Synthesis and biological evaluation of benzoic acid derivatives as potent, orally active VLA-4 antagonists

Article abstract of DOI:10.1016/j.bmc.2006.12.006

A series of benzoic acid derivatives was synthesized as VLA-4 antagonists. Introduction of chlorine or bromine into the 3-position on the central benzene of the diphenylurea portion as in lead compound 2 led to improvement in the pharmacokinetic properties. In particular, 12l demonstrated an acceptable plasma clearance and bioavailability in mice and rats as well as dogs (mice, CL = 18.5 ml/min/kg, F = 28 %; rats, CL = 5.2 ml/min/kg, F = 36 %; dogs, CL = 3.6 ml/min/kg, F = 55 %). Additionally, 12l exhibited potent activity with an IC₅₀ value of 0.51 nM and efficacy by oral administration at a dosage of 10 mg/kg in a rat pleurisy model.

Full text of DOI:10.1016/j.bmc.2006.12.006

Bioorganic & Medicinal Chemistry 15 (2007) 1679–1693
Synthesis and biological evaluation of benzoic acid derivatives as
potent, orally active VLA-4 antagonists
Jun Chiba,a,* Shin Iimura,a Yoshiyuki Yoneda,a Toshiyuki Watanabe,a Fumito Muro,a
Masao Tsubokawa,b Yutaka Iigou,b Atsushi Satoh,b Gensuke Takayama,b
Mika Yokoyama,b Tohru Takashi,b Atsushi Nakayamaa and Nobuo Machinagaa
aMedicinal Chemistry Research Laboratory, Daiichi Pharmaceutical, Co., Ltd, 16-13, Kitakasai 1-chome,
Edogawa-ku, Tokyo 134-8630, Japan
bNew Product Research Laboratories III, Daiichi Pharmaceutical, Co., Ltd, 16-13, Kitakasai 1-chome,
Edogawa-ku, Tokyo 134-8630, Japan
Received 10 November 2006; revised 6 December 2006; accepted 7 December 2006
Available online 9 December 2006
Abstract—A series of benzoic acid derivatives was synthesized as VLA-4 antagonists. Introduction of chlorine or bromine into the
3-position on the central benzene of the diphenylurea portion as in lead compound 2 led to improvement in the pharmacokinetic
properties. In particular, 12l demonstrated an acceptable plasma clearance and bioavailability in mice and rats as well as dogs (mice,
CL ¼ 18:5 ml=min=kg;F ¼ 28%; rats, CL ¼ 5:2 ml=min=kg;F ¼ 36%; dogs, CL ¼ 3:6 ml=min=kg;F ¼ 55%). Additionally, 12l
exhibited potent activity with an IC50 value of 0.51 nM and efficacy by oral administration at a dosage of 10 mg/kg in a rat pleurisy
model.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Idec Inc.), demonstrates promising results in patients
with multiple sclerosis, Crohn’s disease, and rheumatoid
arthritis in clinical trials. Therefore, the development of
small molecular VLA-4 antagonists with acceptable oral
pharmacokinetic profiles is viewed as a reasonable ap-
proach to a novel class of therapeutic agents.
VLA-4 (very late antigen 4; a4b1 integrin; CD49d/CD29)
is a key cell receptor expressed on most leukocytes.1 The
natural ligands include VCAM-1 (vascular cell adhesion
molecule-1) expressed on cytokine-stimulated endotheli-
al cells, the alternatively spliced connecting segment-1
domain of fibronectin on the extracellular matrix,2,3
and junctional adhesion molecule 2 on endothelial cells.4
Through the VLA-4/ligands interactions, VLA-4 plays
an important role in the process of adhesion, migration,
and activation of inflammatory leukocytes at sites of
inflammation. Anti-VLA-4 antibodies or small molecu-
lar VLA-4 antagonists5 have been reported to inhibit
leukocyte infiltration into extravascular tissue and thus
prevent tissue damage in inflammatory disease models
of asthma,6 multiple sclerosis,7 rheumatoid arthritis,8
and inflammatory bowel disease.9 It has recently been
reported that a humanized monoclonal anti-a4 antibody,
natalizumab,10 (Elan Pharmaceuticals Inc. and Biogen
We have reported the identification of morpholinyl-
carboxypiperidinylacetic acid 1 as a potent VLA-4
antagonist with an IC50 value of 4.4 nM11 (Fig. 1).
Compound 1 has, however, been found to show
rapid plasma clearance and low bioavailability in
rats ðCL ¼ 69 ml=min=kg;F < 1%Þ. It has also been
found that 1 is poorly permeable ðPapp values ¼
0:02 ꢀ 10ꢁ6 cm=sÞ in the permeability assay using Ma-
din–Darby canine kidney (MDCK) cell monolayers
owing to its low lipophilicity with a logD value of
ꢁ0.7. In our efforts to improve its rapid clearance and
poor membrane permeability, structural modification
of the morpholine-ring and the piperidinylacetic acid
portion of 1 has resulted in the pyrolidinylmethoxyben-
zoic acid derivative 2 (Fig. 1).12 This compound has
potent activity with an IC50 value of 1.2 nM and
comparatively high lipophilicity with a logD value of
Keywords: VLA-4; Integrin; Oral availability.
*
Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696
8609; e-mail: chiba2mk@daiichipharm.co.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.006

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J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
Morpholinylcarboxypiperidinylacetic acid derivative
Pyrrolidinylmethoxybenzoic acid derivative
F
Diphenylurea portion
O
N
N
A
O
O
N
O
O
CO2H
O
N
H
N
H
O
N
H
N
H
CO2H
3
Me
Me
OMe
2: IC 50 = 1.2 nM, IC 50 (+3%HSA) = 3.7nM, LogD = 1.3
1: IC50 = 4.4 nM, Log D = -0.7
Figure 1. VLA-4 antagonists.
1.3. Thus, we next chose the diphenylurea portion
(Fig. 1) for further exploration.
reduced by hydrogenation to afford anilines 3 and 4
(Scheme 2).
So far, regarding the SAR of the diphenylurea portion,
Biogen research group has reported that the presence of
a methyl group at the 2-position in the terminal benzene
for valine derivatives6 and a methoxy group at the 3-po-
sition in the central benzene for b-alanine derivatives
enhances inhibitory activity.13 Moreover, Glaxo-
SmithKline research group has very recently reported
that the methoxy group at the 3-position in the central
benzene, which seems to form an intra-molecular hydro-
gen bond with acidic urea functionality, contributes to
improved pharmacokinetics.14 From these results, we
decided to investigate the effects of substituents at the
same positions on the activity and pharmacokinetic
properties as a first examination of modifications of
the diphenylurea portion. At this point, we selected
some substituents on the basis of their molecular
refraction index (MR)15 and lipophilicity (p value).15
In addition, since we have found [(4S)-fluoro-(2S)-pyr-
rolidinyl]methoxybenzoic acid (A, Fig. 1) to be a
beneficial motif for maintaining potent activity,12b the
motif was not altered in this study.
Anilines 8 and 9 were prepared from commercially avail-
able phenylacetic acids 16a and 16b by reported proce-
dure17,18 (Scheme 3).
3-Bromophenylacetate 18b was coupled with trimethyl-
silylacetylene by using the Sonogashira reaction19 to
give 19, followed by removal of trimethylsilyl group to
give 20. The C–C triple bond and nitro group of 20 were
reduced at the same time by hydrogenation to afford
aniline 5 (Scheme 4).
Ethyl 4-nitrophenylacetate (21) was converted to 23
according to the reported procedure,20 which was then
subjected to hydrogenation to afford aniline
(Scheme 5).
6
Coupling of compound 24 with tert-butyl ethyl malon-
ate in the presence of NaH gave 25, which was succes-
sively hydrolyzed and decarboxylated in TFA
condition to give 26. Subsequently, the nitro group of
26 was reduced with SnCl2 to afford aniline 7
(Scheme 6).
In this paper, we wish to report the synthesis and SARs
for a variety of substituents in the diphenylurea portion
of benzoic acid derivatives as well as the PK profiles for
representative compounds in rodents and dogs. Further-
more, we also present the result of the evaluation of the
best compound in rat pleurisy model.
3. Results and discussion
3.1. In vitro activity
Benzoic acid derivatives were evaluated for their VLA-4
inhibitory activities in the receptor binding assay, with
or without the addition of 3% human serum albumin
(HSA). These results are summarized in Tables 1 and 2.
2. Chemistry
Benzoic acid derivatives 12a–q were synthesized accord-
ing to the General Procedure shown in Scheme 1. Thus,
treatment of compounds 3–9 with commercially avail-
able isocyanate [2-(R2)PhNCO] and hydrolysis provided
4-ureidophenylacetic acids 10a–q, which were condensed
First, to investigate how replacement of the methyl
group with other substituents (R2) at the 2-position on
the terminal benzene in compound 2 affects the activity,
we introduced several substituents (Cl, Br, MeO, and
CF3) with MR of 5.02–8.88, which are about the same
size as the methyl group ðMR ¼ 5:65Þ, and an isopropyl
group with a larger size ðMR ¼ 14:96Þ into the position
(Table 1).
with
methyl
4-[(4S)-fluoro-(2S)-pyrrolidinylmeth-
oxy]benzoate (11) using EDC and HOBt followed by
basic hydrolysis to afford 12a–q.
Compounds 3–9 in Scheme 1 were prepared as shown in
Schemes 2–6.
As a result, it was found that chlorine (12a,
MR ¼ 6:03) and bromine (12b, MR ¼ 8:88) maintained
the potency (12a, IC50 = 0.87 nM; 12b, IC50 = 1.6 nM)
in comparison with 2 (IC50= 1.2 nM). In contrast, a
methoxy group (12c, MR ¼ 7:87) and a trifluoromethyl
group (12d, MR ¼ 5:02) reduced the potency by 1/5
and 1/96, respectively (12c, IC50 = 9.2 nM; 12d,
Commercially available benzoic acids 13a and 13b were
converted to diazoketone 14a and 14b via the corre-
sponding acid chloride, followed by the Arndt-Eistert
reaction,16 to give tert-butyl phenylacetate 15a and
15b. Subsequently, the nitro groups of 15a and 15b were

