A novel, potent, and orally active VLA-4 antagonist with good aqueous solubility: Trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6- benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy] cyclohexanecarboxylic acid

Article abstract of DOI:10.1016/j.bmc.2012.11.003

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7- fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.

Full text of DOI:10.1016/j.bmc.2012.11.003

Bioorganic & Medicinal Chemistry 21 (2013) 42–61
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel, potent, and orally active VLA-4 antagonist with good aqueous
solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-
benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinyl-
methoxy]cyclohexanecarboxylic acid
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:
We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring
of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving
in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro
activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic
balance of compounds suitable for oral administrative regimen. The modification resulted in the identi-
fication of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which sig-
nificantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a
Received 11 September 2012
Revised 31 October 2012
Accepted 2 November 2012
Available online 10 November 2012
Keywords:
VLA-4
Eosinophil infiltration
Asthma
Aqueous solubility
murine asthma model and showed favorable aqueous solubility (JP1, 89
lg/mL; JP2, 462 lg/mL). Further-
more, this compound showed good oral bioavailability (F = 54%) in monkeys.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Up to date, the humanized monoclonal anti-a4 antibody natal-
izumab was approved for the treatment of relapsing forms of MS
in the US and relapsing and remitting MS in the EU as well as mod-
erately to severely active CD in the US.9 However, none of the small
molecules have reached the marketplace yet. Thus, from a safety
and cost point of view, development of an oral small-molecule
VLA-4 antagonist could be desired in this field.
We have recently reported that starting with trans-4-[(4S)-
substituted-(2S)-pyrrolidinylmethyloxy]cyclohexanecarboxylic
acid derivative 1,10 we worked on structural modification of the
lipophilic moiety in order to improve the poor pharmacokinetic
profile, resulting in the identification of 2-(1-methyl-1H-indol-3-
yl)-1,3-benzoxazole derivative 2 as a potent VLA-4 antagonist.11,12
Compound 2 demonstrates excellent oral efficacy in a bronchial
inflammatory model,12 however, it has been revealed that its ex-
Most of circulating monocytes, granulocytes, and lymphocytes
express the very late antigen-4 (VLA-4, integrin
a4b1, CD49d/
CD29) on their surface.1 The VLA-4 binds to its natural ligands,
the vascular cell adhesion molecule-1 (VCAM-1, CD106) expressed
on cytokine-stimulated endothelial cells and the alternatively
spliced portion of the type III connecting segment of fibronectin
(FN).2,3 It has been well-known that VLA-4 plays an important role
in the process of adhesion, migration, invasion, and proliferation of
inflammatory cells at the site of inflammation through its interac-
tions. 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 rheuma-
toid arthritis,5 multiple sclerosis (MS)6 and Crohn’s disease
(CD).7 Furthermore, 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-1 or FN interaction as a therapeutic target.8
tremely poor aqueous solubility (<0.1 and 8.2 lg/mL in JP1; Japa-
nese Pharmacopeia First Fluid at pH 1.2 and JP2; Japanese
Pharmacopeia Second Fluid at pH 6.8, respectively) is troublesome
for further standard evaluation to see if it could have the potential
as a clinical candidate. In addition, poor solubility of clinical candi-
dates has been recognized as one of the major causes to hamper
drug development.13 Thus, our next objective was to obtain VLA-
4 antagonists possessing potent oral efficacy as well as good
⇑
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 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.003

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
43
aqueous solubility. At this point, since other 2-(1-methyl-1H-in-
2. Chemistry
dol-3-yl)-1,3-benzoxazole derivatives similarly showed poor aque-
ous solubility (data not shown), we concluded that it would be
difficult to obviate the drawback without replacing the 2-(1-
methyl-1H-indol-3-yl)-1,3-benzoxazole structure with another
lipophilic structure. In our continuous work on VLA-4 antagonist,
we have discovered 2-phenylaminobenzoxazole derivative 311
with potent activity with an IC50 value of 2.8 nM and preferable
The general synthesis procedure of designed compounds is
illustrated in Scheme 1. Thus, 2-(2-methylphenylamino)-7-fluoro-
benzoxazolyl acetic acids 4a–b11 were condensed with pyrrol-
idines 5a–n using EDCꢀHCl and HOBt to obtain 6a–k and 6m–q.
