3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 3: Synthesis, metabolic stability, and biological evaluation of optically active analogs

Article abstract of DOI:10.1016/j.bmc.2010.03.028

A series of 3-(2-aminocarbonylphenyl)propanoic acid analogs possessing the (1R)-1-(3,5-dimethylphenyl)-3-methylbutylamine moiety on the carboxyamide side chain were synthesized and evaluated for their binding affinity for the EP1-4 receptors and their ant

Full text of DOI:10.1016/j.bmc.2010.03.028

Bioorganic & Medicinal Chemistry 18 (2010) 3212–3223
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective
EP3 receptor antagonists. Part 3: Synthesis, metabolic stability,
and biological evaluation of optically active analogs
a
a
a
a
Masaki Asada a, , Tetsuo Obitsu , Atsushi Kinoshita , Toshihiko Nagase , Tadahiro Yoshida ,
Yoshiyuki Yamaura a, Hiroya Takizawa a, Ken Yoshikawa a, Kazutoyo Sato a, Masami Narita a, Hisao Nakai a,
Masaaki Toda a, Yoshito Tobe b
*
a Minase Research Institute, Ono Pharmaceutical Co., Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
b Division of Frontier Materials Science, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-(2-aminocarbonylphenyl)propanoic acid analogs possessing the (1R)-1-(3,5-dimethyl-
phenyl)-3-methylbutylamine moiety on the carboxyamide side chain were synthesized and evaluated
for their binding affinity for the EP1-4 receptors and their antagonist activity for the EP3 receptor.
Rational drug design based on the structure of the metabolites in human liver microsomes led us to
the discovery of another series of analogs. Several compounds were further evaluated for their in vivo
efficacy in rats after oral administration and also for their pharmacokinetic profiles including in vitro sta-
bility in the liver microsomes.
Received 25 January 2010
Revised 10 March 2010
Accepted 12 March 2010
Available online 17 March 2010
Keywords:
Prostaglandin
EP3 receptor
Antagonist
Ó 2010 Published by Elsevier Ltd.
Uterine contraction
1. Introduction
(3,5-dimethylphenyl)-3-methylbutylamine moiety on the car-
boxyamide side chain which were synthesized as a racemate, were
It is well known that prostaglandins (PGs) and thromboxane
(TX), both of which are the oxidative metabolites of arachidonic
acid, play important roles in maintaining homeostasis. The chemi-
cal structures of PGs consist of the modified cyclopentane ring and
found to be excellent inhibitors of the PGE2-induced uterine con-
traction in pregnant rats after intraduodenal administration. Based
on the information described above, further investigation of the
optically active analogs was carried out. Herein, we report the
synthesis of a series of 3-(2-aminocarbonylphenyl)propanoic acid
analogs possessing a (1R)-1-(3,5-dimethylphenyl)-3-methylbuty-
lamine moiety on the carboxamide side chain, evaluation of their
in vitro activity for the EP3 receptor subtype, and their stability
in liver microsomes including the identification of two major
metabolites. Several compounds were also evaluated for their
in vivo efficacy after oral administration.
two side chains (a-chain and x-chain) attached to the ring. PGs are
classified into nine series (PGA, PGB, PGC, PGD, PGE, PGF, PGG,
PGH, and PGI) by their structural features of the cyclopentane
ring.1 Among them, PGE2, which is biologically synthesized from
PGH2 by the action of PGE2 synthase, is known to be the most
abundantly produced prostaglandin in humans and exhibits multi-
ple pharmacological actions through four specific receptor sub-
types EP1-4. EP3 receptor subtype is distributed in various
tissues including brain, kidney, uterus, and gastrointestinal tract.
Various actions of PGE2 such as hyperalgesia,2 pyrexia,3 uterine
contraction,4 gastric acid secretion,5 platelet aggregation,6 and
thrombosis7 are also known to be mediated by the EP3 receptor.
As such, the EP3 receptor subtype could be one of the most prom-
ising therapeutic targets in this field.
2. Chemistry
Synthesis of test compounds is outlined in Schemes 1–4. The
optically active amines (S)-27 and (R)-27 were synthesized as
shown in Schemes 1 and 2, respectively. Asymmetric induction
was accomplished by the Ellman’s chiral t-butyl sulfinamide meth-
od.10 As shown in Scheme 1, optically active N-sulfinyl imine 25
was prepared by the dehydrative condensation reaction of (S)-t-
butyl sulfinamide (24) with isovaleraldehyde in the presence of
pyridinium p-toluenesulfonate and anhydrous magnesium sulfate.
While nucleophilic addition of 3,5-dimethylphenylmagnesium
In our previous reports,8,9 a series of 3-(2-aminocarbonyl-4-
phenoxymethylphenyl)propanoic acid analogs possessing a 1-
* Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: m.asada@ono.co.jp (M. Asada).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.03.028

M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
3213
O
S
O
S
a
b
c
NH2
NH+3
Cl
-
S
N
N
H
O
24
25
(S)-27
26
Scheme 1. Synthesis of (S)-27. Reagents: (a) isovaleraldehyde, PPTS, MgSO4, CH2Cl2; (b) 3,5-dimethylphenylmagnesium bromide, THF, CH2Cl2; (c) HCl–dioxane, MeOH.
CHO
a, b
c
NH+3
-
OH
Cl
N
H
HCl
28
29
(R)-27
Scheme 2. Practical synthesis of (R)-27. Reagents: (a) (R)-phenylglycinol, toluene and then 2-methylallylmagnesium chloride; (b) HCl–EtOAc and then recrystallization from
i-PrOH/hexane; (c) H2, PtO2, EtOH.
(a)
COOMe
COOMe
A
O
ArO
O
COOMe
COOH
1-9
and
11-15
a
e
d
HN
HN
MOMO
30
31 A = OMOM
32 A = OH
35a-n
b
c
33 A = OMs
OH
X
OH
l
X = H, Y = H
m X = Me, Y = H
X = H, Y = Me
a R = H
e R = 2-Me
R = 2-OMe
g R = 3-CN
i
j
R = 2-F, 5-F
R = 2-Me, 4-Me
R
b R = 3-F
c R = 2-Cl
d R = 3-Cl
f
ArOH
Y
N
n
k R = 2-Me, 5-Me
h R = 2-F, 4-F
34a-k
34l-n
(b)
COOMe
COOMe
A
O
F
O
O
g
e
f
30
10
HN
HN
F
39
36 A = OMOM
37 A = OH
b
c
38 A = OMs
Scheme 3. Synthesis of 1–15. Reagents: (a) (R)-27, EDCꢀHCl, HOBt, N-methylmorpholine, DMF; (b) HCl-dioxane, MeOH; (c) MsCl, Et3N, THF; (d) 34a–n, NaH, DMF; (e)
NaOHaq, THF, MeOH; (f) (S)-27, EDCꢀHCl, HOBt, N-methylmorpholine, DMF; (g) 2,5-difluorophenol (34i), NaH, DMF.
bromide to the N-sulfinyl imine 25 afforded 26, which was ob-
tained with good diastereomeric selectivity of 93:7, purification
by column chromatography on silica gel was needed to remove
the minor diastereomer. The diastereomeric ratio was determined
by using 1H NMR spectroscopy. Removal of the t-butylsulfinyl
group under acidic conditions afforded (S)-27. Since the acidic
deprotection of the t-butylsulfinyl group proceeded smoothly in
mild acidic condition, the stereochemistry was thought to be re-
tained without affecting the benzylic C–N bond.10 Using essentially
the same procedure as described above, the corresponding enan-
tiomer (R)-27 could be prepared from (R)-t-butyl sulfinamide.
Since (R)-enantiomers were found to show significantly more
potent in vitro activity than the corresponding (S)-enantiomers
for the purposes of our SAR study, we needed a more efficient syn-
thetic method than the one described above to obtain optically
pure (R)-27. An alternative synthetic method is presented in
Scheme 2.11 Optically active (R)-27 was prepared from 3,5-dimeth-
ylbenzaldehyde (28) by the following sequential procedures: (1)
Formation of Schiff base from 3,5-dimethylbenzaldehyde (28)
and (R)-phenylglycinol; (2) the nucleophilic addition of 2-methyl-
allylmagnesium chloride resulting in a free form of 29 with good
diastereomeric selectivity of 92:8 which was determined by using

3214
M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
(a)
COOMe
O
COOMe
COOMe
A
c
b
f
HN
O
MOMO
MOMO
X
a
O
OH
Y
40 X = OH
43 Y = OH
42
d
e
41 X = OMOM
44 Y = OTf
45 Y = COOH
46 A = OMOM
47 A = OH
g
COOEt
R
O
a R = H
b R = 3-F, 5-F
c R = 3-Me, 5-Me
d R = 3-CN
e R = 3-Me
O
B(OH)2
h
i
HN
16-19
R
48a-e
49a-d
(b)
COOEt
COOEt
Br
O
COOEt
H2N
l
k
f
O
O
HN
O2N
O2N
z
H2N
COOH
53
O
O
50 Z = H
51 Z = CH2Ph
52
j
54
COOEt
R
O
N
H
m
i
HN
20-23
55b-e
Scheme 4. Synthesis of 16–23. Reagents: (a) MOMCl, i-Pr2NEt, DMF; (b) NaH, MeOH, THF; (c) H2, Pd–C, MeOH; (d) Tf2O, pyridine, CH2Cl2; (e) CO, Pd(OAc)2, DPPF, KOAc, DMF;
(f) (R)-27, EDCꢀHCl, HOBt, N-methylmorpholine, DMF; (g) HCl-dioxane, MeOH; (h) phenylboronic acids 48a–d, Cu(OAc)2, Et3N, molecular sieves, CH2Cl2; (i) NaOH aq, THF,
MeOH; (j) benzyl bromide, K2CO3, DMF; (k) ethyl acrylate, Pd(OAc)2, DPPF, Et3N, DMSO; (l) H2, Pd–C, EtOH; (m) phenylboronic acids 48b–e, Cu(OAc)2, TEMPO, Et3N, molecular
sieves, CH2Cl2.
1H NMR spectroscopy; (3) treatment of the free form of 29 with
hydrogen chloride in ethyl acetate; (4) fractional crystallization
from i-PrOH/hexane to afford 29 as a single diastereomer; (5) cat-
alytic hydrogenolysis of 29 to afford (R)-27. Exclusive cleavage of
the C–N bond attached to 2-phenylethanol moiety to afford (R)-
27 was accomplished by using platinum oxide as a catalyst with
retention of the stereochemistry of another benzylic carbon. Puri-
fication by tedious column chromatography on silica gel was
needed to remove the minor diastereomer in the synthetic route
shown in Scheme 1, while purification by fractional crystallization
afforded much more effective alternative method. Thus, the syn-
thetic route shown in Scheme 2 was considered to be more useful
for the scale-up synthesis of (R)-27. Absolute configuration of (R)-
27 was determined based on the X-ray crystallographic structure
analysis of 29, as shown in Figure 1.12
resulted in 35a–n, respectively, alkaline hydrolysis of which affor-
ded 1–9 and 11–15, respectively. Synthesis of 10 which is an enan-
4-Aryloxymethyl analogs 1–15 were synthesized as shown in
Schemes 3. All analogs listed in Scheme 3 were synthesized from
the benzoic acid 30, which was prepared from 7-methylcoumarin
as described in our previous paper.9 As shown in Schemes 3a, 31
was synthesized from 30 and (R)-27 using an EDC/HOBt coupling
method. Acidic deprotection of 31 followed by methanesulfonyla-
tion provided 33 which is a common intermediate for the synthe-
ses of 1–9 and 11–15. Nucleophilic substitution reaction of 33 with
the anion prepared from 34a–n in the presence of sodium hydride
Figure 1. Perspective drawing of the X-ray crystal structure of 29.

