H), 7.85 (m,
1H), 7.47 (m, 1H), 7.14 (t, J=8.1Hz, 1H), 6.87 (m,
1H), 3.97 (s, 3H), 2.50 (s, 3H).
5.7. (2E)-3-(2-Methyl-1H-indol-4-yl)acrylic acid (17)
To a stirred solution of methyl ester 16 (950mg,
4.41mmol) in MeOH (10mL) and 1,4-dioxane (10mL)
was added 5M NaOH aq (5mL). After stirring for
16h at room temperature, the reaction mixture was acid-
ified with 2M 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 17 (700mg, 85% yield), which was used for the
next reaction without further purification; TLC
Rf =0.54 (CHCl3/MeOH, 9/1).
5.8. Allyl (2E)-3-(2-methyl-1H-indol-4-yl)acrylate (18)
To a stirred solution of 17 (300mg, 1.60mmol) in DMF
(5mL) were added K2CO3 (332mg, 2.40mmol) and allyl
bromide (0.21mL, 2.40mmol) at room temperature.
After stirring for 2h at 80ꢁC, the reaction mixture was
poured into ice-water and extracted with EtOAc (·3).
The combined organic layers were washed with water,
brine, and dried over Na2SO4. The solvent was removed
by evaporation and the residue was purified by column
chromatography on silica gel to afford 18 (240mg,
5.4. 2-Methyl-1H-indole-4-carboxylic acid (14)
To a stirred solution of methyl ester 13 (4.30g,
22.7mmol) in MeOH (10mL) and 1,4-dioxane (10mL)
was added 5M NaOH aq (10mL). After stirring for
16h at 60ꢁC, the reaction mixture was acidified with
2M HCl aq, and extracted with EtOAc (·2). The com-
bined organiclayers were washed with water, brine, dried
over Na2SO4, and concentrated in vacuo to give a resi-
due, which was purified by column chromatography on
silica gel to yield 14 (1.6g, 40%); TLC Rf =0.48 (CHCl3/
1
62%). TLC Rf =0.43 (n-hexane/EtOAc, 8/2); H NMR
(300MHz, CDCl3) d 8.10–8.00 (m, 2H), 7.35–7.30 (m,
2H), 7.12 (t, J=7.8Hz, 1H), 6.61 (d, J=16.2Hz, 1H),
6.54 (br s, 1H), 6.10–5.95 (m, 1H), 5.95–5.85 (m, 1H),
5.80–5.75 (m, 1H), 4.80–4.70 (m, 2H), 2.50 (s, 3H).
1
MeOH, 9/1); H NMR (300MHz, CD3OD) d 8.14–8.04
(br, 1H), 7.93 (dd, J=8.1,0.9Hz, 1H), 7.52 (m, 1H),
7.18 (dd, J = 8.1, 0.9Hz, 1H), 6.94 (m, 1H), 3.71 (s, 3H).
5.9. Benzyl 1-(4-butoxybenzoyl)-2-methyl-1H-indole-4-
carboxylate (20)
5.5. Benzyl 2-methyl-1H-indole-4-carboxylate (15)
To a stirred solution of 14 (690mg, 3.94mmol) in DMF
(10mL) were added K2CO3 (815mg, 5.91mmol) and ben-
To a stirred solution of 15 (690mg, 2.60mmol) in DMF
(8mL) was added sodium hydride (60% oil dispersion,

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
4693
114mg, 2.86mmol) in several portions at 0ꢁC under
argon atmosphere, and the resulting suspension was stir-
red for 30min at 0ꢁC. After the addition of p-(n-butoxy)
benzoyl chloride (0.54mL, 2.86mmol), the reaction mix-
ture was stirred for 12h at 0ꢁC, quenched with water
and extracted with EtOAc (·3). 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 chromatography on silica gel to yield
20 (1.02g, 89%); TLC Rf =0.61 (n-hexane/EtOAc, 8/2);
1H NMR (300MHz, CDCl3) d 8.10–6.90 (m, 13H),
5.45 (s, 2H), 4.05 (t, J=6.3Hz, 2H), 2.44 (s, 3H), 1.86–
1.74 (m, 2H), 1.60–1.45 (m, 2H), 0.99 (t, J=7.5Hz, 3H).
1H), 7.13 (t, J=8.1Hz, 1H), 6.97 (d, J=9.0Hz, 2H),
4.06 (t, J=6.6Hz, 2H), 2.48 (s, 3H), 1.88–1.76 (m,
2H), 1.60–1.46 (m, 2H), 1.00 (t, J=7.5Hz, 3H); MS
(FAB, Pos.) m/z 352 (M+H)+; HRMS (EI, Pos.) calcd
for C21 H21NO4: 351.1471; found: 351.1493; IR (neat)
2960, 1682, 1604, 1511, 1481, 1432, 1371, 1257, 1171,
912, 804, 757cmꢀ1
.
5.13. 3-[1-(4-Butoxybenzoyl)-2-methyl-1H-indol-4-
yl]propanoic acid (5)
1
8% yield; TLC Rf =0.58 (CHCl3/MeOH, 9/1); H NMR
(300MHz, CDCl3) d 7.70 (d, J=9.3Hz, 2H), 7.00–6.86
(m, 5H), 6.48 (s, 1H), 4.05 (t, J=6.6Hz, 2H), 3.75–
3.65 (br, 1H), 3.19 (t, J=8.4Hz, 2H), 2.79 (t,
J=8.4Hz, 2H), 2.45 (s, 3H), 1.87–1.72 (m, 2H), 1.60–
1.40 (m, 2H), 1.00 (t, J=7.5Hz, 3H); MS (APCI, Neg,
20V.) m/z 378 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C23H25NO4: 379.1784; found: 379.1790; IR (neat)
2960, 2873, 1682, 1605, 1573, 1511, 1432, 1369, 1300,
1256, 1226, 1169, 1115, 1068, 1025, 912, 849, 777, 759,
5.10. Allyl (2E)-3-[1-(4-butoxybenzoyl)-2-methyl-1H-
indol-4-yl]acrylate (22)
98% yield; TLC Rf =0.59 (n-hexane/EtOAc, 8/2); 1H
NMR (300MHz, CDCl3) d 8.10–8.00 (m, 1H), 7.70 (d,
J=11.7Hz, 2H), 7.39 (d, J=7.2Hz, 1H), 7.10–6.90 (m,
4H), 6.72 (s, 1H), 6.58 (d, J=15.9Hz, 1H), 6.10–5.95
(m, 1H), 5.95–5.85 (m, 1H), 5.82–5.75 (m, 1H), 4.80–
4.70 (m, 2H), 4.10–4.00 (m, 2H), 2.47 (s, 3H), 1.90–
1.70 (m, 2H), 1.70–1.40 (m, 2H), 1.10–0.95 (m, 3H).
617cmꢀ1
.
5.14. Benzyl 4-[1-(4-butoxybenzoyl)-2-methyl-1H-indol-
4-yl]butanoate (21)
5.11. (2E)-3-[1-(4-Butoxybenzoyl)-2-methyl-1H-indol-4-
yl]acrylic acid (7)
To a stirred solution of 5 (1.77g, 4.67mmol) in toluene
(20mL) were added oxalyl chloride (0.64mL, 7.4mmol)
at room temperature. After stirring for 1h at room tem-
perature, the reaction mixture was concentrated in va-
cuo to give the corresponding acid chloride, which was
used for the next reaction without further purification.
To a stirred solution of the acid chloride described
above in THF (4mL) and CH3CN (4mL) was added
(trimethylsilyl)diazomethane (2 mol/L in ether,
4.67mL, 9.34mmol) at 0ꢁC. After stirring for 1h at
0ꢁC, the reaction mixture was concentrated in vacuo
to afford a residue which was dissolved with benzyl alco-
hol (4mL) and 2,4,6-collidine (4mL) at room tempera-
ture. After stirring for 30min at 180ꢁC, the reaction
mixture was concentrated in vacuo to afford a residue,
which was directly purified by column chromatography
on silica gel to yield 21 (460mg, 20%); TLC Rf =0.51 (n-
hexane/EtOAc, 8/2); 1H NMR (300MHz, CDCl3) d 7.67
(d, J=9.0Hz, 2H), 7.40–7.20 (m, 5H), 7.03–6.85 (m,
5H), 6.47 (s, 1H), 5.10 (s, 2H), 4.03 (t, J=6.6Hz, 2H),
2.90 (t, J=7.2Hz, 2H), 2.50–2.32 (m, 5H), 2.13–2.00
(m, 2H), 2.00–1.40 (m, 4H), 1.00 (t, J=7.5Hz, 3H).
