A novel and potent VLA-4 antagonist based on trans-4-substituted cyclohexanecarboxylic acid

Article abstract of DOI:10.1016/j.bmc.2008.12.026

During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the diphenylurea structure. However, this modification resulted in a significant decrease (3, IC₅₀ = 19 nM) in VLA-4 inhibitory activity compared to 1 (IC₅₀ = 1.6 nM). To address this discrepancy, we worked on optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discovery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC₅₀ = 2.8 nM) as a novel and potent VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties (CL = 3.3 ml/min/kg, F = 51%) in rats.

Full text of DOI:10.1016/j.bmc.2008.12.026

Bioorganic & Medicinal Chemistry 17 (2009) 1232–1243
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel and potent VLA-4 antagonist based on trans-4-substituted
cyclohexanecarboxylic acid
a
a
a
b
a
Fumihito Muro a, , Shin Iimura , Yoshiyuki Yoneda , Jun Chiba , Toshiyuki Watanabe , Masaki Setoguchi ,
*
Gensuke Takayama c, Mika Yokoyama c, Tohru Takashi d, Atsushi Nakayama a, Nobuo Machinaga a
a Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Biological Research Laboratories III, Daiichi Sankyo, Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
d Biological Research Laboratories IV, Daiichi Sankyo, 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:
During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of
benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced
the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the
diphenylurea structure. However, this modification resulted in a significant decrease (3, IC50 = 19 nM)
in VLA-4 inhibitory activity compared to 1 (IC50 = 1.6 nM). To address this discrepancy, we worked on
optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discov-
ery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC50 = 2.8 nM) as a novel and potent
VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties
(CL = 3.3 ml/min/kg, F = 51%) in rats.
Received 3 November 2008
Revised 9 December 2008
Accepted 10 December 2008
Available online 24 December 2008
Keywords:
VLA-4
Integrin
trans-4-Substituted cyclohexanecarboxylic
acid
Ó 2008 Elsevier Ltd. All rights reserved.
Pharmacophore
1. Introduction
in a murine asthma model by oral dosing at 15 mg/kg. In addition,
during the course of this study it was found that introducing a hal-
VLA-4 (very late antigen-4,
a
4b1 integrin, or CD49d/CD29) is a
ogen atom (Cl or Br) into the 3-position on the central benzene in
the diphenylurea moiety as in 1 tends to improve the pharmacoki-
netic properties in rodents and dogs.11 However, the pharmacoki-
netic profile of 1 (rat, CL = 6.3 ml/min/kg, F = 23%; dog, CL = 5.2 ml/
min/kg, F = 38%) was not fully pursued. From the result that even
the fine tuning in diphenylurea moiety provided the moderate
improvement of the poor pharmacokinetic properties, we consid-
ered that drastic structural modification of the diphenylurea moi-
key cell receptor expressed on most leukocytes.1 The major count-
erligands for VLA-4 are VCAM-1 (Vascular Cell Adhesion Molecule-
1) expressed on cytokine-stimulated endothelial cells and the
alternatively spliced connecting segment-1 domain of fibronectin
on the extracellular matrix.2,3 Through the interaction with these
ligands, VLA-4 plays a significant role in the process of adhesion,
migration, and activation of inflammatory leukocytes at sites of
inflammation. Monoclonal antibodies to the
a
4 subunit and small
ety could lead to
pharmacokinetic properties. On the other hand, piperidinylacetic
acid derivative 2 was reported to show an IC50 of 0.5 M or less
a
substantial improvement of the
molecular VLA-4 antagonists4 have been used to demonstrate the
efficacy in inflammatory animal models of asthma,5 multiple scle-
rosis (MS),6 rheumatoid arthritis,7 and inflammatory bowel disease
l
in a Ramos cell assay,12a in which diphenylurea moiety was
replaced by a 2-(phenylamino)benzoxazole. Therefore, we applied
this diphenylurea variant to 1 by replacing the diphenylurea moi-
ety with a 2-(2-methyamino)benzoxazole to see whether the com-
bination maintained the potent activity. Unfortunately, the
prepared compound 3 was found to be about 12-fold less potent
(IC50 = 19 nM) than 1 in the binding assay and this result implied
that the benzoic acid moiety as in 3 required optimization. For this
reason, we decided to optimize the benzoic acid moiety prior to
further investigation of the diphenylurea moiety.
(IBD).8 Natalizumab,9 a humanized monoclonal antibody to the
a4
subunit, has been approved by the FDA for the treatment of MS and
Crohn’s disease. Because of these excellent proofs of concept, the
development of small molecule VLA-4 antagonists with acceptable
oral pharmacokinetic profiles is viewed as reasonable approach to
a novel class of therapeutic agents.
We recently have reported that benzoic acid derivative 110
(Fig. 1) shows potent VLA-4 inhibitory activity (CHO-K1 cells/hu-
man VCAM binding assay, IC50 = 1.6 nM) and demonstrates efficacy
Herein, we report our optimization efforts on the benzoic acid
moiety in 3 and the discovery of trans-4-substituted cyclohexane-
carboxylic acid derivative 11b showing an IC50 value of single digit
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8609.
E-mail address: muro.fumihito.iy@daiichisankyo.co.jp (F. Muro).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.026

F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
1233
Benzoic acid moiety
Diphenylurea moiety
F
F
N
O
O
CO2H
O
N
H
N
H
Me
Cl
1: VLA-4/VCAM-1 binding assay, IC50 = 1.6 nM
2-(Phenylamino)benzoxazole
F
CO2H
O
H
N
N
O
O
N
N
N
Me
N
H
O
O
CO2H
H
N
O
2: Ramos cell / VCAM-1 binding assay IC50 < 0.5 μM 12a
3: VLA-4/VCAM-1 binding assay, IC50 = 19 nM
Figure 1.
nM in the binding assay. We also report the pharmacokinetics
properties of 11b in rats.
presence of n-BuLi to provide the methyl 2-butynoate 14. After
hydrogenation of the triple bond in 14, the Boc group of the result-
ing methyl butanoate 15 was deprotected by TFA treatment to
yield 5a (Scheme 2).
2. Chemistry
Conversion of the prolinol 12 to methyl 3-[1-(tert-butoxycar-
bonyl)-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]propionate (16) was
achieved by means of a Michael addition with methyl acrylate. Ba-
sic hydrolysis of the ester group of 16 followed by reduction of the
carboxylic acid group with borane–dimethylsulfide complex to af-
ford the alcohol 17, which was converted to the corresponding
aldehyde 18 by oxidation with sulfur trioxide pyridine complex.
Homologation by treatment of the aldehyde 18 with Horner–Em-
mons reagent trimethyl phosphonoacetate, hydrogenation of the
Target compounds were synthesized as depicted in Scheme 1.
Thus, 2-(2-methylphenylamino)-6-benzoxazolylacetic acid (4)
was condensed with (4S)-fluoropyrrolidine derivative 5 using
EDC and HOBt, followed by basic hydrolysis to afford 3 and 6–11.
The intermediates 4 and 5 in Scheme 1 were prepared as shown
in Schemes 2–7. Treatment of 1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-prolinol (12)13 with propargyl bromide gave the propargyl
ether 13, which was coupled with methyl chloroformate in the
F
5
R1
(i)
N
H
F
EDC, HOBt,Et3N
(ii) Hydrolysis
O
N
N
CO2H
O
N
Me
N
H
Me
N
H
R2
O
4
3, 6-11
5a; R1 =
6; R2 =
O
CO2Me
CO2Me
O
O
CO2H
5b; R1 =
5c; R1 =
7; R2 =
8; R2 =
CO2H
O
O
CO2Me
O
CO2H
CO2Et
CO2H
N
N
N
N
N
N
5d; R1 =
5e; R1 =
9; R2 =
O
O
O
O
CO2Et
CO2H
10; R2 =
5f 13b; R1 =
5g; R1 =
3; R2 =
O
O
CO2Me
O
O
CO2H
11; R2 =
CO2Me
CO2H
Scheme 1. Reagents and conditions: (i) 5, EDC, HOBt, DMAP, or Et3N, DMF; (ii) aq NaOH, THF (21% for 6, 64% for 7, 48% for 8, 42% for 9, 40% for 10, 65% for 3, 48% for 11).