In the case of 6k, the methylthiomethyl group in 6k was converted
to the methanesulfonylmethyl group by oxidation using mCPBA to
give 6l. The resulting amides 6a–q were subjected to basic hydro-
lysis to afford the trans-4-substituted cyclohexanecarboxylic acids
7a–q. The syntheses of intermediates 5a–n are presented in
Schemes 2–6.
The preparation of trans-4-[(3S)-methoxy-(2R)-pyrrolidinyl-
methoxy]cyclohexanecarboxylate 5a is depicted in Scheme 2.
Starting from commercially available (3S)-hydroxyproline (8),
methyl esterification of the carboxylic acid and protection of the
nitrogen atom with benzyloxycarbonyl (Z) group gave 9. Ester 9
was converted to silylether 10 by reduction of the ester group
using sodium borohydride and protection of the resulting primary
alcohol with tert-butyldimethylsilyl (TBS) group. Intermediate 10
was subjected to methylation of the secondary alcohol and
desilylation by treatment of tetrabutylammonium fluoride (TBAF)
to afford 11. Condensation of alcohol 11 with methyl 4-hydroxy-
benzoate by Mitsunobu reaction, followed by deprotection of
the Z group by hydrogenation on Pd/C furnished benzoate 12.
Reduction of the benzene ring in 12 was achieved by hydrogena-
tion (10 atm) over Rh/Al2O3 under acidic condition to afford a mix-
ture of the cis- and trans-4-substituted cyclohexanecarboxylate
(cis-rich). Following N-Boc protection, isomerization with sodium
methoxide in refluxing methanol and subsequent esterification
with (trimethylsilyl)diazomethane provided 13 (cis/trans = ca. 1/1).
solubility (11 and 489 lg/mL in JP1 and 2). However, compound
3 did not show a significant inhibition of eosinophil infiltration
into bronchoalveolar lavage fluid (BALF) in the bronchial inflam-
matory model at 15 mg/kg, p.o., bid for 2 days. We assumed that
the cause of the weak efficacy would be low in vitro activity with
an IC50 value of 439 nM in the presence of 3% human serum albu-
min (HSA). Thus, to improve the weak activity, we attempted to
optimize substituents on the pyrrolidine ring in compound 3 while
fixing 2-(phenylamino)-7-fluorobenzoxazolyl group to keep good
aqueous solubility. Regarding substituent at the C-4 position on
the pyrrolidine ring in our study, a fluorine atom gave positive re-
sults leading to promising compounds which showed good in vivo
efficacy in many cases,10–12,14 however, the effect was unfortu-
nately not observed in the case of compound 3. Therefore we
decided to explore introduction of substituents to the C-3 or the
C-5 position (Fig. 1). In addition, we also sought for an appropriate
lipophilic and hydrophilic balance of compounds at the same time,
which balance would be instrumental to make aqueous solubility
compatible with absorption from gastrointestinal tract.
Herein, we report the synthesis, the structure–activity relation-
ships (SAR) and the physicochemical properties of a series of com-
pounds that we synthesized as well as in vivo evaluation results of
some of the representative compounds.
Lipophilic moiety
R1
N
O
O
CO H
2
O
N
H
N
H
Me
Cl
1, trans-4-[(4S)-substituted-(2S)-pyrrolidinylmethoxy]-
cyclohexanecarboxylic acid derivative
Incorporation of the urea into the cyclic structures
2-(phenylamino)benzoxazolyl group
F
X
F
F
F
N
O
N
N
O
N
CO H
O
Me
N
H
2
O
CO H
2
O
N
O
Me
3(X = F); IC50 (-/+3% HSA) = 2.8, 439 nM
Solubility (pH 1.2, 6.8); 11, 489 µg/mL
2; IC50(-/+3% HSA) = 4.7, 156 nM
Solubility (pH 1.2, 6.8); <0.1, 8.2 µg/mL
Estimated serum concentration (10 mg/kg, 15 min); 912 ng/mL
MDCK Papp(pH 6.0, 7.4); 9.18, 3.54 ×10-6 cm/s
LogD; 2.08
Estimated serum concentration (10 mg/kg, 15 min); 2211 ng/mL
MDCK Papp(pH 6.0, 7.4); 11.52, 5.71 ×10-6 cm/s
LogD; 2.78
Conversion to trans-4-[3 or 5-substituted-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic
acid derivative
R
1
R
2
N
N
O
CO H
2
O
CO H
2
Figure 1. Drug designs of 2-(phenylamino)benzoxazole derivatives.