To a stirred solution of 22 (337mg, 1mmol) in THF
(7mL) was added Pd(PPh3)4 (115mg, 0.10mmol) at
room temperature under argon atmosphere. After stir-
ring for 15min, morpholine (0.45mL, 5.0mmol) was
added. After 1h, the reaction mixture was quenched
with water and extracted with EtOAc (·2). The com-
bined organiclayers were washed with water, brine,
dried over Na2SO4 and concentrated in vacuo to give
a residue, which was purified by recrystallization from
EtOActo yield 7 (50mg, 85%); TLC Rf =0.53 (CHCl3/
MeOH, 9/1); 1H NMR (300MHz, CDCl3) d 8.16 (d,
J=16.2Hz, 1H), 7.70 (d, J=9.0Hz, 2H), 7.43 (br d,
J=7.2Hz, 1H), 7.15–7.03 (m, 2H), 6.96 (d, J=9.0Hz,
2H), 6.74 (br s, 1H), 6.59 (d, J=16.2Hz, 1H), 4.06 (t,
J=6.3Hz, 2H), 2.48 (s, 3H), 1.88–1.76 (m, 2H), 1.60–
1.46 (m, 2H), 1.00 (t, J=7.2Hz, 3H); MS (APCI, Neg,
20V.) m/z 376 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C23 H23NO4: 377.1627; found: 377.1642; IR (neat)
2959, 1684, 1626, 1604, 1509, 1427, 1371, 1294, 1256,
1168, 1066, 950, 912, 779, 760, 616cmꢀ1
.
5.12. 1-(4-Butoxybenzoyl)-2-methyl-1H-indole-4-carb-
oxylic acid (4)
5.15. 4-[1-(4-Butoxybenzoyl)-2-methyl-1H-indol-4-yl]but-
anoic acid (6)
To a stirred solution of the compound 20 (1.02g,
2.31mmol) in MeOH (10mL) and EtOAc(5mL) was
added 10% Pd–C (100mg) at room temperature, and
the resulting mixture was stirred for 30min at room tem-
perature under hydrogen atmosphere. The catalyst was
removed by filtration and the filtrate was concentrated
in vacuo to give a residue, which was purified by column
chromatography on silica gel to yield 4 (433mg, 53%);
TLC Rf =0.59 (CHCl3/MeOH, 9/1); 1H NMR
(300MHz, CDCl3) d 7.99 (d, J=8.1Hz, 1H), 7.70 (d,
J=9.0Hz, 2H), 7.36 (d, J=8.1Hz, 1H), 7.21 (br s,
To a stirred solution of the compound 21 (460mg,
0.95mmol) in EtOAc(5mL) and MeOH (5mL) was
added 10wt% Pd–C (45mg) at room temperature. The
resulting suspension was stirred for 1h at room temper-
ature under hydrogen atmosphere. Insoluble substance
was removed by filtration. The filtrate was concentrated
in vacuo to give a crude product, which was purified by
column chromatography on silica gel to yield 6 (170mg,
1
45%); TLC Rf =0.50 (CHCl3/CH3OH, 9/1); H NMR
(300MHz, CDCl3) d 7.71 (d, J=9.0Hz, 2H), 7.00–6.86
(m, 5H), 6.48 (s, 1H), 4.05 (t, J=6.6Hz, 2H), 2.90 (t,

4694
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
J=7.2Hz, 2H), 2.48–2.38 (m, 5H), 2.14–2.00 (m, 2H),
2.00–1.40 (m, 5H), 1.00 (t, J=7.5Hz, 3H); MS (APCI,
Neg 20V) m/z 392 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd
for C24H27NO4: 393.1940; found: 393.1949; IR (neat)
3351, 2959, 1682, 1605, 1573, 1510, 1484, 1432, 1369,
Na2SO4 and concentrated in vacuo to give a residue,
which was purified by column chromatography on silica
gel to yield 26 (800mg, 52%); TLC Rf =0.63 (n-hexane/
EtOAc, 7/3); 1H NMR (300MHz, CDCl3) d 7.69 (d,
J=8.7Hz, 2H), 7.00–6.85 (m, 3H), 6.68 (d, J=8.4Hz,
1H), 6.67 (s, 1H), 6.47 (d, J=7.2Hz, 1H), 6.00–5.87
(m, 1H), 5.40–5.30 (m, 1H), 5.30–5.24 (m, 1H), 4.78 (s,
2H), 4.75–4.68 (m, 2H), 4.05 (t, J=6.3Hz, 2H), 2.42
(s, 3H), 1.87–1.75 (m, 2H), 1.60–1.45 (m, 2H), 1.00 (t,
J=7.5Hz, 3H).
1301, 1257, 1227, 1169, 912, 779, 618cmꢀ1
.
5.16. Methyl [(2-methyl-1H-indol-4-yl)oxy]acetate (23)
To a stirred solution of 2-methyl-4-hydroxy indole 11
(5g, 33.9mmol) in DMF (50mL) were added K2CO3
(11.7g, 84.9mmol) and methyl bromoacetate (3.54mL,
37.4mmol) at room temperature. The reaction mixture
was stirred at 80ꢁC for 2h. The resulting mixture was
quenched with ice water. The resulting precipitates were
collected by filtration to yield 23 (5.4g, 73%) as a gray
powder; TLC Rf =0.75 (n-hexane/EtOAc, 7/3); 1H
NMR (300MHz, CDCl3) d 8.00–7.84 (br, 1H), 7.04–
6.94 (m, 2H), 6.45–5.36 (m, 2H), 4.77 (s, 2H), 3.80 (s,
3H), 2.43(s, 3H).
5.20. {[1-(4-Butoxybenzoyl)-2-methyl-1H-indol-4-
yl]oxy}acetic acid (8)
To a stirred solution of 26 (800mg, 1.90mmol) in THF
(100mL) was added Pd(PPh3)4 (220mg, 0.19mmol) at
room temperature under argon atmosphere. After stir-
ring for 15min, morpholine (0.83mL, 9.54mmol) was
added. After 1h, the reaction mixture was quenched
with water and extracted with EtOAc (·2). The com-
bined organiclayers were washed with water, brine,
dried over Na2SO4 and concentrated in vacuo to give
a residue, which was purified by column chromatogra-
phy on silica gel to yield 8 (570mg, 79%) as a yellow
5.17. [(2-Methyl-1H-indol-4-yl)oxy]acetic acid (24)
To a stirred solution of a methyl ester 23 (5.4g,
24.6mmol) in MeOH (18mL) and 1,4-dioxane (36mL)
was added 5N NaOH aq (15mL). After stirring for 1h
at room temperature, the reaction mixture was acidified
with 2M HCl aq, and the resulting precipitates were col-
lected by filtration to afford 24 (3.5g, 69%) as a white
powder; TLC Rf =0.20 (CHCl3/CH3OH, 9/1); 1H
NMR (300MHz, CDCl3) d 10.89 (s, 1H), 6.90–6.80
(m, 2H), 6.29 (dd, J=1.5,6.9Hz, 1H), 6.11 (s, 1H),
4.68 (s, 2H), 2.33 (s, 3H).
1
powder; TLC Rf =0.38 (CHCl3/MeOH, 9/1); H NMR
(300MHz, CDCl3) d 7.70 (d, J=9.0Hz, 2H), 6.98–6.89
(m, 3H), 6.71 (d, J=8.4Hz, 1H), 6.60–6.57 (m, 1H),
6.51 (d, J=8.4Hz, 1H), 4.84 (s, 2H), 4.05 (t, J=6.6Hz,
2H), 2.43 (s, 3H), 1.87–1.75 (m, 2H), 1.59–1.44 (m,
2H), 1.00 (t, J=7.5Hz, 3H); MS (APCI, Neg, 20V.)
m/z 380 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for C22
H23NO5: 381.1576; found: 381.1577; IR (neat) 2959,
2873, 1734, 1684, 1604, 1571, 1509, 1494, 1438, 1384,
1325, 1295, 1258, 1226, 1170, 1107, 1068, 1029, 952,
5.18. Allyl [(2-methyl-1H-indol-4-yl)oxy]acetate (25)
912, 850, 768, 731, 668, 620cmꢀ1
.
To a stirred solution of 24 (2g, 9.8mmol) in DMF
(20mL) were added K2CO3 (2.02g, 14.6mmol) and allyl
bromide (1.27mL, 14.6mmol) at room temperature.
After stirring for 2h at 80ꢁC, the reaction mixture was
poured into ice water and extracted with EtOAc (·3).
The combined organic layers were washed with water,
brine, and dried over Na2SO4. The solvent was removed
by evaporation and the residue was purified by column
chromatography on silica gel to afford 25 (1.88g, 79%)
as a white solid. TLC Rf =0.50 (n-hexane/EtOAc, 7/3);
1H NMR (300MHz, CDCl3) d 7.88 (s, 1H), 7.03–6.94
(m, 2H), 6.45–6.37 (m, 2H), 6.00–5.85 (m, 1H), 5.34
(dq, J=5.7,1.5Hz, 2H), 5.25 (dq, J=10.2,1.5Hz, 1H),
4.79 (s, 2H), 4.71 (dt, J=5.7,1.5Hz, 2H), 2.43 (s, 3H).
5.21. Benzyl {[1-(4-butoxybenzoyl)-2-methyl-1H-indol-5-
yl]oxy}acetate (28)
A mixture of 27 (2.5g, 15.5mmol) and pyridinium hydro-
chloride (25g) was heated for 2h at 190ꢁC. After cooling
to room temperature, the reaction mixture was poured
into water and extracted with EtOAc (·3). The combined
organiclayers were washed with 1M HCl aq, water,
brine, dried over Na2SO4 and evaporated to give a resi-
due, which was used for the next reaction without further
purification; TLC Rf =0.33 (n-hexane/EtOAc, 7/3).