1234
F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
F
F
F
F
CO2Me
iii
i
ii
O
CO2Me
OH
O
O
N
N
N
N
Boc
Boc
Boc
Boc
15
14
12
13
F
iv
O
CO2Me
N
H
5a
Scheme 2. Reagents and conditions: (i) propargyl bromide, NaH, nBu4NI, THF (85%); (ii) n-BuLi, methyl chloroformate, THF (69%); (iii) H2, 5% Pd/C, MeOH (61%); (iv) TFA,
CH2Cl2 (90%).
F
F
F
F
i
ii, iii
iv
OH
O
O
O
OH
F
CO2Me
vi
CHO
N
Boc
16
N
Boc
N
N
Boc
12
Boc
17
18
F
F
v
vii
O
O
O
CO2Me
CO2Me
N
Boc
N
Boc
CO2Me
N
H
19
20
5b
Scheme 3. Reagents and conditions: (i) methyl acrylate, NaH, THF (48%); (ii) aq NaOH, THF; (iii) BH3ÁMe2S, THF (100% for two steps); (iv) SO3ÁPy, Et3N, DMSO, CH2Cl2 (53%);
(v) trimethyl phosphonoacetate, NaH, THF (86%); (vi) 5% Pd/C, H2, MeOH (78%); (vii) TFA, CH2Cl2 (77%).
F
F
F
F
i
ii
iii
O
O
O
CHO
OH
F
N
Boc
OH
N
Boc
N
N
Boc
12
Boc
OBn
v
21
22
23
F
F
iv
vi
CO2Me
CO2Me
O
O
O
CO2Me
N
Boc
N
Boc
N
H
24
25
5c
Scheme 4. Reagents and conditions: (i) cis-4-benzyloxy-2-butenyl bromide, NaH, n-Bu4NI, THF (78%); (ii) 20% Pd(OH)2, H2, EtOH (67%); (iii) SO3ÁPy, Et3N, DMSO, CH2Cl2
(72%); (iv) trimethyl phosphonoacetate, NaH, THF (93%); (v) 5% Pd/C, H2, MeOH (100%); (vi) TFA, CH2Cl2 (85%).
F
F
F
i
ii
R
CO2Et
R
CO2Et
N
N
CO2H
N
Boc
N
Boc
N
H
O
O
26
27a; R = N
27b; R = C
5d; R = N
5e; R = C
Scheme 5. Reagents and conditions: (i) ethyl 1-piperazinylacetate or ethyl 4-piperidinylacetate, EDC, HOBt, Et3N, DMF (100% for 27a, 32% for 27b); (ii) TFA, CH2Cl2 (86% for
5d, 100% for 5e).
F
F
F
ii
i
O
O
O
N
N
N
H
Boc
Boc
CO2Me
CO2Me
CO2Me
28
29
5g
Scheme 6. Reagents and conditions: (i) 5% Rh on alumina, 10 atm H2, EtOH–AcOH (91%); (ii) TFA, CH2Cl2 (84%).
double bond of the methyl 2-pentenoate 19 and deprotection of
the Boc group in the methyl pentanoate 20 with TFA to afford 5b
(Scheme 3).
The prolinol 12 was converted to the ether 21 by treatment of
cis-4-benzyloxy-2-butenyl bromide14 in the presence of NaH and
a catalytic amount of n-Bu4NI. Hydrogenolysis of 21 resulted in
reduction of the double bond and removal of the benzyl group at
the same time to afford the alcohol 22, which was converted to
methyl 6-[(4S)-fluoro-(2S)-pyrrolidinylmethoxy]hexanoate (5c)
following the same procedure as that described for the preparation
of 5b (Scheme 4).
Commercially available ethyl 1-piperazinylacetate or ethyl 4-
piperidinylacetate was condensed with 1-(tert-butoxycarbonyl)-
(4S)-fluoro-L
-proline (26)15 using EDC and HOBt, followed by

F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
1235
CO2Et
CO2Et
F
BnO
O2N
F
HO
HO
i
ii
iii
iv
CO2H
BnO
O2N
O2N
O2N
30
34
31
32
33
HO
v
vi
CO2Me
O
O
CO2Me
CO2H
Me
N
Me
N
O2N
H
H
N
N
35
4
Scheme 7. Reagents and conditions: (i) BnBr, K2CO3, DMF (91%); (ii) NaH, diethyl malonate, DMF (100%); (iii) concd HCl, AcOH, reflux; (iv) concd H2SO4, MeOH, reflux (84%);
(v) (1) H2, Pd/C, EtOH; (2) 2-methylphenylisothiocyanate; (3) HgO, EtOH, reflux (three steps, 84%); (vi) 0.25 N NaOH, THF (77%).
deprotection of the Boc group of the resulting compounds 27a–b
to afford 5d and 5e (Scheme 5).
Acylic type derivatives showed a significant loss of potency (6,
IC50 = 831 nM, 7, IC50 = 198 nM, 8, IC50 = 164 nM) compared with
3 (IC50 = 19 nM), whereas cyclic type derivatives retained the po-
tency (10, IC50 = 19 nM and 11, IC50 = 15 nM) except for piperazine
derivative (9, IC50 = 50 nM).
The aromatic ring of methyl 4-[1-(tert-butoxycarbonyl)-(4S)-
fluoro-(2S)-pyrrolidinylmethoxy]benzoate (28)13b was reduced by
hydrogenation at 10 atm in the presence of 5% Rh on alumina as
a catalyst, followed by deprotection of the Boc group of the cyclo-
hexane 29 to afford 5g (Scheme 6).
Starting from commercially available 2-nitro-5-fluorophenol
(30), 2-(2-methylphenylamino)-6-benzoxazolylacetic acid (4) was
synthesized by following the reported procedure12 (Scheme 7).
In addition to this structure–activity relationship, during the
medicinal chemistry process, we happened to realize from HPLC
analysis that the cyclohexanecarboxylic acid derivative 11 con-
tained a small amount of a more polar product (11b, ꢀ14%), which
was inseparable by conventional column chromatography. Thus,
we carried out separation of compound 11 utilizing HPLC to afford
the major product 11a as a less polar fraction and the minor prod-
uct 11b as a more polar fraction. From the NMR and MS spectra of
both products, it was inferred that 11a and 11b could be the cis-
and trans-isomer of the 4-substituted cyclohexanecarboxylic acid.
On the other hand, Iguchi et al. have reported that reduction of
methyl 4-propylbenzoate using [RhCl(COD)]2 and hydrogen yields
the cis-isomer (75%) and the trans-isomer (14%) of methyl 4-pro-
pylcylohexanecarboxylate.16 From that result, it was considered
that the isomers should be produced in the Rh catalyzed hydroge-
nation reaction of benzoic acid derivative 28 to cyclohexane
derivative 29 (Scheme 6).
3. Results and discussion
3.1. In vitro activity
Compounds 3 and 6–11 were evaluated for their activities to in-
hibit the binding of CHO (Chinese hamster ovary) cells expressing
VLA-4 to a europium (Eu)-labeled human VCAM-1/Fc chimera.
These results are summarized in Table 1.
Table 1
Next, we evaluated the separated two isomers for their VLA-4
inhibitory activities and the result is shown in Table 2. To our sur-
prise, 11b (the minor product) exhibited single digit activity
(IC50 = 2.8 nM), which was 150-fold more potent than that of 11a
(the major product, IC50 = 443 nM). This result prompted to us to
determine the relative configuration of 11b.
Inhibitory activity of VLA-4 antagonists
F
N
O
N
Me
N
H
R2
O
Compound
R2
IC50 (nM)
831
3.2. Determination of relative configuration of 11b
6
CO2H
O
We attempted to determine the relative configuration of 11a,b
through X-ray crystal structure analysis and comparison of 1H
NMR spectra. At first, in consideration of the ease in providing a
suitable crystal for X-ray crystal analysis, we prepared urea deriv-
CO2H
7
8
198
164
O
O
CO2H
Table 2
Comparison of the inhibitory activity between the separated 11a and 11b
CO2H
9
50
19
N
N
O
O
F
CO2H
10
N
N
O
N
Me
N
H
O
CO2H
O
3
19
15
O
O
CO2H
CO2H
Compound
IC50 (nM)
11a (the major product, less polar)
11b (the minor product, more polar)
443
2.8
11