44
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
X
X
X
R
1
R
R
1
1
F
F
F
c
a
R
R
2
R
2
HN
2
OH
+
N
N
O
N
O
N
O
N
O
O
O
O
O
Me
N
H
N
H
Me
Me
N
H
O
O
O
O
O
O
R
3
R
OH
3
4a, X = H
4b, X = F
5a, R1 = H, R2 = (S)-OMe, R3 = Et
5b, R1 = H, R2 = (R)-OMe, R3 = Et
6a, X = F, R1 = H, R2 = (S)-OMe, R3 = Et
6b, X = F, R1 = H, R2 = (R)-OMe, R3 = Et
7a, X = F, R1 = H, R2 = (S)-OMe
7b, X = F, R1 = H, R2 = (R)-OMe
5c, R1 = (R)-CH2OMe, R2 = H, R3 = Me
5d, R1 = (S)-CH2OMe, R2 = H, R3 = Me
5e, R1 = (S)-CH2OEt, R2 = H, R3 = Me
5f, R1 = (S)- CH2OAc, R2 = H, R3 = Me
5g, R1 = (R)-Me, R2 = H, R3 = Me
6c, X = F, R1 = (R)-CH2OMe, R2 = H, R3 = Me
6d, X = F, R1 = (S)-CH2OMe, R2 = H, R3 = Me
6e, X = H, R1 = (S)-CH2OMe, R2 = H, R3 = Me
6f, X = F, R1 = (S)-CH2OEt, R2 = H, R3 = Me
6g, X = H, R1 = (S)-CH2OEt, R2 = H, R3 = Me
6h, X = F, R1 = (S)-CH2OAc, R2 = H, R3 = Me
6i, X = F, R1 = (R)-Me, R2 = H, R3 = Me
7c, X = F, R1 = (R)-CH2OMe, R2 = H
7d, X = F, R1 = (S)-CH2OMe, R2 = H
7e, X = H, R1 = (S)-CH2OMe, R2 = H
7f, X = F, R1 = (S)-CH2OEt, R2 = H
7g, X = H, R1 = (S)-CH2OEt, R2 = H
7h, X = F, R1 = (S)-CH2OH, R2 = H
7i, X = F, R1 = (R)-Me, R2 = H
5h, R1 = (S)-CH2F, R2 = H, R3 = Me
5i, R1 = (S)-CH2SMe, R2 = H, R3 = Me
5j, R1 = (S)-CH2NMe2, R2 = H, R3 = Me
6j, X = F, R1 = (S)-CH2F, R2 = H, R3 = Me
6k, X = F, R1 = (S)-CH2SMe, R2 = H, R3 = Me
7j, X = F, R1 = (S)-CH2F, R2 = H
7k, X = F, R1 = (S)-CH2SMe, R2 = H
7l, X = F, R1 = (S)-CH2SO2Me, R2 = H
7m, X = F, R1 = (S)-CH2NMe2, R2 = H
7n, X = F, R1 = (S)-CH2N(OMe)Me, R2 = H
5k, R1 = (S)-CH2N(OMe)Me, R2 = H, R3 = Et
b
5l, R1
=
R2 = H, R3 = Et
R2 = H, R3 = Et
R2 = H, R3 = Et
6l, X = F, R1 = (S)-CH2SO2Me, R2 = H, R3 = Me
6m, X = F, R1 = (S)-CH2NMe2, R2 = H, R3 = Me
O
N
O
N
6n, X = F, R1 = (S)-CH2N(OMe)Me, R2 = H, R3 = Et
6o, X = F, R1
6p, X = F, R1
6q, X = F, R1
=
=
=
R2 = H, R3 = Et
R2 = H, R3 = Et
R2 = H, R3 = Et
7o, X = F, R1
7p, X = F, R1
7q, X = F, R1
=
=
=
R2 = H
R2 = H
O
O
5m, R1
5n, R1
=
N
O
N
N
O
N
=
OMe
N
R2 = H
OMe
N
OMe
N
Scheme 1. Reagents and conditions: (a) EDCꢀHCl, HOBt, Et3N, DMF; (b) mCPBA, CH2Cl2, 0 °C; (c) aq NaOH, THF.