To a stirred solution of 2-methyl-5-hydroxy indole de-
scribed above (15.5mmol) in DMF (25mL) were added
K2CO3 (5.35g, 38.77mmol) and benzyl bromoacetate
(2.7mL, 17.06mmol) at room temperature. After stir-
ring for 30min at 80ꢁC, the reaction mixture was poured
into water and extracted with EtOAc (·2). The com-
bined organiclayers were washed with water, brine,
dried over Na2SO4 and concentrated in vacuo to give
a residue, which was purified by column chromatogra-
phy on silica gel to yield an O-alkylated product
(1.17g, 26%); TLC Rf =0.51 (n-hexane/EtOAc, 7/3).
5.19. Allyl {[1-(4-butoxybenzoyl)-2-methyl-1H-indol-4-
yl]oxy}acetate (26)
To a stirred solution of 25 (900mg, 3.67mmol) in DMF
(10mL) was added sodium hydride (60% oil dispersion,
147mg, 3.67mmol) in several portions at 0ꢁC under
argon atmosphere, and the resulting suspension was stir-
red for 30min at 0ꢁC. After the addition of p-(n-butyl-
oxy) benzoyl chloride (0.70mL, 3.67mmol), the
reaction mixture was stirred for 12h at 0ꢁC, quenched
with water and extracted with EtOAc. The combined or-
ganiclayers were washed with water, brine, dried over
To a stirred solution of the above-described product
(300mg, 1.02mmol) and NaOH (powdered, 203mg,
5.08mmol) in CH2Cl2 (4mL) were added TBACl

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
4695
(28mg, 0.102mmol) and p-(n-butyloxy) benzoyl chloride
(0.29mL, 1.53mmol) at room temperature. After stir-
ring for 30min at room temperature, the reaction mix-
ture was purified by column chromatography on silica
gel to yield 28 (220mg, 46%); TLC Rf =0.59 (n-hexane/
EtOAc, 7/3); 1H NMR (300MHz, CDCl3) d 7.68 (d,
J=9.0Hz, 2H), 7.40–7.30 (m, 5H), 6.97–6.88 (m, 4H),
6.68 (dd, J=9.0,2.4Hz, 1H), 6.34–6.28 (m, 1H), 5.24
(s, 2H), 4.68 (s, 2H), 4.05 (t, J=6.6Hz, 2H), 2.42 (s,
3H), 1.88–1.76 (m, 2H), 1.66–1.46 (m, 2H), 1.00 (t,
J=7.5Hz, 3H).
to give a residue, which was purified by column chroma-
tography on silica gel to yield 30 (446g, 100%); TLC
1
Rf =0.80 (n-hexane/EtOAc, 20/1); H NMR (300MHz,
CDCl3) d 8.06 (m, 1H), 7.30 (dd, J=7.5,0.9 Hz, 1H),
7.13 (dd, J=8.1,7.5Hz, 1H), 6.49 (m, 1H), 2.62 (d,
J=0.9Hz, 3H), 1.68 (s, 9H), 0.29 (s, 9H).
5.25. (2-Methyl-1H-indol-4-yl)acetic acid (31)
To a stirred solution of borane-tetrahydrofuran com-
plex (1.0 mol/L in THF, 666mL, 666mmol) in THF
(350mL) was added cyclohexene (135mL, 1332mmol)
at 15ꢁC. After stirring for 1 h at same temperature, a
solution of 30 (117g, 333mmol) in THF (200mL) was
added dropwise. After stirring for 3h at room tempera-
ture, 2M NaOH aq (1000mL, 2mol) was added drop-
wise at 0ꢁC, and then 30% aqueous hydrogen peroxide
(300mL) was added at 50ꢁC. The reaction mixture
was concentrated in vacuo to remove THF. The result-
ing aqueous layer was washed with n-hexane, and acid-
ified by the addition of conc HCl to pH4 and extracted
with EtOAc( ·3). The combined organic layers were
washed with water, brine, dried over Na2SO4 and con-
centrated in vacuo to give a residue, which was used
for the next reaction without further purification; TLC
Rf =0.53 (n-hexane/EtOAc, 1/1).
5.22. {[1-(4-Butoxybenzoyl)-2-methyl-1H-indol-5-
yl]oxy}acetic acid (9)
To a stirred solution of 28 (220mg, 0.47mmol) in
MeOH (3mL) and EtOAc(3mL) was added 10wt%
Pd–C (20mg) at room temperature, and the resulting
suspension was stirred for 20min at room temperature
under hydrogen atmosphere. The resulting mixture
was filtered and the filtrate was concentrated in vacuo
to give a residue, which was recrystallized from EtO-
Ac/n-hexane to yield 9 (80mg, 46%) as pale pink pow-
der; TLC Rf =0.40 (CHCl3/CH3OH, 9/1); 1H NMR
(300MHz, CDCl3) d 7.68 (d, J=8.7Hz, 2H), 7.00–6.92
(m, 4H), 6.70 (dd, J=8.7,2.1Hz, 1H), 6.36 (br s, 1H),
4.68 (s, 2H), 4.05 (t, J=6.6Hz, 2H), 2.42 (s, 3H), 2.00–
1.40 (m, 5H), 1.00 (t, J=7.5Hz, 3H); MS (APCI, Neg.
20V) m/z 380 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C22 H23NO5: 381.1576; found: 381.1556; IR (KBr)
3510, 2952, 1748, 1682, 1608, 1512, 1477, 1432, 1372,
1315, 1254, 1195, 1177, 1150, 1111, 1075, 913, 846,
To a stirred solution of the above-described intermedi-
ate in MeOH (500mL) and 1,4-dioxane (500mL) was
added 5M NaOH aq (1000mL). After stirring for 14h
at 70ꢁC, the reaction mixture was acidified with conc
HCl and extracted with EtOAc (·3). The combined or-
ganiclayers were washed with water, brine, dried over
Na2SO4 and concentrated in vacuo to give a residue,
which was recrystallized from EtOAc/n-hexane to yield
31 (142g, 56%); TLC Rf =0.29 (n-hexane/EtOAc=1/1);
(300MHz, CDCl3/CD3OD) d 9.55 (br s, 1H), 7.20 (d,
J=7.8Hz, 1H), 7.02 (dd, J=7.8,7.8Hz, 1H), 6.93 (d,
J=7.8Hz, 1H), 6.23 (s, 1H), 3.83 (s, 2H), 2.41 (s, 3H).
793, 761, 652cmꢀ1
.
5.23. tert-Butyl 2-methyl-4-{[(trifluoromethyl)sulfon-
yl]oxy}-1H-indole-1-carboxylate (29)
To a stirred solution of 12 (380g, 1.36 mol) in CH3CN
(750mL) were added a solution of Boc2O (326g,
1.50mol) in CH3CN (750mL) and N,N0-dimethylamino-
pyridine (1.66g, 13.6mmol) at room temperature. After
stirring for 1h at room temperature, the resulting mix-
ture was concentrated in vacuo. The residue was dis-
solved with n-hexane/EtOAcand washed with water,
brine, dried over Na2SO4 and concentrated in vacuo
to give a residue, which was used for the next reaction
without further purification; TLC Rf =0.72 (n-hexane/
5.26. Allyl (2-methyl-1H-indol-4-yl)acetate (32)
To a stirred solution of 31 (53mg, 280mmol) in DMF
(500mL) were added K2CO3 (59g, 427mmol) and allyl
bromide (31mL, 358mmol) at room temperature. After
stirring for 1h at 50ꢁC, the reaction mixture was poured
into ice-water containing EtOAc (·3). The combined or-
ganiclayers were washed with water, brine, dried over
Na2SO4 and concentrated in vacuo, to give a residue,
which was purified by column chromatography on silica
gel to yield 32 (43.5g, 68%); TLC Rf =0.50 (n-hexane/Et-
1
EtOAc, 10/1); H NMR (300MHz, CDCl3) d 8.14 (m,
1H), 7.23 (dd, J=8.4, 8.1Hz, 1H), 7.12 (d, J=8.1Hz,
1H), 6.44 (m, 1H), 2.62 (d, J=0.9Hz, 3H), 1.69 (s, 9H).
1
5.24. tert-Butyl 2-methyl-4-[(trimethylsilyl)ethynyl]-1H-
indole-1-carboxylate (30)
OAc, 2/1); H NMR (300MHz, CDCl3) d 7.92 (br s,
1H), 7.19 (d, J=7.0Hz, 1H), 7.06 (t, J=7.0Hz, 1H),
6.96 (dd, J=7.0,1.2Hz, 1H), 6.27 (m, 1H), 5.90 (ddt,
J=17.2, 10.4, 5.4Hz, 1H), 5.25 (d, J=17.2Hz, 1H),
5.18 (d, J=10.4Hz, 1H), 4.59 (m, 2H), 3.88 (s, 2H),
2.43 (s, 3H).
To a stirred solution of 29 (516g, 1.36mol) in DMF
(1000mL) were added CuI (2.59g, 13.6mmol), TEA
(284mL, 2.04mmol), TBAI (5.02g, 13.6mmol),
Pd(PPh3)Cl2 (9.55g, 13.6mmol) and trimethylsilylacety-
lene (160g, 163mol) at room temperature. After stirring
for 7h at 70ꢁC, the reaction mixture was poured into
saturated NH4Cl aq and extracted with EtOAc (·2).
