Discovery of new chemical leads for selective EP1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2006.04.038

A series of 4-({2-[alkyl(phenylsulfonyl)amino]phenoxy}methyl)benzoic acids were identified as functional PGE₂ antagonists with selectivity for the EP1 receptor subtype starting from a chemical lead 1, which was found while screening our in-house compound library. Discovery of the optimized analogs 21-23 is presented here and structure-activity relationships (SAR) are also discussed.

Full text of DOI:10.1016/j.bmc.2006.04.038

Bioorganic & Medicinal Chemistry 14 (2006) 5562–5577
Discovery of new chemical leads for selective EP1
receptor antagonists
Atsushi Naganawa,a,* Tetsuji Saito,a Yuuki Nagao,a Hiromu Egashira,a Maki Iwahashi,a
Tohru Kambe,a Masatoshi Koketsu,b Hiroshi Yamamoto,c Michiyoshi Kobayashi,b
Takayuki Maruyama,a Shuichi Ohuchida,d Hisao Nakai,a
Kigen Kondoa and Masaaki Todaa
aMinase Research Institute, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
bOno Pharmaceutical Co., Ltd, Kyutaro, Chuoh, Osaka 541-0056, Japan
cOno Pharma USA, Inc., Lenox Drive, Lawrenceville, NJ 08648, USA
dFukui Research Institute, Ono Pharmaceutical Co., Ltd, Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
Received 15 March 2006; revised 14 April 2006; accepted 14 April 2006
Available online 12 May 2006
Abstract—A series of 4-({2-[alkyl(phenylsulfonyl)amino]phenoxy}methyl)benzoic acids were identified as functional PGE2 antago-
nists with selectivity for the EP1 receptor subtype starting from a chemical lead 1, which was found while screening our in-house
compound library. Discovery of the optimized analogs 21–23 is presented here and structure–activity relationships (SAR) are also
discussed.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
A few antagonists, such as ZD-4953, ZD-6416, and
ZD-6804, entered clinical development for indications
related to hyperalgesia, but their development was
suspended for unknown reasons.9
Prostanoid receptors are known to be members of the
G-protein coupled receptor superfamily. Recently, eight
prostanoid receptors (EP1, EP2, EP3, EP4, IP, TP, DP,
and FP) have been cloned and characterized.1 The char-
acterization of these receptors at the molecular level has
resulted in renewed interest in this field,2 but selective
agonists and antagonists of human prostanoid receptors
are only available in some limited field.3–6 As a result,
the correlation of specific receptors with various pathol-
ogies is currently being established by using potent but
poorly selective ligands.
Our search for a subtype-selective EP1 receptor antago-
nist started with screening our in-house compound
library. 4-(2-Arylsulfonylaminobenzoyl)amino benzoic
acids 2–4 were the new chemical leads derived from
the initial chemical lead 1, which was identified by
screening. Here we report on the discovery process
(Fig. 1) for a new class of 4-({2-[alkyl(phenylsulfo-
Recent studies have suggested that the EP1 subtype
receptor mediates the induction of pain, pyrexia, allo-
dynia7 and diuresis.8 Based on this information, com-
pounds that are selective antagonists of this receptor
are predicted to be useful as analgesics, antipyretics,
and agents to treat hyperalgesia and pollakisuria.
CO2H
CO2H
O
O
N
R
F3C
O
HN
O
S
O
S
O
Me
O
N
H
21 R = -(CH2)2CH3
O
22 R = -CH(CH)2
3
Keywords: Prostaglandin; EP1 receptor; Antagonist; Sulfonamide.
*
23 R = -CH CH(CH)2
1
2
3
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962
9314; e-mail: naganawa@ono.co.jp
Figure 1. Discovery of new selective EP1 receptor antagonists 21–23.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.038

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5563
nyl)amino]phenoxy}methyl)benzoic acids 21–23 which
are potent subtype-selective EP1 receptor antagonists.
an acid chloride 33 afforded betaine 37, heating of
which provided the propiolic acid derivative 38.10
Reduction of the nitro residue of 38 led to the aniline
39, N-sulfonylation of which with 4-chloro-
benzenesulfonyl chloride afforded the sulfonamide 41.
Alkaline hydrolysis of 41 resulted in the production
of 9.
2. Chemistry
The test compounds listed in Tables 1–5, were synthe-
sized as outlined in Schemes 1–5. Synthesis of 2–5 is
described in Scheme 1. N-Sulfonylation of the aniline
26 with benzenesulfonyl chloride afforded the sulfon-
amide 29a. N-Sulfonylation of the aniline 27 with benze-
nesulfonyl chloride and 4-chlorobensenesulfonyl
chloride gave sulfonamides 29b and 29c, respectively.
Then alkaline hydrolysis of 29a–c led to benzoic acids
30a–c, respectively. N-Sulfonylation of 28 with benze-
nesulfonyl chloride gave the sulfonamide 29d, reduction
of which afforded the aniline 30d. Amidation of 30a–c
with methyl 4-aminobenzoate provided amides 31a–c,
alkaline hydrolysis of which resulted in the production
of 2–4, respectively. N-Acylation of 30d with methyl 4-
(chlorocarbonyl)benzoate gave the amide 31d, alkaline
hydrolysis of which resulted in 5.
Compound 10 was prepared as described in Scheme
2b. Sodium borohydride reduction of 32 afforded
an alcohol 42, methanesulfonylation of which pro-
vided 43. O-Alkylation of methyl 4-hydroxybenzoate
with the methanesulfonate 43 gave 44, reduction of
which led to the aniline 45. N-Sulfonylation of 45
with 4-chlorobenzenesulfonyl chloride, followed by
alkaline, hydrolysis resulted in the production of
10.
Synthesis of 11–12 and 15–25 is described in Scheme 3.
Compounds 11–12 and 15–19 were prepared as outlined
in Scheme 3a. O-Alkylation of nitrophenols 47a–f11 with
methyl 4-(bromomethyl)benzoate in the presence of
potassium carbonate provided benzyl phenyl ethers
48a–f, reduction of which gave anilines 49a–f, respec-
tively. N-Sulfonylation of 49a–f with benzenesulfonyl
chloride, followed by alkaline hydrolysis, resulted in
the production of 12 and 15–19, respectively. N-Sulf-
onylation of 49a with 4-chlorobenzenesulfonyl chloride
led to the sulfonamide 51a, after which alkaline hydroly-
sis produced 11.
Synthesis of 6–9 is outlined in Scheme 2a. Wittig reac-
tion of aldehyde 32 and a phosphonium salt 34 affor-
ded the olefin 35 as a mixture of E- and Z-isomers.
Reduction of the nitro residue of 35, followed by
separation using silica gel column chromatography,
produced Z-isomer 36Z and E-isomer 36E. N-Sulfony-
lation of 36Z and 36E with 4-chlorobenzenesulfonyl
chloride, followed by alkaline hydrolysis resulted in
7 and 8, respectively. Catalytic hydrogenation of the
olefin of 8 gave the phenyl propanoic acid analog 6.
C-Acylation of an anion prepared from the phospho-
nium salt 34 in the presence of tertiary butoxide with
Synthesis of 20–25 is described in Scheme 3b. N-Alkyl-
ation of 51e with appropriate alkyl iodides gave N-alkyl-
ated sulfonamides 52g–l, alkaline hydrolysis of which
resulted in the production of 20–25, respectively.
Table 1. Binding affinities of chemical leads 1–4 for subtype receptors
CO2H
HN
X
O
S
O
O
Ar
N
H
I
Compound
X
Ar
*
Binding Ki (lM)
mEPla
0.18
mEP2a
mEP3a
mEP4a
>10
Me
1
2
3
4
H
>10
>10
>10
>10
b
OPiv
*
H
0.96
0.36
0.40
>10
>10
>10
>10
>10
>10
*
*
Cl
Cl
1.6
Cl
a mEPl-4, mouse EP1-4.
b Piv, pivaloyl.

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
Table 2. Effect of transformation of the carboxyamide moiety on Ki values for subtype receptors and EP1 receptor antagonist activity
CO2H
Cl
X
O
O
S
N
H
II
R
a
Compound
X
R
Binding Ki (lM)
IC50 (lM)
mEPl
mEP2
mEP3
mEP4
mEPl
5
6
–NHCO–
–(CH2)2–
H
3.6
>10
1.4
>10
0.57
1.2
>10
>10
>10
>10
>10
>10
>10
>10
NTb
NTb
NTb
NTb
NTb
NTb
>10
Cl
Cl
Cl
Cl
Cl
Cl
H
0.33
1.2
7
–CH@CH– (Z)
–CH@CH– (E)
0.80
>10
0.047
3.5
8
0.34
0.16
1.8
>10
>10
>10
>10
>10
9
10
11
12
–CH2O–
–OCH2–
–OCH2–
0.076
0.047
0.94
>10
>10
a IC50, mEPl receptor antagonist activity.
b NT, not tested.
Table 3. Effect of transformation of the sulfonamide moiety on Ki values for subtype receptors and EP1 receptor antagonist activity
CO2H
Cl
O
X
III
a
Compound
X
Binding Ki (lM)
IC50 (lM)
mEPl
mEP2
>10
2.9
1.2
mEP3
mEP4
mEPl
12
13
14
–NHSO2–
–SO2NH–
–CH2O–
0.047
0.69
0.28
>10
>10
>10
>10
>10
>10
>10
>10
NTb
a IC50, mEP1 receptor antagonist activity.
b NT, not tested.
Table 4. Effect of the substitution of the aminophenolxy moiety on Ki values for subtype receptors and EP1 receptor antagonist activity
CO2H
5
O
X
O
O
S
4
N
H
IV
a
IC50 (M)
Compound
X
Binding Ki (lM)
mEPl
mEP2
mEP3
mEP4
mEPl
12
15
16
17
18
19
5-C1
5-F
0.047
0.24
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
NTb
NTb
>10
>10
NTb
5-CH3
5-OCH3
5-CF3
4-CH3
0.049
0.14
0.025
0.073
a IC50, mEPl receptor antagonist activity.
b NT, not tested.
Compound 13 was synthesized as shown in Scheme 4.
N-Sulfonylation of aniline with a sulfonyl chloride 53
afforded the sulfonamide 54. Replacement of the fluoro
residue of 54 with an alkoxide prepared from the alcohol
55 in the presence of tertiary butoxide led to the alde-
hyde 56, after which oxidation produced 13.

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5565
Table 5. Effect of N-alkylation on Ki values for subtype receptors and EP1 receptor antagonist activity
CO2H
F3C
O
O
N
R
O
S
V
a
Compound
R
Binding Ki (lM)
IC50 (lM)
mEPl
mEP2
mEP3
mEP4
mEPl
18
20
21
22
23
24
25
–H
–CH2CH3
0.025
>10
>10
>10
>10
>10
>10
6.2
>10
>10
0.72
>10
0.41
0.87
0.46
>10
>10
>10
>10
>10
4.1
>10
0.68
0.30
0.38
0.13
NTb
1.1
0.0029
0.00080
0.0016
0.00050
0.026
–(CH2)2CH3
–CH(CH3)2
–CH2CH(CH3)2
–CH2C(CH3)3
–CH2-cPentyl
0.0066
>10
a IC50, mEPl receptor antagonist activity.
b NT, not tested.
R
R
O
X
linker
X
X
R
d or e
a
O
O
O
S
S
N
H
N
NH2
H
Y
Y
26
X = H, R = CO2Me
29a-c R = CO2Me
30a-c R = CO2H
31a-c linker = -CONH-, R = CO2Me
2-4 linker = -CONH-, R = CO2H
b
c
b
b
27 X = Cl, R = CO2Me
28 X = Cl, R = NO2
29d R = NO2
30d R = NH2
31d linker = -NHCO-, R =CO2Me
5
linker = -NHCO-, R = CO2H
a; X = Y = H, b; X = Cl, Y = H, c; X = Y = Cl, d; X = Cl, Y = H
Scheme 1. Synthesis of 2–5. Reagents: (a) ArSO2Cl, pyridine, CH2Cl2; (b) NaOH, MeOH; (c) Fe, AcOH; (d) methyl 4-aminobenzoate, EDC, THF
for 31a–c; (e) methyl 4-(chlorocarbonyl)benzoate, pyridine, CH2Cl2 for 31d.
a
R
CO2Me
Cl
X
Cl
X
e
a
O
O
X
Y
Cl
S
N
H
NO2
Cl
R = CO2Me, X = -CH=CH- (Z)
R = CO2H, X = -CH=CH- (Z)
R = CO2Me, X = -CH=CH- (E)
R = CO2H, X = -CH=CH- (E)
R = CO2Me, X = -C≡ C-
40(Z)
35(E/Z)
36(Z)
X = -CH=CH- (E+Z), Y = NO
2
f
f
f
32
33
X = CHO
X = COCl
b, c
7
40(E)
8
X = -CH=CH- (Z), Y = NH2
X = -CH=CH- (E), Y = NH2
X = -COC(=PPh3)-, Y = NO2
X = -C ≡C-, Y = NO2
{
36(E)
37
38
39
d
b
41
9
g
Br
CO2Me
R = CO2H, X = -C≡C-
R = CO2H, X = -(CH2)2-
X = -C≡C-, Y = NH2
Ph3P+
6
34
b
R
CO2Me
Cl
e
Cl
X
NO2
j
O
Cl
O
O
O
S
N
H
Y
Cl
32 X = CHO
42 X = CH2OH
X = CH2OMs
44 Y = NO2
h
i
R = CO2Me
R = CO2H
46
10
b
f
Y = NH
45
2
43
Scheme 2. Synthesis of 6–10. Reagents: (a) 34, tBuOK, THF; (b) Fe, HCl, THF, H2O; (c) separation; (d) o-dichlorobenzene, heat; (e) 4-Cl-PhSO2Cl,
pyridine, CH2Cl2; (f) NaOH, MeOH; (g) H2, PtO2, THF; (h) NaBH4, EtOH; (i) MsCl, Et3N, CH2Cl2; (j) methyl 4-hydroxybenzoate, K2CO3, acetone.