OMe
OH
OH
OMe
OH
a, b
c, d
e, f
g, h
OTBS
O
OH
N
H
CO2Me
N
Z
CO2H
N
Z
N
Z
N
H
CO2Me
9
10
11
12
8
OMe
OMe
OMe
i - l
m, n
o
O
O
O
N
Boc
N
H
N
Boc
CO2Me
CO2Et
CO2Et
13
5a
14
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 0 °C to rt; (b) ZCl, Et3N, MeCN/H2O; (c) NaBH4, MeOH, toluene, 0 °C to rt; (d) TBSCl, Et3N, CH2Cl2, 0 °C to rt; (e) MeI, NaH,
DMF, 0 °C to rt; (f) TBAF, THF, 0 °C to rt; (g) methyl 4-hydroxybenzoate, DIAD, Ph3P, THF, reflux; (h) 10% Pd/C, H2, MeOH; (i) 5% Rh/Al2O3, H2 (10 atm), MeOH/AcOH; (j) Boc2O,
DMAP, MeCN; (k) NaOMe, MeOH, reflux; (l) TMSCHN2, benzene/MeOH; (m) 1 N NaOH, THF; (n) EtI, K2CO3, DMF, 70 °C, then separation; (o) TFA, CH2Cl2.
OMe
OH
OMe
OH
O
b
e, f
c, d
a
O
OTBS
OTBS
OH
OTBS
N
H
N
Z
N
Z
N
Z
N
Z
CO2Et
10
15
16
17
18
OMe
OMe
OMe
i, j
k
g, h
O
O
O
N
Boc
N
Boc
N
H
CO2Et
CO2Et
CO2Et
19
20
5b
Scheme 3. Reagents and conditions: (a) (COCl)2, DMSO, CH2Cl2, Et3N, ꢁ78 °C; (b) NaBH4, MeOH, ꢁ10 °C to rt; (c) MeI, NaH, DMF, 0 °C to rt; (d) TBAF, THF, 0 °C to rt; (e) ethyl
4-hydroxybenzoate, DIAD, Ph3P, THF, reflux; (f) Pd(OH)2, H2, MeOH; (g) 5% Rh/Al2O3, H2 (7 atm), MeOH/TFA; (h) Boc2O, MeCN, satd NaHCO3; (i) NaOEt, EtOH, reflux; (j) EtI,
K2CO3, DMF, 60 °C, then separation; (k) TFA, CH2Cl2.
After the methyl ester group in 13 was transformed to an ethyl
ester group via basic hydrolysis and esterification using EtI in order
to easily isolate the trans-isomer, isolation of the trans-isomer was
successfully performed by flash column chromatography to furnish
compound 14. Removal of the Boc group in 14 by TFA treatment
produced the trans-4-substituted cyclohexanecarboxylate 5a.
trans-4-[(3R)-Methoxy-(2R)-pyrrolidinylmethoxy]cyclohexanecar-
boxylate 5b was prepared as shown in Scheme 3. Swern’s oxidation

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
45
a
O
OBn
b
OH
c, d
MeO
OBn
MeO
MeO
HO
N
H
N
Boc
N
Boc
N
Boc
CO Me
2
22
24
23
21
e - i
O
MeO
N
H
CO Me
2
5c
Scheme 4. Reagents and conditions: (a) MeI, NaH, DMF, 0 °C to rt; (b) 10% Pd/C, H2, MeOH; (c) methyl 4-hydroxybenzoate, DIAD, Ph3P, THF, reflux; (d) TFA, CH2Cl2; (e) Rh/
Al2O3, H2 (10 atm), MeOH/TFA; (f) Boc2O, 1,4-dioxane, satd NaHCO3; (g) NaOMe, MeOH, reflux; (h) TMSCHN2, benzene /MeOH, then separation; (i) TFA, CH2Cl2.