J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1681
(a) 2-(R2)PhNCO, Et3N
CO2R
O
CO2H
H2N
N
H
N
H
(b) Hydrolysis
R1
3-9
R2
R1
10a-q
3, R1 = OMe, R = tBu
4, R1 = Me, R = tBu
5, R1 = Et, R = Me
6, R1 = iPr, R = Et
7, R1 = CF3, R = Et
8, R1 = Cl, R = Me
9, R1 = Br, R = Me
F
HN
O
CO2Me
F
11
N
(c) EDC, HOBt, Et3N
O
O
CO2H
O
N
H
N
H
(d) Hydrolysis
R2
R1
12a-q
a, R1 = OMe, R2 = Cl
b, R1 = OMe, R2 = Br
c, R1 = OMe, R2 = OMe
d, R1 = OMe, R2 = CF3
f, R1 = Me, R2 = Me
g, R1 = Me, R2 = Cl
h, R1 = Me, R2 = Br
i, R1 = Et, R2 = Me
j, R1 = iPr, R2 = Me
k, R1 = CF3, R2 = Me
p, R1 = Br, R2 = Cl
q, R1 = Br, R2 = Br
l, R1 = Cl, R2 = Me
m, R1 = Cl, R2 = Cl
n, R1 = Cl, R2 = Br
o, R1 = Br, R2 = Me
e, R1 = OMe, R2 = iPr
Scheme 1.
CO2H
a
COCHN2
b
CO2Bu-t
CO2Bu-t
c
O2N
O2N
X
H2N
O2N
X
X
X
13a, X = OMe
13b, X = Me
15a, X = OMe
15b, X = Me
14a, X = OMe
14b, X = Me
3, X = OMe
4, X = Me
Scheme 2. Reagents: (a) 1-SOCl2, toluene, cat. DMF; 2-TMSCHN2, Et3N, THF; (b) PhCO2Ag, Et3N, tert-BuOH/toluene; (c) H2, 5% Pd/C, MeOH.
OH
OMe
OMe
c
OMe
b
a
O
O
O
O
O2N
H2N
X
X
X
Cl
16a, X = Cl
16b, X = Br
17a, X = Cl
17b, X = Br
18a, X = Cl
18b, X = Br
8, X =Cl
9, X = Br
Scheme 3. Reagents: (a) MeOH, concd H2SO4, ClCH2CH2Cl; (b) concd HNO3, concd H2SO4; (c) Fe, NaOAc, HOAc, MeOH, H2O.
CO2Me
CO2Me
CO2Me
CO2Me
b
a
c
O2N
O2N
H2N
O2N
Br
TMS
5
19
20
18b
Scheme 4. Reagents: (a) TMS–acetylene, Pd(Ph3P)4, CuI, TEA, THF; (b) TBAF, THF; (c) H2, 5% Pd/C, H2, EtOH.
CO2Et
a
CO2Et
CO2Et
b
CO2Et
c
O2N
H2N
O2N
O2N
21
22
23
6
Scheme 5. Reagents: (a) isopropyl magnesium chloride, THF; (b) DDQ, THF; (c) H2, 5% Pd/C, H2, EtOH.
IC50 = 106 nM). In addition, the isopropyl group (12e)
significantly reduced the potency by about 1/700-fold
(IC50 = 769 nM), which was in agreement with the
results of piperidone derivatives reported by Glaxo-
SmithKline research group.14 From these results, it
was found that the size (MR) of the substituent at this
position was a critical factor to maintain the activity;
however, substituents containing a polar atom, such
as a methoxy group, caused a negative effect on the
activity.

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J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
CO2tBu
F
CO2Et
CO2Et
CO2Et
b
a
c
O2N
O2N
O2N
H2N
CF3
CF3
CF3
CF3
26
25
24
7
Scheme 6. Reagents: (a) tert-butyl ethyl malonate, NaH, DMF; (b) TFA, DCM; (c) SnCl2, EtOH, reflux.
Table 1. Inhibitory activity of VLA-4 antagonists
We next investigated the effect of R1 substituents at the
3-position of the central benzene on the activity, where
we retained the methyl group, chlorine, and bromine
showing high potency as R2 substituents (Table 2).
F
N
O
O
CO2H
O
N
H
N
H
In the case of R2 = Me, replacement of the methoxy
group in 2 with chlorine and bromine exhibited
improved activity (12l, R2 = Cl, IC50 = 0.51 nM; 12o,
R2 = Br, IC50 = 0.78 nM), and the replacement by a
methyl group slightly decreased the activity (12f,
IC50 = 2.3 nM).
R2
OMe
Compound
R2
IC50
(nM)
IC50
(+3% HSA)
(nM)
Ratio = IC50
(+3% HSA)/IC50
2
Me
Cl
1.2
3.7
6.2
3.4
7.1
12a
12b
12c
12d
12e
0.87
1.6
Br
16
268
10.0
29.1
Nd
However, ethyl and isopropyl groups reduced the activ-
ities by 1/14 and 1/196 in accordance with the increase of
MR (12i, R2 = ethyl, MR ¼ 10:3, IC50 = 15 nM; 12j,
R2 = isopropyl, MR = 14.96, IC50 = 216 nM). In addi-
tion, the introduction of a trifluoromethyl group
reduced the activity by 1/14 (12k, R2 = CF3,
MR ¼ 5:02, IC50 = 15 nM).
MeO
CF3
iPr
9.2
106
769
>1000
>1000
Nd
Nd, not determined; Nt, not tested.
We also evaluated the activities in the presence of 3%
human serum albumin (HSA) to estimate in vivo
effectiveness.
In the case of R2 = Cl, 12m (R1 = Cl) and 12p (R1 = Br)
showed potent activity (IC50 = 1.3 nM, each); however,
12q (R1 = Me) was 12-fold less potent (IC50 = 13 nM)
than 2. Similar results were observed for compounds
with R2 = Br (12n, R1 = Cl, R2 = Br, IC50 = 1.7 nM;
12q, R1 = Br, R2 = Br, IC50 = 1.4 nM).
As a result, all the compounds 12a–e showed a de-
creased activity by 1/7–1/29 compared with those with-
out the addition of HSA, indicating that these
compounds were more likely to be affected by HSA than
2. The decrease of the activity is probably due to the
high protein binding of these compounds [2, protein
binding (PB) = 95%; 12b, PB = 98%].
As observed in the variations of R2, all of these com-
pounds were found to show decreased affinity against
Table 2. Inhibitory activity of VLA-4 antagonists
F
N
O
O
CO2H
O
N
H
N
H
R2
R1
Compound
R2
R1
IC50 (nM)
IC50 (+3% HSA) (nM)
Ratio = IC50 (+3% HSA)/IC50
2
Me
Me
Me
Me
Me
Me
Me
Cl
MeO
Me
Et
iPr
CF3
Cl
1.2
2.3
15
3.7
49
3.4
21.3
18.8
Nd
12f
12i
12j
12k
12l
12o
12g
12m
12p
12h
12n
12q
282
>1000
486
7.2
8.4
416
81
216
15
32.4
14.1
10.8
32.0
62.3
39.2
63.5
80.6
67.9
0.51
0.78
13
Br
Me
Cl
Cl
Cl
1.3
1.3
9.8
1.7
1.4
Br
51
Br
Br
Me
Cl
622
137
95
Br
Br
Nd, not determined; Nt, not tested.

J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1683
VLA-4 in the presence of HSA, likely due to their high
protein binding (12l, PB = 98%). However, since 12l
(R1 = Cl, R2 = Me) and 12o (R1 = Br, R2 = Me) had
extremely high affinity against VLA-4, these compounds
maintained single digit nM activities even in the pres-
ence of HSA [12l and 12o, IC50 (+HSA) = 7.2 and
8.4 nM].
lipophilicity of the molecule, might be critical for oral
absorption for this series of compounds.
3.3. Pharmacokinetics evaluation
To further clarify the influence of substituent R1 of cen-
tral benzene ring on oral absorption, we evaluated phar-
macokinetic profiles of the three selected compounds
12b (R1 = OMe), 12h (R1 = Me), and 12l (R1 = Cl) in
mice and rats (Table 4).
3.2. Membrane permeability and bio-assay
For the twelve compounds showing potent activity with
IC50 values less than 10 nM, we evaluated their mem-
brane permeability with MDCK cell monolayers and
determined their distribution coefficients [logD, n-octa-
nol–PBS (pH 7.4)] (Table 3).
As a result, 12l exhibited moderate plasma clearance (CL)
of 18.5 ml/min/kg in mice, compared with those of 12h
(CL = 31.7 ml/min/kg) and 12b (CL = 104.5 ml/min/kg),
indicating that substituent R1 improved plasma clearance
in the order of chlorine, methyl group, and methoxygroup.
Additionally, 12l, 12h, and 12b showed F values of 28%,
21%, and 16%, respectively. Furthermore, similar results
were observed in rats ð12l; CL ¼ 5:2 ml=min=kg;
F ¼ 36%;12h; CL ¼ 11:3ml=min=kg; F ¼ 34%;12b; CL ¼
26:5 ml=min=kg; F ¼ 11%Þ. Moreover, 12l was found to
show better pharmacokinetic profiles in dogs
ðCL ¼ 3:6ml=min=kg;F ¼ 55%Þ than rodents (Table 4).
As a result, it was reasonably concluded that the intro-
duction of halogen atom (Cl or Br) into R1 and/or R2
(12a–b, and 12g–q) increased the lipophilicity and en-
hanced membrane permeability. Compounds 12c
(R1 = R2 = OMe) and 12f (R1 = R2 = Me), however,
displayed poor membrane permeability.
Furthermore, to assess oral availability of these com-
pounds, we estimated the serum concentration by mea-
suring the binding activity in the serum at 15 min
post-oral administration at 10 and/or 50 mg/kg in mice
(Table 3).
3.4. In vivo evaluation
On the basis of the activity and pharmacokinetic prop-
erties in rats, we evaluated 12l in the compound 48/80-
inducing rat pleurisy models.21 As a result, it was found
that 12l dose-dependently inhibited the accumulation of
eosinophils in the pleural cavity by oral administration
at 10–30 mg/kg (bid) (Fig. 2).
Compounds 12a–c (R1 = OMe), 12f–h (R1 = Me), and
12l–q (R1 = Cl or Br) showed the estimated concentra-
tions of 100–261 ng/ml, 368–693 ng/ml, and 600–
3659 ng/ml, respectively. Among them, 12l displayed
the highest serum concentration in spite of its relatively
low membrane permeability. Considering that 12b, 12h,
12l, and 12o had about the same logD values ranging
from 1.61 to 1.76, the lipophilicity of substituent R1
(OMe, p = ꢁ0.02; Me, p = 0.56; Cl, p = 0.71; Br,
p = 0.86) and/or the inductive effect, rather than the
4. Conclusion
We have discovered that the introduction of halogen
(Cl and Br) into the 3-position on the central ben-
zene ring significantly improves the pharmacokinetic
Table 3. Plasma concentration and distribution coefficient of VLA-4 antagonists
F
N
O
O
CO2H
O
N
H
N
H
R2
R1
Compound
R2
R1
logD (pH 7.4)
MDCK Papp (·10ꢁ6 cm/s)
Serum concentration (ng/ml)
2
Me
Cl
MeO
MeO
MeO
MeO
Me
1.31
1.87
1.76
1.31
1.09
1.69
1.64
0.70
1.70
2.65
0.44
0.25
3.00
3.00
22b
12a
12b
12c
12f
12g
12h
142b
Br
261a/239b
100a
MeO
Me
Cl
368a
Me
Me
472a
Br
693a
12l
Me
Cl
Cl
Cl
Cl
1.61
2.27
2.34
2.68
5.50
5.25
3659a
2523a
2162a
12m
12n
Br
12o
12p
12q
Me
Cl
Br
Br
Br
1.68
2.17
2.28
1.92
4.49
3.40
>600a
1881a
3514a
Br
a Administered at 10 mg/kg and serum concentration measured after 15 min.
b Administered at 50 mg/kg and serum concentration measured after 30 min.