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
CO2Me
R
X
a
5
5
5
O
Y
O
O
OH
a
c
O
W
4
W
4
W
4
S
N
H
NO2
47a-f
Y = NO2
Y = NH2
48a-f
49a-f
b
X = H, R = CO Me
50a-f
12 and 15-19
2
d
d
X = H, R = CO H
2
a: W = 5-Cl
b: W = 5-F
c: W = 5-Me
d: W = 5-MeO
e: W = 5-CF3
X = Cl, R = CO Me
51a
11
2
X = Cl, R = CO H
2
f: W = 4-Me
CO2Me
b
1
R
F3C
O
F3C
O
O
N
R
e
R2-I
O
O
+
g: R2 = Ethyl
O
S
n
h: R2 = Propyl
S
N
H
i
i: R2 = Propyl
2
i
j: R2 = Butyl
51e
k: R2 = CH2-tButyl
l: R2 = CH2-cPentyl
R1 = CO2Me
R1 = CO2H
52g-l
20-25
d
Scheme 3. Synthesis of 11–12 and 15–25. Reagents: (a) methyl 4-(bromomethyl)benzoate, K2CO3, DMF; (b) Fe, AcOH; (c) ArSO2Cl, pyridine,
CH2Cl2; (d) NaOH, MeOH; (e) K2CO3, DMF.
R
Cl
F
S
Cl
O
S
Cl
F
a
b
H
N
H
N
O
O
SO2Cl
O
O
53
54
R = CHO
R = CO2H
56
13
c
CH(OMe)2
HO
55
Scheme 4. Synthesis of 13. Reagents: (a) aniline, pyridine, CH2Cl2; (b) 55, tBuOK, DMA and then HCl; (c) NaClO2, isobutene, NaH2PO4, CH3CN,
H2O.
R
CO2Me
Cl
O
Cl
OH
X
Cl
O
f
c
O
Y
63
14
X = CO2Me
R = CO2Me
R = CO2H
57
58
59
Y = OTBS
Y = OH
Y = Br
60
61
62
a
b
d
g
X = CH OH
2
e
X = CH OTBS
2
Scheme 5. Synthesis of 14. Reagents: (a) LAH; (b) TBSCl, Et3N, DMAP, THF; (c) methyl 4-(bromomethyl)benzoate, K2CO3, acetone; (d) TBAF,
THF; (e) CBr4, PPh3, CH2Cl2; (f) PhOH, K2CO3, acetone; (g) NaOH, MeOH.
Synthesis of 14 is outlined in Scheme 5. Lithium alumi-
num hydride reduction of the ester 57 provided the
alcohol 58, which was protected as a TBS ether 59.
O-Alkylation of 59 with methyl 4-(bromomethyl)benzo-
ate afforded the ether 60, which was deprotected with
TBAF to give the alcohol 61. Bromination of 61 with
carbon tetrachloride and triphenylphosphine led to the
bromide 62. O-Alkyaltion of phenol with 62 provided
63, after which alkaline hydrolysis resulted in the
production of 14.
3. Results and discussion
The compounds listed in Tables 1–5 were biologically
evaluated for their inhibition of the specific binding of a
radiolabeled ligand [3H]PGE2 to membrane fractions pre-
pared from cells stably expressing each mouse prostanoid
receptor.12 The EP1 antagonist activity of these com-
pounds was determined by a Ca2+ assay using the mouse
EP1 receptor (mEP1) expressed in mouse CHO cells in the
presence of 0.1% of bovine serum albumin (BSA).

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5567
During the course of screening our in-house compound
library, compound 1 was found to show subtype-selec-
tive EP1 receptor affinity (Table 1). Removal of the
metabolically unstable para-ester moiety and the meta-
methyl residue of 1 afforded 2, which showed 5.0-fold
less potent EP1 receptor affinity. Introduction of a
4-chloro residue into the anthranilic acid moiety of 2
afforded 3, which had 2.7-fold more potent receptor
affinity.
5-methoxy analog 17, and 4-methyl analog 19 all
showed a decrease of receptor affinity. Among the com-
pounds tested, the 5-trifluoromethyl analog 18 showed
the most potent EP1 receptor affinity.
The effect of N-alkylation of 18 on receptor affinity and
EP1 receptor antagonist activity was investigated as
shown in Table 5. N-Ethylation of 18 afforded 20, which
had 8.6-fold more potent EP1 receptor affinity. In addi-
tion, 20 exhibited EP1 receptor antagonist activity
(IC50 = 0.68 lM), while 18 did not show antagonist
activity at 10 lM. Replacement of the N-ethyl moiety
of 20 with an N-n-propyl moiety gave 21, with a
significant increase of both EP1 receptor affinity and
antagonist activity. The corresponding N-isopropyl
analog 22 was slightly less potent in terms of both
receptor affinity and antagonist activity. Replacement
of the N-ethyl moiety of 20 with N-isobutyl moiety
afforded 23, which showed 5.8-fold more potent EP1
receptor affinity and 5.2-fold more potent antagonist
activity. Replacement of the N-ethyl moiety of 20 with
an N-neopentyl moiety led to 24, which had reduced
EP1 receptor affinity, and showed weak affinity for the
EP3 and EP4 receptors.
Replacement of the phenylsulfonyl moiety of 3 with a
4-chlorophenylsulfonyl moiety provided 4, which
retained its EP1 receptor affinity and showed more
potent EP2 receptor affinity. Further optimization
was initiated with the chemical modification of 3 and
4. Optimization of the carboxyamide moiety of the
chemical leads was carried out as shown in Table 2.
Transformation of the carboxyamide moiety of 3 into
an aminocarbonyl moiety gave 5, which showed 10-
fold less potent EP1 receptor affinity. Replacement of
the carboxyamide moiety of 4 with an ethylene moiety
produced 6, which retained EP1 receptor affinity and
showed increased affinity for the EP3 receptor.
Replacement of the carboxyamide moiety of 4 with a
cis-double bond and trans-double bond led to 7 and
8, respectively. The trans-isomer 8, which is relatively
close to the carboxyamide structure, showed more
potent EP1 receptor affinity than the cis-isomer 7 and
compound 8 retained potent EP1 receptor affinity.
Replacement of the carboxyamide moiety of 4 with a
triple bond led to 9, which had 2.5-fold more potent
EP1 receptor affinity and showed a marked increase
of EP3 receptor affinity. Replacement of the carboxya-
mide moiety of 4 with methyleneoxy and oxymethylene
moieties provided 10 and 11, respectively, with 4.5-fold
less potent and 5.3-fold more potent EP1 receptor
affinity, respectively. Removal of the chloro residue
from the more potent ether analog 11 gave 12, which
had 1.6-fold more potent EP1 receptor affinity. Com-
pounds 11 and 12 did not exhibit antagonist activity
at 10 lM, although both showed higher EP1 receptor
affinity than the other compounds listed in Tables 1
and 2. Overall, phenylsulfonylamino analogs 3 and 12
tended to show better subtype selectivity than the cor-
responding 4-chlorophenylsulfonyl analogs 4 and 11,
respectively.
N-Cyclopentylmethyl analog 25 exhibited 2.3-fold less
potent receptor affinity and 1.6-fold less potent antago-
nist activity. Among the compounds tested, the N-isobu-
tyl analog 23 showed the most potent EP1 receptor
affinity and antagonist activity.
In summary,
a series of 4-({2-[alkyl(phenylsulfo-
nyl)amino]phenoxy}methyl)benzoic acids 21–23 were
identified as new chemical leads for selective mEP1
receptor antagonists after optimization starting from
the newly found chemical leads 3–4 (Table 1) that
were derived from the initial chemical lead 1. During
this process, the oxymethyl moiety and N-alkylsulf-
onamide moiety were found to be essential for both
enhanced mEP1 receptor affinity and receptor antago-
nist activity. Further optimization will be reported in
due course.
4. Experimental
4.1. General directions
As illustrated in Table 3, the effect on Ki value of trans-
formation of the phenylsulfonylamino moiety of 12
was investigated. The corresponding phenylaminosulfo-
nyl analog 13 demonstrated a 15-fold decrease of
receptor affinity. Replacement of the sulfonylamino
moiety of 12 with an oxymethyl moiety led to 14, with
6.0-fold less potent EP1 receptor affinity and increased
affinity for the EP2 receptor. As a result, the phen-
ylsulfonylamino moiety was confirmed to be an opti-
mum structure.
Analytical samples were homogeneous as confirmed by
TLC, and afforded spectroscopic results consistent with
the assigned structures. Proton nuclear magnetic
resonance spectra (1H NMR) were taken on a Varian
Mercury 300 spectrometer or Varian GEMINI-200 or
VXR-200s spectrometer using deuterated chloroform
(CDCl3) or deuterated dimethylsulfoxide (DMSO-d6)
as the solvent. Fast atom bombardment mass spectra
(FAB-MS, HRMS) and electron ionization (EI) were
obtained on a JEOL JMS-DX303HF spectrometer.
Atmospheric pressure chemical ionization (APCI) was
determined on a HTHACHI MI200H spectrometer.
Infrared spectra (IR) were measured in a Perkin-Elmer
FT-IR 1760X spectrometer. Melting points and results
of elemental analyses were uncorrected. Column
As shown in Table 4, the effect on Ki values of substitut-
ing the aminophenoxy moiety of 12 was investigated.
The 5-methyl analog 16 and the 5-trifluoromethyl ana-
log 18 respectively retained or had slightly more potent
receptor affinity than 12, while the 5-fluoro analog 15,

