Identification of trans-4-[1-[[7-fluoro-2-(1-methyl-3-indolyl)-6- benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid as a potent, orally active VLA-4 antagonist

Article abstract of DOI:10.1016/j.bmc.2011.12.045

For the purpose of obtaining orally potent VLA-4 inhibitors, we have carried out structural modification of the (N′-phenylureido)phenyl group in compound 1, where the group was found to be attributed to poor pharmacokinetic profile in our previous research. Through modification, we have identified several compounds with both potent in vitro activity and improved oral exposure. In particular, compound 7e with 7-fluoro-2-(1-methyl-1H-indol-3- yl)-1,3-benzoxazolyl group as a novel replacement of the (N′-phenylureido) phenyl group significantly inhibited eosinophil infiltration into bronchoalveolar lavage fluid at 15 mg/kg in an Ascaris-antigen-induced murine bronchial inflammatory model, and its efficacy was comparable to that of the anti-mouse α₄ antibody (R1-2).

Full text of DOI:10.1016/j.bmc.2011.12.045

Bioorganic & Medicinal Chemistry 20 (2012) 1201–1212
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of trans-4-[1-[[7-fluoro-2-(1-methyl-3-indolyl)-6-benzoxazol-
yl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid
as a potent, orally active VLA-4 antagonist
Masaki Setoguchi a, , Shin Iimura a, Yuuichi Sugimoto a, Yoshiyuki Yoneda a, Jun Chiba a,
⇑
Toshiyuki Watanabe a, Fumihito Muro a, Yutaka Iigo b, Gensuke Takayama a, Mika Yokoyama a,
Tomoe Taira a, Misato Aonuma b, Tohru Takashi b, Atsushi Nakayama a, Nobuo Machinaga a
a R&D Division, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Daiichi Sankyo RD Novare Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
For the purpose of obtaining orally potent VLA-4 inhibitors, we have carried out structural modification of
the (N0-phenylureido)phenyl group in compound 1, where the group was found to be attributed to poor
pharmacokinetic profile in our previous research. Through modification, we have identified several com-
pounds with both potent in vitro activity and improved oral exposure. In particular, compound 7e with
7-fluoro-2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazolyl group as a novel replacement of the (N0-pheny-
lureido)phenyl group significantly inhibited eosinophil infiltration into bronchoalveolar lavage fluid at
15 mg/kg in an Ascaris-antigen-induced murine bronchial inflammatory model, and its efficacy was com-
parable to that of the anti-mouse a4 antibody (R1–2).
Received 24 October 2011
Revised 21 December 2011
Accepted 21 December 2011
Available online 30 December 2011
Keywords:
VLA-4
Integrin
Eosinophil infiltration
Asthma
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
natalizumab has recently been a therapeutic option approved for
adult patients with moderately to severely active CD in the US.9
The very late antigen-4 (VLA-4, integrin
a
4b1, CD49d/CD29)
Several small-molecule VLA-4 antagonists have been advanced
into clinical trials for the treatment of MS and CD, however, none
of these candidates has yet reached the marketplace. Thus, from
a safety and cost point of view, the next hurdle to overcome in this
field should be to develop an oral small-molecule VLA-4 antagonist
with similar efficacy to natalizumab and an appropriate half-life.
We have previously reported that we identified trans-4-[(4S)-
substituted-(2S)-pyrrolidinylmethyloxy]cyclohexanecarboxylic
acid derivative 1 as a potent VLA-4 antagonist10 and worked on
structural modification of the lipophilic moiety for the purpose of
improvement of poor pharmacokinetic (PK) profile. As a result,
we made clear that modifications to reduce the polar surface area
(PSA) value and the number of hydrogen bond donors (HBD) in the
lipophilic moiety lead to improvement of PK profile, resulting in
the discovery of 1-methyl-3-indolyl based compound 2 as a clinical
candidate which exhibits potent oral efficacy in animal models of
asthma with good oral bioavailability.11
expressed on the surface of most leukocytes is a heterodimeric
glycoprotein receptor consisting of a4 and b1 chains.1 It has been
well-established that VLA-4 plays a pivotal role in the process of
adhesion, migration, invasion, and proliferation of inflammatory
cells at the site of inflammation via interactions with its natural li-
gands, the vascular cell adhesion molecule-1 (VCAM-1, CD106)
expressed on cytokine-stimulated endothelial cells and the alter-
natively spliced portion of the type III connecting segment of fibro-
nectin (FN).2,3 The inappropriate continuation of the cellular
process causes tissue damage and dysfunction, resulting in the
pathogenesis of inflammatory and autoimmune diseases such as
asthma,4 rheumatoid arthritis,5 multiple sclerosis (MS)6 and Cro-
hn’s disease (CD).7 So far, anti-a4 antibodies and small-molecule
VLA-4 antagonists have demonstrated efficacy in a wide variety
of inflammatory animal models, validating the blockade of the
VLA-4/VCAM or FN interaction as a therapeutic target.8 Further-
more, the humanized monoclonal anti-a4 antibody natalizumab
was approved for the treatment of relapsing forms of MS in the
US and relapsing and remitting MS in the EU. On top of that,
From these results, we decided to conduct further structural
modifications of the lipophilic moiety in compound 1. In the
aforementioned modifications, we incorporated the urea in
(N0-phenylureido)phenyl group into the cyclic structures leading
to 2-(arylamino)benzoxazolyl (I), 4-(2-benzoxazolylamino)phenyl
(II), and 4-(heteroarylcarboxamide)phenyl (III) groups as depicted
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5740 3640.
E-mail address: setoguchi.masaki.d4@daiichisankyo.co.jp (M. Setoguchi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.045

1202
M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
Lipophilic moiety
R1
I
II
N
O
O
CO2H
O
N
H
N
H
Me
III
Cl
1, trans-4-[(4S)-substituted-(2S)-pyrrolidinylmethyloxy]-
cyclohexanecarboxylic acid derivative
(HBD, 2; PSA, 36.6 Å2)
Cyclization at I, II, or III position
R5
OMe
R4
R3
R2
O
Cl
Cl
O
O
N
N
N
H
N
O
N
H
N
H
Cl
O
CO2H
N
Me
O
Cl
N
H
N
Me
I
II
III
(HBD, 1; PSA, 29.2 Å2;
for R5 = H )
(HBD, 1; PSA, 31.5 Å2;
(HBD, 1; PSA, 34.0 Å2;
2, clinical candidate; IC50 = 5.4 nM
for R4 = H )
for R2 = R3 = H
Further modifications of the lipophilic moiety
R8
R7
R6
S
O
Ar
N
N
N
H
Cl
IV (I + III)
II'
(HBD, 0; PSA, 24.0 Å2;
for Ar = 1-methyl-3-indolyl;
R6 = H )
(HBD, 1; PSA, 19.9 Å2;
for R7 = R8 = H )
Figure 1. Drug designs of lipophilic moiety.
in Figure 1.11 In this study, we attempted the following two
modifications as shown in Figure 1, that one modification leads
to 2-aryl-1,3-benzoxazolyl (IV) groups double cyclized at I and
III positions, and the other leads to 4-(2-benzothiazolylami-
no)phenyl (II0) groups cyclized at II position and replaced the oxy-
gen atom with a sulfur atom, which PSA values (24.0 Å2 for IV,
Ar = 1-methyl-3-indolyl, R6 = H; 19.9 Å2 for II0, R7 = R8 = H) are
less than that of the lipophilic moiety III used for compound 2
(29.2 Å2). In addition, we fixed a fluorine atom as R1 of 1 in the
modification based on the structure–activity relationships (SAR)
that we previously reported.10
were condensed with pyrrolidine 610,11 using EDCꢀHCl and HOBt.
The resulting amides were subjected to basic hydrolysis to afford
trans-4-substituted cyclohexanecarboxylic acids 7–9. The synthe-
ses of the intermediates 3–5 in Scheme 1 are fully presented in
Schemes 2–4.
The preparation of (6-benzoxazolyl)acetic acids 3a–g is
depicted in Scheme 2. 1-Methylindole-3-carboxylic acid (10) was
refluxed in xylene with methyl 4-amino-3-hydroxyphenylacetate
(11a)12 in the presence of boric acid to give the corresponding
benzoxazole, which was hydrolyzed under a basic condition to pro-
vide [2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazol-6-yl]acetic acid
(3a). Indoles 12a–c or azaindole 12d were subjected to N-methyl-
ation using NaH and MeI, followed by Vilsmeier–Haack reaction to
afford the aldehydes 13a–d.13,14 Treatment of the aldehydes 13a–e
with methyl 4-amino-3-hydroxyphenylacetate (11a) or methyl
4-amino-2-fluoro-3-hydroxyphenylacetate (11b)11 in the presence
of iodobenzene diacetate furnished the corresponding benzoxaz-
oles, which were hydrolyzed under a basic condition to provide
the (2-aryl-6-benzoxazolyl)acetic acids 3b–g.
Herein, we report the synthesis and SAR of a series of com-
pounds that we modified the lipophilic moiety as well as in vivo
evaluation results of some representative compounds.
2. Chemistry
The target compounds were synthesized according to the gen-
eral procedure outlined in Scheme 1. Thus, arylacetic acids 3–5
F
F
a, b
CO2H
O
Ar
CO2H
+
N
Ar
N
H
CO2Me
O
O
3-5
6
7-9
Scheme 1. Reagents and conditions: (a) EDCꢀHCl, HOBt, DMAP or Et3N, DMF; (b) aq NaOH, THF.