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J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
18.00
16.00
Total cell
Eosinophil
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
saline
Control
10 mg/kg
30 mg/kg
12l
Figure 2. Effect of compound 12l on pleural eosinophils accumulation
in rats induced by compound 48/80. Compound 12l was orally
administered to rats twice at ꢁ0.25 and 8 h (10 mg/kg · 2 or 30 mg/
kg · 2, respectively). *p < 0.05: Significant difference versus control
(Dunnett’s test).
properties. Notably, 12l (R1 = Cl, R2 = Me) demon-
strated an acceptable plasma clearance and bioavail-
ability in mice and rats as well as dogs (mice,
CL ¼ 28:5 ml=min=kg;F ¼ 28%;
rats,
CL ¼ 5:3 ml=min=kg; F ¼ 36%; dog, CL ¼ 3:6 ml=
min=kg;F ¼ 55%Þ. We have also found that these
substitutions maintain the potent inhibitory activity
against VLA-4. Compound 12l exhibited single digit
nanomolar IC50 values for VLA-4 even in the pres-
ence of HSA and demonstrated efficacy in a rat
pleurisy model. Further synthetic modification of
the diphenylurea portion will be presented in forth-
coming publications.
5. Experimental
5.1. General
Melting points were determined on a YANACO MP-J3
and are uncorrected. Column chromatography was per-
formed with Merck silica gel 60 (particle size 0.060–0.200
or 0.040–0.063). Flash column chromatography was per-
formed with Biotage Si and YAMAZEN Hi-Flash
packed columns. Thin-layer chromatography (TLC)
was performed on Merck pre-coated TLC glass sheets
1
with silica gel 60 F254. H NMR spectra were recorded
on a JEOL JNM-EX-400 spectrometer, and chemical
shifts are given in ppm (d) from tetramethylsilane as
the internal standard. IR spectra were recorded on a
HITACHI 270-30 spectrometer. ESI mass spectra were
recorded on a SCIEX API-150EX spectrometer; FAB
mass spectra were recorded on a JEOL JMS-HX110
spectrometer. HR-FAB mass spectra were recorded on
a JEOL JMS-700 spectrometer.
5.2. General procedure A: preparation of 4-[N0-(2-chloro-
phenyl)ureido]-3-methoxyphenylacetic acid (10a)
To a stirred solution of tert-butyl 4-amino-3-meth-
oxyphenylacetate (3) (29.9 g, 126 mmol) in THF
(200 ml) were added 2-chlorophenyl isocyanate (19.4 g,

J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1685
126 mmol) and a catalytic amount of Et3N at room tem-
perature. After 12 h of stirring, the mixture was poured
into water (300 ml), and the resulting precipitate was
collected by filtration, followed by drying in vacuo, to
give tert-butyl 4-[N0-(2-chlorophenyl)ureido]-3-meth-
oxyphenylacetate (46.7 g, 94%) as a pale yellow crystal-
line powder.
5.6. 4-[N0-(2-Isopropylphenyl)ureido]-3-methoxyphenyl-
acetic acid (10e)
Yield 74% (two steps). Colorless crystalline material
recrystallized from EtOH–n-hexane. Mp: 144–147 ꢁC;
1H NMR (CDCl3) d 1.17 (6H, d, J = 7.2 Hz), 3.16–
3.23 (1H, m), 3.49 (2H, s), 3.86 (3H, s), 6.74–6.71 (6H,
m), 8.00–8.03 (1H, m), 8.48 (2H, d, J = 4.0 Hz); MS
(ESI) m/z , 343 (M++H).
To a stirred solution of tert-butyl 4-[N0-(2-chlorophe-
nyl)ureido]-3-methoxyphenylacetate (46.7 g, 120 mmol)
in CH2Cl2 (100 ml) was added TFA (100 ml) at room
temperature. After 12 h of stirring, the mixture was con-
centrated and poured into water. The resulting precipi-
tate was collected by filtration. The solid was
recrystallized from CHCl3–MeOH to give the com-
pound 10a (30.5 g, 76%) as a pale yellow crystalline
5.7. 4-[N0-2-(Methylphenyl)ureido]-3-methylphenylacetic
acid (10f)
Yield 92% (two steps). Pale yellow solid. 1H NMR
(DMSO-d6) d 2.24 (3H, s), 2.26 (3H, s), 3.47 (2H, s),
6.84–7.27 (5H, m), 7.70 (1H, d, J = 8.1 Hz), 7.80 (1H,
d, J = 8.1 Hz), 8.15–8.25 (2H, m); MS (ESI), m/z 297
(M++1).
1
powder. Mp: 177–180 ꢁC; H NMR (DMSO-d6) d 3.51
(2H, s), 3.87 (3H, s), 6.79 (1H, dd, J = 8.3, 1.5 Hz),
6.94 (1H, d, J = 1.5 Hz), 7.02 (1H, t, J = 7.3 Hz), 7.28
(1H, t, J = 8.3 Hz), 7.44 (1H, d, J = 7.8 Hz), 7.98 (1H,
d, J = 8.3 Hz), 8.09 (1H, dd, J = 8.3, 1.5 Hz), 8.90 (1H,
s), 8.95 (1H, s), 12.27 (1H, s); MS (FAB), m/z 335
(M++1); Anal. Calcd for C16H15ClN2O4: C, 57.41; H,
4.52; N, 8.37; Cl, 10.59. Found: C, 57.41; H, 4.47; N,
8.28; Cl, 10.69.
5.8. 4-[N0-(2-Chlorophenyl)ureido]-3-methylphenylacetic
acid (10g)
Yield 93% (two steps). Yellow powder. Mp: 243–245 ꢁC
1
(dec); H NMR (CDCl3) d 2.24 (3H, s), 3.47 (2H, s),
6.99–7.08 (3H, m), 7.28 (1H, t, J = 7.6 Hz), 7.44 (1H,
dt, J = 8.0, 2.4 Hz), 7.66 (1H, dd, J = 8.3, 1.9 Hz), 8.13
(1H, dd, J = 6.1, 1.7 Hz), 8.61 (2H, d, J = 6.3 Hz); MS
(ESI), m/z 319 (M++1), 321 (M++3).
Compounds 10b–h were prepared according to general
procedure A.
5.3. 4-[N0-(2-Bromophenyl)ureido]-3-methoxyphenylacetic
acid (10b)
5.9. 4-[N0-(2-Bromophenyl)ureido]-3-methylphenylacetic
acid (10h)
Yield 61% (two steps). Pale yellow powder recrystallized
from MeOH–CHCl3–n-hexane. Mp: 187–191 ꢁC; 1H
NMR (DMSO-d6) d 3.51 (2H, s), 3.87 (3H, s), 6.77–
6.81 (1H, m), 6.93–7.02 (2H, m), 7.30–7.35 (1H, m),
7.59–7.64 (1H, m), 7.94–7.98 (2H, m), 8.75 (1H, s),
8.95 (1H, s), 12.31 (1H, s); MS (FAB), m/z 381
(M++2); Anal. Calcd for C16H15BrN2O4: C, 50.68; H,
3.99; Br, 21.07; N, 7.39. Found: C, 50.59; H, 4.04; Br,
21.20; N, 7.25.
Yield 90% (two steps). Pale yellow powder. Mp: 245–
248 ꢁC (dec); H NMR (CDCl3) d 2.24 (3H, s), 3.48
1
(2H, s), 6.96 (1H, dt, J = 7.3, 1.5 Hz), 7.02 (1H, d,
J = 8.3 Hz), 7.07 (1H, s), 7.32 (1H, t, J = 8.1 Hz),
7.59–7.66 (2H, m), 8.44 (1H, s), 8.62 (1H, s); MS
(ESI), m/z 363 (M++1), 365 (M++3); Anal. Calcd for
C16H15BrN2O3Æ0.75H2O: C, 51.01; H, 4.41; Br, 21.72;
N, 7.44. Found: C, 50.84; H, 4.02; Br, 21.72; N, 7.18.
5.10. General procedure B: preparation of 3-chloro-4-[N0-
(2-methylphenyl)ureido]phenylacetic acid (10l)
5.4. 3-Methoxy-4-[N0-(2-methoxyphenyl)ureido]phenyl-
acetic acid (10c)
To a mixture of methyl 4-amino-3-chlorophenylacetate (8)
(1.00 g, 5.01 mmol) and 2-methylphenyl isocyanate
(0.60 ml, 5.01 mmol) in THF (20 ml) was added Et3N
(0.14 ml, 1.00 mmol) at room temperature. After 1 day of
stirring, 2-methylphenyl isocyanate (0.60 ml, 5.01 mmol)
was re-added to the reaction mixture and stirred for 17 h.
The reaction mixture was concentrated in vacuo. The resi-
due was triturated by the addition of n-hexane to give meth-
yl 3-chloro-4-[N0-(2-methylphenyl)ureido]phenylacetate
(1.23 g, 74%) as a colorless powder.
Yield 74% (two steps). Colorless powder (1.40 g, 82%).
1
Mp: 182–185 ꢁC; H NMR (CD3OD) d 3.55 (2H, s),
3.88 (3H, s), 3.89 (3H, s), 6.80–6.99 (5H, m), 7.94 (1H,
d, J = 8.4 Hz), 8.00 (1H, d, J = 7.2 Hz); MS (ESI) m/z ,
330 (M+).
5.5. 3-Methoxy-4-[N0-(2-trifluoromethylphenyl)ureido]phenyl-
acetic acid (10d)
Yield 49% (two steps). Colorless crystalline powder
recrystallized from MeOH–CHCl3–IPE. Mp: 218–
220 ꢁC; H NMR (DMSO-d6) d 3.51 (2H, s), 3.87 (3H,
s), 6.76–6.79 (1H, m), 6.93–6.94 (1H, m), 7.27–7.30
(1H, m), 7.61–7.69 (2H, m), 7.82–7.84 (1H, m), 7.97–
7.99 (1H, m), 8.71 (1H, s), 8.89 (1H, s), 12.30 (1H, s);
Anal. Calcd for C17H15F3N2 O4: C, 55.44; H, 4.11; N,
7.61; F, 15.47. Found: C, 55.30; H, 4.08; N, 7.63; F,
15.13.
To a stirred solution of methyl 3-chloro-4-[N0-(2-meth-
ylphenyl)ureido]phenylacetate (1.23 g, 3.70 mmol) in
THF (30 ml) was added 0.25 M NaOH (30 ml). After
stirring at room temperature for 14 h, the reaction mix-
ture was concentrated in vacuo. The residue was tritu-
rated by the addition of 1 M HCl. The resulting
precipitate was collected and dried under a reduced pres-
sure to give the compound 10l (1.22 g, 100%) as a color-
1