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
chromatography was carried out on silica gel [Merck sil-
ica gel 60 (0.063–0.200 mm), Wako gel C200 or Fuji
Silysia FL60D]. Thin layer chromatography was per-
formed on silica gel (Merck TLC or HPTLC plates, sil-
ica gel 60 F254). The following abbreviations for
solvents and reagents are used; tetrahydrofuran
(THF), diethylether (Et2O), dimethylsulfoxide (DMSO),
ethyl acetate (EtOAc), dimethylformamide (DMF),
dichloromethane (CH2Cl2), chloroform (CHCl3), meth-
anol (MeOH), acetic acid (AcOH), hydrochloric acid
(HCl), and triethylamine (TEA).
According to the same procedure as described above,
30b–c were prepared from the corresponding esters
29b–c, respectively.
4.3.2. 5-Chloro-2-[(phenylsulfonyl)amino]benzoic acid
(30b). Yield 100%; 1H NMR (200 MHz, CDCl3) d
10.31 (s, 1H), 7.99 (d, J = 2.5 Hz, 1H), 7.87 (m, 2H),
7.70 (d, J = 9.0 Hz, 1H), 7.63–7.42 (m, 4H).
4.3.3. 5-Chloro-2-{[(4-chlorophenyl)sulfonyl]amino}-
benzoic acid (30c). Yield 98%; TLC Rf = 0.24 (MeOH/
1
CHCl3, 1:4); H NMR (300 MHz, CDCl3) d 11.06 (br
s, 1H), 7.84–7.76 (m, 3H), 7.64–7.56 (m, 3H), 7.49 (d,
J = 8.7 Hz, 1H).
4.2. General procedure for the preparation of methyl
2-[(phenylsulfonyl)amino]benzoate analogs (29a–c)
4.2.1. Methyl 2-[(phenylsulfonyl)amino]benzoate (29a).
To a stirred solution of methyl 2-aminobenzoate 26
(1.51 g, 10 mmol) and pyridine (2.4 mL, 30 mmol) in
CH2Cl2 (10 ml) was added a solution of benzenesulfonyl
chloride (2.3 g, 13 mmol) in CH2Cl2 (5 ml) at room tem-
perature under argon atmosphere. The reaction mixture
was stirred for 3 h, the reaction mixture was quenched
with water and 1 M HCl, and extracted with CH2Cl2,
dried over MgSO4. The organic layer was concentrated
in vacuo. The resulting residue was recrystallized
from EtOAc and hexane to yield 29a (2.5 g, 86%).
TLC Rf = 0.40 (EtOAc/hexane, 1:2); 1H NMR
(300 MHz, CDCl3) d 10.65 (br s, 1H), 7.91 (dd, J = 7.8,
1.8 Hz, 1H), 7.88–7.83 (m, 2H), 7.70 (dd, J = 8.4,
1.2 Hz, 1H), 7.560–7.40 (m, 4H), 7.04 (dt, J = 8.4,
1.8 Hz, 1H), 3.87 (s, 3H).
4.4. N-(4-Chloro-2-nitrophenyl)benzenesulfonamide (29d)
The title compound 29d was prepared from 28 in 19%
yield according to the same procedure as described for
the preparation of 29a from 26. TLC Rf = 0.37
(EtOAc/hexane, 1:5); 1H NMR (200 MHz, CDCl3) d
9.85–9.65 (br s, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.90–
7.78 (m, 3H), 7.68–7.44 (m, 4H).
4.5. N-(2-Amino-4-chlorophenyl)benzenesulfonamide
(30d)
To a stirred solution of 29d (172 mg, 0.55 mmol) in
AcOH (4 ml) was added iron (325 mesh, 154 mg,
2.75 mmol) at room temperature. The reaction mix-
ture was heated at reflux for 2 h, cooled to room
temperature, and diluted with EtOAc. Insoluble sub-
stance was removed by filtration through a pad of
Celite. The filtrate was concentrated in vacuo. The
resulting residue was purified by column chromatog-
raphy on silica gel to yield 30d (92 mg, 59%). TLC
Rf = 0.34 (EtOAc/hexane, 1:2); 1H NMR (200 MHz,
CDCl3) d 7.80–7.70 (m, 2H), 7.70–7.44 (m, 3H),
6.73 (d, J = 2.0 Hz, 1H), 6.46 (dd, J = 8.0, 2.0 Hz,
1H), 6.33 (d, J = 8.0 Hz, 1H), 6.15 (br s, 1H), 4.50–
3.90 (br s, 2H).
According to the same procedure as described above,
29b–c were prepared from the aniline 27.
4.2.2. Methyl 5-chloro-2-[(phenylsulfonyl)amino]benzoate
(29b). Yield 94%; TLC Rf = 0.30 (EtOAc/hexane, 1:4);
1H NMR (200 MHz, CDCl3) d 10.51 (s, 1H), 7.90–
7.78 (m, 3H), 7.68 (d, J = 9.0 Hz, 1H), 7.60–7.37 (m,
4H), 3.88 (s, 3H).
4.2.3. Methyl 5-chloro-2-{[(4-chlorophenyl)sulfonyl]-
amino}benzoate (29c). Yield 88%; TLC Rf = 0.41
(EtOAc/hexane, 1:4); 1H NMR (200 MHz, CDCl3) d
10.53 (s, 1H), 7.90 (d, J = 2.5 Hz, 1H), 7.76 (d,
J = 8.5 Hz, 2H), 7.49–7.37 (m, 3H), 3.89 (s, 3H).
4.6. General procedure for the preparation of methyl
4-({2-[(phenylsulfonyl)amino]benzoyl}amino)benzoate
analogs (30a–c)
4.6.1. Methyl 4-({2-[(phenylsulfonyl)amino]benzoyl}ami-
no)benzoate (31a). To a stirred suspension of 30a (2 g,
7.21 mmol) and methyl 4-aminobenzoate (1.2 g,
7.94 mmol) in CH2Cl2 (40 ml) were added EDCÆHCl
(1.3 g, 7.21 mmol) and 4-DMAP (44 mg, 0.361 mmol).
The reaction mixture was stirred for 4 days at room
temperature, quenched with water and extracted with
EtOAc. The organic layer was washed with water,
then brine, dried over MgSO4 and concentrated in
vacuo to afford a residue, which was purified by col-
umn chromatography on silica gel to yield 31a
(722 mg, 21%). TLC Rf = 0.47 (EtOAc/hexane, 2:1);
1H NMR (200 MHz, CDCl3) d 10.14 (br s, 1H),
8.05 (d, J = 6.8 Hz, 2H), 7.74 (m, 4H), 7.59 (d,
J = 6.8 Hz, 2H), 7.26–7.51 (m, 5H), 7.15 (m, 1H),
3.93 (s, 3H).
4.3. General procedure for the preparation of 2-[(phen-
ylsulfonyl)amino]benzoic acids (30a–c)
4.3.1. 2-[(Phenylsulfonyl)amino]benzoic acid (30a). To a
stirred solution of 29a (2 g, 6.87 mmol) in MeOH
(20 ml)—DME (10 ml)—THF (5 ml) was added 2 M
NaOH (13.8 ml, 27.6 mmol) at 50 ꢁC. The reaction mix-
ture was stirred for 5 h, cooled to room temperature and
acidified with 2 M HCl. The resulting precipitates were
collected by filtration and dried in vacuo at 70 ꢁC for
overnight to yield 30a (1.9 g, 95%). TLC Rf = 0.20
1
(MeOH/CHCl3, 1:3); H NMR (300 MHz, DMSO-d6)
d 13.83 (br s, 1H), 11.11 (br s, 1H), 7.87 (d,
J = 7.2 Hz, 1H), 7.82–7.74 (m, 2H), 7.63–7.44 (m, 5H),
7.10–7.03 (m, 1H).

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5569
1
According to the same procedure as described above,
31b–c were prepared from the corresponding carboxylic
acids 30b–c, respectively.
CHCl3, 3:17); H NMR (200 MHz, DMSO-d6) d 12.74
(br s, 1H), 10.61 (s, 1H), 10.40 (s, 1H), 7.95 (2H, d,
J = 8.6 Hz, 2H), 7.85–7.71 (m, 5H), 7.64–7.35 (m, 5H);
IR (KBr) 3323, 3153, 1689, 1595, 1533, 1480, 1421,
1386, 1323, 1290, 1253, 1167 cmꢀ1; MS (APCI, Neg.)
m/e 429 (MꢀH)ꢀ.
4.6.2. Methyl 4-({5-chloro-2-[(phenylsulfonyl)amino]ben-
zoyl}amino)benzoate (31b). Yield 40%; TLC Rf = 0.29
1
(EtOAc/benzene, 1:9); H NMR (200 MHz, CDCl3) d
10.40 (s, 1H), 9.889 (m, 1H), 8.03 (dd, J = 8.5, 2.5 Hz,
2H), 7.83–7.70 (m, 4H), 7.63 (dd, J = 8.5, 2.5 Hz, 1H),
7.53–7.27 (m, 5H), 3.93 (s, 3H).
4.8.3. 4-[(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]amino}ben-
zoyl)amino]benzoic acid (4). Yield 100%; TLC Rf = 0.27
1
(MeOH/CHCl3, 3:17); H NMR (200 MHz, DMSO-d6)
d 12.70 (br s, 1H), 10.59 (s, 1H), 10.30 (s, 1H), 7.83–
7.65 (m, 5H), 7.62–7.47 (m, 3H), 734 (d, J = 9.0 Hz,
2H); IR (KBr) 3332, 3150, 1689, 1595, 1532, 1478, 1421,
1387, 1325, 1285, 1254, 1166 cmꢀ1; MS (APCI, Neg.) m/
e 463 (MꢀH)ꢀ.
4.6.3. Methyl 4-[(5-chloro-2-{[(4-chlorophenyl)sulfo-
nyl]amino}benzoyl)amino]benzoate (31c). Yield 48%;
TLC Rf = 0.32 (EtOAc/hexane, 3:7); 1H NMR
(200 MHz, CD3OD) d 8.05 (d, J = 8.5 Hz, 2H), 7.74–
7.58 (m, 6H), 7.47 (dd, J = 8.5, 2.5 Hz, 1H), 7.27 (d,
J = 8.5 Hz, 2H), 3.95 (s, 3H).
4.8.4. 4-[({5-Chloro-2-[(phenylsulfonyl)amino]phenyl}ami-
no)carbonyl]benzoic acid (5). Yield 100%; TLC Rf = 0.36
(EtOAc/hexane/AcOH, 8:10:1); 1H NMR (200 MHz,
CDCl3) d 13.00 (br s, 1H), 9.80 (br s, 1H), 9.65 (s,
1H), 8.09 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H),
7.81 (d, J = 2.5, 1H9), 7.65–7.50 (m, 3H), 7.40 (t,
J = 7.5 Hz, 2H), 7.22 (dd, J = 8.5, 5.2 Hz, 1H), 7.14 (d,
J = 8.5 Hz, 1H).IR (KBr) 3321, 1693, 1657, 1590,
1524, 1491, 1425, 1330, 1924, 1163, 1097 cmꢀ1; MS
(FAB, Pos.) 431 (M+H)+.
4.7. Methyl 4-[({5-chloro-2-[(phenylsulfonyl)amino]phen-
yl}amino)carbonyl]benzoate (31d)
To a stirred solution of 30d (90 mg, 0.319 mmol) and
pyridine (0.05 ml, 0.637 mmol) in CH2Cl2 (5 ml) was
added methyl 4-(chlorocarbonyl)benzoate (70 mg,
0.35 mmol) at room temperature under argon atmo-
sphere. The reaction mixture was stirred for 6 h,
quenched with water and extracted with EtOAc (3·).
The combined organic layers were washed with water,
brine, dried over MgSO4 and evaporated. The resulting
residue was crystallized from EtOAc and hexane to yield
31d (112 mg, 79%). TLC Rf = 0.55 (EtOAc/hexane, 1:1);
1H NMR (200 MHz, CDCl3 + DMSO-d6) d 9.41 (br s,
1H), 8.93 (br s, 1H), 8.22 (d, J = 2.0 Hz, 1H), 8.15 (d,
J = 9.0 Hz, 2H), 7.98 (d, J = 9.0 Hz, 2H), 7.72–7.62
(m, 2H), 7.58–7.45 (m, 1H), 7.44–7.32 (m, 2H), 6.96
(dd, J = 8.5, 2.0 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H),
3.98 (s, 3H).
4.9. Methyl 4-[(E/Z)-2-(5-chloro-2-nitrophenyl)vinyl]-
benzoate (35E) and (35Z)
To a stirred suspension of [4-(methoxycarbonyl)ben-
zyl](trimethyl)phosphonium bromide 34 (4.83 g,
10 mmol) in THF (20 ml) and MeOH (0.2 ml) was add-
ed potassium tert-butoxide (1.12 g, 10 mmol) at room
temperature under argon atmosphere. After 1.5 h, 5-
chloro-2-nitrobenzaldehyde 33 (742 mg, 4.00 mmol)
was added to the reaction mixture and stirring was con-
tinued for additional 30 min at room temperature. The
reaction mixture was quenched with 1 M HCl and
extracted with EtOAc (2·). The combined organic layers
were washed with water, brine, dried over MgSO4 and
evaporated. The resulting residue was purified by col-
umn chromatography on silica gel to yield a mixture
of 35E and 35Z (680 mg, 56%). TLC Rf = 0.44
(EtOAc/hexane, 1:4); MS (EI, Pos.) m/e 317 (M+).
4.8. General procedure for the preparation of 4-({2-[(phen-
ylsulfonyl)amino]benzoyl}amino)benzoic acids (2–5)
4.8.1. 4-({2-[(Phenylsulfonyl)amino]benzoyl}amino)benzo-
ic acid (2). To a stirred solution of 31a (715 mg,
1.74 mmol) in EtOH (8 ml) was added 5 M NaOH
(1.1 ml) at room temperature. The reaction mixture
was stirred for 3.5 days, acidified with 2 M HCl
(3.0 ml) and extracted with EtOAc. The organic layer
was washed with water, then brine, dried over MgSO4
and concentrated in vacuo to yield 2 (677 mg, 98%).
TLC Rf = 0.50 (MeOH/CHCl3/AcOH, 10:100:1); 1H
NMR (200 MHz, DMSO-d6) d 12.76 (br s, 1H), 10.57
(s, 1H), 10.49 (s, 1H), 7.95 (d, J = 8.8 Hz, 2H),
7.77 (m, 5H), 7.62–7.28 (m, 5H), 7.24 (m, 1H); IR
(KBr) 3390, 2989, 1686, 1595, 1532, 1501, 1331, 1288,
1253, 1175, 1092 cmꢀ1; MS (APCI, Neg.) m/e 395
(MꢀH)ꢀ.
4.10. Methyl 4-[(E)-2-(2-amino-5-chlorophenyl)vinyl]ben-
zoate (36E) and methyl 4-[(Z)-2-(2-amino-5-chloro-phen-
yl)vinyl]benzoate (36Z)
To a stirred solution of a mixture of 35E and 35Z
(525 mg, 1.62 mmol) in THF (4 ml) and water
(1.5 ml) were added 2 M HCl (0.4 ml) and iron (325
mesh, 884 mg, 15.8 mmol) at room temperature under
argon. After 4 days, the reaction mixture was diluted
with EtOAc. Insoluble substance was removed by fil-
tration. The filtrate was washed with NaHCO3aq,
brine, dried over MgSO4 and concentrated in vacuo.
The resulting residue was purified by column chroma-
tography on silica gel to yield 36E (116 mg, 25%) and
36Z (321 mg, 69%). 36E: TLC Rf = 0.37 (EtOAc/
benzene, 1:19); 1H NMR (200 MHz, CDCl3) d 8.03
(d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.39
According to the same procedure as described above,
3–5 were prepared from the corresponding esters 31b–
d, respectively.
4.8.2. 4-({2-[(5-Chlorophenylsulfonyl)amino]benzoyl}ami-
no)benzoic acid (3). Yield 69%; TLC Rf = 0.32 (MeOH/

