Further optimization of sulfonamide analogs as EP1 receptor antagonists: Synthesis and evaluation of bioisosteres for the carboxylic acid group

Article abstract of DOI:10.1016/j.bmc.2006.06.067

4-{[2-[(2-Furylsulfonyl)(isobutyl)amino]-5-(trifluoromethyl)phenoxy]methyl}benzoic acid analogs 2a and b and a series of the acid analogs, in which the carboxylic acid residue of 2b was replaced with various kinds of carboxylic acid bioisosteres, were synthesized and evaluated as EP1 receptor antagonists. Compound 2b and its monocyclic acid analogs, in which the carboxylic acid residue of 2b was replaced with monocyclic acid bioisosteres, were found to show potent EP1 receptor antagonist activity. Optimization of the linker Y between the phenyl moiety and the carboxylic acid residue of 2b was also carried out (Table 5). Compounds 2b and 16 and 17 possessing conformationally restricted linker Y were found to show the most optimized potency among the tested compounds. Cytochrome P450 inhibition of optimized compounds was also investigated. Details of the structure-activity relationship study are presented.

Full text of DOI:10.1016/j.bmc.2006.06.067

Bioorganic & Medicinal Chemistry 14 (2006) 7121–7137
Further optimization of sulfonamide analogs as EP1 receptor
antagonists: Synthesis and evaluation of bioisosteres for the
carboxylic acid group
Atsushi Naganawa,a,* Toshiaki Matsui,b Masaki Ima,a Tetsuji Saito,a Masayuki Murota,a
Yoshiyuki Aratani,a Hideomi Kijima,a Hiroshi Yamamoto,c Takayuki Maruyama,a
Shuichi Ohuchida,b Hisao Nakaia and Masaaki Todaa
aMinase Research Institute, Ono Pharmaceutical Co. Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
bFukui Research Institute, Ono Pharmaceutical Co. Ltd, Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
cOno Pharma USA, Inc., Lenox Drive, Lawrenceville, NJ 08648, USA
Received 5 June 2006; revised 29 June 2006; accepted 30 June 2006
Available online 1 August 2006
Abstract—4-{[2-[(2-Furylsulfonyl)(isobutyl)amino]-5-(trifluoromethyl)phenoxy]methyl}benzoic acid analogs 2a and b and a series
of the acid analogs, in which the carboxylic acid residue of 2b was replaced with various kinds of carboxylic acid bioisosteres, were
synthesized and evaluated as EP1 receptor antagonists. Compound 2b and its monocyclic acid analogs, in which the carboxylic acid
residue of 2b was replaced with monocyclic acid bioisosteres, were found to show potent EP1 receptor antagonist activity. Optimi-
zation of the linker Y between the phenyl moiety and the carboxylic acid residue of 2b was also carried out (Table 5). Compounds 2b
and 16 and 17 possessing conformationally restricted linker Y were found to show the most optimized potency among the tested
compounds. Cytochrome P450 inhibition of optimized compounds was also investigated. Details of the structure–activity relation-
ship study are presented.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In one of our previous papers,5 we reported on the dis-
covery of 1 as a new lead compound in the EP1 receptor
antagonists. Compound 1 demonstrated remarkably
weak antagonist activity (IC50, 0.13 lM) for its strong
receptor affinity (Ki, 0.0005 lM). In an effort to improve
this weak antagonist activity, we incorporated bioisos-
teres into our further optimization process because this
methodology has been frequently used for such a pur-
pose in conventional medicinal chemistry. Accordingly,
the phenylsulfonyl moiety and the carboxylic acid resi-
due were replaced with a furan-2-sulfonyl moiety and
acid residue, respectively (Fig. 1). We here report on fur-
ther optimization efforts for the newly found EP1 recep-
tor antagonist 1.
Cyclooxygenases metabolize arachidonic acid to five
primary prostanoids, PGE2, PGF2, PGI2, TXA2 (TX),
and PGD2. These lipid mediators interact with specific
members of a family of distinct G-protein-coupled pro-
stanoid receptors.1 Coleman et al. proposed the exis-
tence of specific receptors for TX, PGI, PGE, PGF,
and PGD, named TP, IP, EP, FP, and DP receptors,
respectively.2 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 and their therapeu-
tic potential have already been described in the litera-
ture. Discovery of selective EP1 receptor antagonists
would offer an opportunity to elucidate the role of this
receptor in various pathological conditions such as
hyperalgesia3 and pollakisuria.4
2. Chemistry
The synthesis of the test compounds listed in Tables 1–5
is outlined in Schemes 1–3. Compounds 2b, 4, and 15–17
were synthesized as described in Schemes 1a and b.
O-Alkylation of 196 with methyl 4-(bromomethyl)
Keywords: Prostaglandin; EP1 receptor; Antagonist; Bioisostere.
*
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962
9314; e-mail: naganawa@ono.co.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.067

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
acidic residue
CO2H
F3C
O
O
N
F3C
O
O
N
O
O
S
S
O
Me
Me
Me
Me
Me
I
1
Figure 1. Transformation of a phenylsulfonyl aminobenzoic acid analog 1 to 5-methylfuran-2-sulfonyl aminobenzoic acid analogs I.
Table 1. Effect of transformation of the aryl moiety Ar on Ki values and antagonist activities
CO2H
CF3
O
O
N
O
S
Ar
Me
II
Me
Compound
Ar
Binding Ki (lM)
Function IC50 (lM)
EP1
EP2
>10
EP3
0.41
EP4
>10
EP1
0.13
*
1
0.00050
O
O
*
*
2a
2b
0.00040
0.00020
8.1
1.7
>10
2.1
0.17
Me
>10
0.82
0.0056
benzoate in the presence of potassium carbonate yielded
20a, reduction of which, with lithium borohydride, gave
alcohol 21. Oxidation of 21 with DMSO yielded an alde-
hyde 22, C2 homologation of which, using malonic acid
reaction followed by esterification, gave an a,b-unsatu-
rated methyl ester 20b.7 Reduction of the nitro residue
of 20a and b yielded anilines 24a and b, respectively.
Hydrogenation of 24b gave 24c.8 N-Sulfonylation of
24a–c with 5-methylfuran-2-sulfonyl chloride 29, which
was prepared from 27 according to a previously report-
ed method9 (Scheme 1b), gave sulfonamides 25a–c,
respectively. N-Alkylation of 25a–c with isobutyl iodide
in the presence of potassium carbonate yielded 26a–c,
respectively. Alkaline hydrolysis of 26a gave a carboxyl-
ic acid 2b. Compound 2b was converted to the corre-
sponding acid chloride with thionyl chloride. Wittig
reaction of the resulting acid chloride with methyl (tri-
phenylphosphoranylidene) acetate, followed by heating,
yielded 26d.10 Alkaline hydrolysis of 26d gave the cor-
responding carboxylic acid 17. Condensation of 2b with
methanesulfonamide in the presence of EDC yielded 4.
Alkaline hydrolysis of 26b and c gave 16 and 15,
respectively.
um carbonate yielded 33. Acidic deprotection of 33 gave
34, O-alkylation of which with 4-(bromomethyl)benzo-
nitrile, in the presence of potassium carbonate, pro-
duced the nitrile 3. Reaction of the nitrile 3 with
sodium azide, in the presence of triethylamine, resulted
in the tetrazole 11.11 Reaction of the nitrile 3 with
hydroxylamine, in the presence of triethylamine, pro-
duced the N-hydroxy amidine 35. Reaction of 35 with
thionyl chloride yielded 3H-1,2,3,5-oxathiadiazole 2-ox-
ide 8.12 Reaction of 35 with 2-ethylhexyl chloroformate,
followed by heating in xylene, gave oxadiazol-5-one 9.12
Reaction of 35 with thiocarbonyl diimidazole, followed
by cyclization with boron trifluoride etherate, yielded
thiadiazol-5-one 10.12
Synthesis of 5–7, 12–14, and 18 is described in Scheme 3.
Compounds 5–7 and 18 were prepared as outlined in
Scheme 3a. Reaction of 4-bromomethylbenzoic acid
(36) with thionyl chloride yielded the corresponding acid
chloride, which was converted to the corresponding
amide 40a by the usual ammonolysis with aqueous
ammonia. O-Alkylation of 34 (Scheme 2) with 40a gave
41a. N-Acylation of the amide function of 41a yielded
N-acetylcarboxyamide 5. Ammonolysis of 4-bromom-
ethyl benzenesulfonyl chloride (37) gave the sulfonamide
40b. O-Alkylation of 34 (Scheme 2) with 40b, in the
presence of potassium carbonate, yielded 41b.
N-Methanesulfonylation of the primary sulfonamide res-
idue of 41b resulted in N-methanesulfonylsulfonamide 6.
N-Acetylation of the sulfonamide residue of 41b
Synthesis of 3 and 8–11 is outlined in Scheme 2. Catalyt-
ic hydrogenation of a protected O-nitrophenol 30, which
was prepared by O-alkylation of 19 with methoxymethyl
chloride, yielded 31. N-Sulfonylation of 31 with
5-methylfuran-2-sulfonyl chloride gave 32, N-alkylation
of which with isobutyl iodide in the presence of potassi-

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7123
Table 2. Effect of transformation of the carboxylic acid moiety X on Ki values and antagonist activities
X
CF3
O
O
N
O
S
O
Me
Me
III
Me
Compound
X
Binding Ki (lM)
Function IC50 (lM)
EP1
EP2
>10
EP3
0.82
EP4
2.1
EP1
2b
0.00020
0.0056
CO2H
*
*
CN
O
3
4
5
6
0.30
>10
>10
>10
>10
>10
>10
0.61
>10
0.61
>10
>10
>10
>10
>10
>10
>10
0.0022
0.0073
0.026
0.058
0.038
0.36
NTa
0.83
NHMs
*
*
O
NHAc
O
*
O
S
S
NHMs
O
*
O
7
NHAc
a NT, not tested.
Table 3. Activity profiles of monocyclic acid analogs
Het
H
CF3
O
O
N
O
S
O
Me
Me
Me
IV
Compound
Binding Ki (lM)
Function IC50 (lM)
Het
N
H
O
EP1
EP2
EP3
0.39
EP4
>10
EP1
8
0.0029
7.5
0.0026
O
S
N
*
*
*
*
H
O
N
N
N
O
O
9
10
11
0.0031
0.0069
0.0026
4.5
3.3
2.5
0.48
0.54
0.026
>10
2.4
0.0016
0.0035
0.00074
N
H
S
N
H
N
N
>10
N
H
produced N-acetyl sulfonamide 7. O-Alkylation of the
phenol residue of 38 with methyl chloroacetate yielded
39. Bromination reaction of benzyl alcohol 39 with phos-
phorus tribromide provided 40c. O-Alkylation of 34
(Scheme 2) with 40c yielded 41c, alkaline hydrolysis of
which resulted in the corresponding carboxylic acid 18.