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J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1
less powder. H NMR (DMSO-d6) d 2.26 (3H, s), 3.40
(2H, s), 6.95 (1H, t, J = 7.3 Hz), 7.11 (2H, d,
J = 7.6 Hz), 7.16 (1H, d, J = 7.3 Hz), 7.32 (1H, s), 7.76
(1H, d, J = 8.0 Hz), 7.94 (1H, dd, J = 9.3, 1.0 Hz), 8.72
(2H, s); MS (ESI), m/z 319 (M++1), 321 (M++3).
5.17. 3-Bromo-4-[N0-(2-chlorophenyl)ureido]phenylacetic
acid (10p)
Yield 70% (two steps). Colorless powder. 1H NMR
(DMSO-d6) d 3.56 (2H, s), 7.03–7.07 (1H, m), 7.21
(1H, dd, J = 8.6, 1.7 Hz), 7.29 (1H, t, J = 7.8 Hz), 7.46
(1H, d, J = 8.1 Hz), 7.53 (1H, d, J = 1.7 Hz), 7.83 (1H,
d, J = 8.3 Hz), 8.06 (1H, d, J = 7.6 Hz), 8.86 (1H, s),
8.89 (1H, s), 12.40 (1H, s); MS (ESI), m/z 382 (M++1),
384 (M++3).
Compounds 10i–q were prepared according to general
procedure B.
5.11. 3-Ethyl-4-[N0-(2-methylphenyl)ureido]phenylacetic
acid (10i)
5.18. 3-Bromo-4-[N0-(2-bromophenyl)ureido]phenylacetic
acid (10q)
1
Yield 67% (two steps). Colorless crystalline solid. H
NMR (DMSO-d6) d 1.17 (3H, t, J = 7.6 Hz), 2.26 (3H,
s), 2.61 (2H, q, J = 7.6 Hz), 3.49 (2H, s), 6.91–7.17
(6H, m), 7.63 (1H, d, J = 8.3 Hz), 7.78 (1H, d,
J = 8.0 Hz), 8.24 (1H, d, J = 5.4 Hz).
Yield 69% (two steps). Colorless powder. 1H NMR
(DMSO-d6) d 3.56 (2H, s), 6.99 (1H, dt, J = 7.8,
1.5 Hz), 7.21 (1H, dd, J = 8.3, 1.7 Hz), 7.33 (1H, dt,
J = 7.1, 1.5 Hz), 7.53 (1H, d, J = 1.7 Hz), 7.62 (1H, dd,
J = 8.1, 1.5 Hz), 7.82 (1H, d, J = 8.3 Hz), 7.93 (1H, dd,
J = 8.1, 1.5 Hz), 8.82 (1H, s), 8.86 (1H, s), 12.39 (1H,
s); MS (ESI), m/z 428 (M++1), 430 (M++3).
5.12. 3-Isopropyl-4-[N0-(2-methylphenyl)ureido]phenyl-
acetic acid (10j)
1
Yield 98% (two steps). Colorless crystalline solid. H
NMR (DMSO-d6) d 1.19 (6H, d, J = 6.8 Hz), 2.26
(3H, s), 3.18–3.22 (1H, m), 3.52 (2H, s), 6.90–7.17
(6H, m), 7.48–7.52 (1H, m), 7.79–7.83 (1H, m), 8.25
and 8.36 (total 1H, each s, amide isomers).
5.19. General procedure C: preparation of 4-[1-[3-chloro-4-
[N0-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]benzoic acid (12l)
A
mixture of 3-chloro-4-[N0-(2-methylphenyl)ure-
5.13. 4-[N0-(2-Methylphenyl)ureido]-3-trifluoromethylphenyl-
acetic acid (10k)
ido]phenylacetic acid (10l) (319 mg, 1.00 mmol), methyl
4-[(4S)-fluoro-(2S)-pyrrolidinyl]methoxybenzoate (11)
(253 mg, 1.00 mmol), EDCÆHCl (288 mg, 1.50 mmol),
HOBT (203 mg, 1.50 mmol), and Et3N (0.70 ml,
5.00 mmol) in DMF (4 ml) was stirred at room tempera-
ture for 15 h. The mixture was poured into ice water and
extracted with EtOAc. The combined extracts were
washed with ice water and brine. After drying over
Na2SO4, the extracts were concentrated in vacuo.
The residue was purified on TLC [CHCl3–acetone
(5/1, v/v)] to give methyl 4-[1-[3-chloro-4-[N0-(2-methyl-
phenyl) ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidi-
nyl]methoxybenzoate (480 mg, 87%) as a colorless
Yield 40% (two steps). Colorless solid recrystallized with
CHCl3–n-hexane. 1H NMR (CDCl3) d 2.30 (3H, s), 3.66
(2H, s), 7.00–7.04 (1H, m), 7.16–7.20 (2H, m), 7.53–7.57
(3H, m), 7.78–7.82 (1H, m); MS (ESI) m/z 353 (M++1).
5.14. 3-Chloro-4-[N0-(2-chlorophenyl)ureido]phenylacetic
acid (10m)
Yield 65% (two steps). Colorless powder. 1H NMR
(DMSO-d6) d 3.52 (2H, s), 7.03–7.07 (1H, m), 7.17
(1H, d, J = 8.5 Hz), 7.30 (1H, d, J = 7.6 Hz), 7.37 (1H,
s), 7.46 (1H, dd, J = 8.0, 1.5 Hz), 7.95 (1H, dd, J = 8.3,
1.2 Hz), 8.07 (1H, d, J = 8.3 Hz), 9.00 (2H, d,
J = 8.0 Hz); MS (FAB), m/z 339 (M++1), 341 (M++3),
343 (M++5).
1
amorphous solid. H NMR (CDCl3) d 2.10–2.60 (2H,
m), 2.29 (3H, s), 3.56 (1H, d, J = 6.8 Hz), 3.71–3.84
(1H, m), 3.87 and 3.89 (total 3H, each s, amide isomers),
3.91–4.20 (3H, m), 4.49–4.60 (2H, m), 5.32 (1H, dt,
J = 53.0, 4.2 Hz), 6.80 (1H, br), 6.89 and 6.95 (total 2H,
each d, each J = 8.8 Hz, amide isomers), 7.09–7.26 (6H,
m), 7.50 (1H, d, J = 7.3 Hz), 7.94 and 8.00 (total 2H, each
d, each J = 8.8 Hz, amide isomers), 8.10 and 8.15 (total
1H, each d, J = 8.1 and 8.3 Hz, respectively, amide
isomers); FAB-MS, m/z 554 (M++1), 556 (M++3).
5.15. 4-[N0-(2-Bromophenyl)ureido]-3-chlorophenylacetic
acid (10n)
Yield 54% (two steps). Colorless powder. 1H NMR
(DMSO-d6) d 3.56 (2H, s), 6.98–7.02 (1H, m), 7.17
(1H, dd, J = 9.0, 1.7 Hz), 7.32–7.40 (2H, m), 7.62 (1H,
dd, J = 8.0, 1.2 Hz), 7.95 (2H, m), 8.83 (1H, s), 9.01
(1H, s), 12.41 (1H, br); MS (FAB), m/z 385 (M++2),
386 (M++4), 388 (M++6).
To a solution of methyl 4-[1-[3-chloro-4-[N0-(2-methyl-
phenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolid-
inyl]methoxybenzoate (480 mg, 0.866 mmol) in THF
(30 ml) was added 0.25 N NaOH (30 ml). After 2 days
of stirring at room temperature, the mixture was concen-
trated under a reduced pressure and acidified with 1 N
HCl. The precipitates were collected, washed with water,
and dried under a reduced pressure to give 12 l (374 mg,
80%) as a colorless solid. IR (KBr) 3354, 3060, 2976,
5.16. 3-Bromo-4-[N0-(2-methylphenyl)ureido]phenylacetic
acid (10o)
Yield 72% (two steps). Colorless powder. 1H NMR
(DMSO-d6) d 2.26 (3H, s), 3.32 (2H, s), 6.91–6.95 (2H,
m), 7.10–7.17 (4H, m), 7.76 (2H, d, J = 8.1 Hz), 8.52
(1H, s); MS (ESI), m/z 385 (M++Na), 387 (M++2+Na).
1709, 1604, 1244 cmꢁ1 1H NMR (DMSO-d6) d 2.27
;
(3H, s), 2.31 (2H, s), 3.66 (2H, d, J = 7.2 Hz), 3.71–4.67
(5H, m), 5.32–5.53 (1H, m), 6.97 (1H, t, J = 7.3 Hz),