M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
1203
CO2H
Z
a, b
HO
CO2Me
O
N
+
CO2H
N
Me
N
H
2N
Me
10
11a, Z = H
3a
Y
CHO
X
Z
Y
X
c, d
e, b
Y
Z
HO
CO2Me
+
W
O
N
N
H
CO2H
W
N
Me
X
W
H
2N
N
Me
12a, W = C, X = F, Y = H
12b, W = C, X = H, Y = F
12c, W = C, X = H, Y = Cl
12d, W = N, X = H, Y = H
13a, W = C, X = F, Y = H
13b, W = C, X = H, Y = F
13c, W = C, X = H, Y = Cl
13d, W = N, X = H, Y = H
13e, W = C, X = H, Y = H
11a, Z = H
11b, Z = F
3b, W = C, X = F, Y = H, Z = H
3c, W = C, X = H, Y = F, Z = H
3d, W = C, X = H, Y = Cl, Z = H
3e, W = C, X = H, Y = H, Z = F
3f, W = C, X = H, Y = F, Z = F
3g, W = N, X = H, Y = H, Z = H
Scheme 2. Reagents and conditions: (a) B(OH)3, xylene, reflux; (b) 0.25 N NaOH, THF; (c) NaH, MeI, DMF; (d) POCl3, DMF; (e) methyl 4-amino-3-hydroxyphenylacetate or
methyl 4-amino-2-fluoro-3-hydroxyphenylacetate, PhI(OAc)2, EtOH.
HO
a, b
CO2H
O
N
CO2Me
CO2H
+
H2N
12f
11a
4a
CHO
HO
c
d, b
CO2Me
Y
N
O
N
+
CO2H
H
X
2N
X
Y
12g
13f, X = C, Y = N
13g, X= Y = C
13h, X=N, Y = C
11a
4b, X = C, Y = N
4c, X= Y = C
4d, X=N, Y = C
Scheme 3. Reagents and conditions: (a) B(OH)3, xylene, reflux; (b) 0.25 N NaOH, THF; (c) SeO2, dioxane, reflux; (d) PhI(OAc)2, EtOH.
Z
Z
X
a
b, c
S
N
S
N
CO2R
S
CO2H
X
X
NH2
Y
+
N
H2N
N
H
Cl
Cl
14b, X = 6-Me
14c, X = 6-F
14d, X = 6-Cl
14e, X = 5-F
14f, X = 4-Cl
14g, X = H
15a, X = H, Y = Cl
15b, X = 6-Me, Y = Br
15c, X = 6-F, Y = Br
15d, X = 6-Cl, Y = Br
15e, X = 5-F, Y = Br
15f, X = 4-Cl, Y = Br
15g, X = H, Y = Br
16a, Z = H, R = Me
16b, Z = F, R = Et
5a, X = H, Z = H
5b, X = 6-Me, Z = H
5c, X = 6-F, Z = H
5d, X = 6-Cl, Z = H
5e, X = 5-F, Z = H
5f, X = 4-Cl, Z = H
5g, X = H, Z = F
5h, X = 5-F, Z = F
Scheme 4. Reagents and conditions: (a) CuBr, tBuONO, MeCN, 60 °C; (b) PPTS, xylene, reflux; (c) 0.5 N or 1 N NaOH, THF.
(6-Benzoxazolyl)acetic acids 4a–d were synthesized as shown
in Scheme 3. Intermediate 4a15 was prepared from 11a and ben-
zoic acid (12f) in two steps as described for the preparation of 3a
in Scheme 2. On the other hand, aldehydes 13f–h in which alde-
hyde 13f16 was prepared by oxidation of 12g using SeO2, were con-
verted to 4b–d by the same procedure as that used for the
preparation of 3b–g in Scheme 2.
4-aminophenylacetates 16a18 or 16b19 in the presence of PPTS
and subsequent basic hydrolysis afforded [4-(2-benzothiazol-
yl)aminophenyl]acetic acids 5a–h.
3. Results and discussion
3.1. In vitro activity
The preparation of [4-(2-benzothiazolyl)aminophenyl]acetic
acids 5a–h was shown in Scheme 4. When 2-Aminobenzothiaz-
oles 14b–g were treated with tert-butyl nitrite, followed by
CuBr, the 2-bromobenzothiazoles 15b–g were successfully
produced.17 Nucleophilic displacement of the resulting 15b–g
or commercially available 2-chlorobenzothiazole 15a with
The synthesized compounds (7–9) were evaluated for their
VLA-4 inhibitory activities in an established receptor binding assay
(VLA-4 overexpressed Chinese hamster ovary cells/Human
VCAM-1 labeled with Europium) with or without the addition of
3% human serum albumin (HSA).

1204
M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
Table 1
Table 2
Inhibitory activity of 2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazole derivatives 7a–f
Inhibitory activity of 2-aryl-1,3-benzoxazole derivatives 8a–e
F
F
X
X
N
N
O
N
O
N
Ar
Ar
O
CO2H
O
CO2H
O
O
7a-f
8a-e
Compound
Ar
X
IC50 (nM) ( 3% HSA)
2.3/121
Ratio ( )
53
Compound
Ar
X
IC50 (nM) ( 3% HSA)
200/n.d.
Ratio ( )
n.d.
N
7a
H
H
8a
H
N
N
Me
F
Me
8b
8c
H
H
487/n.d.
43/n.d.
n.d.
n.d.
7b
7c
22/977
7.3/350
44
48
N
N
Me
Me
F
H
8d
8e
H
H
900/n.d.
59/1468
n.d.
25
N
Cl
N
7d
7e
7f
H
F
30/n.d.
4.7/156
10/432
n.d.
33
N
N
n.d., not determined.
Me
Me
Table 3
Inhibitory activity of 4-(2-benzothiazolyl)aminophenyl derivatives 9a–h
F
Y
F
Z
X
F
43
N
S
W
N
O
CO2H
N
O
N
H
Me
Cl
n.d., not determined.
9a-h
Compound
W
X
Y
Z
IC50 (nM) ( 3% HSA)
Ratio ( )
9a
9b
9c
9d
9e
9f
H
H
H
H
H
Cl
H
H
H
H
H
H
F
H
H
F
H
Me
F
Cl
H
H
H
H
H
H
H
H
F
2.5/137
65/36% inhibition at 1
9.2/619
9.0/514
4.0/190
7.0/293
2.6/62
3.6/127
55
n.d.
67
57
48
42
24
35
The evaluation results of 2-(1-methyl-1H-indol-3-yl)-1,3-benz-
oxazole derivative 7a–f are shown in Table 1. At first, compound 7a
with 2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazole as a lipophilic
moiety showed potent activity with IC50 values of 2.3 and
121 nM (+3% HSA). We next investigated the effect of substituents
(F or Cl) on the benzene rings of 7a on the inhibitory activity. The
introduction of a fluorine atom at the C-6 position in the indole
ring (7b) decreased the activity by 1/10 as compared with 7a.
The introduction of a fluorine atom at the C-5 position in the indole
ring (7c) relatively retained the potency, however, a chlorine atom
at the same position (7d) caused a significant loss of the potency.
Concerning the introduction of a fluorine atom at the C-7 position
in the benzoxazole ring, 7e and 7f relatively retained the activity as
compared with the corresponding compounds 7a and 7c. In addi-
tion, of all the 2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazole deriva-
tives, compound 7a and 7e were found to show acceptable
activities with IC50s of less than 200 nM even in the presence of
3% HSA.
lg/mL
9g
9h
H
H
F
n.d., not determined.
ring based on a positive effect on pharmacokinetic properties in ro-
dents and dogs that we previously reported.20
Compound 9a showed preferable activity with IC50 values of 2.5
and 137 nM (+3% HSA). The introduction of a methyl group at the
C-6 position in the benzothiazole ring (9b) caused a significant loss
of the potency as compared with 9a. In the case of a fluorine (9c) or
chlorine atom (9d) at the same position, those compounds were
slightly less potent than 9a. The introduction of a fluorine atom
at the C-5 position (9e) and a chlorine atom at the C-4 position
(9f) in the benzothiazole ring relatively retained the potency. On
the other hand, the introduction of a fluorine atom at the C-2 posi-
tion in the central benzene ring (9g and 9h) retained comparable
activity to the corresponding compounds 9a and 9e, which seem
to be in line with the SAR we mentioned in Table 1. Additionally,
it was found that compound 9a, 9e, 9g, and 9h in the
4-(2-benzothiazolyl)aminophenyl derivatives exhibited promising
Next, we examined replacement of the 1-methyl-1H-indole ring
in 7a with 1-methyl-1H-pyrrolo[2,3-b]pyridine, benzene, naphtha-
lene, quinoline and isoquinoline (Table 2). However, all of the mod-
ifications were detrimental to the potency.
The evaluation results of 4-(2-benzothiazolyl)aminophenyl
derivatives 9a–h are summarised in Table 3. In this modification,
we fixed a chlorine atom at the 3-position on the central benzene

M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
1205
Table 4
Estimated serum concentration of compounds 7a, 7e, 9e, 9g, and 9h
F
N
Ar
O
CO2H
O
Compound
Ar
Estimated serum concentration (ng/mL) 15 min/60 min
O
N
7a
461/Not tested
2211/406
N
N
Me
F
O
N
7e
9e
Me
F
S
3196/284
3316/691
8551/619
N
S
N
H
Cl
F
9g
9h
N
N
H
Cl
F
F
S
N
N
H
Cl
activities with IC50s of less than 200 nM even in the presence of 3%
HSA.
but might be attributed to retention of the serum concentration
after oral dosing.
We selected compounds 7a, 7e, 9e, 9g, and 9h based on the
potency in the presence of 3% HSA and structural character, and
measured the activities in the serum at 15 and 60 min after oral
dosing of those compounds at 10 mg/kg in mice, calculating the
estimated serum concentrations to predict the oral absorption.
These results are summarized in Table 4.
Almost all compounds except for 7a exhibited relatively high
estimated serum concentrations. Among them, compound 9h with
two fluorine atoms in the lipophilic moiety showed the highest
concentration values of 8551 ng/ml (15 min) and 619 ng/ml
(60 min). From the results previously reported,21 we consider that
the introduction of a fluorine atom into the lipophilic moiety
would make the compound easier to use certain serum protein
as carrier, resulting in the high serum concentration.
3.3. Pharmacokinetic properties
Concerning compound 7e exhibiting excellent efficacy in a bron-
chial inflammatory model, we determined the pharmacokinetic pro-
file in rats in order to make sure how this modification of the
lipophilicmoietyin 7e affectspharmacokinetic properties. Asshown
in Table 5, compound 7e showed good oral exposure (AUC = 1000
ng h/ml) and iv plasma clearance (CL = 3.6 ml/min/kg). In addition,
the profiles seemed to be more preferable as compared with those
of compound 2 (AUC = 975 ng h/ml, CL = 19.3 ml/min/kg).11
9.0
8.0
Total cells
Eosinophil
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
3.2. In vivo efficacy
On the basis of the values of the estimated serum concentration,
we selected compounds 7e, 9g, and 9h for further biological eval-
uation. These compounds were evaluated for their anti-inflamma-
tory effect in an Ascaris-antigen-induced murine bronchial
inflammatory model by measuring the level of eosinophils in bron-
choalveolar lavage fluid (BALF) at 48 h after the antigen challenge.
The compounds were administered orally to the mice at 5 mg/kg,
bid and 15 mg/kg, bid. As shown in Figure 2, compound 7e signif-
icantly reduced eosinophil infiltration into BALF by 46% at 15 mg/
kg, and its efficacy was comparable to that of compound 211 and
the anti-mouse a4 antibody22 (R1–2, 5 mg/kg once a day) used as
the positive control in this experiment. However, compounds 9g
and 9h were not effective in this inflammatory model (data not
shown). The basis of the discrepancy remains currently unresolved,
Saline
Control
5mg/kg
15mg/kg
5mg/kg
RR1-2
sc
7e
7e
-
po
Figure 2. Effect of compound 7e in an Ascaris-antigen-induced Murine Bronchial
Inflammatory Model. Compound 7e was given to the mice twice a day for 2 days.
The anti-mouse
a4 antibody (R1–2) was given to the mice once a day for 2 days.
Each column represents mean S.D. of seven animals. $p <0.05, $$p <0.01, $$$p
<0.001; significant difference from the control (Aspin-Welch’s t-test), ⁄p <0.05, ⁄⁄p
<0.01; significant difference from the control (Dunnett’s multiple comparison test).