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
Table 4. Activity profiles of fused bicyclic acid analogs
Het
Me
H
CF3
O
O
N
O
S
O
Me
V
Me
Compound
Binding Ki (lM)
Function IC50 (lM)
Het
O
H
EP1
EP2
>10
EP3
>10
EP4
>10
EP1
0.68
*
*
12
0.0061
NH
O
OH
N
13
14
0.0013
0.0013
>10
>10
>10
>10
>10
>10
0.19
0.56
N
*
*
OH
N
OH
N
Table 5. Effect of transformation of the linker Y on Ki values and antagonist activities
Y
CO2H
CF3
O
O
N
O
S
O
Me
Me
VI
Me
Compound
Y
Binding Ki (lM)
Function IC50 (lM)
EP1
EP2
3.0
EP3
EP4
EP1
0.17
15
0.0034
1.1
2.9
*
*
*
16
17
18
0.0004
0.0011
0.00042
4.8
3.1
3.5
1.9
2.0
0.0053
0.0058
0.021
*
*
0.79
>10
4.5
*
O
*
>10
*
Compound 12 was prepared as shown in Scheme 3b.
Bromination of 42 with N-bromosuccinimide yielded
43. O-Alkylation of 34 (Scheme 2) with the bromide
43 produced 44, alkaline hydrolysis of which yielded
45. Heating of 45 in the presence of acetic anhydride,
followed by ammonolysis and then heating in toluene,
resulted in the phthalimide analog 12.
Cyclization of 51 with imidoformamide hydrochloride,
with heating, resulted in 4-hydroxyquinazoline 13.13
Cyclization of 51 with urea, with heating, resulted in
2,4-dihydroxyquinzoline 14.14
3. Results and discussion
Synthesis of 13 and 14 is described in Scheme 3c. Partial
alkaline hydrolysis of 46 yielded 47, reduction of which
with diborane-dimethylsulfide complex produced 48.
O-Alkylation of 34 (Scheme 2) with 48, using the
Mitsunobu reaction, gave 49. Reduction of 49 with iron
powder, followed by alkaline hydrolysis, produced 51.
The test compounds listed in Tables 1–5 were biological-
ly evaluated for their inhibition of the specific binding of
a radiolabeled ligand, [3H]PGE2, to membrane fractions
prepared from cells stably expressing each mouse
prostanoid receptor. The EP1 antagonist properties of
these compounds were determined by a Ca2+ assay using

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7125
a
R
R
OH
F3C
a
f
h
F3C
O
O
F3C
NO2
NH2
NO2
19
20a R = CO2Me
R = CO2Me
R =
24a
24b
b
21
R = CH2OH
R = CHO
R =
CO2Me
CO2Me
c
d
e
g
22
24c R =
CO2H
23
CO2Me
20b
R =
R
R
O
O
N
F3C
i
O
O
F3C
O
S
O
O
S
O
Me
N
Me
Me
H
Me
26a R = CO2Me
25a R = CO2Me
j
2b
R = CO2H
R =
CO2Me
CO2Me
25b
25c
R =
R =
l, m, n
j
CO2Me
CO2H
26d
k
R =
17
4
R = CONHMs
CO2Me
CO2H
R=
26b
j
j
16 R =
26c R =
CO2Me
CO2H
15
R =
NH+
O
b
p
O
o
O
Me
SO2Cl
Me
SO3-
28
Me
29
27
Scheme 1. Synthesis of 2b, 4, and 15–17. Reagents and condition: (a) methyl 4-(bromomethyl)benzoate K2CO3, acetone; (b) LiBH4, MeOH, Et2O; (c)
SO3ÆPy, iPr2NEt, DMSO, EtOAc; (d) malonic acid, piperidine, pyridine; (e) MeI, K2CO3, DMF; (f) Fe, AcOH, H2O; (g) NaBH4, NiCl2Æ6H2O, EtOH,
H2O; (h) 29, pyridine; (i) iBuI, K2CO3, DMF; (j) NaOH, MeOH, dioxane; (k) MsNH2, EDC, DMAP, DMF; (l) SOCl2, EtOAc; (m)
Ph3P = CHCO2Me, Et3N, toluene; (n) o-dichlorobenzene, heat; (o) SO3ÆPy, CH3CN; (p) SOCl2, DMF.
mouse EP1 receptor expressed on CHO cells in the pres-
ence of 0.1% of bovine serum albumin.
reason, synthesis and biological evaluation of the acid
analogs listed in Tables 2–5 are of great interest to
medicinal chemists.
Furan-2-sulfonyl analogs 2a15 and b were synthesized and
evaluated (Table 1). Among these, 5-methylfuran-2-sul-
fonyl analog 2b showed much stronger antagonist activity
relative to the chemical lead 1, while demethylated analog
2a showed nearly the same potency as 1 in terms of both
receptor affinity and antagonist activity. Based on these
results, further optimization of 5-methylfuran-2-sulfonyl
analogs, instead of the phenylsulfonyl and furan-2-sulfo-
nyl analogs, was carried out as shown in Tables 2–5.
The bioisostere is known as one of the useful transfor-
mations for improving the activity and/or pharmacoki-
netic profiles of chemical leads in medicinal
chemistry.16 In an effort to further optimize the acid
residue of the chemical lead 2b, the carboxylic acid
was replaced by various bioisosteres, as illustrated in
Tables 2–5. As shown in Table 2, replacement of the
carboxylic acid of 2b with an N-acylsulfonamide and
an N-acylacetoamide (imide) yielded 4 and 5, respectively.
Compound 4 exhibited 11-fold less EP1 receptor affinity
and 6.8-fold less antagonist activity relative to 2b, while
5 exhibited less receptor affinity and antagonist activity.
Replacement of the carboxylic acid residue of 2b with a
N-sulfonyl methanesulfonamide and a N-sulfonyl ace-
toamide yielded 6 and 7, respectively, which showed less
receptor affinity relative to 4 and 5, respectively.
In proceeding the optimization process, focus was
placed on the nitrile analog 3 (Table 2), in which a car-
boxylic acid residue was replaced by a nitrile residue.
The marked reduction in the activity of this transforma-
tion strongly suggested a structural requirement for an
acid residue at this position, which was supposed to cor-
respond to the carboxylic acid residue of PGE2. For this

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
F3C
OW
O
e
b
OMOM
X
F3C
O
S
O
N
Y
Me
30
31
X = NO
32
W = MOM, Y = H
2
c
a
i
X = NH
33 W = MOM, Y = Bu
2
d
i
34
W = H, Y = Bu
R
F3C
O
O
N
O
S
O
Me
Me
h
Me
8
9
i, j
k, l
3
R = CN
f
N
N
N
11 R =
10
g
N
H
35
R = C(=NOH)NH2
i
Scheme 2. Synthesis of 3 and 8–11. Reagents and condition: (a) H2, Pd–C, MeOH; (b) 29, pyridine, CH2Cl2; (c) BuI, K2CO3, DMF; (d) HCl,
MeOH; (e) 4-(bromomethyl)benzonitrile, K2CO3, acetone; (f) NaN3, Et3NÆHCl, toluene; (g) NH2OHÆHCl, Et3N, EtOH; (h) SOCl2, pyridine, THF;
(i) 2-ethylhexyl chloroformate, pyridine; (j) xylene, heat; (k) thiocarbonyl diimidazole (TCDI), THF; (l) BF3ÆOEt2, THF.
As illustrated in Table 3, monocyclic acid analogs were
synthesized and evaluated.12 2-Oxido-3H-1,2,3,5-oxa-
thiadiazol analog 8 showed 15-fold less potent EP1
receptor affinity relative to 2b, while it showed 2.2-fold
more potent antagonist activity. Oxadiazole-5-one ana-
log 9 exhibited nearly equipotent receptor affinity and
antagonist activity with 8. A thiadiazole-5-one analog
10 showed 2.4-fold less potent EP1 receptor affinity
and 1.3-fold less potent antagonist activity relative to
8, while it also showed weak affinity for the EP4 recep-
tor. A tetrazole analog 11 demonstrated the strongest
antagonist activity among this series of analogs,
although it showed nearly the same receptor affinity as
8–10. Besides, 11 showed an increased affinity for EP3
receptor relative to 8–10.
of the carboxylic acid residue with a propenoic acid res-
idue and a propynoic acid residue gave 16 and 17,
respectively, with 2-fold and 5.5-fold less potent EP1
receptor affinity, while both showed nearly the same
antagonistic activity as 2b. Oxyacetic acid analog 18
showed nearly the same EP1 receptor affinity as 16,
while it showed 4-fold less antagonist activity, which
was weaker than its receptor affinity.
Based on the relatively stronger antagonist activity of
2b, 16, and 17, conformational restriction of the acid
residue was found to be one of the required partial struc-
tures for increased antagonist activity. Optimized com-
pounds 2b, 8–11, and 16 and 17 were also evaluated
for their inhibition of P450 enzymes, to predict their
potential for drug–drug interactions.17 As shown in
Table 6, monocyclic acid analogs 8–11 exhibited rela-
tively stronger inhibition. Tetrazole analog 11 demon-
strated especially strong inhibitory activities against
2C9, 2C19, and 3A4. Propynoic acid analog 17 showed
increased inhibitory activities against 2C9 and 3A4 com-
pared with 2b.
Activity profiles of fused bicyclic acid analogs are shown
in Table 4. Analogs 12–14 showed weaker antagonist
activity relative to the series of monocyclic acids 8–11,
although they showed equipotency in their EP1 receptor
affinity and better subtype selectivity. Phthalimide ana-
log 12 showed 31-fold less EP1 receptor affinity and
121-fold less potent antagonist activity relative to 2b.
4-Hydroxyquinazoline analog 13 and 2,4-dihydroxyqui-
nazoline analog 14 exhibited equipotency in their EP1
receptor affinity, while 13 showed 2.9-fold more potent
antagonist activity than 14.
In summary, we synthesized and evaluated 5-methylfu-
ran-2-sulfonyl analog 2b, which exhibited much stronger
antagonist activity than 1 and 2a. Based on the data, the
5-methyl residue of 2b was considered to play an impor-
tant role in transmitting functional activity via the EP1
receptor. To improve antagonist activity, we also syn-
thesized and evaluated acid analogs of 2b by incorporat-
ing the concept of the bioisostere into further
optimization. Among the tested compounds, monocyclic
acid analogs 8–11 listed in Table 3 exhibited stronger
EP1 receptor antagonist activity relative to 2b, although
all of them showed weaker receptor affinity. In
Second, the effect of transformation of the linker Y
(Table 5) between the carboxylic acid residue and the
phenyl moiety on receptor affinity and antagonist activ-
ity was investigated. Replacement of the carboxylic acid
residue of 2b with a propionic acid residue yielded 15,
which showed 17-fold less potent EP1 receptor affinity
and 30-fold less potent antagonist activity. Replacement

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7127
a
R
F3C
O
O
N
R
O
e
S
O
X
Me
Me
36 R = CO2H, X = Br
Me
a,b
R = CONH , X = Br
40a
41a
5
R = CONH
2
2
f
R = CONHAc
R = SO2NH2
37 R = SO2Cl, X = Br
40b R = SO2NH2, X = Br
41b
b
g
h
6
7
R = SO2NHMs
R = SO2NHAc
38 R = X = OH
c
R = OCH CO2Me
41c
18
2
39 R = OCH CO2Me, X = OH
2
i
d
R = OCH CO2H
2
40c R = OCH CO2Me, X = Br
2
O
b
R
NH
F3C
O
O
N
F3C
O
O
N
CO2Me
e
f, b, k
R
O
O
S
O
Me
O
S
X
O
Me
CO2Me
Me
Me
Me
44
Me
12
42
43
X = H
R = CO2Me
j
i
X = Br
45 R = CO2H
c
OH
R
N
F3C
O
o or p
F3C
O
CO2Me
NO2
X
N
W
m
O
O
N
O
O
N
S
O
S
O
Me
Y
Me
Me
Me
Me
R = CO2Me, X = NO
Me
Y = CO2Me
46
47 Y= CO2H
48
i
49
50
51
2
o; 13 W = H
W = OH
n
i
l
R = CO2Me, X = NH
Y = CH2OH
2
p; 14
R = CO2H, X = NH
2
Scheme 3. Synthesis of 5–7, 12–14, and 18. Reagents and conditions: (a) SOCl2, EtOAc; (b) aqueous NH3; (c) methyl chloroacetate, K2CO3, KI,
acetone; (d) PBr3, Et2O; (e) 34, K2CO3, DMF; (f) Ac2O, heat; (g) MsCl, NaOH, THF; (h) Ac2O, pyridine; (i) NaOH, dioxane; (j) NBS, (BzO)2, CCl4;
(k) toluene, heat; (l) BH3ÆSMe2, THF; (m) 34, DEAD, PPh3, THF; (n) Fe, AcOH, H2O; (o) formamidineÆHCl, heat; (p) urea, heat.
Table 6. Cytochrome P450 inhibitory activities of optimized analogs
Compound
P-450 inhibition (%, 3 lM)
1A2
2C9
2C19
2D6
3A4
2b
8
2.6
22.2
10.2
5.9
13.7
8.5
0.4
32.7
28.9
33.2
97.9
ꢀ3.8
18.8
ꢀ5.1
2.0
11.7
67.6
32.2
14.8
93.8
10.9
42.2
9
32.6
ꢀ9.4
98.0
8.9
4.4
1.1
10
11
16
17
12.1
2.2
2.6
ꢀ3.4
5.8
11.5
47.2
particular, tetrazole analog 11 showed the strongest
antagonist activity among the tested compounds.
1, 2a and b (Table 1), while the monocyclic acid equiva-
lents showed the most optimized antagonist activity as
illustrated by the analogs 8–11 (Table 3). Based on the
above-described results, the acidic equivalents of the
analogs 4–7 (Table 2) were considered to show
As a result, the carboxylic acid residue showed the most
optimized receptor affinity as illustrated by the analogs