J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1687
1
7.04–7.22 (5H, m), 7.32 and 7.35 (total 1H, each d, each
J = 1.7 Hz, amide isomers), 7.77 (1H, d, J = 7.6 Hz),
7.87 and 7.90 (total 2H, each d, J = 8.8 and 9.0 Hz,
respectively, amide isomers), 8.01 and 8.03 (total 1H, each
d, J = 7.5 and 8.5 Hz, respectively, amide isomers), 8.57
and 8.59 (total 1H, each s, amide isomers), 8.63 and
8.65 (total 1H, each s, amide isomers), 12.63 (1H, s);
MS (ESI), m/z 540 (M++1), 542 (M++3). Anal. Calcd
for C28H27ClFN3O5Æ0.5H2O: C, 61.26; H, 5.14; N, 7.65;
Cl, 6.46; F, 3.46. Found: C, 61.16; H, 5.27; N, 7.25; Cl,
6.47; F, 3.39.
2893, 1709, 1685, 1628, 1604 cmꢁ1; H NMR (DMSO-
d6) d 2.23–2.29 (2H, m), 3.54–4.38 (8H, m), 4.40–4.61
(2H, m), 5.30–5.49 (1H, m), 6.72–6.91 (2H, m), 7.02–
7.08 (2H, m), 7.25–7.29 (1H, m), 7.59–7.67 (2H, m),
7.81–7.99 (4H, m), 8.69–8.70 (1H, m), 8.87–8.90 (1H,
m), 12.67 (1H, s); FAB, m/z 590 (M++1); HR-MS
(FAB) calcd for C29H27F4N3O6: 590.1914. Found:
590.1898.
5.24. 4-[(4S)-Fluoro-1-[4-[N0-(2-isopropylphenyl)ureido]-3-
methoxyphenylacetyl]-(2S)-pyrrolidinylmethoxy]benzoic
acid (12e)
Compounds 12a–q were prepared according to general
procedure C.
Yield 89% (two steps). Colorless amorphous solid.
IR (ATR) 3338, 3060. 2964. 2870, 1685, 1657,
5.20. 4-[1-[4-[N0-(2-Chlorophenyl)ureido]-3-methoxyphe-
nylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic
acid (12a)
1603, 1530, 1448, 1417 cmꢁ1 1H NMR (CD3OD)
;
d
1.23 (6H, d, J = 6.8 Hz), 2.15–2.49 (2H, m),
3.19–3.25 (1H, m), 3.62–4.76 (7H, m), 3.87 and
3.93 (total 3H, each s, amide isomers), 5.23–5.40
(1H, m), 6.77–8.01 (11H, m); MS (ESI) m/z , 564
(M++H); C31H34FN3O6Æ1.25H2O: C, 63.52; H, 6.28;
N, 7.17; F, 3.24. Found: C, 63.46; H, 6.02; N,
7.11; F, 3.06.
Yield 24% (two steps), as a pale yellow amorphous solid.
IR (KBr) 3338, 3059, 2960, 1705, 1685, 1628, 1533, 1466,
1
1421 cmꢁ1; H NMR (DMSO-d6) d: 2.24–2.50 (2H, m),
3.48–4.65 (10H, m), 5.30–5.50 (1H, m), 6.75–7.08 (5H,
m), 7.29 (1H, t, J = 7.3 Hz), 7.43–7.45 (1H, m), 7.89–
7.98 (2H, m), 7.99 (1H, d, J = 8.3 Hz), 8.09 (1H, d,
J = 7.1 Hz), 8.90–8.96 (2H, m); MS (FAB) m/z , 556
(M++1); Anal. Calcd for C28H27ClFN3O6Æ0.25H2O: C,
60.00; H, 4.95; N, 7.50. Found: C, 59.67; H, 5.08; N, 7.10.
5.25. 4-[(4S)-Fluoro-1-[3-methyl-4-[N0-(2-methylphenyl)ure-
ido]phenylacetyl]-(2S)-pyrrolidinylmethoxy]benzoic acid
(12f)
Yield 65% (two steps). Colorless amorphous solid. IR
(KBr) 3356, 2974, 1604, 1537, 1454, 1252 cmꢁ1 1H
;
5.21. 4-[1-[4-[N0-(2-Bromophenyl)ureido]-3-methoxyphe-
nylacetyl]-(4S)-fluoro-(2S)- pyrrolidinylmethoxy]benzoic
acid (12b)
NMR (DMSO-d6) d 2.24 (3H, s), 2.26 (3H, s), 3.60
(2H, d, J = 3.7 Hz), 3.65–4.65 (8H, m), 5.31–5.50
(1H, m), 6.92–7.18 (7H, m), 7.67–7.92 (4H, m),
8.22–8.32 (2H, m); MS (FAB), m/z 520 (M++1); Anal.
Calcd for C29H30FN3 O5ÆH2O: C, 64.79; H, 6.00; F,
3.53; N, 7.82. Found: C, 64.71; H, 5.90; F, 3.24; N,
7.51.
Yield 67% (two steps), as a colorless amorphous solid.
IR (KBr) 3332, 2956, 1705, 1687, 1604, 1581, 1529,
1
1464, 1435, 1298 cmꢁ1; H NMR (DMSO-d6) d: 2.24–
2.31 (2H, m), 3.21–4.63 (10H, m), 5.31–5.51 (1H, m),
6.74–7.10 (5H, m), 7.32 (1H, t, J = 7.8 Hz), 7.60 (2H,
d, J = 7.8 Hz), 7.87–7.99 (4H, m), 8.74–8.75 (1H, m),
8.92–8.94 (1H, m); MS (FAB) m/z , 601 (M++1); Anal.
Calcd for C28H27BrFN3O6Æ2H2O: C, 52.84; H, 4.91; N,
6.60. Found: C, 52.38; H, 4.62; N, 5.99.
5.26. 4-[1-[4-[N0-(2-Chlorophenyl)ureido]-3-methylphenyl-
acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid
(12g)
Yield 42% (two steps). Colorless amorphous solid. IR
5.22. 4-[(4S)-Fluoro-1-[4-[N0-(2-methoxyphenyl)ureido]-
3-methoxyphenylacetyl]-(2S)- pyrrolidinylmethoxy]ben-
zoic acid (12c)
(KBr) 3346, 2976, 1709, 1685, 1604, 1533, 1439 cmꢁ1
;
1H NMR (DMSO-d6) d 2.20 and 2.24 (total 3H, each
s, amide isomers), 2.28–2.32 (1H, m), 3.60 (2H, s),
3.71–4.62 (6H, m), 5.30–5.50 (1H, m), 7.01–7.09 (5H,
m), 7.28 (1H, t, J = 7.8 Hz), 7.44 (1H, d, J = 8.1 Hz),
7.66 (1H, t, J = 8.1 Hz), 7.87 (2H, d, J = 7.1 Hz), 8.13
(1H, d, J = 7.9 Hz), 8.62 (2H, d, J = 6.1 Hz); MS
(FAB), m/z 540 (M++1), 542 (M++3); Anal. Calcd for
C28H27ClFN3O7Æ0.5H2O: C, 61.26; H, 5.14; N, 7.65.
Found: C, 61.22; H, 5.30; N, 7.33.
Yield 59% (two steps). Colorless amorphous solid. IR
(ATR) 3336, 2937, 2839, 1682, 1628, 1599, 1525, 1458,
1417, 1333, 1250, 1232 cmꢁ1 1H NMR (CD3OD) d
;
2.14–2.48 (2H, m), 3.69–4.20 (5H, m), 3.88 (3H, s),
3.89 (3H, s), 4.46–4.57 (2H, m), 5.27–5.41 (1H, m),
6.79–7.04 (7H, m), 7.90–8.02 (4H, m); MS (ESI) m/z ,
552 (M++H); Anal. Calcd for C29H30FN3O7Æ1.5H2O:
C, 60.20; H, 5.75; N, 7.26; F, 3.28. Found: C, 60.03;
H, 5.40; N, 7.27; F, 3.14.
5.27. 4-[1-[4-[N0-(2-Bromophenyl)ureido]-3-methylphenylace-
tyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid (12h)
5.23. 4-[(4S)-Fluoro-1-[3-methoxy-4-[N0-(2-trifluoromethyl-
phenyl)ureido]phenylacetyl]-(2S)-pyrrolidinylmethoxy]ben-
zoic acid (12d)
Yield 49% (two steps). Colorless amorphous solid. IR
(KBr) 3325, 2972, 1709, 1604, 1529, 1252 cmꢁ1 1H
;
NMR (DMSO-d6) d 2.25 (3H, s), 2.29–2.33 (1H, m),
3.17 (1H, s), 3.60 (2H, d, J = 4.7 Hz), 3.83–4.67 (5H,
m), 5.31–5.51 (1H, m), 6.97 (1H, t, J = 7.3 Hz), 7.02–
7.09 (5H, m), 7.33 (1H, t, J = 8.0 Hz), 7.61 (1H, d,
Yield 42% (two steps). Colorless crystalline powder.
Mp: 129–132 ꢁC; IR (KBr) 3354, 3072, 2972, 2939,

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J = 7.8 Hz), 7.64 (1H, d, J = 8.3 Hz), 7.87 (2H, d,
J = 8.3 Hz), 7.90 (1H, d, J = 8.8 Hz), 8.44–8.65 (2H,
m); MS (ESI), m/z 585 (M++1), 587 (M++3); Anal.
Calcd for C28H27BrFN3O5Æ0.5H2O: C, 56.67; H, 4.76;
Br, 13.46; F, 3.20; N, 7.08. Found: C, 56.91; H, 4.93;
Br, 13.23; F, 3.15; N, 6.88.
57.41; H, 4.37; N, 7.44; Cl, 12.55; F, 3.36. Found: C,
57.72; H, 4.47; N, 7.14; Cl, 12.44; F, 3.44.
5.32. 4-[1-[4-[N0-(2-Bromophenyl)ureido]-3-chloropheny-
lacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid
(12n)
5.28. 4-[1-[3-Ethyl-4-[N0-(2-methylphenyl)ureido]phenylace-
tyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid (12i)
Yield 9% (two steps). Colorless amorphous solid. IR
(KBr) 3329, 3060, 2976, 1712, 1526, 1435 cmꢁ1 1H
;
NMR (DMSO-d6) d 2.28–2.33 (2H, m), 3.48–4.68 (7H,
m), 5.32–5.53 (1H, m), 6.99–7.19 (4H, m), 7.36 (1H, s),
7.63 (1H, dd, J = 6.7, 1.2 Hz), 7.86–8.18 (4H, m), 8.83
(1H, s), 9.02 (1H, s), 12.67 (1H, br); MS (ESI), m/z
604 (M++1), 606 (M++3), 608 (M++5); Anal. Calcd
for C27H24BrClFN3O5Æ0.5H2O: C, 52.83; H, 4.10; N,
6.85; Cl, 5.78; F, 3.09. Found: C, 53.243; H, 4.32; N,
6.43; Cl, 6.01; F, 3.07.
Yield 70% (two steps). Colorless solid. IR (KBr) 3353,
1
2967, 1604, 1536, 1249, 1166 cmꢁ1; H NMR (DMSO-
d6) d 1.10–1.19 (3H, m), 2.15–2.32 (5H, m), 2.55–2.65
(2H, m), 3.55–4.20 (5H, m), 4.32–4.65 (2H, m), 5.28–
5.52 (1H, m), 6.90–7.20 (7H, m), 7.60–7.95 (4H, m),
8.17–8.20 (2H, m); MS (FAB) m/z 533 (M+H)+; Anal.
calcd for C30H32N3O5FÆ0.5 H2O: C, 66.41; H, 6.13; N,
7.74. Found: C, 66.14; H, 6.11; N, 7.63.
5.33. 4-[1-[3-Bromo-4-[N0-(2-methylphenyl)ureido]phenylace-
tyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid (12o)
5.29. 4-[1-[3-Isopropyl-4-[N0-(2-methylphenyl)ureido]pheny-
lacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid
(12j)
Yield 85% (two steps). Colorless solid. IR (KBr) 3313,
3060, 2976, 1687, 1604, 1525, 1244 cmꢁ1 1H NMR
;
Yield 91% (two steps). Colorless solid. IR (KBr) 3372,
(DMSO-d6) d 2.27 (3H, s), 2.29 (2H, m), 3.66 (2H, d,
J = 8.1 Hz), 3.72–4.68 (5H, m), 5.31–5.53 (1H, m),
6.92–6.99 (1H, m), 7.04 and 7.07 (total 2H, each d,
J = 9.3 and 8.3 Hz, respectively, amide isomers), 7.11–
7.21 (3H, m), 7.48 and 7.51 (total 1H, each s, amide iso-
mers), 7.75 and 7.79 (total 1H, each d, each J = 8.1 Hz,
amide isomers), 7.86–7.92 (3H, m), 8.45 and 8.47 (total
1H, each s, amide isomers), 8.59 (1H, s), 12.64 (1H, s);
MS (FAB), m/z 584 (M++1), 586 (M++3); Anal. Calcd
for C28H27BrFN3O5: C, 57.54; H, 4.66; N, 7.19; Br,
13.67; F, 3.25. Found: C, 57.93; H, 4.97; N, 7.04; Br,
13.35; F, 2.89.
2964, 1604, 1533, 1249, 1166, 1112 cmꢁ1 1H NMR
;
(DMSO-d6) d 1.08–1.21 (6H, m), 2.18–2.40 (5H, m),
3.10–3.25 (1H, m), 3.45–4.21 (5H, m), 4.35–4.68 (2H,
m), 5.30–5.55 (1H, m), 6.90–7.17 (7H, m), 7.47–7.52
(1H, m), 7.80–7.90 (3H, m), 8.12–8.23 (2H, m); MS
(FAB) m/z 547 (M+H)+; Anal. calcd for C31H34N3O5-
FÆ0.5H2O: C, 66.89; H, 6.34; N, 7.55. Found: C, 66.57;
H, 6.30; N, 7.39.
5.30. 4-[1-[4-[N0-(2-Methylphenyl)ureido]-3-trifluoromethyl-
phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic
acid (12k)
5.34. 4-[1-[3-Bromo-4-[N0-(2-chlorophenyl)ureido]phenylace-
tyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid (12p)
Yield 65% (two steps). Colorless solid. Mp: 155–160 ꢁC;
IR (KBr) 3354, 3300, 3060, 2958, 2875, 1709, 1687,
1641, 1604, 1541 cmꢁ1
;
1H NMR (CD3OD) d 2.10–
Yield 86% (two steps). Pale yellow solid. IR (KBr) 3317,
2.50 (5H, m), 3.60–4.30 (5H, m), 4.45–4.90 (2H, m),
5.27–5.40 (1H, m), 6.95–7.05 (3H, m), 7.12–7.21 (2H,
m), 7.42–7.64 (3H, m), 7.80–7.99 (3H, m); MS (ESI)
m/z , 574 (M++1); Anal. Calcd for C29H27F4N3O5Æ0.25-
H2O: C, 60.26; H, 4.80; N, 7.27. Found: C, 60.20; H,
5.12; N, 7.07.
3072, 1709, 1685, 1604, 1529, 1290 cmꢁ1 1H NMR
;
(DMSO-d6)
d 2.24–2.50 (2H, m), 3.67 (2H, d,
J = 8.3 Hz), 3.73–4.68 (5H, m), 5.31–5.52 (1H, m),
7.03–7.09 (3H, m), 7.22 (1H, dt, J = 8.3, 1.7 Hz), 7.30
(1H, d, J = 7.3 Hz), 7.46 (1H, dd, J = 8.0, 1.4 Hz), 7.49
and 7.52 (total 1H, each d, J = 1.7 and 2.0 Hz, respec-
tively, amide isomers), 7.80–7.91 (3H, m), 8.07 (1H,
dd, J = 8.3, 1.2 Hz), 8.85 and 8.86 (total 1H, each s,
amide isomers), 8.96 and 8.97 (total 1H, each s, amide
isomers), 12.62 (1H, s); MS (FAB), m/z 605 (M++1),
607 (M++3), 609 (M++3), 626 (M++1+Na); Anal. Calcd
for C27H24BrClFN3O5Æ0.75H2O: C, 52.44; H, 4.16; N,
6.80; F, 3.07. Found: C, 52.63; H, 4.12; N, 6.62; F, 2.97.
5.31. 4-[1-[3-Chloro-4-[N0-(2-chlorophenyl)ureido]pheny-
lacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid
(12m)
Yield 80% (two steps). Colorless solid. IR (KBr) 3348,
3072, 2954, 1703, 1604,1529, 1439 cmꢁ1 1H NMR
;
(DMSO-d6)
d 2.25–2.42 (2H, m), 3.67 (2H, d,
J = 8.3 Hz), 3.81–4.68 (5H, m), 5.39 and 5.46 (total 1H,
each d, J = 54.0 and 54.4 Hz, respectively, amide iso-
mers), 7.04–7.10 (3H, m), 7.18 (1H, d, J = 8.3 Hz), 7.31
(1H, t, J = 8.3 Hz), 7.33 and 7.37 (total 1H, each s, amide
isomers), 7.47 (1H, d, J = 8.1 Hz), 7.88 (2H, dd, J = 9.0,
3.2 Hz), 7.98 (1H, dd, J = 8.5, 3.0 Hz), 8.09 (1H, d,
J = 8.3 Hz), 8.99 (1H, d, J = 2.9 Hz), 9.02 (1H, s), 12.64
(1H, s); MS (ESI), m/z 560 (M++1), 562 (M++3), 564
(M++5); Anal. Calcd for C27H24Cl2FN3O5Æ0.25H2O: C,
5.35. 4-[1-[3-Bromo-4-[N0-(2-bromophenyl)ureido]phenylace-
tyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid (12q)
Yield 74% (two steps). Colorless amorphous solid. IR
(KBr) 3450, 3313, 3070, 1709, 1684, 1525, 1435 cmꢁ1
;
1H NMR (DMSO-d6) d 2.25–2.50 (2H, m), 3.67 (2H,
d, J = 8.3 Hz), 3.73–4.68 (5H, m), 5.31–5.53 (1H, m),
6.98–7.08 (3H, m), 7.21 (1H, d, J = 8.2 Hz), 7.34 (1H,
t, J = 8.8 Hz), 7.50 and 7.53 (total 1H, each s, amide iso-