M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
3215
Table 1
Effect of the aryloxymethyl side chain on activity profiles and metabolic stability in liver microsomes
COOH
6
5
O
O
R
4
.
HN
2
X
3
a
Compound
R
X
Absolute configuration
Binding affinity Ki (nM)
Function IC50 (nM)
Metabolic stability (% remaining)
EP1
EP2
EP3
EP4
75
170
130
790
100
55
EP3
HLMb
RLMc
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
H
3-F
2-Cl
3-Cl
2-Me
2-OMe
3-CN
2-F, 4-F
2-F, 5-F
2-F, 5-F
2-Me, 4-Me
2-Me, 5-Me
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
>10,000
>10,000
4400
4400
>10,000
4100
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
0.41
0.37
0.29
0.16
0.19
0.67
0.28
0.29
0.068
1.4
5.5
6.6
2.9
1.8
1.9
7.3
2.7
3.3
1.6
19
4.3
1.7
2.0
4.4
5.4
51
46
62
50
73
70
57
65
37
56
93
61
80
68
60
27
31
40
43
58
90
70
38
21
19
69
83
100
57
70
72
110
230
3000
85
1.4
0.33
0.50
0.55
0.48
92
460
460
600
2-Me
4-Me
N
N
a
b
c
EP3 antagonist activity was evaluated in the presence of 1% BSA.
HLM: human liver microsomes.
RLM: rat liver microsomes.
Table 2
Activity profiles and metabolic stability in liver microsomes of phenoxy analogs 16–19 and anilino analogs 20–23
5
6
4
3
COOH
R.
O
X
2
HN
Compound
R
X
Binding affinity Ki (nM)
Function IC50 (nM)a
EP3
Metabolic stability (% remaining)
EP1
EP2
EP3
EP4
HLMb
RLMc
16
17
18
19
20
21
22
23
H
O
O
O
O
NH
NH
NH
NH
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
8700
>10,000
>10,000
>10,000
>10,000
>10,000
3000
7.6
250
410
120
43
430
300
220
180
39
11
96
100
85
73
82
97
95
93
76
89
74
63
72
93
71
91
3-F, 5-F
3-Me, 5-Me
3-CN
3-F, 5-F
3-Me, 5-Me
3-CN
0.85
0.28
0.28
0.77
0.23
0.36
0.39
2.9
3.8
6.9
2.0
4.9
5.5
>10,000
5200
3-Me
>10,000
a
b
c
EP3 antagonist activity was evaluated in the presence of 1% BSA.
HLM: human liver microsomes.
RLM: rat liver microsomes.
tiomer of 9 is shown in Scheme 3b. Dehydrative condensation reac-
tion of 30 with (S)-27 afforded 36, acidic deprotection of which
provided 37. Methanesulfonylation of 37 followed by substitution
of 38 with the phenoxide prepared from 2,5-difluorophenol (34i)
in the presence of sodium hydride provided 39, alkaline hydrolysis
of which resulted in the corresponding carboxylic acid 10.
Synthesis of 4-phenoxy analogs 16–19 is outlined in Scheme 4a.
Protection of the hydroxy group of 7-hydroxycoumarin (40) as the
corresponding methoxymethyl ether afforded 41, ring-opening
reaction of which resulted in unsaturated ester 42.13 Catalytic
hydrogenation of 42 afforded methyl propanoate 43. Trifluorome-
thanesulfonylation of 43 afforded 44, which was converted to the
carboxylic acid 45 by the palladium-catalyzed insertion reaction
of carbon monoxide. Dehydrative condensation of 45 with (R)-27
followed by acidic deprotection gave phenol 47. Cross-coupling
reaction of phenol 47 and the phenylboronic acid (48a) in the pres-
ence of a stoichiometric amount of copper(II) acetate resulted in
diphenyl ether 49a.14 Using optionally substituted phenylboronic
acids 48b–d instead of 48a, 49b–d were prepared from 47, respec-
tively. Alkaline hydrolysis of 49a–d resulted in the carboxylic acids
16–19, respectively.
4-Anilino analogs 20–23 were synthesized from 2-bromo-5-
nitrobenzoic acid (50) as described in Scheme 4b. Heck reaction
of 51, which was prepared from 50, with ethyl acrylate afforded

3216
M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
Table 3
unsaturated ester 52. Catalytic hydrogenation of 52 provided 53,
condensation reaction of which with (R)-27 afforded 54. The cou-
pling reaction of aniline 54 with optimally substituted phenylbo-
ronic acids 48b–e in the presence of copper (II) acetate and
TEMPO afforded 55b–e,15 alkaline hydrolysis of which provided
the corresponding carboxylic acids 20–23, respectively.
In vivo efficacies of 5, 9, 12, 18, and 21 after oral administration
Compound
In vivo efficacya (%inh., po)
0.3 mg/kg
0.1 mg/kg
1 mg/kg
5
9
12
18
21
29 13
47 11
NTb
53 10
85 8.0
60 6.3
42 9.1
67 5.9
98 6.4
NTb
84 4.4
79 5.4
77 6.0
NTb
3. Results and discussion
52 15c
a
b
c
Test compounds listed in Tables 1 and 2 were biologically eval-
uated for their inhibition of the specific binding of a radiolabeled
ligand, [3H]PGE2 to membrane fractions prepared from cells stably
Evaluation at 4 h after oral administration.
NT: Not tested.
Evaluation at 2 h after oral administration.
expressing each mouse EP1, EP2, EP3
receptor antagonist activity was determined by a Ca2+ assay using
mouse EP3 receptors expressed on CHO cells in the presence of
a, and EP4 receptors. The EP3
affinity for the EP3 receptor and decreased affinity for EP4 receptor
relative to 1. Analog 11 tended to show reduced affinity for the EP3
receptor relative to 1 while it showed retained affinity for the EP4
receptor. Analog 12 again showed nearly equipotent binding affin-
ity for EP3 and EP4 receptors relative to 1. The corresponding
(S)-enantiomer 10 was reconfirmed to be less active than the
(R)-enantiomer 9. Replacement of the phenoxy moiety of 1 with
pyridin-3-yloxy, 2-methylpyridine-3-yloxy, and 6-methylpyri-
dine-3-yloxy moieties afforded 13–15, respectively, with nearly
equipotent affinity for the EP3 receptor and reduced affinity for
the EP4 receptor. As a result, these analogs 13–15 exhibited im-
proved selectivity for the EP3 receptor with retained antagonist
activity for the EP3 receptor.
a
1% bovine serum albumin (BSA).
We previously reported a series of 3-(2-aminocarbonyl-4-phen-
oxymethylphenyl)propanoic acid analogs possessing the 1-(3,5-
dimethylphenyl)-3-methylbutylamine moiety on the carboxyam-
ide side chain, which were synthesized as racemates for the SAR
study.9 Since (R)-enantiomers possessing carboxyamide side
chains derived from the (R)-a-substituted benzylamine was
thought to show stronger activity relative to the corresponding
(S)-enantiomers derived from the (S)-a-substituted benzylamine
from our previous results,8 we focused on the SAR study of the
(R)-enantiomers. Results are summarized in Table 1. Unsubstituted
phenoxymethyl analog
1 exhibited potent in vitro activities
Since good oral activity should be derived from good pharmaco-
kinetic profiles, we focused on the improvement of the pharmaco-
kinetic profiles of these analogs. To propose a synthetic strategy to
improve their pharmacokinetic profiles, in vitro stability of 1–15 in
human liver microsomes (HLM) and rat liver microsomes (RLM)
was evaluated. Results are summarized in Table 1. Most of the
tested phenoxymethyl analogs, in which one or two substituents
were introduced into the phenoxy moieties, tended to show mod-
erate stability in both HLM and RLM. Replacement of the phenoxy
moiety with a pyridine-3-yloxy moiety was also effective for pro-
viding in vitro stabilization.
Qualitative analysis of the in vitro metabolites of several phen-
oxymethyl analogs incubated in the presence of HLM was also car-
ried out. As shown in Scheme 5, treatment of test compound A
with HLM afforded an aldehyde 56 and the corresponding carbox-
ylic acid 57 as the main metabolites, both of which were estimated
to be produced via presumed hemiacetal B. Treatment of test com-
(Ki = 0.41 nM and IC50 = 5.5 nM for the EP3 receptor) and relatively
less potent binding affinity for EP4 receptor (Ki = 75 nM), while it
exhibited no affinity for the EP1 and EP2 receptors at concentra-
tions tested up to 10,000 nM. Replacement of the phenoxy moiety
of 1 with 2-methoxyphenoxy and 3-cyanophenoxy moieties affor-
ded 6 and 7, respectively, with a similar retention of the binding
affinity and subtype selectivity for other EP receptors. Replacement
of the phenoxy moiety of 1 with 3-fluorophenoxy, 2-chlorophen-
oxy, 3-chlorophenoxy, and 2-methylphenoxy moieties afforded
2–5, respectively, with a tendency of retained affinity for the EP3
receptor and reduced affinity for the EP4 receptor. Thus, analogs
2–5 exhibited more subtype selectivity for EP3 relative to 1 (1:
EP4/EP3 = 180; 2–5: EP4/EP3 = 460, 450, 4900, 530). Replacement
of the phenoxy moiety of 1 with disubstituted phenoxy moieties
such as 2,4-difluorophenoxy, 2,5-difluorophenoxy, 2,4-dimethyl-
phenoxy, and 2,5-dimethylphenoxy moieties afforded 8, 9, 11,
and 12, respectively. Analogs 8 and 9 tended to show an increased
COOH
COOH
COOH
human liver
microsomes
O
O
O
O
OHC
HOOC
R
HN
HN
HN
+
A
57
56
COOH
O
O
R
OH
HN
B
Scheme 5. Identification of the metabolites of phenoxymethyl analogs in human liver microsomes.