1206
M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
Table 5
Pharmacokinetic properties of 7e in rats (n = 3)
Fa (%)
po (1 mg/kg)
iv (1 mg/kg)
CLe (ml/min/kg)
3.6
c
d
f
AUCb (ng h/ml)
1000
Cmax (ng/ml)
T1/2 (h)
AUC (ng h/ml)
4668
Vdss (l/kg)
T1/2 (h)
21
643
2.2
0.173
0.7
a
b
c
d
e
f
Oral bioavailability.
Pharmacokinetic area under curve.
Pharmacokinetic maximum concentration.
Plasma half-life.
Pharmacokinetic clearance.
Volume of distribution.
anecarboxylate (431 mg, 96%) as a yellow amorphous solid. 1H NMR
(CDCl3) d 1.21–1.34 (m, 2H), 1.40–1.53 (m, 2H), 1.96–2.15 (m, 4H),
2.19–2.50 (m, 3H), 3.26 (m, 1H), 3.35 and 3.50 (each m, total 1H),
3.64 and 3.66 (each s, total 3H), 3.69–3.87 (m, 3H), 3.90 (s, 3H),
3.94–4.06 (m, 2H), 4.25 and 4.39 (each m, total 1H), 5.23 (m, 1H),
7.19 (m, 1H), 7.34–7.41 (m, 3H), 7.49 (m, 1H), 7.64 (m, 1H), 7.93
(s, 1H), 8.44 (m, 1H); MS (ESI) m/z 548 [M+H]+.
4. Conclusion
For the purpose of obtaining orally potent VLA-4 inhibitors, we
have worked on structural exploration of the lipophilic moiety in
compound 1 based on our previous result that reduction of PSA
value and HBD in the moiety improved PK profile. As a result, we
found out several novel lipophilic moieties maintaining potent
VLA-4 inhibitory activity. Moreover, this study has led to the iden-
tification of compound 7e with 7-fluoro-2-(1-methyl-1H-indol-
3-yl)-1,3-benzoxazolyl group as
demonstrated excellent efficacy at oral dosing in a bronchial
inflammatory model as well as favorable PK profile.
To a solution of methyl trans-4-[1-[[2-(1-methyl-3-indolyl)-6-
benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclo-
hexanecarboxylate (431 mg, 0.79 mmol) in THF (8.0 ml) was added
0.25 N NaOH (5.00 ml, 1.25 mmol) at room temperature. After
being stirred for 5 h, the reaction mixture was poured into 1 N
HCl (10 ml), and the precipitate was collected, washed with water,
and dried to give the title compound (396 mg, 94%) as a colorless
solid. IR (ATR) 2938, 2861, 1718, 1644, 1627, 1575, 1523,
a lipophilic moiety, which
5. Experimental
5.1. General
1423 cmꢁ1 1H NMR (DMSO-d6) d 1.15–1.27 (m, 2H), 1.32–1.45
;
(m, 2H), 1.89–2.11 (m, 4H), 2.14–2.29 (m, 3H), 3.23 (m, 1H), 3.55
(m, 1H), 3.75–3.95 (m, 6H), 3.97 (s, 3H), 4.11 and 4.38 (each m, to-
tal 1H), 5.35 and 5.40 (each m, total 1H), 7.22 (m, 1H), 7.31–7.39
(m, 2H), 7.55–7.67 (m, 3H), 8.35 (s, 1H), 12.03 (br s, 1H); MS
(ESI) m/z 534 [M+H]+; Anal. Calcd for C30H32FN3O5: C, 67.53; H,
6.04; N, 7.87. Found: C, 67.33; H, 6.06; N, 7.70.
All starting materials and synthesis reagents were obtained
commercially. Column chromatography was performed with a
Merck Silica Gel 60 (particle size 0.060–0.200 or 0.040–0.063).
Flash column chromatography was performed with Biotage FLASH
Si or YAMAZEN Hi-Flash packed columns. Thin-layer chromatogra-
phy (TLC) was performed on Merck precoated TLC glass sheets with
Silica Gel 60 F254. Yields were of purified products and were not
optimized. The 1H NMR spectra were recorded on a JEOL JNM-
EX-400 spectrometer, and chemical shifts are given in ppm (d)
from tetramethylsilane as an internal standard. The special split-
ting patterns are designated as follows: s, singlet; d, doublet; dd,
double of doublet; t, triplet; td, triple of doublet; q, quartet; m,
multiple. The IR spectra were recorded on a HORIBA FT-720 spec-
trometer. The mass spectra were recorded on a SCIEX API-150EX
spectrometer (ESI). The high-resolution mass (HRMS) spectra were
recorded on a JEOL JMS-100LP spectrometer. Elemental analysis
Compound 7b–d and 8a were prepared according to general
procedure A.
5.3. trans-4-[1-[[2-(6-Fluoro-1-methyl-3-indolyl)-6-benzoxaz
olyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexane
carboxylic acid (7b)
Yield 84% (two steps). A yellow solid. IR (ATR) 2938, 1712, 1616,
1577, 1434 cmꢁ1 1H NMR (DMSO-d6) d 1.09–1.39 (m, 4H), 1.84–
;
1.93 (m, 4H), 2.06–2.44 (m, 3H), 3.16–3.99 (m, 10H, including sin-
glet, 3H, at d 3.89), 4.13 and 4.33 (each m, total 1H), 5.21–5.46 (m,
1H), 7.12–7.21 (m, 2H), 7.49 (dd, J = 9.8, 2.2 Hz, 1H), 7.51 and 7.53
(each s, total 1H), 7.60 and 7.61 (each d, J = 8.1 Hz, total 1H), 8.28
(dd, J = 8.1, 5.6 Hz, 1H), 8.30 and 8.31 (each s, total 1H), 12.03 (br
s, 1H); HRMS (ESI) Calcd for C30H31F2N3O5+H: 552.23100. Found:
552.23598.
was performed using
CHNS-932 and a YOKOKAWA analysis IC7000RS.
a PerkinElmer CHNS/O 2400II, a Leco
5.2. General procedure A: preparation of trans-4-[1-[[2-(1-
methyl-3-indolyl)-6-benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7a)
5.4. trans-4-[1-[[2-(5-Fluoro-1-methyl-3-indolyl)-6-benzoxaz
olyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexane
carboxylic acid (7c)
To a solution of [2-(1-methyl-3-indolyl)-6-benzoxazolyl]acetic
acid (3a) (251 mg, 0.82 mmol), methyl trans-4-[(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (6)10,11 (212 mg,
0.82 mmol), HOBt (22.0 mg, 0.16 mmol), and DMAP (20.0 mg,
0.16 mmol) in DMF (5.0 mL) was added EDCꢀHCl (204 mg,
1.07 mmol). After being stirred for 4 h, the reaction mixture was di-
luted with water and extracted with EtOAc. The extract was washed
with brine, dried over Na2SO4, and concentrated to dryness. The
residue was purified by column chromatography with EtOAc as
eluent to give methyl trans-4[-1-[[2-(1-methyl-3-indolyl)-6-benz-
oxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohex-
Yield 74% (two steps). A yellow solid. IR (ATR) 2938, 1716, 1616,
1631, 1577, 1484, 1434 cmꢁ1 1H NMR (DMSO-d6) d 1.09–1.39 (m,
;
4H), 1.84–1.97 (m, 4H), 2.03–2.28 (m, 3H), 3.15–4.00 (m, 7H), 3.93
(s, 3H), 4.15 and 4.34 (each m, total 1H), 5.30 and 5.36 (each m,
total 1H), 7.16–7.21 (m, 2H), 7.52 and 7.54 (each s, total 1H),
7.61–7.64 (m, 2H), 7.97 (dd, J = 9.8, 2.9 Hz, 1H), 8.36 and 8.37 (each
s, total 1H), 12.03 (br s, 1H); HRMS (ESI) Calcd for C30H31F2N3O5+H:
552.23100. Found: 552.23543.