J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1689
mers), 7.62 (1H, d, J = 8.0 Hz), 7.80–7.96 (4H, m), 8.82
(1H, s), 8.85 and 8.86 (total 1H, each s, amide isomers),
12.63 (1H, s); MS (FAB), m/z 650 (M++1), 652 (M++3),
654 (M++3), 672 (M++Na); Anal. Calcd for
C27H24Br2FN3O5ÆH2O: C, 48.60; H, 3.93; N, 6.30; F,
2.85. Found: C, 48.96; H, 3.98; N, 5.92; F, 2.77.
5.37.2. tert-Butyl 4-amino-3-methylphenylacetate (4).
Yield 82%. Brown solid. 1H NMR (CDCl3) d 1.43
(9H, s), 2.15 (3H, s), 3.39 (2H, s), 3.47–3.64 (2H, m),
6.62 (1H, d, J = 7.8 Hz), 6.89–6.96 (2H, m); MS (ESI),
m/z 222 (M++1).
5.38. General procedure E: preparation of methyl 4-amino-
3-chlorophenylacetate (8)
5.36. General procedure D: preparation of tert-butyl 4-
amino-3-methoxyphenylacetate (3)
5.38.1. Methyl 3-chlorophenylacetate (17a). To a stirred
solution of 3-chlorophenylacetic acid (16a) (21.76 g,
127.6 mmol) in dichloroethane (100 ml) were added
MeOH (15.6 ml, 383 mmol) and H2SO4 (1 ml) at room
temperature. After 20 min of stirring, the mixture was
heated at 80 ꢁC for 2 h. The reaction mixture was poured
into ice water and extracted with CHCl3. The combined
extracts were washed with aq NaHCO3 and brine. After
drying over Na2SO4, the extract was concentrated in vac-
uo to give the compound 17a (25.4 g, 100%) as a colorless
oil. 1H NMR (CDCl3) d 3.60 (2H, s), 3.70 (3H, s), 7.15–
7.26 (4H, m).
5.36.1. tert-Butyl 3-methoxy-4-nitrophenylacetate (15a).
To a stirred suspension of 13a (19.9 g, 0.101 mol) in tol-
uene (150 ml) were added SOCl2 (14 ml, 0.191 mol) and
a catalytic amount of DMF at room temperature. The
mixture was refluxed for 3 h with stirring. After cooling
to room temperature, the reaction mixture was concen-
trated under reduced pressure. The resulting residue was
dissolved in THF (250 ml), and then Et3N (23 ml,
0.165 mol) and 2.0 M TMSCHN2 in hexane solution
(83 ml, 0.165 mol) were added at 0 ꢁC. After 12 h of stir-
ring, the mixture was poured into sat. NaHCO3 solution
and extracted with EtOAc. The combined extracts were
washed with brine, dried over MgSO4, and evaporated
to give crude 14a as a yellow solid.
5.38.2. Methyl 3-chloro-4-nitrophenylacetate (18a). To a
stirred mixture of methyl 3-chlorophenylacetate (17a)
(25.4 g, 128 mmol) in H2SO4 (44 ml) was added concd
HNO3 (5.5 ml, 138 mmol) at 0 ꢁC. The reaction mixture
was gradually raised to room temperature for 4 h. The
reaction mixture was poured into ice water and extracted
with EtOAc. The combined extracts were washed with aq
NaHCO3 and brine. After drying over Na2SO4, the ex-
tracts were concentrated in vacuo. The residue was puri-
fied by column chromatography with n-hexane–EtOAc
(40/1, v/v) as an eluent to give the compound 18a
To a refluxed solution of Et3N (42 ml) and PhCO2Ag
(6.97 g, 30 mmol) in tert-butanol–toluene (1/1, v/v,
400 ml) was added dropwise a solution of crude 14a in
tert-butanol (300 ml) with stirring. After 1 h of reflux-
ing, the reaction mixture was cooled to room tempera-
ture. Activated carbon powder was added to the
reaction mixture, which was then filtered through Celite.
The filtrate was diluted with EtOAc and washed with
brine. After drying over MgSO4, the resulting solution
was concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel
with n-hexane–EtOAc (20/1, v/v) as an eluent to afford
compound 15a (17.4 g, two steps, 65%) as a brown oil.
1H NMR (CDCl3) d 1.45 (9H, s), 3.58 (2H, s), 3.97
(3H, s), 6.93 (1H, dd, J = 8.3, 2.2 Hz), 7.03 (1H, d,
J = 2.0 Hz), 7.83 (1H, d, J = 8.3 Hz).
1
(11.4 g, 36%) as a yellow oil. H NMR (CDCl3) d 3.69
(2H, s), 3.74 (3H, s), 7.33 (1H, dd, J = 8.3, 1.5 Hz),
7.49 (1H, d, J = 1.5 Hz), 7.87 (1H, d, J = 8.3 Hz).
5.38.3. Methyl 4-amino-3-chlorophenylacetate (8). A mix-
ture of methyl 3-chloro-4-nitrophenylacetate (18a)
(10.9 g, 47.5 mmol), reduced iron powder (8.58 g,
153.6 mmol), NaOAcÆ3H2O (6.05 g, 44.5 mmol), and
HOAc (17.6 ml) in MeOH–H2O (100/400 ml) was heated
at 110 ꢁC for 1 h. After cooling to room temperature, the
reaction mixture was filtered through Celite and the fil-
tered cake was washed with MeOH. The filtrate was
evaporated and extracted with EtOAc. The combined ex-
tracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by col-
umn chromatography with CHCl3–EtOAc (10/1, v/v)
as an eluent to give the compound 8 (4.58 g, 48%) as a
5.36.2. tert-Butyl 4-amino-3-methoxyphenylacetate (3). A
suspension of 15a (17.4 g, 65.1 mmol) and 5% Pd/C
(3.5 g) in EtOH–HOAc (1/1, v/v, 200 ml) was hydroge-
nated for 1 day at room temperature. After the catalyst
was removed by filtration, the filtrate was concentrated.
The residue was purified by column chromatography on
silica gel with n-hexane–EtOAc (4/1, v/v) as an eluent to
1
afford compound 3 (13.8 g, 89%) as a brown oil. H
NMR (CDCl3) d 1.43 (9H, s), 3.41 (2H, s), 3.85 (3H,
1
red oil. H NMR (CDCl3) d 3.49 (1H, s), 3.68 (3H, s),
4.01 (2H, br), 6.70 (1H, d, J = 7.4 Hz), 6.96 (1H, dd,
J = 8.1, 2.0 Hz), 7.17 (1H, d, J = 2.0 Hz).
s), 6.64–6.73 (3H, m); MS (ESI), m/z 238 (M++1).
Compound 4 was prepared according to general proce-
dure D.
Compound 9 was prepared according to general proce-
dure E.
5.37. Preparation of tert-butyl 4-amino-3-methylphenylace-
tate (4)
5.39. Preparation of methyl 4-amino-3-bromophenylace-
tate (9)
5.37.1. tert-Butyl 3-methyl-4-nitrophenylacetate (15b).
Yield 55% (three steps). Yellow oil. H NMR (CDCl3)
d 1.45 (9H, s), 2.61 (3H, s), 3.57 (2H, s), 7.22–7.27
(2H, m), 7.95 (1H, d, J = 8.8 Hz).
1
5.39.1. Methyl 3-bromophenylacetate (17b). Yield 99%.
Colorless oil. H NMR (CDCl3) d 3.60 (2H, s), 3.71
(3H, d, J = 1.0 Hz), 7.18–7.44 (4H, m).
1

