Development of a prostaglandin D2 receptor antagonist: Discovery of a new chemical lead

Article abstract of DOI:10.1016/j.ejmech.2004.11.011

A series of N-(p-alkoxy)benzoyl-5-methoxy-2-methylindole-3-acetic acids and N-(p-butoxy)benzoyl-2-methylindole-4-acetic acid were discovered as new chemical leads for a prostaglandin D₂ (PGD₂) receptor antagonist. Most of them exhibi

Full text of DOI:10.1016/j.ejmech.2004.11.011

European Journal of Medicinal Chemistry 40 (2005) 505–519
www.elsevier.com/locate/ejmech
Preliminary communication
Development of a prostaglandin D2 receptor antagonist:
discovery of a new chemical lead
Kazuhiko Torisu a,*, Kaoru Kobayashi a, Maki Iwahashi a, Hiromu Egashira a,Yoshihiko Nakai a,
Yutaka Okada a, Fumio Nanbu a, Shuichi Ohuchida b, Hisao Nakai a, Masaaki Toda a
a Minase Research Institute, Ono Pharmaceutical Co., Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
b Fukui Research Institute, Ono Pharmaceutical Co., Ltd., Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
Received 8 June 2004; received in revised form 24 November 2004; accepted 29 November 2004
Available online 04 February 2005
Abstract
A series of N-(p-alkoxy)benzoyl-5-methoxy-2-methylindole-3-acetic acids and N-(p-butoxy)benzoyl-2-methylindole-4-acetic acid were
discovered as new chemical leads for a prostaglandin D2 (PGD2) receptor antagonist. Most of them exhibited PGD2 receptor binding and
blocked cyclic adenosine 3′,5′-monophosphate (cAMP) formation in vitro. In particular, 2-methylindole-4-acetic acid analog 1 showed mark-
edly increased receptor affinity and cAMP antagonist activity. Chemistry and structure activity relationship (SAR) data are also presented.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Prostaglandin; DP receptor; Antagonist
1. Introduction
PGD2 also promotes the migration of Th2 cells, eosino-
phils, and basophils by binding to the chemoattractant recep-
tor homologous molecule expressed on Th2 cells (CRTH2)
[13,14]. Thus, PGD2 is an endogenous ligand that binds to
both the DP receptor and CRTH2. Accordingly, the discov-
ery of selective DP receptor antagonists [15–18] would offer
a significant opportunity to elucidate the role of this receptor
in various pathologies such as allergic diseases. However, the
role of the DP receptor remains to be clarified because of the
lack of potent and subtype-selective ligands. Here we report
on the discovery of a chemical lead 1 for a new class of DP
receptor antagonists starting from the chemical modification
of Indomethacin analog 2d, which could be an excellent
chemical lead for developing an orally active drug (Fig. 1).
Prostanoids (prostaglandins and thromboxanes) are prod-
ucts of cyclooxygenase derived from arachidonic acid.
Cyclooxygenases metabolize arachidonic acid to five pri-
mary prostanoids, which are PGE2, PGF2a, PGI2, TXA2(TX),
and PGD2. These lipid mediators interact with specific mem-
bers of a family of distinct G-protein-coupled prostanoid
receptors.
Coleman et al. [1,2] proposed the existence of specific
receptors for TX, PGI, PGE, PGF, and PGD that are named
TP, IP, EP, FP, and DP receptors, respectively. They further
classified the EP receptor into four subtypes (EP1, EP2, EP3
and EP4), all of which respond to PGE2 in different ways. A
number of specific ligands for these receptors have already
been described in the literature [3]. Among them, the DP
receptor was the most recent prostanoid receptor to be cloned
and is perhaps the least well characterized [4,5]. PGD2 is the
major prostanoid released from mast cells after challenge with
IgE [6,7] and it has also been shown to affect the sleep cycle
[8–11] and body temperature [12].
2. Chemistry
The synthesis of N-benzoyl-5-methoxy-2-methylindole-3-
acetic acids 2a–j is outlined in Scheme 1. Formation of a
hydrazone of 12 with acetaldehyde in toluene, followed by
N-acylation of another NH moiety with acid chlorides 13a–j,
afforded the N-acyl hydrazones 14a–j, respectively. Acidic
hydrolysis of 14a–j, followed by conventional indole synthe-
* Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: torisu@ono.co.jp (K. Torisu).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.11.011

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K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
Fig. 1. Discovery of a new chemical lead 1.
Scheme 1. Synthesis of 2a–j. Reagents: (a) CH3CHO, toluene; (b) 13a–j, pyridine, CH2Cl2; (c) 4 N HCl, 1,4-dioxane; (d) levulinic acid, AcOH.
sis using levulinic acid, gave the N-benzoyl-5-methoxy-2-
methylindole-3-acetic acids 2a–j, respectively [19–21].
As shown in Scheme 2, compounds 3a–e were prepared
from their corresponding 4-subsituted phenylhydrazines
(15a–e, respectively) according to the same procedures
described above.
Synthesis of 4 and 5 is shown in Scheme 3. Replacement
of the methoxy moiety of 17 with a benzyloxy moiety was
successfully achieved as follows. Demethylation of the
5-methoxy group of 17 with pyridinium chloride at 180 °C,
followed by benzylation of a formed phenol derivative 18 with
benzyl bromide, afforded a benzyl ether 19, N-acylation of
which followed by catalytic hydrogenation gave 4. Com-
pound 5 was prepared from the corresponding indole deriva-
tive 21 according to the N-acylation procedure described
above.
Synthesis of 6–10 is described in Scheme 4. Compounds
6 and 8–10 were prepared according to the usual indole syn-
thesis procedure using N-acyl hydrazine 22 and the corre-
sponding keto carboxylic acids 24 and 25a–c, respectively.
Compound 7 was prepared by deprotection of the allyl ester
23, which was prepared from 22 and 26 by the indole synthe-
sis procedure described above. An a,b-unsaturated carboxy-
lic acid derivative 11 was prepared from 5-methoxy-2-
methylindole 27 as outlined in Scheme 5. Treatment of 27
with dimethylformamide in the presence of phosphorus oxy-
chloride gave 28, C2 homologation of which by the Wittig
reaction resulted in 29. Alkaline hydrolysis of 29 provided
the corresponding carboxylic acid 30, N-acylation of which
with 4-nbutoxybenzoylchloride in the presence of n-BuLi
afforded 11. Synthesis of 2-methylindole-4-acetic acid 1 is
outlined in Scheme 6. Methoxycarbonylation of 32, which
Scheme 2. Synthesis of 3a–e. Reagents: (a) CH3CHO, toluene; (b) 13d, pyridine, CH2Cl2; (c) 4 N HCl, 1,4-dioxane; (d) levulinic acid, AcOH.

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
507
Scheme 3. Synthesis of 4 and 5. Reagents: (a) HCl-pyridine, 180 °C; (b) benzyl bromide, K2CO3, DMF (c) NaH, 13d, DMF; (d) H2, Pd–C, i-PrOH, EtOAc.
Scheme 4. Synthesis of 6–10. Reagents: (a) 24 or 25a–c or 26, AcOH; (b) morpholine, Pd(PPh3)4, THF.
Scheme 5.
Synthesis of 11. Reagents: (a) POCl3, DMF, –78 °C; (b) Br–Ph3P+CHCO2Me, benzene; (c) NaOH, H2O; (d) n-BuLi, 13d, THF.
was prepared from 31 by the usual method, was carried out
by the palladium-catalyzed carbon monoxide insertion reac-
tion in the presence of methanol. Alkaline hydrolysis of 33
[22–25], followed by O-benzylation, provided 35, after which
N-acylation of 35 with an acid chloride 13d and deprotection
with hydrogenolysis afforded a carboxylic acid 37. Com-
pound 37 was converted to 38 by the conventional C-1 ho-
mologation procedure [26], followed by O-benzylation.
Deprotection of 38 with hydrogenolysis gave 1.
beled ligand, [3H]PGD2, to membrane fractions prepared from
cells stably expressing each prostanoid receptor and for their
effect on cyclic adenosine 3′,5′-monophosphate (cAMP) for-
mation evoked by PGD2 in CHO cells [27] in the presence of
bovine serum albumin (BSA). The compounds were also
evaluated binding to all the subtypes of mouse PGE2 (mEP1,
mEP2, mEP3 and mEP4) [3].
In the course of screening our targeted library for lipid
mediators, 2b derived from Indomethacin was found to show
weak binding to the mouse DP (mDP) receptor while In-
domethacin did not show any binding at 10 µM. Based on
this finding, structural optimization was initiated with chemi-
cal modification of the p-alkoxy substituent of the N-benzoyl
residue of 2b. As illustrated in the biological evaluation of
3. Results and discussion
The test compounds listed in Tables 1–5 were biologically
evaluated for inhibition of the specific binding of a radiola-

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K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
Scheme 6
.
Synthesis of 1. Reagents: (a) Tf2O, 2,6-lutidine, CH2Cl2, –78 °C; (b) Pd(PPh3)4, CO, TEA, MeOH, DMF; (c) NaOH, MeOH, 1,4-dioxane; (d) benzyl
bromide, K2CO3, DMF; (e) NaH, 13d, DMF; (f) Pd–C, H2, MeOH, EtOAc; (g) (COCl)2, DMF, toluene; (h) TMSCHN2, THF, CH3CN; (i) 2,4,6-collidine,
benzyl alcohol.
Table 1
Effect of the chain length of the p-substitutent of the N-benzoyl moiety on Ki values
Compound
R
Binding Ki (µM)
IC50 (µM)
mDP
NT
mEP1
>10
mEP2
>10
mEP3
>10
mEP4
>10
mDP
>10
1 Indomethacin
Cl
2a
>10
>10
>10
>10
>10
NT
2b
2c
2d
2e
2f
>10
>10
>10
>10
>10
>10
4.5
>10
4.1
4.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.7
3.4
NT
5.9
1.6
0.77
2.5
1.4
3.0
0.61
0.27
0.19
0.50
0.54
0.28
0.020
1.5
1.9
2g
2h
0.58
1.3
2.3
3.1
Table 2
Table 3
Effect of a 5-substituent on Ki values
Effect of the substitution pattern of the N-benzoyl moiety on Ki values
Compound
R
Binding Ki (µM)
IC50
Compound Position
Binding Ki (µM)
IC50 (µM)
(µM)
mDP
1.6
mEP1 mEP2 mEP3 mEP4 mDP mDP
mEP1 mEP2 mEP3 mEP4 mDP
2i
2j
2d
m-
o-
p-
>10
6.5
1.7
>10
4.7
>10
>10
>10
>10
>10
>10
0.41
>10 NT
0.27 1.6
2.5
2d
3a
3b
3c
3d
3e
4
OMe >10
4.7
>10
>10
>10
4.0
>10
>10
2.2
0.27
0.11
0.15
0.045
0.11
0.33
1.9
H
5.2
5.0
2.1
3.9
>10
Me
1.7
2.9
2a–g (Table 1), the chain length of the p-alkoxy moiety was
optimized at the n-pentyloxy of 2e with regard to both mDP
receptor affinity and antagonist activity, showing increased
mEP2 receptor affinity and mEP4 receptor affinity.
i-Pr 1.1
0.75
2.8
1.1
5.1
F
>10
7.4
>10
>10
>10
>10
2.4
1.6
Cl
0.98
3.3
2.9
OH >10
>10
>10