7128
A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
decreased antagonist activities because of the more influ-
ence of bovine serum albumin (BSA)18 relative to those
of 8–11. Fused bicyclic acid equivalents of the analogs
12–14 were also considered to show much more de-
creased antagonist activities for their relatively potent
binding affinities because of the more influence of BSA
such as a presumed increased protein binding.
under argon atmosphere. The resulting mixture was
stirred at 50 ꢁC for 4 h, then quenched with water and
extracted with EtOAc. The organic layer was washed
with water, and brine, dried over MgSO4, and concen-
trated in vacuo. The resulting residue was recrystallized
from EtOH and hexane to yield 20a (7.6 g, 89%). TLC
Rf = 0.38 (EtOAc/hexane, 1:5); 1H NMR (300 MHz,
CDCl3) d 8.10 (d, J = 6.3 Hz, 2H), 7.95 (d, J = 8.7 Hz,
1H), 7.54 (d, J = 6.3 Hz, 2H), 7.40–7.25 (m, 2H), 5.33
(s, 2H), 3.94 (s, 3H).
The effect of transformation of the linker Y between the
carboxylic acid residue and the phenyl moiety of 2b was
also investigated, and is illustrated in Table 5. Analogs
2b, 16, and 17, possessing conformationally restricted
carboxylic acid residues, tended to show stronger antag-
onist activity than analogs 15 and 18 which possess the
more flexible carboxylic acid residues. Cytochrome P450
inhibition by the optimized compounds was also investi-
gated. As a result, 2b and 16 were found to possess the
most desired in vitro profiles, based on their antagonist
activity and P450 inhibition.
4.3. Methyl 4-{[2-amino-5-(trifluoromethyl)phen-
oxy]methyl}benzoate (24a)
To a stirred solution of 20a (7.6 g, 21.4 mmol) in AcOH
(30 mL) and water (2 mL) was added iron (325 mesh,
6 g, 107 mmol) under argon atmosphere. After being
stirred for 30 min at 50 ꢁC, the reaction mixture was fil-
tered through a pad of Celite. The filtrate was concen-
trated in vacuo, diluted with aqueous NaHCO3, 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
iPr2O and hexane to yield 24a (5.88 g, 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).
4. Experimental
4.1. General directions
Analytical samples were homogeneous as confirmed by
TLC and afforded spectroscopic results consistent with
the assigned structures. Proton nuclear magnetic reso-
nance 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.
The matrix-assisted laser desorption ionization-time of
flight high-resolution mass spectra (MALDI-TOF) were
obtained on a PerSeptive voyager Elite 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 uncorrected. Column chromatog-
raphy was carried out on silica gel [Merck silica gel 60
(0.063–0.200 mm), Wako gel C200 or Fuji Silysia
FL60D]. Thin-layer chromatography was performed
on silica gel (Merck TLC or HPTLC plates, silica gel
60 F254). The following abbreviations for solvents
and reagents are used; tetrahydrofuran (THF), diethyl
ether (Et2O), diisopropylether (iPr2O), dimethylsulfox-
ide (DMSO), ethyl acetate (EtOAc), dimethylformam-
ide (DMF), dichloromethane (CH2Cl2), chloroform
(CHCl3), carbon tetrachloride (CCl4), methanol
(MeOH), ethanol (EtOH), isopropanol (iPrOH), acetic
acid (AcOH), hydrochloric acid (HCl), diisopropylethyl-
amine (iPr2NEt), and triethylamine (TEA).
4.4. Methyl 4-{[2-{[(5-methyl-2-furyl)sulfonyl]amino}-5-
(trifluoromethyl)phenoxy]methyl}benzoate (25a)
To a stirred solution of 24a (1.05 g, 3.23 mmol) and pyr-
idine (0.52 mL, 6.46 mmol) in CH2Cl2 (10 mL) was add-
ed 29 (874 mg, 4.84 mmol) at room temperature under
argon atmosphere. The reaction mixture was stirred
for 9 h, quenched with water, and extracted with EtOAc
(2·). The organic layers were washed with water, brine,
dried over MgSO4, and evaporated. The resulting resi-
due was recrystallized from EtOH to yield 25a (1.2 g,
80%). TLC Rf = 0.33 (EtOAc/hexane, 1:2); 1H NMR
(200 MHz, CDCl3) d 8.09 (d, J = 8.2 Hz, 2H), 7.64 (d,
J = 8.4 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.35 (s, 1H),
7.22 (m, 1H), 7.07 (m, 1H), 7.00 (d, J = 3.4 Hz, 1H),
6.06 (d, J = 3.4 Hz, 1H), 5.16 (s, 2H), 3.95 (s, 3H),
2.29 (s, 3H).
4.5. Methyl 4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}benzoate
(26a)
To a stirred solution of 25a (550 mg, 1.17 mmol) in
DMF (2 mL) were added K2CO3 (484 mg, 3.51 mmol)
and isobutyl iodide (0.20 mL, 1.76 mmol) under argon
atmosphere. After being stirred at 80 ꢁC overnight, the
reaction mixture was poured into water and extracted
with EtOAc (2·). The combined organic layers were
washed with water, brine, dried over MgSO4, and con-
centrated in vacuo. The resulting residue was purified
by column chromatography on silica gel to yield 26a
(577 mg, 94%). TLC Rf = 0.45 (EtOAc/hexane, 1:2);
1H NMR (200 MHz, CDCl3) d 8.06 (d, J = 8.6 Hz,
4.2. Methyl 4-{[2-nitro-5-(trifluoromethyl)phenoxy]meth-
yl}benzoate (20a)
To a stirred suspension of 196 (5 g, 24.1 mmol) and
K2CO3 (6.7 g, 48.3 mmol) in acetone (30 mL) was added
methyl 4-(bromomethyl)benzoate (6 g, 26.6 mmol)

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7129
2H), 7.41 (d, J = 8.6 Hz, 2H), 7.50–7.40 (m, 1H), 7.22–
7.30 (m, 1H), 7.16 (m, 1H), 6.74 (d, J = 3.6 Hz, 1H),
5.96 (m, 1H), 5.07 (s, 2H), 3.94 (s, 3H), 3.50 (d,
J = 7.2 Hz, 2H), 2.14 (s, 3H), 1.72–1.62 (m, 1H), 0.89
(d, J = 6.6 Hz, 6H).
(2·), brine, dried over MgSO4, and evaporated. The
resulting residue was purified by column chromatogra-
phy on silica gel to yield 21 (14.4 g, 85%). TLC
Rf = 0.28 (EtOAc/hexane, 1:2); 1H NMR (300 MHz,
CDCl3) d 7.91 (d, J = 7.5 Hz, 1H), 7.47–7.31 (m, 6H),
5.28 (s, 2H), 4.27 (s, 2H), 1.69 (br s, 1H).
4.6. 4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]amino}-5-
(trifluoromethyl)phenoxy]methyl}benzoic acid (2b)
4.9. 4-{[2-Nitro-5-(trifluoromethyl)phenoxy]methyl}benz-
aldehyde (22)
To a stirred solution of 26a (577 mg, 1.10 mmol) in
MeOH (2 mL) and dioxane (2 mL) was added 2 M
NaOH (2 mL) at room temperature. After being stirred
overnight, the reaction mixture was acidified with 1 M
HCl and extracted with EtOAc (2·). The combined
organic layers were washed with water, brine, dried over
MgSO4, and concentrated in vacuo. The resulting residue
To a stirred solution of 21 (13.9 g, 42.6 mmol), DMSO
(93 mL) and Pr2NEt (44.5 mL, 256 mmol) in EtOAc
i
(67 mL) was added SO3ÆPy (20.3 g, 128 mmol) at room
temperature. The reaction mixture was stirred for
30 min, quenched with water, and extracted with EtOAc
(2·). The combined organic layers were washed with
water (3·), brine, dried over MgSO4, and evaporated
to yield 22 (11.7 g, 81%). TLC Rf = 0.56 (EtOAc/hexane,
1:2); 1H NMR (300 MHz, CDCl3) d 10.05 (s, 1H),
8.00–7.29 (m, 3H), 7.65 (d, J = 8.1 Hz, 2H), 7.40–7.36
(m, 2H), 5.36 (s, 2H).
i
was recrystallized from PrOH and hexane to yield 2b
(495 mg, 88%). TLC Rf = 0.46 (MeOH/CHCl3, 1:9); H
1
NMR (200 MHz, CDCl3) d 8.15 (d, J = 8.6 Hz, 2H),
7.46 (d, J = 8.6 Hz, 2H), 7.41 (m, 1H), 7.29 (m, 1H),
7.18 (m, 1H), 6.76 (d, J = 3.4 Hz, 1H), 6.03–5.93 (m,
1H), 5.10 (s, 2H), 3.51 (d, J = 6.2 Hz, 2H), 2.16 (s, 3H),
1.64 (m, 1H), 0.90 (d, J = 6.8 Hz, 6H); IR (KBr) 2963,
4.10. (2E)-3-(4-{[2-Nitro-5-(trifluoromethyl)phen-
oxy]methyl}phenyl)acrylic acid (23)
1694, 1588, 1427, 1360, 1331, 1173, 1128, 1020 cmꢀ1
;
MS (APCI, Neg.) m/e 510 (M–H)ꢀ; HRMS (Pos.) calcd
for C24H25F3NO6S: 512.1355; found: 512.1349.
A solution of 22 (11.3 g, 34.9 mmol), malonic acid
(7.26 g, 69.8 mmol), and piperidine (6.9 mL, 69.8 mmol)
in pyridine (70 mL) was stirred at 100 ꢁC under argon
atmosphere. After being stirred for 30 min, the reaction
mixture was concentrated in vavuo, quenched with
water, and extracted with EtOAc (2·). The combined
organic layers were washed with 1 M HCl, water, brine,
and dried over MgSO4, and evaporated to afford a res-
idue, which was recrystallized from EtOAc and hexane
4.7. N-Isobutyl-5-methyl-N-[2-[(4-{[(methylsulfonyl)ami-
no]carbonyl}benzyl)oxy]- 4-(trifluoromethyl)phenyl]furan-
2-sulfonamide (4)
To a stirred solution of 2b (130 mg, 0.25 mmol) in DMF
(1 mL) were added methanesulfonamide (121 mg,
1.27 mmol), DMAP (37 mg, 0.31 mmol), and EDC
(58 mg, 0.31 mmol) under argon atmosphere. After
being stirred overnight, the reaction mixture was
quenched with 0.5 M HCl and extracted with EtOAc.
The organic layer was washed with water, brine, dried
over Na2SO4, and concentrated in vacuo. The resulting
residue was purified by column chromatography on
silica gel to yield 4 (38 mg, 25%). TLC Rf = 0.23
1
to yield 23 (11.6 g, 90%). H NMR (300 MHz, CDCl3)
d 8.64 (m, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.77 (d,
J = 15.6 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.51 (d,
J = 8.4 Hz, 2H), 7.40-7.30 (m, 1H), 6.48 (d,
J = 15.6 Hz, 1H), 5.30 (s, 2H).
4.11. Methyl (2E)-3-(4-{[2-nitro-5-(trifluoromethyl)phen-
oxy]methyl}phenyl)acrylate (20b)
1
(EtOAc/hexane, 1:1); H NMR (300 MHz, DMSO-d6)
d 12.13 (br s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.46 (d,
J = 8.4 Hz, 2H), 7.46–7.43 (m, 2H), 7.38 (d,
J = 8.4 Hz, 1H), 6.90 (d, J = 3.3 Hz, 1H), 6.18 (dd,
J = 3.3, 0.9 Hz, 1H), 5.22 (br s, 2H), 3.39 (d,
J = 6.9 Hz, 2H), 3.35 (s, 3H), 2.15 (s, 3H), 1.51 (m,
1H), 7.82 (d, J = 6.9 Hz, 6H); IR (KBr) 3266, 2962,
2933, 2874, 1696, 1613, 1591, 1509, 1430, 1332, 1257,
1212, 1164, 1132, 1083, 1019, 972 cmꢀ1; MS (APCI,
Pos.) m/e 611 (M + Na)+, 589 (M + H)+ ; HRMS
(Pos.) calcd for C25H28F3N2O7S: 589.1290; found:
589.1281.
To a stirred solution of 23 (11.5 g, 31.4 mmol) and
K2CO3 (6.6 g, 47.2 mmol) in DMF (125 mL) was added
methyl iodide (2.9 mL, 47.2 mmol) under argon atmo-
sphere. After being stirred for 1.5 h at 65 ꢁC, the reac-
tion mixture was quenched with water and extracted
with EtOAc (2·). The combined organic layers were
washed with water (3·), brine, dried over MgSO4, and
evaporated to yield 20b (12.1 g, 100%). TLC Rf = 0.76
(EtOAc/hexane, 1:2); 1H NMR (300 MHz, CDCl3) d
7.93 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 15.9 Hz, 1H),
7.57 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H),
7.39–7.32 (m, 2H), 6.46 (d, J = 15.9 Hz, 1H), 5.29 (s,
2H), 3.82 (s, 3H).
4.8. (4-{[2-Nitro-5-(trifluoromethyl)phenoxy]meth-
yl}phenyl)methanol (21)
To a stirred solution of 20a (18.5 g, 52.1 mmol) in Et2O
(200 mL) and MeOH (3.2 mL) was added LiBH4
(1.72 g, 78.2 mmol) under argon atmosphere. The reac-
tion mixture was refluxed, stirred at 0 ꢁC overnight,
quenched with water, and extracted with EtOAc (2·).
The combined organic layers were washed with water
4.12. Methyl (2E)-3-(4-{[2-amino-5-(trifluorometh-
yl)phenoxy]methyl}phenyl)acrylate (24b)
Compound 24b was prepared from 20b according to the
same procedure as described for the preparation of 24a
from 20a. Yield 91%; TLC Rf = 0.34 (EtOAc/hexane,