1236
F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
separation by
F
F
F
F
flash column
i
O
O
O
O
+
N
H
N
Cbz
N
Cbz
N
Cbz
CO2Me
CO2Me
CO2Me
CO2Me
5g
37
37a (61%)
less polar
37b (15%)
more polar
ii
F
F
F
iv
O
O
iii
O
N
N
N
H
CO2H
CO2Me
O
O
N
N
CO2Me
H
H
Me
Me
40
39
38
F
O
N
CO2H
O
N
H
Me
40
Scheme 8. Reagents and conditions: (i) Z–Cl, aq NaHCO3, CH2Cl2 (85%); (ii) H2, Pd(OH)2/C, MeOH (97%); (iii) 2-methylphenylisocyanate, Et3N, THF (56%); (iv) aq NaOH, THF,
MeOH (93%).
ative 40 utilizing the intermediate 5g shown in Scheme 8. Thus,
after protection of the free amine of 5g with benzyloxycarbonyl
(Cbz) group, we investigated the resulting compound 37 by 1H
NMR and HPLC. According to the results, it turned out to be a mix-
ture of the cis- and trans-isomer, as expected above. Next, the sep-
aration of the two isomers was successfully achieved using flash
column chromatography, affording 37a (61%, a less polar fraction)
and 37b (15%, a more polar fraction). After removal of the Cbz
group of 37b, treatment of the resulting amine 38 with 2-meth-
ylphenylisocyanate and basic hydrolysis afforded the urea deriva-
tive 40, which was recrystallized from ethyl acetate. X-ray crystal
structure analysis of the obtained crystal proved that the relative
configuration of the 1 and 4-substituents on the cyclohexane ring
was trans form (Fig. 2). Furthermore, starting from the methyl
trans-4-substituted cyclohexanecarboxylate 38, we synthesized
compound 11 according to the procedure shown in Scheme 1,
making sure that the 1H NMR and MS data were completely iden-
tical with those of 11b. At the same time, we also synthesized
compound 11 starting from methyl cis-4-substituted cyclohexane-
carboxylate 37a, finding that the 1H NMR and MS data were com-
pletely identical with those of 11a and no isomerization occured in
this basic hydrolysis condition. Consequently, we successfully
established the relative configuration of 11b to be trans. On top
of that, it was clarified that the trans-4-substituted cyclohexane-
carboxylic acid skeleton should be the critical pharmacophore
responsible for VLA-4 inhibitory activity.
Figure 2. X-ray structure of 40.
In an effort to elucidate the structure–activity relationship of
the carboxylic acid moiety, we examined the structural compari-
son between benzoic acid derivative (3, IC50 = 19 nM), trans-4-
substituted cyclohexanecarboxylic acid (11b, IC50 = 2.8 nM), and
cis-4-substituted cyclohexanecarboxylic acid (11a, IC50 = 443 nM)
by using the molecular modeling technique. At first, the lowest en-
ergy conformations of these compounds were generated using
‘Macromodel version 9.1113’17 (Fig. 3). We previously reported
that the distance between the nitrogen in the pyrrolidine ring
and the carbon in the carboxylic acid significantly influences on
VLA-4 inhibitory activity.18 Therefore, we measured the corre-
sponding distance of each conformer. However, we were unable
to make the SAR explicit from the results (3, 8.108 Å; 11b,
8.480 Å; 11a, 8.543 Å). On the other hand, we also attempted to
superimpose those carboxylic acid parts using the oxygen at the
4-position in the cyclohexanecarboxylic acid and comparing the
direction of the carboxylic acid group from the oxygen (Fig. 4).
As a result, compound 3 and 11b displayed a quite similar direc-
tion. But the direction of compound 11a was obviously different
from those of 3 and 11b, implying that the direction is responsible
for the activity. As for the 7-fold more potent activity of 11b than 3,
we speculate that the higher activity of 11b could be attributed to
structural flexibility of the cyclohexane ring in 11b, in comparison
with the benzene ring in 3.

F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
1237
3.3. Pharmacokinetic properties of 11b
that these profiles were preferable compared to those of compound
1 (AUC = 1438 ng h/ml at 2 mg/kg oral dosing, CL = 6.3 ml/min/kg,
F = 23%).10
In order to make sure how the replacement of the diphenylurea
moiety with 2-(2-methylphenylamino)benzoxazole moiety affects
pharmacokinetic properties, we determined the pharmacokinetic
profile of 11b in rats. As can be seen in Table 3, compound 11b
showed excellent exposure (AUC = 14,932 ng h/ml), plasma clear-
ance (CL = 3.3 ml/min/kg) and oral bioavailability (F = 51%), finding
4. Conclusion
To improve the pharmacokinetic properties of the potent VLA-4
antagonist 1 (IC50 = 1.6 nM), we firstly replaced the diphenylurea
moiety in 1 with a 2-(phenylamino)benzoxazole moiety. This mod-
ification resulted in compound 3 showing less potent activity
(IC50 = 19 nM) than 1. Therefore, we focused on optimization of
the benzoic acid moiety of 3 prior to getting around to the modifi-
cation of the diphenylurea moiety. Consequently, we discovered
the trans-4-substituted cyclohexanecarboxylic acid 11b, which
exhibits comparable activity with an IC50 value of 2.8 nM to 1.
On top of that, compound 11b shows preferable pharmacokinetic
properties (CL = 3.3 ml/min/kg, F = 51%) compared to those of 1 in
rats, indicating that the structural modification of the diphenylurea
moiety should lead to improvement of pharmacokinetic properties.
Further biological evaluation of 11b and structural modification of
the 2-(phenylamino)benzoxazole moiety, while keeping the trans-
4-substituted cyclohexanecarboxylic acid skeleton as a valuable
pharmacophore to retain VLA-4 inhibitory activity, will be reported
in a forthcoming publication.
5. Experimental
5.1. General
The melting points were determined on a YANACO MP-J3 and
were uncorrected. 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 pre-coated TLC glass sheets
with Silica Gel 60 F254. 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 spec-
tral splitting patterns are designated as follows: s, singlet; d, dou-
blet; dd, doublet of doublets; t, triplet; q, quartet; m, multiple. The
IR spectra were recorded on a HORIBA FT-720 spectrometer. The
mass spectra were recorded on a SCIEX API-150EX spectrometer
(ESI) or a JEOL JMS-HX110 spectrometer (FAB). The HRMS spectra
were recorded on a JEOL JMS-100LP spectrometer. Elemental anal-
ysis was performed using a Perkin–Elmer CHNS/O 2400II, a Leco
CHNS-932 and a YOKOKAWA analysis IC7000RS.
Figure 3. Stable conformations of 3, 11b and 11a.
5.2. General procedure A: preparation of 4-[(4S)-fluoro-1-[2-(2-
methylphenylamino)-6-benzoxazolylacetyl]-(2S)-
pyrrolidinylmethoxy]benzoic acid (3)
A mixture of [2-(2-methylphenylamino)-6-benzoxazolyl]acetic
acid (4) (300 mg, 1.06 mmol), methyl 4-[(4S)-fluoro-(2S)-pyrrolid-
inylmethoxy]benzoate13b (5f) (269 mg, 1.06 mmol), EDCÁHCl
(305 mg, 1.59 mmol), HOBt (5 mg, 0.04 mmol) and DMAP (5 mg,
0.04 mmol) in DMF (10 ml) was stirred at room temperature for
15 h. The mixture was diluted with H2O and extracted with EtOAc.
Figure 4. Comparison of the direction of the carboxylic acid moiety (gray: 3, green:
11b, yellow: 11a).
Table 3
Pharmacokinetic properties of 11b in rats (n = 4)
F (%)
PO (5 mg/kg)
Cmax (ng/ml)
10,441
IV (1 mg/kg)
AUC (ng h/ml)
14,932
T1/2 (h)
AUC (ng h/ml)
5804
CL (ml/min/kg)
3.3
Vdss (l/kg)
0.16
T1/2 (h)
51
1.5
1.1