M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
1207
5.5. trans-4-[1-[[2-(5-Chloro-1-methyl-3-indolyl)-6-benzoxaz
olyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexane
carboxylic acid (7d)
7.90 (m, 2H), 8.19 (d, J = 7.3 Hz, 1H), 8.30 (dd, J = 1.5, 4.5 Hz, 1H),
9.16 (d, J = 4.5 Hz, 1H), 9.42 (d, J = 8.6 Hz, 1H); MS (ESI) m/z 532
[M+H]+; Anal. Calcd for C30H30FN3O5ꢀ0.5H2O: C, 66.65; H, 5.78; N,
7.77. Found: C, 66.53; H, 5.61; N, 7.63.
Yield 65% (two steps). A yellow solid. IR (ATR) 2938, 2863, 1720,
1629, 1581, 1434 cmꢁ1
;
1H NMR (DMSO-d6) d 1.13–1.40 (m, 4H),
5.9. General procedure C: preparation of trans-4-[1-[[7-fluoro-
2-(1-methyl-3-indolyl)-6-benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7e)
1.85–2.07 (m, 4H), 2.11–2.44 (m, 3H), 3.19–3.52 (m, 3H), 3.72–
3.90 (m, 4H), 3.94 (s, 3H), 4.07 and 4.35 (each m, total 1H), 5.31
and 5.37 (each m, total 1H), 7.20 (m, 1H), 7.35 (dd, J = 8.8, 2.0 Hz,
1H), 7.53–7.57 (m, 1H), 7.64–7.66 (m, 2H), 8.29–8.31 (m, 1H),
8.38–8.41 (m, 1H), 12.02 (br s, 1H); MS (ESI) m/z 568 [M+H]+;
A mixture of [7-fluoro-2-(1-methyl-3-indolyl)-6-benzoxazol-
yl]acetic acid (3e) (350 mg, 1.08 mmol), methyl trans-4-[(4S)-flu-
HRMS (ESI) Calcd for
568.19918.
C
30H31ClFN3O5+H: 568.20145. Found:
oro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate
(6)
(280 mg, 1.08 mmol), EDCꢀHCl (311 mg, 1.62 mmol), HOBt
(219 mg, 1.62 mmol) and Et3N (0.75 ml, 5.40 mmol) in DMF
(10 ml) was stirred at room temperature for 17 h. The mixture
was poured into ice water and extracted with EtOAc. The combined
extracts were washed with ice water and brine. After dried over
Na2SO4, the extracts were concentrated in vacuo. The residue
was purified by column chromatography on silica gel with n-hex-
ane–EtOAc (3:1, v/v) as eluent to give methyl trans-4-[1-[[7-fluoro-
2-(1-methyl-3-indolyl)-6-benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (360 mg, 59%) as a
brown oil. 1H NMR (CDCl3) d 1.21–1.65 (m, 4H), 1.95–2.56 (m,
7H), 3.21–3.39 (m, 2H), 3.56 (dd, J = 8.8, 6.8 Hz, 1H), 3.64 and
3.67 (each s, total 3H, amide isomers), 3.68–3.90 (m, 3H), 3.92 (s,
3H), 4.00 and 4.03 (each s, total 1H), 4.31–4.42 (m, 1H), 5.19–
5.39 (m, 1H), 7.15–7.23 (m, 1H), 7.36–7.48 (m, 4H), 7.98 (s, 1H),
8.45 (m, 1H); MS (ESI) m/z 566 [M+H]+.
5.6. trans-4-[1-[[2-[1-Methyl-3-(1H-pyrrolo[2,3-b]pyridinyl)]-6-
benzoxazolyl]acethyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (8a)
Yield 66% (two steps). A yellow solid. IR (ATR) 2940, 2861, 1716,
1631, 1569, 1525, 1486, 1442 cmꢁ1 1H NMR (DMSO-d6) d 1.13–
;
1.37 (m, 4H), 1.85–2.09 (m, 4H), 2.11–2.21 (m, 3H), 3.17–3.31
(m, 2H), 3.45 and 3.58 (each m, total 1H), 3.72–3.92 (m, 4H),
3.96 (s, 3H), 4.15 and 4.35 (each m, total 1H), 5.32 and 5.37 (each
m, total 1H), 7.21 (m, 1H), 7.35 and 7.36 (each d, J = 8.1 Hz, total
1H), 7.55 and 7.57 (each s, total 1H), 7.61 and 7.62 (each d,
J = 8.1 Hz, total 1H), 8.44 (dd, J = 4.7, 1.5 Hz, 1H), 8.53 (s, 1H),
8.61 (dd, J = 7.8, 1.5 Hz, 1H), 11.99 (br s, 1H); MS (ESI) m/z 535
[M+H]+; Anal. Calcd for C29H31FN4O5ꢀ0.75H2O: C, 63.55; H, 5.98;
N, 10.22. Found: C, 63.67; H, 5.91; N, 10.22.
To a solution of methyl trans-4-[1-[[7-fluoro-2-(1-methyl-3-
indolyl)-6-
benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinyl-
5.7. General procedure B: preparation of trans-4-[1-[(2-phenyl-
6-benzoxazolyl)acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (8b)
methoxy]cyclohexanecarboxylate (130 mg, 0.23 mmol) in THF–
MeOH (15 ml, 2:1, v/v) was added 1 N NaOH (4 ml). After stirring
at room temperature for 13 h, the mixture was concentrated under
reduced pressure and acidified with 1 N HCl. The precipitate was
collected, washed with water and dried under reduced pressure
to give the title compound (93 mg, 74%) as a brown solid. IR
To
a solution of 2-phenyl-6-benzoxazolylacetic acid (4a)
(57 mg, 0.23 mmol) and methyl trans-4-[(4S)-fluoro-(2S)-pyrrolid-
inylmethoxy]cyclohexanecarboxylate (6) (58 mg, 0.23 mmol) in
DMF (5 ml) were added HOBt (58 mg, 0.43 mmol), DMAP (catalytic
amount) and EDCꢀHCl (65 mg, 0.34 mmol). The reaction mixture
was stirred at room temperature for 18 h. The reaction mixture
was poured in 1 N HCl and extracted with EtOAc three times. The
combined extracts were washed with brine and dried over anhy-
drous MgSO4. The solvent was evaporated under reduced pressure.
The residue was dissolved in THF–MeOH (4 ml, 1:1, v/v) then 1 N
NaOH (1 ml) was added. The reaction mixture was stirred for
18 h at room temperature and poured in 1 N HCl. The mixture
was extracted with CHCl3–MeOH and the combined extracts were
dried over anhydrous MgSO4. The mixture was evaporated and the
residue was purified with TLC (CHCl3–MeOH, 20:1, v/v) to give the
title compound (42 mg, 39%) as a colorless solid. 1H NMR (DMSO-
d6) d 1.15–1.40 (m, 4H), 1.90 (m, 3H), 2.20 (m, 2H), 3.20–3.60 (m,
6H), 3.70–4.00 (m, 3H), 4.20 and 4.40 (m, 1H), 5.32 and 5.48 (each
d, J = 7.0 Hz, total 1H), 7.30 (m, 1H), 7.70 (s, 4H), 7.74 (d, J = 2.0 Hz,
1H), 8.20 (m, 2H). MS (ESI) m/z 481 [M+H]+; Anal. Calcd for
(ATR) 2941, 2864, 1716, 1628, 1583, 1504, 1442, 1369 cmꢁ1 1H
;
NMR (DMSO-d6) d 1.13–1.43 (m, 5H), 1.82–2.36 (m, 7H), 3.24 (m,
1H), 3.46–3.93 (m, 4H), 3.95 (s, 3H), 4.11 and 4.15 (each s, total
1H, amide isomers), 4.42 (m, 1H), 5.25–5.50 (m, 1H), 7.22 (m,
1H), 7.35 (m, 2H), 7.48 and 7.50 (each t, J = 4.4 and 3.6 Hz respec-
tively, total 1H, amide isomers), 7.63 (d, J = 6.6 Hz, 1H), 8.29 (d,
J = 7.3 Hz, 1H), 8.44 and 8.45 (each s, total 1H, amide isomers);
MS (ESI) m/z 552 [M+H]+; Anal. Calcd for C30H31F2N3O5ꢀ1H2O: C,
63.26; H, 5.84; N, 7.38. Found: C, 63.52; H, 5.77; N, 7.25.
Compound 8c, 8e and 9a–h were prepared according to general
procedure C.
5.10. trans-4-[1-[[2-(1-Naphthyl)-6-benzoxazolyl]acetyl]-(4S)-
fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid
(8c)
Yield 29% (two steps). A colorless solid. 1H NMR (DMSO-d6) d
1.11–1.36 (m, 4H), 1.83–2.20 (m, 7H), 3.19–4.01 (m, 7H), 4.09–
4.19 and 4.32–4.42 (each m, total 1H), 5.23–5.47 (m, 1H), 7.28–
7.32 (m, 1H), 7.64–7.85 (m, 5H), 8.09 (d, J = 8.0 Hz, 1H), 8.21 (d,
J = 8.0 Hz, 1H), 8.42–8.44 (m, 1H), 9.40–9.42 (m, 1H); MS (ESI),
m/z 531 [M+H]+; Anal. Calcd for C31H31FN2O5ꢀ0.5H2O: C, 69.00; H,
5.98; N, 5.19. Found: C, 69.11; H, 5.76; N, 5.17.
C
27H29FN2O5ꢀ1.0H2O: C, 65.05; H, 6.27; N, 5.62. Found: C, 64.86;
H, 5.89; N, 5.37.
Compound 8d was prepared according to general procedure B.
5.8. trans-4-[1-[[2-(4-Quinolinyl)-6-benzoxazolyl]acetyl]-(4S)-
fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid
(8d)
5.11. trans-4-[1-[[2-(1-Isoquinolinyl)-6-benzoxazolyl]acetyl]-
(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic
acid (8e)
Yield 56%. A colorless solid. 1H NMR (DMSO-d6) d 1.20 (m, 2H),
1.32 (m, 2H), 1.90 (m, 4H), 2.18 (m, 4H), 3.20 (m, 1H), 3.45–3.60
(m, 1H), 3.72–4.12 (m, 4H), 4.15 and 4.40 (each m, total 1H),
5.35 (m, 1H), 7.36 (m, 1H), 7.75 (d, J = 9.3 Hz, 1H), 7.85 (m, 1H),
Yield 72% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
1.12–1.37 (m, 4H), 1.84–2.11 (m, 4H), 2.14–2.21 (m, 3H), 3.17–4.04