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
1:2); 1H NMR (300 MHz, CDCl3) d 7.71 (d, J = 15.9 Hz,
1H), 7.56 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H),
7.09 (m, 1H), 7.04 (d, J = 1.2 Hz, 1H), 6.73 (dd,
J = 8.1, 0.6 Hz, 1H), 6.47 (d, J = 15.9 Hz, 1H), 5.12 (s,
2H), 4.12 (br s, 2H), 3.88 (s, 3H).
with brine, dried over MgSO4, and concentrated in vac-
uo. The resulting residue was purified by column chro-
matography on silica gel to yield 24c (370 mg, 74%).
TLC Rf = 0.39 (EtOAc/hexane, 1:2); 1H NMR
(300 MHz, CDCl3) d 7.37 (d, J = 8.1 Hz, 2H), 7.24 (d,
J = 8.1 Hz, 2H), 7.11–7.04 (m, 2H), 6.73 (d,
J = 8.1 Hz, 1H), 5.06 (s, 2H), 4.09 (br, 2H), 3.68 (s,
3H), 2.98 (t, J = 7.8 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H);
MS (APCI, Pos.) m/e 354 (M + H)+.
4.13. Methyl (2E)-3-(4-{[2-{[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}phen-
yl)acrylate (25b)
Compound 25b was prepared from 24b according to the
same procedure as described for the preparation of 25a
from 24a. Yield 80%; TLC Rf = 0.59 (EtOAc/benzene,
4.17. Methyl 3-(4-{[2-{[(5-methyl-2-furyl)sulfonyl]ami-
no}-5-(trifluoromethyl)phenoxy]methyl}phenyl)propano-
ate (25c)
1:9); 1H NMR (200 MHz, CDCl3)
d
7.72 (d,
J = 16.2 Hz, 1H), 7.67–7.53 (m, 3H), 7.44–7.32 (m,
3H), 7.22 (d, J = 7.6 Hz, 1H), 7.10 (m, 1H), 7.00 (d,
J = 3.4 Hz, 1H), 6.48 (d, J = 16.2 Hz, 1H), 6.06 (d,
J = 3.4 Hz, 1H), 5.11 (s, 2H), 3.83 (s, 3H), 2.28 (s, 3H).
Compound 25c was prepared from 24c according to the
same procedure as described for the preparation of 25a
from 24a. Yield 100%; TLC Rf = 0.33 (EtOAc/hexane,
1:2); 1H NMR (300 MHz, CDCl3)
d
7.60 (d,
J = 8.7 Hz, 1H), 7.34 (s, 1H), 7.32–7.16 (m, 5H), 7.09
(d, J = 1.8 Hz, 1H), 6.98 (d, J = 2.7 Hz, 1H), 6.05 (dq,
J = 3.3, 0.9 Hz, 1H), 5.03 (s, 2H), 3.68 (s, 3H), 2.99 (t,
J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.28 (d,
J = 0.9 Hz, 3H); MS (APCI, Pos.) m/e 498 (M + H)+.
4.14. Methyl (2E)-3-(4-{[2-{isobutyl[(5-methyl-2-
furyl)sulfonyl]amino}-5-(trifluoromethyl)phenoxy]meth-
yl}phenyl)acrylate (26b)
Compound 26b was prepared from 25b according to the
same procedure as described for the preparation of 26a
from 25a. Yield 94%; TLC Rf = 0.44 (EtOAc/hexane,
4.18. Methyl 3-(4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}phen-
yl)propanoate (26c)
1:3); 1H NMR (200 MHz, CDCl3)
d
7.70 (d,
J = 16.0 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.45–7.15
(m, 3H), 7.37 (d, J = 8.2 Hz, 2H), 6.73 (d, J = 3.4 Hz,
1H), 6.47 (d, J = 16.0 Hz, 1H), 5.96 (dq, J = 3.4, 1.0 Hz,
1H), 5.03 (s, 2H), 3.82 (s, 3H), 3.51 (d, J = 7.2 Hz, 2H),
2.14 (s, 3H), 1.75–1.50 (m, 1H), 0.89 (d, J = 6.6 Hz, 6H).
Compound 26c was prepared from 25c according to the
same procedure as described for the preparation of 26a
from 25a. Yield 70%; TLC Rf = 0.55 (EtOAc/hexane,
1
1:1); H NMR (300 MHz, CDCl3) d 7.44 (dd, J = 8.1,
0.6 Hz, 1H), 7.30–7.15 (m, 6H), 6.69 (dd, J = 3.3,
0.3 Hz, 1H), 5.94 (dq, J = 3.6, 1.2 Hz, 1H), 4.95 (br s,
2H), 3.68 (s, 3H), 3.52 (d, J = 7.5 Hz, 2H), 2.97 (t,
J = 7.5 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H), 2.10 (d,
J = 0.3 Hz, 3H), 1.62 (sept, J = 6.9 Hz, 1H), 0.90 (d,
J = 6.9 Hz, 6H); MS (APCI, Pos.) m/e 554 (M + H)+.
4.15. (2E)-3-(4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}phen-
yl)acrylic acid (16)
Compound 16 was prepared from 26b according to the
same procedure as described for the preparation of 2b
from 26a. Yield 91%; TLC Rf = 0.51 (EtOAc/hexane/
4.19. 3-(4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]ami-
no}-5-(trifluoromethyl)phenoxy]methyl}phenyl)propanoic
acid (15)
1
AcOH, 1:1:0.02); H NMR (200 MHz, CDCl3) d 7.80
(d, J = 16.2 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H),
7.45–7.36 (m, 3H), 7.26 (dd, J = 8.2, 1.8 Hz, 1H), 7.18
(d, J = 1.8 Hz, 1H), 7.00–5.00 (br, 1H), 6.75 (d,
J = 3.4 Hz, 1H), 6.49 (d, J = 16.2 Hz, 1H), 5.98 (dq,
J = 3.4, 0.8 Hz, 1H), 5.05 (br s, 2H), 3.51 (d,
J = 7.4 Hz, 2H), 2.16 (q, J = 0.8 Hz, 3H), 1.75–1.50
(m, 1H), 0.90 (d, J = 6.8 Hz, 6H); IR (KBr) 2967,
1683, 1630, 1510, 1424, 1380, 1331, 1219, 1127, 1086,
1066, 1041, 1019 cmꢀ1; MS (FAB, Pos.) m/e 538
(M + H)+; Anal. Calcd for C26H26F3NO6 S: C, 58.09;
H, 4.88; N, 2.61; S, 5.96. Found: C, 58.11; H, 4.77; N,
2.56; S, 6.20.
Compound 15 was prepared from 26c according to the
same procedure as described for the preparation of 2
from 26a. Yield 98%; TLC Rf = 0.39 (EtOAc/hexane,
1
1:1); H NMR (300 MHz, CDCl3) d 7.43 (dd, J = 8.1,
1.8 Hz, 1H), 7.27–7.22 (m, 5H), 7.16 (d, J = 1.8 Hz,
1H), 6.69 (d, J = 3.3 Hz, 1H), 5.94 (dd, J = 3.3, 1.2 Hz,
1H), 4.95 (s, 2H), 3.51 (d, J = 6.9 Hz, 2H), 2.99 (t,
J = 7.5 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H), 2.10 (s, 3H),
1.60 (sept, J = 6.9 Hz, 1H), 0.89 (d, J = 6.9 Hz, 6H);
IR (neat) 2979, 1712, 1611, 1590, 1513, 1427, 1330,
1129, 1038, 903, 698 cmꢀ1; MS (APCI, Neg.) m/e 538
(M–H)ꢀ; HRMS (Pos.) calcd for C26H29F3NO6 S:
540.1668; found: 540.1684.
4.16. Methyl 3-(4-{[2-amino-5-(trifluoromethyl)phen-
oxy]methyl}phenyl)propanoate (24c)
To a stirred solution of 24b (500 mg, 1.43 mmol) in
EtOH (2 mL) and THF (2 mL) were added NiCl2Æ6H2O
(34 mg, 0.14 mmol) and NaBH4 (59 mg, 1.57 mmol) un-
der argon atmosphere. The reaction mixture was stirred
for 1 h at room temperature, quenched with water, and
extracted with EtOAc. The organic layer was washed
4.20. Methyl 3-(4-{[2-[[(5-methyl-2-furyl)sulfonyl](isobu-
tyl)amino]-5-(trifluoromethyl)phenoxy]methyl}phen-
yl)prop-2-ynoate (26d)
To a stirred solution of 2b (250 mg, 0.49 mmol) in
EtOAc (4 mL) was added SOCl2 (0.071 mL, 0.98 mmol)