1238
F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
The combined extracts were washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel with CHCl3–EtOAc (9:1, v/v) as an
eluent to give methyl 4-[(4S)-fluoro-1-[2-(2-methylphenylami-
no)-6-benzoxazolylacetyl]-(2S)-pyrrolidinylmethoxy]benzoate
(573 mg, 100%) as a pale yellow foam. 1H NMR (CDCl3) d 2.06–2.22
(1H, m), 2.36 (3H, s), 2.43–2.59 (1H, m), 3.69–4.15 (8H, m), 4.52–
4.65 (2H, m), 5.24–5.37 (1H, m), 6.86–7.10 (4H, m), 7.21–7.32
(4H, m), 7.39–7.42 (1H, m), 7.95–8.01 (2H, m), 8.05–8.07 (1H, d,
J = 8.1 Hz); MS (ESI) m/z 518 [M+H]+.
To a stirred solution of methyl 4-[(4S)-fluoro-1-[2-(2-methyl-
phenylamino)-6-benzoxazolylacetyl]-(2S)-pyrrolidinylmeth-
oxy]benzoate (573 mg, 1.11 mmol) in THF (10 ml) was added
0.25 N NaOH (8.8 ml, 2.22 mmol) and the reaction mixture was stir-
red at room temperature for 15 h. The mixture was acidified with
1 N HCl and extracted with CHCl3–MeOH (10:1, v/v). The combined
extracts were dried over MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel with
CHCl3–MeOH (20:1 to 10:1, v/v) as an eluent to give the title com-
pound (365 mg, 65%) as a colorless amorphous solid. 1H NMR
(DMSO-d6) d 2.25–2.51 (2H, m), 2.30 (3H, s), 3.70–4.67 (7H, m),
5.30–5.50 (1H, m), 7.03–7.09 (4H, m), 7.24–7.34 (4H, m), 7.81–
7.91 (3H, m), 9.61 (1H, br s); MS (ESI) m/z 504 [M+H]+; HRMS
(ESI) Calcd for C28H26FN3O5+H: 504.19347. Found: 504.19076.
Anal. Calcd for C28H26FN3O5Á0.5H2O: C, 65.62; H, 5.31; N, 8.20.
Found: C, 65.97; H, 5.61; N, 7.73.
N, 8.25; F, 3.73. Found: C, 65.66; H, 6.43; N, 8.02; F, 3.59; Major
(1,4-cis isomer): minor (1,4-trans isomer) = 6:1.
5.6. Purification of 11 by HPLC
HPLC analysis and purification of 11 were performed on a SHI-
MADZU 10A series with
a Waters Symmetry C18 column
(4.6 Â 250 mm) using MeCN–0.02 N NaOAc buffer (1:1, v/v) as an
eluent.
5.6.1. cis-4-[[2-(2-Methylphenylamino)-6-benzoxazolylacetyl]-
(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic
acid (11a)
HPLC retention time: 4.4 min (less polar); IR (KBr) 2933, 1693,
1641, 1575 cmÀ1 1H NMR (CDCl3) d 1.45–1.55 (2H, m), 1.68–2.02
;
(6H, m), 2.11–2.52 (3H, m), 2.34 (3H, s), 3.30–4.11 (7H, m), 4.22–
4.38 (1H, m), 5.14–5.29 (1H, m), 7.05–7.12 (2H, m), 7.20–7.33 (5H,
m), 7.72 (1H, dd, J = 8.0, 10.6 Hz); MS (FAB) m/z 510 [M++H]+; HRMS
(ESI) Calcd for C28H32FN3O5+H: 510.24042. Found: 510.23849.
5.6.2. trans-4-[[2-(2-Methylphenylamino)-6-
benzoxazolylacetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (11b)
HPLC retention time: 3.3 min (more polar); IR (KBr) 2937, 1699,
1641, 1574 cmÀ1 1H NMR (CDCl3) d 1.26–1.30 (2H, m), 1.45–1.52
;
(2H, m), 2.05–2.10 (5H, m), 2.21–2.50 (2H, m), 2.36 (3H, s), 3.22–
3.26 (1H, m), 3.32–3.98 (6H, m), 4.20–4.42 (1H, m), 5.17–5.31
(1H, m), 7.09–7.11 (2H, m), 7.22–7.36 (5H, m), 7.81–7.82 (1H,
m); MS (FAB) m/z 510 [M++H]+; Anal. Calcd for C28H32FN3O5: C,
66.00; H, 6.33; N, 8.25. Found: C, 65.71; H, 6.42; N, 8.08.
Compound 9–11 were prepared according to general procedure
A.
5.3. 4-[(4S)-Fluoro-1-[2-(2-methylphenylamino)-6-
benzoxazolylacetyl]-(2S)-pyrrolidinylcarbonyl]-1-
piperazinylacetic acid (9)
5.7. General procedure B: preparation of 4-[(4S)-fluoro-1-[2-(2-
methylphenylamino)-6-benzoxazolylacetyl]-(2S)-
pyrrolidinylmethoxy]butanoic acid (6)
Yield 42% (two steps). Colorless amorphous solid. IR (ATR) 1633,
1571, 1434, 1242, 1223, 982, 752 cmÀ1 1H NMR (DMSO-d6) d
;
1.90–2.22 (2H, m), 2.30 (3H, s), 2.46–2.67 (4H, m), 3.14 (2H, d,
J = 5.9 Hz), 3.37–4.04 (8H, m), 4.90–5.12 (1H, m), 5.18–5.38 (1H,
m), 6.97–7.10 (2H, m), 7.22–7.28 (3H, m), 7.40 (1H, d, J = 1.2 Hz),
7.83 (1H, t, J = 7.1 Hz), 9.62 (1H, br s); MS (ESI) m/z 524 [M+H]+;
HRMS (ESI) Calcd for C27H30FN5O5+H: 524.23092. Found:
524.22953. Anal. Calcd for C27H30FN5O5Á2H2O: C, 57.95; H, 6.12;
N, 12.52. Found: C, 58.01; H, 5.84; N, 12.46.
A mixture of [2-(2-methylphenylamino)-6-benzoxazolyl]acetic
acid (4) (282 mg, 1.00 mmol), methyl 4-[(4S)-fluoro-(2S)-pyrrolid-
inyl]methoxybutanoate (5a) (219 mg, 1.00 mmol), EDCÁHCl
(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 24 h. The mixture was diluted with H2O and extracted with
EtOAc. The combined extracts were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
preparative TLC (CHCl3–acetone = 10:1, v/v) to give methyl 4-
[(4S)-fluoro-1-[2-(2-methylphenylamino)-6-benzoxazolylacetyl]-
(2S)-pyrrolidinylmethoxy]butanoate (378 mg, 78%) as a yellow oil.
1H NMR (CDCl3) d 1.88–2.30 (5H, m), 2.35 (3H, s), 2.37–2.45 (3H,
m), 3.28–4.00 (9H, m), 4.12–4.48 (1H, m), 5.15–5.37 (1H, m),
7.06–7.10 (2H, m), 7.22–7.32 (4H, m), 7.38 (1H, dd, J = 8.1,
4.9 Hz), 8.05 (1H, d, J = 8.1 Hz); MS (ESI) m/z 484 [M+H]+.
To a solution of methyl 4-[(4S)-fluoro-1-[2-(2-methylphenyla-
mino)-6-benzoxazolylacetyl]-(2S)-pyrrolidinylmethoxy]butanoate
(378 mg, 0.78 mmol) in THF (20 ml) was added 0.25 N NaOH
(20 ml) and the reaction mixture was stirred at 50 °C for 17 h. After
being cooled to room temperature, the mixture was acidified with
1 N HCl and extracted with CHCl3–MeOH (10:1, v/v). The combined
extracts were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by preparative TLC
(CHCl3–MeOH = 10:1, v/v) to give the title compound (99 mg,
27%) as a yellow oil. To a solution of the compound (99 mg,
0.21 mmol) in EtOH (3 ml) was added 1 N NaOH (0.21 ml,
0.21 mmol) and stirred. The solution was concentrated in vacuo
and triturated with diethyl ether to give the title compound as a
pale yellow amorphous solid. Sodium salt. 1H NMR (CDCl3) d
1.68–2.30 (5H, m), 2.24 (3H, s), 2.41–2.46 (2H, m), 3.30–4.00 (7H,
m), 4.24–4.40 (1H, m), 5.14–5.28 (1H, m), 7.07–7.16 (2H, m),
5.4. 1-[(4S)-Fluoro-1-[2-(2-methylphenylamino)-6-
benzoxazolylacetyl]-(2S)-pyrrolidinylcarbonyl]-4-
piperidinylacetic acid (10)
Yield 40% (two steps). Colorless amorphous solid. IR (ATR) 1712,
1639, 1573 cmÀ1 1H NMR (CDCl3) d 1.21–1.26 (2H, m), 1.79–1.87
;
(2H, m), 2.04–2.09 (1H, m), 2.28–2.30 (3H, m), 2.35 (3H, s), 2.44–
2.64 (3H, m), 3.06–3.15 (1H, m), 3.75–4.01 (4H, m), 4.58–4.61
(1H, m), 5.02–5.04 (1H, m), 5.17–5.31 (1H, m), 7.06–7.12 (2H,
m), 7.21–7.35 (5H, m), 7.88 (1H, d, J = 8.8 Hz); MS (ESI) m/z 523
[M+H]+; HRMS (ESI) Calcd for C28H31FN4O5+H: 523.23567. Found:
523.23313.
5.5. 4-[[2-(2-Methylphenylamino)-6-benzoxazolylacetyl]-(4S)-
fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid
(11)
Yield 48% (two steps). Colorless amorphous solid. 1H NMR
(CDCl3) d 1.24–2.28 (9H, m), 2.35 (3H, s), 2.38–2.51 (2H, m),
3.34–4.16 (7H, m), 4.22–4.41 (1H, m), 5.14–5.30 (1H, m), 7.06–
7.16 (2H, m), 7.21–7.39 (5H, m), 7.79–7.94 (1H, m); MS (ESI) m/z
510 [M+H]+; HRMS (ESI) Calcd for C28H32FN3O5+H: 510.24042.
Found: 510.24033. Anal. Calcd for C28H32FN3O5: C, 66.00; H, 6.33;