5570
A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
(d, J = 2.5 Hz, 1H), 7.18 (d, J = 16.0 Hz, 1H), 7.07 (dd,
J = 8.5, 2.5 Hz, 1H), 7.01 (d, J = 16.0 Hz, 1H), 6.56 (d,
J = 8.5 Hz, 1H), 3.93 (s, 3H), 3.83 (br s, 2H); 36Z:
TLC Rf = 0.41 (EtOAc/benzene, 5:95); 1H NMR
(200 MHz, CDCl3) d 7.88 (d, J = 8.5 Hz, 2H), 7.27
(d, J = 8.5 Hz, 2H), 7.05 (dd, J = 8.5, 2.0 Hz, 1H),
7.00 (d, J = 2.0 Hz, 1H), 6.71 (d, J = 12.0 Hz, 1H),
6.62 (d, J = 8.5 Hz, 1H), 6.54 (d, J = 12.0 Hz, 1H),
3.88 (s, 3H), 3.71 (br s, 2H).
J = 16.0 Hz, 1H); IR (KBr) 3220, 1688, 1606, 1478,
1425, 1330, 1293, 1092 cmꢀ1; MS (APCI, Neg.) m/e
446 (MꢀH)ꢀ.
4.15. 4-[2-(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]amino}-
phenyl)ethyl]benzoic acid (6)
A suspension of 8 (54 mg, 0.12 mmol) and platinum diox-
ide (3 mg, 0.013 mmol) in THF (4 ml) was stirred vigor-
ously at room temperature under hydrogen atmosphere.
After 2 h, the catalyst was removed by filtration through
a pad of Celite. The filtrate was concentrated in vacuo.
The resulting residue was washed with a mixture of
CH2Cl2 and hexane to yield 6 (46 mg, 85%). TLC
Rf = 0.42 (MeOH/CHCl3, 3:17); 1H NMR (200 MHz,
DMSO-d6) d 12.75 (br s, 1H), 9.88 (s, 1H), 7.84 (d,
J = 8.5 Hz, 2H), 7.72–7.57 (m, 4H), 7.32 (d, J = 2.5 Hz,
1H), 7.23 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H),
6.88 (d, J = 9.0 Hz, 1H). IR (KBr) 3285, 29676, 2667,
2538, 1684, 1611, 1484, 1398, 1337, 1288, 1169,
1095 cmꢀ1; MS (APCI, Neg) m/e 448 (MꢀH)ꢀ.
4.11. Methyl 4-[(Z)-2-(5-chloro-2-{[(4-chlorophenyl)sul-
fonyl]amino}phenyl)vinyl]benzoate (40Z)
The title compound 40Z was prepared from 36Z in 95%
yield according to the same procedure as described for
the preparation of 29a from 26 using 4-chloro-
benzenesulfonyl chloride instead of benzenesulfonyl
chloride. TLC Rf = 0.23 (EtOAc/benzene, 1:24); 1H
NMR (200 MHz, CDCl3) d 7.82 (d, J = 8.0 Hz, 2H),
7.57 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 9.0 Hz, 1H), 7.33
(d, J = 9.0 Hz, 2H), 7.24 (dd, J = 9.0, 2.0 Hz, 1H),
7.06 (d, J = 2.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 2H), 6.72
(d, J = 12.0 Hz, 1H), 6.48 (s, 1H), 6.20 (d, J = 12.0 Hz,
1H), 3.90 (s, 3H).
4.16. Methyl 4-[2-(5-chloro-2-nitrophenyl)-2-oxo-1-(tri-
phenylphosphoranylidene)ethyl]benzoate (37)
4.12. 4-[(Z)-2-(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]ami-
no}phenyl)vinyl]benzoic acid (7)
To a stirred suspension of [4-(methoxycarbonyl)benzyl]-
(triphenyl)phosphonium bromide (1.17 g, 2.38 mmol) in
THF (8 ml) was added potassium butoxide (246 mg,
t
The title compound 7 was prepared from 40Z in 46%
yield according to the same procedure as described for
the preparation of 2 from 31a. TLC Rf = 0.46 (MeOH/
2.19 mmol) at 0 ꢁC under argon atmosphere. After
30 min, a solution of 5-chloro-2-nitrobenzoyl chloride
33 (218 mg, 0.992 mmol) in THF (4 ml)was added drop-
wise. The reaction mixture was allowed to warm up to
room temperature and stirred for additional 3 h. The
reaction mixture was diluted with CHCl3, washed with
NH4Claq, water, brine and dried over MgSO4. The
organic layer was concentrated in vacuo. The resulting
residue was purified by column chromatography on sil-
ica gel to yield 37 (619 mg, 100%). TLC Rf = 0.26
1
CHCl3, 3:17); H NMR (200 MHz, DMSO-d6) d 10.05
(br s, 1H), 7.79–7.67 (m, 4H), 7.55 (d, J = 8.5 Hz, 2H),
7.30 (dd, J = 8.5, 2.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H),
7.00 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 2.5 Hz, 1H), 6.64
(m, 2H); IR (KBr) 3336, 3096, 1692, 1608, 14808,
1412, 1323, 1293, 1167 cmꢀ1; MS (APCI, Neg.) m/e
446 (MꢀH)ꢀ.
1
(MeOH/CHCl3, 1:100); H NMR (200 MHz, CDCl3) d
4.13. Methyl 4-[(E)-2-(5-chloro-2-{[(4-chlorophenyl)sulfo-
nyl]amino}phenyl)vinyl]benzoate (40E)
7.80–7.40 (m, 18H), 7.07 (m, 2H), 6.93 (dd, J = 8.0 Hz,
2H), 3.80 (s, 3H); IR (neat) 3065, 1715, 1521, 1436,
1348, 1275, 1103 cmꢀ1; MS (APCI, Pos.) m/e 594
(M+H)+.
The title compound 40E was prepared from 36E in 98%
yield according to the same procedure as described for
the preparation of 29a from 26 using 4-chloro-
benzenesulfonyl chloride instead of benzenesulfonyl
chloride. TLC Rf = 0.15 (EtOAc/benzene, 1:24); 1H
NMR (200 MHz, CDCl3) d 8.02 (d, J = 8.5 Hz, 2H),
7.63 (d, J = 8.5 Hz, 2H), 7.51 (s, 1H), 7.41–7.30 (m,
4H), 7.26–7.22 (m, 2H), 6.91 (d, J = 16.0 Hz, 1H), 6.81
(d, J = 16.0 Hz, 1H), 6.63 (s, 1H), 3.95 (s, 3H).
4.17. Methyl 4-[(5-chloro-2-nitrophenyl)ethynyl]benzoate
(38)
A solution of 37 (513 mg, 0.865 mmol) was heated at
180 ꢁC with stirring. After 9 h, the reaction mixture
was concentrated in vacuo and the resulting residue
was purified by column chromatography on silica gel
to yield 38 (189 mg, 69%). TLC Rf = 0.40 (EtOAc/hex-
ane, 1:7); 1H NMR (200 MHz, CDCl3) d 8.09 (d,
J = 8.8 Hz, 1H), 7.72 (d, J = 2.4 Hz, 1H), 7.66 (d,
J = 8.6 Hz, 2H), 7.46 (dd, J = 8.8, 2.4 Hz, 1H), 3.94 (s,
3H); IR (KBr) 3424, 2225, 1713, 1606, 1516, 1346,
1278, 1108 cmꢀ1; MS (FAB, Pos.) m/e 316 (M+H)+.
4.14. 4-[(E)-2-(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]ami-
no}phenyl)vinyl]benzoic acid (8)
The title compound 8 was prepared from 40E in 92%
yield according to the same procedure as described for
the preparation of 2 from 31a. TLC Rf = 0.46 (MeOH/
1
CHCl3, 3:17); H NMR (200 MHz, DMSO-d6) d 10.11
4.18. Methyl 4-[(2-amino-5-chloro-phenyl)ethynyl]benzo-
ate (39)
(br s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.80 (d,
J = 2.5 Hz, 1H), 7.59 (d, J = 9.0 Hz, 2H), 7.52–7.41
(m, 4H), 7.36 (dd, J = 8.5, 2.5 Hz, 1H), 7.20 (d,
J = 8.5 Hz, 1H), 7.15 (d, J = 16.0 Hz, 1H), 7.08 (d,
The title compound 39 was prepared from 38 in 88%
yield according to the same procedure as described for