M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
3217
Table 4
Pharmacokinetic profiles of 5, 9, 12, 18, and 21 in rats
Compound
Route
Dose (mg/kg)
AUC (
l
g h/mL)
t1/2 (h)
CLtot (mL/min/kg)
51.5
Vss (L/kg)
Cmax
(
lg/mL)
F (%)
5
iv
po
iv
po
iv
po
iv
po
iv
po
2.7
10
1.2
10
1.8
10
2
10
2
10
0.89
1.01
0.681
1.02
1.37
4.10
1.37
1.43
2.67
3.27
0.4
1.6
0.3
5.0
0.4
2.2
0.4
4.6
2.0
2.7
1.24
0.33
0.19
1.35
0.95
1.37
31
18
54
21
25
9
12
18
21
29.8
22.0
24.1
11.5
0.39
0.67
0.56
1.03
pound A with RLM afforded mainly the same metabolites as de-
scribed above. Structure determination of metabolites 56 and 57
was carried out by LC-NMR.
effect of 21 was 57 3.9%. As such, compound 9 was found to be
more potent than 21. Based on the results described above, com-
pound 9 was estimated to show nearly same efficacy as that of
21, which possesses better pharmacokinetic profiles after their oral
administration.
Based on the results described above, we directed our efforts to-
wards synthesizing a series of 4-phenoxy analogs 16–19 and 4-ani-
lino analogs 20–23 because these analogs do not have a benzylic
moiety which may be susceptible to metabolism. Introduction of
one or two substituents into the terminal phenyl moiety was also
carried out as illustrated by 17–23. All of the analogs listed in Ta-
ble 2 exhibited good stability in the presence of liver microsomes.
These analogs were also evaluated for their binding affinity for
EP1-4 receptor and antagonist activity for the EP3 receptor. Com-
pounds 17–23, which showed excellent subtype selectivity, exhib-
ited potent binding affinity and antagonist activity for the EP3
receptor, while 16 showed weaker antagonist activity relative to
the other tested compounds.
Further biological evaluation of above-mentioned analogs was
carried out. The inhibitory effect of PGE2-induced uterine contrac-
tion in pregnant rats was investigated as a beneficial effect. The
efficacy of 5, 9, 12, 18, and 21 was expressed as % inhibition at
4 h after oral administration, as shown in Table 3. All analogs
exhibited efficacy after their oral administration in dose-depen-
dent manner. The inhibitory effect was more than 50% at less than
1 mg/kg of oral administration. Among the tested compounds, 2,5-
difluorophenoxymethyl analog 9 and 3,5-dimethylanilino analog
21 showed nearly 50% inhibition at 0.1 mg/kg of oral administra-
tion, while they showed 85% inhibition and 67% inhibition, respec-
tively, at 0.3 mg/kg of oral administration.
4. Conclusion
Further optimization of 3-(2-aminocarbonylphenyl)propanoic
acid analogs possessing a (1R)-1-(3,5-dimethylphenyl)-3-methyl-
butylamine moiety on the carboxyamide side chain was carried
out. A series of analogs possessing the 4-aryloxymethyl side chain
were found to exhibit potent antagonist activity for the EP3 recep-
tor with high subtype selectivity. Identification of the metabolites
of 4-phenoxymethyl analogs in the presence of HLM resulted in the
discovery that these analogs are metabolically susceptible at their
benzylic position. Thus, rational design and synthesis of 4-phenoxy
and 4-anilino analogs both of which do not have the benzylic car-
bon atom, led us to the discovery of another series of selective EP3
receptor antagonists. Several compounds were evaluated for their
inhibitory effect against the PGE2-induced uterine contraction in
pregnant rats after their oral administration. Both series of analogs
showed potent efficacy in this animal model. Among the tested
analogs, compounds 9 and 21 exhibited relatively potent oral
activity. Based on the results described above, these potent and
selective EP3 receptor antagonists are expected to have potential
for the treatment of diseases mediated by the EP3 receptor
subtype.
The pharmacokinetic profiles of 5, 9, 12, 18, and 21 were also
investigated. Results are summarized in Table 4. Compound 12,
which showed improved stability in the presence of HLM and
5. Experimental
5.1. Chemistry
RLM, exhibited a good maximum concentration (Cmax = 1.35 lg/
mL at 10 mg/kg po) and good bioavailability (F = 54%). Compound
21, which also showed improved stability in the presence of HLM
and RLM, had the lowest total clearance (CLtot = 11.5 mL/min/kg)
among the tested compounds. 21 also showed a good maximum
concentration (Cmax = 1.37 lg/mL at 10 mg/kg po) and moderate
bioavailability (F = 25%). Compounds 5, 9, and 18 exhibited moder-
5.1.1. General procedures
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, Varian GEM-
INI-200 or VXR-200s spectrometer using deuterated chloroform
(CDCl3), deuterated dimethylsulfoxide (DMSO-d6) or deuterated
methanol (CD3OD) as the solvent. Fast atom bombardment (FAB-
MS, HRMS) and electron ionization (EI) mass spectra were obtained
on a JEOL JMS-DX303HF spectrometer. Atmospheric pressure
chemical ionization (APCI) mass spectra were determined on a
HITACHI MI200H spectrometer. Infrared spectra (IR) were mea-
sured in a Perkin-Elmer FT-IR 1760X spectrometer. Melting points
and results of elemental analyses were uncorrected. Optical rota-
tions were measured in a JASCO DIP-1000 digital polarimeter.
The enantiomer excess (% ee) was determined by the following
HPLC system: Agilent Technologies 1200 Series with a CHIRALCEL
ate bioavailability with F% of 31%, 18%, and 21%, respectively, and
maximum concentration (Cmax) of 0.33, 0.19, and 0.95 lg/mL at
10 mg/kg po, respectively. Compound 9, which showed the most po-
tent in vivo efficacy among the tested compounds, showed the low-
est Cmax with the longest half time (t1/2) after oral administration.
The in vivo efficacies of 9 and 21 seemed to be close, while
pharmacokinetic profiles of 9 and 21 did not always reflect the
pharmacological efficacies. To explain the relationship between
the in vivo efficacy and the pharmacokinetic profiles of these two
compounds, their inhibitory effects on the PGE2-induced uterine
contraction in pregnant rats after their intravenous administration
were evaluated. The inhibitory effect of 9 was 96 5.2% after 5 min
of intravenous administration at 0.03 mg/kg, while the inhibitory

3218
M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
OD-RH (4.6 ꢁ 150 mm), eluting with 0.15 M aqueous KPF6/aceto-
nitrile = 60/40, flow rate of 1.0 mL/min, column temperature of
40 °C, detection with UV (210 nm). Column chromatography
was carried out on silica gel [Merck Silica Gel 60 (0.063–
0.200 mm), Wako gel C-200, 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; t-butylmethylether (TBME),
N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), etha-
nol (EtOH), ethyl acetate (EtOAc), methanol (MeOH), tetrahy-
drofuran (THF), 1,10-bis(diphenylphosphino)ferrocene (DPPF),
N,N-diisopropylethylamime (DIPEA), 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (EDCꢀHCl), 1-hydroxyben-
zotriazole (HOBt), isopropanol (i-PrOH), methanesulfonyl
chloride (MsCl), methanol (MeOH), methoxymethyl chloride
(MOMCl), pyridinium p-toluenesulfonate (PPTS), 2,2,6,6-tetrame-
thylmorpholine oxide (TEMPO), triethylamine (TEA), and trifluo-
romethanesulfonic anhydride (Tf2O).
propene (76.7 g, 0.847 mol) and magnesium (51.5 g, 2.12 mol) in
THF (1700 mL) over 3 h at 0 °C under an argon atmosphere. After
being stirred for 30 min at 0 °C, the reaction was quenched with
aqueous NH4Cl and water. The organic layer was separated and
then the aqueous layer was extracted with EtOAc. The combined
organic layers were washed with brine, dried over MgSO4, and con-
centrated in vacuo. The resultant residue was dissolved in EtOAc
(500 mL) and then 4 M HCl in dioxane (100 mL) was added. After
concentration in vacuo, the resultant residue was recrystallized
from i-PrOH/hexane to yield 29 (77.1 g, 61% in three steps) as an
off-white powder. 1H NMR (300 MHz, CDCl3) d 9.52 (br s, 2H),
7.39–7.20 (m, 5H), 6.94 (s, 2H), 6.81 (s, 1H), 5.44 (br s, 1H), 4.70
(s, 1H), 4.63 (s, 1H), 4.40–4.20 (m, 2H), 4.19–4.09 (m, 1H), 3.88–
3.78 (m, 1H), 3.11 (dd, J = 14, 4.4 Hz, 1H), 2.94 (dd, J = 14, 11 Hz,
1H), 2.17 (s, 6H), 1.49 (s, 3H).
5.1.6. (1R)-1-(3,5-Dimethylphenyl)-3-methylbutylamine
hydrochloride ((R)-27)
A suspension of 29 (33.0 g, 95.4 mmol) and PtO2 (4.60 g) in
EtOH (330 mL) was stirred for 40 h at 60 °C under an atmosphere
of hydrogen. The reaction mixture was filtered through a pad of
Celite and the filtrate was concentrated in vacuo. The resultant res-
idue was recrystallized from EtOH/EtOAc to yield (R)-27 (12.8 g,
58%) as a white powder. 1H NMR (300 MHz, DMSO-d6) d 8.41 (br
s, 3H), 7.11 (s, 2H), 7.01 (s, 1H), 4.15–4.05 (m, 1H), 2.27 (s, 6H),
1.82–1.66 (m, 2H), 1.37–1.21 (m, 1H), 0.86 (d, J = 6.6 Hz, 3H),
5.1.2. (SS)-2-Methyl-N-[(1E)-3-methylbutylidene]propane-2-
sulfinamide (25)
A solution of 24 (1.75 g, 14.4 mmol), isovaleraldehyde (3.10 mL,
28.9 mmol), PPTS (181 mg, 0.720 mmol) and MgSO4 (8.67 g,
72.0 mmol) in CH2Cl2 (30 mL) was stirred for 5 h at room temper-
ature under an argon atmosphere. The reaction mixture was con-
centrated in vacuo and the resultant residue was purified by
column chromatography on silica gel (EtOAc/hexane, 100/0 to
50/50) to yield 25 (2.61 g, 96%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) d 8.05 (t, J = 5.3 Hz, 1H), 2.47–2.38 (m, 2H),
2.16–1.98 (m, 1H), 1.20 (s, 9H), 0.99 (d, J = 6.6 Hz, 6H).
0.82 (d, J = 6.6 Hz, 3H); Optical rotation ½a D22
ꢃ
-21.1 (c 2.08, MeOH);
HPLC retention time 3.99 min, 99% ee.
5.1.7. Methyl 3-{2-({[(1R)-1-(3,5-dimethylphenyl)-3-methylbutyl]
amino}carbonyl)-4-(hydroxymethyl)phenyl} propanoate (32)
A solution of 30 (4.00 g, 14.2 mmol), (R)-27 (3.23 g, 14.2 mmol),
N-methylmorpholine (1.72 mL, 15.6 mmol), EDCꢀHCl (2.99 g,
15.6 mmol) and HOBt (3.84 g, 28.4 mmol) in DMF (15 mL) was stir-
red for 12 h at room temperature under an argon atmosphere. The
reaction was quenched with 1 M aqueous solution of HCl and then
the resultant mixture was extracted with EtOAc/hexane = 1/1 solu-
tion. The organic layer was washed with aqueous NaHCO3, water
and brine, dried over Na2SO4, and concentrated in vacuo to yield
31, which was used for the next step without purification. To a stir-
red solution of 31 in MeOH (10 mL) was added 4 M HCl in dioxane
(10 mL) at room temperature and the reaction mixture was stirred
for 30 min. After concentration in vacuo, the resultant residue was
recrystallized from EtOAc/hexane to yield 32 (5.21 g, 89% in two
steps) as an off-white powder. 1H NMR (300 MHz, CDCl3) d 7.35–
7.27 (m, 2H), 7.20 (d, J = 7.8 Hz, 1H), 6.97 (s, 2H), 6.90 (s, 1H),
6.59 (d, J = 8.4 Hz, 1H), 5.22–5.10 (m, 1H), 4.64 (s, 2H), 3.62 (s,
3H), 3.07–2.92 (m, 2H), 2.76–2.56 (m, 2H), 2.31 (s, 6H), 1.86–
1.52 (m, 3H), 0.99 (d, J = 6.0 Hz, 3H), 0.98 (d, J = 6.0 Hz, 3H).
5.1.3. (SS)-N-[(1S)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-2-
methylpropane-2-sulfinamide (26)
To a stirred solution of 25 (250 mg, 1.32 mmol) in CH2Cl2 (5 mL)
was added dropwise 3,5-dimethylphenylmagnesium bromide (2 M
in THF, 1.32 mL, 2.64 mmol) at ꢂ78 °C under an argon atmosphere.
After being stirred for ꢂ78 °C at 12 h, the reaction was quenched
with aqueous NH4Cl and then the resultant mixture was extracted
with EtOAc. The organic layer was washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. The resultant resi-
due was purified by column chromatography on silica gel
(EtOAc/hexane, 1/9 to 1/1) to yield 26 (320 mg, 82%) as a white so-
lid. 1H NMR (300 MHz, CDCl3) d 6.91 (s, 3H), 4.35–4.26 (m, 1H),
3.25 (d, J = 3.9 Hz, 1H), 2.31 (s, 6H), 1.87–1.76 (m, 1H), 1.68–1.55
(m, 1H), 1.52–1.38 (m, 1H), 1.21 (s, 9H), 0.92 (d, J = 6.6 Hz, 3H),
0.86 (d, J = 6.6 Hz, 3H).
5.1.4. (1S)-1-(3,5-Dimethylphenyl)-3-methylbutylamine
hydrochloride ((S)-27)
To a stirred solution of 26 (200 mg, 0.677 mmol) in MeOH
(1 mL) was added 4 M HCl in dioxane (1 mL) at room temperature.
After being stirred for 1 h, the reaction mixture was concentrated
in vacuo. The resultant residue was washed with EtOAc/hexane
to yield (S)-27 (136 mg, 88%) as a white powder. 1H NMR
(300 MHz, DMSO-d6) d 8.41 (br s, 3H), 7.11 (s, 2H), 7.00 (s, 1H),
4.17–4.04 (m, 1H), 2.27 (s, 6H), 1.82–1.64 (m, 2H), 1.38–1.22 (m,
1H), 0.86 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H); Optical rotation
5.1.8. 3-[2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]amino}-
carbonyl)-4-(phenoxymethyl)phenyl]propanoic acid (1)
To a stirred solution of 32 (333 mg, 0.810 mmol) and TEA
(169 lL, 1.22 mmol) in THF (3 mL) was added MsCl (75 lL,
0.973 mmol) at 0 °C under an argon atmosphere. After being stir-
red for 30 min at 0 °C, the reaction mixture was diluted with EtOAc,
washed with water and brine, dried over MgSO4, and concentrated
in vacuo to yield 33, which was used for the next step without
purification. To a stirred solution of 34a (91 mg, 0.972 mmol) in
DMF (1 mL) was added NaH (60% in oil, 34 mg, 0.891 mmol) at
0 °C under an argon atmosphere and the reaction mixture was stir-
red for 20 min at room temperature. To the reaction mixture was
added 33 in DMF (1 mL) at 0 °C. After being stirred for 1 h at room
temperature, the reaction was quenched with aqueous NH4Cl and
then the resultant mixture was extracted with TBME. The organic
layer was washed with 0.5 M aqueous solution of NaOH, water
½
a 2D3
+20.7 (c 1.94, MeOH); HPLC retention time 5.50 min, 94% ee.
ꢃ
5.1.5. (2R)-2-{[(1R)-1-(3,5-Dimethylphenyl)-3-methyl-3-buten-
1-yl]amino}-2-phenylethanol hydrochloride (29)
A solution of 28 (49.4 g, 0.368 mol) and (R)-phenylglycinol
(50.5 g, 0.368 mol) in toluene (350 mL) was refluxed for 15 h to ob-
tain the imine by azeotropic distillation to remove water. To the
stirred solution of the imine was added dropwise 2-allylmethyl-
magnesium chloride which was prepared from 3-chloro-2-methyl-