1208
M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
(m, 7H), 4.10–4.20 and 4.33–4.43 (each m, total 1H), 5.23–5.47 (m,
1H), 7.33–7.37 (m, 1H), 7.72–7.74 (m, 1H), 7.86–7.93 (m, 3H),
8.11–8.15 (m, 2H), 8.74–8.76 (m, 1H), 9.51–9.53 (m, 1H), 12.02
(br s, 1H); MS (ESI) m/z 532 [M+H]+; Anal. Calcd for
C
27H28ClF2N3O4SꢀH2O: C, 55.71; H, 5.19; N, 7.22; S, 5.51. Found:
C, 56.00; H, 4.99; N, 6.89; S, 5.38.
5.17. trans-4-[1-[[3-chloro-4-(4-Chloro-2-benzothiazolyl)
aminophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (9f)
C30H30FN3O5ꢀH2O: C, 65.56; H, 5.87; N, 7.65. Found: C, 65.39; H,
5.94; N, 7.51.
5.12. trans-4-[1-[[4-(2-Benzothiazolyl)amino-3-chlorophenyl]
acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecar
boxylic acid (9a)
Yield 88% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
1.13–1.37 (m, 4H), 1.86–1.95 (m, 4H), 2.08–2.33 (m, 3H), 3.16–3.88
(m, 7H), 4.08–4.18 and 4.30–4.40 (each m, total 1H), 5.23–5.48 (m,
1H), 7.11–7.15 (m, 1H), 7.23–7.26 (m, 1H), 7.38–7.41 (m, 2H),
7.75–7.77 (m, 1H), 8.14–8.18 (m, 1H), 10.23 (br s, 1H), 12.05 (br
s, 1H); MS (ESI) m/z 580 [M+H]+; HRMS (ESI) Calcd for
C27H28Cl2FN3O4S+H: 580.12398. Found: 580.12176.
Yield 70% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
1.10–1.39 (m, 4H), 1.84–1.94 (m, 4H), 2.06–2.20 (m, 3H), 3.15–3.87
(m, 7H), 4.07–4.17 and 4.28–4.38 (each m, total 1H), 5.22–5.47 (m,
1H), 7.11–7.15 (m, 1H), 7.19–7.23 (m, 1H), 7.27–7.31 (m, 1H),
7.36–7.38 (m, 1H), 7.50–7.52 (m, 1H), 7.77–7.79 (m, 1H), 8.06–
8.10 (m, 1H); MS (ESI) m/z 546 [M+H]+; HRMS (ESI) Calcd for
5.18. trans-4-[1-[[4-(2-Benzothiazolyl)amino-5-chloro-2-
fluorophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (9g)
C27H29ClFN3O4S+H: 546.16296. Found: 546.15858.
Yield 62% (two steps). A colorless amorphous solid. 1H NMR
(DMSO-d6) d 1.16–1.38 (m, 4H), 1.89–2.23 (m, 7H), 3.17–3.93 (m,
7H), 4.08–4.18 and 4.30–4.40 (each m, total 1H), 5.24–5.50 (m,
1H), 7.17–7.21 (m, 1H), 7.32–7.36 (m, 1H), 7.40–7.45 (m, 1H),
7.61–7.63 (m, 1H), 7.82–7.84 (m, 1H), 8.32–8.34 (m, 1H); MS
(ESI) m/z 564 [M+H]+; HRMS (ESI) Calcd for C27H28ClF2N3O4S+H:
564.15354. Found: 564.15126.
5.13. trans-4-[1-[[3-Chloro-4-(6-methyl-2-benzothiazolyl)
aminophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (9b)
Yield 59% (two steps). A brown solid. 1H NMR (DMSO-d6) d
1.16–1.36 (m, 4H), 1.76–1.94 (m, 4H), 2.14–2.40 (m, total 6H,
including s, 3H, at d 2.34), 3.15–3.86 (m, 7H), 4.07–4.17 and
4.27–4.37 (each m, total 1H), 5.22–5.47 (m, 1H), 7.09–7.11 (m,
1H), 7.19–7.22 (m, 1H), 7.35–7.49 (m, 3H), 7.57 (s, 1H), 8.10–
8.12 (m, 1H); MS (ESI) m/z 560 [M+H]+; Anal. Calcd for
5.19. trans-4-[1-[[5-Chloro-2-fluoro-4-(5-fluoro-2-benzothiaz
olyl)aminophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmeth
oxy]cyclohexanecarboxylic acid (9h)
C
28H31ClFN3O4Sꢀ1.5H2O: C, 57.28; H, 5.84; N, 7.16; S, 5.46. Found:
C, 57.20; H, 5.66; N, 6.91; S, 5.39.
Yield 70% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
1.18–1.38 (m, 4H), 1.86–1.99 (m, 4H), 2.10–2.21 (m, 3H), 3.17–4.02
(m, 7H), 4.08–4.18 and 4.31–4.41 (each m, total 1H), 5.24–5.50 (m,
1H), 7.03–7.08 (m, 1H), 7.41–7.48 (m, 2H), 7.83–7.87 (m, 1H),
8.25–8.27 (m, 1H); MS (ESI) m/z 582 [M+H]+; Anal. Calcd for
5.14. trans-4-[1-[[3-Chloro-4-(6-fluoro-2-benzothiazolyl)
aminophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (9c)
Yield 73% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
1.17–1.38 (m, 4H), 1.74–2.21 (m, 7H), 3.18–3.87 (m, 7H), 4.09–4.19
and 4.29–4.39 (each m, total 1H), 5.23–5.47 (m, 1H), 7.12–7.23 (m,
2H), 7.37–7.38 (m, 1H), 7.51–7.54 (m, 1H), 7.71–7.74 (m, 1H),
8.09–8.13 (m, 1H), 9.94 (br s, 1H); MS (ESI) m/z 564 [M+H]+; HRMS
(ESI) Calcd for C27H28ClF2N3O4S+H: 564.15354. Found: 564.14881.
C
27H27ClF3N3O4Sꢀ0.5H2O: C, 54.87; H, 4.77; N, 7.11; S, 5.43. Found:
C, 54.68; H, 4.62; N, 6.85; S, 5.45.
5.20. trans-4-[1-[[2-(5-Fluoro-1-methyl-3-indolyl)-7-fluoro-6-
benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (7f)
To a stirred solution of [2-(5-fluoro-1-methylindolyl)-7-fluoro-
6-benzoxazolyl]acetic acid (3f) (202 mg, 0.59 mmol), methyl
5.15. trans-4-[1-[[3-chloro-4-(6-Chloro-2-benzothiazolyl)
aminophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (9d)
trans-4-[(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecar-
boxylate (6) (153 mg, 0.59 mmol) and EDCꢀHCl (124 mg,
0.65 mmol) in DMF (15 ml) was added HOBt (16.0 mg, 0.12 mmol),
and the resulting mixture was stirred at room temperature over-
night. The mixture was poured into water and extracted with
EtOAc. The extracts were washed thrice with water, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by thin layer chromatography on silica gel with n-hex-
ane–EtOAc (5:1, v/v) to give methyl trans-4-[1-[[2-(5-fluoro-1-
methylindolyl)-7-fluoro-6-benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (295 mg, 86%) as a
colorless amorphous solid. 1H NMR (CDCl3) d 1.20–1.31 (m, 2H),
1.39–1.55 (m, 2H), 1.99–2.55 (series of m, 8H), 3.21–3.40 and
3.52–3.59 (series of m, 2H), 3.64 and 3.67 (s, total 3H), 3.68–4.08
(series of m, 7H, including s, 3H, at d 3.90), 4.30–4.44 (m, 1H),
5.19–5.40 (m, 1H), 7.08–7.13 (m, 1H), 7.20 (dd, J = 8.8, 15.2 Hz,
1H), 7.32 (dd, J = 4.0, 8.8 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.98 (s,
1H), 8.11 (dd, J = 2.4, 9.6 Hz, 1H); MS (ESI) m/z 584 [M+H]+.
Yield 79% (two steps). A pale yellow amorphous solid. 1H NMR
(DMSO-d6) d 1.16–1.36 (m, 4H), 1.87–2.20 (m, 7H), 3.15–3.87 (m,
7H), 4.08–4.18 and 4.28–4.38 (each m, total 1H), 5.22–5.47 (m,
1H), 7.21–7.23 (m, 1H), 7.29–7.32 (m, 1H), 7.37–7.38 (m, 1H),
7.48–7.51 (m, 1H), 7.92 (s, 1H), 8.05–8.09 (m, 1H); MS (ESI) m/z
580 [M+H]+; HRMS (ESI) Calcd for C27H28Cl2FN3O4S+H:
580.12398. Found: 580.11988.
5.16. trans-4-[1-[[3-Chloro-4-(5-fluoro-2-benzothiazolyl)
aminophenyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (9e)
Yield 80% (two steps). A colorless solid. 1H NMR (DMSO-d6) d
1.16–1.38 (m, 4H), 1.78–1.95 (m, 4H), 2.15–2.21 (m, 3H), 3.16–
3.88 (m, 7H), 4.08–4.18 and 4.29–4.39 (each m, total 1H), 5.24–
5.48 (m, 1H), 6.98–7.02 (m, 1H), 7.23–7.24 (m, 1H), 7.34–7.39
(m, 2H), 7.78–7.82 (m, 1H), 8.01–8.05 (m, 1H), 10.11 (br s, 1H),
12.06 (br s, 1H); MS (ESI) m/z 564 [M+H]+; Anal. Calcd for
To a stirred solution of methyl trans-4-[1-[[2-(5-fluoro-1-methy-
lindolyl)-7-fluoro-6-benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrro-
lidinylmethoxy]cyclohexanecarboxylate (288 mg, 0.49 mmol) in