e
b or c
d
OH
O
h - n
OBn
OBn
R
1
R
1
R
1
R
1
O
N
N
N
N
OR
Boc
Boc
Boc
Boc
2
25a, R1 = OH
25b, R1 = OMs
26a, R1 = OMe
26b, R1 = OEt
26c, R1 = F
27a, R1 = OMe
27b, R1 = OEt
25a, R1 = OBn
27c, R1 = H
28a, R1 = OMe, R2 = Me
28b, R1 = OEt, R2 = Me
28c, R1 = OBn, R2 = Me
28d, R1 = OH, R2 = Me
28e, R1 = H, R2 = Me
28f, R1 = F, R2 = Me
a
f
27d, R1 = F
28g, R1 = OBn, R2 = Et
28h, R1 = OH, R2 = Et
g
q
O
O
R
1
R
1
O
O
N
Boc
N
H
OR
OR
2
2
29a, R1 = OMe, R2 = Me
29b, R1 = OEt, R2 = Me
29c, R1 = OH, R2 = Me
29d, R1 = OAc, R2 = Me
29e, R1 = H, R2 = Me
29f, R1 = F, R2 = Me
5d, R1 = OMe, R2 = Me
5e, R1 = OEt, R2 = Me
5f, R1 = OAc, R2 = Me
5g, R1 = H, R2 = Me
5h, R1 = F, R2 = Me
5i, R1 = SMe, R2 = Me
o
a
29g, R1 = OMs, R2 = Me
29h, R1 = SMe, R2 = Me
29i, R1 = OBn, R2 = Et
29j, R1 = OH, R2 = Et
p
g
Scheme 5. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, 0 °C; (b) MeI (for 26a) or EtI (for 26b), NaH, DMF, 0 °C to rt; (c) Bu4NF, THF, reflux (for 26c); (d) Pd/C, H2, MeOH; (e)
methyl 4-hydroxybenzoate (for 28a–c and 28e–f) or ethyl 4-hydroxybenzoate (for 28g), DIAD, Ph3P, THF, rt or heating; (f) Pd/C, H2, EtOH; (g) Pd(OH)2, H2, EtOH/AcOH; (h)
TFA, CH2Cl2 (for 29b, 29e and 29i); (i) Rh/AlO3, H2 (3.5–10 atm), EtOH/AcOH (for 29a and 29c), MeOH/AcOH (for 29b and 29f), MeOH (for 29e) or EtOH/AcOH/TFA (for 29i); (j)
Boc2O, Et3N, CH2Cl2 (for 29b and 29i) or MeCN/H2O (for 29e); (k) BnBr, NaH, DMF (only for 29i); (l) NaOMe, MeOH, reflux (for 29a, 29c, 29e and 29f); (m) NaH, MeOH (for 29b)
or NaH, EtOH (for 29i), DMF, 0 °C to rt; (n) TMSCHN2, benzene or toluene/MeOH (for 29a–c, 29e and 29f) or H2O, K2CO3, EtI (for 29i), then separation; (o) (AcO)2O, pyridine;
(p) NaSMe, DMF, 60 °C, then TMSCHN2, benzene/MeOH, 0 °C to rt; (q) TFA, CH2Cl2 or 4 N HCl/dioxane.
O
R2
R
H O
O
a
O
b
O
c
c
O
2
a
b
O
O
O
O
N
Boc
N
Boc
N
Boc
N
H
OR
OR
OR
OR1
1
1
1
29c, R1 = Me
29j, R1 = Et
31a, R1 = Me, R2 = NMe2
5j, R1 = Me, R2 = NMe2
30a, R1 = Me
30b, R1 = Et
31b, R1 = Et, R2 = N(OMe)Me
5k, R1 = Et, R2 = N(OMe)Me
31c, R1 = Et, R2 =
5l, R1 = Et, R2 =
O
N
O
N
O
O
31d, R1 = Et, R2 =
31e, R1 = Et, R2 =
5m, R1 = Et, R2 =
5n, R1 = Et, R2 =
N
N
OMe
OMe
N
N
Scheme 6. Reagents and conditions: (a) TEMPO, trichloroisocyanuric acid, CH2Cl2, 0 °C to rt; (b) Me2NH (for 31a), N-methoxymethanamine hydrochloride (for 31b),
isoxazolidine hydrochloride (for 31c), morpholine (for 31d), or 3-methoxyazetidine hydrochloride (for 31e), NaBH(OAc)3, THF; (c) 4 N HCl/dioxane or TFA, CH2Cl2.

46
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
of the hydroxyl group in intermediate 10, followed by stereo spe-
cific reduction of the resulting ketone 15 using NaBH4 afforded
(3R)-hydroxypyrrolidine 16. Alcohol 16 was subjected to methyla-
tion, desilylation, Mitsunobu reaction, and deprotection of the Z
group to provide 18. In a procedure similar to that described for
the preparation of 5a, benzoate 18 was converted to trans-4-
substituted cyclohexanecarboxylate 5b.
sponding (5S)-aminomethyl derivatives 31a–e, in which the Boc
group was removed under acidic conditions to furnish 5j–n.