The combined organic layers were washed with water,
brine, dried over Na2SO4 and concentrated in vacuo
5.27. Benzyl (2-methyl-1H-indol-4-yl)acetate (33)
To a stirred solution of compound 31 (650mg,
3.44mmol) in DMF (7mL) were added K2CO3 (1.02g,
7.38mmol) and benzyl bromide (0.49mL, 4.12mmol)

4696
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
at room temperature under argon atmosphere, and stir-
ring was continued for 4h. The reaction mixture was
quenched with 1M HCl aq and extracted two times 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 col-
umn chromatography on silica gel to yield 33 (600mg,
62%); TLC Rf =0.60 (n-hexane/EtOAc, 3/1); (300MHz,
CDCl3/CD3OD) d 7.93 (br s, 1H), 7.30 (m, 5H), 7.15
(d, J=7.8Hz, 1H), 7.04 (dd, J=7.8,7.8Hz, 1H), 6.96
(d, J=7.8Hz, 1H), 6.22 (s, 1H), 5.12 (s, 2H), 3.89 (s,
2H), 2.37 (s, 3H).
CDCl3) d 7.70 (d, J=8.8Hz, 2H), 7.10–6.90 (m, 5H),
6.49 (s, 1H), 6.02–5.60 (m, 1H), 5.32–5.19 (m, 2H),
4.65–4.60 (m, 2H), 4.07 (t, J=6.8Hz, 2H), 3.85 (s,
2H), 2.45 (s, 3H), 1.85–0.90 (m, 13H).
5.28.6. Allyl {1-[4-(but-3-en-1-yloxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetate (34i). 89% yield; TLC Rf =0.60
1
(n-hexane/EtOAc, 1/1); H NMR (200MHz, CDCl3) d
7.72–7.68 (m, 2H), 7.08–6.90 (m, 5H), 6.49 (s, 1H),
6.03–5.60 (m, 2H), 5.32–5.10 (m, 4H), 4.63–4.59 (m,
2H), 4.10 (t, J=6.6Hz, 2H), 3.86 (s, 2H), 2.70–2.53 (s,
2H), 2.45 (s, 3H).
5.28. General procedure for the preparation of N-benzoyl-
2-methyl-indole-4-acetic acids (34c, e–j, l–n, p–v)
5.28.7. Allyl {1-[4-(benzyloxy)benzoyl]-2-methyl-1H-in-
dol-4-yl}acetate (34j). 78% yield; TLC Rf =0.32 (n-hex-
ane/EtOAc, 7/3); H NMR (200MHz, CDCl3) d 7.70–
1
5.28.1.
Allyl
{1-[4-(2-cyclopropylethoxy)benzoyl]-2-
7.50 (m, 2H), 7.50–7.35 (m, 5H), 7.06–6.90 (m, 5H),
6.45 (s, 1H), 6.02–5.60 (m, 1H), 5.32–5.10 (m, 4H),
4.63–4.60 (m, 2H), 3.85 (s, 2H), 2.42 (s, 3H).
methyl-1H-indol-4-yl}acetate (34h). To a stirred solution
of 32 (230mg, 1.0mmol) and NaOH (powdered, 200mg,
5.0mmol) in CH2Cl2 (5mL) were added TBACl (28mg,
0.102mmol) and p-(2-cyclopropylethoxy) benzoyl chlo-
ride (450mg, 2mmol) 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 organic layers 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 34h (380mg, 91%); TLC Rf =0.78
5.28.8. Allyl {2-methyl-1-[4-(3-phenylpropoxy)benzoyl]-
1H-indol-4-yl}acetate (34l). 85% yield; TLC Rf =0.67
1
(n-hexane/EtOAc, 7/3); H NMR (200MHz, CDCl3) d
7.70 (d, J=9.0Hz, 2H), 7.35–7.16 (m, 5H), 7.08–6.85
(m, 5H), 6.51 (s, 1H), 6.02–5.62 (m, 1H), 5.32–5.20 (m,
2H), 4.63–4.59 (m, 2H), 4.05 (t, J=6.5Hz, 2H), 3.87
(s, 2H), 2.84 (t, J=6.5Hz, 2H), 2.45 (s, 3H), 2.20–2.09
(m, 2H).
1
(n-hexane/EtOAc, 1/1); H NMR (200MHz, CDCl3) d
7.73–7.69 (m, 2H), 7.06–6.93 (m, 5H), 6.51 (s, 1H),
6.02–5.62 (m, 1H), 5.32–5.19 (m, 2H), 4.63–4.60 (m,
2H), 4.16–4.09 (m, 2H), 3.87 (s, 2H), 2.45 (s, 3H), 1.73
(q, J=7.0Hz, 2H), 0.98–0.78 (m, 1H), 0.58–0.49 (m,
2H), 0.20–0.13 (m, 2H).
5.28.9. Allyl {1-[4-(2-methoxyethoxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetate (34m). 64% yield; TLC Rf =0.20
(n-hexane/EtOAc, 7/3); H NMR (200MHz, CDCl3) d
7.70 (d, J=8.8Hz, 2H), 7.10–6.90 (m, 5H), 6.50 (s,
1H), 6.05–5.60 (m, 1H), 5.35–5.20 (m, 2H), 4.63–4.60
(m, 2H), 4.20 (t, J=5.0Hz, 2H), 3.85 (s, 2H), 3.80 (t,
J=5.0Hz, 2H), 3.46 (s, 3H), 2.45 (s, 3H).
1
5.28.2. Allyl {1-[4-(hexyloxy)benzoyl]-2-methyl-1H-indol-
4-yl}acetate (34c). 90% yield; TLC Rf =0.36 (n-hexane/
1
EtOAc, 7/3); H NMR (200MHz, CDCl3) d 7.72–7.68
5.28.10.
Allyl
{1-[4-(3-methoxypropoxy)benzoyl]-2-
(m, 2H), 7.08–6.85 (m, 5H), 6.50 (s, 1H), 6.02–5.60 (m,
1H), 5.33–5.18 (m, 2H), 4.63–4.58 (m, 2H), 4.04 (t,
J=6.8Hz, 2H), 3.87 (s, 2H), 2.45 (s, 3H), 1.91–1.82
(m, 2H), 1.62–1.33 (m, 6H), 1.00–0.90 (m, 3H).
methyl-1H-indol-4-yl}acetate (34n). 63% yield; TLC
Rf =0.25 (n-hexane/EtOAc, 2/1); 1H NMR (200MHz,
CDCl3) d 7.72 (d, J=8.7Hz, 2H), 7.09–6.90 (m, 5H),
6.50 (s, 1H), 6.02–5.60 (m, 1H), 5.35–5.20 (m, 2H),
4.65–4.60 (m, 2H), 4.15 (t, J=6.7Hz, 2H), 3.87 (s,
2H), 3.58 (t, J=6.0Hz, 2H), 3.37 (s, 3H), 2.43 (s, 3H),
2.20–2.05 (m, 2H).
5.28.3. Allyl {1-[4-(3,3-dimethylbutoxy)benzoyl]-2-methyl
2H}-1H-indol-4-yl}acetate (34e). 92% yield; TLC
Rf =0.36 (n-hexane/EtOAc, 7/3); 1H NMR (200MHz,
CDCl3) d 7.69 (d, J=8.7Hz, 2H), 7.08–6.85 (m, 5H),
6.48 (s, 1H), 6.03–5.62 (m, 1H), 5.32–5.18 (m, 2H),
4.64–4.58 (m, 2H), 4.09 (t, J=7.0Hz, 2H), 3.83 (s,
2H), 2.43 (s, 3H), 1.75 (t, J=7.0Hz, 2H), 1.02 (s, 9H).
5.28.11. Allyl {1-[4-(3-ethoxypropoxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetate (34p). 83% yield; TLC Rf =0.33 (n-
hexane/EtOAc, 2/1); 1H NMR (200MHz, CDCl3) d 7.71
(d, J=9.0Hz, 2H), 7.10–6.85 (m, 5H), 6.52 (s, 1H), 6.05–
5.60 (m, 1H), 5.37–5.18 (m, 2H), 4.65–4.60 (m, 2H), 4.17
(t, J=6.5Hz, 2H), 3.85 (s, 2H), 3.62 (t, J=6.5Hz, 2H),
3.50 (q, J=6.8Hz, 2H), 2.45 (s, 3H), 2.08 (m, 2H),
1.21 (t, J=6.8Hz, 3H).
5.28.4. Allyl (2-methyl-1-{4-[(4-methylpentyl)oxy]ben-
zoyl}-1H-indol-4-yl)acetate (34f). 83% yield; TLC
Rf =0.35 (n-hexane/EtOAc, 7/3); 1H NMR (200MHz,
CDCl3) d 7.72 (d, J=8.8Hz, 2H), 7.08–6.88 (m, 5H),
6.49 (s, 1H), 6.04–5.60 (m, 1H), 5.33–5.19 (m, 2H),
4.65–4.60 (m, 2H), 4.03 (t, J=6.8Hz, 2H), 3.87 (s,
2H), 2.45 (s, 3H), 1.95–1.50 (m, 3H), 1.45–1.10 (m,
2H), 0.95 (d, J=6.8Hz, 6H).