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
509
Table 4
Effect of transformation of 2-methyl or 3-acetic acid residues on Ki values
Compound
R
Binding Ki (µM)
mEP3
IC50 (µM)
mEP1
>10
mEP2
8.9
mEP4
>10
mDP
2.2
mDP
NT
5
>10
6
7
3.8
2.9
>10
>10
>10
>10
1.5
NT
4.0
>10
>10
0.94
8
>10
>10
>10
>10
5.7
2.7
5.2
2.2
>10
2.4
3.0
1.5
4.2
3.0
0.28
0.19
0.40
1.4
1.5
9
0.91
4.5
10
11
>10
>10
NT
Table 5
tively lower affinity for mEP2 and mEP4 receptors compared
with their mDP receptor.As a result, compound 2d was found
to be most promising as a chemical lead for further chemical
modification because of its activity profile and subtype selec-
tivity.
Activity profiles of N-(p-butoxy)benzoyl-2-methylindole-4-acetic acid (1)
Compound
Binding Ki (µM)
IC50 (µM)
mDP
mEP1 mEP2 mEP3 mEP4 mDP
2d
1
>10
>10
4.7
2.0
>10
3.3
>10
>10
0.27
1.6
0.010
0.30
Replacement of the ethoxy moiety of 2b with an n-pentyl
moiety provided 2h, with a marked increase of mDP receptor
affinity and increased affinity for the mEP1, mEP2 and
mEP4 receptors. The mDP receptor antagonist activity of
2c–2h was also evaluated in the presence of 0.1% of BSA.
When the structure activity relationship (SAR) of the o-
and m- isomers of 2d was also investigated, as shown in
Table 2, m-isomer 2i had slightly lower mDP receptor affin-
As shown in Table 1, the increased mEP2 receptor affinity
of 2e was especially marked. But, subtype selectivity of 2e
remarkably decreased because compound 2e showed nearly
10-fold higher mEP2 receptor affinity relative to its mDP
receptor affinity. Compounds 2c, 2d, 2f and 2g also showed
higher mDP receptor affinity than 2b, while they showed rela-

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K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
Table 6
Pharmacokinetic data for 1 in rats
Dose (mg kg–1
)
AUCinf (µg h ml–1
)
Tmax (h) Cmax (µg ml–1
1.0 1.1
)
CLtot (ml min –1kg–1
7.6
)
T1/2 (h)
1.2
Vss (ml kg–1
)
BA (%)
95
iv
1
1
2.1
2.0
665
po
1.5
ity than p-isomer 2d with subtype selectivity close to that of
p-isomer 2d. The o-isomer 2j did not show any mDP recep-
tor affinity at 10 µM, while it showed weak affinity for the
mEP1 receptor. Thus, the p-isomer was found to be the best
of the three isomers.
As shown in Table 3, the effect of a 5-substituent on the
indole ring was investigated. Replacement of the 5-methoxy
moiety of 2d with hydrogen afforded 3a, which had increased
affinity for the mEP1, mEP2 and mDP receptors, while 3a
showed weaker mDP receptor antagonist activity.
moiety afforded 8–10, respectively, which showed nearly
equal affinity, slightly stronger affinity and slightly weaker
affinity for the mDP receptor, respectively, while 8–10 were
nearly equipotent, slightly stronger, and nearly threefold
weaker with respect to antagonist activity. The subtype selec-
tivity of 8–10 was reduced relative to that of 2d. Compounds
8 and 10 showed weak affinity for the mEP2 and mEP4 recep-
tors, while 9 showed weak affinity for the mEP2, mEP3 and
mEP4 receptors.
The a,b-unsaturated acid derivative 11 showed nearly five-
fold lower affinity for the mDP receptor relative to 2d, while
it showed nearly twofold higher affinity for the mEP2 recep-
tor and also showed weak affinity for the mEP4 receptor.
An acidic function such as a carboxylic acid could be con-
sidered to play an important role in the binding of these ligands
to PG receptors because of the similarity of these Indometha-
cin analogs to prostanoid structures consisting of an acidic
a-chain and hydrophobic x-chain. Based on this consider-
ation, transfer of the 3-acetic acid moiety of the
2-methylindole-3-acetic acids was carried out to detect its
optimized position.
A breakthrough was achieved by structural transforma-
tion of the 2-methylindole-3-acetic acid template to a
2-methylindole-4-acetic acid template. As described in
Table 5, N-(p-butoxy)benzoyl-2-methylindole-4-acetic acid
(1) demonstrated markedly increased mDP receptor affinity
and antagonist activity. Compound 1 demonstrated 27-fold
stronger mDP receptor affinity and nearly fivefold more potent
antagonist activity relative to that of 2d. These compounds
were also evaluated for their TP receptor affinity, because
PGD2 has been known to be a TP agonist. All of the com-
pounds listed in Tables 1–5 showed less than 1000-fold potent
affinity to TP receptor.
The result of pharmacokinetic (PK) study of compound 1
was shown in Table 6 as a representative example. When 1
was administered orally to male rats at a dose of 10 mg kg–1
under fasting condition. Compound 1 had a good PK profile
in rats (Table 6). Administration of this compound to rats
(1 mg kg–1, iv; 1 mg kg–1, po; n = 3) led to detectable plasma
levels, with a T1/2 of 1.2 h (iv) and 1.5 h (po). The volume of
distribution (Vss) was calculated to be 665 ml kg–1, indicat-
ing distribution to the tissues. Systemic clearance (CL) was
7.6 ml min–1 kg–1. The peak plasma level (Cmax) was
1.1 µg ml–1 at 1.0 h, and the oral bioavailability (BA) was
95% at a dose of 1 mg kg–1. The AUC was 2.1 µg h ml–1 (iv)
and 2.0 µg h ml–1 (po).
Replacement of the methoxy moiety of 2d with a methyl
moiety gave 3b, with increased affinity for the mEP1, mEP2,
mEP4 and mDP receptors, but weaker antagonist activity.
Replacement of the methoxy moiety of 2d with an
i-propyloxy moiety provided 3c, which had increased affin-
ity for the mEP1, mEP2, mEP3, mEP4 and mDP receptors,
but showed weaker antagonist activity than 2d despite its
higher mDP receptor affinity.
Replacement of the methoxy moiety of 2d with fluoro and
chloro moieties afforded 3d and 3e, respectively, with
increased affinity and slightly decreased affinity for the mDP
receptor, respectively. In addition, 3d and 3e were equipo-
tency and nearly twofold less potency relative to 2d, respec-
tively, with regard to mDP antagonist activity. Compound 3d
had a subtype selectivity analogous to that of 2d, and showed
weak affinity for the mEP2 receptor. Compound 3e also
showed weak affinity for the mEP1 and mEP4 receptors.
Demethylation of the methoxy moiety of 2d afforded a
hydroxy derivative 4, with lower affinity and slightly increased
affinity for the mDP receptor and mEP2 receptor, respec-
tively, while it showed no antagonist activity at 10 µM.
Thus, mDP receptor affinity was optimized by the
5-ipropyloxy derivative 3c, but subtype selectivity was much
lower than that of 2d. The mDP receptor antagonist activity
and subtype selectivity of 2d and 3d were greater than those
of the other derivatives listed in Table 3.
Further modification of the 5-methoxy-2-methylindole-3-
acetic acid skeleton was performed.
As shown in Table 4, removal of the 2-methyl moiety of
2d afforded 5, with nearly 10-fold and nearly twofold lower
affinity for the mDP and mEP2 receptors, respectively.
Replacement of the 2-methyl moiety of 2d with an ethyl
moiety gave 6, which had lower affinity and slightly higher
affinity for the mDP receptor and mEP2 receptor, respec-
tively, while 6 showed low affinity for the mEP1 receptor.
Replacement of the 3-acetic acid moiety of 2d with a car-
boxy moiety gave 7, with nearly fourfold lower affinity for
the mDP receptor and threefold less potent antagonist activ-
ity. Replacement of the 3-acetic acid moiety of 2d with a pro-
pionic acid moiety, butanoic acid moiety, and pentanoic acid
4. Conclusion
In summary, structural optimization of an mDP receptor
antagonist was initiated by chemical modification of 2b