M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
3219
and brine, and dried over MgSO4. Concentration in vacuo afforded
35a which was used for the next step without purification. To a
stirred solution of 35a in THF (2 mL) and MeOH (2 mL) was added
2 M aqueous solution of NaOH (2 mL) at room temperature. After
being stirred for 12 h at room temperature, the reaction mixture
was acidified with 1 M aqueous solution of HCl and then extracted
with EtOAc. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. The resultant residue
was recrystallized from EtOAc/hexane to yield 1 (201 mg, 52% in
three steps) as a white powder. 1H NMR (300 MHz, CDCl3) d
7.46–7.38 (m, 2H), 7.34–7.25 (m, 3H), 7.02–6.93 (m, 5H), 6.91 (s,
1H), 6.28 (d, J = 8.2 Hz, 1H) 5.22–5.11 (m, 1H), 5.02 (s, 2H), 3.09–
2.97 (m, 2H), 2.72 (t, J = 6.6 Hz, 2H), 2.21 (s, 6H), 1.83–1.54 (m,
3H), 0.98 (d, J = 6.3 Hz, 6H); MS (FAB, Pos.) m/e 474 (M+H)+; HRMS
(Pos.) calcd for C30H36NO4: 474.2644; found: 474.2645; Optical
(d, J = 6.6 Hz, 3H); MS (FAB, Pos.) m/e 488 (M+H)+; HRMS (Pos.)
calcd for C31H38NO4: 488.2801; found: 488.2802; Optical rotation
½
a 2D4
+17.9 (c 0.90, MeOH).
ꢃ
5.1.13. 3-{2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-[(2-methoxyphenoxy)methyl]phenyl}-
propanoic acid (6)
The titled compound was synthesized in the same manner as
described for 1 using 34f instead of 34a as a white powder. Yield
47% in three steps; 1H NMR (300 MHz, CDCl3) d 7.44–7.39 (m,
2H), 7.28–7.23 (m, 1H), 7.00–6.80 (m, 7H), 6.32 (d, J = 8.7 Hz,
1H), 5.21–5.11 (m, 1H), 5.09 (s, 2H), 3.86 (s, 3H), 3.10–2.95 (m,
2H), 2.71 (t, J = 7.5 Hz, 2H), 2.30 (s, 6H), 1.80–1.55 (m, 3H), 0.97
(d, J = 6.3 Hz, 6H); MS (FAB, Pos.) m/e 504 (M+H)+; HRMS (Pos.)
calcd for C31H38NO5: 504.2750; found: 504.2750; Optical rotation
rotation ½a 2D4
ꢃ
+18.3 (c 1.00, MeOH).
½
a 2D4
+16.3 (c 0.99, MeOH).
ꢃ
5.1.9. 3-{2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]amino}-
carbonyl)-4-[(3-fluorophenoxy)methyl]phenyl}propanoic acid (2)
The titled compound was synthesized in the same manner as
described for 1 using 34b instead of 34a as a white powder. Yield
80% in three steps; 1H NMR (300 MHz, CDCl3) d 7.44–7.38 (m, 2H),
7.33–7.18 (m, 2H), 6.95 (s, 2H), 6.91 (s, 1H), 6.76–6.63 (m, 3H), 6.31
(d, J = 8.2 Hz, 1H), 5.23–5.14 (m, 1H), 5.00 (s, 2H), 3.08–2.95 (m,
2H), 2.72 (t, J = 7.5 Hz, 2H), 2.31 (s, 6H), 1.85–1.55 (m, 3H), 0.98
(d, J = 6.3 Hz, 6H); MS (FAB, Pos.) m/e 492 (M+H)+; HRMS (Pos.)
calcd for C30H35FNO4: 492.2550; found: 492.2553; Optical rotation
5.1.14. 3-[4-[(3-Cyanophenoxy)methyl]-2-({[(1R)-1-(3,5-dimeth-
ylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]propanoic
acid (7)
The titled compound was synthesized in the same manner as
described for 1 using 34g instead of 34a as a white powder. Yield
59% in three steps; 1H NMR (300 MHz, CDCl3) d 7.42–7.35 (m,
3H), 7.32–7.25 (m, 2H), 7.21–7.15 (m, 2H), 6.96 (s, 2H), 6.91 (s,
1H), 6.37 (d, J = 8.6 Hz, 1H), 5.22–5.12 (m, 1H), 5.03 (s, 2H), 3.07–
2.99 (m, 2H), 2.76–2.68 (m, 2H), 2.31 (s, 6H), 1.85–1.55 (m, 3H),
0.99 (d, J = 6.6 Hz, 6H); MS (FAB, Pos.) m/e 499 (M+H)+; HRMS
(Pos.) calcd for C31H35N2O4: 499.2597; found: 499.2595; Optical
½
a 2D4
+17.2 (c 1.00, MeOH).
ꢃ
rotation ½a 2D4
ꢃ
+15.4 (c 1.06, MeOH).
5.1.10. 3-[4-[(2-Chlorophenoxy)methyl]-2-({[(1R)-1-(3,5-dime-
thylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]propanoic
acid (3)
5.1.15. 3-[4-[(2,4-Difluorophenoxy)methyl]-2-({[(1R)-1-(3,5-
dimethylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]-
propanoic acid (8)
The titled compound was synthesized in the same manner as
described for 1 using 34c instead of 34a as a white powder. Yield
The titled compound was synthesized in the same manner as
described for 1 using 34h instead of 34a as a white powder. Yield
28% in three steps; 1H NMR (300 MHz, CDCl3) d 7.43–7.37 (m, 2H),
7.27 (d, J = 7.8 Hz, 1H), 6.98–6.83 (m, 5H), 6.81–6.72 (m, 1H), 6.34
(d, J = 8.2 Hz, 1H), 5.21–5.12 (m, 1H), 5.04 (s, 2H), 3.08–2.95 (m,
2H), 2.74–2.67 (m, 2H), 2.31 (s, 6H), 1.84–1.55 (m, 3H), 0.99 (d,
J = 6.3 Hz, 6H); MS (FAB, Pos.) m/e 510 (M+H)+; HRMS (Pos.) calcd
for C30H34F2NO4: 510.2456; found: 510.2458; Optical rotation
74% in three steps; 1H NMR (300 MHz, CDCl3)
d 7.52 (d,
J = 1.7 Hz, 1H), 7.45–7.37 (m, 2H), 7.28 (d, J = 8.1 Hz, 1H), 7.24–
7.17 (m, 1H), 6.99–6.90 (m, 5H), 6.32 (d, J = 8.7 Hz, 1H), 5.21–
5.13 (m, 1H), 5.12 (s, 2H), 3.10–2.98 (m, 2H), 2.72 (t, J = 7.1 Hz,
2H), 2.31 (s, 6H), 1.85–1.57 (m, 3H), 0.99 (d, J = 6.2 Hz, 3H), 0.98
(d, J = 6.2 Hz, 3H); MS (FAB, Pos.) m/e 508 (M+H)+; HRMS (Pos.)
calcd for C30H35ClNO4: 508.2255; found: 508.2255; Optical rota-
tion ½a 2D4
ꢃ
+20.3 (c 1.14, MeOH).
½
a 2D4
+17.4 (c 1.11, MeOH).
ꢃ
5.1.11. 3-[4-[(3-Chlorophenoxy)methyl]-2-({[(1R)-1-(3,5-dimeth-
ylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]propanoic
acid (4)
5.1.16. 3-[4-[(2,5-Difluorophenoxy)methyl]-2-({[(1R)-1-(3,5-
dimethylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]-
propanoic acid (9)
The titled compound was synthesized in the same manner as
described for 1 using 34d instead of 34a as a white powder. Yield
55% in three steps; 1H NMR (300 MHz, CDCl3) d 7.43–7.37 (m, 2H),
7.31–7.17 (m, 2H), 6.98–6.94 (m, 2H), 6.91 (s, 1H), 6.87–6.81 (m,
1H), 6.30 (d, J = 8.9 Hz, 1H), 5.22–5.12 (m, 1H), 5.00 (s, 2H), 3.09–
2.96 (m, 2H), 2.73 (t, J = 7.2 Hz, 2H), 2.31 (s, 6H), 1.85–1.55 (m,
3H), 0.99 (d, J = 6.6 Hz, 6H); MS (FAB, Pos.) m/e 508 (M+H)+; HRMS
(Pos.) calcd for C30H35ClNO4: 508.2255; found: 508.2256; Optical
The titled compound was synthesized in the same manner as
described for 1 using 34i instead of 34a as a white powder. Yield
58% in three steps; 1H NMR (300 MHz, CDCl3) d 7.45–7.38 (m,
2H), 7.29 (d, J = 7.8 Hz, 1H), 7.07–6.97 (m, 1H), 6.96 (s, 2H), 6.91
(s, 1H), 6.76–6.68 (m, 1H), 6.65–6.56 (m, 1H), 6.31 (d, J = 8.4 Hz,
1H), 5.21–5.11 (m, 1H), 5.06 (s, 2H), 3.11–2.93 (m, 2H), 2.72 (t,
J = 6.0 Hz, 2H), 2.31 (s, 6H), 1.85–1.56 (m, 3H), 0.99 (d, J = 6.3 Hz,
6H); MS (FAB, Pos.) m/e 510 (M+H)+; HRMS (Pos.) calcd for
rotation ½a 2D4
ꢃ
+15.9 (c 1.00, MeOH).
C30H34F2NO4: 510.2456; found: 510.2456; Optical rotation ½a D24
ꢃ
+14.9 (c 2.04, MeOH).
5.1.12. 3-{2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-[(2-methylphenoxy)methyl]phenyl}-
propanoic acid (5)
The titled compound was synthesized in the same manner as
described for 1 using 34e instead of 34a as an off-white powder.
Yield 30% in three steps; 1H NMR (300 MHz, CDCl3) d 7.44–7.38
(m, 2H), 7.27 (d, J = 8.1 Hz, 1H), 7.19–7.11 (m, 2H), 6.95 (s, 2H),
6.92–6.83 (m, 3H), 6.28 (d, J = 8.4 Hz, 1H), 5.22–5.12 (m, 1H),
5.03 (s, 2H), 3.09–2.97 (m, 2H), 2.71 (t, J = 7.2 Hz, 2H), 2.30 (s,
6H), 2.26 (s, 3H), 1.83–1.57 (m, 3H), 0.99 (d, J = 6.6 Hz, 3H), 0.98
5.1.17. 3-[4-[(2,4-Dimethylphenoxy)methyl]-2-({[(1R)-1-(3,5-
dimethylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]-
propanoic acid (11)
The titled compound was synthesized in the same manner as
described for 1 using 34j instead of 34a as an off-white powder.
Yield 48% in three steps; 1H NMR (300 MHz, CDCl3) d 7.45–7.37
(m, 2H), 7.30–7.25 (m, 1H), 7.00–6.87 (m, 5H), 6.75 (d, J = 8.2 Hz,
1 H) 6.24 (d, J = 8.8 Hz, 1H), 5.22–5.13 (m, 1H), 5.01 (s, 2H), 3.09–
2.99 (m, 2H), 2.73 (t, J = 6.9 Hz, 2H), 2.31 (s, 6H), 2.26 (s, 3H),