3. Results and discussion
3.1. In vitro activity and physicochemical properties
The preparation of methyl trans-4-[(5R)-methoxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5c) is depicted in
Scheme 4. Methylation of the hydroxyl group in compound 2115
and removal of the benzyl group provided alcohol 23, which was
subjected to Mitsunobu reaction with methyl 4-hydroxybenzoate
and deprotection of the Boc group to furnish 24. In a procedure
similar to that described for the preparation of 5a, benzoate 24
was converted to 5c.
The synthesized compounds (7a–q) 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% HSA.
In addition, we determined their solubility in the Japanese Pharma-
copoeia fluids (JP1, at pH 1.2 and JP2, at pH 6.8) and distribution
coefficient (LogD at pH 6.8) to predict the oral absorption in this
study, because compound 3 exhibits acceptable oral exposure
4-(2,5-trans-Substituted-pyrrolidinylmethoxy)cyclohexane-
carboxylates 5d–i were synthesized as shown in Scheme 5.
Alkylation or fluorination via mesylation of the hydroxyl group
in 25a16 gave 26a–c, in which the benzyl group was removed by
hydrogenation to provide 27a–b and 27d. Condensation of 25a
and 27a–d17 with methyl or ethyl 4-hydroxybenzoate by Mitsun-
obu reaction gave 28a–c and 28e–g, in which 28c and 28g were
subjected to hydrogenation to give the alcohols 28d and 28h. In
a procedure similar to that described for the preparation of 5a,
those benzoates were converted to the corresponding trans-
cyclohexanecarboxylates 29a–c, 29e–f, and 29i. Compound 29c
was further converted to 29d by acetylation of the hydroxyl group
and 29h by substitution with a methylthio group via mesylation. In
addition, 29i was hydrogenated to give alcohol 29j. The resulting
N-Boc derivatives were treated with TFA or hydrochloride to fur-
nish the target intermediates 5d–i.
The preparation of (5S)-acyclic or cyclicaminomethylpyrroli-
dine derivatives 5j–5n was shown in Scheme 6. Oxidation of
(5S)-hydroxymethylpyrrolidine derivatives 29c and 29j by
treatment with 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)
and trichloroisocyanuric acid, followed by reductive amination
of the resulting aldehyde 30a–b with dimethylamine, N-meth-
oxymethanamine hydrochloride, isoxazolidine hydrochloride,
morpholine, or 3-methoxyazetidine hydrochloride gave the corre-
due
to
good
membrane
permeability
(Papp
value = 3.54 ꢂ 10ꢁ6 cm/s) in a permeability assay using Madin-
Darby canine kidney (MDCK) cell monolayers,18 which is likely
based on its acceptable LogD value of 2.08.
The evaluation results are shown in Tables 1 and 2. At first, we
examined the effect of a substituent at the C-3 position in the pyr-
rolidine ring on inhibitory activity. (3S)-Methoxypyrrolidine 7a
was less potent than (3R)-methoxypyrrolidne 7b (7a, IC50 = 16 nM;
7b, IC50 = 4.3 nM), however, in the presence of 3% HSA, both of
them showed relatively low activity with IC50 values of more than
100 nM (Table 1).