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
511
derived from indomethacin, which is a well-known orally
active drug. Optimization of the N-benzoyl moiety, 5-methoxy
moiety, 2-methyl moiety, and 3-acetic acid moiety was car-
ried out. As a result, N-(p-butoxy)benzoyl-5-methoxy-2-
methylindole-3-acetic acid 2d was found to be the best among
this series of compounds with regard to mDP receptor affin-
ity and antagonist activity. Synthesis and evaluation of N-(p-
butoxy)benzoyl-2-methylindole-4-acetic acid (1) provided a
breakthrough for further optimization. These findings strongly
suggested that the discovery of a new class of more potent
and orally active DP receptor antagonists could be achieved
by continuing this approach. Further optimization of 1 will
be reported in the following paper.
After stirring for 2 h at room temperature, the reaction mix-
ture was concentrated in vacuo to give an oily residue, which
was purified by column chromatography on silica gel to yield
14a (720 mg, 79% yield) as an oil. TLC Rf = 0.42 (n-
hexane/EtOAc, 1:1); 1H NMR (300 MHz, CDCl3) d 7.79 (d,
J = 9.0 Hz, 2H), 7.10 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz,
2H), 6.89 (d, J = 9.0 Hz, 2H), 6.82 (q, J = 5.1 Hz, 1H), 3.85
(s, 3H), 3.84 (s, 3H), 1.91 (d, J = 5.1 Hz, 3H).
5.2.2. 4-Ethoxy-N′-[(1E)-ethylidene]-N-(4-methoxyphenyl)-
benzohydrazide (14b)
81% yield; TLC Rf = 0.50 (n-hexane/EtOAc, 1:1); 1H NMR
(300 MHz, CDCl3) d 7.78 (d, J = 8.7 Hz, 2H), 7.10 (d,
J = 8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz,
2H), 6.82 (q, J = 5.1 Hz, 1H), 4.08 (q, J = 7.2 Hz, 2H), 3.84
(s, 3H), 1.91 (d, J = 5.1 Hz, 3H), 1.43 (t, J = 7.2 Hz, 3H).
5. Experimental
5.2.3. N′-[(1E)-Ethylidene]-N-(4-methoxyphenyl)-4-pro-
poxybenzohydrazide (14c)
5.1. General directions
74% yield; TLC Rf = 0.17 (n-hexane/EtOAc, 3:1); 1H NMR
(200 MHz, CDCl3) d 7.77 (d, J = 9.0 Hz, 2H), 7.10 (d,
J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz,
2H), 6.81 (q, J = 5.5 Hz, 1H), 3.96 (t, J = 6.6 Hz, 2H), 3.83 (s,
3H), 1.90 (d, J = 5.5 Hz, 3H), 1.89–1.72 (m, 2H), 1.04 (t,
J = 7.3 Hz, 3H).
Analytical samples were homogeneous as confirmed by
TLC, and afforded spectroscopic results consistent with the
assigned structures. Proton nuclear magnetic resonance spec-
tra (1H NMR) were taken on aVarian Mercury 300 spectrom-
eter orVarian GEMINI-200 orVXR-200s spectrometer using
deuterated chloroform (CDCl3) or deuterated methanol
(CD3OD) or deuterated dimethylsulfoxide (DMSO-d6) as the
solvent. Fast atom bombardment mass spectra (FAB-MS), fast
atom bombardment high-resolution mass spectra (FAB-
HRMS) and electron ionization mass spectra (EI-MS, HRMS)
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 PerSeptiveVoyager Elite spectrometer.Atmospheric pres-
sure chemical ionization mass spectra (APCI-MS) were
obtained on a HITACHI M1200H spectrometer. Infrared (IR)
spectra were measured on a Perkin–Elmer FT-IR 1760X spec-
trometer. Melting points and results of elemental analyses
were uncorrected. Column chromatography was carried out
on silica gel [Merck silica gel 60 (0.063–0.200 mm), Wako
gel C200 or Fuji Silysia BW235]. Thin layer chromatogra-
phy 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), dichlo-
romethane (CH2Cl2), chloroform (CHCl3), methanol
(MeOH), acetic acid (AcOH), triethylamine (TEA).
5.2.4. 4-Butoxy-N′-[(1E)-ethylidene]-N-(4-methoxyphenyl)-
benzohydrazide (14d)
73% yield; TLC Rf = 0.20 (n-hexane/EtOAc, 3:1); 1H NMR
(200 MHz, CDCl3) d 7.77 (d, J = 9.0 Hz, 2H), 7.10 (d,
J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz,
2H), 6.81 (q, J = 5.5 Hz, 1H), 4.05 (t, J = 7.0 Hz, 2H), 3.83 (s,
3H), 1.90 (d, J = 5.3 Hz, 3H), 1.88–1.74 (m, 2H), 1.60–1.43
(m, 2H), 1.00 (t, J = 7.5 Hz, 3H).
5.2.5. N′-[(1E)-Ethylidene]-N-(4-methoxyphenyl)-4-(penty-
loxy)benzohydrazide (14e)
73% yield; TLC Rf = 0.28 (n-hexane/EtOAc, 7:3); 1H NMR
(200 MHz, CDCl3) d 7.77 (d, J = 8.4 Hz, 2H), 7.10 (d,
J = 8.4 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz,
2H), 6.81 (q, J = 4.8 Hz, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.84 (s,
3H), 1.90 (d, J = 4.8 Hz, 3H), 1.86–1.74 (m, 2H), 1.52–1.30
(m, 4H), 0.93 (t, J = 6.6 Hz, 3H).
5.2.6. N′-[(1E)-Ethylidene]-4-(hexyloxy)-N-(4-methoxy-
phenyl)benzohydrazide (14f)
92% yield; TLC Rf = 0.32 (n-hexane/EtOAc, 2:1); 1H NMR
(200 MHz, CDCl3) d 7.77 (d, J = 8.8 Hz, 2H), 7.10 (d,
J = 9.2 Hz, 2H), 7.01 (d, J = 9.2 Hz, 2H), 6.86 (d, J = 8.8 Hz,
2H), 6.81 (q, J = 5.4 Hz, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.83 (s,
3H), 1.90 (d, J = 5.4 Hz, 3H), 1.79 (m, 2H), 1.50–1.29 (m,
6H), 0.91 (t, J = 6.6 Hz, 3H).
5.2. General procedure for the preparation
of N-benzoyl-2-methyl-5-methoxyindole-3- acetic acids
(2a–j)
5.2.1. 4-Methoxy-N′-[(1E)-ethylidene]-N-(4-methoxyphe-
nyl)benzohydrazide (14a)
To a stirred solution of 12 (500 mg, 3.04 mmol) and pyri-
dine (0.37 ml, 4.57 mmol) in CH2Cl2 (4 ml) was added drop-
wise p-methoxybenzoyl chloride (519 mg, 3.04 mmol) at 0 °C.
5.2.7. N′-[(1E)-Ethylidene]-4-(heptyloxy)-N-(4-methoxy-
phenyl)benzohydrazide (14g)
82% yield; TLC Rf = 0.32 (n-hexane/EtOAc, 2:1); 1H NMR
(200 MHz, CDCl3) d 7.77 (d, J = 9.0 Hz, 2H), 7.10 (d,

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K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.86 (d, J = 9.0 Hz,
2H), 6.81 (q, J = 5.4 Hz, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.83 (s,
3H), 1.90 (d, J = 5.4 Hz, 3H), 1.79 (m, 2H), 1.50–1.25 (m,
8H), 0.90 (t, J = 6.6 Hz, 3H).
(d, J = 9.0 Hz, 2H), 6.98–6.90 (m, 3H), 6.88 (d, J = 9.0 Hz,
1H), 6.65 (dd, J = 2.4, 9.0 Hz, 1H), 4.12 (q, J = 6.9 Hz, 2H),
3.83 (s, 3H), 3.70 (s, 2H), 2.41 (s, 3H), 1.46 (t, J = 6.9 Hz,
3H); MS (APCI, Neg, 40 V) m/z 366 (M – H)–, 322 (M–
CO2H)–; HRMS (EI, Pos) Calc. for C21H21NO5: 367.1420;
found: 367.1435; IR (neat) 2932, 1713, 1682, 1604, 1509,
1478, 1357, 1315, 1256, 1173, 1147, 1068, 1039, 921, 842,
763, 733 cm–1.
5.2.8. N′-[(1E)-Ethylidene]-N-(4-methoxyphenyl)-4-pentyl-
benzohydrazide (14h)
80% yield; TLC Rf = 0.25 (n-hexane/EtOAc, 3:1); 1H NMR
(200 MHz, CDCl3) d 7.60 (d, J = 9.0 Hz, 2H), 7.27 (d,
J = 9.0 Hz, 2H), 6.92 (d, J = 9.0 Hz, 1H), 6.86 (d, J = 9.0 Hz,
1H), 6.62 (m, 1H), 3.79 (s, 3H), 3.65 (s, 2H), 2.66 (t, 2H),
2.38 (s, 3H), 1.63 (m, 2H), 1.32 (m, 4H), 0.89 (t, 3H).
5.2.13. [5-Methoxy-2-methyl-1-(4-propoxybenzoyl)-1H-
indol-3-yl]acetic acid (2c)
63% yield; TLC Rf = 0.42 (CHCl3/CH3OH, 9:1); 1H NMR
(200 MHz, CDCl3) d 7.69 (d, J = 9.0 Hz, 2H), 6.98–6.91 (m,
3H), 6.88 (d, J = 9.0 Hz, 1H), 6.65 (dd, J = 9.0, 2.4 Hz, 1H),
4.00 (t, J = 6.4 Hz, 2H), 3.82 (s, 3H), 3.70 (s, 2H), 2.40 (s,
3H), 1.92–1.78 (m, 2H), 1.06 (t, J = 7.3 Hz, 3H); MS (APCI,
Neg, 20 V) m/z 380 (M – H)–; HRMS (EI, Pos) Calc. for
C22H23NO5: 381.1576; found: 381.1581; IR (KBr) 3700–
2800, 1698, 1605, 1513, 1473, 1380, 1322, 1220, 1181, 1154,
1079, 1039, 1008, 929, 846, 787, 763, 706, 613 cm–1.
5.2.9. 3-Butoxy-N′-[(1E)-ethylidene]-N-(4-methoxyphenyl)-
benzohydrazide (14i)
96% yield; TLC Rf = 0.19 (n-hexane/EtOAc, 3:1); 1H NMR
(200 MHz, CDCl3) d 7.45–7.05 (m, 6H), 7.00 (d, J = 9.0 Hz,
2H), 6.81 (q, J = 5.4 Hz, 1H), 3.95 (t, J = 7.0 Hz, 2H), 3.82 (s,
3H), 1.91 (d, J = 5.4 Hz, 3H), 1.83–1.40 (m, 4H), 0.95 (t,
J = 7.4 Hz, 3H).
5.2.14. [1-(4-Butoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acid (2d)
5.2.10. 2-Butoxy-N′-[(1E)-ethylidene]-N-(4-methoxyphe-
nyl)benzohydrazide (14j)
14% yield; TLC Rf = 0.43 (n-hexane/AcOEt, 3:1); 1H NMR
(200 MHz, CDCl3) d 7.62–7.48 (m, 2H), 7.26–7.12 (m, 2H),
7.10 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.81 (q,
J = 5.4 Hz, 1H), 3.82 (s, 3H), 3.72 (t, J = 6.2 Hz, 2H), 1.90 (d,
J = 5.4 Hz, 3H), 1.40–1.18 (m, 2H), 1.10–0.90 (m, 2H), 0.65
(t, J = 7.4 Hz, 3H).
58% yield; m.p. 131–132 °C; TLC Rf = 0.49
(CHCl3/CH3OH, 10:1); 1H NMR (200 MHz, CDCl3) d 7.72–
7.65 (m, 2H), 6.97–6.87 (m, 4H), 6.65 (dd, J = 9.0, 2.4 Hz,
1H), 4.04 (t, J = 7.0 Hz, 2H), 3.82 (s, 3H), 3.70 (s, 2H), 2.40
(s, 3H), 1.88–1.74 (m, 2H), 1.61–1.43 (m, 2H), 1.00 (t,
J = 7.4 Hz, 3H); MS (APCI, Neg, 20 V) m/z 394 (M – H)–;
HRMS (EI, Pos) Calc. for C23H25NO5: 395.1733; found:
395.1811; IR (KBr) 2958, 1703, 1605, 1513, 1471, 1359,
1320, 1261, 1220, 1180, 1153, 1079, 1039, 929, 851, 786,
763, 614 cm–1.
5.2.11. [1-(4-Methoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acid (2a)
To a stirred solution of 14a (720 mg, 2.41 mmol) in tolu-
ene (7 ml) and CH3OH (7 ml) was added 4 M HCl dioxane
(3.5 ml). After stirring for 40 min at room temperature, the
reaction mixture was concentrated in vacuo to give a hydrazide
, which was used for the next reaction without further purifi-
cation. To a stirred solution of the above-described hydrazide
(2.41 mmol) in AcOH (25 ml) was added levulinic acid
(0.30 ml, 2.89 mmol) at room temperature. After stirring for
3 h at 80 °C, the reaction mixture was concentrated in vacuo
to give a crude solid, which was purified by column chroma-
tography on silica gel to yield 2a (403 mg, 47% yield) as a
white powder; m.p. 154–156 °C, TLC Rf = 0.50
5.2.15. {5-Methoxy-2-methyl-1-[4-(pentyloxy)benzoyl]-1H-
indol-3-yl}acetic acid (2e)
52% yield; m.p. 117–119 °C; TLC Rf = 0.56
1
(CHCl3/CH3OH, 9:1); H NMR (300 MHz, CDCl3) d 7.69
(d, J = 9.0 Hz, 2H), 6.97–6.91 (m, 3H), 6.89 (d, J = 9.0 Hz,
1H), 6.66 (dd, J = 2.4, 9.0 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H),
3.83 (s, 3H), 3.70 (s, 2H), 2.41 (s, 3H), 1.90–1.76 (m, 2H),
1.54–1.33 (m, 4H), 0.95 (t, J = 6.6 Hz, 3H); MS (APCI, Neg,
20 V) m/z 408 (M – H)–; HRMS (EI, Pos) Calc. for
C24H27NO5: 409.1889; found: 409.1893; IR (neat) 2933,
1710, 1682, 1604, 1510, 1478, 1357, 1314, 1257, 1172, 1147,
1068, 1037, 927, 840, 763, 734, 671, 637 cm–1.
1
(CHCl3/CH3OH, 9:1); H NMR (300 MHz, CDCl3) d 7.71
(d, J = 9.0 Hz, 2H), 6.99–6.92 (m, 3H), 6.88 (d, J = 9.0 Hz,
1H), 6.66 (dd, J = 2.7, 9.0 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H),
3.71 (s, 2H), 2.40 (s, 3H); MS (APCI, Neg, 40 V) m/z 352 (M
– H)–, 308 (M-CO2H)–; HRMS (MALDI-TOF, Pos) Calc.
for C20H19NO5 + H+: 354.1342; found: 354.1341; IR (neat)
2932, 1682, 1604, 1511, 1478, 1357, 1315, 1258, 1173, 1147,
1068, 1030, 913, 840, 763, 733 cm–1.
5.2.16. {1-[4-(Hexyloxy)benzoyl]-5-methoxy-2-methyl-1H-
indol-3-yl}acetic acid (2f)
69% yield; TLC Rf = 0.40 (CHCl3/CH3OH, 10:1); 1H NMR
(300 MHz, CDCl3) d 7.69 (d, J = 8.6 Hz, 2H), 6.96–6.87 (m,
4H), 6.65 (dd, J = 9.0, 3.0 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H),
3.82 (s, 3H), 3.70 (s, 2H), 2.40 (s, 3H), 1.89–1.75 (m, 2H),
1.56–1.30 (m, 6H), 0.95–0.88 (m, 3H); MS (APCI, Neg, 20V)
m/z 422 (M – H)–; HRMS (MALDI-TOF, Pos) Calc. for
C25H29NO5 + H+: 424.2125; found: 424.2124; IR (KBr) 2931,
1684, 1606, 1479, 1359, 1312, 1254, 1149, 1069 cm–1.
5.2.12. [1-(4-Ethoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acid (2b)
69% yield; m.p. 142–143 °C; TLC Rf = 0.50
1
(CHCl3/CH3OH, 9:1); H NMR (300 MHz, CDCl3) d 7.69