3220
M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
2.23 (s, 3H), 1.81–1.57 (m, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.98 (d,
J = 6.3 Hz, 3H); MS (FAB, Pos.) m/e 502 (M+H)+; HRMS (Pos.) calcd
5.1.22. Methyl 3-{2-({[(1S)-1-(3,5-dimethylphenyl)-3-methyl-
butyl]amino}carbonyl)-4-(hydroxymethyl)phenyl} propanoate
(37)
The titled compound was synthesized in the same manner as
described for 32 using (S)-27 instead of (R)-27 as a white powder.
Yield 75% in two steps; 1H NMR (300 MHz, CDCl3) d 7.38–7.30 (m,
2H), 7.23 (d, J = 7.8 Hz, 1H), 6.97 (s, 2H), 6.90 (s, 1H), 6.46 (d,
J = 9.3 Hz, 1H), 5.22–5.12 (m, 1H), 4.67 (d, J = 5.7 Hz, 2H), 3.62 (s,
3H), 3.08–2.97 (m, 2H), 2.71–2.60 (m, 2H), 2.31 (s, 6H), 1.86–1.59
(m, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H).
for C32H40NO4: 502.2957; found: 502.2951; Optical rotation ½a D24
ꢃ
+16.8 (c 1.01, MeOH).
5.1.18. 3-[4-[(2,5-Dimethylphenoxy)methyl]-2-({[(1R)-1-(3,5-
dimethylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]-
propanoic acid (12)
The titled compound was synthesized in the same manner as
described for 1 using 34k instead of 34a as an off-white powder.
Yield 33% in three steps; 1H NMR (300 MHz, CDCl3) d 7.45–7.37
(m, 2H), 7.28 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 7.2 Hz, 1H), 6.95 (s,
2H), 6.90 (s, 1H), 6.73–6.68 (m, 2H), 6.27 (d, J = 8.8 Hz, 1H),
5.22–5.12 (m, 1H), 5.02 (s, 2H), 3.10–2.96 (m, 2H), 2.72 (t,
J = 7.2 Hz, 2H), 2.31 (s, 3H), 2.30 (s, 6H), 2.22 (s, 3H), 1.84–1.54
(m, 3H), 0.99 (d, J = 6.2 Hz, 3H), 0.98 (d, J = 6.2 Hz, 3H); MS
(FAB, Pos.) m/e 502 (M+H)+; HRMS (Pos.) calcd for C32H40NO4:
5.1.23. 3-[4-[(2,5-Difluorophenoxy)methyl]-2-({[(1S)-1-(3,5-
dimethylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]-
propanoic acid (10)
The titled compound was synthesized in the same manner as
described for 1 using 37 and 34i instead of 32 and 34a, respec-
tively, as a white powder. Yield 89% in three steps; 1H NMR
(300 MHz, CDCl3) d 7.45–7.38 (m, 2H), 7.29 (d, J = 7.8 Hz, 1H),
7.09–6.98 (m, 1H), 6.96 (s, 2H), 6.91 (s, 1H), 6.78–6.69 (m, 1H),
6.66–6.56 (m, 1H), 6.31 (d, J = 8.4 Hz, 1H), 5.22–5.12 (m, 1H),
5.06 (s, 2H), 3.11–2.93 (m, 2H), 2.72 (t, J = 6.0 Hz, 2H), 2.31 (s,
6H), 1.84–1.52 (m, 3H), 0.99 (d, J = 6.3 Hz, 6H); MS (FAB, Pos.) m/
e 510 (M+H)+; HRMS (Pos.) calcd for C30H34F2NO4: 510.2456;
502.2957; found: 502.2947; Optical rotation ½a D24
ꢃ
+18.9 (c 1.02,
MeOH).
5.1.19. 3-{2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-[(pyridin-3-yloxy)methyl]phenyl}propanoic
acid (13)
found: 510.2457; Optical rotation ½a D24
ꢂ15.8 (c 1.02, MeOH).
ꢃ
The titled compound was synthesized in the same manner as
described for 1 using 34l instead of 34a as a white powder. Yield
36% in three steps; 1H NMR (300 MHz, DMSO-d6) d 12.08 (s, 1H),
8.76 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H), 8.17 (d, J = 4.0 Hz, 1H), 7.46–
7.40 (m, 2H), 7.37–7.28 (m, 3H), 6.95 (s, 2H), 6.84 (s, 1H), 5.17
(s, 2H), 5.02–4.92 (m, 1H), 2.84 (t, J = 7.5 Hz, 2H), 2.45 (t,
J = 7.5 Hz, 2H), 2.24 (s, 6H), 1.78–1.56 (m, 2H), 1.46–1.35 (m,
1H), 0.92 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H); MS (FAB,
Pos.) m/e 475 (M+H)+; HRMS (Pos.) calcd for C29H35N2O4:
5.1.24. 7-(Methoxymethoxy)-2H-chromen-2-one (41)
To a stirred solution of 40 (100 g, 0.617 mol) and DIPEA
(161 mL, 0.925 mol) in DMF (500 mL) was added dropwise
methoxymethyl chloride (70.3 mL, 0.925 mol) at 0 °C under an ar-
gon atmosphere. After being stirred for 4 h at room temperature,
the reaction mixture was poured into cold aqueous NaHCO3 and
EtOAc/hexane = 1/1 solution. The organic layer was separated
and then the aqueous layer was extracted with EtOAc. The com-
bined organic layers were washed with water and brine, dried over
MgSO4, and concentrated in vacuo to yield 41 as a pale yellow so-
lid, which was used for the next step without purification. 1H NMR
(300 MHz, CDCl3) d 7.64 (d, J = 9.6 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H),
7.01 (d, J = 2.4 Hz, 1H), 6.96 (dd, J = 8.7, 2.4 Hz, 1H), 6.28 (d,
J = 9.6 Hz, 1H), 5.24 (s, 2H), 3.49 (s, 3H).
475.2597; found: 475.2602; Optical rotation ½a D22
ꢃ
+17.4 (c 0.45,
MeOH).
5.1.20. 3-(2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-{[(2-methylpyridin-3-yl)oxy]methyl}-
phenyl)propanoic acid (14)
5.1.25. Methyl (2E)-3-[2-hydroxy-4-(methoxymethoxy)phenyl]-
acrylate (42)
The titled compound was synthesized in the same manner as
described for 1 using 34m instead of 34a as a white powder. Yield
29% in three steps; 1H NMR (300 MHz, DMSO-d6) d 12.08 (s, 1H),
8.79 (d, J = 8.8 Hz, 1H), 8.02–7.98 (m, 1H), 7.45–7.28 (m, 4H), 7.17
(dd, J = 8.1, 4.8 Hz, 1H), 6.95 (s, 2H), 6.84 (s, 1H), 5.15 (s, 2H),
5.02–4.91 (m, 1H), 2.84 (t, J = 7.5 Hz, 2H), 2.44 (t, J = 7.5 Hz, 2H),
2.39 (s, 3H), 2.25 (s, 6H), 1.80–1.60 (m, 2H), 1.45–1.32 (m, 1H),
0.93 (d, J = 6.2 Hz, 3H), 0.89 (d, J = 6.2 Hz, 3H); MS (FAB, Pos.)
m/e 489 (M+H)+; HRMS (Pos.) calcd for C30H37N2O4: 489.2753;
To a stirred suspension of NaH (63% in oil, 46.9 g, 1.23 mol) in
THF (300 mL) was added dropwise MeOH (60.0 mL, 1.46 mol) at
0 °C under an argon atmosphere and the reaction mixture was stir-
red for 20 min at room temperature. To the resultant suspension
was added dropwise 41 in THF (1000 mL) and MeOH (100 mL) at
0 °C. After being stirred for 40 min at 60 °C, the reaction was
quenched with aqueous NH4Cl and successively 2 M aqueous solu-
tion of HCl at 0 °C, and then the resultant mixture was extracted
with EtOAc. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. The resultant residue
was washed with EtOAc (500 mL) and hexane (1000 mL) to yield
42 (100 g, 68% in two steps) as a yellow powder. 1H NMR
(300 MHz, CDCl3) d 7.92 (d, J = 16 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H),
6.62 (dd, J = 8.5, 2.2 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H), 6.51 (d,
J = 16 Hz, 1H), 6.01 (s, 1H), 5.17 (s, 2H), 3.81 (s, 3H), 3.47 (s, 3H).
found: 489.2755; Optical rotation ½a D22
ꢃ
+20.6 (c 0.48, MeOH).
5.1.21. 3-(2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-{[(6-methylpyridin-3-yl)oxy]methyl}-
phenyl)propanoic acid (15)
The titled compound was synthesized in the same manner as
described for 1 using 35n instead of 35a as a white powder. Yield
26% in three steps; 1H NMR (300 MHz, DMSO-d6) d 12.07 (s, 1H),
8.80 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 4.8 Hz, 1H), 7.45–7.33 (m, 3H),
7.31 (d, J = 8.1 Hz, 1H), 7.17 (dd, J = 8.4, 4.8 Hz, 1H), 6.95 (s, 2H),
6.84 (s, 1H), 5.15 (s, 2H), 5.02–4.91 (m, 1H), 2.84 (t, J = 7.8 Hz,
2H), 2.48–2.41 (m, 2H), 2.39 (s, 3H), 2.24 (s, 6H), 1.80–1.60 (m,
2H), 1.45–1.31 (m, 1H), 0.93 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.4 Hz,
3H); MS (FAB, Pos.) m/e 489 (M+H)+; HRMS (Pos.) calcd for
5.1.26. Methyl 3-[2-hydroxy-4-(methoxymethoxy)phenyl]-
propanoate (43)
A suspension of 42 (90.0 g, 0.378 mol) and Pd–C (10% wet type,
8.40 g) in MeOH (1000 mL) was stirred for 7 h at room temperature
under an atmosphere of hydrogen. The reaction mixture was fil-
tered through a pad of Celite and the filtrate was concentrated in
vacuo to yield 43 (92.1 g, quant.) as a brown oil. 1H NMR
(300 MHz, CDCl3) d 7.24 (s, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.61 (d,
C30H37N2O4: 489.2753; found: 489.2750; Optical rotation ½a D23
ꢃ
+20.1 (c 0.56, MeOH).