1690
J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
5.39.2. Methyl 3-bromo-4-nitrophenylacetate (18b). Yield
(4.18 g, 20.0 mmol) in THF (100 ml) was added 2.0 M
isopropylmagnesium chloride in diethyl ether solution
(12 ml, 24 ml) at ꢁ15 ꢁC and stirred for 1.5 h. DDQ
(5.9 g, 26 mmol) was added to the reaction mixture at
the same temperature. After 4 h of stirring at room tem-
perature, the reaction mixture was poured into water
and extracted with CH2Cl2. The combined extracts were
washed with water, dried over Na2SO4, and concentrat-
ed in vacuo. The residue was purified by column chro-
matography on silica gel with n-hexane–EtOAc (7/1,
v/v) as an eluent to give the compound 23 (1.6 g, 32%)
as a yellow oil. 1H NMR (CDCl3) d 1.24–1.30 (9H,
m), 3.42–3.46 (1H, m), 3.67 (2H, s), 4.15–4.20 (2H, m),
7.23 (1H, d, J = 8.3 Hz), 7.37 (1H, s), 7.68 (1H, d,
J = 8.3 Hz).
1
29%. Yellow oil. H NMR (CDCl3) d 3.68 (2H, s), 3.73
(3H, s), 7.38 (1H, dd, J = 8.3, 1.2 Hz), 7.67 (1H, d,
J = 1.3 Hz), 7.83 (1H, d, J = 8.3 Hz).
5.39.3. Methyl 4-amino-3-bromophenylacetate (9). Yield
69%. Brown oil. 1H NMR (CDCl3) d 3.48 (2H, s),
3.68 (3H, s), 4.05 (2H, br), 6.69 (1H, d, J = 8.3 Hz),
7.00 (1H, dd, J = 8.1, 2.0 Hz), 7.32 (1H, d, J = 2.0 Hz).
5.40. Preparation of methyl 4-amino-3-ethylphenylacetate
(5)
5.40.1. Methyl 3-nitro-4-trimethylsilylethynylphenylace-
tate (19). To a stirred solution of methyl 3-bromo-4-
nitrophenylacetate (18b) (5.0 g, 18.2 mmol), trimethylsilyl-
acetylene (3.9 ml, 27.4 mmol), CuI (693 mg, 3.7 mmol),
and TEA (15 ml, 109 mmol) in THF (40 ml) was added a
solution of Pd(Ph3P)4 (1.0 g, 0.9 mmol) in THF (20 ml) at
room temperature. After stirring at room temperature for
18 h, the reaction mixture was concentrated in vacuo.
The reaction mixture was poured into water and extracted
with EtOAc. The combined extracts were washed with sat.
NaHCO3, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography on sil-
ica gel with n-hexane–EtOAc (5/1, v/v) as an eluent to give
5.41.2. Ethyl 4-amino-3-isopropylphenylacetate (6). A
mixture of ethyl 4-nitro-3-isopropylphenylacetate (23)
(1.6 g, 6.4 mmol) and 5% Pd/C (0.8 g) in EtOH (20 ml)
was hydrogenated at room temperature for 3 h. The cat-
alyst was filtered off, and the filtrate was concentrated in
vacuo. The residue was purified by column chromatog-
raphy on silica gel with n-hexane–EtOAc (3/1, v/v) as
an eluent to give the compound 6 (1.4 g, quant.) as a
pale yellow oil. 1H NMR (CDCl3) d 1.22–1.27 (9H,
m), 2.84–2.89 (1H, m), 3.49 (2H, s), 3.61 (2H, s), 4.09–
4.15 (2H, m), 6.62 (1H, d, J = 8.0 Hz), 6.93 (1H, dd,
J = 1.9, 8.0 Hz), 7.03 (1H, d, J = 1.9 Hz).
1
the compound 19 (4.5 g, 85%) as a dark yellow oil. H
NMR (CDCl3) d 0.25–0.29 (9H, m), 3.68 (2H, s), 3.73
(3H, s), 7.37 (1H, dd, J = 1.9, 8.5 Hz), 7.57 (1H, d,
J = 1.9 Hz), 7.99 (1H, d, J = 8.5 Hz).
5.42. Preparation of ethyl 4-amino-3-trifluoromethylphe-
nylacetate (7)
5.40.2. Methyl 3-ethynyl-4-nitrophenylacetate (20). To a
3-nitro-4-trim-
stirred
solution
of
methyl
5.42.1. Ethyl 4-nitro-3-trifluoromethylphenylacetate (26).
To a stirred solution of butyl ethyl malonate (4.53 ml,
23.9 mmol) in DMF (200 ml) was added NaH (60% in
oil, 2.87 g, 71.7 mmol) at room temperature. After
20 min, 4-fluoro-3-trifluoromethylnitrobenzene (24)
(5 g, 23.9 mmol) in DMF (50 ml) was added dropwise
via a dropping funnel. After 3 h of stirring, the reaction
mixture was poured into ice water and extracted with
EtOAc. The combined extracts were washed with brine,
dried over MgSO4, filtered, and concentrated to give
tert-butyl 2-ethoxycarbonyl-4-nitro-3-trifluoromethyl-
phenylacetate (25).
ethylsilylethynylphenylacetate (19) (4.5 g, 15.5 mmol) in
THF (30 ml) was added 1.0 M TBAF in THF solution
(18.5 ml, 18.5 mmol) at 0 ꢁC. The reaction mixture was
stirred at room temperature for 2 h. The reaction mixture
was diluted with water and extracted with EtOAc. The
combined extracts were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography with n-hexane–
EtOAc (1/2, v/v) as an eluent to give the compound 20
1
(1.5 g, 44%) as a colorless oil. H NMR (CDCl3) d 3.52
(1H, s), 3.70 (2H, s), 3.73 (3H, s), 7.42 (1H, dd, J = 1.9,
8.3 Hz), 7.61 (1H, d, J = 1.9 Hz), 8.03 (1H, d, J = 8.3 Hz).
To a solution of tert-butyl 2-ethoxycarbonyl-4-nitro-3-
trifluoromethylphenylacetate (25) in CH2Cl2 (20 ml)
was added TFA (20 ml) at room temperature. The reac-
tion mixture was refluxed for 18 h. The reaction mixture
was evaporated in vacuo and then co-evaporated with
toluene. The residue was purified by middle pressure
chromatography system (YAMAZEN YFLC-5404-FC,
B 50 mm · 300 mm, 15 ml/min) with linear gradient n-
hexane–EtOAc (10/0 to 1/1, v/v) as an eluent to afford
the compound 26 (6.6 g, 94%) as a yellow oil. 1H
NMR (CD3OD) d 1.30 (3H, t, J = 7.3 Hz), 3.79 (2H,
s), 4.22 (2H, q, J = 7.3 Hz), 7.68 (1H, dd, J = 8.3,
1.6 Hz), 7.76 (1H, s), 7.87 (1H, d, J = 8.3 Hz); MS
(ESI) m/z , 277 (M+).
5.40.3. Methyl 4-amino-3-ethylphenylacetate (5). A sus-
pension of methyl 3-ethynyl-4-nitrophenylacetate (20)
(1.5 g, 6.8 mmol) and 5% Pd/C (1.5 g) in EtOH
(15.0 ml) was hydrogenated at room temperature for
18 h. The catalyst was filtered off, and the filtrate was
concentrated in vacuo. The residue was purified by col-
umn chromatography with n-hexane–EtOAc (3/1, v/v)
as an eluent to give the compound 5 (1.0 g, 77%) as a pale
1
yellow oil. H NMR (CDCl3) d 1.24 (3H, t, J = 7.6 Hz),
2.50 (2H, t, J = 7.6 Hz), 3.51 (2H, s), 3.59 (2H, s), 3.67
(3H, s), 6.63 (1H, d, J = 7.8 Hz), 6.93–6.97 (2H, m).
5.41. Preparation of ethyl 4-amino-3-isopropylphenylac-
etate (6)
5.42.2. Ethyl 4-amino-3-trifluoromethylphenylacetate (7).
To a stirred solution of ethyl 3-trifluoromethyl-4-
nitrophenylacetate (26) (6.6 g, 23.8 mmol) in EtOH
5.41.1. Ethyl 3-isopropyl-4-nitrophenylacetate (23). To a
stirred solution of ethyl 4-nitrophenylacetate (21)