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
513
5.2.17. {1-[4-(Heptyloxy)benzoyl]-5-methoxy-2-methyl-1H-
indol-3-yl}acetic acid (2g)
trated in vacuo to give a residue, which was used for the next
reaction without further purification. To a stirred solution of
the above-described product (425 mg, 2.87 mmol) in pyri-
dine (0.35 ml, 4.31 mmol) and CH2Cl2 (2 ml) at 0 °C was
added dropwise a solution of p-butoxybenzoyl chloride
(0.54 ml, 2.87 mmol) in CH2Cl2 (2 ml). After stirring for 2 h
at room temperature, the reaction mixture was concentrated
in vacuo to give a crude oil, which was purified by column
chromatography on silica gel to yield 16b (480 mg, 52% yield)
65% yield; m.p. 93–95 °C; TLC Rf = 0.42 (CHCl3/CH3OH,
1
10:1); H NMR (300 MHz, CDCl3) d 7.69 (d, J = 8.6 Hz,
2H), 6.96–6.87 (m, 4H), 6.65 (dd, J = 9.0, 3.0 Hz, 1H), 4.04
(t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.70 (s, 2H), 2.40 (s, 3H),
1.89–1.75 (m, 2H), 1.56–1.24 (m, 8H), 0.93–0.87 (m, 3H);
MS (APCI, Neg, 20 V) m/z 436 (M – H)–; HRMS (EI, Pos)
Calc. for C26H31NO5: 437.2202; found: 437.2179; IR (KBr)
2931, 1699, 1671, 1608, 1513, 1463, 1355, 1321, 1262, 1225,
1178, 1153, 1073, 1038, 915, 846, 793, 763, 636 cm–1.
1
as an oil; TLC Rf = 0.38 (n-hexane/EtOAc, 7:3); H NMR
(300 MHz, CDCl3) d 7.78 (d, J = 9.0 Hz, 2H), 7.29 (d,
J = 8.1 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 6.87 (d, J = 9.0 Hz,
2H), 6.80 (q, J = 5.4 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H), 2.39 (s,
3H), 1.90 (d, J = 5.4 Hz, 3H), 1.85–1.72 (m, 2H), 1.58–1.40
(m, 2H), 0.98 (t, J = 7.5 Hz, 3H).
5.2.18. [5-Methoxy-2-methyl-1-(4-pentylbenzoyl)-1H-
indol-3-yl]acetic acid (2h)
80% yield; m.p. 119–120 °C; TLC Rf = 0.30
1
(MeOH/CHCl3, 1:20); H NMR (200 MHz, CDCl3) d 7.60
(d, J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 6.92 (d,
J = 9.0 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 6.62 (m, 1H), 3.79
(s, 3H), 3.65 (s, 2H), 2.66 (t, 2H), 2.38 (s, 3H), 1.63 (m, 2H),
1.32 (m, 4H), 0.89 (t, 3H); MS (APCI, Neg, 20 V) m/z 392
(M – H)–; HRMS (EI, Pos) Calc. for C24H27NO4: 393.1940;
found: 393.1918.
5.3.2. 4-Butoxy-N′-[(1E)-ethylidene]-N-phenylbenzohy-
drazide (16a)
79% yield; TLC Rf = 0.43 (n-hexane/EtOAc, 2:1); 1H NMR
(200 MHz, CDCl3) d 7.82–7.77 (m, 2H), 7.54–7.36 (m, 3H),
7.22–7.17 (m, 2H), 6.90–6.86 (m, 2H), 6.79 (q, J = 5.0 Hz,
1H), 4.01 (t, J = 6.2 Hz, 2H), 1.91 (d, J = 5.0 Hz, 3H), 1.85–
1.71 (m, 2H), 1.55–1.41 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H).
5.2.19. [1-(3-Butoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acid (2i)
5.3.3. 4-Butoxy-N′-[(1E)-ethylidene]-N-(4-isopropyl-
phenyl)benzohydrazide (16c)
48% yield; m.p. 133–134 °C; TLC Rf = 0.40
(CHCl3/CH3OH, 10:1); 1H NMR (200 MHz, CDCl3) d 7.40–
7.10 (m, 4H), 6.90 (m, 2H), 6.65 (m, 1H), 3.97 (t, J = 7.0 Hz,
2H), 3.82 (s, 3H), 3.70 (s, 2H), 2.39 (s, 3H), 1.82–1.65 (m,
2H), 1.60–1.40 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H); MS (EI,
Pos) m/z 395 (M)+; HRMS (EI, Pos) Calc. for C23H25NO5:
395.1733; found: 395.1752; IR (KBr) 2941, 1698, 1667, 1600,
1471, 1439, 1362, 1332, 1234, 1147, 1037 cm–1.
60% yield; TLC Rf = 0.53 (n-hexane/EtOAc, 2:1); 1H NMR
(200 MHz, CDCl3) d 7.79 (d, J = 8.8 Hz, 2H), 7.35 (d,
J = 8.0 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.0 Hz,
2H), 6.80 (q, J = 5.4 Hz, 1H), 4.01 (t, J = 6.6 Hz, 2H), 2.95
(septet, J = 6.8 Hz, 1H), 1.90 (d, J = 5.4 Hz, 3H), 1.79 (m,
2H), 1.49 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H).
5.3.4. 4-Ethoxy-N′-[(1E)-ethylidene]-N-(4-fluorophenyl)-
benzohydrazide (16d)
5.2.20. [1-(2-Butoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acid (2j)
82% yield; TLC Rf = 0.39 (n-hexane/EtOAc, 3:1); 1H NMR
(200 MHz, CDCl3) d 7.80 (d, J = 8.7 Hz, 2H), 6.79 (q,
J = 5.1 Hz, 1H), 4.01 (t, J = 6.6 Hz, 2H), 1.91 (d, J = 5.1 Hz,
3H), 1.85–1.72 (m, 2H), 1.58–1.44 (m, 2H), 0.98 (t,
J = 7.5 Hz, 3H).
16% yield; TLC Rf = 0.38 (CHCl3/CH3OH, 9:1); 1H NMR
(300 MHz, CDCl3) d 7.54–7.42 (m, 2H), 7.18–7.01 (m, 2H),
6.95–6.88 (m, 2H), 6.64 (dd, J = 9.0, 2.8 Hz, 1H), 3.81 (s,
3H), 3.77 (t, J = 6.2 Hz, 2H), 3.65 (s, 2H), 2.30 (s, 3H), 1.39–
1.23 (m, 2H), 1.11–0.90 (m, 2H), 0.64 (t, J = 7.4 Hz, 3H);
MS (APCI, Neg, 20 V) m/z 394 (M – H)–; HRMS (EI, Pos)
Calc. for C23H25NO5: 395.1733; found: 395.1811; IR (neat)
2958, 2933, 2872, 1710, 1681, 1601, 1478, 1455, 1361, 1325,
1223, 1150, 1067, 1038, 914, 756 cm–1.
5.3.5. 4-Butoxy-N-(4-chlorophenyl)-N′-[(1E)-ethylidene]-
benzohydrazide (16e)
16% yield; TLC Rf = 0.47 (n-hexane/EtOAc, 2:1); 1H NMR
(200 MHz, CDCl3) d 7.82–7.77 (m, 2H), 7.50–7.46 (m, 2H),
7.16–7.12 (m, 2H), 6.91–6.87 (m, 2H), 6.85–6.76 (m, 1H),
4.01 (t, J = 6.0 Hz, 1H), 1.92 (d, J = 5.0 Hz, 3H), 1.86–1.72
(m, 2H), 1.6–1.4 (m, 2H), 0.98 (t, J = 7.0 Hz, 3H).
5.3. General procedure for the preparation of N-benzoyl-
2-methyl-5-substituted indole-3-acetic acids (3a–e)
5.3.1. 4-Butoxy-N′-[(1E)-ethylidene]-N-(4-methylphenyl)-
benzohydrazide (16b)
5.3.6. [1-(4-Butoxybenzoyl)-2,5-dimethyl-1H-indol-3-yl]-
acetic acid (3b)
To a stirred solution of p-tolylhydrazine 15b (3.51 g,
28.7 mmol) in toluene (100 ml) was added acetaldehyde
(1.6 ml, 28.7 mmol) at room temperature. After stirring for
2 h at room temperature, the reaction mixture was concen-
To a stirred solution of 16b (480 mg, 1.48 mmol) in tolu-
ene (5 ml) and CH3OH (5 ml) was added 4 M HCl dioxane
(3 ml).After stirring for 40 min at room temperature, the reac-
tion mixture was concentrated in vacuo to give a hydrazide,