Next, we examined the introduction of several substituents at
the C-5 position in the pyrrolidine ring (Table 1). Compound 7c–
7h with an alkoxymethyl or hydroxymethyl group exhibited good
potent activities with IC50 values of 1.1–3.7 nM and less than
100 nM (+3% HSA), finding that a fluorine atom at the C-5 position
(X) in the terminal benzene ring was not critical for the activity. In
comparison between compound 7c and 7d, it was shown that the
absolute configuration at the C-5 position (R1) did not affect the
activity. Alternatively, introduction of a methyl (7i), a fluoromethyl
(7j), a methylthiomethyl (7k) or a methylsulfonylmethyl group (7l)
resulted in
a slight loss of potency (7i, IC50 = 3.6 nM; 7j,
IC50 = 3.7 nM; 7k, IC50 = 2.9 nM; 7l, IC50 = 5.5 nM) as well as activ-
ities with IC50 values of more than 100 nM in the presence of 3%
Table 1
Inhibitory activity, solubility and distribution coefficient of 2-(phenylamino)benzoxazole derivatives 7a–7l
X
R
1
F
R
2
N
O
N
Me
N
H
O
CO H
2
O
Compound
X
R1
R2
IC50 (nM) ( 3% HSA)
Solubility (
lg/mL)
LogD (pH 6.8)
JP1a
JP2b
2
3
—
—
F
F
F
F
H
F
H
F
F
—
—
H
H
—
—
4.7/156
2.8/439
16/132
4.3/144
3.7/36
1.1/42
1.8/37
1.7/79
1.7/90
<0.1
11
31
28
11
11
66
3.5
35
49
5
8.2
2.78
2.08
1.95
2.04
2.54
2.49
2.17
2.94
2.57
1.8
489
1711
865
560
833
1200
340
440
1599
292
194
111
1109
7a
7b
7c
7d
7e
7f
7g
7h
7i
(S)-OMe
(R)-OMe
(R)-CH2OMe
(S)-CH2OMe
(S)-CH2OMe
(S)-CH2OEt
(S)-CH2OEt
(S)-CH2OH
(R)-Me
(S)-CH2F
(S)-CH2SMe
(S)-CH2SO2Me
H
H
H
H
H
H
H
H
H
H
1.7/31
3.6/184
3.7/137
2.9/202
5.5/132
2.81
2.6
3.2
7j
7k
7l
F
F
F
3
1
20
1.5
a
JP1; Japanese Pharmacopeia First Fluid at pH 1.2.
JP2; Japanese Pharmacopeia Second Fluid at pH 6.8.
b

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
47
Table 2
Inhibitory activity, solubility and distribution coefficient of 2-(phenylamino)benzoxazole derivatives 7m–7q
F
R
O
F
N
O
N
Me
N
H
O
CO H
2
Compound
R
IC50 (nM) ( 3% HSA)
Solubility (lg/mL)
LogD (pH 6.8)
JP1
JP2
Me
Me
Me
O
N
N
7m
7n
6.8/21
1.7/86
1945
89
1875
462
0.6
Me
2.57
O
7o
7p
2.2/75
1.2/9
>2000
1900
1225
1300
2.26
1.85
N
O
N
OMe
7q
4.2/35
>670
>670
1.1
N
HSA. Regarding aqueous solubility, all compounds exhibited pref-
erable solubility in JP2, however, 7f and 7i–7k showed low solubil-
ity of less than 10 lg/mL in JP1. On the other hand, all the
duction of basic substituents at this position as described in Table 2.
In this modification, we fixed the fluorine atom at the 5-position on
the terminal benzene ring in order to maintain the moderate lipo-
philicity. All the compounds exhibited potent activities with IC50
values of less than 100nM even in the presence of 3% HSA, and
showed preferable solubility in both JP1 and JP2 due to zwitter-
ionic character of the compounds. On the other hand, 7n and 7o
showed acceptable logD values of 2.57 and 2.26, respectively, how-
ever, the others showed likely insufficient logD values (7m,
LogD = 0.6; 7p, LogD = 1.85; 7q, LogD = 1.1).
compounds except 7a, 7h and 7l showed relatively appropriate
LogD values of more than 2.0, suggesting that the compounds
would have appropriate cell permeability leading to good oral
exposure. In addition, it was found that compounds 7e and 7g
without a fluorine atom at the C-5 position (X) in the terminal ben-
zene ring showed a decrease in logD value by 0.32 and 0.37,
respectively, in comparison with the corresponding compounds
7d and 7f, implying that the introduction of a fluorine atom at that
position should have a positive effect to enhance lipophilicity.
Since introduction of the substituents into the C-5 position in
the pyrrolidine ring gave relatively good results for both activity
and physicochemical properties, we next investigated the intro-
3.2. In vivo efficacy
On the basis of the potency in the presence of 3% HSA (IC50
<100 nM) and lipophilicity (LogD >2.5), we selected compounds
7c, 7f, 7g and 7n, evaluating their anti-inflammatory effect in an
Ascaris-antigen-induced murine bronchial inflammatory model
by measuring the level of eosinophils in bronchoalveolar lavage
fluid (BALF) at 48 h after the antigen challenge. The results are
summarized in Table 3. Oral administration of 7n significantly re-
duced eosinophil infiltration into BALF by 68% at 12.5 mg/kg and
44% at 5 mg/kg. On the other hand, compound 7c, 7f and 7g didn’t
show significant efficacy in this model, implying that the concen-
tration in blood and/or the duration might be less than that of 7n.