5.28.12. Allyl {2-methyl-1-[4-(2-propoxyethoxy)benzoyl]-
1H-indol-4-yl}acetate (34q). 62% yield; TLC Rf =0.39 (n-
hexane/EtOAc, 2/1); 1H NMR (300MHz, CDCl3) d 7.72
(d, J=8.9Hz, 2H), 7.05–6.90 (m, 5H), 6.49 (s, 1H), 6.02–
5.60 (m, 1H), 5.32–5.20 (m, 2H), 4.65–4.58 (m, 2H),
4.23–4.20 (m, 2H), 3.86 (s, 2H), 3.86–3.80 (m, 2H),
3.50 (t, J=7.0Hz, 2H), 2.44 (s, 3H), 1.65 (m, 2H), 0.95
(t, J=7.5Hz, 3H).
5.28.5.
methyl-1H-indol-4-yl}acetate (34g). 80% yield; TLC
Allyl
{1-[4-(2-cyclohexylethoxy)benzoyl]-2-
Rf =0.41 (n-hexane/EtOAc, 7/3); 1H NMR (200MHz,

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
4697
1
5.28.13. Allyl {1-[4-(2-isopropoxyethoxy)benzoyl]-2-
methyl-1H-indol-4-yl}acetate (34r). 100% yield; TLC
Rf =0.65 (n-hexane/EtOAc, 1/1); 1H NMR (200MHz,
CDCl3) d 7.72–7.68 (m, 2H), 7.08–6.80 (m, 5H), 6.50
(s, 1H), 6.02–5.62 (m, 1H), 5.32–5.19 (m, 2H), 4.63–
4.59 (m, 2H), 4.22–4.14 (m, 2H), 3.86 (s, 2H), 3.86–
3.65 (m, 3H), 2.44 (s, 3H), 1.21 (d, J=6.0Hz, 6H).
ane/EtOAc, 4/1); H NMR (300MHz, CDCl3) d 7.72–
7.65 (m, 2H), 7.39–7.28 (m, 5H), 7.05–6.88 (m, 5H),
6.45 (s, 1H), 5.15 (s, 2H), 4.04 (t, J=6.6Hz, 2H), 3.87
(s, 2H), 2.45 (s, 3H), 1.87–1.20 (m, 6H), 0.95 (t,
J=6.9Hz, 3H).
5.29.3. Benzyl {2-methyl-1-[4-(3-methylbutoxy)benzoyl]-
1H-indol-4-yl}acetate (34d). 80% yield; TLC Rf =0.58 (n-
hexane/EtOAc, 4/1); 1H NMR (200MHz, CDCl3) d
7.73–7.67 (m, 2H), 7.42–7.20 (m, 5H), 7.06–6.90 (m,
5H), 6.45 (s, 1H), 5.15 (s, 2H), 4.06 (t, J=6.6Hz, 2H),
3.87 (s, 2H), 2.45 (s, 3H), 1.85 (m, 1H), 1.72 (m, 2H),
0.99 (t, J=6.2Hz, 6H).
5.28.14. Allyl {1-[4-(2-tert-butoxyethoxy)benzoyl]-2-
methyl-1H-indol-4-yl}acetate (34s). 80% yield; TLC
Rf =0.68 (n-hexane/EtOAc, 1/1); 1H NMR (200MHz,
CDCl3) d 7.70 (d, J=9.0Hz, 2H), 7.04–6.93 (m, 5H),
6.50 (s, 1H), 6.01–5.82 (m, 1H), 5.32–5.19 (m, 2H),
4.61 (dt, J=5.8,1.6Hz, 2H), 4.17 (t, J=5.2Hz, 2H),
3.86 (s, 2H), 3.76 (t, J=5.2Hz, 2H), 2.45 (s, 3H), 1.25
(s, 9H); MS (APCI, Pos, 20V.) m/z 450 (M+H)+.
5.29.4. Benzyl {2-methyl-1-[4-(2-phenylethoxy)benzoyl]-
1H-indol-4-yl}acetate (34k). 100% yield; TLC Rf =0.64
(n-hexane/EtOAc, 8/1); H NMR (200MHz, CDCl3) d
1
5.28.15. Allyl (1-{4-[2-(2-methoxyethoxy)ethoxy]ben-
zoyl}-2-methyl-1H-indol-4-yl)acetate (34t). 97% yield;
TLC Rf =0.49 (n-hexane/EtOAc, 1/2); 1H NMR
(300MHz, CDCl3) d 7.71–7.68 (m, 2H), 7.10–6.80 (m,
5H), 6.50 (s, 1H), 5.98–5.70 (m, 1H), 5.62–5.40 (m,
2H), 4.63–4.60 (m, 2H), 4.25–4.11 (m, 2H), 3.92–3.89
(m, 2H), 3.86 (s, 2H), 3.78–3.73 (m, 2H), 3.60–3.58 (m,
2H), 3.40 (s, 3H), 2.45 (s, 3H).
8.07 (d, J=9.0Hz, 1H), 7.69 (d, J=8.6Hz, 2H), 7.40–
7.24 (m, 9H), 7.10–6.90 (m, 5H), 6.45 (s, 1H), 5.15 (s,
2H), 4.26 (t, J=7.0Hz, 2H), 3.88 (s, 2H), 3.15 (t,
J=7.0Hz, 2H), 2.42 (s, 3H).
5.29.5. Benzyl {1-[4-(2-ethoxyethoxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetate (34o). 100% yield; TLC Rf =0.35
1
(n-hexane/EtOAc, 4/1); H NMR (200MHz, CDCl3) d
7.70 (d, J=9.0Hz, 2H), 7.39–7.28 (m, 5H), 7.05–6.88
(m, 5H), 6.45 (s, 1H), 5.15 (s, 2H), 4.21 (t, J=4.8Hz,
2H), 3.87 (s, 2H), 3.83 (t, J=4,8Hz, 2H), 3.62 (q,
J=6.6Hz, 2H), 2.42 (s, 3H), 1.26 (t, J=6.6Hz, 3H).
5.28.16. Allyl {2-methyl-1-[4-(2-phenoxyethoxy)benzoyl]-
1H-indol-4-yl}acetate (34u). 97% yield; TLC Rf =0.50 (n-
hexane/EtOAc, 7/3); 1H NMR (300MHz, CDCl3) d 7.72
(d, J=9.0Hz, 2H), 7.32 (t, J=7.7Hz, 2H), 7.10–6.90 (m,
8H), 6.50 (s, 1H), 6.00–5.70 (m, 1H), 5.40–5.20 (m, 2H),
4.63–4.60 (m, 2H), 4.45–4.30 (m, 4H), 3.86 (s, 2H), 2.45
(s, 3H).
5.30. General procedure for the preparation of N-benzoyl-
2-methylindole-4-acetic acids (3c, e–j, l–n)
5.30.1.
{1-[4-(2-Cyclopropylethoxy)benzoyl]-2-methyl-
5.28.17. Allyl
(1-{4-[2-(ethylthio)ethoxy]benzoyl}-2-
1H-indol-4-yl}acetic acid (3h). To a stirred solution of
34h (380mg, 0.91mmol) in THF (5mL) was added
Pd(PPh3)4 (50mg, 0.05mmol) at room temperature un-
der argon atmosphere. After stirring for 15min, morph-
oline (0.40mL, 4.55mmol) 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 3h
(214mg, 62%); TLC Rf =0.37 (EtOAc); 1H NMR
(200MHz, CDCl3) d 7.73–7.69 (m, 2H), 7.08–6.94 (m,
5H), 6.49 (s, 1H), 4.12 (t, J=6.5Hz, 2H), 3.87 (s, 2H),
2.45 (s, 3H), 1.72 (q, J=6.5Hz, 2H), 0.96–0.80 (m,
1H), 0.56–0.47 (m, 2H), 0.18–0.13 (m, 2H); MS (APCI,
Neg, 20V.) m/z 376 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C23H23NO4: 377.1627; found: 395.1811; IR (neat) 2944,
1703, 1604, 1510, 1436, 1372, 1302, 1214, 1173, 1010,
methyl-1H-indol-4-yl)acetate (34v). 85% yield; TLC
Rf =0.46 (n-hexane/EtOAc, 7/3); 1H NMR (200MHz,
CDCl3) d 7.71 (d, J=9.0Hz, 2H), 7.05–6.90 (m, 5H),
6.50 (s, 1H), 6.00–5.75 (m, 1H), 5.35–5.19 (m, 2H),
4.63–4.58 (m, 2H), 4.23 (t, J=7.0Hz, 2H), 3.86 (s,
2H), 2.95 (t, J=7.0Hz, 2H), 2.67 (q, J=7.4Hz, 2H),
2.45 (s, 3H), 1.30 (t, J=7.4Hz, 3H).
5.29. General procedure for the preparation of N-benzoyl-
2-methylindole-4-acetic acids (34a, b, d, k, o)
5.29.1. Benzyl [1-(4-butoxybenzoyl)-2-methyl-1H-indol-4-
yl]acetate (34a). To a stirred solution of 33 (285mg,
1.02mmol) and NaOH (powdered, 203mg, 5.08mmol)
in CH2Cl2 (4mL) were added TBACl (28mg,
0.102mmol) and p-(n-butyloxy) benzoyl chloride
(0.29mL, 1.53mmol) at room temperature. After stir-
ring for 30min at room temperature, the reaction mix-
ture was purified by column chromatography on silica
gel to yield 3b (395mg, 85%); TLC Rf =0.56 (n-hexane/
EtOAc, 4/1); 1H NMR (300MHz, CDCl3) d 7.70 (d,
J=9.0Hz, 2H), 7.40–7.25 (m, 5H), 7.08–6.88 (m, 5H),
6.45 (s, 1H), 5.15 (s, 2H), 4.05 (t, J=6.6Hz, 2H), 3.87
(s, 2H), 2.45 (s, 3H), 1.90–1.74 (m, 2H), 1.60–1.45 (m,
2H), 1.00 (t, J=7.5Hz, 3H).