514
K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
which was used for the next reaction without further purifi-
cation. To a stirred solution of the above-described hydrazide
(1.48 mmol) in AcOH (10 ml) was added levulinic acid
(0.30 ml, 2.96 mmol) at room temperature. After stirring for
3 h at 80 °C, the reaction mixture was concentrated in vacuo
to give a crude solid, which was purified by column chroma-
tography on silica gel to yield 3b (153 mg, 27% yield) as a
white powder; m.p. 132–134 °C; TLC Rf = 0.57
5.3.10. [1-(4-Butoxybenzoyl)-5-chloro-2-methyl-1H-indol-
3-yl]acetic acid (3e)
1% yield; m.p. 165–166 °C; TLC Rf = 0.32 (AcOEt); 1H
NMR (300 MHz, CDCl3) d 7.71–7.67 (m, 2H), 7.47 (m, 1H),
7.02–6.90 (m, 4H), 4.05 (t, J = 6.5 Hz, 2H), 3.71 (s, 2H), 2.41
(s, 3H), 1.86–1.77 (m, 2H), 1.58–1.46 (m, 2H), 1.00 (t,
J = 6.0 Hz, 3H); MS (APCI, Neg, 20 V) m/z 398 (M – H)–;
HRMS (MALDI-TOF, Pos) Calc. for C22H22ClNO4 + H+:
400.1316; found: 400.1316; IR (neat) 3470, 1706, 1684, 1604,
1510, 1460, 1418, 1371, 1349, 1311, 1260, 1239, 1198, 1176,
1065, 926, 884, 859, 844, 800, 763, 710, 655 cm–1.
1
(CHCl3/CH3OH, 9:1); H NMR (300 MHz, CDCl3) d 7.70
(d, J = 9.0 Hz, 2H), 7.30–7.25 (m, 1H), 6.94 (d, J = 9.0 Hz,
2H), 6.85 (s, 2H), 4.05 (t, J = 6.3 Hz, 2H), 3.71 (s, 2H), 2.41
(s, 3H), 2.40 (s, 3H), 1.87–1.75 (m, 2H), 1.59–1.45 (m, 2H),
1.00 (t, J = 7.5 Hz, 3H); MS (APCI, Neg, 20 V) m/z 378 (M –
H)–; HRMS (MALDI-TOF, Pos) Calc. for C23H25NO4 + H+:
380.1863; found: 380.1862; IR (neat) 2960, 2932, 2873, 1710,
1683, 1604, 1576, 1510, 1467, 1421, 1395, 1349, 1311, 1257,
1231, 1172, 1068, 1027, 970, 926, 840, 801, 763, 734, 670,
646, 592 cm–1.
5.4. (5-Hydroxy-2-methyl-1H-indol-3-yl)acetic acid (18)
A mixture of 17 (5 g, 26.4 mmol) and pyridinium hydro-
chloride (50 g) was heated for 3 h at 190 °C. After cooling to
room temperature, the reaction mixture was poured into water
and extracted with EtOAc (×3). The combined organic layers
were washed with 1 M HClaq, water, brine, dried over Na2SO4
and evaporated to yield 18 (2.32 g, 43% yield), which was
used for the next reaction without further purification; TLC
Rf = 0.10 (CHCl3/CH3OH, 9:1); MS (EI, Pos) m/z 205 (M)+;
HRMS (EI, Pos) Calc. for C11H11NO3: 205.0739; found:
205.0747; IR (KBr) 3476, 3405, 2908, 1693, 1597, 1506,
1462, 1421, 1359, 1341, 1319, 1229, 1187, 1120, 1105, 925,
846, 831, 793, 648, 617, 596, 567 cm–1.
5.3.7. [1-(4-Butoxybenzoyl)-2-methyl-1H-indol-3-yl]acetic
acid (3a)
8% yield; m.p. 118–120 °C; TLC Rf = 0.39
(CHCl3/CH3OH, 20:1); 1H NMR (200 MHz, CDCl3) d 7.75–
7.68 (m, 2H), 7.50 (d, J = 7.6 Hz, 1H), 7.20–7.12 (m, 1H),
7.09–6.92 (m, 4H), 4.05 (t, J = 6.8 Hz, 2H), 3.74 (s, 2H), 2.42
(s, 3H), 1.88–1.75 (m, 2H), 1.60–1.43 (m, 2H), 1.00 (t,
J = 7.4 Hz, 3H); MS (APCI, Neg, 20 V) m/z 364 (M – H)–;
HRMS (EI, Pos) Calc. for C22H23NO4: 365.1627; found:
365.1611; IR (KBr) 2980, 1681, 1606, 1511, 1459, 1318,
1176, 1067, 843, 742, 621 cm–1.
5.5. Benzyl [5-(benzyloxy)-2-methyl-1H-indol-3-yl]acetate
(19)
To a stirred solution of compound 18 (2.32 g, 11.4 mmol)
in DMF (12 ml) were added K2CO3 (9.1 g, 66 mmol) and
benzyl bromide (3.9 ml, 33 mmol) at room temperature under
argon atmosphere. After stirring for 15 h, the reaction mix-
ture was quenched with 1 M HClaq and extracted twice with
EtOAc. The combined organic layers were washed with water,
brine, dried over Na2SO4 and concentrated in vacuo to give
an oily residue, which was purified by column chromatogra-
phy on silica gel to yield 19 (2.14 g, 42%); TLC Rf = 0.19
(n-hexane/EtOAc, 3:1); 1H NMR (200 MHz, CDCl3) d 8.00
(brs, 1H), 7.50–7.20 (m, 10H), 6.90–6.80 (m, 2H), 6.60 (m,
1H), 5.10 (s, 2H), 4.96 (s, 2H), 3.68 (s, 2H), 2.35 (s, 3H); MS
(EI, Pos) m/z 385 (M)+; HRMS (EI, Pos) Calc. for
C25H23NO3: 385.1678; found: 385.1675; IR (KBr) 3399,
3060, 3032, 2942, 1720, 1621, 1591, 1482, 1455, 1379, 1317,
1299, 1274, 1235, 1201, 1166, 1126, 1100, 1017, 1000, 939,
915, 861, 831, 797, 744, 711, 695, 634, 618 cm–1.
5.3.8. [1-(4-Butoxybenzoyl)-5-isopropyl-2-methyl-1H-
indol-3-yl]acetic acid (3c)
15% yield; TLC Rf = 0.38 (CHCl3/CH3OH, 10:1); 1H NMR
(200 MHz, CDCl3) d 7.70 (m, 2H), 7.32 (s, 1H), 6.93 (m,
4H), 4.05 (t, J = 6.6 Hz, 2H), 3.73 (s, 2H), 2.96 (septet,
J = 6.8 Hz, 1H), 2.41 (s, 3H), 1.82 (m, 2H), 1.52 (m, 2H),
1.26 (d, J = 6.8 Hz, 6H), 1.00 (t, J = 7.4 Hz, 3H); MS (APCI,
Neg, 20 V) m/z 406 (M – H)–; HRMS (MALDI-TOF, Pos)
Calc. for C25H29NO4 + H+: 408.2176; found: 408.2175; IR
(neat) 2960, 1683, 1604, 1510, 1475, 1310, 1256, 1174, 1062,
843, 763 cm–1.
5.3.9. [5-Fluoro-2-methyl-1-(4-pentylbenzoyl)-1H-indol-3-
yl]acetic acid (3d)
3% yield; m.p. 146–147 °C; TLC Rf = 0.61
1
(CHCl3/CH3OH, 9:1); H NMR (200 MHz, CDCl3) d 7.69
(d, J = 9.0 Hz, 2H), 7.16 (dd, J = 8.7, 2.4 Hz, 1H), 7.00–6.90
(m, 3H), 6.77 (td, J = 9.3, 2.4 Hz, 1H), 4.05 (t, J = 6.4 Hz,
2H), 3.70 (s, 2H), 2.40 (s, 3H), 1.88–1.75 (m, 2H), 1.66–1.44
(m, 2H), 1.00 (t, J = 7.3 Hz, 3H); MS (APCI, Neg, 20 V) m/z
382 (M – H)–; HRMS (EI, Pos) Calc. for C22H22FNO4:
383.1533; found: 383.1611; IR (KBr) 3700–3300, 2941, 1714,
1682, 1510, 1473, 1375, 1355, 1313, 1260, 1175, 1137, 1069,
933, 845, 802, 764, 709, 671, 599 cm–1.
5.6. Benzyl [5-(benzyloxy)-1-(4-butoxybenzoyl)-2-methyl-
1H-indol-3-yl]acetate (20)
To a stirred solution of 19 (1.77 g, 4.60 mmol) in DMF
(30 ml) was added sodium hydride (60% oil dispersion,
184 mg, 4.60 mmol) in several portions at 0 °C under argon
atmosphere, and the resulting suspension was stirred for 1 h
at 0 °C. After the addition of p-(n-butoxy) benzoyl chloride