M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
3221
J = 2.5 Hz, 1H), 6.57 (dd, J = 8.2, 2.5 Hz, 1H), 5.13 (s, 2H), 3.69 (s,
3H), 3.46 (s, 3H), 2.84 (t, J = 6.1 Hz, 2H), 2.69 (t, J = 6.1 Hz, 2H).
3H), 0.96 (d, J = 6.3 Hz, 3H); MS (FAB, Pos.) m/e 460 (M+H)+; HRMS
(Pos.) calcd for C29H34NO4: 460.2488; found: 460.2497; Optical
rotation ½a 2D4
ꢃ
+19.6 (c 0.94, MeOH).
5.1.27. Methyl 3-(4-(methoxymethoxy)-2-{[(trifluoromethyl)sul-
fonyl]oxy}phenyl)propanoate (44)
5.1.31. 3-[4-(3,5-Difluorophenoxy)-2-({[(1R)-1-(3,5-dimethyl-
phenyl)-3-methylbutyl]amino}carbonyl)phenyl]propanoic acid
(17)
To a stirred solution of 43 (82.8 g, 0.345 mol) and pyridine
(33.5 mL, 0.414 mol) in CH2Cl2 (300 mL) was added dropwise
Tf2O (63.8 mL, 0.379 mol) at 0 °C under an argon atmosphere. After
being stirred for 10 min at 0 °C, the reaction was quenched with
water and then the resultant mixture was extracted with EtOAc.
The organic layer was washed with water and brine, dried over
MgSO4, and concentrated in vacuo to yield 44, which was used
for the next step without purification. 1H NMR (300 MHz, CDCl3)
d 7.24 (d, J = 8.4 Hz, 1H), 7.04–6.96 (m, 2H), 5.16 (s, 2H), 3.68 (s,
3H), 3.47 (s, 3H), 2.98 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H).
The titled compound was synthesized in the same manner as
described for 16 using 48b instead of 48a as a beige powder. Yield
59% in two steps; 1H NMR (300 MHz, CDCl3) d 7.30–7.25 (m, 1H),
7.06–6.98 (m, 2H), 6.94 (s, 2H), 6.90 (s, 1H), 6.59–6.43 (m, 3H),
6.28 (d, J = 9.0 Hz, 1H), 5.20–5.09 (m, 1H), 3.07–2.96 (m, 2H),
2.72 (t, J = 7.5 Hz, 2H), 2.30 (s, 6H), 1.83–1.52 (m, 3H), 0.97 (d,
J = 6.4 Hz, 6H); MS (FAB, Pos.) m/e 496 (M+H)+; HRMS (Pos.) calcd
for C29H32F2NO4: 496.2299; found: 496.2301; Optical rotation
½
a 2D4
+16.7 (c 1.02, MeOH).
ꢃ
5.1.28. 5-(Methoxymethoxy)-2-(3-methoxy-3-oxopropyl)-
benzoic acid (45)
5.1.32. 3-[4-(3,5-Dimethylphenoxy)-2-({[(1R)-1-(3,5-dimethyl-
phenyl)-3-methylbutyl]amino}carbonyl)phenyl]propanoic acid
(18)
A suspension of 44, potassium acetate (169 g, 1.73 mol), DPPF
(7.65 g, 13.9 mmol) and Pd(OAc)2 (1.55 g, 6.90 mmol) in DMF
(400 mL) was stirred for 24 h at 90 °C under an atmosphere of car-
bon monoxide. The reaction mixture was filtered through a pad of
Celite. Water was added to the filtrate and then the resultant solu-
tion was extracted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4, and concentrated in vacuo. The
resultant residue was passed through the column chromatography
on silica gel (EtOAc/hexane, 1/1) and then above obtained residue
was recrystallized from EtOAc/hexane to yield 45 (51.4 g, 56% in
two steps) as a white powder. 1H NMR (300 MHz, CDCl3) d 7.71
(d, J = 2.7 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 8.7, 2.7 Hz,
1H), 5.20 (s, 2H), 3.67 (s, 3H), 3.49 (s, 3H), 3.27 (t, J = 7.6 Hz, 2H),
2.68 (t, J = 7.6 Hz, 2H).
The titled compound was synthesized in the same manner as de-
scribed for 16 using 48c instead of 48a as a white powder. Yield 89%
in two steps; 1H NMR (300 MHz, CDCl3) d 7.20 (d, J = 8.4 Hz, 1H), 7.00
(d, J = 2.6 Hz, 1H), 6.97–6.88 (m, 4H), 6.77 (s, 1H), 6.61 (s, 2H), 6.19
(d, J = 8.6 Hz, 1H), 5.19–5.08 (m, 1H), 3.04–2.96 (m, 2H), 2.72
(t, J = 7.2 Hz, 2H), 2.29 (s, 6H), 2.28 (s, 6H), 1.82–1.52 (m, 3H), 0.96
(d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H); MS (FAB, Pos.) m/e 488
(M+H)+; HRMS (Pos.) calcd for C31H38NO4: 488.2801; found:
488.2801; Optical rotation ½a D22
ꢃ
+16.3 (c 0.49, MeOH).
5.1.33. 3-[4-(3-Cyanophenoxy)-2-({[(1R)-1-(3,5-dimethylphenyl)-
3-methylbutyl]amino}carbonyl)phenyl]propanoic acid (19)
The titled compound was synthesized in the same manner as
described for 16 using 48d instead of 48a as a white powder. Yield
37% in two steps; 1H NMR (300 MHz, CDCl3) d 7.47–7.36 (m, 2H),
7.30–7.25 (m, 1H), 7.24–7.16 (m, 2H), 7.04–6.95 (m, 2H), 6.94 (s,
2H), 6.90 (s, 1H), 6.32 (d, J = 8.8 Hz, 1H), 5.19–5.09 (m, 1H), 3.07–
2.96 (m, 2H), 2.73 (t, J = 7.5 Hz, 2H), 2.29 (s, 6H), 1.85–1.51 (m,
3H), 0.97 (d, J = 6.6 Hz, 6H); MS (FAB, Pos.) m/e 485 (M+H)+; HRMS
(Pos.) calcd for C30H33N2O4: 485.2440; found: 485.2440; Optical
5.1.29. Methyl 3-[4-hydroxy-2-({[(1R)-3-methyl-1-phenylbutyl]-
amino}carbonyl)phenyl]propanoate (47)
The titled compound was synthesized in the same manner as de-
scribed for 32 using 45 instead of 30 as a white powder. Yield 95% in
two steps; 1H NMR (300 MHz, CDCl3) d 7.01 (d, J = 8.2 Hz, 1H), 6.96
(s, 2H), 6.88 (s, 1H), 6.85 (d, J = 9.6 Hz, 1H), 6.81 (d, J = 2.7 Hz, 1H),
6.74 (dd, J = 8.2, 2.7 Hz, 1H), 6.68 (br s, 1H), 5.18–5.08 (m, 1H),
3.62 (s, 3H), 2.97–2.84 (m, 2H), 2.72–2.54 (m, 2H), 2.29 (s, 6H),
1.84–1.52 (m, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H).
rotation ½a 2D4
ꢃ
+10.8 (c 0.57, MeOH).
5.1.34. Benzyl 2-bromo-5-nitrobenzoate (51)
To a stirred suspension of 2-bromo-5-benzoic acid (50) (25.0 g,
0.102 mol), K2CO3 (21.2 g, 0.153 mol) in DMF (150 mL) was added
benzyl bromide (12.7 mL, 0.107 mol) at 0 °C under an argon atmo-
sphere. After being stirred for 30 min at 60 °C, the reaction mixture
was poured into water and then extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and concentrated
in vacuo. The resultant residue was washed with EtOAc/hexane to
yield 51 (30.9 g, 90%) as a yellow powder. 1H NMR (300 MHz,
CDCl3) d 8.64 (d, J = 2.7 Hz, 1H), 8.16 (dd, J = 8.7, 2.7 Hz, 1H), 7.86
(d, J = 8.7 Hz, 1H), 7.49–7.39 (m, 5H), 5.42 (s, 2H).
5.1.30. 3-[2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-phenoxyphenyl]propanoic acid (16)
A
suspension of 47 (367 mg, 0.922 mmol), 48a (338 mg,
2.77 mmol), TEA (643 L, 4.61 mmol) Cu(OAc)2 (168 mg, 0.922
l
mmol) and molecular sieves (60 mg) in CH2Cl2 (2 mL) was stirred
for 12 h at room temperature. The reaction mixture was diluted
with EtOAc and then filtered through a pad of Celite. The filtrate
was separated and the organic layer was washed with water and
brine, dried over MgSO4, and concentrated in vacuo. The resultant
residue was purified by column chromatography on silica gel
(EtOAc/hexane, 1/3) to yield 49a which was dissolved in THF
(2 mL) and MeOH (2 mL) and then 2 M aqueous solution of NaOH
(2 mL) was added at room temperature. After being stirred for
12 h at room temperature, the reaction mixture was acidified with
1 M aqueous solution of HCl and then extracted with EtOAc. The or-
ganic layer was washed with water and brine, dried over MgSO4,
and concentrated in vacuo. The resultant residue was recrystal-
lized with EtOAc/hexane to yield 16 (293 mg, 71% in two steps) as
a white powder. 1H NMR (300 MHz, CDCl3) d 7.40–7.30 (m, 2H),
7.17 (d, J = 8.1 Hz, 1H), 7.16–7.09 (m, 1H), 7.04–6.82 (m, 7H), 6.19
(d, J = 8.4 Hz, 1H), 5.18–5.08 (m, 1H), 3.08–2.92 (m, 2H), 2.71 (t,
J = 6.9 Hz, 2H), 2.29 (s, 6H), 1.80–1.50 (m, 3H), 0.97 (d, J = 6.3 Hz,
5.1.35. Benzyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-5-
nitrobenzoate (52)
A solution of 51 (30.9 g, 91.8 mmol), ethyl acrylate (19.9 mL,
0.184 mol), Pd(OAc)2 (2.06 g, 9.18 mmol), DPPF (5.09 g, 9.18 mmol)
and TEA (64.0 mL, 0.459 mol) in DMSO (185 mL) was stirred for
1.5 h at 80 °C under an argon atmosphere. The reaction mixture
was diluted with EtOAc/hexane = 1/1 and 1 M aqueous solution
of HCl, and then filtered through a pad of Celite. The filtrate was
separated and the organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. The resultant residue
was purified by column chromatography on silica gel (EtOAc/hex-