F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
1239
7.20–7.34 (5H, m), 7.69–7.75 (1H, m); MS (ESI), m/z 470 [M+H]+;
HRMS (ESI) Calcd for C25H28FN3O5+H: 470.20912. Found:
470.20604. Anal. Calcd for C25H27FN3O5NaÁ3H2O: C, 55.04; H,
6.10; N, 7.70. Found: C, 55.26; H, 5.94; N, 7.38.
Compound 7 and 8 were prepared according to general proce-
dure B.
Na2SO4, the extracts were concentrated in vacuo. The residue
was purified by preparative TLC (n-hexane–EtOAc = 2:1, v/v) to
give the title compound (149 mg, 69%) as a yellow oil. 1H NMR
(CDCl3) d 1.48 (9H, s), 2.01–2.28 (1H, m), 2.36–2.41 (1H, m),
3.45–3.73 (3H, m), 3.78 (3H, s), 3.83 (1H, dd, J = 8.5, 4.6 Hz),
3.98–4.34 (3H, m), 5.14–5.27 (1H, m); MS (ESI) m/z 316 [M+H]+.
5.8. 5-[(4S)-Fluoro-1-[2-(2-methylphenyl)amino-6-
5.12. Methyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
benzoxazolylacetyl]-(2S)-pyrrolidinylmethoxy]pentanoic acid
pyrrolidinylmethoxy]butanoate (15)
(7)
A suspension of methyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]-2-butynoate (195 mg, 0.618 mmol) and
5% Pd/C (wet) (200 mg) in MeOH (4 ml) was stirred at room tem-
perature under a hydrogen atmosphere for 8 h. After removing
the catalyst by filtration, the filtrate was concentrated in vacuo.
The residue was purified by column chromatography on silica gel
with n-hexane–EtOAc (2:1, v/v) as an eluent to give the title com-
pound (120 mg, 61%) as a colorless oil. 1H NMR (CDCl3) d 1.47 (9H,
s), 1.89 (2H, t, J = 6.8 Hz), 1.93–2.17 (1H, m), 2.3–2.42 (3H, m), 3.32
(1H, t, J = 8.5 Hz), 3.42–3.60 (5H, m), 3.67 (3H, s), 3.94–4.12 (1H,
m), 5.13–5.27 (1H, m); MS (ESI) m/z 320 [M+H]+.
Yield 64% (two steps). Light brown amorphous solid. Sodium
salt. IR (KBr) 3425, 3251, 2939, 2868, 1643, 1574, 1439 cmÀ1
;
1H NMR (DMSO-d6) d 1.01–1.30 (3H, m), 1.45–1.55 (4H, m),
1.83–1.92 (2H, m), 2.03–2.23 (2H, m), 2.28 (3H, s), 3.15–4.43
(5H, m), 4.18–4.36 (1H, m), 5.23–5.41 (1H, m), 6.99–7.03 (2H,
m), 7.18–7.30 (5H, m), 7.82–7.86 (1H, m); MS (ESI) m/z 484
[M+H]+, 506 [M+Na]+; HRMS (ESI) Calcd for C26H30FN3O5+H:
484.22477. Found: 484.22139. Anal. Calcd for C26H29FN3O5-
NaÁ2H2O: C, 57.66; H, 6.14; N, 7.76; F, 3.51. Found: C, 57.61;
H, 6.20; N, 7.26; F, 3.34.
5.13. Methyl 4-[(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]butanoate (5a)
5.9. 6-[(4S)-Fluoro-1-[2-(2-methylphenyl)amino-6-
benzoxazolylacetyl]-(2S)-pyrrolidinylmethoxy]hexanoic acid
(8)
To a solution of methyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]butanoate (120 mg, 0.376 mmol) in
CH2Cl2 (2 ml) was added TFA (0.5 ml) at room temperature. After
stirring for 1 h, the mixture was concentrated in vacuo. The residue
was diluted with CH2Cl2 and 1 N NaOH, and extracted with CH2Cl2.
The combined extracts were washed with brine. After being dried
over Na2SO4, the extract was concentrated in vacuo to give the title
compound (74 mg, 90%) as a yellow oil. 1H NMR (CDCl3) d 1.73–
1.86 (1H, m), 1.90–1.94 (2H, m), 2.18–2,23 (2H, m), 2.41 (2H, t,
J = 7.3 Hz), 2.82–2.95 (1H, m), 3.27–3.35 (2H, m), 3.43–3.53 (4H,
m), 3.67 (3H, s), 5.11–5.25 (1H, m).
Yield 48% (two steps). Pale yellow amorphous solid. Sodium salt.
IR (KBr) 3423, 3211, 2937, 2864, 1643, 1574, 1485, 1574 cmÀ1 1H
;
NMR (DMSO-d6) d 1.36–1.72 (6H, m), 1.92–2.28 (4H, m), 2.34 (3H,
s), 3.26–4.00 (8H, m), 4.20–4.43 (1H, m), 5.13–5.28 (1H, m), 7.04–
7.11 (2H, m), 7.21–7.34 (5H, m), 7.76 (1H, t, J = 7.3 Hz); MS (ESI) m/
z 498 [M+H]+; HRMS (ESI) Calcd for C27H32FN3O5+H: 498.24042.
Found: 498.23922. Anal. Calcd for C27H31FN3O5NaÁ1.0H2O: C, 60.33;
H, 6.19; N, 7.82. Found: C, 60.11; H, 6.19; N, 7.41.
5.10. 1-(tert-Butoxycarbonyl)-(4S)-fluoro-(2S)-
propargyloxymethylpyrrolidine (13)
5.14. General procedure C: preparation of methyl 5-[(4S)-
fluoro-(2S)-pyrrolidinylmethoxy]pentanoate (5b)
To a solution of 1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-pro-
linol13 (12) (660 mg, 3.01 mmol) in THF (20 ml) were added NaH
(181 mg, 4.52 mmol, 60% in oil), n-Bu4NI (cat.) and propargyl bro-
mide (0.40 ml, 3.62 mmol) at 0 °C under an atmosphere of nitro-
gen. The stirring mixture was allowed to warm up to room
temperature for 7 h. The reaction mixture was cooled in an ice
bath, poured into ice water and extracted with EtOAc. The com-
bined extracts were washed with satd NaHCO3 and brine. After
being dried over Na2SO4, the extracts were concentrated in vacuo.
The residue was purified by column chromatography on silica gel
with n-hexane–EtOAc (4:1, v/v) as an eluent to give the title com-
pound (660 mg, 85%) as a yellow oil. 1H NMR (CDCl3) d 1.48 (9H, s),
1.99–2.17 (1H, m), 2.37–2.44 (2H, m), 3.44 (1H, br s), 3.50–3.72
(2H, m), 3.83 (1H, br s), 4.05 (1H, br s), 4.17 (2H, s), 5.14–5.30
(1H, m); MS (ESI) m/z 258 [M+H]+.
5.14.1. Methyl 3-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]propionate (16)
To a cooled (À50 °C), stirred suspension of NaH (60% in oil,
365 mg, 9.12 mmol) in THF (20 ml) was added 1-(tert-butoxycar-
bonyl)-(4S)-fluoro-(2S)-prolinol (1.00 g, 4.56 mmol) in THF
(15 ml) for 5 min. After 15 min stirring, methyl acrylate (0.95 ml,
10.5 mmol) was added to the reaction mixture at À50 °C and stir-
red for 15 min. The reaction mixture was warmed up to À40 °C and
kept for 14 h. The reaction mixture was acidified with AcOH (1 ml),
diluted with ice water and extracted with EtOAc. The combined ex-
tracts were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by column
chromatography on silica gel with n-hexane–EtOAc (2:1, v/v) as
an eluent to give the title compound (670 mg, 48%) as a colorless
oil. 1H NMR (CDCl3) d 1.47 (9H, s), 1.98–2.15 (1H, m), 2.36 (1H,
dd, J = 20.5, 5.6 Hz), 2.58 (2H, t, J = 6.6 Hz), 3.35 (1H, t, J = 9.0 Hz),
3.48–3.80 (8H, m), 3.94–4.17 (1H, m), 5.13–5.26 (1H, m); MS
(ESI) m/z 306 [M+H]+.
5.11. Methyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]-2-butynoate (14)
To a solution of 1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-prop-
argyloxymethylpyrrolidine (177 mg, 0.688 mmol) in THF (6 ml)
was added n-BuLi (0.76 ml, 1.17 mol, 1.54 M solution in n-hexane)
at À50 °C under a nitrogen atmosphere. After 20 min stirring at the
5.14.2. 3-[1-(tert-Butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]propanol (17)
same temperature, methyl chloroformate (80
ll, 1.03 mmol) was
To a stirred solution of methyl 3-[1-(tert-butox-ycarbonyl)-
(4S)-fluoro-(2S)-pyrrolidinylmethoxy]propionate (2.84 g, 9.30 mmol)
in THF (40 ml) was added 0.25 N NaOH (40 ml) and the reaction
mixture was stirred at room temperature for 19 h. After removal
of the solvent, the mixture was acidified with 1 N HCl and ex-
added to the reaction mixture at À40 °C and allowed to À20 °C
for 1 h. To the reaction mixture was added satd NH4Cl, and the
mixture was extracted with EtOAc. The combined extracts were
washed with satd NaHCO3 and brine. After being dried over