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
515
(0.96 ml, 5.06 mmol) at 0 °C, the reaction mixture was stirred
for 16 h, 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 crude
product, which was purified by column chromatography on
silica gel to yield 20 (916 mg, 35%); TLC Rf = 0.48 (n-
hexane/EtOAc, 3:1); 1H NMR (300 MHz, CDCl3) d 7.68 (d,
J = 9.0 Hz, 2H), 7.50–7.20 (m, 10H), 7.03 (d, J = 2.7 Hz,
1H), 6.97–6.86 (m, 3H), 6.73 (m, 1H), 5.13 (s, 2H), 4.99 (s,
2H), 4.05 (t, J = 6.6 Hz, 2H), 3.71 (s, 2H), 2.39 (s, 3H), 1.93–
1.77 (m, 2H), 1.75–1.45 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H);
MS (EI, Pos) m/z 561 (M)+; HRMS (EI, Pos) Calc. for
C36H35NO5: 561.2515; found: 561.2521; IR (KBr) 3028,
2935, 2871, 1732, 1674, 1604, 1509, 1475, 1461, 1382, 1352,
1324, 1254, 1236, 1166, 1146, 1066, 1042, 1027, 1001, 917,
841, 793, 762, 737, 696, 630 cm–1.
MS (FAB, Pos) m/z 382 (M + H)+; HRMS (EI, Pos) Calc. for
C22H23NO5: 381.1576; found: 381.1552; IR (neat) 2959,
1714, 1681, 1605, 1510, 1478, 1451, 1374, 1334, 1309, 1256,
1174, 1046, 912, 844, 760, 648 cm–1.
5.9. General procedure for the preparation
of 4-nbutoxybenzoyl-2-methyl-5-methoxy indole-3-alkanoic
acids derivatives 6, 8–10 and 23
5.9.1. 3-[1-(4-Butoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl] propanoic acid (8)
To a stirred solution of 14d (2.72 g, 7.99 mmol) in toluene
(20 ml) and CH3OH (20 ml) was added 4 M HCl-dioxane
(2 ml).After stirring for 30 min at room temperature, the reac-
tion mixture was concentrated in vacuo to give 22 (4.92 g),
which was used for the next reaction without further purifi-
cation.
To a stirred solution of 22 (376 mg, 1.07 mmol) in AcOH
(10 ml) was added 24a (0.158 ml, 1.28 mmol) at room tem-
perature. After stirring for 2 h at 80 °C, the reaction mixture
was concentrated in vacuo to give a crude solid, which was
purified by column chromatography on silica gel to yield 8
(320 mg, 73% yield) as a pale yellow powder; m.p. 105–
5.7. [1-(4-Butoxybenzoyl)-5-hydroxy-2-methyl-1H-indol-3-
yl]acetic acid (4)
To a stirred solution of 20 (795 mg, 1.42 mmol) in EtOAc
(3 ml) was added 10% Pd–C (100 mg) at room temperature.
The resulting suspension was stirred for 2 h at room tempera-
ture under hydrogen atmosphere. Insoluble substance was
removed by filtration. The filtrate was concentrated in vacuo
to give a crude product, which was purified by recrystalliza-
tion from EtOAc/n-hexane to yield 4 (390 mg, 72%) as a white
powder; m.p. 185–187 °C; TLC Rf = 0.16 (CHCl3/CH3OH,
1
106 °C; TLC Rf = 0.31 (CHCl3/CH3OH, 20:1); H NMR
(300 MHz, CDCl3) d 7.70–7.65 (m, 2H), 6.97–6.89 (m, 4H),
6.66 (dd, J = 9.4, 2.8 Hz, 1H), 4.05 (t, J = 6.4 Hz, 2H), 3.84
(s, 3H), 3.03 (t, J = 7.8 Hz, 2H), 2.68 (m, 2H), 2.38 (s, 3H),
1.88–1.74 (m, 2H), 1.61–1.43 (m, 2H), 0.99 (t, J = 7.4 Hz,
3H); MS (APCI, Neg, 20 V) m/z 408 (M – H)–; HRMS (EI,
Pos) Calc. for C24H27NO5: 409.1889; found: 409.1889; IR
(KBr) 2929, 1703, 1606, 1471, 1357, 1259, 1224, 1174, 1149,
1049, 794, 761 cm–1.
1
10:1); H NMR (200 MHz, CDCl3/CD3OD) d 7.69 (d,
J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 2.0 Hz,
1H), 6.84 (d, J = 8.8 Hz, 1H), 6.58 (dd, J = 8.8, 2.0 Hz, 1H),
4.05 (t, J = 6.4 Hz, 2H), 3.62 (s, 2H), 2.38 (s, 3H), 1.82 (m,
2H), 1.52 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H); MS (FAB, Pos)
m/z 382 (M + H)+; HRMS (EI, Pos) Calc. for C22H23NO5:
381.1576; found: 381.1589; IR (KBr) 3239, 2960, 1706, 1669,
1605, 1576, 1512, 1468, 1387, 1351, 1310, 1256, 1180, 1148,
1074, 1027, 968, 931, 836, 799, 762, 707, 673, 637, 601 cm–1.
5.9.2. [1-(4-Butoxybenzoyl)-2-ethyl-5-methoxy-1H-indol-3-
yl]acetic acid (6)
1% yield; TLC Rf = 0.43 (CHCl3/MeOH, 10:1); 1H NMR
(200 MHz, CDCl3) d 7.72 (d, J = 9.0 Hz, 2H), 6.98–6.92 (m,
3H), 6.71–6.60 (m, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.83 (s,
3H), 3.74 (s, 2H), 2.96 (q, J = 7.6 Hz, 2H), 1.89–1.74 (m,
2H), 1.62–1.42 (m, 2H), 1.18 (t, J = 7.6 Hz, 3H), 1.00 (t,
J = 7.6 Hz, 3H); MS (APCI, Neg, 20 V) m/z 408 (M – H)–;
HRMS (EI, Pos) Calc. for C24H27NO5: 409.1889; found:
409.1888; IR (neat) 2931, 1680, 1604, 1478, 1259, 1042,
802 cm–1.
5.8. [1-(4-Butoxybenzoyl)-5-methoxy-1H-indol-3-yl]acetic
acid (5)
To a stirred solution of 21 (0.205 g, 1 mmol) in THF (3 ml)
was added n-butyl lithium (1.6 M in n-hexane, 1.25 ml,
2 mmol) at –78 °C under argon atmosphere, and the resulting
suspension was stirred for 1 h at –78 °C. After the addition of
p-(n-butoxy) benzoyl chloride (0.255 g, 1.2 mmol) at –78 °C,
the reaction mixture was stirred for 1 h at –20 °C, then
quenched with saturated NH4Claq and extracted with EtOAc
(×2). The combined organic layers were washed with water,
brine, dried over Na2SO4 and concentrated in vacuo to give a
crude product, which was purified by column chromatogra-
phy on silica gel to yield 20 (0.016 mg, 4%); TLC Rf = 0.41
(CHCl3/CH3OH, 10:1); 1H NMR (200 MHz, CDCl3) d 8.25
(d, J = 9.8 Hz, 1H), 7.70 (d, J = 9.0 Hz, 2H), 7.35 (s, 1H),
7.06–6.93 (m, 4H), 4.05 (t, J = 6.6 Hz, 2H), 3.88 (s, 3H), 3.72
(s, 2H), 1.82 (m, 2H), 1.52 (m, 2H), 1.00 (t, J = 8.0 Hz, 3H) ;
5.9.3. 4-[1-(4-Butoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]butanoic acid (9)
84% yield; TLC Rf = 0.34 (CHCl3/MeOH, 20:1); 1H NMR
(200 MHz, CDCl3) d 7.70–7.65 (m, 2H), 6.98–6.87 (m, 4H),
6.64 (dd, J = 9.0, 2.4 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.84
(s, 3H), 2.74 (dd, J = 7.8, 7.0 Hz, 2H), 2.44 (dd, J = 7.4,
7.0 Hz, 2H), 2.35 (s, 3H), 1.98 (dddd, J = 7.8, 7.4, 7.0, 7.0 Hz,
2H), 1.88–1.74 (m, 2H), 1.52 (qt, J = 7.4, 7.6 Hz, 2H), 1.00
(t, J = 7.4 Hz, 3H); MS (APCI, Neg, 20 V) m/z 422 (M – H)–;
HRMS (EI, Pos) Calc. for C25H29NO5: 423.2046; found:
423.2064; IR (neat) 2958, 1708, 1605, 1477, 1256, 1173,
763 cm–1.