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7131
under argon atmosphere. After being stirred for 1.5 h at
80 ꢁC, the reaction mixture was concentrated in vacuo.
To the resulting residue were added toluene (4 mL),
methyl (triphenylphosphoranylidene)acetate (167 mg,
0.50 mmol), and Et3N (0.07 mL, 0.50 mmol) under
argon atmosphere. After being stirred for 3 h at 125 ꢁC,
the reaction mixture was quenched with water and
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and concentrated in vac-
uo. A solution of the resulting residue in o-dichloroben-
zene (2 mL) was stirred at 200 ꢁC for 3.5 h and then
concentrated in vacuo. The resulting residue was puri-
fied by column chromatography on silica gel to yield
26d (170 mg, 63%). TLC Rf = 0.52 (EtOAc/hexane,
MgSO4, and evaporated to yield 29 (31 g, 41%), which
was used for the next step without further purification.
TLC Rf = 0.32 (EtOAc/hexane, 1:2); 1H NMR
(200 MHz, CDCl3) d 7.23-7.21 (m, 1H), 6.27–6.25 (m,
1H), 2.47 (s, 3H).
4.24. 2-(Methoxymethoxy)-4-(trifluoromethyl)aniline (31)
A suspension of 30 (2.32 g, 9.24 mmol) and 10% Pd–C
(232 mg) in MeOH (10 mL) was stirred for 1 h under
hydrogen 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
chromatography on silica gel to yield 31 (1.88 g, 88%).
TLC Rf = 0.49 (EtOAc/hexane, 1:2); 1H NMR
(200 MHz, CDCl3) d 7.24 (m, 1H), 7.10 (m, 1H), 6.73
(m, 1H), 5.22 (s, 2H), 3.51 (s, 3H).
1:2); 1H NMR (300 MHz, CDCl3)
d
7.61 (d,
J = 8.4 Hz, 2H), 7.40 (d, J = 8.1 Hz, 1H), 7.31 (d,
J = 8.4 Hz, 2H), 7.27 (m, 1H), 7.15 (d, J = 1.5 Hz,
1H), 6.74 (d, J = 3.3 Hz, 1H), 5.98 (dd, J = 3.3, 0.9 Hz,
1H), 5.09 (br s, 2H), 3.86 (s, 3H), 3.49 (d, J = 6.9 Hz,
2H), 2.16 (s, 3H), 1.60 (sept, J = 6.9 Hz, 1H), 0.88 (d,
J = 6.9 Hz, 6H).
4.25. N-[2-(Methoxymethoxy)-4-(trifluoromethyl)phenyl]-
5-methylfuran-2-sulfonamide (32)
To a stirred solution of 31 (1.88 g, 8.50 mmol) and pyr-
idine (1.7 mL, 20.4 mmol) in CH2Cl2 (10 mL) was added
29 (1.84 g, 10.2 mmol) at 0 ꢁC under argon atmosphere.
After being stirred for 6 h at room temperature, the
reaction mixture was quenched with water and extracted
with EtOAc (2·). The combined organic layers were
washed with 0.5 M HCl, water, and brine, dried over
MgSO4, and concentrated in vacuo to yield 32 (2.86 g,
92%). TLC Rf = 0.51 (EtOAc/hexane, 1:1); 1H NMR
(200 MHz, CDCl3) d 7.61 (d, J = 8.4 Hz, 1H), 7.43 (br
s, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.24 (dd, J = 8.4,
1.8 Hz, 1H), 7.02 (d, J = 3.4 Hz, 1H), 6.07 (d,
J = 3.4 Hz, 1H), 5.21 (s, 2H), 3.47 (s, 3H), 2.31 (s, 3H).
4.21. 3-(4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]ami-
no}-5-(trifluoromethyl)phenoxy]methyl}phenyl)prop-2-
ynoic acid (17)
Compound 17 was prepared from 26c according to the
same procedure as described for the preparation of 2b
from 26a. Yield 91%; TLC Rf = 0.24 (MeOH/CHCl3,
1:4); 1H NMR (300 MHz, CDCl3)
d
7.64 (d,
J = 8.1 Hz, 2H), 7.43–7.37 (m, 3H), 7.27 (m, 1H), 7.16
(d, J = 1.5 Hz, 1H), 6.76 (d, J = 3.3 Hz, 1H), 5.99 (dd,
J = 3.3, 0.9 Hz, 1H), 5.06 (br s, 2H), 3.48 (d,
J = 6.9 Hz, 2H), 2.18 (s, 3H), 1.63 (sep, J = 6.9 Hz,
1H), 0.88 (d, J = 6.9 Hz, 6H); IR (KBr) 3437, 3085,
2967, 2582, 2227, 2202, 1679, 1513, 1427, 1358, 1331,
1212, 1127, 909 cmꢀ1; MS (FAB, Pos.) m/e 558
(M + Na)+, 536 (M + H)+; Anal. Calcd for
C26H24F3NO6 S: C, 58.31; H, 4.52; N, 2.62; S, 5.99.
Found: C, 58.51; H, 4.53; N, 2.60; S, 5.96.
4.26. N-Isobutyl-N-[2-(methoxymethoxy)-4-(trifluoro-
methyl)phenyl]-5-methylfuran-2- sulfonamide (33)
Compound 33 was prepared from 32 according to the
same procedure as described for the preparation of
26a from 25a. Yield 63%; TLC Rf = 0.63 (EtOAc/hex-
ane, 1:3); 1H NMR (300 MHz, CDCl3) d 7.41 (d,
J = 1.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.28–7.22
(m, 1H), 6.76 (d, J = 3.6 Hz, 1H), 6.08 (d, J = 3.6 Hz,
1H), 5.03 (br s, 2H), 3.48 (q, J = 7.5 Hz, 2H), 3.44 (s,
3H), 2.39 (s, 3H), 1.70–1.50 (m, 1H), 0.91 (d,
J = 6.9 Hz, 6H).
4.22. Pyridinium 5-methylfuran-2-sulfonate (28)
To a stirred solution of 27 (46 g, 560 mmol) in CH3CN
(60 mL) was added SO3ÆPy (116 g, 728 mmol). After
being stirred at 40 ꢁC for 23 h under argon atmosphere,
the reaction mixture was diluted with EtOAc (120 mL)
and stirred for 2 h at 5 ꢁC. The resulting precipitates
1
were collected by filtration to yield 28 (126 g, 93%). H
4.27. N-[2-Hydroxy-4-(trifluoromethyl)phenyl]-N-isobu-
tyl-5-methylfuran-2-sulfonamide (34)
NMR (300 MHz, DMSO-d6) d 8.95 (dd, J = 6.3,
1.5 Hz, 2H), 8.61 (tt, J = 8.1, 1.5 Hz, 1H), 8.08 (dd,
J = 8.1, 6.3 Hz, 2H), 6.27 (d, J = 3.0 Hz, 1H),
5.98–5.94 (m, 1H), 2.24 (d, J = 0.6 Hz, 3H).
To a stirred solution of 33 (2.08 g, 4.94 mmol) in MeOH
(10 mL) was added 4 M HCl in dioxane (4 mL) at room
temperature. After being stirred for 6 h, the reaction
mixture was quenched with aqueous NaHCO3 and
extracted with EtOAc. The organic layer was washed
with water (2·), brine, dried over MgSO4 and concen-
trated in vacuo to yield 34 (1.75 g, 94%). TLC
Rf = 0.27 (EtOAc/Hex, 1:3); 1H NMR (200 MHz,
4.23. 5-Methylfuran-2-sulfonyl chloride (29)
To a stirred suspension of 28 (102 g, 420 mmol) in DME
(300 mL) were added oxalyl chloride (55 mL, 631 mmol)
and then DMF (33 mL) at 0 ꢁC under argon atmo-
sphere. After being stirred for 3 h at room temperature,
the reaction mixture was quenched with ice-water and
extracted with toluene. The organic layer was washed
with aqueous NaHCO3, water, and brine, dried over
DMSO-d6)
d 7.31 (d, J = 8.7 Hz, 1H), 7.14 (d,
J = 6.6 Hz, 2H), 6.89 (d, J = 3.3 Hz, 1H), 6.29 (d,
J = 3.3 Hz, 1H), 3.39 (d, J = 7.5 Hz, 2H), 2.35 (s, 3H),
1.50 (qn, J = 6.9 Hz, 1H), 0.85 (d, J = 6.9 Hz, 6H).