3222
M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
ane, 1/7 to 1/5) to yield 52 (11.8 g, 36%) as a brown solid. 1H NMR
(300 MHz, CDCl3) d 8.82 (d, J = 2.1 Hz, 1H), 8.45 (d, J = 16 Hz, 1H),
8.36 (dd, J = 8.7, 2.1 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.49–7.39
(m, 5H), 6.38 (d, J = 16 Hz, 1H), 5.42 (s, 2H), 4.29 (q, J = 7.2 Hz,
2H), 1.35 (t, J = 7.2 Hz, 3H).
5.1.39. 3-(4-[(3,5-Dimethylphenyl)amino]-2-{[(1R)-1-(3,5-
dimethylphenyl)-3-methylbutyl]carbamoyl}phenyl)propanoic
acid (21)
The titled compound was synthesized in the same manner as
described for 20 using 48c instead of 48b as an off-white powder.
Yield 78% in two steps; 1H NMR (300 MHz, CDCl3) d 7.12 (d,
J = 8.4 Hz, 1H), 7.07–6.98 (m, 2H), 6.93 (s, 2H), 6.89 (s, 1H), 6.67
(s, 2H), 6.61 (s, 1H), 6.25 (d, J = 8.6 Hz, 1H), 5.27–5.04 (m, 1H),
3.08–2.84 (m, 2H), 2.69 (t, J = 7.6 Hz, 2H), 2.29 (s, 6H), 2.26 (s,
6H), 2.00–1.37 (m, 3H), 0.97 (d, J = 6.2 Hz, 3H), 0.96 (d, J = 6.2 Hz,
3H); MS (FAB, Pos.) m/e 487 (M+H)+; HRMS (Pos.) calcd for
5.1.36. 5-Amino-2-(3-ethoxy-3-oxopropyl)benzoic acid (53)
A suspension of 52 (11.8 g, 33.2 mmol) and Pd–C (10% wet
type, 2.36 g) in EtOH (300 mL) was stirred for 5 h at room tem-
perature under an atmosphere of hydrogen. The reaction mixture
was filtered through a pad of Celite and the filtrate was concen-
trated in vacuo to yield 53 (7.72 g, 98%) as a pale yellow powder.
1H NMR (300 MHz, CDCl3) d 7.35 (d, J = 2.7 Hz, 1H), 7.10 (d,
J = 8.1 Hz, 1H), 6.81 (dd, J = 8.1, 2.7 Hz, 1H), 4.11 (q, J = 7.2 Hz,
2H), 3.20 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 1.23 (t,
J = 7.2 Hz, 3H).
C31H39N2O3: 487.2961; found: 487.2958; Optical rotation ½a D20
ꢃ
ꢂ8.91 (c 0.66, CHCl3).
5.1.40. 3-[4-[(3-Cyanophenyl)amino]-2-({[(1R)-1-(3,5-dimethyl-
phenyl)-3-methylbutyl]amino}carbonyl)phenyl]propanoic acid
(22)
The titled compound was synthesized in the same manner as
described for 20 using 48d instead of 48b as a pale yellow powder.
Yield 57% in two steps; 1H NMR (300 MHz, DMSO-d6) d 8.77 (d,
J = 8.8 Hz, 1H), 8.62 (s, 1H), 7.43–7.32 (m, 3H), 7.23–7.17 (m, 2H),
7.08 (dd, J = 8.3, 5.3 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 6.93 (s, 2H),
6.83 (s, 1H), 5.00–4.90 (m, 1H), 2.78 (t, J = 7.9 Hz, 2H), 2.42 (t,
J = 7.9 Hz, 2H), 2.23 (s, 6H), 1.78–1.59 (m, 2H), 1.44–1.32 (m, 1H),
0.92 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H); MS (FAB, Pos.) m/e
484 (M+H)+; HRMS (Pos.) calcd for C30H34N3O3: 484.2600; found:
5.1.37. Ethyl 3-[4-amino-2-({[(1R)-3-methyl-1-
phenylbutyl]amino}carbonyl)phenyl]propanoate (54)
A
solution of 53 (600 mg, 2.52 mmol), (R)-27 (633 mg,
2.78 mmol), N-methylmorpholine (305
lL, 2.78 mmol), EDCꢀHCl
(531 mg, 2.77 mmol), and HOBt (681 mg, 5.04 mmol) in DMF
(7 mL) was stirred for 12 h at room temperature under argon
atmosphere. The reaction mixture was diluted with water and
then extracted with TBME. The organic layer was washed with
aqueous NaHCO3, water and brine, dried over MgSO4, and concen-
trated in vacuo. The resultant residue was purified by column
chromatography on silica gel (EtOAc/hexane, 1/1) to yield 54
(659 mg, 64%) as a pale brown powder. 1H NMR (300 MHz, CDCl3)
d 7.00 (d, J = 8.7 Hz, 1H), 6.96 (s, 2H), 6.89 (s, 1H), 6.69–6.61 (m,
2H), 6.48 (d, J = 9.0 Hz, 1H), 5.20–5.07 (m, 1H), 4.07 (q, J = 7.2 Hz,
2H), 3.63 (s, 2H), 2.90 (t, J = 7.4 Hz, 2H), 2.69–2.50 (m, 2H), 2.31
(s, 6H), 1.86–1.53 (m, 3H), 1.20 (t, J = 7.2 Hz, 3H), 0.99 (d,
J = 6.0 Hz, 3H), 0.98 (d, J = 6.0 Hz, 3H).
484.2600; Optical rotation ½a D23
ꢂ33.2 (c 0.80, CH3CN).
ꢃ
5.1.41. 3-{2-({[(1R)-1-(3,5-Dimethylphenyl)-3-methylbutyl]-
amino}carbonyl)-4-[(3-methylphenyl)amino]phenyl}propanoic
acid (23)
The titled compound was synthesized in the same manner as
described for 20 using 48e instead of 48b as a white powder. Yield
71% in two steps; 1H NMR (300 MHz, CDCl3) d 7.19–7.10 (m, 2H),
7.06–6.99 (m, 2H), 6.93 (s, 2H), 6.89 (s, 1H), 6.88–6.82 (m, 2H),
6.78 (d, J = 7.5 Hz, 1H), 6.24 (d, J = 8.4 Hz, 1H), 5.18–5.09 (m, 1H),
2.99–2.91 (m, 2H), 2.70 (t J = 7.7 Hz, 2H), 2.30 (s, 3H), 2.29 (s,
6H), 1.81–1.53 (m, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.96 (d, J = 6.4 Hz,
3H); MS (FAB, Pos.) m/e 473 (M+H)+; HRMS (Pos.) calcd for
5.1.38. 3-[4-[(3,5-Difluorophenyl)amino]-2-({[(1R)-1-(3,5-
dimethylphenyl)-3-methylbutyl]amino}carbonyl)phenyl]-
propanoic acid (20)
A suspension of 54 (80 mg, 0.195 mmol), 48b (61 mg, 0.389
mmol), TEA (54 lL, 0.389 mmol), Cu(OAc)2 (7.1 mg, 38.9 lmol),
C30H37N2O3: 473.2804; found: 473.2804; Optical rotation ½a D22
ꢃ
ꢂ8.08 (c 0.71, CHCl3).
and molecular sieves (20 mg) in CH2Cl2 (2 mL) was stirred for
24 h at room temperature. The reaction mixture was diluted with
EtOAc and then filtered through a pad of Celite. The filtrate was
separated and the organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. The resultant residue
was purified by column chromatography on silica gel (EtOAc/hex-
ane, 1/4 to 1/3) to yield 55b which was dissolved in THF (1 mL) and
MeOH (1 mL) and then 2 M aqueous solution of NaOH (1 mL) was
added at room temperature. After being stirred for 12 h at room
temperature, the reaction mixture was neutralized with 1 M aque-
ous solution of HCl and then extracted with EtOAc. The organic
layer was washed with water and brine, dried over MgSO4 and con-
centrated in vacuo. The resultant residue was recrystallized from
EtOAc/hexane to yield 20 (60 mg, 62% in two steps) as a white
powder. 1H NMR (300 MHz, DMSO-d6) d 12.05 (s, 1H), 8.79
(d, J = 8.1 Hz, 1H), 8.72 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.09 (dd,
J = 8.4, 2.3 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.94 (s, 2H), 6.83
(s, 1H), 6.72–6.63 (m, 2H), 6.57–6.48 (m, 1H), 5.01–4.89 (m, 1H),
2.78 (t, J = 7.9 Hz, 2H), 2.47–2.40 (m, 2H), 2.23 (s, 6H), 1.78–1.61
(m, 2H), 1.44–1.32 (m, 1H), 0.92 (d, J = 6.4 Hz, 3H), 0.90 (d,
J = 6.4 Hz, 3H); MS (FAB, Pos.) m/e 495 (M+H)+; HRMS (Pos.) calcd
for C29H33F2N2O3: 495.2459; found: 495.2455; Optical rotation
5.2. Pharmacology
5.2.1. mEP1-4 receptor binding assay
Competitive binding studies were conducted using radiolabeled
ligands and membrane fractions prepared from Chinese hamster
ovary (CHO) cells stably expressing the prostanoid receptors
mEP1-4. Membranes from CHO cells expressing prostanoid recep-
tors were incubated with radiolabeled ligand (2.5 nM [3H]PGE2)
and test compounds at various concentrations in assay buffer
(10 mM KH2PO4–KOH buffer containing 1 mM EDTA, 10 mM MgCl2
and 0.1 mM NaCl, pH 6.0). Incubation was carried out at 25 °C for
60 min except for mEP1 which was incubated for 20 min. Incuba-
tion 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 filter was measured in 6 mL of liquid scintilla-
tion (ACSII) mixture with a liquid scintillation counter. Nonspecific
binding was achieved by adding excess amounts of unlabeled PGE2
in assay buffer. The half maximal inhibitory concentration of spe-
cific binding (IC50 value) was estimated from the regression curve.
The Ki value (M) was calculated according to the following
equation:
½
a 2D2
ꢃ
ꢂ14.8 (c 1.22, DMF).