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5571
the preparation of 36Z and 36E from the mixture of
35E and 35Z. TLC Rf = 0.22 (EtOAc/hexane, 1:5);
1H NMR (200 MHz, CDCl3) d 8.03 (d, J = 8.8 Hz,
2H), 7.57 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 2.6 Hz,
1H), 7.11 (dd, J = 8.8, 2.6 Hz, 1H), 6.66 (d,
J = 8.8 Hz, 1H), 4.29 (br s, 2H), 23.93 (s, 3H); IR
(KBr) 3481, 3382, 2203, 1713, 1620, 1604, 1488,
1438, 1279, 1252, 1150, 1111 cmꢀ1; MS (APCI, Neg.)
m/e 284 (MꢀH)ꢀ.
with 2 M HCl, brine, dried over MgSO4 and evapo-
rated. The resulting residue was concentrated in
vacuo to yield 43 (600 mg). TLC Rf = 0.36 (EtOAc/
hexane, 1:2); 1H NMR (200 MHz, CDCl3) d 8.17
(d, J = 8.6 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H),
7.53 (dd, J = 8.6, 2.4 Hz, 1H), 5.66 (s, 2H), 3.16
(s, 3H).
4.23. Methyl 4-[(5-chloro-2-nitrobenzyl)oxy]benzoate (44)
4.19. Methyl 4-[(5-chloro-2-{[(4-chlorophenyl)sulfonyl]-
amino}phenyl)ethynyl]benzoate (41)
To a stirred solution of 43 (600 mg) in acetone (20 ml)
were added methyl 4-hydroxybenzoate (425 mg,
2.79 mmol) and K2CO3 (900 mg, 6.51 mmol). After
being stirred for 22 h at room temperature under argon
atmosphere, the reaction mixture was filtered through a
pad of Celite and the filtrate was concentrated in vacuo.
The resulting residue was purified by column chroma-
tography on silica gel to yield 44 (464 mg, 67% in 2
The title compound 41 was prepared from 39 in 94%
yield according to the same procedure as described for
the preparation of 29a from 26 using 4-chloro-
benzenesulfonyl chloride instead of benzenesulfonyl
chloride. TLC Rf = 0.50 (EtOAc/hexane, 1:3); 1H
NMR (200 MHz, CDCl3) d 8.07 (d, J = 8.4 Hz, 2H),
7.67 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.8 Hz, 1H), 7.49
(d, J = 8.4 Hz, 2H), 7.39 (d, J = 2.4 Hz, 1H), 7.34 (d,
J = 8.6 Hz, 2H), 7.32 (dd, J = 8.8, 2.4 Hz, 1H), 7.07
(br s, 1H), 3.96 (s, 3H).; MS (APCI, Pos.) m/e 460
(M+H)+.
1
steps). TLC Rf = 0.26 (EtOAc/hexane, 1:2); H NMR
(200 MHz, CDCl3) d 8.18 (d, J = 8.8 Hz, 1H), 8.04 (d,
J = 9.2 Hz, 2H), 7.91 (d, J = 2.2 Hz, 1H), 7.48 (dd,
J = 8.8, 2.2 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 5.53 (s,
2H), 3.90 (s, 3H).
4.24. Methyl 4-[(2-amino-5-chlorobenzyl)oxy]benzoate
(45)
4.20. 4-[(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]amino}-
phenyl)ethynyl]benzoic acid (9)
The title compound 45 was prepared from 44 according
to the same procedure as described for the preparation
of 36E and 36Z from the mixture of 35E and 35Z.
TLC Rf = 0.23 (EtOAc/hexane 1:2); 1H NMR
(200 MHz, CDCl3) d 8.01 (d, J = 8.8 Hz, 2H), 7.19 (d,
J = 2.4 Hz, 1H), 7.13 (dd, J = 8.4, 2.4 Hz, 1H), 7.00 (d,
J = 8.8 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 5.02 (s, 2H),
3.89 (s, 3H).
The title compound 9 was prepared from 41 in 94% yield
according to the same procedure as described for the
preparation of 2 from 31a. TLC Rf = 0.43 (MeOH/
1
CHCl3/AcOH, 5:100:1); H NMR (200 MHz, DMSO-
d6) d 13.16 (br s, 1H), 10.32 (br s, 1H), 8.00 (d,
J = 8.4 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.59 (d,
J = 2.6 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.50 (dd,
J = 8.5 Hz, 2.6 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.35
(d, J = 8.5 Hz, 1H); IR (KBr) 3455, 3270, 2845, 2545,
1688, 1608, 1481, 1425, 1392, 1347, 1314, 1285, 1172,
1093 cmꢀ1; MS (EI, Pos.) m/e 445 (M+), 382, 368, 356,
270.
4.25. Methyl 4-[(5-chloro-2-{[(4-chlorophenyl)sulfo-
nyl]amino}benzyl)oxy]benzoate (46)
The title compound 46 was prepared from 45 in 47%
yield according to the same procedure as described for
the preparation of 29a from 26 using 4-chloro-
benzenesulfonyl chloride instead of benzenesulfonyl
chloride. TLC Rf = 0.45 (EtOAc/benzene, 1:9); 1H
NMR (200 MHz, CDCl3) d 8.01 (d, J = 8.8 Hz, 1H),
7.62 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H),
7.32–7.26 (m, 3H), 7.11 (br s, 1H), 6.90 (d, J = 8.8 Hz,
2H), 4.80 (s, 2H), 3.90 (s, 3H).
4.21. (5-Chloro-2-nitrophenyl)methanol (42)
To a stirred solution of methyl 5-chloro-2-nitrobenzal-
dehyde 32 (800 mg, 4.31 mmol) in EtOH (20 ml) was
added NaBH4 (170 mg, 4.49 mmol) at 0 ꢁC. After
30 min, the reaction mixture was quenched with 1 M
HCl and extracted with EtOAc. The organic layer was
washed with NaHCO3aq, brine, dried over MgSO4
and concentrated in vacuo to yield 42 (792 mg, 97%).
TLC Rf = 0.47 (EtOAc/hexane, 1:2); 1H NMR
(200 MHz, CDCl3) d 8.09 (d, J = 8.6 Hz, 1H), 7.82 (d,
J = 2.4 Hz, 1H), 7.34 (dd, J = 8.6, 2.4 Hz, 1H), 5.08–
4.98 (br s, 2H), 2.50–2.34 (br s, 1H).
4.26. 4-[(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]ami-
no}benzyl)oxy]benzoic acid (10)
The title compound 10 was prepared from 46 in 67%
yield according to the same procedure as described for
the preparation of 2 from 31a. TLC Rf = 0.51 (EtOAc);
1H NMR (200 MHz, DMSO-d6) d 10.05 (br s, 1H), 7.90
(d, J = 8.7 Hz, 2H), 7.69 (d, J = 9.0 Hz, 2H), 7.61
(d, J = 9.0 Hz, 2H), 7.49 (d, J = 2.4 Hz, 1H), 7.36
(dd, J = 8.5, 2.4 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H),
6.92 (d, J = 8.7 Hz, 2H), 5.02 (s, 2H); IR (KBr) 3500,
3215, 1679, 1607, 1431, 1330, 1295, 1247, 1173, 1155,
1092, 1042, 1015 cmꢀ1; MS (APCI, Neg.) m/e 450
(MꢀH)ꢀ.
4.22. 5-Chloro-2-nitrobenzyl methanesulfonate (43)
To a stirred solution of 42 (400 mg, 2.13 mmol) in
CH2Cl2 (6 ml) were added Et3N (0.6 ml, 4.30 mmol)
and methanesulfonyl chloride (0.25 ml, 3.2 mmol) at
ꢀ10 ꢁC under argon atmosphere. After 15 min, the
reaction mixture was quenched with water and
extracted with EtOAc. The organic layer was washed

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
4.27. General procedure for the preparation of methyl
4-(2-nitrophenoxy)methylbenzoate analogs (48a–f)
mesh, 6.41 g, 115 mmol) under argon atmosphere. The
reaction mixture was stirred for 3 h at 50 ꢁC, the reac-
tion mixture was filtered through a pad of Celite. The fil-
trate was concentrated in vacuo, diluted with
NaHCO3aq and extracted with EtOAc. The organic
layer was washed with water, brine and dried over
MgSO4 and concentrated in vacuo to yield 49a (6.45 g,
100%). TLC Rf = 0.27 (EtOAc/benzene, 1:19); 1H
NMR (200 MHz, CDCl3) d 8.07 (d, J = 8.0 Hz, 2H),
7.49 (d, J = 8.0 Hz, 2H), 6.84–6.75 (m, 2H), 6.65 (d,
J = 9.0 Hz, 1H), 5.12 (s, 2H), 3.93 (s, 3H), 3.83 (br s, 2H).
4.27.1. Methyl 4-[(5-chloro-2-nitrophenoxy)methyl]benzo-
ate (48a). To a stirred suspension of 5-chloro-2-nitro-
phenol 47a (4.0 g, 23.1 mmol) and K2CO3 (3.83 g,
27.7 mmol) in DMF (40 ml) was added methyl 4-(bro-
momethyl)benzoate (5.83 g, 25.4 mmol) under argon
atmosphere. The resulting mixture was stirred for 1 h
at 50 ꢁC, then quenched by the addition of water, and
extracted with EtOAc. The organic layer was washed
with water, brine, dried over MgSO4 and concentrated
in vacuo. The resulting residue was recrystallized from
EtOH yielded 48a (7.11 g, 99%). TLC Rf = 0.49
According to the same procedure as described above,
49b–f were prepared from 48b–f, respectively.
1
(EtOAc/hexane, 1:10); H NMR (200 MHz, CDCl3) d
8.10 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.2 Hz, 1H), 7.54
(d, J = 8.6 Hz, 2H), 7.12–7.02 (m, 2H), 5.28 (s, 2H),
3.93 (s, 3H).
4.28.2. Methyl 4-[(2-amino-5-fluorophenoxy)methyl]ben-
zoate (49b). Yield 28%; TLC Rf = 0.65 (EtOAc/hexane,
1:1); 1H NMR (200 MHz, CDCl3)
d
8.07 (d,
J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 6.72–6.46
(m, 3H), 5.12 (s, 2H), 3.93 (s, 3H); MS (APCI, Pos.)
m/e 276 (M+H)+.
According to the same procedure as described above,
48b–f were prepared from 47b–f, respectively.
4.27.2. Methyl 4-[(5-fluoro-2-nitrophenoxy)methyl]benzo-
ate (48b). Yield 93%; TLC Rf = 0.26 (EtOAc/hexane,
4.28.3. Methyl 4-[(2-amino-5-methylphenoxy)methyl]ben-
zoate (49c). Yield 100%; TLC Rf = 0.47 (EtOAc/hexane,
1:2); 1H NMR (200 MHz, CDCl3) d 8.06 (d, J = 8.5 Hz,
2H), 7.51 (d, J = 8.5 Hz, 2H), 6.65 (s, 3H), 5.13 (s, 2H),
3.93 (s, 3H), 3.75 (br s, 2H), 2.24 (s, 3H).
1:5); 1H NMR (300 MHz, CDCl3)
d
8.09 (d,
J = 8.4 Hz, 2H), 8.05 (dd, J = 9.0, 6.0 Hz, 1H), 7.55 (d,
J = 8.4 Hz, 2H), 6.81 (dd, J = 9.6, 2.1 Hz, 1H), 6.76
(ddd, J = 9.0, 7.2, 2.1 Hz, 1H), 5.28 (s, 2), 3.93 (s, 3H).
4.28.4. Methyl 4-[(2-amino-5-methoxyphenoxy)meth-
yl]benzoate (49d). Yield 100%; TLC Rf = 0.39 (EtOAc/
hexane, 1:1); H NMR (300 MHz, CDCl3) d 8.05 (d,
J = 8.7 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 6.69 (d,
J = 8.7 Hz, 1H), 6.47 (d, J = 2.7 Hz, 1H), 3.79 (dd,
J = 8.7, 2.7 Hz, 1H), 3.93 (s, 3H), 3.72 (s. 3H), 1.19 (s,
2H).
4.27.3. Methyl 4-[(5-methyl-2-nitrophenoxy)methyl]ben-
zoate (48c). Yield 77%; TLC Rf = 0.24 (EtOAc/hexane,
1
1:5); 1H NMR (200 MHz, CDCl3)
d
8.07 (d,
J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.56 (d,
J = 8.0 Hz, 2H), 6.90 (s, 1H), 6.86 (d, J = 8.0 Hz, 1H),
5.27 (s, 2H), 3.93 (s, 3H), 2.40 (s, 3H).
4.27.4. Methyl 4-[(5-methoxy-2-nitrophenoxy)meth-
yl]benzoate (48d). Yield 97%; TLC Rf = 0.45 (EtOAc/
hexane, 1:1); H NMR (300 MHz, CDCl3) d 8.10–8.00
(m, 3H), 7.56 (d, J = 8.1 Hz, 2H), 6.60–6.50 (m, 2H),
5.26 (s, 2H), 3.92 (s, 3H), 3.85 (s, 3H).
4.28.5. Methyl 4-{[2-amino-5-(trifluoromethyl)pheno-
lxy]methyl}benzoate (49e). Yield 85%; TLC Rf = 0.50
(EtOAc/hexane, 1:5); 1H NMR (200 MHz, CDCl3) d
8.08 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.10
(d, J = 8.0 Hz, 1H), 7.04 (s, 1H), 6.75 (d, J = 8.0 Hz,
1H), 5.16 (s, 2H), 4.13 (br s, 2H), 3.94 (s, 3H).
1
4.27.5.
Methyl
4-{[2-nitro-5-(trifluoromethyl)phen-
oxy]methyl}benzoate (48e). Yield 89%; TLC Rf = 0.38
(EtOAc/hexane, 1:5); 1H NMR (300 MHz, CDCl3) d
8.10 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.7 Hz, 1H), 7.54
(d, J = 8.4 Hz, 2H), 7.40–7.25 (m, 2H), 5.33 (s, 2H),
3.94 (s, 3H).
4.28.6. Methyl 4-[(2-amino-4-methylphenoxy)methyl]ben-
zoate (49f). Yield 100%; TLC Rf = 0.52 (EtOAc/hexane,
1:2); 1H NMR (200 MHz, CDCl3) d 8.05 (d, J = 8.0 Hz,
2H), 7.50 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.0 Hz, 1H),
6.59 (d, J = 1.5 Hz, 1H), 6.49 (dd, J = 8.0, 1.5 Hz, 1H),
5.11 (s, 2H), 5.00–3.50 (br s, 2H), 3.92 (s, 3H), 2.22 (s, 3H).
4.27.6. Methyl 4-[(4-methyl-2-nitrophenoxy)methyl]ben-
zoate (48f). Yield 77%; TLC Rf = 0.24 (EtOAc/hexane,
4.29. General procedure for methyl 4-({2-[(phenylsulfo-
nyl)amino]phenoxy}methyl)benzoate analogs (50a–f)
1:5); 1H NMR (200 MHz, CDCl3)
d
8.06 (d,
J = 8.5 Hz, 2H), 7.69 (d, J = 1.5 Hz, 1H), 7.54 (d,
J = 8.5 Hz, 2H), 7.30 (dd, J = 8.5, 1.5 Hz, 1H), 7.98
(d, J = 8.5 Hz, 1H), 5.26 (s, 2H), 3.92 (s, 3H), 2.35 (s,
3H).
According to the same procedure as described for the
preparation of 29a–c from the corresponding anilines,
50a–f were prepared from the corresponding anilines
49a–f, respectively.
4.28. General procedure for the preparation of methyl
4-(2-aminophenoxy)methylbenzoate analogs (49a–f)
4.29.1.
no]phenoxy}methyl)benzoate (50a). Yield 85%; TLC
Methyl
4-({5-chloro-2-[(phenylsulfonyl)ami-
1
Rf = 0.31 (EtOH/benzene, 1:19); H NMR (200 MHz,
4.28.1. Methyl 4-[(2-amino-5-chlorophenoxy)methyl]ben-
zoate (49a). To a stirred solution of 48a (7.1 g, 23 mmol)
in AcOH (70 ml) and water (4.7 ml) was added iron (325
CDCl3) d 8.03 (d, J = 8.5 Hz, 2H), 7.70 (m, 2H), 7.56–
6.50 (m, 2H), 7.40 (m, 2H), 7.16 (d, J = 8.5 Hz, 2H),