1240
F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
tracted with EtOAc. The combined extracts were washed with
brine, dried over Na2SO4, and concentrated in vacuo to give 3-
[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]-
propionic acid (2.48 g, 92%) as a colorless oil. To a stirred solu-
tion of the product in THF (100 ml) was added 10.0 M BH3ÁMe2S
(2.79 ml, 27.9 mmol) at room temperature and the reaction mix-
ture was stirred at 60 °C for 30 min. After being cooled to room
temperature, the reaction mixture was poured into ice 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 on silica gel with
CHCl3–MeOH (20:1, v/v) as an eluent to give the title compound
(2.84 g, 100%) as a colorless oil. 1H NMR (CDCl3) d 1.47 (9H, s),
1.64–1.73 (1H, m), 1.83 (2H, br s), 2.00–2.41 (2H, m), 3.37 (1H,
t, J = 9.1 Hz), 3.50–3.85 (6H, m), 4.02–4.18 (1H, m), 5.14–5.28
(1H, m); MS (ESI) m/z 278 [M+H]+.
5.14.6. Methyl 5-[(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]pentanoate (5b)
To a solution of methyl 5-[1-(tert-butoxycarbonyl)-(4S)-flu-
oro-(2S)-pyrrolidinylmethoxy]pentanoate (1.09 g, 3.27 mmol) in
CH2Cl2 (50 ml) was added TFA (10 ml) and the reaction mixture
was stirred at room temperature for 3.5 h. After removal of the
solvent, the residue was diluted with CH2Cl2 and 1 N NaOH,
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo to give
the title compound (590 mg, 77%) as a yellow oil. 1H NMR
(CDCl3) d 1.59–1.96 (5H, m), 2.09–2.20 (1H, m), 2.34 (2H, t,
J = 7.1 Hz), 2.81–2.94 (1H, m), 3.27–3.35 (2H, m), 3.42–3.53
(4H, m), 3.67 (3H, s), 5.11–5.25 (1H, m); MS (ESI) m/z 234
[M+H]+.
5.15. (Z)-(2S)-(4-Benzyloxy-2-butenyloxy)methyl-1-(tert-
butoxycarbonyl)-(4S)-fluoropyrrolidine (21)
5.14.3. 3-[1-(tert-Butoxycarbonyl)-(4S)-fluoro-(2S)-
To a solution of 1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-pro-
linol (2.46 g, 11.2 mmol) in THF (100 ml) was added NaH
(896 mg, 22.4 mmol, 60% in oil) at 0 °C under an atmosphere of
nitrogen. After 20 min stirring, 4-benzyloxy-2-butenyl bromide
(2.76 g, 11.2 mmol) in THF (100 ml) was added to the reaction mix-
ture for 10 min and after 5 min, n-Bu4NI (100 mg, 0.27 mmol) was
added and stirred for 15 min at the same temperature. The reaction
mixture was allowed to warm up to room and stirred for 19 h. The
reaction mixture was poured into ice water and the mixture was
extracted with EtOAc. The combined extracts were washed with
brine. After being dried over Na2SO4, the extracts were concen-
trated in vacuo. The residue was purified by column chromatogra-
phy on silica gel with n-hexane–EtOAc (4:1, v/v) as an eluent to
give the title compound (3.30 g, 78%) as a colorless oil. 1H NMR
(CDCl3) d 1.47 (9H, s), 1.98–2.25 (1H, m), 2.35–2.43 (1H, m), 3.31
(1H, t, J = 9.3 Hz), 3.48–3.81 (3H, m), 3.95–4.18 (5H, m), 4.51 (2H,
s), 5.13–5.26 (1H, m), 5.70–5.84 (2H, m), 7.27–7.53 (5H, m); MS
(ESI) m/z 380 [M+H]+.
pyrrolidinylmethoxy]propanal (18)
To a stirred solution of 3-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]propanol (2.84 g, 9.30 mmol), Et3N
(7.78 ml, 55.8 mmol) and DMSO (6.61 ml, 93.7 mmol) in CH2Cl2
(43 ml) was added SO3Ápyridine (4.44 g, 27.9 mmol) at 0 °C and
the reaction mixture was stirred at 0 °C to room temperature for
2 h. After removal of the solvent, the mixture was diluted with
water and extracted with EtOAc. The combined extracts were
washed with brine, dried over Na2SO4, and condensed in vacuo.
The residue was purified by column chromatography on silica gel
with n-hexane–EtOAc (4:1, v/v) as an eluent to give the title com-
pound (1.35 g, 53%) as a colorless oil. 1H NMR (CDCl3) d 1.47 (9H, s),
1.95–2.16 (1H, m), 2.30–2.39 (1H, m), 2.65 (2H, br s), 3.36 (1H, t,
J = 9.8 Hz), 3.52–3.87 (5H, m), 4.00–4.13 (1H, m), 5.13–5.26 (1H,
m), 9.79 (1H, s).
5.14.4. Methyl (E)-5-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]-2-pentenoate (19)
To a cooled (0 °C), stirred solution of trimethyl phosphono-
acetate (0.95 ml, 5.88 mmol) in THF (40 ml) was added NaH
(60% in oil, 235 mg, 5.88 mmol) under a nitrogen atmosphere
and the resulting mixture was stirred for 10 min. To the result-
ing mixture was added 3-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]propanal (1.35 g, 4.90 mmol) in THF
(40 ml) and the reaction mixture was stirred at 0 °C for 2 h.
The mixture was poured into the ice water and extracted with
EtOAc. The combined extracts were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel with CHCl3–acetone
(10:1, v/v) as an eluent to give the title compound (1.40 g, 86%)
as a colorless oil. 1H NMR (CDCl3) d 1.47 (9H, s), 2.02–2.21 (1H,
m), 2.30–2.58 (2H, m), 3.34 (1H, t, J = 10.0 Hz), 3.45–3.71 (7H,
m), 3.73 (3H, s), 5.13–5.28 (1H, m), 5.89 (1H, d, J = 15.9 Hz),
6.95 (1H, dt, J = 15.9, 7.1 Hz).
5.16. 4-[1-(tert-Butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]-1-butanol (22)
A suspension of (Z)-(2S)-(4-benzyloxy-2-butenyloxy)methyl-1-
(tert-butoxycarbonyl)-(4S)-fluoropyrrolidine (4.58 g, 12.1 mmol)
and 20% Pd(OH)2 (dry) (4.0 g) in EtOH (200 ml) was stirred at room
temperature under a hydrogen atmosphere for 14 h. After remov-
ing the catalyst by filtration, the filtrate was concentrated in vacuo.
The residue was purified by column chromatography on silica gel
with CHCl3–acetone (10:1, v/v) as an eluent to give the title com-
pound (2.42 g, 67%) as a colorless oil. 1H NMR (CDCl3) d 1.47 (9H,
s), 1.62 (2H, s), 1.98–2.23 (2H, m), 2.32–2.40 (1H, m), 3.33–3.38
(1H, m), 3.52–3.82 (8H, m), 4.01–4.13 (1H, m), 5.14–5.27 (1H,
m); MS (ESI) m/z 292 [M+H]+.
Compounds 23–25 and 5c were prepared according to general
procedure C.
5.14.5. Methyl 5-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]pentanoate (20)
5.17. 4-[1-(tert-Butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]butyraldehyde (23)
A
suspension of (E)-5-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]-2-pentenoate (1.40 g, 4.22 mmol) and
5% Pd/C (wet) (700 mg) in MeOH (50 ml) was stirred at room tem-
perature under hydrogen atmosphere for 20 h. After removing the
catalyst by filtration, the filtrate was concentrated in vacuo. The
residue was purified by column chromatography on silica gel with
n-hexane–EtOAc (4:1, v/v) as an eluent to give the title compound
(1.09 g, 78%) as a colorless oil. 1H NMR (CDCl3) d 1.47 (9H, s), 1.61–
1.74 (3H, m), 1.98–2.18 (2H, m), 2.31–2.42 (3H, m), 3.31 (1H, dt,
J = 7.9, 1.5 Hz), 3.40–3.73 (5H, m), 3.67 (3H, s), 4.09 (1H, br s),
5.13–5.27 (1H, m).
Yield 72%. Colorless oil. 1H NMR (CDCl3) d 1.47 (9H, s), 1.91 (2H,
t, J = 6.6 Hz), 1.96–2.17 (1H, m), 2.31–2.40 (1H, m), 2.52 (2H, t,
J = 7.1 Hz), 3.32 (1H, t, J = 9.8 Hz), 3.49–4.14 (6H, m), 5.13–5.27
(1H, m), 9.77 (1H, s).
5.18. Methyl (E)-6-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]-2-hexenoate (24)
Yield 93%. Colorless oil. 1H NMR (CDCl3) d 1.47 (9H, s), 1.73 (1H,
br s), 1.99–2.51 (4H, m), 3.29–3.34 (1H, m), 3.41–3.71 (6H, m), 3,73