516
K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
5.9.4. 5-[1-(4-Butoxybenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl]pentanoic acid (10)
EtOAc (×2). The combined organic layers were washed with
water, brine, dried over Na2SO4 and evaporated. The residue
was purified by recrystallization from EtOAc to yield 28
(1.03 g, 92% yield) as a pale pink powder; m.p. 191–194 °C;
TLC Rf = 0.28 (n-hexane/EtOAc, 1:2); 1H NMR (200 MHz,
CDCl3) d 9.93 (s, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.21 (d,
J = 8.8 Hz, 1H), 6.80 (dd, J = 8.8, 2.4 Hz, 1H), 3.81 (s, 3H),
2.65 (s, 3H); MS (EI, Pos) m/z 189 (M)+; HRMS (EI, Pos)
Calc. for C11H11NO2: 189.0790; found: 189.0773; IR (KBr)
3068, 2817, 2334, 1638, 1585, 1457, 1365, 1303, 1271, 1208,
1180, 1127, 1098, 1031, 982, 893, 844, 801, 720, 644, 621,
577 cm–1.
87% yield; TLC Rf = 0.28 (CHCl3/MeOH, 20:1); 1H NMR
(200 MHz, CDCl3) d 7.71–7.64 (m, 2H), 6.95–6.89 (m, 4H),
6.64 (dd, J = 9.0, 2.4 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.84
(s, 3H), 2.69 (m, 2H), 2.40 (m, 2H), 2.35 (s, 3H), 1.88–1.65
(m, 6H), 1.60–1.43 (m, 2H), 1.00 (t, J = 7.6 Hz, 3H); MS
(APCI, Neg, 20 V) m/z 436 (M – H)–; HRMS (EI, Pos) Calc.
for C26H31NO5: 437.2202; found: 437.2208; IR (KBr) 2936,
1675, 1606, 1512, 1478, 1373, 1324, 1259, 1217, 1175, 1069,
1039, 927, 847, 761, 633, 609 cm–1.
5.9.5. Allyl-1-(4-butoxybenzoyl)-5-methoxy-2-methyl-1H-
indole-3-carboxylate (23)
5.12. Methyl (2E)-3-(5-methoxy-2-methyl-1H-indol-3-yl)-
acrylate (29)
85% yield; TLC Rf = 0.30 (n-hexane/EtOAc, 5:1); 1H NMR
(200 MHz, CDCl3) d 7.74–7.66 (m, 3H), 7.00–6.91 (m, 3H),
6.73 (dd, J = 9.0, 2.8 Hz, 1H), 6.22–6.02 (m, 1H), 5.47 (m,
1H), 5.31 (m, 1H), 4.90 (m, 2H), 4.05 (t, J = 6.4 Hz, 2H),
3.86 (s, 3H), 2.72 (s, 3H), 1.88–1.74 (m, 2H), 1.57–1.42 (m
2H), 0.99 (t, J = 7.2 Hz, 3H); MS (EI, Pos.) m/z 421 (M)+;
HRMS (EI, Pos) Calc. for C25H27NO5: 421.1889; found:
421.1895; IR (neat) 2957, 2873, 1695, 1602, 1556, 1509,
1475, 1455, 1393, 1356, 1311, 1258, 1198, 1164, 1112, 1038,
969, 925, 846, 764, 738, 649 cm–1.
To a stirred solution of 28 (0.992 g, 5.66 mmol) in ben-
zene (35 ml) was added methyl (triphenylphosphoranylidene)
acetate (5.68 g, 17.0 mmol) at room temperature. After stir-
ring for 50 h at 85 °C, the reaction mixture was concentrated
in vacuo to give a residue, which was purified by column
chromatography on silica gel to yield 29 (1.22 g, 89% yield)
as a white powder; TLC Rf = 0.50 (n-hexane/EtOAc, 1:1); 1H
NMR (200 MHz, CDCl3) d 8.34 (brs, 1H), 7.94 (d,
J = 16.0 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 7.20 (d, J = 8.8 Hz,
1H), 6.84 (dd, J = 8.8, 2.4 Hz, 1H), 6.33 (d, J = 16.0 Hz, 1H),
3.88 (s, 3H), 3.82 (s, 3H), 2.52 (s, 3H); MS (EI, Pos) m/z 245
(M)+; HRMS (EI, Pos) Calc. for C14H15NO3: 245.1052;
found: 245.1058; IR (KBr) 3276, 3001, 2940, 2836, 1693,
1605, 1483, 1456, 1427, 1315, 1284, 1224, 1150, 1105, 1032,
968, 879, 836, 818, 790, 728, 699, 643 cm–1.
5.10. 1-(4-Butoxybenzoyl)-5-methoxy-2-methyl-1H-indole-
3-carboxylic acid (7)
To a stirred solution of compound 23 (220 mg,
0.522 mmol) in THF (3 ml) was added Pd(PPh3)4 (60 mg,
0.052 mmol) at room temperature under argon atmosphere,
and stirring was continued for 15 min. To the stirred reaction
mixture, morpholine (0.23 ml, 2.61 mmol) was added. After
stirring for 1 h, reaction mixture was quenched with 1 M
HClaq and extracted with EtOAc (×2). The combined organic
layers were washed with water, brine, dried over Na2SO4 and
concentrated in vacuo, to give an oily residue, which was puri-
fied by column chromatography on silica gel to yield 7
(139 mg, 34% in two steps) as a pale yellow powder; TLC
5.13. (2E)-3-(5-Methoxy-2-methyl-1H-indol-3-yl)acrylic
acid (30)
To a stirred solution of methyl ester 29 (1.08 g, 4.39 mmol)
in MeOH (5 ml) and 1,4-dioxane (15 ml) was added 2 M
NaOHaq (5 ml). After stirring for 5 h at 50 °C, the reaction
mixture was acidified with 1 M HClaq, 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 recrystallization from
EtOAc to yield 30 (344 mg, 96%) as a pale yellow powder;
TLC Rf = 0.36 (CHCl3/MeOH, 10:1); 1H NMR (200 MHz,
CD3OD) d 7.97 (d, J = 16.0 Hz, 1H), 7.32 (s, 1H), 7.21 (d,
J = 8.8 Hz, 1H), 7.21 (s, 1H), 6.79 (dd, J = 8.8, 2.1 Hz, 1H),
6.21 (d, J = 16.0 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 2.50 (s,
3H); MS (EI, Pos) m/z 231 (M)+; HRMS (EI, Pos) Calc. for
C13H13NO3: 231.0895; found: 231.0896; IR (KBr) 3349,
2942, 2835, 1674, 1602, 1482, 1408, 1316, 1284, 1219, 1192,
1176, 1103, 1032, 972, 884, 843, 794, 689, 607 cm–1.
1
Rf = 0.39 (CHCl3/CH3OH, 20:1); H NMR (200 MHz,
CDCl3) d 7.75–7.69 (m, 3H), 6.99–6.93 (m, 3H), 6.75 (dd,
J = 9.0, 2.4 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.91 (s, 3H),
2.79 (s, 3H), 1.89–1.75 (m, 2H), 1.52 (qt, J = 7.2, 7.6 Hz,
2H), 1.00 (t, J = 7.2 Hz, 3H); MS (APCI, Neg, 20 V) m/z 380
(M – H)–; HRMS (EI, Pos) Calc. for C22H23NO5: 381.1576;
found: 381.1601; IR (KBr) 2957, 1698, 1604, 1476, 1358,
1260, 1208, 1170, 926, 848, 754 cm–1.
5.11. 5-Methoxy-2-methyl-1H-indole-3-carbaldehyde (28)
Vilsmeier Reagent was prepared from POCl3 (0.85 ml,
9.67 mmol) and DMF (3 ml) according to a reported proce-
dure [11].
To a stirred solution of 27 (1.04 g, 6.45 mmol) in DMF
(3 ml) was added dropwise above-described Vilsmeier
Reagent at 0 °C. After stirring for 30 min at 0 °C, the reaction
mixture was poured into 2 M NaOHaq and extracted with
5.14. (2E)-3-[1-(4-Butoxybenzoyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acrylic acid (31)
To a stirred solution of 30 (0.250 g, 1.08 mmol) in THF
(4 ml) was added n-butyl lithium (1.53 M in n-hexane, 1.4 ml,

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
517
2.16 mmol) at –60 °C under argon atmosphere, and the result-
ing suspension was stirred for 1 h at –60 °C. After the addi-
tion of p-(n-butoxy) benzoyl chloride (0.246 ml, 1.3 mmol)
at –78 °C, the reaction mixture was stirred for 2 h at –78 °C
and for 1 h at –20 °C, then quenched with 1 M HClaq and
extracted with EtOAc (×2). The combined organic layers were
washed with water, brine, dried over Na2SO4 and concen-
trated in vacuo to give a residue, which was purified by col-
umn chromatography on silica gel to yield 31 (0.198 g, 45%);
m.p. 153–155 °C; TLC Rf = 0.53 (CHCl3/MeOH, 10:1); 1H
NMR (200 MHz, CDCl3) d 8.07 (d, J = 16.0 Hz, 1H), 7.71 (d,
J = 8.8 Hz, 2H), 7.30 (d, J = 2.4 Hz, 1H), 7.70–6.94 (m, 3H),
6.75 (dd, J = 8.8, 2.4 Hz, 1H), 6.50 (d, J = 16.0 Hz, 1H), 4.06
(t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 2.56 (s, 3H), 1.89–1.75 (m,
2H), 1.61–1.47 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H) ; MS (APCI,
Neg. 20V) m/z 406 (M – H)–; HRMS (EI, Pos) Calc. for
C24H25NO5: 407.1733; found: 407.1740; IR (KBr) 2958,
1690, 1604, 1510, 1477, 1399, 1352, 1307, 1256, 1172, 1059,
927, 850, 802, 764, 671 cm–1.
C11H11NO2: 189.0790; found: 189.0792; IR (KBr) 3339,
3065, 2951, 2845, 1698, 1615, 1577, 1552, 1494, 1464, 1442,
1407, 1388, 1355, 1337, 1293, 1234, 1195, 1172, 1136, 1058,
999, 944, 781, 752, 697, 682 cm–1.
5.17. 2-Methyl-1H-indole-4-carboxylic acid (34)
To a stirred solution of methyl ester 33 (4.30 g, 22.7 mmol)
in MeOH (10 ml) and 1,4-dioxane (10 ml) was added 5 M
NaOHaq (10 ml). After stirring for 16 h at 60 °C, the reaction
mixture was acidified with 2 M HClaq 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 chromatogra-
phy on silica gel to yield 34 (1.6 g, 40%); m.p. 218–220 °C;
1
TLC Rf = 0.48 (CHCl3/MeOH, 9:1); H NMR (300 MHz,
CD3OD) d 8.14–8.04 (br, 1H), 7.93 (dd, J = 8.1, 0.9 Hz, 1H),
7.52 (m, 1H), 7.18 (dd, J = 8.1, 0.9 Hz, 1H), 6.94 (m, 1H),
3.71 (s, 3H); MS (EI, Pos) m/z 175 (M)+; HRMS (EI, Pos)
Calc. for C10H9NO2: 175.0633; found: 175.0641; IR (KBr)
3384, 2917, 2681, 2578, 1658, 1614, 1577, 1558, 1497, 1442,
1405, 1357, 1335, 1307, 1273, 1226, 1186, 1146, 1065, 1002,
918, 794, 749, 777, 749, 688, 647, 571, 549 cm–1.
5.15. 2-Methyl-1H-indol-4-yl trifluoromethanesulfonate
(32)
To a stirred solution of 31 (2-methyl-1H-indol-4-ol)
(10.0 g, 67.9 mmol) in CH2Cl2 (100 ml) were added 2,6-
lutidine (10.3 ml, 88.3 mmol) and Tf2O (13.72 ml,
81.54 mmol) at 0 °C. After stirring for 1 h, the reaction mix-
ture was poured into water and extracted with EtOAc (×2).
The combined organic layers were washed with water, brine,
dried over Na2SO4 and evaporated to afford 32, which was
used for the next reaction without further purification; m.p.
69–70 °C; TLC Rf = 0.57 (n-hexane/EtOAc, 7:3); 1H NMR
(200 MHz, CDCl3) d 8.21 (brs, 1H), 7.27 (m, 1H), 7.09 (dd,
J = 7.8, 7.5 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 6.33 (m, 1H),
2.47 (d, J = 0.6 Hz, 3H; MS (EI, Pos) m/z 279 (M)+; HRMS
(EI, Pos) Calc. for C10H8NF3O3S: 279.0177; found: 279.0197;
IR (KBr) 3951, 3444, 1892, 1633, 1585, 1552, 1496, 1450,
1405, 1351, 1336, 1286, 1251, 1206, 1151, 1054, 1010, 990,
886, 837, 786, 759, 729, 659, 621, 523 cm–1.
5.18. Benzyl 2-methyl-1H-indole-4-carboxylate (35)
To a stirred solution of 34 (690 mg, 3.94 mmol) in DMF
(10 ml) were added K2CO3 (815 mg, 5.91 mmol) and benzyl
bromide (0.7 ml, 5.91 mmol) at room temperature. After stir-
ring for 2 h at 80 °C, 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 chromatography on silica gel
to afford 35 (610 mg, 58%); TLC Rf = 0.44 (n-hexane/EtOAc,
8:3); 1H NMR (300 MHz, CDCl3) d 8.05 (brs, 1H), 7.91 (d,
J = 7.2 Hz, 1H), 7.54–7.24 (m, 7H), 6.88 (m, 1H), 5.44 (s,
2H), 2.48 (s, 3H); MS (EI, Pos) m/z 265 (M)+; HRMS (EI,
Pos) Calc. for C17H15NO2: 265.1103; found: 265.1112; IR
(KBr) 3356, 3330, 1698, 1681, 1611, 1577, 1553, 1496, 1455,
1436, 1407, 1377, 1342, 1271, 1232, 1190, 1142, 1020, 952,
795, 755, 738, 695, 666, 632, 591 cm–1.
5.16. Methyl 2-methyl-1H-indole-4-carboxylate (33)
To a stirred solution of 32 (6.34 g, 22.7 mmol) in MeOH
(33 ml) and DMF (200 ml) were added TEA (6.3 ml,
45.29 mmol) and Pd(PPh3)4 (2.6 g, 2.26 mmol) at room tem-
perature. After stirring over night at 60 °C under an atmo-
sphere of carbon monoxide, the reaction mixture was filtered
through a pad of Celite. The filtrate 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 residue, which was purified by column
chromatography on silica gel to yield 33 (100% yield); m.p.
126–129 °C; TLC Rf = 0.18 (toluene); 1H NMR (200 MHz,
CDCl3) d 8.14–8.00 (brs, 1H), 7.85 (m, 1H), 7.47 (m, 1H),
7.14 (t, J = 8.1 Hz, 1H), 6.87 (m, 1H), 3.97 (s, 3H), 2.50 (s,
3H); MS (EI, Pos) m/z 189 (M)+; HRMS (EI, Pos) Calc. for
5.19. Benzyl 1-(4-butoxybenzoyl)-2-methyl-1H-indole-4-
carboxylate (36)
To a stirred solution of the 35 (690 mg, 2.60 mmol) in
DMF (8 ml) was added sodium hydride (60% oil dispersion,
114 mg, 2.86 mmol) in several portions at 0 °C under argon
atmosphere, and the resulting suspension was stirred for
30 min at 0 °C. After the addition of p-(n-butoxy) benzoyl
chloride (0.54 ml, 2.86 mmol), the reaction mixture was stirred
for 12 h at 0 °C, 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 chromatogra-