911, 757, 707, 638cmꢀ1
.
5.30.2. {1-[4-(Hexyloxy)benzoyl]-2-methyl-1H-indol-4-
yl}acetic acid (3c). 45% yield; TLC Rf =0.44 (EtOAc);
1H NMR (300MHz, CDCl3) d 7.72–7.69 (m, 2H),
7.06–6.93 (m, 5H), 6.49 (s, 1H), 4.04 (t, J=6.5Hz,
2H), 3.87 (s, 2H), 2.45 (s, 3H), 1.9–1.8 (m, 2H), 3.87
(s, 2H), 2.45 (s, 3H), 1.9–1.8 (m, 2H), 1.6–1.4 (m, 2H),
1.4–1.3 (m, 4H), 1.0–0.9 (m, 3H); MS (APCI, Neg,
20V.) m/z 392 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C24H27NO4: 393.1940; found: 393.1949; IR (KBr)
5.29.2. Benzyl {2-methyl-1-[4-(pentyloxy)benzoyl]-1H-in-
dol-4-yl}acetate (34b). 91% yield; TLC Rf =0.58 (n-hex-

4698
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
2953, 1678, 1606, 1511, 1436, 1374, 1322, 1260, 1211,
1172, 1020, 911, 839, 812, 775, 759, 706, 640cmꢀ1
(m, 5H), 6.50 (s, 1H), 4.05 (t, J=6.3Hz, 2H), 3.88 (s,
2H), 2.84 (t, J=6.3Hz, 2H), 2.45 (s, 3H), 2.20–2.10
(m, 2H); MS (APCI, Neg, 20V.) m/z 426 (MꢀH)ꢀ;
HRMS (EI, Pos.) calcd for C27H25NO4: 427.1784;
found: 427.1772; IR (neat) 3369, 2922, 1682, 1604,
1510, 1435, 1370, 1300, 1257, 1169, 1031, 912, 837,
.
5.30.3. {1-[4-(3,3-Dimethylbutoxy)benzoyl]-2-methyl-1H-
indol-4-yl}acetic acid (3e). 68% yield; TLC Rf =0.45
1
(CHCl3/MeOH, 10/1); H NMR (200MHz, CDCl3) d
7.68 (d, J=8.8Hz, 2H), 7.08–6.86 (m, 5H), 6.47 (s,
1H), 4.09 (t, J=7.2Hz, 2H), 3.83 (s, 2H), 2.43 (s, 3H),
1.76 (t, J=7.2Hz, 2H), 1.00 (s, 9H); MS (APCI, Neg,
20V.) m/z 392 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C24H27NO4: 393.1940; found: 393.1934; IR (KBr)
2955, 1686, 1605, 1573, 1510, 1435, 1369, 1299, 1259,
743, 701, 590cmꢀ1
.
5.30.9. {1-[4-(2-Methoxyethoxy)benzoyl]-2-methyl-1H-
indol-4-yl}acetic acid (3m). 67% yield; TLC Rf =0.44
1
(CHCl3/MeOH, 10/1); H NMR (200MHz, CDCl3) d
7.70 (d, J=8.8Hz, 2H), 7.03–6.93 (m, 5H), 6.48 (s,
1H), 4.21 (t, J=5.0Hz, 2H), 3.85 (s, 2H), 3.79 (t,
J=5.0Hz, 2H), 3.47 (s, 3H), 2.44 (s, 3H); MS (APCI,
Neg, 20V.) m/z 366 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd
for C21H21NO5: 367.1420; found: 367.1425; IR (KBr)
2941, 1687, 1609, 1437, 1373, 1318, 1259, 1216, 1175,
1169cmꢀ1
.
5.30.4. (2-Methyl-1-{4-[(4-methylpentyl)oxy]benzoyl}-
1H-indol-4-yl)acetic acid (3f). 84% yield; TLC Rf =0.43
1
(n-hexane/EtOAc, 1/3); H NMR (200MHz, CDCl3) d
7.70 (d, J=8.6Hz, 2H), 7.07–6.88 (m, 5H), 6.48 (s,
1H), 4.03 (t, J=6.8Hz, 2H), 3.86 (s, 2H), 2.45 (s, 3H),
1.92–1.73 (m, 2H), 1.73–1.52 (m, 1H), 1.43–1.11 (m,
2H), 0.94 (d, J=6.8Hz, 6H); MS (FAB, Pos.) m/z 394
(M+H)+; HRMS (EI, Pos.) calcd for C24H27NO4:
393.1940; found: 393.1950; IR (KBr) 2962, 1704, 1683,
1607, 1577, 1510, 1471, 1436, 1373, 1317, 1260, 1214,
1170, 1114, 1047, 1006, 913, 861, 837, 812, 776, 759,
1123cmꢀ1
.
5.30.10. {1-[4-(3-Methoxypropoxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetic acid (3n). 74% yield; TLC Rf =0.12
1
(EtOAc); H NMR (300MHz, CDCl3) d 7.72–7.69 (m,
2H), 7.08–6.94 (m, 5H), 6.48 (s, 1H), 4.15 (t,
J=6.5Hz, 2H), 3.86 (s, 2H), 3.58 (t, J=6.0Hz, 2H),
3.37 (s, 3H), 2.44 (s, 3H), 2.13–2.05 (m, 2H); MS (APCI,
Neg, 20V.) m/z 380 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C22H23NO5: 381.1576; found: 381.1575; IR (KBr) 2931,
1677, 1608, 1435, 1337, 1253, 1174, 964, 852, 759,
743, 708, 642, 621, 609cmꢀ1
.
5.30.5. {1-[4-(2-Cyclohexylethoxy)benzoyl]-2-methyl-1H-
indol-4-yl}acetic acid (3g). 65% yield; TLC Rf =0.50
(CHCl3/MeOH, 9/1); 1H NMR (200MHz, CDCl3) d
7.70 (d, J=8.8Hz, 2H), 7.06–6.91 (m, 5H), 6.48 (s,
1H), 4.08 (t, J=6.8Hz, 2H), 3.86 (s, 2H), 2.44 (s, 3H),
1.81–0.95 (m, 13H); MS (APCI, Neg, 20V.) m/z 418
(MꢀH)ꢀ; HRMS (EI, Pos.) calcd for C26H29NO4:
419.2097; found: 419.2108; IR (KBr) 3436, 2923, 2851,
1684, 1605, 1574, 1510, 1435, 1370, 1300, 1257, 1222,
622cmꢀ1
.
5.30.11. {1-[4-(3-Ethoxypropoxy)benzoyl]-2-methyl-1H-
indol-4- yl}acetic acid (3p). 87% yield; TLC Rf =0.22
1
(n-hexane/EtOAc, 1/2); H NMR (200MHz, CDCl3) d
7.70 (d, J=9.0Hz, 2H), 7.10–6.86 (m, 5H), 6.49 (s,
1H), 4.16 (t, J=6.4Hz, 2H), 3.86 (s, 2H), 3.62 (t,
J=6.4Hz, 2H), 3.51 (q, J=6.8Hz, 2H), 2.45 (s, 3H),
2.09 (m, 2H), 1.21 (t, J=6.8Hz, 3H); MS (APCI, Neg,
20V.) m/z 394 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C23H25NO5: 395.1733; found: 395.1762; IR (neat)
2974, 2873, 1684, 1604, 1573, 1510, 1434, 1370, 1300,
1258, 1221, 1169, 1116, 959, 911, 838, 761, 641,
1169, 1043, 960, 911, 837, 760, 642cmꢀ1
.
5.30.6. {1-[4-(But-3-en-1-yloxy)benzoyl]-2-methyl-1H-in-
dol-4-yl}acetic acid (3i). 43% yield; TLC Rf =0.28 (EtO-
1
Ac); H NMR (300MHz, CDCl3) d 7.72–7.69 (m, 2H),
7.08–6.94 (m, 5H), 6.49 (s, 1H), 5.98–5.85 (m, 1H),
5.23–5.13 (m, 2H), 4.10 (t, J=6.5Hz, 2H), 3.86 (s,
2H), 2.64–2.56 (m, 2H), 2.45 (s, 3H); MS (APCI, Neg,
20V.) m/z 362 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C22H21NO4: 363.1471; found: 363.1467; IR (KBr)
2928, 1705, 1675, 1603, 1438, 1373, 1300, 1258, 1214,
617cmꢀ1
.