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5573
6.95 (dd, J = 8.5, 2.0 Hz, 1H), 6.86 (s, 1H), 6.73 (d,
J = 2.0 Hz, 1H), 4.88 (s, 2H), 3.95 (s, 3H).
red solution of 51e (250 mg, 0.54 mmol) in DMF
(3 ml) were added K2CO3 (89 mg, 0.64 mmol) and ethyl
iodide (0.51 ml, 0.64 mmol). The reaction mixture was
stirred overnight at room temperature, then poured into
water and extracted with EtOAc (2·). The combined
organic layers were washed with water, brine, dried over
MgSO4 and concentrated in vacuo. The resulting residue
was purified by column chromatography on silica gel to
yield 52g (280 mg, 100%). TLC Rf = 0.64 (EtOAc/ben-
zene, 1:9); 1H NMR (200 MHz, CDCl3) d 8.00 (d,
J = 8.4 Hz, 2H), 7.70–7.60 (m, 2H), 7.50–7.10 (m, 8H),
4.89 (s, 2H), 3.95 (s, 3H), 3.67 (q, J = 7.2 Hz, 2H), 1.09
(t, J = 7.2 Hz, 3H); MS (APCI, Pos) m/e 494 (M+H)+.
4.29.2.
Methyl
4-({5-fluoro-2-[(phenylsulfonyl)ami-
no]phenoxy}methyl)benzoate (50b). Yield 88%; TLC
Rf = 0.59 (EtOAc/hexane, 2:3); 1H NMR (200 MHz,
CDCl3) d 8.03 (d, J = 8.5 Hz, 2H), 7.70–7.50 (m, 4H),
7.45–7.32 (m, 2H), 7.11 (d, J = 8.0 Hz, 2H), 6.73 (br s,
1H), 6.75–6.63 (m, 1H), 6.45 (dd, J = 10.0, 2.5 Hz,
1H), 4.82 (s, 2H), 3.95 (s, 3H).
4.29.3. Methyl 4-({5-methyl-2-[(phenylsulfonyl)ami-
no]phenoxy}methyl)benzoate (50c). Yield 87%; TLC
Rf = 0.43 (EtOAc/hexane, 1:2); 1H NMR (200 MHz,
CDCl3) d 8.01 (d, J = 8.0 Hz, 2H), 7.74–7.64 (m, 2H),
7.60–7.45 (m, 2H), 7.44–7.32 (m, 2H), 7.14 (d,
J = 8.0 Hz, 2H), 6.81 (br s, 1H), 6.85–6.73 (m 1H),
6.53 (s, 1H), 4.84 (s, 2H), 3.95 (s, 3H), 2.25 (s, 3H).
According to the same procedure as described above,
52h–l were prepared from 51e.
4.31.2. Methyl 4-{[2-[(phenylsulfonyl)(propyl)amino]-5-
(trifluoromethyl)phenoxy]methyl}benzoate (52h). Yield
1
100%; TLC Rf = 0.73 (EtOAc/hexane, 1:9); H NMR
4.29.4. Methyl 4-({5-methoxy-2-[(phenylsulfonyl)ami-
no]phenoxy}methyl)benzoate (50d). Yield 88%; TLC
Rf = 0.25 (EtOAc/hexane, 1:2); 1H NMR (200 MHz,
CDCl3) d 7.99 (d, J = 8.5 Hz, 2H), 7.66–7.45 (m, 4H),
7.35 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.61
(s, 1H), 6.50 (dd, J = 9.0, 2.0 Hz, 1H), 6.26 (d,
J = 2.5 Hz, 1H), 4.76 (s, 2H), 3.95 (s, 3H), 3.74 (s, 3H).
(200 MHz, CDCl3) d 8.01 (d, J = 8.4 Hz, 2H), 7.70–
7.60 (m, 2H), 7.50–7.10 (m, 8H), 4.87 (s, 2H), 3.95 (s,
3H), 3.58 (t, J = 7.6 Hz, 2H), 1.60–1.40 (m, 2H), 0.8 (t,
J = 7.6 Hz, 3H); MS (APCI, Pos.) m/e 508 (M+H)+.
4.31.3. Methyl {[2-[isopropyl(phenylsulfonyl)amino]-5-
(trifluoromethyl)phenoxy]methyl}benzoate (52i). Yield
75%; TLC Rf = 0.58 (EtOAc/hexane, 1:2); 1H NMR
(200 MHz, CDCl3) d 8.07 (d, J = 8.2 Hz, 2H), 7.79 (m
2H), 7.56–7.32 (m, 5H), 7.23 (m, 3H), 5.09 (s, 2H), 4.37
(sept, J = 6.6 Hz, 1H), 3.94 (s, 3H), 1.05 (d, J = 6.6 Hz, 6H).
4.29.5. Methyl 4-{[2-[(phenylsulfonyl)amino]-5-(trifluoro-
methyl)phenoxy]methyl}benzoate (50e). Yield 86%; TLC
1
Rf = 0.76 (acetone/benzene, 1:9); H NMR (200 MHz,
CDCl3) d 8.05 (d, J = 8.2 Hz, 2H), 7.77 (m, 2H), 7.69 (d,
J = 8.6 Hz, 1H), 7.58 (m, 1H), 7.45 (m, 2H), 7.25 (m,
3H), 7.18 (m, 1H), 6.99 (m, 1H), 5.02 (s, 2H), 3.95 (s, 3H).
4.31.4. Methyl {[2-[isobutyl(phenylsulfonyl)amino]-5-(tri-
fluoromethyl)phenoxy]methyl}benzoate
(52j).
Yield
1
4.29.6. Methyl 4-({4-methyl-2-[(phenylsulfonyl)ami-
no]phenoxy}methyl)benzoate (50f). Yield 88%; TLC
Rf = 0.43 (EtOAc/hexane, 1:2); 1H NMR (200 MHz,
CDCl3) d 8.00 (d, J = 8.5 Hz, 2H), 7.75–7.65 (m, 2H),
7.60–7.40 (m, 1H), 7.46–7.30 (m, 3H), 7.14 (d,
J = 8.5 Hz, 2H), 6.90 (br s, 1H), 6.81 (dd, J = 8.5,
2.0 Hz, 1H), 6.58 (d, J = 8.5 Hz, 1H), 4.85 (s, 2H),
3.94 (s, 3H), 2.28 (s, 3H).
100%; TLC Rf = 0.70 (EtOAc/benzene, 1:9); H NMR
(200 MHz, CDCl3) d 8.01 (d, J = 8.2 Hz, 2H), 7.70–
7.60 (m, 2H), 7.50–7.20 (m, 5H), 7.17 (d, J = 8.2 Hz,
2H), 7.09 (d, J = 1.2 Hz, 1H), 4.90–4.70 (m, 2H), 3.95
(s, 3H), 3.50–3.40 (m, 2H), 1.70–1.50 (m, 1H), 0.89 (d,
J = 6.6 Hz, 6H); MS (APCI, Pos.) m/e 522 (M+H)+.
4.31.5. Methyl {[2-[neopentyl (phenylsulfonyl)amino]-5-
(trifluoromethyl)phenoxy]methyl}benzoate (52k). Yield
39%; TLC Rf = 0.64 (EtOAc/hexane, 3:7); 1H NMR
(200 MHz, CDCl3) d 8.04 (d, J = 8.4 Hz, 2H), 7.60–7.40
(m, 4H), 7.30–7.20 (m, 5H), 7.07 (d, J = 1.6 Hz, 1H),
4.98 (d, J = 12.2 Hz, 1H), 4.67 (d, J = 12.2 Hz, 1H), 3.95
(s, 3H), 3.57 (d, J = 14.4 Hz, 1H), 3.48 (d, J = 14.4 Hz,
1H), 0.85 (s, 9H); MS (APCI, Pos.) m/e 536 (M+H)+.
4.30. Methyl 4-[(5-chloro-2-{[(4-chlorophenyl)sulfo-
nyl]amino}phenoxy)methyl]benzoate (51a)
The title compound 51a was prepared from 49a in 98%
yield according to the same procedure as described for
the preparation of 29a from 26 using 4-chloro-
benzenesulfonyl chloride instead of benzenesulfonyl
chloride. TLC Rf = 0.30 (EtOAc/benzene, 1:24); 1H
NMR (200 MHz, CDCl3) d 8.06 (d, J = 8.5 Hz, 2H),
7.59 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 9.0 Hz, 1H), 7.34
(d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 6.96 (dd,
J = 9.0, 2.0 Hz, 1H), 6.82 (br s, 1H), 6.76 (d, J = 2.0
Hz, 1H), 4.89 (s, 2H), 3.96 (s, 3H).
4.31.6. Methyl {[2-[(cyclopentylmethyl)(phenylsulfo-
nyl)amino]-5-(trifluoromethyl)phenoxy]methyl}benzoate
(52l). Yield 100%; TLC Rf = 0.51 (EtOAc/hexane, 1:3);
1H NMR (200 MHz, CDCl3) d 8.01 (d, J = 8.4 Hz, 2H),
7.63–7.58 (m, 2H), 7.48–7.25 (m, 5H), 7.17 (d,
J = 8.4 Hz, 2H), 7.09 (d, J = 1.4 Hz, 1H), 4.83 (br s,
2H), 3.95 (s, 3H), 3.50–3.40 (m, 2H), 1.92–1.09
(m, 9H); MS (APCI, Pos.) m/e 548 (M+H)+.
4.31. General procedure for methyl 4-{[2-[alkyl(phen-
ylsulfonyl)amino]-5-(trifluoromethyl)phenoxy]methyl}-
benzoate analogs (52g–l)
4.32. Synthesis of 11–12 and 15–25
4.31.1. Methyl 4-{[2-[ethyl(phenylsulfonyl)amino]-5-(tri-
fluoromethyl)phenoxy]methyl}benzoate (52g). To a stir-
Compounds 11–12 and 15–25 were prepared by the usu-
al alkaline hydrolysis of their corresponding esters.