M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
1209
THF–MeOH (20 ml, 1:1, v/v) was added aqueous 0.25 N NaOH
(9.9 ml, 2.45 mmol), and the resulting mixture was stirred at room
temperature overnight. The mixture was poured into aqueous 1 N
HCl and extracted with CHCl3–MeOH (10:1, v/v). The extracts were
dried over anhydrous Na2SO4, and concentrated in vacuo. The resi-
due was purified by column chromatography on silica gel with
CHCl3–MeOH (10:1, v/v) as eluent to give the title compound
(296 mg, quant.) as a colorless amorphous solid. 1H NMR (CDCl3) d
1.18–1.38 (m, 2H), 1.39–1.58 (m, 2H), 1.96–2.56 (series of m, 7H),
3.21–3.60 (series of m, 2H), 3.66–4.10 (series of m, 8H, including
s, 3H, at d 3.89), 4.30–4.46 (m, 1H), 5.20–5.40 (m, 1H), 7.06–7.13
(m, 1H), 7.16–7.24 (m, 1H), 7.30–7.32 (m, 1H), 7.46 (t, J = 8.0 Hz,
1H), 7.99 (s, 1H), 8.10 (dd, J = 2.0, 9.2 Hz, 1H); MS (ESI) m/z 570
[M+H]+.
POCl3 (5.87 ml, 63.0 mmol) was added dropwise to DMF (45 ml)
at 0 °C, and the mixture was stirred for 20 min at same tempera-
ture. A solution of 6-fluoro-1-methylindole (6.71 g, 45.0 mmol) in
DMF (45 ml) was added slowly to the mixture at 0 °C. After being
stirred at room temperature for 1.5 h, the reaction mixture was
poured into ice, and the resulting mixture was neutralized with
1 N NaOH and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated to dryness. The residue
was purified by column chromatography on silica gel with EtOAc
as eluent to give the title compound (4.18 g, 52%) as a colorless so-
lid. 1H NMR (CDCl3) d 3.85 (s, 3H), 7.03–7.11 (m, 2H), 7.68 (s, 1H),
8.25 (dd, J = 8.8, 5.6 Hz, 1H), 9.97 (s, 1H).
5.23.2. [2-(6-Fluoro-1-methyl-3-indolyl)-6-benzoxazolyl]acetic
acid (3b)
A mixture of 6-fluoro-1-methylindole-3-carbaldehyde (13a)
(500 mg, 2.82 mmol) and methyl 4-amino-3-hydroxyphenylace-
tate (11a) (767 mg, 4.23 mmol) in EtOH (15 ml) was stirred for
22 h, and iodobenzene diacetate (1.09 g, 3.38 mmol) was added
portionwise to the mixture. After being stirred for 1 h, the mixture
was concentrated, and the residue was purified column chroma-
tography with n-hexane–AcOEt (1:1, v/v) as eluent to give methyl
[2-(6-fluoro-1-methyl-3-indolyl)-6-benzoxazolyl]acetate (889 mg,
93%) as a brown solid. 1H NMR (CDCl3) d 3.72 (s, 3H), 3.76 (s,
2H), 3.85 (s, 3H) 7.05–7.12 (m, 2H), 7.22 (dd, J = 8.1, 1.5 Hz, 1H),
7.49 (d, J = 1.5 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.89 (s, 1H), 8.38
(dd, J = 8.9, 5.4 Hz, 1H); MS (ESI) m/z 339 [M+H]+.
To a solution of methyl [2-(6-fluoro-1-methyl-3-indolyl)-6-
benzoxazolyl]acetate (889 mg, 2.63 mmol) in THF (25 ml) was
added 0.25 N NaOH (15.8 ml, 3.95 mmol) at room temperature.
After being stirred for 4 h, the reaction mixture was poured into
1 N HCl (30 ml). The precipitate was collected, washed with water,
and dried to give the title compound (741 mg, 87%) as a brown so-
lid. 1H NMR (DMSO-d6) d 3.71 (s, 2H), 3.94 (s, 3H), 7.18 (td, J = 9.5,
2.5 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.59–7.64 (m, 3H), 7.97 (dd,
J = 9.5, 2.5 Hz, 1H), 8.39 (s, 1H), 12.35 (br s, 1H); MS (ESI) m/z
325 [M+H]+.
5.21. General procedure D: preparation of [2-(1-methyl-3-
indolyl)-6-benzoxazolyl]acetic acid (3a)
A mixture of 1-methylindole-3-carboxylic acid (10) (1.00 g,
5.71 mmol) and methyl 4-amino-3-hydroxyphenylacetate (11a)12
(1.03 g, 5.71 mmol) in xylene (20 ml) was refluxed in the presence
of boric acid (1.05 g, 17.1 mmol) for 24 h, during which time water
was removed with Dean–Stark water trap. The precipitate was fil-
tered off and the filtrate was concentrated to dryness. The residue
was purified by column chromatography with n-hexane–EtOAc
(4:1, v/v) as eluent to give methyl [2-(1-methyl-3-indolyl)-6-benz-
oxazolyl]acetate (0.20 g, 11%) as a yellow oil. 1H NMR (CDCl3) d
3.72 (s, 3H), 3.76 (s, 2H), 3.88 (s, 3H), 7.21 (dd, J = 8.1, 1.5 Hz,
1H), 7.32–7.40 (m, 3H), 7.49 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.91
(s, 1H), 8.45 (m, 1H); MS (ESI) m/z 321 [M+H]+.
To a solution of methyl [2-(1-methyl-3-indolyl)-6-benzoxazol-
yl]acetate (200 mg, 0.62 mmol) in THF (6.5 ml) was added 0.25 N
NaOH (3.75 ml, 0.93 mmol). The reaction mixture was stirred for
2 h and poured into 1 N HCl (10 ml). The precipitate was collected,
washed with water, and dried to give the title compound (189 mg,
99%) as a colorless solid. 1H NMR (DMSO-d6) d 3.73 (s, 2H), 3.94 (s,
3H), 7.24 (dd, J = 7.8, 1.5 Hz, 1H), 7.31 (td, J = 7.8, 1.5 Hz, 1H), 7.35
(td, J = 7.8, 1.5 Hz, 1H), 7.60–7.64 (m, 3H), 8.32 (dd, J = 8.1, 1.5 Hz,
1H), 8.34 (s, 1H), 12.37 (br s, 1H); MS (ESI) m/z 307 [M+H]+.
Compound 4a was prepared according to general procedure D.
Compound 3c–g, 4b–d and 13b–d were prepared according to
general procedure E.
5.24. 5-Fluoro-1-methylindole-3-carbaldehyde (13b)
Quantitative yield (two steps). A colorless solid. 1H NMR (CDCl3)
d 3.88 (s, 3H), 7.09 (m, 1H), 7.29 (d, J = 9.3, 4.2 Hz, 1H), 7.71 (s, 1H),
7.98 (dd, J = 9.3, 2.4 Hz, 1H), 9.96 (s, 1H); MS (ESI) m/z 178 [M+H]+.
5.22. 2-Phenyl-6-benzoxazolylacetic acid (4a)
Yield 11% (two steps). A colorless solid. 1H NMR (DMSO-d6) d
3.76 (s, 2H), 7.32 (dd, J = 8.3, 1.1 Hz, 1H), 7.60–7.66 (m, 3H), 7.70
(d, J = 1.1 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 8.18–8.23 (m, 2H),
12.42 (br s, 1H); MS (ESI) m/z 254 [M+H]+.
5.25. [2-(5-Fluoro-1-methyl-3-indolyl)-6-benzoxazolyl]acetic
acid (3c)
Yield 24% (two steps). A brown solid. 1H NMR (DMSO-d6) d 3.72
(s, 2H), 3.90 (s, 3H), 7.15 (td, J = 9.8, 2.2 Hz, 1H), 7.23 (dd, J = 8.1,
1.2 Hz, 1H), 7.50 (dd, J = 9.8, 2.2 Hz, 1H), 7.59 (d, J = 1.2 Hz, 1H),
7.62 (d, J = 8.1 Hz, 1H), 8.29 (dd, J = 8.8, 5.4 Hz, 1H), 8.33 (s, 1H),
12.37 (br s, 1H); MS (ESI) m/z 325 [M+H]+.
5.23. General procedure E: preparation of [2-(6-fluoro-1-
methyl-3-indolyl)-6-benzoxazolyl]acetic acid (3b)
5.23.1. 6-Fluoro-1-methylindole-3-carbaldehyde (13a)
To a solution of 6-fluoroindole (12a) (6.10 g, 45.1 mmol) in DMF
(65 ml) was added NaH (60% in oil, 1.45 g, 36.3 mmol) at 0 °C. After
being stirred for 30 min at 0 °C, MeI (2.46 ml, 39.6 mmol) was
added to the mixture, which was stirred for further 2.5 h and
quenched by the addition of saturated aqueous NH4Cl. The result-
ing mixture was diluted with water and extracted with EtOAc. The
extract was washed brine, dried over MgSO4, and concentrated to
dryness. The residue was purified by column chromatography on
silica gel with n-hexane–EtOAc (5:1, v/v) as eluent to give 6-flu-
oro-1-methylindole (6.71 g, 99%) as a yellow oil. 1H NMR (CDCl3)
d 3.73 (s, 3H), 6.45 (d, J = 3.2 Hz, 1H), 6.86 (ddd, J = 9.8, 8.5,
2.2 Hz, 1H), 6.98 (dd, J = 9.8, 2.2 Hz, 1H), 7.01 (d, J = 3.2 Hz, 1H),
7.51 (dd, J = 8.5, 5.3 Hz, 1H); MS (ESI) m/z 150 [M+H]+.
5.26. 5-Chloro-1-methylindole-3-carbaldehyde (13c)
Yield 80% (two steps). A colorless solid. 1H NMR (CDCl3) d 3.87
(s, 3H), 7.27 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 8.5, 1.9 Hz, 1H), 7.69 (s,
1H), 8.30 (d, J = 1.9 Hz, 1H), 9.96 (s, 1H).
5.27. [2-(5-Chloro-1-methyl-3-indolyl)-6-benzoxazolyl]acetic
acid (3d)
Yield 9% (two steps). A brown solid. 1H NMR (DMSO-d6) d 3.73
(s, 2H), 3.94 (s, 3H), 7.25 (dd, J = 8.1, 1.7 Hz, 1H), 7.36 (dd, 1H,