5.30.12. {2-Methyl-1-[4-(2-propoxyethoxy)benzoyl]-1H-
indol-4-yl}acetic acid (3q). 82% yield; TLC Rf =0.20
1
(EtOAc); H NMR (300MHz, CDCl3) d 7.71–7.69 (m,
2H), 7.04–6.92 (m, 5H), 6.48 (s, 1H), 4.23-4.19 (m,
2H), 3.86 (s, 2H), 3.86–3.81 (m, 2H), 3.51 (t,
J=7.0Hz, 2H), 2.44 (s, 3H), 1.65 (tq, J=7.0,7.5Hz,
2H), 0.94 (t, J=7.5Hz, 3H); MS (APCI, Neg, 20V.)
m/z 394 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C23H25NO5: 395.1733; found: 395.1740; IR (KBr)
2965, 1704, 1607, 1512, 1436, 1374, 1322, 1211, 1175,
1174, 911, 757, 639cmꢀ1
.
5.30.7. {1-[4-Benzyloxy)benzoyl]-2-methyl-1H-indol-4-
yl}acetic acid (3j). 56% yield; TLC Rf =0.55 (CHCl3/
1
MeOH, 10/1); H NMR (200MHz, CDCl3) d 7.65–7.33
(d, J=8.8Hz, 2H), 7.45–7.36 (m, 5H), 7.05–6.90 (m,
5H), 6.48 (s, 1H), 5.14 (s, 2H), 3.85 (s, 2H), 2.43 (d,
J=1.0Hz, 3H); MS (APCI, Neg, 20V.) m/z 398
(MꢀH)ꢀ; HRMS (EI, Pos.) calcd for C25H21NO4:
399.1471; found: 399.1494; IR (KBr) 3437, 1698, 1686,
1047, 916, 760, 621cmꢀ1
.
5.30.13. {1-[4-(2-Isopropoxyethoxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetic acid (3r). 50% yield; TLC Rf =0.35
1671, 1602, 1509, 1435, 1372, 1320, 1303cmꢀ1
.
(EtOAc); H NMR (200MHz, CDCl3) d 7.72–7.67 (m,
1
2H), 7.04–6.90 (m, 5H), 6.48 (s, 1H), 4.22–4.17
(m, 2H), 3.86 (s, 2H), 3.84–3.80 (m, 2H), 3.78–3.62
(m, 1H), 2.44 (s, 3H), 1.21 (d, J=6.0Hz, 6H);
MS (APCI, Neg, 20V.) m/z 394 (MꢀH)ꢀ; HRMS
(EI, Pos.) calcd for C23H25NO5: 395.1733; found:
5.30.8. {2-Methyl-1-[4-(3-phenylpropoxy)benzoyl]-1H-in-
dol-4-yl}acetic acid (3l). 86% yield; TLC Rf =0.41
(CHCl3/MeOH, 9/1); 1H NMR (200MHz, CDCl3) d
7.70 (d, J=9.3Hz, 2H), 7.34–7.16 (m, 5H), 7.08–6.88

K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
4699
395.1725; IR (KBr) 2976, 1687, 1604, 1508, 1436, 1372,
1316, 1257, 1212, 1172, 1036, 912, 808, 757, 642cmꢀ1
2H), 2.45 (s, 3H), 1.88–1.74 (m, 2H), 1.80–1.40 (br,
1H), 1.60–1.45 (m, 2H), 1.00 (t, J=7.5Hz, 3H); MS
(APCI, Neg 20V) m/z 364 (MꢀH)ꢀ; HRMS (EI, Pos.)
calcd for C22H23NO4: 365.1627; found: 365.1619; IR
(neat) 3369, 2959, 1682, 1604, 1574, 1511, 1472, 1434,
1369, 1299, 1257, 1222, 1169, 1016, 912, 837, 779,
.
5.30.14. {1-[4-(2-tert-Butoxyethoxy)benzoyl]-2-methyl-
1H-indol-4-yl}acetic acid (3s). 58% yield; TLC Rf =0.50
(CHCl3/MeOH, 9/1); 1H NMR (200MHz, CDCl3) d
7.69 (d, J=8.8Hz, 2H), 7.04–6.94 (m, 5H), 6.48 (s,
1H), 4.17 (t, J=5.2Hz, 2H), 3.86 (s, 2H), 3.76 (t,
J=5.2Hz, 2H), 2.44 (s, 3H), 1.25 (s, 9H); MS (APCI,
Neg, 20V.) m/z 408 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd
for C24H27NO5: 409.1889; found: 409.1891; IR (KBr)
2975, 2927, 1684, 1605, 1574, 1510, 1434, 1368, 1300,
1260, 1222, 1194, 1170, 1099, 961, 912, 779, 761, 641,
641cmꢀ1
.
5.31.2. {2-Methyl-1-[4-(pentyloxy)benzoyl]-1H-indol-4-
yl}acetic acid (3b). 58% yield; TLC Rf =0.64 (CHCl3/
CH3OH, 10/1); 1H NMR (300MHz, CDCl3) d 7.73–
7.66 (m, 2H), 7.07–6.92 (m, 5H), 6.49 (s, 1H), 4.04 (t,
J=6.6Hz, 2H), 3.87 (s, 2H), 2.44 (s, 3H), 1.86–1.23
(m, 6H), 0.95 (t, J=6.9Hz, 3H); MS (APCI, Neg,
20V.) m/z 378 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C23H25NO4: 379.1784; found: 379.1800; IR (KBr)
2945, 1707, 1679, 1607, 1511, 1437, 1374, 1320, 1304,
1261, 1214, 1174, 1049, 1014, 913, 839, 808, 760, 743,
615cmꢀ1
.
5.30.15. (1-{4-[2-(2-Methoxyethoxy)ethoxy]benzoyl}-2-
methyl-1H-indol-4-yl)acetic acid (3t). 75% yield; TLC
Rf =0.20 (EtOAc); 1H NMR (300MHz, CDCl3) d
7.71–7.68 (m, 2H), 7.06–6.91 (m, 5H), 6.48 (s, 1H),
4.24–4.21 (m, 2H), 3.92–3.89 (m, 2H), 3.85 (s, 2H),
3.76–3.73 (m, 2H), 3.61–3.58 (m, 2H), 3.40 (s, 3H),
2.44 (s, 3H); MS (APCI, Neg, 20V.) m/z 410 (MꢀH)ꢀ;
HRMS (EI, Pos.) calcd for C23H25NO6: 411.1682;
found: 411.1679; IR (KBr) 2935, 1681, 1608, 1511,
707, 642cmꢀ1
.
5.31.3. {2-Methyl-1-[4-(3-methylbutoxy)benzoyl]-1H-in-
dol-4-yl}acetic acid (3d). 64% yield; TLC Rf =0.34
1
(CHCl3/CH3OH, 10/1); H NMR (200MHz, CDCl3) d
7.73–7.67 (m, 2H), 7.06–6.91 (m, 5H), 6.48 (s, 1H),
4.07 (t, J=6.6Hz, 2H), 3.86 (s, 2H), 2.45 (s, 3H), 1.84
(m, 1H), 1.72 (m, 2H), 0.98 (d, J=6.4Hz, 6H); MS
(FAB Pos.) m/z 380 (M+H)+; HRMS (EI, Pos.) calcd
for C23H25NO4: 379.1784; found: 379.1799; IR (KBr)
2958, 1704, 1680, 1606, 1438, 1374, 1320, 1304, 1259,
1373, 1319, 912, 759cmꢀ1
.
5.30.16. {2-Methyl-1-[4-(2-phenoxyethoxy)benzoyl]-1H-
indol-4-yl}acetic acid (3u). 70% yield; TLC Rf =0.55
(CHCl3/MeOH, 9/1); 1H NMR (300MHz, CDCl3) d
7.72 (d, J=9.0Hz, 2H), 7.32 (t, J=7.8Hz, 2H), 7.08–
6.92 (m, 8H), 6.49 (s, 1H), 4.46–4.34 (m, 4H), 3.87 (s,
2H), 2.45 (s, 3H); MS (APCI, Neg, 20V.) m/z 428
(MꢀH)ꢀ; HRMS (EI, Pos.) calcd for C26H23NO5:
429.1576; found: 429.1567; IR (neat) 3369, 1684, 1602,
1216, 1174, 913, 760, 641cmꢀ1
.
5.31.4. {2-Methyl-1-[4-(2-phenylethoxy)benzoyl]-1H-in-
dol-4-yl}acetic acid (3k). 58% yield; TLC Rf =0.60
1
(CHCl3/MeOH, 10/1); H NMR (300MHz, CDCl3) d
1497, 1477, 1434, 1370, 1301, 1244, 1170, 912, 756cmꢀ1
.
7.72–7.66 (m, 2H), 7.38–7.23 (m, 5H), 7.07–6.92 (m,
5H), 6.49 (s, 1H), 4.26 (t, J=6.9Hz, 2H), 3.86 (s, 2H),
3.14 (t, J=6.9Hz, 2H), 2.44 (s, 3H); MS (APCI, Neg,
20V.) m/z 412 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for
C26H23NO4: 413.1627; found: 413.1637; IR (KBr)
1684, 1604, 1510, 1434, 1370, 1300, 1258, 1169, 911,
5.30.17. (1-{4-[2-(Ethylthio)ethoxy]benzoyl}-2-methyl-
1H-indol-4-yl)acetic acid (3v). 75% yield; TLC Rf =0.50
1
(CHCl3/MeOH, 10/1); H NMR (200MHz, CDCl3) d
7.71 (d, J=9.0Hz, 2H), 7.05–6.93 (m, 5H), 6.48 (s,
1H), 4.22 (t, J=6.8Hz, 2H), 3.86 (s, 2H), 2.95 (t,
J=6.8Hz, 2H), 2.67 (q, J=7.4Hz, 2H), 2.44 (s, 3H),
1.31 (t, J=7.4Hz, 3H); MS (APCI, Neg, 20V.) m/z
396 (MꢀH)ꢀ; HRMS (EI, Pos.) calcd for C22H23NO4S:
397.1348; found: 397.1321; IR (KBr) 3405, 2928, 1702,
1603, 1509, 1436, 1372, 1320, 1256, 1213, 1174,
758, 701cmꢀ1
.