F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
1241
(3H, s), 3.97–4.20 (1H, m), 5.14–5.27 (1H, m), 5.83 (1H, d,
2.24–2.38 (3H, m), 2.66–2.78 (2H, m), 3.03–3.06 (1H, m), 3.46–
3.51 (1H, m), 3.74–3.80 (1H, m), 3.90–3.91 (1H, m), 4.14 (2H, q,
J = 7.2 Hz), 4.60–4.62 (1H, m), 5.10–5.24 (1H, m).
J = 15.4 Hz), 6.94–7.01 (1H, m); MS (ESI) m/z 346 [M+H]+.
5.19. Methyl 6-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]hexanoate (25)
5.24. Methyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (29)
Yield 100%. Colorless oil. 1H NMR (CDCl3) d 1.37–1.39 (2H, m),
1.47 (9H, s), 1.57–1.70 (4H, m), 1.98–2.18 (1H, m), 2.31 (3H, t,
J = 7.3 Hz), 2.35–2.44 (1H, m), 3.31 (1H, t, J = 8.8 Hz), 3.41–3.60
(3H, m), 3.67 (3H, s), 3.68–4.18 (2H, m), 5.13–5.26 (1H, m).
A mixture of methyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]benzoate13b (28) (550 mg, 1.56 mmol)
and 5% Rh on alumina (300 mg) in EtOH–AcOH (11 ml, 10:1, v/v)
was hydrogenated at room temperature at 10 atm for 24 h. After
removing the catalyst by filtration, the filtrate was concentrated
in vacuo. The residue was purified by column chromatography
on silica gel with n-hexane–EtOAc (4:1, v/v) as an eluent to give
the title compound (510 mg, 91%) as a colorless oil. 1H NMR
(CDCl3) d 1.47 (9H, s), 1.49–1.67 (4H, m), 1.78–2.13 (5H, m),
2.28–2.48 (2H, m), 3.26–3.33 (1H, m), 3.47–3.75 (7H, m), 3.96–
4.13 (1H, m), 5.13–5.26 (1H, m); MS (ESI) m/z 382 [M+Na]+.
5.20. Methyl 6-[(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]hexanoate (5c)
Yield 85%. Yellow oil. 1H NMR (CDCl3) d 1.38–1.70 (6H, m), 2.21–
2.30 (1H, m), 2.34 (2H, t, J = 6.8, Hz), 2.46–2.61 (1H, m), 3.50–3.74
(9H, m), 4.15 (1H, m), 5.30–5.45 (1H, m); MS (ESI) m/z 248 [M+H]+.
5.21. General procedure D: preparation of ethyl 4-[(4S)-fluoro-
(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetate (5d)
5.25. Methyl 4-[(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5g)
5.21.1. Ethyl 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylcarbonyl]-1-piperazinylacetate (27a)
To a stirred solution of 4-[1-(tert-butoxycarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (200 mg, 0.56
mmol) in CH2Cl2 (5 ml) was added TFA (5 ml) and the reaction
mixture was stirred at room temperature for 16 h. The mixture
was made basic by satd NaHCO3 and extracted with CHCl3. The
combined extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo to give the title compound (121 mg,
84%) as a colorless oil. 1H NMR (CDCl3) d 1.26–2.38 (11H, m),
2.82–2.95 (1H, m), 3.25–3.57 (6H, m), 3.67 (3H, s), 5.11–5.25
(1H, m); MS (ESI) m/z 260 [M+H]+.
To a stirred solution of ethyl 1-piperazinylacetate (222 mg,
1.29 mmol) and 1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-pyrrolidi-
nylcarboxylic acid15 (26) (300 mg, 1.29 mmol) in THF (10 ml) was
added EDCÁHCl (370 mg, 1.93 mmol), HOBt (198 mg, 1.29 mmol),
and Et3N (540 lg, 3.87 mmol), and the resulting mixture was stirred
at room temperature for 16 h. After removal of the solvent, the resi-
due was purified by flash column chromatography (Biotage 25M)
with CHCl3–MeOH (95:5, v/v) as an eluent to give the title compound
(500 mg, 100%) as a colorless oil. 1H NMR (CDCl3) d 1.28 (3H, t,
J = 7.2 Hz), 1.42–1.47 (9H, m), 2.15–2.26 (1H, m), 2.39–2.67 (5H,
m), 3.23–3.24 (2H, m), 3.46–3.91 (6H, m), 4.19 (2H, q, J = 7.2 Hz),
4.60–4.75 (1H, m), 5.13–5.29 (1H, m); MS (ESI) m/z 388 [M+H]+.
5.26. Methyl 2-(2-methylphenylamino)-6-benzoxazolylacetate
(35)
5.21.2. Ethyl 4-[(4S)-fluoro-(2S)-pyrrolidinylcarbonyl]-1-
piperazinylacetate (5d)
A solution of methyl 3-hydroxy-4-nitrophenylacetate12 (34)
(1.00 g, 4.74 mmol) in EtOH (20 ml) was hydrogenated over 5%
Pd/C (500 mg) for 15 h. The mixture was filtered to remove the cat-
To a stirred solution of ethyl 4-[1-(tert-butoxycarbonyl)-(4S)-
fluoro-(2S)-pyrrolidinylcarbonyl]-1-piperazinylacetate
(670 mg,
alyst and filtrate was added o-tolylisothiocyanate (765 ll,
1.73 mmol) in CH2Cl2 (5 ml) was added TFA (5 ml) and the reaction
mixture was stirred at room temperature for 15 h. The mixture was
condensed in vacuo and the residue was made basic with satd
NaHCO3. The mixture was extracted with CHCl3–MeOH (10:1, v/
v). The combined extracts were washed with brine, dried over
K2CO3, and evaporated to give the title compound (427 mg, 86%)
as a yellow oil. 1H NMR (DMSO-d6) d 1.25 (3H, t, J = 7.2 Hz),
2.22–2.33 (1H, m), 2.67–2.81 (1H, m), 3.17–3.50 (5H, m), 3.57–
3.71 (6H, m), 4.13 (2H, br s), 4.22 (2H, q, J = 7.2 Hz), 4.78 (1H, br
s), 5.37–5.50 (1H, m); MS (ESI) m/z 288 [M+H]+.
5.69 mmol) and the mixture was stirred for 15 h. HgO (1.72 g,
7.94 mmol) was added to this mixture and the mixture was heated
under reflux for 4 h. After being cooled to room temperature, the
mixture was filtered through a Celite pad. The filtrate was concen-
trated in vacuo and the residue was purified by column chroma-
tography on silica gel with CHCl3–EtOAc (10:1, v/v) as an eluent
to give the title compound (1.18 g, 84%) as a colorless solid. Mp
78–80 °C; 1H NMR (CDCl3) d 2.35 (3H, s), 3.69 (2H, s), 3.70 (3H,
s), 7.07–7.33 (6H, m), 7.40 (1H, d, J = 8.1 Hz), 8.06 (1H, d,
J = 8.1 Hz); MS (ESI) m/z 297 [M+H]+; HRMS (ESI) Calcd for
C17H16N2O3+H: 297.12392. Found: 297.12028.
Compounds 27b and 5e were prepared according to general
procedure D.
5.27. 2-(2-Methylphenylamino)-6-benzoxazolylacetic acid (4)
5.22. Ethyl 1-[1-(tert-butoxycarbonyl)-(4S)-fluoro-(2S)-
pyrrolidinylcarbonyl]-4-piperidinylacetate (27b)
To a stirred solution of methyl 2-(2-methylphenylamino)-6-
bezoxazolylacetate (1.18 g, 3.98 mmol) in THF (30 ml) was added
0.25 N NaOH (32 ml, 7.96 mmol) and the reaction mixture was stir-
red at room temperature for 15 h. The mixture was acidified with
1 N HCl and extracted with CHCl3–MeOH (5:1, v/v). The combined
extracts were dried over MgSO4, and concentrated in vacuo to give
the title compound (867 mg, 77%) as a pale yellow solid. Mp 198–
200 °C; 1H NMR (DMSO-d6) d 2.31 (3H, s), 3.64 (2H, s), 7.09–7.13
(2H, m), 7.24–7.30 (3H, m), 7.39 (1H, d, J = 1.0 Hz), 7.80 (1H, dd,
J = 7.3, 1.5 Hz); MS (ESI) m/z 283 [M+H]+; HRMS (ESI) Calcd for
C16H14N2O3+H: 283.10823. Found: 283.10425.
Yield 32%. Yellow oil. 1H NMR (CDCl3) d 1.13–1.22 (2H, m), 1.26
(3H, t, J = 7.2 Hz), 1.47 (9H, s), 1.70 (2H, s), 1.75–1.81 (2H, m), 2.03–
2.24 (4H, m), 2.43–2.57 (2H, m), 3.02–3.10 (1H, m), 3.75–3.83 (2H,
m), 4.14 (2H, q, J = 7.2 Hz), 4.60 (1H, d, J = 9.6 Hz), 5.13–5.27 (1H, m).
5.23. Ethyl 1-[(4S)-fluoro-(2S)-pyrrolidinylcarbonyl]-4-
piperidinylacetate (5e)
Yield 100%. Yellow oil. 1H NMR (CDCl3) d 1.13–1.22 (2H, m),
1.26 (3H, t, J = 7.2 Hz), 1.76–1.82 (2H, m), 1.87–2.02 (3H, m),