J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
1691
(125 ml) was added SnCl2 (16 g, 71.4 mmol) at room
5.44. Bio-assay
temperature. Stirring was continued for 18 h at reflux.
The reaction mixture was evaporated in vacuo. The res-
idue was dissolved in CHCl3 and 4 M NaOH at 0 ꢁC,
and then extracted with CHCl3. The combined extracts
were washed with sat. NaHCO3 and brine, and then
dried over MgSO4. After removal of the solvent, the res-
idue was purified by middle pressure chromatography
Female BALB/c mice (18–23 g, 8 weeks of age, Charles
River Japan, Inc.) were used. Each group consisted of 4
animals. Mice were orally administered the compound
dissolved in 0.5% (w/v) carboxymethylcellulose (CMC)
at a dose of 10 mg/1 ml/kg (or 50 mg/1 ml/kg). After
15 min (or 30 min), blood samples were collected via
inferior vena cava from the animals under ether anesthe-
sia. Blood samples were stored at room temperature and
centrifuged at 2000 rpm for 10 min at 4 ꢁC. Serums were
subsequently stored in a ꢁ20 ꢁC freezer prior to analy-
sis. According to the VLA-4/VCAM-1 binding assay, in-
stead of the compound solution, 50 ll of plasma samples
at various concentrations was distributed into each well
(final concentration: 0.01–10%). As for the metage line,
each diluted compound solution was also evaluated.
system (YAMAZEN YFLC-5404-FC,
U 50 mm ·
300 mm, 15 ml/min) with linear gradient n-hexane–
EtOAc (9/1–4/1, v/v) as an eluent to afford the com-
pound 7 (4.37 g, 74%) as a colorless oil. 1H NMR
(CDCl3) d 1.28 (3H, t, J = 8.8 Hz), 3.52 (2H, s), 4.14
(2H, q, J = 8.8 Hz), 6.70 (1H, d, J = 8.3 Hz), 7.22 (1H,
d, J = 8.8 Hz), 7.32 (1H, s); MS (ESI) m/z 248 (M++1).
5.43. VLA-4/VCAM-1 binding assay
A human VLA-4-expressing cell line, 4B4, was estab-
lished at Pharmacopeia, by transfecting both the a4
gene and b1 gene of VLA-4 into CHO-K1 cells. The
4B4 cells were maintained in Ham’s F-12 medium
(Sigma) supplemented with 10% (v/v) fetal calf serum
(REHATUIN Fetal Bovine Serum, Serologicals
Corporation), 100 U/ml penicillin (Invitrogen Corpo-
ration), 100 lg/ml streptomycin (Invitrogen Corpora-
tion), 2 mM L-glutamine (Invitrogen Corporation),
and 1 mg/ml G-418 (Geneticin, Invitrogen Corpora-
tion). An Eu-labeling Reagent (Perkin-Elmer Inc.)
was used to label the human VCAM-1/Fc chimeric
antibody (R&D Systems Inc.). The Eu-labeled protein
was purified with a PD-10 column (Amersham Biosci-
ences KK.) and stored at ꢁ80 ꢁC until used. All assays
were performed in duplicate. In preparation for the as-
say, the 4B4 cells were suspended at 3 · 105 cells/ml in
Ham’s F-12 medium. One hundred microliters of the
4B4 cell suspension was distributed into each well of
a 96-well culture plate (Costar). The plates were incu-
bated at 37 ꢁC in a 95% air/5% CO2 atmosphere
humidified incubator (Themo Forma, model 3120;
Forma Scientific) for 2 days. Prior to the assay the
medium was discarded, and each well was washed
twice with 300 ll of chilled Wash Buffer (25 mM
HEPES, pH 7.5; 150 mM NaCl; 1 mM CaCl2; 1 mM
MgCl2; 4 mM MnCl2). Then, 50 ll of compound solu-
tions was added to a well, followed by 50 ll of 2 nM
Eu-labeled Human VCAM-1/Fc Chimera diluted with
the Wash Buffer (final concentration: 1 nM). For as-
says conducted in the presence of human serum albu-
min, 50 ll of compound at various concentrations and
an equal volume of 2 nM Eu-labeled human VCAM-1/
Fc Chimera in 3% (w/v) human serum albumin (Sig-
ma) were distributed into each well (final concentra-
tion: 1 nM). The plates were incubated for 60 min at
room temperature, and the wells were washed 4 times
with 300 ll of chilled Wash Buffer. Finally, 100 ll of
the enhancement solution (Perkin-Elmer Inc.) was
added to each well. The plates were placed on a shaker
for 5 min Eu fluorescence was then measured using a
time-resolved fluorometer (DELFIA Research fluo-
rometer, model 1234-001; Perkin-Elmer Inc.). The
concentration of compound required for 50% inhibi-
tion in the assay was determined.
5.45. Distribution coefficient
The distribution coefficients (logD) were determined by
the shake-flask method.22 Four hundred lM of com-
pound solution of each compound in a 2 ml n-octa-
nol–2 ml PBS solution was placed on a shaker for
30 min. After centrifuging each solution separately at
3000 rpm for 10 min, an LC/MS method was used to
assay each layer. The LC/MS system consisted of an
1100 Series LC/MSD (Agilent) and an X Terraꢂ
MSC18 3.5lm, 3.0 · 30 mm column (Waters). The mo-
bile phase was a 10 mM ammonium acetate buffer (pH
4.5)/0.05% (v/v) acetic acid mixture in acetonitrile; the
gradient condition (95/5–10/90). Analyst software pro-
gram (version 1.4, Applied Bio. Systems) was used to
calculate the logD.
5.46. Madin–Darby canine kidney cell permeability
The cell permeability of selected compounds was deter-
mined with Madin–Darby canine kidney (MDCK,
American Type Culture Collection) cells. MDCK cells
were maintained in Minimum Essential Medium (GIB-
CO) containing 10% (v/v) fetal bovine serum (Bioprod-
ucts Inc.), a penicillin–streptomycin mixture (GIBCO),
and L-glutamine. For the transport assay, cells were
seeded into HTS 24-well transwells (Costar) at 3 · 105
cells/ml and grown for 6 days after seeding to allow
the formation of a cell monolayer. Transport Buffer
was prepared using NaHCO3 (final 0.35 g/l), D-glucose
(final 3.5 g/l), HEPES (Sigma; final 10 mM), CaCl2 (final
0.14 g/l), and MgSO4 (final 0.098 g/l) in 10· Hanks’ Bal-
anced Salt Solution (GIBCO) and adjusted to pH 6.0 or
7.4 with 1 M HCl or 1 M NaOH. For each test com-
pound, a dosing solution containing one of the com-
pounds at a concentration of 10 lM in Transport
Buffer (pH 6.0) (100 ll) was added to the apical (A) side
of a monolayer. A blank solution containing 4% (w/v)
BSA in Transport Buffer (pH 7.4) (600 ll), which was
re-adjusted to pH 7.4, was added to the basolateral
(B) side of the monolayer. Metoprolol was used as a po-
sitive control. After 1 h of incubation at 37 ꢁC, aliquots
of the apical and basal solutions were separately ana-
lyzed on an LC/MS/MS system comprising an Alliance
2790 HPLC (Waters), Atlantis dC18, 2.1 mm ID

1692
J. Chiba et al. / Bioorg. Med. Chem. 15 (2007) 1679–1693
·20 mm L, 3 lm particle size column (Waters), and
TSQ7000 mass spectrometer (ThermoQuest). The mo-
bile phase consisted of a 10 mM HCO2NH4–acetonitrile
step gradient 100/0–80/20–100/0. The concentrations of
each compound in the apical and the basolateral solu-
tions were determined from a peak area versus concen-
tration standard curve. For each compound, Eq. 1 was
used to calculate an apparent permeability coefficient
(Papp) from the LC/MS/MS-determined concentration
in the basolateral compartment (Cb, lM) and the initial
10 lM concentration in the donor compartment. In the
following equation, 3600 s is the total time for the mea-
surement of compound flux, and 0.33 cm2 is the area of
the transwell filter.
5.49. Pharmacokinetic studies on rats
Male Sprague–Dawley rats [Crj: CD(SD) IGS, 6 weeks
of age, Charles River Laboratories] were used. Animals
were fasted for 18 h prior to dosing. Each group consist-
ed of 3–4 animals. Rats were orally administered the test
compound at the doses of 10 mg/5 ml/kg dissolved in
0.5% (w/v) MC aqueous solution or 20 mg/ml/kg dis-
solved in 20% (w/v) PEG aqueous solution. Rats were
intravenously administered the test compound at a dose
of either 10 mg/5 ml/kg or 10 mg/ml/kg dissolved in sal-
ine. Blood samples (0.4 ml) were collected at 0.08, (or
0.25 for po), 0.5, 1, 2, and 6 h after the administration.
These analytical samples were prepared and analyzed
according to the Pharmacokinetic Studies on mice.
Pappð10ꢁ6cm=sÞ ¼ ðCb ꢀ 600 llÞ=ð10 lM
5.50. Pharmacokinetic studies on dogs
ꢀ 3600 s ꢀ 0:33 cm2Þ
ð1Þ
Male Beagle dogs (10–12 kg, Gokitabreeding and Ni-
honnousannkou) were used. Animals were fasted for
18 h prior to dosing. Each group consisted of 2 animals.
Dogs were orally administered the test compound dis-
solved in 0.5% (w/v) MC at a dose of 2 mg/5 ml/kg or
intravenously dissolved in saline at a dose of 2 mg/
5 ml/kg. Blood samples (1 ml) were collected after
0.08, (or 0.25 for po), 0.5, 1, 2, 4, 8, and 24 h. After
the 4-h sampling, animals were provided food. These
analytical samples were prepared and analyzed accord-
ing to the Pharmacokinetic Studies on mice.
5.47. Protein binding
Protein binding was determined by the ultra filtration
method. One lM of compound dissolved in DMSO
and 1458 ll of human serum solution (CONSERA,
lyophilized Human serum, Nissui Pharmaceutical) were
distributed into a Deep well plate (Waters) and mixed
by pipetting. Three hundred microliters of the mixed
solution was distributed into each well of a 96-well filter
plate (MultiScreenꢂ Filter Plate with Ultracel, Milli-
pore) and was centrifuged at 3000 rpm for 60 min at
37 ꢁC. The filtrate (50 ll) was collected and subjected
to LC/MS analysis. The LC/MS system consisted of
Alliance 2795 (Waters), ZQ (Waters), and an X Terraꢂ
MS C18 3.5 lm, 3.0 · 30 mm column (Waters). The
5.51. Evaluation of compound 48/80-induced rat pleurisy
models (in vivo evaluation)
Male Sprague–Dawley rats (Slc: SD, 8 weeks of age,
Nihon SLC) were used. Each group consisted of 7 ani-
mals. Rats received were orally administered the test com-
pound dissolved in 0.5% (w/v) methylcellulose (MC) at a
dose of 30 mg/5 ml/kg or 10 mg/5 ml/kg. After 15 min,
rats were injected intercostally with compound 48/80 (Sig-
ma, Lot No. 79H4070, 50 lg/0.1 ml/cavity). In the nega-
tive control group, rats were injected with saline instead
of compound 48/80. The test compound was re-orally
administered 8 h later at the same dosage. Sixteen hours
later, the rats were euthanized by cutting the abdominal
aorta while the animals were under ether anesthesia.
The plural cavity was opened and rinsed with 2 · 2.5 ml
Hanks’ balanced salt solution supplemented with 0.2%
EDTA. The pleural exudate was sampled and measured
(with the volume of the rinse fluid subtracted). The cells
were counted in a particle analyzer CDA-500 (Sysmex).
Cytocentrifuged preparations (Cytospin 2; Shandon)
were stained with Wright’s stain solution (Muto Chemi-
cals) for differential counts, based on standard morpho-
logical criteria. The eosinophil and total cell numbers of
all groups were analyzed by Dunnett’s method.
mobile phase was
a 10 mM ammonium format
buffer–MeOH; the gradient condition, 80/20–10/90
(v/v). Protein binding was analyzed using QuantLynx
(Waters).
5.48. Pharmacokinetic studies on mice
Female BALB/c mice (8–10 weeks of age, Charles River
Japan, Inc.) were used. Each group consisted of 3–4 ani-
mals. Mice were orally or intravenously administered
the test compound at a dose of 5 mg/5 ml/kg dissolved
in 0.5% (w/v) MC aqueous solution and in saline for
the oral and intravenous doses, respectively. Blood sam-
ples were collected at 0.25, 0.5, 1, 2, and 6 h after the
administration. Blood samples were stored on ice, fol-
lowed by centrifugation at 2000 rpm (1500g)for 10 min
at 4 ꢁC. Plasma fractions were subsequently stored in
a ꢁ20 ꢁC freezer until analyzed. Concentrations of test
compounds were determined by an LC/MS/MS method,
comprising an Alliance 2695 HPLC (Waters), Symme-
try Shield RP8, 2.1 · 50 mm, 3.5 lm column (Waters),
and TSQ-700 (Thermo Electron, Waltham, MA). The
mobile phase consisted of 10 mM HCOONH4 in
water–methanol; the gradient condition was 90/10–10/
90. Plasma concentrations versus time data were ana-
lyzed by non-compartmental approaches using Win-
Nonlin software program (version 1.13.1 Pharsight,
Mountain View, CA).
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