5.31.5. {1-[4-(2-Ethoxyethoxy)benzoyl]-2-methyl-1H-in-
dol-4-yl}acetic acid (3o). 90% yield; TLC Rf =0.50
1
(CHCl3/CH3OH, 10/1); H NMR (300MHz, CDCl3) d
7.70 (d, J=8.7Hz, 2H), 7.08–6.92 (m, 5H), 6.49 (s,
1H), 4.21 (t, J=4.8Hz, 2H), 3.87–3.81 (m, 4H), 3.62
(q, J=7.5Hz, 2H), 2.45 (s, 3H), 1.26 (t, J=7.5Hz,
3H); MS (APCI, Pos, 20V.) m/z 382 (M+H)+; HRMS
(EI, Pos.) calcd for C22H23NO5: 381.1576; found:
381.1590; IR (KBr) 2979, 1703, 1676, 1607, 1513,
1439, 1374, 1321, 1305, 1264, 1214, 1176, 1127, 1046,
912cmꢀ1
.
5.31. General procedure for the preparation of N-benzoyl-
2-methyl-indole-4-acetic acids (3a, b, k, d, o)
5.31.1. [1-(4-Butoxybenzoyl)-2-methyl-1H-indol-4-yl]ace-
tic acid (3a). To a stirred solution of the compound 34a
(110mg, 239mmol) in EtOAc(4mL) was added 10%
Pd–C (15mg) at room temperature. The resulting sus-
pension was stirred for 2h at room temperature under
hydrogen atmosphere. Insoluble substance was removed
by filtration. The filtrate was concentrated in vacuo to
give a crude product, which was purified by recrystalli-
zation from EtOAc/n-hexane to yield 3a (25mg, 28%);
TLC Rf =0.46 (CHCl3/CH3OH, 9/1); 1H NMR
(300MHz, CDCl3) d 7.70 (d, J=9.0Hz, 2H), 7.08–6.90
(m, 5H), 6.49 (s, 1H), 4.05 (t, J=6.6Hz, 2H), 3.87 (s,
913, 842, 806, 759, 743cmꢀ1
.
5.31.6. (1-{4-[2-(Ethylsulfonyl)ethoxy]benzoyl}-2-methyl-
1H-indol-4-yl)acetic acid (10). To a stirred solution of 3v
(0.050g, 0.126mmol) in CH2Cl2 (5mL) was added
mCPBA (0.0870g, 0.504mmol) at room temperature.
After stirring 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 concentrated in vacuo to give a
residue, which was purified by column chromatography

4700
K. Torisu et al. / Bioorg. Med. Chem. 12 (2004) 4685–4700
on silica gel to yield 10 (0.044g, 81%); TLC Rf =0.40
(CHCl3/CH3OH, 10/1); H NMR (200MHz, CDCl3) d
For the assessment of the antagonist activity of test com-
pounds, a suspension of CHO cells expressing mDP
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 approximately 10min
at 37ꢁC and the culture medium was removed. The as-
say medium (MEM containing 0.1% BSA, 1mM IBMX
and 2lM Diclofenac) was added to each well and the
cells were incubated for approximately 10min at 37ꢁC.
The assay medium, assay medium containing 10 nM
of PGD2, or assay medium containing various concen-
trations of test compounds and 10nM of PGD2 was
added to each well and 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 incubation mixture was frozen at ꢀ80ꢁC until
the assay for cAMP.
1
7.73 (d, J=8.8Hz, 2H), 7.07–6.90 (m, 5H), 6.49 (s,
1H), 4.52 (t, J=5.6Hz, 2H), 3.86 (s, 2H), 3.47 (t,
J=5.6Hz, 2H), 3.20 (q, J=7.4Hz, 2H), 2.44 (s, 3H),
1.48 (t, J=7.4Hz, 3H); MS (APCI, Neg, 20V.) m/z
428 (MꢀH)ꢀ; IR (KBr) 2927, 1681, 1606, 1435, 1317,
1256, 1172, 1127cmꢀ1
.
5.32. Prostanoid mEP, mDP, and hIP receptor binding
assay
Competitive binding studies were conducted using radi-
olabeled ligands and membrane fractions prepared from
Chinese hamster ovary (CHO) cells stably expressing the
respective prostanoid receptors, mEP1, mEP2, mEP3a,
mEP4, mDP, and hIP.
Membranes from CHO cells expressing prostanoid re-
ceptors were incubated with radioligand (2.5nM of
[3H]PGE2 for mEP1-4 or 2.5nM of [3H]PGD2 for
mDP or 5.0nM of [3H]Iloprost for IP) and the test com-
pounds at various concentrations in assay buffer (10mM
KH2PO4–KOH buffer containing 100mM NaCl, pH6.0
for mEP1-4, 25mM HEPES-NaOH buffer containing
1mM EDTA, 5mM MgCl2 and 10mM MnCl2, pH7.4
for mDP, 50mM Tris–HCl buffer containing 1mM
EDTA and 10mM MgCl2, pH7.5, for IP-receptor).
Incubation was carried out at 25ꢁC for 60min except
for mEP1 and mDP (20min), IP (30min). The incuba-
tion was terminated by filtration through Whatman
GF/B filters. The filters were then 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 0.01w/v% BSA,
pH7.4 for mDP, 50mM Tris–HCl buffer containing
1mM EDTA and 10mM MgCl2, pH7.5, for IP-recep-
tor), and the radioactivity on the filter was measured
in 6 ml of liquid scintillation (ACSII) mixture with a liq-
uid scintillation counter. Nonspecific binding was
achieved by adding excess amounts of unlabeled PGE2
(for mEP1-4) or unlabeled PGD2 (for mDP) or unlabe-
led Iloprost (for IP) with assay buffer. The concentra-
tions 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.
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 tri-noctylamine 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 determined by radioimmunoassay using a
cAMP assay kit (Amersham). The relative responsive-
ness (%) 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.
References and notes
1. (a) Coleman, R. A. Prostanoids Receptors. In The IU-
PHAR Compendium of Receptor Characterization and
Classification; Girdlestne, D., Ed.; Burlington: Cambridge,
1998, pp 229–244; (b) Coleman, R. A.; Smith, W. L.;
Narumiya, S. Pharmacol. Rev. 1994, 46, 205–229.
2. Narumiya, S.; Sugimoto, Y.; Ushikubi, F. Physiol. Rev.
1999, 79, 1193–1226.
3. (a) Mitsumori, S.; Tsuri, T.; Honma, T.; Hiramatsu, Y.;
Okada, T.; Hashizume, H.; Inagaki, M.; Arimura, A.;
Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M. J. Med.
Chem. 2003, 46, 2436–2445; (b) Mitsumori, S.; Tsuri, T.;
Honma, T.; Hiramatsu, Y.; Okada, T.; Hashizume, H.;
Inagaki, M.; Arimura, A.; Yasui, K.; Asanuma, F.;
Kishino, J.; Ohtani, M. J. Med. Chem. 2003, 46, 2446–2455.
4. Torisu, K.; Kobayashi, K.; Iwahashi, M.; Egashira, H.;
Nakai, Y.; Okada, Y.; Nanbu, F.; Ohuchida, S.; Nakai, H.;
Toda, M. Bioorg. Med. Chem. Lett., accepted for publica-
tion. DOI:10.1016/j.bmcl.2004.06.006.
Ki ¼ IC50=ð1 þ ½LŠ=KdÞ
[L]: Concentration of radiolabeled ligand; Kd: Dissocia-
tion constant of radiolabeled ligand towards the prosta-
noid receptors.
5. Knoelker, H. J.; Knoell J. Chem. Commun. 2003, 10,
1170–1171.
6. Cacchi, S.; Ciattini, P. G.; Morera, E.; Ortar, G. Tetra-
hedron Lett. 1986, 27, 3931.
5.33. Measurement of the mDP receptor antagonist
activity
7. (a) Lee, V.; Newman, M. S. Org. Synth. 1970, 50, 77; (b)
Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29, 3249.
8. Powell, N. A.; Rychnovsky, S. D. Tetrahedron Lett. 1996,
37, 7901–7904.
9. Kiriyama, M.; Ushikubi, F.; Kobayashi, T.; Hirata, M.;
Sugimoto, Y.; Narumiya, S. Br. J. Pharmacol. 1997, 122,
217–224.
To confirm that test compounds antagonize the mDP re-
ceptor and to estimate potencies of antagonism for the
mDP receptor, a functional assay was performed by
measuring PGD2-stimulated changes in intracellular sec-
ond messenger cAMP (cyclic adenosine 30, 50-mono-
phosphate) as an indicator of receptor function.