518
K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
phy on silica gel to yield 36 (1.02 g, 89%); TLC Rf = 0.61
(n-hexane/EtOAc, 8:2); 1H NMR (300 MHz, CDCl3) d 8.10–
6.90 (m, 13H), 5.45 (s, 2H), 4.05 (t, J = 6.3 Hz, 2H), 2.44 (s,
3H), 1.86–1.74 (m, 2H), 1.60–1.45 (m, 2H), 0.99 (t,
J = 7.5 Hz, 3H).
room temperature. The resulting suspension was stirred for
2 h at room temperature under hydrogen atmosphere. In-
soluble substance was removed by filtration. The filtrate was
concentrated in vacuo to give a crude product, which was
purified by recrystallization from EtOAc/n-hexane to yield 1
(25 mg, 28%); m.p. 95–97 °C; TLC Rf = 0.46 (CHCl3/
1
5.20. 1-(4-Butoxybenzoyl)-2-methyl-1H-indole-4-
carboxylic acid (37)
CH3OH, 9:1); H NMR (300 MHz, CDCl3) d 7.70 (d,
J = 9.0 Hz, 2H), 7.08–6.90 (m, 5H), 6.49 (s, 1H), 4.05 (t,
J = 6.6 Hz, 2H), 3.87 (s, 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 = 7.5 Hz, 3H); MS (APCI, Neg 20 V) m/z 364 (M – H)–;
HRMS (EI, Pos) Calc. 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, 641 cm–1.
To a stirred solution of the compound 36 (1.02 g,
2.31 mmol) in MeOH (10 ml) and EtOAc (5 ml) was added
10% Pd–C (100 mg) at room temperature, and the resulting
mixture was stirred for 30 min at room temperature 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 37 (433 mg, 53%); m.p. 127–128 °C; TLC Rf = 0.59
(CHCl3/MeOH, 9:1); 1H NMR (300 MHz, CDCl3) d 7.99 (d,
J = 8.1 Hz, 1H), 7.70 (d, J = 9.0 Hz, 2H), 7.36 (d, J = 8.1 Hz,
1H), 7.21 (brs, 1H), 7.13 (t, J = 8.1 Hz, 1H), 6.97 (d,
J = 9.0 Hz, 2H), 4.06 (t, J = 6.6 Hz, 2H), 2.48 (s, 3H), 1.88–
1.76 (m, 2H), 1.60–1.46 (m, 2H), 1.00 (t, J = 7.5 Hz, 3H);
MS (FAB, Pos) m/z 352 (M + H)+; HRMS (EI, Pos) Calc. for
C21H21NO4: 351.1471; found: 351.1493; IR (neat) 2960,
1682, 1604, 1511, 1481, 1432, 1371, 1257, 1171, 912, 804,
757 cm–1.
5.23. Prostanoid mEP and mDP receptor binding assay
Competitive binding studies were conducted using radio-
labeled ligands and membrane fractions prepared from Chi-
nese hamster ovary (CHO) cells stably expressing the respec-
tive prostanoid receptors, mEP1, mEP2, mEP3a, mEP4 and
mDP.
Membranes from CHO cells expressing prostanoid recep-
tors were incubated with radioligand (2.5 nM of [3H]PGE2
for mEP1-4 or 2.5 nM of [3H]PGD2 for mDP) and the test
compounds at various concentrations in assay buffer (10 mM
KH2PO4–KOH buffer containing 100 mM NaCl, pH 6.0 for
mEP1-4, 25 mM HEPES–NaOH buffer containing 1 mM
EDTA, 5 mM MgCl2 and 10 mM MnCl2, pH 7.4 for mDP).
Incubation was carried out at 25 °C for 60 min except for
mEP1 and mDP (20 min). The incubation was terminated by
filtration through Whatman GF/B filters. The filters were then
washed with ice-cold buffer (10 mM KH2PO4–KOH buffer
containing 100 mM NaCl, pH 6.0 for mEP1-4, 10 mM Tris–
HCl buffer containing 100 mM NaCl and 0.01 w/v% BSA,
pH 7.4 for mDP), and the radioactivity on the filter was mea-
sured in 6 ml of liquid scintillation (ACSII) mixture with a
liquid scintillation counter. Nonspecific binding was achieved
by adding excess amounts of unlabeled PGE2 (for mEP1-4)
or unlabeled PGD2 (for mDP) with assay buffer. The concen-
trations 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.
5.21. Benzyl 4-[1-(4-butoxybenzoyl)-2-methyl-1H-indol-4-
yl]acetate (38)
To a stirred solution of 37 (290 mg, 0.825 mmol) in tolu-
ene (5 ml) were added oxalyl chloride (0.12 ml, 1.32 mmol)
and DMF (a few drops) at room temperature. After stirring
for 1 h at room temperature, the reaction mixture was con-
centrated in vacuo to give an acid chloride, which was used
for the next reaction without further purification. To a stirred
solution of the above-described acid chloride in THF (4 ml)
and CH3CN (4 ml) was added (trimethylsilyl)diazomethane
(2 M in ether, 0.83 ml, 1.65 mmol) at 0 °C. After stirring for
1 h at 0 °C, the reaction mixture was concentrated in vacuo to
afford a residue.
The residue described above was dissolved in benzyl alco-
hol (4 ml) and 2,4,6-collidine (4 ml) at room temperature.
After stirring for 30 min at 180 °C, the reaction mixture was
concentrated in vacuo to afford a residue, which was purified
by column chromatography on silica gel to yield 38 (110 mg,
1
29%); TLC Rf = 0.56 (n-hexane/EtOAc, 4:1); H NMR
K = IC / 1 + L /K
͓ ͔ ͒
d
͑
i
50
(300 MHz, CDCl3) d 7.70 (d, J = 9.0 Hz, 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 = 6.6 Hz, 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 = 7.5 Hz, 3H).
[L]: concentration of radiolabeled ligand, Kd: dissociation
constant of radiolabeled ligand towards the prostanoid recep-
tors.
5.22. [1-(4-Butoxybenzoyl)-2-methyl-1H-indol-4-yl]acetic
acid (1)
5.24. Measurement of the mDP receptor antagonist
activity
To a stirred solution of the compound 38 (110 mg,
239 mmol) in EtOAc (4 ml) was added 10% Pd-C (15 mg) at
To confirm that test compounds antagonize the mDP recep-
tor and to estimate potencies of antagonism for the mDP

K. Torisu et al. / European Journal of Medicinal Chemistry 40 (2005) 505–519
519
receptor, a functional assay was performed by measuring
PGD2-stimulated changes in intracellular second messenger
cAMP as an indicator of receptor function.
AUCpo: AUC after oral dosing; AUCiv: AUC after intrave-
nous dosing; Dpo: dosage of oral administration; Div: dosage
of intravenous administration.
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 per well and
cultivated for 2 days. The cells in each well were rinsed with
minimum essential medium (MEM), and MEM containing
2 µM of Diclofenac was added to each well. The cells were
incubated for approximately 10 min at 37 °C and the culture
medium was removed. The assay medium (MEM containing
0.1% BSA, 1 mM IBMX and 2 µM Diclofenac) was added to
each well and the cells were incubated for approximately
10 min at 37 °C. The assay medium, assay medium contain-
ing 10 nM of PGD2, or assay medium containing various con-
centrations of test compounds and 10 nM of PGD2 was added
to each well and the cells were further incubated for 10 min
at 37 °C. The reaction was terminated by the addition of ice-
cold trichloroacetic acid (TCA; 10 w/v%) and the incubation
mixture was frozen at –80 °C until the assay 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
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 responsiveness (%) of cAMP production was cal-
culated relative to the maximum increase in cAMP that
occurred in the absence of test compound (100%) to estimate
of the IC50 values.
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Single dose PK of 1 was studied in rats. Formulation for
intravenous injection was prepared using 30% HP-b-CD con-
taining 5% DMSO (1 mg ml–1 kg–1). Formulation for oral
dosing was prepared using 0.5% MC (10 mg/5 ml kg–1). Test
compounds (1 mg kg–1) were dosed intravenously to the fasted
male rats (n = 3). Test compounds (1 mg kg–1) were dosed
orally to the fasted male rats (n = 3). After dosing, blood
samples (250 µl) were collected from the jugular vein using a
heparinized syringe at the selected time points (iv: pre-
dosing, 2, 5, 15 and 30 min, 1, 2, 4, 6 and 8 h; po: pre-dosing,
15 and 30 min 1, 2, 4, 6 and 8 h, respectively). The blood
samples were ice-chilled and then centrifuged at 12,000 rpm
for 2 min at 4 °C to obtain plasma, which was preserved at
–80 °C in a freezer. The AUC, Cmax, Tmax, T1/2, Vss and CLtot
were obtained by measuring the time course of the plasma
concentration of the test compounds. BA was calculated
according to the following equation:
[26] V. Lee, M.S. Newman, Org. Synth. 50 (1970) 77.
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S. Narumiya, Br. J. Pharmacol. 122 (1997) 217–224.
BA % = AUC /D /AUC /D ) × 100
͑ ͒ ͑
͒
po
po
iv
iv