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
4.32.1. 4-[(5-Chloro-2-{[(4-chlorophenyl)sulfonyl]ami-
no}phenoxy)methyl]benzoic acid (11). Yield 97%; TLC
Rf = 0.43 (MeOH/CHCl3, 3:17); 1H NMR (200
MHz, DMSO-d6) d 9.89 (br s, 1H), 7.93 (d, J = 8.0
Hz, 2H), 7.60 (d, J = 8.5Hz, 2H), 7.42 (d, J = 8.5 Hz,
2H), 7.34 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz,
1H), 7.06 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.0,
2.0 Hz, 1H), 4.98 (s, 2H); IR (KBr) 3331, 2877,
1689, 1595, 1498, 1424, 1395, 1353, 1252, 1172,
1123, 1095 cmꢀ1; MS (APCI, Neg.) m/e 450 (MꢀH)ꢀ.
4.32.7. 4-({4-Methyl-2-[(phenylsulfonyl)amino]phenolxy}-
methyl)benzoic acid (19). Yield 82%; TLC Rf = 0.43
(EtOAc/hexane/AcOH, 7:12:1); 1H NMR (200 MHz,
DMSO-d6) d 7.89 (d, J = 8.0 Hz, 2H), 7.66 (d, J =
7.0 Hz, 2H), 7.60–7.48 (m, 1H), 7.41 (d, J = 8.0 Hz, 2H),
7.35 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 2.0 Hz, 1H), 6.90
(dd, J = 8.0, 2.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 4.88 (s,
2H), 2.19 (s, 3H); IR (KBr) 3289, 2919, 1687, 1615, 1579,
1511, 1458, 1424, 1390, 1335, 1295, 1222, 1175, 1127,
1092, 1035, 1018 cmꢀ1; MS (FAB, Pos.) m/e 398 (M+H)+.
4.32.2.
4-({5-Chloro-2-[(phenylsulfonyl)amino]pheno-
4.32.8.
4-{[2-[Ethyl(phenylsulfonyl)amino]-5-(trifluoro-
lxy}methyl)benzoic acid (12). Yield 89%; TLC Rf = 0.39
(MeOH/CHCl3, 1:4); 1H NMR (200 MHz, DMSO-d6) d
12.98 (s, 1H), 9.78 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.65
(d, J = 8.5 Hz, 2H), 7.55 (t, J = 8.5 Hz, 1H), 7.41 (t,
J = 8.5 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.28 (d,
J = 8.5 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.98 (dd,
J = 8.5, 2.0 Hz, 1H), 4.98 (s, 2H); IR (KBr) 3260, 2996,
methyl)phenoxy]methyl}benzoic acid (20). Yield 76%;
TLC Rf = 0.43 (MeOH/CHCl3, 1:9); 1H NMR
(200 MHz, CDCl3) d 8.09 (d, J = 7.4 Hz, 2H), 7.70–
7.60 (m, 2H), 7.50–7.20 (m, 7H), 7.15 (d, J = 1.6 Hz,
1H), 4.94 (s, 2H), 3.69 (q, J = 7.4 Hz, 2H), 1.11 (t,
J = 7.4 Hz, 3H); IR (KBr) 2981, 2360, 1694, 1614,
1510, 1429, 1332, 1219, 1172, 1127, 1087, 1019 cmꢀ1
;
2887, 1687, 1598, 1426, 1339, 1290, 1265, 1168 cmꢀ1
;
MS (FAB, Pos.) m/e 480 (M+H)+.
MS (APCI, Neg.) m/e 416 (MꢀH)ꢀ.
4.32.9. {[2-[(Phenylsulfonyl)(propyl)amino]-5-(trifluoro-
methyl)phenoxy]methyl}benzoic acid (21). Yield 70%;
TLC Rf = 0.50 (MeOH/CHCl3, 1:9); 1H NMR
(200 MHz, CDCl3) d 8.09 (d, J = 8.2 Hz, 2H), 7.70–
7.60 (m, 2H), 7.50–7.20 (m, 7H), 7.14 (s, 1H), 4.92 (s,
2H), 3.59 (t, J = 7.4 Hz, 2H), 1.60–1.40 (m, 2H), 0.88
(t, J = 7.4 Hz, 3H); IR (KBr) 2970, 1694, 1614,
150,1428, 1332, 1171, 1127, 1087, 1018 cmꢀ1; MS
(FAB, Pos.) m/e 494 (M+H)+.
4.32.3.
4-({5-Fluoro-2-[(phenylsulfonyl)amino]pheno-
lxy}methyl)benzoic acid (15). Yield 85%; TLC Rf = 0.42
(EtOAc/hexane/AcOH, 6:13:1); 1H NMR (200 MHz,
DMSO-d6) d 12.95 (br s, 1H), 9.65 (br s, 1H), 7.91 (d,
J = 8.5 Hz, 2H), 7.60 (d, J = 7.0 Hz, 2H), 7.52 (t,
J = 7.0 Hz, 1H), 7.39 (d, J = 7.5 Hz, 2H), 7.35 (d,
J = 7.5 Hz, 2H), 7.26 (dd, J = 7.0, 6.5 Hz, 1H), 6.84
(dd, J = 11.0, 2.5 Hz, 1H), 6.75 (dt, J = 8.5, 2.5 Hz,
1H), 4.90 (s, 2H); IR (KBr) 3267, 1686, 1616, 1459,
1449, 1398, 1336, 1284, 1163, 1093, 1041, 1019 cmꢀ1
;
4.32.10. {[2-[Isopropyl(phenylsulfonyl)amino]-5-(trifluoro-
methyl)phenoxy]methyl}benzoic acid (22). Yield 78%; TC
Rf = 0.44 (MeOH/CHCl3/AcOH, 5:100:1); 1H NMR
(200 MHz, CDCl3) d 8.15 (d, J = 8.6 Hz, 2H), 7.81 (m,
2H), 7.52 (m, 3H), 7.38 (m, 2H), 7.24 (m 3H), 5.13 (s, 2H),
4.4 0 (sept, J = 6.8 Hz, 1H9, 1.06 (d, J = 6.8 Hz, 6H); IR
(KBr) 3423, 2982, 1698, 1426, 1332, 1294, 1218, 1172, 1127,
1088, 1034 cmꢀ1, MS (FAB, Pos.) m/e 494 (M+H)+, 353.
MS (FAB, Pos.) m/e 402 (M+H)+.
4.32.4. 4-({5-Methyl-2-[(phenylsulfonyl)amino]phenolxy}-
methyl)benzoic acid (16). Yield 86%; TLC Rf = 0.43
(EtOAc/hexane/AcOH, 7:12:1); 1H NMR (200 MHz,
DMSO-d6) d 7.91 (d, J = 8.5 Hz, 2H), 7.62 (d, J =
7.0 Hz, 2H), 7.57–7.45 (m, 1H), 7.44–7.30 (m, 4H),
7.14 (d, J = 8.0 Hz, 1H), 6.75 (s, 2H), 6.71 (d,
J = 8.0 Hz, 1H), 4.89 (s, 2H), 2.21 (s, 3H); IR (KBr)
3266, 2867, 1685, 1614, 1579, 1510, 1449, 1415, 1337,
1286, 1160, 1122, 1093, 1048, 1020 cmꢀ1; MS (FAB,
Pos.) m/e 398 (M+H)+.
4.32.11. {[2-[Isobutyl(phenylsulfonyl)amino]-5-(trifluoro-
methyl)phenoxy]methyl}benzoic acid (23). Yield 80%;
TLC Rf = 0.53 (MeOH/CHCl3, 1:9); 1H NMR
(200 MHz, CDCl3) d 8.10 (d, J = 8.4 Hz, 2H), 7.70–
7.60 (m, 2H), 7.50–7.20 (m, 7H), 7.11 (d, J = 1.6 Hz,
1H), 5.00–4.80 (m, 2H), 3.44 (d, J = 7.4 Hz, 2H), 1.70–
1.50 (m, 1H), 0.90 (d, J = 6.6 Hz, 6H); IR (KBr) 2963,
1694, 1510, 1448, 1427, 1331, 1289, 1257, 1213, 1169,
1127, 1086, 1019 cmꢀ1; MS (FAB, Pos.) m/e 508 (M+H)+.
4.32.5. 4-({5-Methoxy-2-[(phenylsulfonyl)amino]phenolxy}-
methyl)benzoic acid (17). Yield 89%; TLC Rf = 0.40
1
(MeOH/CHCl3, 1:4); H NMR (200 MHz, DMSO-d6) d
7.90 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.0 Hz, 1H), 7.51
(t, J = 8.0 Hz, 1H), 7.44–7.28 (m, 4H), 7.15 (d, J = 8.5
Hz, 1H), 6.54–6.47 (m, 2H), 4.86 (s, 2H), 3.69 (s, 3H);
IR (KBr) 3293, 3006, 1686, 1614, 1510, 1450, 1398, 1337,
1200, 1166 cmꢀ1; MS (APCI, Neg.) m/e 412 (MꢀH)ꢀ.
4.32.12. {[2-[Neopentyl (phenylsulfonyl)amino]-5-(trifluo-
romethyl)phenoxy]methyl}benzoic acid (24). Yield 80%;
TLC Rf = 0.50 (MeOH/CHCl3, 1:9); 1H NMR
(200 MHz, CDCl3) d 8.12 (d, J = 8.0 Hz, 2H), 7.60–7.40
(m, 4H), 7.40–7.20 (m, 5H), 7.09 (d, J = 1.8 Hz, 1H),
5.02 (d, J = 12.4 Hz, 1H), 4.72 (d, J = 12.4 Hz, 1H), 3.53
8s, 2H), 0.86 (s, 9H); IR (KBr) 2961, 1694, 1614, 1581,
1510, 1478, 1448, 1427, 1329, 1279, 1234, 1201, 1168,
1129, 100, 1019 cmꢀ1; MS (EI, Pos.) m/e 521 (M+).
4.32.6.
4-{[2-[(Phenylsulfonyl)amino]-5-(trifluorometh-
yl)phenoxy]methyl}benzoic acid (18). Yield 99%; TLC
Rf = 0.52 (MeOH/CHCl3/AcOH, 5:100:1); 1H NMR
(200 MHz, DMSO-d6) d 12.95 (s, 1H), 10.10 (br s,
1H), 7.93 (d, J = 8.0 Hz, 2H), 7.75 (m, 2H), 7.59 (m,
1H), 7.53–7.40 (m, 5H), 7.27 (m, 2H), 5.14 (s, 2H); IR
(KBr) 3379, 3297, 3072, 2671, 1694, 1415, 1522, 1443,
1353, 1334, 1286, 1169, 1123, 1090, 1011 cmꢀ1; MS
(FAB, Pos.) m/e 452 (M+H)+.
4.32.13. {[2-[(Cyclopentylmethyl)(phenylsulfonyl)amino]-
5-(trifluoromethyl)phenoxy]methyl}benzoic acid (25).
Yield 100%; TLC Rf = 0.40 (MeOH/CHCl3/H2O,