1210
M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
J = 8.8, 1.7 Hz, 1H), 7.62–7.68 (m, 3H), 8.30 (d, J = 1.7 Hz, 1H), 8.41
(s, 1H), 12.38 (br s, 1H); MS (ESI) m/z 341 [M+H]+.
2H), 7.88–7.93 (m, 2H), 8.74–8.76 (m, 1H), 9.30–9.33 (m, 1H),
10.39 (s, 1H); MS (ESI), m/z 158 [M+H]+.
5.28. [7-Fluoro-2-(1-methyl-3-indolyl)-6-benzoxazolyl]acetic
acid (3e)
5.36. General procedure F: preparation of [3-chloro-4-(6-
methyl-2-benzothiazolyl)aminophenyl]acetic acid (5b)
Yield 22% (two steps). A dark green solid. 1H NMR (DMSO-d6) d
3.79 (s, 2H), 3.95 (s, 3H), 7.26–7.39 (m, 3H), 7.50 (d, J = 8.3 Hz, 1H),
7.61–7.66 (m, 1H), 8.27–8.31 (m, 1H), 8.46 (s, 1H); MS (ESI), m/z
325 [M+H]+.
5.36.1. 2-Bromo-6-methylbenzothiazole (15b)
To a stirred suspension of CuBr (1.57 g, 10.9 mmol) in MeCN
t
(25 ml) was added BuONO (1.63 ml, 13.7 mmol) and the mixture
was stirred at 60 °C for 10 min. Then 2-amino-6-methylbenzothia-
zole (14b) (1.50 g, 9.13 mmol) was added to the mixture, and the
reaction mixture was stirred at 60 °C for 1 h. After cooled to room
temperature, the mixture was poured into 1 N HCl. The resulting
precipitate was dissolved into EtOAc, washed with 1 N HCl, brine,
dried over Na2SO4, and evaporated. The residue was purified by
column chromatography on silica gel with n-hexane–EtOAc (7:1,
v/v) as eluent to give the title compound (703 mg, 34%) as a brown
oil. 1H NMR (CDCl3) d 2.47 (s, 3H), 7.27–7.29 (m, 1H), 7.58–7.59 (m,
1H), 7.84–7.87 (m, 1H).
5.29. [2-(5-Fluoro-1-methyl-3-indolyl)-7-fluoro-6-
benzoxazolyl]acetic acid (3f)
Yield 21% (two steps). A brown solid. 1H NMR (DMSO-d6) d 3.78
(s, 2H), 3.95 (s, 3H), 7.19–7.24 (m, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.50
(d, J = 8.4 Hz, 1H), 7.66 (dd, J = 8.8, 4.4 Hz, 1H), 7.94 (br d, J = 9.6 Hz,
1H), 8.50 (br s, 1H), 12.52 (br s, 1H).
5.30. 1-Methyl-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (13d)
Yield 72% (two steps). A yellow solid. 1H NMR (CDCl3) d 3.98 (s,
3H), 7.28 (dd, J = 7.8, 4.7 Hz, 1H), 7.85 (s, 1H), 8.44 (d, J = 4.7 Hz,
1H), 8.55 (d, J = 7.8 Hz, 1H), 9.97 (s, 1H).
5.36.2. [3-Chloro-4-(6-methyl-2-benzothiazolyl)aminophenyl]
acetic acid (5b)
A mixture of 2-bromo-6-methylbenzothiazole (15b) (703 mg,
3.08 mmol),
methyl
4-amino-3-chlorophenylacetate
(16a)
(615 mg, 3.08 mmol), and PPTS (232 mg, 0.92 mmol) in xylene
(10 ml) was refluxed for 10 h. After cooled to room temperature,
the solvent was evaporated. The residue was purified by column
chromatography on silica gel with n-hexane–EtOAc (6:1, v/v) as
eluent to give methyl [3-chloro-4-(6-methyl-2-benzothiazol-
yl)aminophenyl]acetate (274 mg, 26%) as a pale yellow oil. 1H
NMR (CDCl3) d 2.43 (s, 3H), 3.59 (s, 2H), 3.71 (s, 3H), 7.17–7.19
(m, 1H), 7.23–7.25 (m, 1H), 7.35 (m, 1H), 7.46 (s, 1H), 7.58 (d,
J = 8.3 Hz, 1H), 8.35 (d, J = 8.3 Hz, 1H); MS (ESI) m/z 347 [M+H]+.
To a stirred solution of methyl [3-chloro-4-(6-methyl-2-benzo-
thiazolyl)aminophenyl]acetate (274 mg, 0.79 mmol) in THF (5 ml)
was added 0.5 N NaOH (5.0 ml, 2.50 mmol), and the reaction mix-
ture was stirred at room temperature for 15 h. The mixture was
poured into ice-1 N HCl. The resulting precipitate was collected
with suction, and dried under vacuum to give the title compound
(202 mg, 77%) as a pale yellow solid. 1H NMR (DMSO-d6) d 2.35
(s, 3H), 3.60 (s, 2H), 7.10–7.13 (m, 1H), 7.25–7.27 (m, 1H), 7.41–
7.42 (m, 2H), 7.59 (s, 1H), 8.13–8.15 (m, 1H).
5.31. [2-[1-Methyl-3-(1H-pyrrolo[2,3-b]pyridinyl)]-6-
benzoxazolyl]acetic acid (3g)
Yield 62% (two steps). A brown solid. 1H NMR (DMSO-d6) d 3.74
(s, 2H), 3.96 (s, 3H), 7.26 (dd. J = 8.1, 1.5 Hz, 1H), 7.36 (dd, J = 7.8,
4.7 Hz, 1H), 7.62 (d, J = 1.5 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 8.45
(dd, J = 4.7, 1.5 Hz, 1H), 8.53 (s, 1H), 8.62 (dd, J = 7.8, 1.5 Hz, 1H),
12.37 (br s, 1H); MS (ESI) m/z 308 [M+H]+.
5.32. [2-(1-Isoquinolinyl)-6-benzoxazolyl]acetic acid (4b)
Yield 29% (two steps). A pale brown solid. 1H NMR (DMSO-d6) d
3.82 (s, 2H), 7.41–7.43 (m, 1H), 7.82 (s, 1H), 7.88–7.95 (m, 3H),
8.13–8.17 (m, 2H), 8.76–8.78 (m, 1H), 9.53–9.55 (m, 1H), 12.45
(br s, 1H); MS (ESI), m/z 305 [M+H]+.
5.33. [2-(1-Naphthyl)-6-benzoxazolyl]acetic acid (4c)
Yield 38% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.78 (s, 2H), 7.34–7.36 (m, 1H), 7.65–7.78 (m, 4H), 7.84 (d,
J = 8.1 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 8.21–8.23 (m, 1H), 8.43–
8.45 (m, 1H), 9.40–9.42 (m, 1H).
Compound 5a, 5c–h and 15c–g were prepared according to gen-
eral procedure F.
5.37. [4-(2-Benzothiazolyl)amino-3-chlorophenyl]acetic acid
(5a)
5.34. [2-(4-Quinolinyl)-6-benzoxazolyl]acetic acid (4d)
Yield 30% (two steps). A colorless solid. 1H NMR (DMSO-d6) d
3.59 (s, 2H), 7.11–7.15 (m, 1H), 7.25–7.31 (m, 2H), 7.42 (d,
J = 2.0 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H),
8.11–8.13 (m, 1H); MS (ESI) m/z 319 [M+H]+.
Yield 85% (two steps). A brown solid. 1H NMR (DMSO-d6) d 3.82
(s, 2H), 7.41 (dd, J = 8.1, 1.0 Hz, 1H), 7.82 (s, 1H), 7.85 (d, J = 8.3 Hz,
1H), 7.92 (m, 2H), 8.19 (d, J = 7.8 Hz, 1H), 8.31 (d, J = 4.4 Hz, 1H),
9.14 (d, J = 4.4 Hz, 1H), 9.42 (d, J = 8.1 Hz, 1H); MS (ESI) m/z 305
[M+H]+.
5.38. 2-Bromo-6-fluorobenzothiazole (15c)
Yield 40%. A brown solid. 1H NMR (CDCl3) d 7.19–7.24 (m, 1H),
5.35. 1-Isoquinolinecarbaldehyde (13f)
7.49–7.52 (m, 1H), 7.92–7.96 (m, 1H).
To a stirred solution of 1-methylisoquinoline (12g) (300 mg,
2.10 mmol) in dioxane (20 ml) was added SeO2 (323 mg,
2.91 mmol), and the reaction mixture was refluxed for 1.5 h under
N2 atmosphere. After cooled to room temperature, the mixture was
filtered through a celite pad, and the filtrate was evaporated. The
residue was purified by column chromatography on silica gel with
n-hexane–EtOAc (5:1, v/v) as eluent to give the title compound
(252 mg, 77%) as a white solid. 1H NMR (CDCl3) d 7.73–7.79 (m,
5.39. [3-Chloro-4-(6-fluoro-2-
benzothiazolyl)aminophenyl]acetic acid (5c)
Yield 27% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.59 (s, 2H), 7.11–7.15 (m, 1H), 7.25–7.27 (m, 1H), 7.41–7.42 (m,
1H), 7.50–7.53 (m, 1H), 7.70–7.73 (m, 1H), 8.12–8.14 (m, 1H),
9.97 (br s, 1H), 12.42 (br s, 1H); MS (ESI) m/z 337 [M+H]+.

M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
1211
5.40. 2-Bromo-6-chlorobenzothiazole (15d)
streptomycin (Invitrogen Corp.), 2 mM
L
-glutamine (Invitrogen
Corp.) and 1 mg/ml G-418 (Geneticin, Invitrogen Corp.). A Eu-label-
ling Reagent (PerkinElmer Inc.) was used to label the human
VCAM-1/Fc chimera (R&D Systems Inc.). The Eu-labelled protein
was purified with a PD-10 column (Amersham Biosciences KK.)
and stored at ꢁ80 °C until use. All assays were performed in dupli-
cate. In preparation for the assay, the 4B4 cells were suspended at
3 ꢂ 105 cells/ml in Ham’s F-12 medium. One hundred microliter of
the 4B4 cell suspension was placed into each well of a 96-well-cul-
ture plate (Costar Inc.). The plates were incubated at 37 °C in a 5%
CO2 atmosphere for 2 days. Prior to the assay, the medium was dis-
Yield 69%. A yellow solid. 1H NMR (CDCl3) d 7.42–7.46 (m, 1H),
7.76–7.90 (m, 2H).
5.41. [3-chloro-4-(6-Chloro-2-benzothiazolyl)aminophenyl]
acetic acid (5d)
Yield 23% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.61 (s, 2H), 7.26–7.33 (m, 2H), 7.43–7.44 (m, 1H), 7.50–7.52 (m,
1H), 7.92–7.93 (m, 1H), 8.08–8.10 (m, 1H).
carded and each well was washed twice with 300
Wash Buffer (25 mM HEPES, pH 7.5; 150 mM NaCl; 1 mM CaCl2;
1 mM MgCl2; 4 mM MnCl2). Then, 50 l of compound solution
was added to a well, followed by 50 l of 2 nM of Eu-labelled hu-
ll of chilled
5.42. 2-Bromo-5-fluorobenzothiazole (15e)
l
Yield 63%. A yellow solid. 1H NMR (CDCl3) d 7.18–7.23 (m, 1H),
l
7.64–7.77 (m, 2H).
man VCAM-1/Fc chimera diluted with the Wash Buffer (final con-
centration: 1 nM). For assays conducted in the presence of human
5.43. [3-Chloro-4-(5-fluoro-2-benzothiazolyl)aminophenyl]
acetic acid (5e)
serum albumin, 50 ll of compound at various concentrations and
an equal volume of 2 nM Eu-labelled human VCAM-1/Fc chimera
in 6% (w/v) human serum albumin (Sigma Corp.) were distributed
into each well (final concentration: 1 nM). The plates were incu-
bated for 60 min at room temperature and the wells were washed
Yield 27% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.61 (s, 2H), 6.98–7.03 (m, 1H), 7.27 (dd, J = 8.3, 2.0 Hz, 1H), 7.34–
7.37 (m, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.78–7.82 (m, 1H), 8.05 (d,
J = 8.3 Hz, 1H).
four times with 300 ll of chilled Wash Buffer. Finally, 100 ll of the
enhancement solution (PerkinElmer 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 Re-
search fluorometer, model 1234–001; PerkinElmer Inc.). The con-
centration of compound required for 50% inhibition in the assay
was determined.
5.44. 2-Bromo-4-chlorobenzothiazole (15f)
Yield 65%. A pale yellow solid. 1H NMR (CDCl3) d 7.33–7.38 (m,
1H), 7.49–7.53 (m, 1H), 7.67–7.71 (m, 1H).
5.45. [3-chloro-4-(4-Chloro-2-benzothiazolyl)aminophenyl]
acetic acid (5f)
5.50. Estimated serum concentration
Female BALB/c mice (8 weeks old, Charles River Japan, Inc.)
were used. Each group consisted of four animals. The mice were or-
ally administered the compound dissolved in 0.5% (w/v) methyl-
cellulose (MC) at a dose of 10 mg/ml/kg. After 15 or 60 min,
blood samples were collected via inferior vena cava from the ani-
mals under ether anesthesia. The blood samples were stored at
room temperature and centrifuged at 2000 rpm for 10 min at
4 °C. The serum samples were subsequently stored in a ꢁ20 °C
freezer prior to analysis. According to the VLA-4/VCAM-1 binding
Yield 51% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.63 (s, 2H), 7.12–7.16 (m, 1H), 7.29–7.31 (m, 1H), 7.38–7.41 (m,
1H), 7.46–7.47 (m, 1H), 7.76–7.78 (m, 1H), 8.18–8.21 (m, 1H),
10.26 (br s, 1H), 12.45 (br s, 1H).
5.46. 2-Bromobenzothiazole (15g)
Yield 57%. A brown oil. 1H NMR (CDCl3) d 7.39–7.51 (m, 2H),
7.77–7.82 (m, 1H), 7.94–8.00 (m, 1H).
assay, instead of the compound solution, 50 ll of serum samples
at various concentrations were added into each well (final concen-
tration: 0.01–10%). As for the calibration curve, each diluted com-
pound solution was also assayed in the presence of the same
concentration of untreated mouse serum.
5.47. [4-(2-Benzothiazolyl)amino-5-chloro-2-fluorophenyl]
acetic acid (5g)
Yield 36% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.63 (s, 2H), 7.17–7.21 (m, 1H), 7.32–7.39 (m, 1H), 7.52–7.54 (m,
1H), 7.61–7.63 (m, 1H), 7.82–7.84 (m, 1H), 8.38 (m, 1H), 10.17
(br s, 1H), 12.52 (br s, 1H).
5.51. Murine bronchial inflammatory model
Female BALB/c AnNCrj mice (8 weeks old, Charles River Japan,
Inc.) received an oral administration of cyclophosphamide dis-
solved in water at a dose of 150 mg/kg (day 0). On day 2 and 14,
5.48. [5-Chloro-2-fluoro-4-(5-fluoro-2-benzothiazolyl)
aminophenyl]acetic acid (5h)
five-hundred
lg protein of Ascaris suum extract (LSL Co., Ltd) in
0.2 ml saline containing 4.5 mg aluminum hydroxide was injected
intraperitoneally. On day 22, the mice were challenged intratrach-
eally under anesthesia with 300 lg (30 ll) protein of Ascaris suum
extract. In the negative control group, sensitized mice were chal-
Yield 28% (two steps). A pale yellow solid. 1H NMR (DMSO-d6) d
3.64 (s, 2H), 7.03–7.08 (m, 1H), 7.45–7.55 (m, 2H), 7.83–7.86 (m,
1H), 8.28–8.31 (m, 1H), 10.26 (br s, 1H), 12.56 (br s, 1H).
lenged with saline instead of the antigen.
5.49. VLA-4/VCAM-1 binding assay
5.52. Effect on eosinophil infiltration
A human VLA-4-expressing cell line, 4B4, was established at
Pharmacopeia Inc., by transfecting both the
a
4 gene and b1 gene
Test compounds, which were dissolved in 0.5% MC containing
0.03% Tween 80, were orally administered 15 min before and 8, 24,
and 32 h after the antigen challenge at a dose of 5 or 15 mg/kg.
Forty-eight hours after antigen challenge, the mice were sacrificed
and BALF was collected using tracheal polyethylene cannula with
of VLA-4 into CHO-K1 cells. The 4B4 cells were maintained in
Ham’s F-12 medium (Sigma Corp.) supplemented with 10% (v/v)
fetal calf serum (REHATUIN Fetal Bovine Serum, Serologicals
Corp.), 100 U/ml penicillin (Invitrogen Corp.), 100 lg/ml