M. Asada et al. / Bioorg. Med. Chem. 18 (2010) 3212–3223
3223
Ki ¼ IC50=ð1 þ ½Lꢃ=KdÞ
where [L] is the concentration of radiolabeled ligand, and Kd is the
dissociation constant of radiolabeled ligand for the prostanoid
receptor of interest.
where
D
S (test compound) is the increased uterine contraction of
S (vehicle) is the increased
administration of a test compound, and
uterine contraction of administration of vehicle.
D
5.2.4. The stability in human and rat microsomes
The test compound (5
195 L of 50% acetonitrile in water to make a 250
the test compound. Phosphate buffer (0.1 M, 245
0.5 mg/mL rat or human liver microsomes and NADPH-Co-factor
was added to a reaction container, pre-warmed to 37 °C in a water
bath, and incubated for 5 min. The reaction was initiated by the
l
L, 10 mM in DMSO) was diluted with
M solution of
L) containing
5.2.2. mEP3 receptor antagonist activity
l
l
l
To confirm that test compounds antagonized the mEP3 receptor
and to estimate the extent of antagonism for the mEP3 receptor, a
functional assay was performed by measuring PGE2-stimulated in-
creases in intracellular Ca2+. The cells expressing mEP3
a receptor
were seeded at 1 ꢁ 104 cells/well in 96 well plates and cultured
addition of 5
acetonitrile with 0.05% DMSO, final concentration of 5
the initiation of the reaction, a 20 L aliquot was taken from the
solution immediately and transferred to 180 L of acetonitrile con-
taining the internal standard (warfarin) to terminate the reaction.
A 20 L aliquot of the mixture was stirred with 180 L of 50% ace-
lL of the solution of the test compound (in 0.975%
for two days with 10% FBS (fetal bovine serum)/minimum essential
l
M). After
medium Eagle alpha modification (
5% CO2). The cells in each well were rinsed with phosphate buffer
(PBS(ꢂ)), and load buffer (10% FBS/ MEM containing 5 M of Fura
2/AM, 20 M of indomethacin, and 2.5 mM of probenecid) was
aMEM) in an incubator (37 °C,
l
l
a
l
l
l
l
added. After incubation for 1 h, the cells in each well were rinsed
with assay buffer (Hank’s balanced salt solution (HBSS) containing
tonitrile on a plate with a filter for deproteinization and filtered by
suction. The filtrate was used as the standard sample. After incuba-
tion for 15 min, a 20 lL aliquot was taken from the solution and
then treated with the same procedure described above to obtain
the reaction sample. The obtained samples were measured by an
LC-MS/MS system. The percent remaining (%) was calculated by
dividing the peak area ratio (i.e., test compound/I.S.) for the reac-
tion sample by the peak area ratio for the standard sample and
multiplying by 100.
1% (w/v) BSA, 2
10 mM of HEPES-NaOH) twice. Then 90
l
M of indomethacin, 2.5 mM of probenecid, and
L of assay buffer was
l
added to each well and the cells were incubated in the dark at
room temperature for 1 h. After the addition of a solution contain-
ing test compound (30 lL) and PGE2 (30 lL), which were prepared
with assay buffer, the intracellular calcium concentration was
measured with a fluorescence drug screening system (FDSS-3000,
Hamamatsu Photonics). The fluorescence intensities emitted at
500 nm using excitation wavelengths of 340 nm and 380 nm were
measured. The percent inhibition from the increase in the intracel-
lular Ca2+ concentration induced by PGE2 (10 nM) was calculated
relative to the maximum Ca2+ concentration that occurred in the
absence of the test compound (100%). This was then used to esti-
mate the IC50 value.
Acknowledgments
We thank Mr. Kazuhide Masago, Dr. Rie Oumi, and Dr. Yoshi-
hiko Odagaki for the X-ray crystallographic analysis of compound
29 and their technical assistance.
References and notes
5.2.3. Inhibitory effect of test compounds on the PGE2-induced
uterine contraction in pregnant rats
1. Narumiya, S.; Sugimoto, Y.; Ushikubi, F. Physiol. Rev. 1999, 79, 1193.
2. Minami, T.; Nakano, H.; Kobayashi, T.; Sugimoto, Y.; Ushikubi, F.; Ichikawa, A.;
Narumiya, S.; Ito, S. Br. J. Pharmacol. 2001, 133, 438.
3. Ushikubi, F.; Segi, E.; Sugimoto, Y.; Murata, T.; Matsuoka, T.; Kobayashi, T.;
Hizaki, H.; Tuboi, K.; Katsuyama, M.; Ichikawa, A.; Tanaka, T.; Yoshida, N.;
Narumiya, S. Nature 1998, 395, 281.
4. (a) Senior, J.; Marshall, K.; Sangha, R.; Baxter, G. S.; Clayton, J. K. Br. J. Pharmacol.
1991, 102, 747; (b) Goureau, O.; Tanfin, Z.; Marc, S.; Harbon, S. Am. J. Physiol.
1992, 263, C257.
5. Chen, M. C. Y.; Amirian, D. A.; Toomey, M.; Sanders, M. J.; Soll, A. H.
Gastroenterology 1988, 94, 1121.
6. Ma, H.; Hara, A.; Xiao, C. Y.; Okada, Y.; Takahata, O.; Nakaya, K.; Sugimoto, Y.;
Ichikawa, A.; Narumiya, S.; Ushikubi, F. Circulation 2001, 104, 1176.
7. Gross, S.; Tilly, P.; Hentsch, D.; Vonesch, J. L.; Fabre, J. E. J. Exp. Med. 2007, 204,
311.
8. Asada, M.; Obitsu, T.; Nagase, T.; Tanaka, M.; Yamaura, Y.; Takizawa, H.;
Yoshikawa, K.; Sato, K.; Narita, M.; Ohuchida, S.; Nakai, H.; Toda, M. Bioorg.
Med. Chem. 2010, 18, 80.
9. Asada, M.; Iwahashi, M.; Obitsu, T.; Kinoshita, A.; Nakai, Y.; Onoda, T.; Nagase,
T.; Tanaka, M.; Yamaura, Y.; Takizawa, H.; Yoshikawa, K.; Sato, K.; Narita, M.;
Ohuchida, S.; Nakai, H.; Toda, M. Bioorg. Med. Chem. 2010, 18, 1641.
10. Corgan, D. E.; Liu, G.; Ellman, J. Tetrahedron 1999, 55, 8883.
11. Takahashi, H.; Chida, Y.; Yoshii, T.; Suzuki, T.; Yanaura, S. Chem. Pharm. Bull.
1986, 34, 2071.
12. CCDC 768381 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, or by e-mailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12, Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Fed pregnant rats were anesthetized with pentobarbital (20 mg/
20 mL/kg ip) and urethane (1.5 g/5 mL/kg sc) then fixed in the dor-
sal position. A midline incision was made in the lower abdomen
and a small incision was made near the cervical area of the right
or left uterine horn, and a balloon catheter (Okamoto Medical
Industry, for rats) was inserted between the uterine wall and the
amnion. After suturing the abdominal incision, the intraballoon
pressure was loaded to approximately 10 mmHg. Uterine motility
was recorded on a recticoder (WR3320 or WR3701, GRAPHTEC)
via a pressure transducer (Life Kit DX-360, Nihon Kohden Corp.)
and a strain pressure amplifier (AP-601G, Nihon Kohden Corp.).
After spontaneous uterine motility was kept at a stable level, an in-
creased uterine contraction was elicited by an intravenous admin-
istration of PGE2 (30 lg/kg). Test compound (5 mL/kg in 0.5%
methylcellulose (MC)) or vehicle (0.5% MC) was orally adminis-
trated 4 h prior to PGE2 administration. Subtraction of the area un-
der the uterus pressure-time curve (AUC) for 10 min before PGE2
administration from the AUC for 10 min after PGE2 administration
was determined to be the increased uterine contraction (=DS). The
inhibitory effect of a test compound (% inhibition) was calculated
according to the following equation:
13. Ganguly, N.; Sukai, A. K.; De, S. Synth. Commun. 2001, 31, 301.
14. Evans, D. A.; Katz, J. L.; West, T. R. Tetrahedron Lett. 1998, 39, 2937.
15. Lam, P. Y. S.; Vincent, G.; Clark, C. G.; Deudon, S.; Jadhav, P. K. Tetrahedron Lett.
2001, 42, 3415.
Inhibitory effect ð% inhibitionÞ
¼ f½
D
SðvehicleÞ ꢂ
D
Sðtest compoundÞꢃ= SðvehicleÞg ꢄ 100
D