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
4.28. N-[2-[(4-Cyanobenzyl)oxy]-4-(trifluoromethyl)phen-
yl]-N-isobutyl-5-methylfuran-2-sulfonamide (3)
4.31. N-Isobutyl-5-methyl-N-[2-{[4-(2-oxido-3H-1,2,3,5-
oxathiadiazol-4-yl)benzyl]oxy}-4-(trifluoromethyl)phen-
yl]furan-2-sulfonamide (8)
A solution of 34 (800 mg, 2.12 mmol), 4-(bromometh-
yl)benzonitrile (424 mg, 2.12 mmol), and K2CO3
(293 mg, 2.12 mmol) in DMF (8 mL) was stirred at
65 ꢁC under argon atmosphere. After being stirred for
40 min, the reaction mixture was quenched with water
and extracted with EtOAc. The organic layer was
washed with water, brine, dried over Na2SO4, and con-
centrated in vacuo. The resulting residue was purified
by column chromatography on silica gel to yield 3
(777 mg, 74%). TLC Rf = 0.84 (EtOAc/hexane, 1:1);
1H NMR (300 MHz, CDCl3) d 7.70 (d, J = 8.4 Hz,
2H), 7.53 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 7.8 Hz,
1H), 7.27 (dd, J = 7.8, 1.5 Hz, 1H), 7.17 (d,
J = 1.5 Hz, 1H), 6.79 (d, J = 3.3 Hz, 1H), 6.02 (dd,
J = 3.3, 0.9 Hz, 1H), 5.13 (s, 2H), 3.46 (d, J = 6.9 Hz,
2H), 2.23 (s, 3H), 1.61 (m, 1H), 0.87 (d, J = 6.9 Hz,
6H); IR (KBr) 3450, 2962, 1700, 1593, 1569, 1429,
1332, 1182, 1128, 1019 cmꢀ1; MS (FAB, Pos.) m/e
670 (M + H)+; Anal. Calcd for C24H23F3N2 O4S: C,
58.53; H, 4.71; N, 5.69; S, 6.51. Found: C, 58.36; H,
4.76; N, 5.59; S, 6.79.
To a stirred solution of 35 (150 mg, 0.29 mmol) and pyr-
idine (0.047 mL, 0.58 mmol) in THF (6 mL) was added
SOCl2 (0.022 mL, 0.29 mmol) at 0 ꢁC under argon
atmosphere. After being stirred for 30 min, the reaction
mixture was quenched with water and extracted with
EtOAc. The organic layer was washed with water, brine,
dried over Na2SO4, and concentrated in vacuo. The
resulting residue was purified by column chromatogra-
phy on silica gel to yield 8 (83 mg, 51%). TLC
Rf = 0.56 (EtOAc/hexane, 1:1); 1H NMR (300 MHz,
DMSO-d6) d 12.18 (br s, 1H), 7.86 (d, J = 8.4 Hz, 2H),
7.53 (d, J = 8.4 Hz, 2H), 7.50 (s, 1H), 7.44 (d,
J = 8.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 6.91 (d,
J = 3.3 Hz, 1H), 6.18 (dd, J = 3.3, 1.2 Hz, 1H), 5.24
(br, 2H), 3.39 (d, J = 6.6 Hz, 2H), 2.15 (s, 3H), 1.52
(sep, J = 6.6 Hz, 1H), 0.82 (d, J = 6.6 Hz, 6H); IR
(KBr) 3252, 2961, 2930, 2873, 1613, 1592, 1508, 1332,
1211, 1173, 1130, 1020 cmꢀ1; MS (FAB, Pos.) m/e 572
(M + H)+; HRMS (Pos.) calcd for C24H25F3N3 O6S2:
572.1137; found: 572.1116.
4.29. N-Isobutyl-5-methyl-N-[2-{[4-(1H-tetraazol-5-
yl)benzyl]oxy} -4-(trifluoromethyl)phenyl]furan-2-sulfon-
amide (11)
4.32. N-Isobutyl-5-methyl-N-[2-{[4-(5-oxo-4,5-dihydro-
1,2,4-oxadiazol-3-yl)benzyl]oxy}-4-(trifluoromethyl)phen-
yl]furan-2-sulfonamide (9)
A solution of 3 (230 mg, 0.47 mmol), sodium azide
(84 mg,
To a stirred solution of 35 (200 mg, 0.38 mmol) and pyr-
idine (0.034 mL, 0.42 mmol) in DMF (1 mL) was added
2-ethylhexyl chloroformate (0.075 mL, 0.38 mmol) at
0 ꢁC under argon atmosphere. After being stirred for
30 min, the reaction mixture was quenched with water
and extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, and concentrat-
ed in vacuo. To a stirred solution of the resulting residue
in xylene (3 mL) was stirred at 140 ꢁC for 2 h, the reac-
tion mixture was concentrated and purified by column
chromatography on silica gel to yield 9 (77 mg, 36%).
TLC Rf = 0.50 (EtOAc/hexane, 1:1); 1H NMR
(300 MHz, DMSO-d6) d 12.95 (br s, 1H), 7.82 (d,
J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.50 (s, 1H),
7.44 (d, J = 8.4 Hz, 1H) 7.38 (d, J = 8.4 Hz, 1H), 6.90
(d, J = 3.3 Hz, 1H), 6.18 (dd, J = 3.3, 1.2 Hz, 1H), 5.23
(br, 2H), 3.39 (d, J = 6.6 Hz, 2H), 2.15 (s, 3H), 1.51
(sept, J = 6.6 Hz, 1H), 0.81 (d, J = 6.6 Hz, 6H); IR
(KBr) 3490, 3138, 2957, 2930, 2871, 1766, 1508, 1428,
(40 mg,
0.61 mmol),
and
Et3NÆHCl
0.61 mmol) in toluene (2 mL) was stirred at 120 ꢁC
under reflux condition. After being stirred overnight,
the reaction mixture was quenched with 0.5 N HCl,
extracted with EtOAc. The organic layer was washed
with water, brine, dried over Na2SO4, and concen-
trated in vacuo. The resulting residue was purified
by column chromatography on silica gel to yield 11
(49 mg, 20%). TLC Rf = 0.38 (MeOH/CHCl3, 1:4);
1H NMR (300 MHz, CDCl3) d 8.08 (d, J = 8.4 Hz,
2H), 7.53 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz,
1H), 7.26 (m, 1H), 7.20 (s, 1H), 6.78 (d, J = 3.3
Hz, 1H), 6.02 (d, J = 3.3 Hz, 1H), 5.11 (br, 2H),
3.49 (d, J = 6.3 Hz, 2H), 2.21 (s, 3H), 1.63 (m, 1H),
0.88 (d, J = 6.3 Hz, 6H); IR (KBr) 2962, 1592,
1510, 1428, 1332, 1256, 1212, 1173, 1128, 1084,
1064, 1022, 912 cmꢀ1; MS (FAB, Pos.) m/e 536
(M + H)+; HRMS (Pos.) calcd for C24H25F3N5 O4S:
536.1579; found: 536.1582.
1369, 1328, 1255, 1214, 1173, 1136, 1020, 910 cmꢀ1
;
MS (FAB, Pos.) m/e 552 (M + H)+ ; HRMS (Pos.) calcd
for C25H25F3N3 O6S: 552.1416; found: 552.1403.
4.30. N0-Hydroxy-4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}benzene-
carboximidamide (35)
4.33. N-Isobutyl-5-methyl-N-[2-{[4-(5-oxo-4,5-dihydro-
1,2,4-thiadiazol-3-yl)benzyl]oxy}-4-(trifluoromethyl)phen-
yl]furan-2-sulfonamide (10)
A solution of 3 (547 mg, 1.11 mmol), NH2OHÆHCl
(155 mg, 2.22 mmol), and Et3N (0.31 mL, 2.22 mmol)
in EtOH (6 mL) was stirred at 78 ꢁC under argon atmo-
sphere. After being stirred for 2.5 h, the reaction mix-
ture was quenched with water and extracted with
EtOAc. The organic layer was washed with water,
brine, dried over Na2SO4, and concentrated in vacuo
to yield 35 (584 mg, quant). TLC Rf = 0.53 (EtOAc/
hexane, 1:1).
To a stirred solution of 35 (400 mg, 0.78 mmol) and
DBU (432 mg, 3.13 mmol) in CH3CN (7 mL) was added
TCDI (233 mg, 1.17 mmol) under argon atmosphere.
After being stirred for 1 h, the reaction mixture was
quenched with 1 M HCl and extracted with EtOAc.
The organic layer was washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. To the

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7133
stirred solution of the resulting residue in THF (5 mL)
was added BF3ÆOEt2 (0.51 mL, 3.91 mmol) under argon
atmosphere. After being stirred for 1 h, the reaction
mixture was quenched with water and extracted with
EtOAc. The organic layer was washed with 1 M HCl,
water, and brine, dried over Na2SO4, and concentrated
in vacuo. The resulting residue was purified by column
chromatography on silica gel to yield 10 (49 mg, 20%).
TLC Rf = 0.74 (EtOAc/hexane, 1:1); 1H NMR
(300 MHz, DMSO-d6) d 13.38 (br s, 1H), 7.95 (d,
J = 8.1 Hz, 2H), 7.50 (s, 1H), 7.48 (d, J = 8.1 Hz, 2H),
7.44 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 6.90
(d, J = 3.3 Hz, 1H), 6.18 (d, J = 3.3 Hz, 1H), 5.21 (br,
2H), 3.39 (d, J = 6.9 Hz, 2H), 2.15 (s, 3H), 1.51 (sept,
J = 6.9 Hz, 1H), 0.81 (d, J = 6.9 Hz, 6H); IR (KBr)
3423, 3271, 2973, 2930, 2875, 1712, 1687, 1413, 1360,
1258, 1165, 1117, 1016, 911 cmꢀ1; MS (FAB, Pos.) m/e
568 (M + H)+; HRMS (Pos.) calcd for C25H25F3N3
O5S2: 568.1188; found: 568.1168.
atmosphere. After being stirred for 3 h, the reaction
mixture was concentrated in vacuo, quenched with
aqueous NaHCO3, and extracted with EtOAc. The
organic layer was washed with water, brine, dried over
Na2SO4, and concentrated in vacuo. The resulting resi-
due was purified by column chromatography on silica
gel to yield 5 (50 mg, 26%). TLC Rf = 0.61 (EtOAc/hex-
ane, 1:1); 1H NMR (300 MHz, CDCl3) d 8.55 (br s, 1H),
7.87 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.36
(d, J = 7.8 Hz, 1H), 7.26 (m, 1H), 7.16 (d, J = 1.5 Hz,
1H), 6.78 (d, J = 3.3 Hz, 1H), 6.01 (dd, J = 3.3, 1.2 Hz,
1H), 5.13 (br, 2H), 3.48 (d, J = 6.9 Hz, 2H), 2.63 (s,
3H), 2.22 (s, 3H), 1.59 (sept, J = 6.9 Hz, 1H), 0.88 (d,
J = 6.9 Hz, 6H); IR (KBr) 3423, 3271, 2973, 2930,
2875, 1712, 1687, 1413, 1360, 1258, 1165, 1117, 1016,
911 cmꢀ1; MS (FAB, Pos.) m/e 553 (M + H)+; HRMS
(Pos.) calcd for C26H28F3N2 O6S: 553.1620; found:
553.1611.
4.37. 4-(Bromomethyl)benzenesulfonamide (40b)
4.34. 4-(Bromomethyl)benzamide (40a)
To a stirred solution of 37 (3.0 g, 11.1 mmol) in THF
(40 mL) was added 28% NH3 (2.8 mL) at 0 ꢁC. After
being stirred for 1 h, the reaction mixture was quenched
with water and extracted with EtOAc. The organic layer
was washed with water, brine, dried over Na2SO4, and
concentrated to yield 40b (2.39 g, 86%). 1H NMR
(300 MHz, CDCl3) d 7.79 (d, J = 8.4 Hz, 2H), 7.62 (d,
J = 8.4 Hz, 2H), 7.37 (br s, 2H), 4.75 (s, 2H).
To a stirred solution of 36 (2.15 g, 10.0 mmol) in EtOAc
(40 mL) was added SOCl2 (1.1 mL, 15.0 mmol). After
being stirred overnight at 75 ꢁC, the reaction mixture
was concentrated in vacuo and the resulting residue was
dissolved with CH2Cl2 (40 mL). To this stirred solution
was added 28% NH3 (1.2 mL, 20.0 mmol) at 0 ꢁC under
argon. After being stirred for 10 min, the reaction mixture
was quenched with water and filtered through a pad of
Celite. The filtrate was extracted with EtOAc and the
organic layer was washed with 1 M HCl, water, and brine,
dried over Na2SO4, andconcentratedin vacuotoyield 40a
4.38. 4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]amino}-5-
(trifluoromethyl)phenoxy]methyl}benzenesulfonamide (41b)
1
(1.39 g, 65%). TLC Rf = 0.44 (EtOAc/hexane, 1:1); H
Compound 41b was prepared from 34 and 40b according
to the same procedure as described for the preparation of
41a from 34 and 40a. Yield 69%; TLC Rf = 0.27 (EtOAc/
NMR (300 MHz, DMSO-d6) d 7.97 (br s, 1H), 7.83 (d,
J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.38 (br s, 1H),
4.73 (s, 2H); MS (APCI, Neg.) m/e 214 and 212 (M–H)ꢀ.
1
hexane, 2:3); H NMR (300 MHz, CDCl3) d 7.97 (d,
J = 8.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 7.37 (m, 1H),
7.26 (m, 1H), 7.18 (m, 1H), 6.78 (m, 1H), 6.03 (m, 1H),
5.14 (s, 2H), 4.90 (m, 2H), 3.47 (d, J = 7.2 Hz, 2H), 2.23
(s, 3H), 1.62 (m, 1H), 0.87 (d, J = 6.6 Hz, 6H).
4.35. 4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]amino}-5-
(trifluoromethyl)phenoxy]methyl}benzamide (41a)
A stirred solution of 34 (150 mg, 0.40 mmol), 40a
(88 mg, 0.40 mmol) and K2CO3 (57 mg, 0.40 mmol), in
DMF (2 mL) was stirred at 70 ꢁC under argon atmo-
sphere. After being stirred for 1.5 h, the reaction mixture
was quenched with water and extracted with EtOAc.
The organic layer was washed with water, brine, dried
over Na2SO4, and concentrated in vacuo. The resulting
residue was purified by column chromatography on sil-
ica gel to yield 41a (179 mg, 88%). 1H NMR (300 MHz,
CDCl3) d 7.85 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.1 Hz,
2H), 7.39 (d, J = 8.1 Hz, 1H), 7.26 (dd, J = 8.1, 1.8 Hz,
1H), 7.17 (d, J = 1.8 Hz, 1H), 6.76 (d, J = 3.3 Hz, 1H),
6.17 (br, 1H), 5.99 (dd, J = 3.3, 0.6 Hz, 1H), 5.75 (br,
1H), 5.09 (br s, 2H), 3.49 (d, J = 6.9 Hz, 2H), 2.18 (s,
3H), 1.63 (m, 1H), 0.88 (d, J = 6.9 Hz, 6H).
4.39. N-Isobutyl-5-methyl-N-[2-[(4-{[(methylsulfo-
nyl)amino]sulfonyl}benzyl)oxy]-4- (trifluoromethyl)phen-
yl]furan-2-sulfonamide (6)
To a stirred solution of 41b (365 mg, 0.67 mmol) in
THF (5 mL) were added 1 M NaOH (2.68 mL,
2.68 mmol) and methanesulfonyl chloride (0.124 mL,
1.6 mmol) at room temperature under argon atmo-
sphere. After being stirred for 1 h, the reaction mixture
was quenched with 2 M HCl and extracted with EtOAc.
The organic layer was washed with water, brine, dried
over MgSO4, and concentrated in vacuo. The resulting
residue was purified by column chromatography on sil-
ica gel to yield 6 (374 mg, 90%). TLC Rf = 0.33 (MeOH/
1
CHCl3, 1:4); H NMR (300 MHz, CDCl3) d 8.02 (d,
4.36. N-Acetyl-4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5- (trifluoromethyl)phenoxy]methyl}benzam-
ide (5)
J = 8.7 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 7.27 (m,
1H), 7.20 (m, 1H), 7.11 (m, 1H), 6.69 (d, J = 3.3 Hz,
1H), 5.94 (m, 1H), 5.10–4.88 (br, 2H), 3.37 (d,
J = 7.5 Hz, 2H), 2.67 (s, 3H), 2.19 (s, 3H), 1.51
(m, 1H), 0.76 (d, J = 6.9 Hz, 6H); IR (KBr) 3545,
1509, 1431, 1353, 1333, 1281, 1257, 1125, 1088,
A solution of 41a (179 mg, 0.35 mmol) in acetic anhy-
dride (2 mL) was stirred at 140 ꢁC under argon