1212
M. Setoguchi et al. / Bioorg. Med. Chem. 20 (2012) 1201–1212
4. (a) Barthel, S. R.; Johansson, M. W.; McNamee, D. M.; Mosher, D. F. J. Leukocyte
Biol. 2008, 83, 1; (b) Abonia, J. P.; Hallgren, J.; Jones, T.; Shi, T.; Xu, Y.; Koni, P.;
Flavell, R. A.; Boyce, J. A.; Austen, K. F.; Gurish, M. F. Blood 2006, 108, 1588.
5. (a) Silverman, M. D.; Hass, C. S.; Rad, A. M.; Arbab, A. S.; Koch, A. E. Arthritis
Rheum. 2007, 56, 1817; (b) Carter, R. A.; Wicks, I. P. Arthritis Rheum. 2001, 44,
985; (c) Seiffge, D. J. Rheumatol. 1996, 23, 2086.
6. (a) Rice, G. P. A.; Hartung, H. P.; Calabresi, P. A. Neurology 2005, 64, 1336; (b)
Yednock, T. A.; Cannon, C.; Fritz, L. C.; Sanchez-Madrid, F.; Steinman, L.; Karin,
N. Nature 1992, 356, 63; (c) Piraino, P. S.; Yednock, T. A.; Freedman, S. B.;
Messersmith, E. K.; Pleiss, M. A.; Vandevert, C.; Thorsett, E. D.; Karlik, S. J. J.
Neuroimmunol. 2002, 131, 147.
7. (a) Podolsky, D. K. N. Eng. J. Med. 1991, 325, 928; (b) Bischoff, S. C.; Wedemeyer,
J.; Herrmann, A.; Meier, P. N.; Trautwein, C.; Cetin, Y.; Maschek, H.; Stolte, M.;
Gebel, M.; Manns, M. P. Histopathology 1996, 28, 1; (c) Hogan, S. P.; Rothenberg,
M. E. Aliment. Pharmacol. Ther. 2004, 20, 1231.
8. (a) Tilley, J. W. Expert Opin. Ther. Pat. 2008, 18, 841; (b) Yang, G. X.; Hagmann,
W. K. Med. Res. Rev. 2003, 23, 369; (c) Jackson, D. Y. Curr. Pharm. Des. 2002, 8,
1229; (d) Vanderslice, P.; Biediger, R. J.; Woodside, D. G.; Berens, K. L.; Holland,
G. W.; Dixon, R. A. F. Pulmon. Pharmacol. Ther. 2004, 17, 1.
9. (a) Miller, D. H.; Khan, O. A.; Sheremata, W. A.; Blumhardt, L. D.; Rice, G. P. A.;
Libonati, M. A.; Willmer-Hulme, A. J.; Dalton, C. M.; Miszkiel, K. A.; O’Connor, P.
W. N. Eng. J. Med. 2003, 348, 15; (b) Ghosh, S.; Goldin, E.; Gordon, F. H.;
Malchow, H. A.; Madsen, J. R.; Rutgeerts, P.; Vynálek, P.; Zádorová, Z.; Palmer,
T.; Donoghue, S. N. Eng. J. Med. 2003, 348, 24; (c) Steinman, L. Nat. Rev. Drug
Disc. 2005, 4, 510; (d) Elan-Biogen official home page: http://www.tysabri.com/
.
10. Muro, F.; Iimura, S.; Yoneda, Y.; Chiba, J.; Watanabe, T.; Setoguchi, M.;
Takayama, G.; Yokoyama, M.; Takashi, T.; Nakayama, A.; Machinaga, N. Bioorg.
Med. Chem. 2009, 17, 1232.
2 ꢂ 0.5 ml Hanks’ balanced salt solution. The cells in the BALF were
counted in a particle analyzer CDA-500 (Sysmex Corp.). Cytocentri-
fuged preparations (Cytospin 2; Shandon) were stained with
Wright’s stain solution (Muto Chemical Co., Ltd) for differential
counts, based on standard morphologic criteria. The number of
eosinophils was calculated by multiplying the total cell number by
the percentage of eosinophils in the cytocentrifuged preparations.
5.53. Pharmacokinetic studies on rats
Male Sprague–Dawley rats [7 weeks old, SLC Japan] were used.
The animals were fasted for 16 h prior to drug administration,
whereas access to water was provided ad libitum. Each group
consisted of three animals. For the oral administration, the test
compound was suspended in 0.5% (w/v) MC and administrated in
rats at the dose of 1 mg/kg. For the intravenous administration,
the test compound was dissolved in saline with 3 equiv NaOH solu-
tion and administrated to the jugular vein at the dose of 1 mg/kg.
Blood samples (0.5 ml) were collected at 0.5, 1, 2, 4, and 6 h for
po, or 0.08, 0.5, 1, 2, and 4 h for iv after the administration. These
analytical samples were stored at room temperature, followed by
centrifugation at 3000 rpm for 10 min at 4 °C. The plasma fractions
were subsequently stored in a ꢁ20 °C freezer until analysis. The
concentrations of the test compounds were measured by an
LC/MS/MS method consisting of an Alliance 2695 HPLC (Waters),
11. Muro, F.; Iimura, S.; Sugimoto, Y.; Yoneda, Y.; Chiba, J.; Watanabe, T.;
Setoguchi, M.; Iigou, Y.; Matsumoto, K.; Satoh, A.; Takayama, G.; Taira, T.;
Yokoyama, M.; Takashi, T.; Nakayama, A.; Machinaga, N. J. Med. Chem. 2009, 52,
7974.
Symmetry Shield RP8, 2.1 ꢂ 50 mm, 3.5
lm column (Waters),
12. (a) Fischer, R.; Lehmann, T.; Mueller, G.; Hessler, G.; Tajimi, M.; Ziegelbauer, K.;
Okigami, H.; Hasegawa, H.; Komura, H.; Mizoguchi, M. WO2001058871.; (b)
Clark, D. E.; Eastwood, P. R.; Harris, N. V.; McCarthy, C.; Morley, A. D.; Pickett, S.
D. WO2000061580.; (c) Brittain, D. R.; Johnstone, C.; Davies, G. M.; Large, M. S.
WO200005223.; (d) Brittain, D. R.; Johnstone, C.; Davies, G. M.; Large, M. S.
WO200005224.
13. Pedras, M. S. C.; Ahiahonu, P. W. K. Bioorg. Med. Chem. 2002, 10, 3307.
14. Kato, M.; Ito, K.; Nishino, S.; Yamakuni, H.; Takasugi, H. Chem. Pharm. Bull.
1995, 43, 1351.
and TSQ-700 (Thermo Electron, Waltham, MA). The mobile phase
consisted of 10 mM HCOONH4 in water/methanol; the gradient
condition was 90:10 to 10:90 (the plasma concentrations versus
the time data were analyzed by non-compartmental approaches
using the WinNonlin software program (version 1.13.1 Pharsight,
Mountain View, CA)). The pharmacokinetics parameters after oral
and intravenous administration were calculated using the
15. Evans, D.; Dunwell, D. W.; Hicks, T. A. US3962441A1.
16. Barrows, R. S.; Lindwall, H. G. J. Am. Chem. Soc. 1942, 64, 2430.
17. Carr, A. A.; Maynard, G. D.; Kane, J. M.; Cheng, H. C.; Dudley, M. W.
US5371093A1.
non-compartmental method without extrapolation using
validated program developed in-house.
a
18. RajanBabu, T. V.; Chenard, B. L.; Petti, M. A. J. Org. Chem. 1986, 51, 1704.
19. Muro, F.; Iimura, S.; Yoneda, Y.; Chiba, J.; Watanabe, T.; Setoguchi, M.; Iigou, Y.;
Takayama, G.; Yokoyama, M.; Takashi, T.; Nakayama, A.; Machinaga, N. Bioorg.
Med. Chem. 2008, 16, 9991.
All the animal experiments were conducted with the approval
of the Animal Experiment Ethics Committee of Daiichi Pharmaceu-
tical Co., Ltd.
20. Chiba, J.; Iimura, S.; Yoneda, Y.; Watanabe, T.; Muro, F.; Tsubokawa, M.; Iigou,
Y.; Satoh, A.; Takayama, G.; Taira, T.; Yokoyama, M.; Takashi, T.; Nakayama, A.;
Machinaga, N. Bioorg. Med. Chem. 2007, 15, 1679.
References and notes
21. (a) Ito, T.; Takahashi, M.; Sudo, K.; Sugiyama, Y. J. Pharmacol. Exp. Ther. 2007,
320, 124; (b) Ito, T.; Takahashi, M.; Sudo, K.; Sugiyama, Y. J. Pharm. Sci. 2009, 98,
1545; (c) Ito, T.; Takahashi, M.; Okazaki, O.; Sugiyama, Y. Mol. Pharm. 2010, 7,
1254.
1. Hynes, R. O. Cell 1987, 48, 549.
2. Elices, M. J.; Osborn, L.; Takeda, Y.; Crouse, C.; Luhowskyj, S.; Hemler, M. E.;
Lobb, R. R. Cell 1990, 60, 577.
3. (a) Guan, J. L.; Hyens, R. O. Cell 1990, 60, 53; (b) Wayner, E. A.; Garcia-Pardo, A.;
Humphries, M. J.; McDonald, J. A.; Carter, W. G. J. Cell. Biol. 1989, 109, 1321.
22. Holzmann, B.; Mclntyre, B. W.; Weissman, I. L. Cell 1989, 56, 37.