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
5575
1:9:0.1); 1H NMR (200 MHz, CDCl3) d 8.09 (d,
4.36. 5-Chloro-2-(hydroxymethyl)phenol (58)
J = 8.2 Hz, 2H), 7.65–7.61 (m, 2H), 7.47–7.20 (m, 7H),
7.11 (d, J = 1.8 Hz, 1H), 4.89 (br s, 2H), 3.59–3.51 (m,
2H), 1.93–1.10 (m, 9H), 7.11 (d, J = 1.8 Hz, 1H), 4.89
(br s, 2H), 3.59–3.51 (m, 2H), 1.93–1.10 (m, 9H); IR
(KBr) 2953, 2871, 1697, 1580, 1510, 1448, 1429, 1333,
1289, 1215, 1167, 1128, 1085, 1019 cmꢀ1; MS (APCI,
Neg.) m/e 532 (MꢀH)ꢀ.
To a stirred solution of methyl 4-chloro-2-hydrox-
ybenzoate 57 (2.16 g, 11.6 mmol) in THF (20 ml) was
added lithium aluminum hydride (440 mg, 11.6 mmol)
at 0 ꢁC under argon atmosphere. The reaction mixture
was allowed to warm up to room temperature and stir-
red for additional 30 min, diluted with Et2O, acidified
with 2 M HCl and extracted with EtOAc. The organic
layer was washed with water, brine, dried over MgSO4
and concentrated in vacuo to afford 58 (1.85 g, 100%).
TLC Rf = 0.50 (EtOAc/hexane, 1:1); 1H NMR
(200 MHz, CDCl3) d 7.51 (s, 1H), 6.95 (d, J = 8.2 Hz,
1H), 6.90 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.2, 2.0 Hz,
1H), 4.85 (d, J = 3.0 Hz, 1H), 2.21 (br s, 1H).
4.33. 4-Chloro-2-fluoro-N-phenylbenzenesulfonamide (54)
To a stirred solution of aniline (372 mg, 4.0 mmol) in
CH2Cl2 (10 ml) was added pyridine (0.40 ml, 4.0 mmol)
and benzenesulfonyl chloride 53 (1.37 g, 6.0 mmol) at
0 ꢁC under argon atmosphere. The reaction mixture was
allowed to warm up to room temperature, stirred for
15 min and concentrated in vacuo. The resulting residue
was purified by column chromatography on silica gel to
yield 54 (964 mg, 85%); TLC Rf = 0.24 (EtOAc/hexane,
1:6); 1H NMR (300 MHz, CDCl3) d 7.75 (m, 1H), 7.29–
7.07 (m, 7H), 6.87 (br s, 1H); MS (APCI, Neg.) m/e 286
(MꢀH)ꢀ.
4.37. 2-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-5-chloro-
phenol (59)
To a stirred solution of 58 (1.85 g, 11.6 mmol) in THF
(20 ml) were added Et3N (4.85 ml, 34.8 mmol), 4-
DMAP (28 mg, 0.232 mmol) and TBSCl (1.92 g,
12.8 mmol) under argon atmosphere. The reaction mix-
ture was stirred for 24 h, diluted with EtOAc. The
organic layer was washed with water and then brine.
The organic layer was dried over MgSO4 and evaporat-
ed. The resulting residue was purified by column chro-
matography on silica gel to yield 59 (1.89 g, 60%);
TLC Rf = 0.61 (EtOAc/hexane, 1:10); 1H NMR
(200 MHz, CDCl3) d 8.23 (s, 1H), 6.88 (d, J = 2.6 Hz,
1H), 6.85 (d, J = 8.0 Hz, 1H), 6.78 (dd, J = 8.0, 3.6 Hz,
1H), 4.86 (s, 2H), 0.91 (s, 9H), 0.13 (s, 6H).
4.34. 4-Chloro-2-[(4-formylbenzyl)oxy]-N-phenylbenzene-
sulfonamide (56)
To a stirred solution of [4-(dimethoxymethyl)phen-
yl]methanol 55 (866 mg, 12 mmol) in DMA (3 ml) was
added potassium tbutoxide (462 mg, 4.12 mmol) at
80 ꢁC under argon atmosphere. After 20 min, 54
(392 mg, 1.37 mmol) was added to the reaction mixture
and stirring was continued for additional 2 h at 110 ꢁC.
The reaction mixture was cooled to room temperature
and quenched by the addition of water. The aqueous layer
was extracted with EtOAc (2·) and the combined organic
layers were washed with 2 M HCl (3·) and then brine. The
organic layer was dried over MgSO4 and evaporated. The
resulting residue was purified by column chromatography
on silica gel to yield 56 (415 mg, 76%). TLC Rf = 0.24
(EtOAc/hexane, 1:6); 1H NMR (300 MHz, CDCl3) d
10.06 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.80 (d,
J = 8.7 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.19 (m, 2H),
7.11–6.93 (m, 5H), 6.69 (s, 1H), 5.34 (s, 2H).
4.38. Methyl 4-[(5-chloro-2-{[(tert-butyl(dimethyl)silyl)-
oxy]methyl}phenoxy)methyl]benzoate (60)
To a stirred solution of 59 (1.85 g, 6.80 mmol) in ace-
tone (20 ml) were added methyl 4-(bromomethyl)benzo-
ate (2.03 g, 8.84 mmol) and K2CO3 (1.41 g, 10.2 mmol)
under argon atmosphere. After stirred for 4 h at 50 ꢁC,
the reaction mixture was diluted with EtOAc and fil-
tered through a pad of Celite. The filtrate was concen-
trated in vacuo to yield 60 (3.47 g), which was used
without further purification; TLC Rf = 0.32 (EtOAc/
hexane, 1:20).
4.35. 4-{[2-(Anilinosulfonyl)-5-chlorophenoxy]methyl}-
benzoic acid (13)
4.39. Methyl 4-{[5-chloro-2-(hydroxymethyl)phenoxy]-
methyl}benzoate (61)
To astirred solution of 56(413 mg, 1.03 mmol) in CH3CN
(5 ml) and water (5 ml) were added 2-methyl-2-butene
(0.545 ml, 5.15 mmol), NaH2PO4 (122 mg, 1.03 mmol)
and NaClO2 (452 mg, 4.12 mmol) at room temperature.
The reaction mixture was stirred for 1.5 h under argon,
diluted with water, acidified with 1 M HCl and extracted
with Et2O. The organic layer was dried over MgSO4 and
concentrated in vacuo. The resulting residue was purified
by column chromatography on silica gel to yield 13
(127 mg, 30%). TLC Rf = 0.35 (MeOH/CHCl3, 1:10); 1H
NMR (300 MHz, DMSO-d6) d 7.87 (d, J = 8.4 Hz, 2H),
7.80 (d, J = 8.7Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.24–
7.17 (m, 3H), 7.13–7.05 (m, 3H), 6.97 (m, 1H), 5.48 (s,
2H); IR (KBr) 3215, 1707, 1587, 1499, 1479, 1408, 1386,
1320, 1239, 1156, 1136, 1064 cmꢀ1; MS (APCI, Neg.) m/
e 416 (MꢀH)ꢀ.
To a stirred solution of 60 (3.47 g, <6.80 mmol, crude)
in THF (10 ml) was added 1.0 M solution of TBAF in
THF (6.8 ml, 6.8 mmol). The reaction mixture was stir-
red for 10 min and diluted with EtOAc. The organic
layer was washed with water, brine, dried over MgSO4
and concentrated in vacuo. The resulting residue was
recrystallized from EtOAc and hexane to yield 61
(1.76 g, 84% in 2 steps); TLC Rf = 0.58 (EtOAc/hexane,
1
1:1); H NMR (200 MHz, CDCl3) d 7.99 (d, J = 8.4 H,
2H), 7.59 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.0 Hz, 1H),
7.09 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.0, 2.0 Hz, 1H),
5.25 (s, 2H), 5.13 (t, J = 5.6 Hz, 1H), 4.55 (d,
J = 5.6 Hz, 1H), 3.86 (s, 3H).

5576
A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 5562–5577
4.40. Methyl 4-{[2-(bromomethyl)-5-chlorophenoxy]-
methyl}benzoate (62)
were then washed with ice-cold buffer (10 mm
KH2PO4ꢀ–KOH buffer containing 0.1 mm NaCl, pH
6.0), and the radioactivity on the filter was measured
in 6 ml of liquid scintillation (ACSII) mixture with a
liquid scintillation counter. Nonspecific binding was
achieved by adding excess amount of unlabeled
PGE2 with assay buffer. The concentration of the test
compound required to inhibit the amount of the spe-
cific 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.
To a stirred solution of 61 (200 mg, 0.654 mmol) in
CH2Cl2 (2 ml) were added triphenylphosphine
(206 mg, 0.784 mmol) and carbon tetrabromide
(325 mg, 0.98 mmol) at room temperature under argon.
The reaction mixture was stirred for 5 min, the reaction
mixture was diluted with EtOAc. The organic layer was
washed with NaHCO3aq, water, brine, dried over
MgSO4 and concentrated in vacuo to yield 62 (759 mg,
100%), which was used without further purification.
TLC Rf = 0.71 (EtOAc/hexane, 1:2).
Ki ¼ IC50=ðl þ ½Lꢁ=KdÞ;
where [L] is concentration of radiolabeled ligand; Kd,
dissociation constant of radiolabeled ligand for the pro-
stanoid receptors.
4.41. Methyl 4-{[5-chloro-2-(phenoxymethyl)phenoxy]-
methyl}benzoate (63)
To a stirred solution of 62 (759 mg, 0.654 mmol) in
acetone (2 ml) were added phenol (0.057 ml,
0.654 mmol) and K2CO3 (116 mg, 0.85 mmol) at room
temperature under argon atmosphere. The reaction
mixture was stirred for 2 days, diluted with EtOAc
and filtered through a pad of Celite. The filtrate was
concentrated in vacuo. The resulting residue was puri-
fied by column chromatography on silica gel to yield
63 (240 mg, 96%, in 2 steps); TLC Rf = 0.35 (ben-
zene/hexane, 7:3); 1H NMR (200 MHz, CDCl3) d
8.04 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H),
7.41 (d, J = 8.9 Hz, 1H), 7.29 (m, 2H), 6.97 (m, 5H),
5.17 (s, 2H), 5.12 (s, 2H), 3.92 (s, 3H); IR (KBr)
5.2. Measurement of the mEP1 receptor antagonist
activity
To confirm that test compounds antagonize the mEP1
receptor and estimate potencies of antagonism for
mEP1 receptor, a functional assay was performed by
measuring PGE2-stimulated changes in intracellular
Ca2+ as an indicator of receptor function. The cells
expressing mEP1 receptor were seeded at 1 · 104 cells/
well in 96 well plates and cultured for 2 days with 10%
FBS (fetal bovine serum)/minimum essential medium
Eagle alpha modification (aMEM) in the incubator
(37 ꢁC, 5% CO2). The cells in each well were rinsed with
phosphate buffer (PBS(minus)), and load buffer was add-
ed. After incubation for 1 h, the load buffer (10% FBS/
aMEM containing 5 lM of Fura 2/AM, 20 lM of indo-
methacin, 2.5 mm of probenecid) was discarded. After
the addition of the assay buffer (Hank’s balanced salt
solution (HBSS) containing 0.1% (w/v) BSA, 2 lM of
indomethacin, 2.5 mm of probenecid and 10 mm of
HEPES–NaOH) to each well, the cells were incubated
in a dark place at room temperature for 1 h. After the
addition of a test compound (10 ll) and PGE2 (10 ll)
which were prepared with assay buffer, intracellular cal-
cium concentration was measured with Fluorescence
drug screening system (FDSS-4000, Hamamatsu Pho-
tonics). A pair of fluorescence intensities emitted
500 nm by an excitation wavelength of each 340 and
380 nm was measured. The percent inhibition of the in-
crease of the intracellular Ca2+ concentration induced
by PGE2 (100 nM) was calculated relative to the maxi-
mum Ca2+ concentration that occurred in the absence
of test compound (100%) to estimate the IC50 value.
1722, 1599, 1495, 1281, 1243, 1109 cmꢀ1
.
4.42. 4-{[5-Chloro-2-(phenoxymethyl)phenoxy]methyl}-
benzoic acid (14)
The title compound 14 was prepared from 63 in 100%
yield according to the same procedure as described for
the preparation of 2 from 31a. TLC Rf = 0.49 (MeOH/
1
CHCl3/AcOH, 5:100:1); H NMR (200 MHz, CDCl3) d
8.11 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.42
(d, J = 8.0 Hz, 1H), 7.30 (m, 2H), 6.98 (m, 5H), 5.19 (s,
2H), 5.14 (s, 2H); IR (KBr) 3449, 2901, 2361, 1703,
1602, 1499, 1407, 1377, 1292 cmꢀ1; MS (EI, Pos.) m/e
368 (M+), 275, 135, 107.
5. Biological assay method
5.1. Prostanoid mEP1-4 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
prostanoid receptors mouse EP1-4.
References and notes
Membranes from CHO cells expressing prostanoid
receptors were incubated with redioligand (2.5 nM of
[3H]PGE2) and the test compounds at various concen-
trations in assay buffer (10 mm KH2PO4–KOH buffer
containing 1 mm EDTA and 0.1 mm NaCl, pH 6.0).
Incubation was carried out at 25 ꢁC for 60 min except
for mEP1 (20 min). Incubation was terminated by fil-
tration through Whatman GF/B filters. The filters
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