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A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
1061 cmꢀ1; MS (FAB, Pos.) m/e 647 (M + Na)+, 625
(M + H)+; HRMS (Pos.) calcd for C24H27F3N2
O8S3Na: 647.0779; found: 647.0764.
of 41a from 34 and 40a. Yield 90%; TLC Rf = 0.40
(EtOAc/hexane, 1:2); 1H NMR (300 MHz, CDCl3) d
7.42 (d, J = 7.8 Hz, 1H), 7.30–7.15 (m, 4H), 6.91 (d,
J = 9.0 Hz, 2H), 6.70 (d, J = 3.3 Hz, 1H), 6.00–5.95
(m, 1H), 4.93 (s, 2H), 4.66 (s, 2H), 3.82 (s, 3H), 3.49
(d, J = 6.9 Hz, 2H), 2.15 (s, 3H), 1.70–1.50 (m, 1H),
0.88 (d, J = 6.6 Hz, 6H).
4.40. N-[2-({4-[(Acetylamino)sulfonyl]benzyl}oxy)-4-(tri-
fluoromethyl)phenyl]-N-isobutyl-5-methylfuran-2-sulfon-
amide (7)
To a stirred solution of 41b (190 mg, 0.35 mmol) in pyr-
idine (1.5 mL) was added acetic anhydride (0.040 mL,
0.42 mmol) at room temperature under argon atmo-
sphere. After being stirred for 1 h at 70 ꢁC, the reaction
mixture was quenched with water and extracted with
EtOAc. The organic layer was washed with water, brine,
dried over Na2SO4, and concentrated in vacuo. The
resulting residue was purified by column chromatogra-
phy on silica gel to yield 7 (124 mg, 65%). TLC
Rf = 0.63 (MeOH/CHCl3, 1:4); 1H NMR (300 MHz,
DMSO-d6) d 12.11 (s, 1H), 7.92 (d, J = 8.1 Hz, 2H),
7.56 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 1.5 Hz, 1H), 7.47
(d, J = 8.1 Hz, 1H), 7.39 (dd, J = 8.1, 1.5 Hz, 1H), 6.88
(d, J = 3.3 Hz, 1H), 6.10 (dd, J = 3.3, 0.9 Hz, 1H), 5.24
(br s, 2H), 3.42 (d, J = 7.2 Hz, 2H), 2.03 (s, 3H), 1.93
(s, 3H), 1.54 (m, 1H), 0.82 (d, J = 7.2 Hz, 6H); IR
(KBr) 3447, 3255, 2963, 2874, 1726, 1592, 1508, 1430,
1332, 1213, 1165, 1129, 1020 cmꢀ1; MS (APCI, Neg.)
m/e 587 (M–H)ꢀ; HRMS (Pos.) calcd for C25H28F3N2
O7S2: 589.1290; found: 589.1281.
4.44. (4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]amino}-
5-(trifluoromethyl)phenoxy]methyl}phenoxy)acetic acid
(18)
Compound 18 was prepared from 41c according to
the same procedure as described for the preparation
of 2b from 26a. Yield 93%; TLC Rf = 0.28 (MeOH/
1
CHCl3, 1:9); H NMR (300 MHz, CDCl3) d 7.41 (d,
J = 8.1 Hz, 1H), 7.35–7.15 (m, 4H), 6.94 (d,
J = 8.7 Hz, 2H), 6.71 (d, J = 3.3 Hz, 1H), 6.00–5.95
(m, 1H), 4.94 (br, 2H), 4.71 (s, 2H), 3.48 (d,
J = 6.9 Hz, 2H), 2.16 (s, 3H), 1.70–1.50 (m, 1H),
0.88 (d, J = 6.6 Hz, 6H); IR (KBr) 3448, 2961, 2930,
2874, 1740, 1613, 1591, 1515, 1429, 1358, 1332,
1212, 1177, 1127, 1082, 1012, 909 cmꢀ1; MS (MALDI,
Pos.) m/e 580 (M + K)+, 564 (M + Na)+; HRMS
(Pos.) calcd for C25H27F3NO7S: 542.1460; found:
542.1455.
4.45. Dimethyl 4-(bromomethyl)phthalate (43)
4.41. Methyl [4-(hydroxymethyl)phenoxy]acetate (39)
To a stirred solution of 42 (10.4 g, 50 mmol) in CCl4
(40 mL) were added N-bromo succinimide (8.90 g,
50 mmol) and dibenzoyl peroxide (847 mg, 3.5 mmol).
After being stirred for 1 h at 76 ꢁC under argon atmo-
sphere, the reaction mixture was concentrated in vacuo,
diluted with EtOAc, and filtered through a pad of Celite.
The filtrate was purified by column chromatography on
silica gel to yield 43 (9.0 g, 63%). TLC Rf = 0.30
(EtOAc/hexane, 1:4); 1H NMR (300 MHz, CDCl3) d
7.74 (d, J = 1.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.52
(dd, J = 7.8, 1.8 Hz, 1H), 4.48 (s, 2H), 3.92 (s, 3H),
3.91 (s, 3H).
To a stirred solution of 38 (2.48 g, 20 mmol) and methyl
chloroacetate (2.60 g, 24 mmol) in acetone (40 mL) were
added K2CO3 (5.53 g, 40 mmol) and KI (332 mg, 2.0
eq). After being stirred for 4.5 h at 65 ꢁC, the reaction
mixture was quenched with water and extracted with
EtOAc. The organic layer was washed with water, brine,
dried over Na2SO4, and concentrated in vacuo. The
resulting residue was purified by column chromatogra-
phy on silica gel to yield 39 (2.0 g, 51%). TLC
Rf = 0.46 (MeOH/CHCl3, 1:4); 1H NMR (300 MHz,
CDCl3) d 7.30 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz,
2H), 4.64 (s, 2H), 4.63 (s, 2H), 3.81 (s, 3H).
4.46. Dimethyl 4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}phthalate
(44)
4.42. Methyl [4-(bromomethyl)phenoxy]acetate (40c)
To a stirred solution of 39 (435 mg, 2.22 mmol) in
CH2Cl2 (2 mL) was added
Compound 44 was prepared from 34 and 43 according
to the same procedure as described for the preparation
of 41a from 34 and 40a. Yield 80%; TLC Rf = 0.31
(EtOAc/hexane, 1:2); 1H NMR (300 MHz, CDCl3) d
7.77 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 1.2 Hz, 1H), 7.56
(m, 1H), 7.42 (dd, J = 7.8, 0.6 Hz, 1H), 7.28 (m, 1H),
6.73 (d, J = 3.3 Hz, 1H), 5.96 (dd, J = 3.3, 0.9 Hz, 1H),
5.06 (br s, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.58 (d,
J = 6.6 Hz, 2H), 2.14 (s, 3H), 1.63 (m, 1H), 0.89 (d,
J = 6.6 Hz, 6H); MS (APCI, Pos.) m/e 552 (M + H)+.
a solution of PBr3
(0.24 mL, 2.66 mmol) in Et2O (2 mL). After being stir-
red for 30 min, the reaction mixture was quenched with
water and extracted with EtOAc. The organic layer was
washed with water, brine, dried over Na2SO4, and con-
centrated in vacuo to yield 40c (523 mg, 91%). TLC
Rf = 0.66 (EtOAc/hexane, 1:2); 1H NMR (300 MHz,
CDCl3) d 7.33 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz,
2H), 4.64 (s, 2H), 4.49 (s, 2H), 3.81 (s, 3H).
4.43. Methyl (4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}phen-
oxy)acetate (41c)
4.47. 4-{[2-{Isobutyl[(5-methyl-2-furyl)sulfonyl]amino}-5-
(trifluoromethyl)phenoxy]methyl}phthalic acid (45)
Compound 45 was prepared from 44 according to the
same procedure as described for the preparation of 2b
from 26a. Yield 99%; TLC Rf = 0.25 (MeOH/CHCl3/
Compound 41c was prepared from 34 and 40c according
to the same procedure as described for the preparation