1242
F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
5.28. Methyl 4-[1-benzyloxycarbonyl-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (37)
5.31. trans-4-[1-(2-Methylphenylaminocarbonyl)-(4S)-fluoro-
(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid (40)
To a stirred solution of methyl trans-4-[1-(2-methylphenylami-
nocarbonyl)-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexaneca-
rboxylate (200 mg, 0.51 mmol) in THF–MeOH (10 ml, 1:1, v/v) was
added 0.25 N NaOH (10.2 ml, 2.55 mmol) and the reaction mixture
was stirred at room temperature for 5 h. The mixture was poured
into 1 N HCl and extracted with CHCl3–MeOH (10:1, v/v). The ex-
tract was washed with brine, dried over Na2SO4, and evaporated.
The residue was purified by recrystallization from EtOAc–n-hexane
to give the title compound (179 mg, 93%) as a colorless powder. 1H
NMR (CDCl3) d 1.21–1.31 (2H, m), 1.39–1.51 (2H, m), 2.00–2.13
(5H, m), 2.20–2.41 (2H, m), 2.26 (3H, s), 3.31–3.38 (1H, m), 3.60–
3.63 (1H, m), 3.68–3.77 (2H, m), 3.90–4.04 (1H, m), 4.17–4.22
(1H, m), 5.16–5.32 (1H, m), 7.00 (1H, t, J = 7.6 Hz), 7.14–7.19 (2H,
m), 7.75 (1H, d, J = 8.4 Hz), 8.03 (1H, s); MS (ESI) m/z 379 [M+H]+.
To a stirred solution of methyl 4-[(4S)-fluoro-(2S)-pyrrolidi-
nylmethoxy]cyclohexanecarboxylate (529 mg, 2.04 mmol) and
carbobenzyloxychloride (30–35% in toluene, 1.21 ml, 2.04 mmol)
in CH2Cl2 (20 ml) was added saturated NaHCO3 (5 ml) at room
temperature, and the resulting mixture was stirred for 5.5 h.
The mixture was poured into water and extracted with EtOAc.
The extract was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by chromatogra-
phy on silica-gel with n-hexane–EtOAc (2:1, v/v) as an eluent to
give a diastereomeric mixture of methyl 4-[1-benzyloxycarbonyl-
(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (37,
684 mg, 85%) as a colorless oil. The two diastereomers were sep-
arated by middle pressure silica gel column chromatography
(YAMAZEN YFLC-5404-FC,
U
26 Â 300 mm) with n-hexane–EtOAc
(3:1, v/v) as an eluent to give cis-isomer (37a, 489 mg, 61%) as a
colorless oil and trans-isomer (37b, 123 mg, 15%) as a colorless
oil.
5.32. X-ray crystallography
Crystal of 40 suitable for X-ray analysis was obtained by recrys-
tallization from EtOAc. The diffraction data were collected on
First fraction (37a) (less polar, cis-isomer): 1H NMR (CDCl3) d
1.45–1.52 (2H, m), 1.60–1.65 (2H, m), 1.80–1.90 (4H, m), 2.01–
2.18 (1H, m), 2.31–2.37 (1H, m), 2.42–2.51 (1H, m), 3.33–3.51
(2H, m), 3.60–3.75 (3H, m), 3.67 (3H, s), 4.11–4.15 (1H, m),
5.10–5.29 (3H, m), 7.30–7.37 (5H, m); MS (ESI) m/z 394
[M+H]+.
AFC7R diffractometer with Cu Ka radiation (k = 1.54178 Å). The
structure of 40 was solved by direct methods. The refinement
was made with anisotropic displacement factors for all non-hydro-
gen atoms. All the hydrogen atoms were refined isotropically. The
hydrogen atoms bound to carbon atoms were generated geometri-
cally and their coordinates were fixed while they were refined. On
the other hand, coordinates of hydrogen atoms bound to nitrogen
or oxygen atoms were altered while they were refined.
CCDC 707340 contains the supplementary crystallographic data
for this paper. The data can be obtained free of charge from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK (fax: +44 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
Second fraction (37b) (more polar, trans-isomer): 1H NMR
(CDCl3) d 1.15–1.30 (2H, m), 1.40–1.50 (2H, m), 1.99–2.13 (5H,
m), 2.25 (1H, br s), 2.39–2.48 (1H, m), 3.18–3.36 (2H, m), 3.61–
3.86 (3H, m), 3.67 (3H, s), 4.05–4.14 (1H, m), 5.10–5.29 (3H, m),
7.31–7.37 (5H, m); MS (ESI) m/z 394 [M+H]+.
5.29. Methyl trans-4-[(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (38)
A mixture of methyl trans-4-[1-benzyloxycarbonyl-(4S)-flu-
oro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (121 mg,
0.31 mmol) and Pd(OH)2 (20% on carbon, 19 mg) in MeOH
(5 ml) was stirred under a hydrogen atmosphere (1 atm) at room
temperature overnight. The mixture was filtered to remove the
catalyst. The filtrate was concentrated in vacuo to give the title
compound (77 mg, 97%) as a colorless oil. 1H NMR (CDCl3) d
1.21–1.31 (2H, m), 1.41–1.51 (2H, m), 1.74–1.87 (1H, m), 1.98–
2.09 (5H, m), 2.11–2.30 (2H, m), 2.48 (1H, br s), 2.85–2.98 (1H,
m), 3.22–3.59 (4H, m), 3.66 (3H, s), 5.12–5.26 (1H, m); MS
(ESI) m/z 260 [M+H]+.
5.33. Molecular modeling
3D structure building and molecular modeling were performed
using Ligprep 2.0113.17 All the conformation searches were per-
formed using Macromodel 9.1113.17 A total of 10,000 conformers
were generated for each molecule using the serial torsional-low
mode sampling method and subsequent single-cycle minimization
with truncated Newton conjugate gradient was performed for each
conformer. The OPLS2005 forcefield was used for the energy calcu-
lation. All the calculations were carried out on a Hewlett-Packard
workstation xw8200 running Red Hat Linux 9 and equipped with
two 3.2 GHz Xeon processors and 2GB RAM.
5.30. Methyl trans-4-[1-(2-methylphenylaminocarbonyl)-(4S)-
fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (39)
5.34. VLA-4/VCAM-1 binding assay
To a stirred solution of methyl trans-4-[(4S)-fluoro-(2S)-pyrro-
lidinylmethoxy]cyclohexanecarboxylate (1.33 g, 5.13 mmol) and
The inhibitory activity of the compounds against VLA-4 was
determined by the method described in a reported publication.10
Briefly, CHO cells expressing VLA-4 were used. The binding of the
human VCAM-1/Fc chimeric antibody (R&D Systems Inc.) labeled
with europium was measured using a time-resolved fluorometer
(DELFIA Research fluorometer, model 1234-001; Perkin–Elmer
Inc.). The concentration of the compound required for 50% inhibi-
tion in the assay was determined.
o-tolyl isocyanate (636
ll, 5.13 mmol) in THF (50 ml) was added
Et3N (143 l, 1.03 mmol) at room temperature and the resulting
l
mixture was stirred for 15 h. The mixture was concentrated in
vacuo and the residue was poured into 1 N HCl. The mixture
was extracted with EtOAc, dried over Na2SO4, and evaporated.
The residue was purified by recrystallization from EtOAc–n-hex-
ane to give the title compound (1.13 g, 56%) as a pale yellow
powder. 1H NMR (CDCl3) d 1.21–1.31 (2H, m), 1.39–1.48 (2H,
m), 1.98–2.39 (8H, m), 2.26 (3H, s), 3.32–3.37 (1H, m), 3.60–
3.77 (2H, m), 3.65 (3H, s), 3.90–4.04 (1H, m), 4.17–4.21 (1H,
m), 5.17–5.32 (1H, m), 7.00 (1H, t, J = 6.8 Hz), 7.14–7.22 (2H,
m), 7.77 (1H, d, J = 7.6 Hz), 8.03 (1H, s); MS (ESI) m/z 393
[M+H]+.
5.35. Pharmacokinetic studies on rats
Male Sprague-Dawley rats [Crj: CD(SD) IGS, 7 weeks old,
Charles River Laboratories] were used. The animals were fasted
for 18 h prior to dosing. Each group consisted of 4 animals. The rats
were orally administered compound 11b at the doses of 5 mg/kg

F. Muro et al. / Bioorg. Med. Chem. 17 (2009) 1232–1243
1243
5. Lin, K.; Ateenq, H. S.; Hsiunig, S. H.; Chong, L. T.; Zimmerman, C. N.; Castro, A.;
Lee, W. C.; Hammond, C. E.; Kalkunte, S.; Chen, L. L.; Pepinsky, R. B.; Leone, D.
R.; Sprague, A. G.; Abraham, W. M.; Gill, A.; Lobb, R. R.; Adams, S. P. J. Med.
Chem. 1999, 42, 920.
6. (a) Yednock, T. A.; Cannon, C.; Fritz, L. C.; Sanchez-Madrid, F.; Steinman, L.;
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Messersmith, E. K.; Pleiss, M. A.; Vandevert, C.; Thorsett, E. D.; Karlik, S. J. J.
Neuroimmunol. 2002, 131, 147.
dissolved in 0.5% (w/v) MC with 3 equiv NaOH aqueous solution. The
rats were intravenously administered compound 11b at a dose of
1 mg/kg dissolved in saline with 3 equiv NaOH solution. Blood sam-
ples (0.4 ml) were collected at 0.08 (or 0.25 for p.o.), 0.5, 1, 2, and 6 h
after the administration. These analytical samples were stood at
room temperature, followed by centrifugation at 15,000 rpm for
10 min at 4 °C. The plasma fractions were subsequently stored in a
À20 °C freezer until being analyzed. The concentrations of com-
pounds 11b were determined by an LC/MS/MS method, comprised
of an Alliance 2695 HPLC (Waters), Symmetry Shield RP8,
7. Seiffge, D. J. Rheumatol. 1996, 23, 2086.
8. (a) Podolsky, D. K. N. Eng. J. Med. 1991, 325, 928; (b) Powrie, F.; Leach, M. W.
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2.1 Â 50 mm, 3.5
lm column (Waters), and TSQ-700 (Thermo Elec-
tron, 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 ana-
lyzed by non-compartmental approaches using the WINNONLIN soft-
ware program (version 1.13.1 Pharsight, Mountain View, CA).
11. Chiba, J.; Iimura, S.; Yoneda, Y.; Watanabe, T.; Muro, F.; Tsubokawa, M.; Iigou,
Y.; Satoh, A.; Takayama, G.; Yokoyama, M.; Takashi, T.; Nakayama, A.;
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Acknowledgments
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.
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WO2000005223.; (d) Brittain, D. R.; Johnstone, C.; Davies, G. M.; Large, M. S.
WO2000005224.
The authors thank Mr. Makoto Suzuki for X-ray crystal structure
analysis. We also thank Mr. Takashi Shimada for technical support
of molecular modeling and helpful discussions.
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