A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
7135
1
AcOH, 2:20:1); H NMR (300 MHz, CDCl3) d 7.94 (d,
(300 MHz, DMSO-d6) d 7.94 (d, J = 1.0 Hz, 1H), 7.84
(d, J = 8.0 Hz, 1H), 7.75 (dd, J = 8.0, 1.0 Hz, 1H),
5.61 (t, J = 6.0 Hz, 1H), 4.64 (d, J = 6.0 Hz, 2H), 3.83
(s, 3H).
J = 7.8 Hz, 1H), 7.87 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H),
7.42 (d, J = 8.1 Hz, 1H), 7.32–7.13 (m, 1H), 7.19 (s,
1H), 6.77 (d, J = 3.3 Hz, 1H), 5.97 (dd, J = 3.3, 1.2 Hz,
1H), 5.12 (br, 2H), 3.48 (d, J = 6.9 Hz, 2H), 2.16 (s,
3H), 1.67 (m, 1H), 0.90 (d, J = 6.6 Hz, 6H); MS (APCI,
Pos.) m/e 556 (M + H)+.
4.51. Methyl 4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5- (trifluoromethyl)phenoxy]methyl}-2-nitro-
benzoate (49)
4.48. N-[2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-5-
yl)methoxy]-4-(trifluoromethyl)phenyl]-N-isobutyl-5-
methylfuran-2-sulfonamide (12)
To a stirred solution of 34 (520 mg, 1.38 mmol), 48
(580 mg, 2.75 mmol), and PPh3 (725 mg, 2.76 mmol) in
THF (10 mL) was added 40% solution of DEAD in
toluene (1.25 mL, 2.76 mmol) at room temperature un-
der argon atmosphere. After being stirred for 3 h, the
reaction mixture was concentrated in vacuo. The result-
ing residue was purified by column chromatography on
silica gel to yield 49 (625 mg, 80%). TLC Rf = 0.42
A solution of 45 (480 mg, 0.86 mmol) in Ac2O (10 mL)
was stirred at 140 ꢁC under argon atmosphere. After
being stirred for 30 min, the reaction mixture was con-
centrated and diluted with THF (10 mL). To this stir-
red solution was added 28% NH3 (1 mL) at 0 ꢁC
under argon atmosphere. After being stirred for 1 h,
the reaction mixture was quenched with 1 N HCl
and extracted with Et2O. The organic layer was
washed with brine, dried over MgSO4, and concentrat-
ed in vacuo. A solution of the resulting residue in tol-
uene (10 mL) was stirred at 125 ꢁC for 3 h, the
reaction mixture was concentrated and purified by col-
umn chromatography on silica gel to yield 12 (65 mg,
13%). TLC Rf = 0.39 (EtOAc/hexane, 1:2); 1H NMR
(300 MHz, CDCl3) d 7.92 (d, J = 8.4 Hz, 1H), 7.88–
7.83 (m, 2H), 7.65 (br s, 1H), 7.36 (d, J = 8.1 Hz,
1H), 7.32–7.25 (m, 1H), 7.19 (d, J = 1.8 Hz, 1H),
6.82 (d, J = 3.3 Hz, 1H), 6.04 (dd, J = 3.3, 0.9 Hz,
1H), 5.20 (s, 2H), 3.46 (d, J = 7.5 Hz, 2H), 2.24 (s,
3H), 1.63 (m, 1H), 0.88 (d, J = 6.6 Hz, 6H); IR
(KBr) 3297, 1776, 1724, 1510, 1427, 1360, 1332,
1213, 1175, 1136, 1043, 860 cmꢀ1; MS (APCI, Pos.)
m/e 537 (M + H)+; HRMS (Pos.) calcd for
C25H24F3N2 O6S: 537.1307; found: 537.1301.
1
(EtOAc/hexane, 1:2); H NMR (300 MHz, DMSO-d6)
d 8.02 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.79 (d,
J = 8.0 Hz, 1H), 7.51 (s, 1H), 7.47 (d, J = 8.5 Hz, 1H),
7.41 (d, J = 8.5 Hz, 1H), 6.89 (d, J = 3.0 Hz, 1H), 6.10
(d, J = 3.0 Hz, 1H), 5.32 (br s, 2H), 3.86 (s, 3H), 3.39
(d, J = 7.0 Hz, 2H), 2.11 (s, 3H), 1.67–1.47 (m, 1H),
0.83 (d, J = 7.0 Hz, 6H).
4.52. Methyl 2-amino-4-{[2-{isobutyl[(5-methyl-2-
furyl)sulfonyl]amino}-5-(trifluoromethyl)phenoxy]meth-
yl}benzoate (50)
To a stirred solution of 49 (509 mg, 0.89 mmol) in
AcOH (5 mL) and water (0.5 mL) was added iron (325
mesh, 250 mg, 4.46 mmol) under argon atmosphere.
After being stirred for 3 h at 50 ꢁC, the reaction mixture
was filtered through a pad of Celite. The filtrate was
concentrated in vacuo, diluted with aqueous NaHCO3,
and extracted with EtOAc. The organic layer was
washed with water, brine, dried over MgSO4, and con-
centrated in vacuo. The resulting residue was purified
by column chromatography on silica gel to yield 50
(376 mg, 78%). TLC Rf = 0.64 (EtOAc/hexane, 1:2);
1H NMR (300 MHz, DMSO-d6) d 7.68 (d, J = 8.0 Hz,
1H), 7.45 (d, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.36 (d,
J = 8.0 Hz, 1H), 6.88 (d, J = 3.0 Hz, 1H), 6.71 (s, 2H),
6.67 (s, 1H), 6.48 (d, J = 8.0 Hz, 1H), 6.16 (d,
J = 3.0 Hz, 1H), 5.00 (br s, 2H), 3.78 (s, 3H), 3.42 (d,
J = 7.0 Hz, 2H), 2.14 (s, 3H), 1.60–1.40 (m, 1H), 0.83
(d, J = 7.0 Hz, 6H).
4.49. 4-(Methoxycarbonyl)-3-nitrobenzoic acid (47)
To a stirred solution of 46 (10.1 g, 42.1 mmol) in MeOH
(350 mL) was added 2 M NaOH (21 mL, 42.0 mmol).
After being stirred for 1 day at room temperature, the
reaction mixture was quenched with 2 M HCl and
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and concentrated in vac-
uo. The resulting residue was washed with hexane to
yield 47 (6.22 g, 66%). TLC Rf = 0.42 (MeOH/CHCl3,
1:4); 1H NMR (300 MHz, DMSO-d6) d 13.92 (br s,
1H), 8.45 (d, J = 1.5 Hz, 1H), 8.32 (dd, J = 8.0, 1.5 Hz,
1H), 7.97 (d, J = 8.0 Hz, 1H), 3.87 (s, 3H).
4.53. 2-Amino-4-{[2-{isobutyl[(5-methyl-2-furyl)sulfo-
nyl]amino}-5-(trifluoromethyl)phenoxy]methyl}benzoic
acid (51)
4.50. Methyl 4-(hydroxymethyl)-2-nitrobenzoate (48)
Compound 51 was prepared from 49 according to the
same procedure as described for the preparation of 2b
from 26a. Yield 80%; TLC Rf = 0.55 (MeOH/CHCl3,
To a stirred solution of 47 (2.0 g, 8.88 mmol) in THF
(20 mL) was added 2 M solution of BH3ÆSMe2 in
THF (4.5 mL, 9.0 mmol) under argon atmosphere.
After being stirred for 3 days at room temperature,
the reaction mixture was quenched with water and
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and concentrated in
vacuo. The resulting residue was purified by column
chromatography on silica gel to yield 48 (1.94 g,
1
1:9); H NMR (300 MHz, DMSO-d6) d 9.50–8.00 (br
s, 2H), 7.67 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz,
1H), 7.44 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 6.88 (d,
J = 3.0 Hz, 1H), 6.63 (s, 1H), 6.45 (d, J = 8.0 Hz, 1H),
6.17 (d, J = 3.0 Hz, 1H), 4.98 (br s, 2H), 3.42 (d,
J = 7.0 Hz, 2H), 2.15 (s, 3H), 1.51 (sept, J = 7.0 Hz,
1H), 0.83 (d, J = 7.0 Hz, 6H); MS (APCI, Neg.) m/e
525 (M–H)ꢀ.
1
100%). TLC Rf = 0.43 (MeOH/CHCl3, 1:9); H NMR

7136
A. Naganawa et al. / Bioorg. Med. Chem. 14 (2006) 7121–7137
4.54. N-[2-[(4-Hydroxyquinazolin-7-yl)methoxy]-4-(tri-
fluoromethyl)phenyl]- N-isobutyl-5-methylfuran-2-sulfon-
amide (13)
taining 1 mM EDTA and 0.1 mM NaCl, pH 6.0).
Incubation was carried out at 25 ꢁC for 60 min except
for mEP1 that was incubated for 20 min. Incubation
was terminated by filtration through a Whatman
GF/B filter. The filter was subsequently washed with
ice-cold buffer (10 mM KH2PO4-KOH buffer containing
0.1 mM NaCl, pH 6.0), and the radioactivity on the fil-
ter was measured in 6 mL of liquid scintillation (ACSII)
mixture with a liquid scintillation counter. Nonspecific
binding was achieved by adding excess amounts of unla-
beled PGE2 in assay buffer. The concentration of the test
compounds required for the inhibition of specific bind-
ing in the vehicle group by 50% (IC50 value) was estimat-
ed from the regression curve. The Ki value (M) was
calculated according to the following equation.
A mixture of 51 (100 mg, 0.18 mmol) in formamidine
hydrochloride (290 mg, 3.62 mmol) was stirred at
210 ꢁC. After being stirred for 1 h, the reaction mixture
was cooled to room temperature, quenched with water,
and extracted with EtOAc. The organic layer was
washed with brine, dried over MgSO4, and concentrated
in vacuo. The resulting residue was purified by column
chromatography on silica gel to yield 13 (82 mg, 80%).
TLC Rf = 0.57 (MeOH/CHCl3, 1:9); 1H NMR
(300 MHz, DMSO-d6) d 12.27 (br s, 1H), 8.12 (d,
J = 8.0 Hz, 1H), 8.11 (s, 1H), 7.66 (s, 1H), 7.52 (s,
1H), 7.46 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H),
7.39 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 3.0 Hz, 1H), 6.12
(d, J = 3.0 Hz, 1H), 5.31 (br s, 2H), 3.43 (d,
J = 7.0 Hz, 2H), 2.08 (s, 3H), 1.63–1.43 (m, 1H), 0.83
(d, J = 7.0 Hz, 6H); IR (KBr) 3443, 1690, 1621, 1426,
1333, 1175, 1135 cmꢀ1; MS (APCI, Pos.) m/e 536
(M + H)+; Anal. Calcd for C25H24F3N3 O5S: C, 56.07;
H, 4.52; N, 7.85; S, 5.99. Found: C, 55.89; H, 4.77; N,
7.92; S, 6.16.
Ki ¼ IC50=ðl þ ½Lꢁ=KdÞ
[L]: Concentration of radiolabeled ligand; Kd; Dissocia-
tion constant of radiolabeled ligand for the prostanoid
receptors.
5.2. Measurement of the mEP1 receptor antagonist
activity
To confirm that test compounds antagonized the mEP1
receptor and estimate potencies of antagonism for the
mEP1receptor, 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 an incubator
(37 ꢁC, 5% CO2). The cells in each well were rinsed with
phosphate buffer (PBS(ꢀ)), and load buffer was added.
After incubation for 1 h, the load buffer (10% FBS/
aMEM containing 5 lM of Fura 2/AM, 20 lM of indo-
methacin, and 2.5 mM of probenecid) was discarded.
After the addition of assay buffer (Hanks’ 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
the dark at room temperature for 1 h. After the addition
of a solution containing test compound (10 ll) and
PGE2 (10 ll), which was prepared with an assay buffer,
intracellular calcium concentration was measured with a
Fluorescence drug screening system (FDSS-4000,
Hamamatsu Photonics). The fluorescence intensities
emitted at 500 nm by an excitation wavelength of 340
and 380 nm were measured. The percent inhibition on
the increase of the intracellular Ca2+ concentration
induced by PGE2 (100 nM) was calculated relative to
the maximum Ca2+ concentration that occurred in the
absence of the test compound (100%) to estimate the
IC50 value.
4.55. N-[2-[(2,4-Dihydroxyquinazolin-7-yl)methoxy]-4-
(trifluoromethyl)phenyl]- N-isobutyl-5-methylfuran-2-sul-
fonamide (14)
A mixture of 51 (88 mg, 0.16 mmol) and urea (100 mg,
1.67 mmol) was stirred at 200 ꢁC. After being stirred
for 1 h, the reaction mixture was cooled to room temper-
ature, quenched with water, and extracted with EtOAc.
The organic layer was washed with brine, dried over
MgSO4, and concentrated in vacuo. The resulting resi-
due was purified by column chromatography on silica
gel to yield 14 (80 mg, 87%). TLC Rf = 0.55 (MeOH/
1
CHCl3, 1:9); H NMR (300 MHz, DMSO-d6) d 11.32
(br s, 2H), 7.88 (d, J = 8.0 Hz, 1H), 7.51 (s, 1H), 7.48
(d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.11 (d,
J = 8.0 Hz, 1H), 7.06 (s, 1H), 6.88 (d, J = 3.0 Hz, 1H),
6.08 (d, J = 3.0 Hz, 1H), 5.19 (br s, 2H), 3.45 (br s,
2H), 2.04 (s, 3H), 1.66–1.46 (m, 1H), 0.85 (d,
J = 7.0 Hz, 6H); IR (KBr) 3461, 3061, 2962, 1718,
1633, 1428, 1360, 1332, 1174, 1135 cmꢀ1; MS (APCI,
Pos.) m/e 552 (M + H)+; Anal. Calcd for C25H24F3N3
O6S: C, 54.44; H, 4.39; N, 7.62; S, 5.81. Found: C,
54.41; H, 4.68; N, 7.68; S, 5.93.
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 mEP1-4.
References and notes
Membranes from CHO cells expressing prostanoid
receptors were incubated with radioligand (2.5 nM
[3H]PGE2) and test compounds at various concentra-
tions in assay buffer (10 mM KH2PO4-KOH buffer con-
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