Next, compound 7n showing excellent efficacy in the inflamma-
tion model was evaluated in an actively sensitized murine asthma
model, assessing its ability to reduce bronchial hyper-responsiveness
(BHR) to acetylcholine chloride (ACh) at 48 h after the antigen
challenge. As shown in Figure 2, intratracheal antigen challenge
induced BHR. Compound 7n reduced the response in a dose-
dependent manner, and higher dose of the compound reduced
the response to the level of the antigen non-challenged group.
Table 3
Anti-inflammatory effect in an Ascaris-antigen-induced murine bronchial inflamma-
tory model
X
R
F
N
O
N
Me
N
H
O
CO H
2
O
Compound
X
R
Inhibition of the eosinophil infiltration (%)
5 mg/kg
15 mg/kg
7c
7f
7g
F
F
H
(R)-MeOCH2
(S)-EtOCH2
(S)-EtOCH2
14
24
8
12
23
42
Me
O
N
Me
7n
F
44*
68a,**
3.3. Pharmacokinetic properties
a
Compound 7n was administered at 12.5 mg/kg.
p <0.05, significant difference from the control (Dunnett’s multiple comparison
*
Concerning compound 7n exhibiting favorable efficacy in the
murine asthma model, we investigated the pharmacokinetic pro-
file in monkeys. As shown in Table 4, compound 7n showed good
test).
**
p <0.01; significant difference from the control (Dunnett’s multiple comparison
test).

48
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
300
250
200
150
100
50
EX-400 spectrometer, and chemical shifts are given in ppm (d)
ACh 100 μg/kg
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; tt, triple of 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). The high-resolution mass (HRMS)
spectra were recorded on a JEOL JMS-T100CS spectrometer. Ele-
mental analysis was performed using a PerkinElmer CHNS/O
2400II, a Leco CHNS-932 and a YOKOKAWA analysis IC7000RS.
++
**
0
Challenge with
saline
Vehicle
Ascaris
Vehicle
Ascaris
2 mg/kg
Ascaris
12.5 mg/kg
Treated with
5.2. General procedure A: preparation of ethyl trans-4-[1-[7-
fluoro-2-(5-fluoro-2-methylphenylamino)-6-benzoxazolyl]
acetyl]-(3S)-methoxy-(2R)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6a)
7n
Figure 2. Effect of compound 7n on antigen induced bronchial hyper-responsive-
ness (BHR) to acetylcholine chloride (ACh) in Ascaris suum sensitized mice. Female
BALB/c mice sensitized with Ascaris suum extract were used. The mice were
challenged intratracheally with saline or Ascaris suum extract. Vehicle or 7n were
given to the mice p.o. four times (0.25 h before and 8, 24 and 32 h after the
A mixture of [7-fluoro-2-(5-fluoro-2-methylphenylamino)-6-
benzoxazolyl]acetic acid (4b) (199 mg, 0.63 mmol), ethyl trans-4-
[(3S)-methoxy-(2R)-pyrrolidinylmethoxy]cyclohexanecarboxylate
(5a) (170 mg, 0.63 mmol), EDCꢀHCl (180 mg, 0.94 mmol), HOBt
challenge). Then BHR to ACh was measured 2 days after the antigen challenge. Each
++
column represents mean S.D. of eight animals.
p <0.01; significant difference
from the control (Dunnett’s multiple comparison test), ⁄⁄p <0.01; significant
difference from the control (student’s t-test).
(127 mg, 0.94 mmol), and Et3N (130 ll, 0.93 mmol) in DMF
(5 mL) was stirred at room temperature for 2 h. The mixture was
diluted with H2O and extracted with EtOAc. The extract was
washed with brine, dried over Na2SO4, and evaporated. The residue
was purified by column chromatography on silica gel with CHCl3–
MeOH (30:1, v/v) as eluent to give the title compound (323 mg,
88%) as a brown oil. 1H NMR (CDCl3) d 1.14–1.28 (m, 5H), 1.37–
1.48 (m, 2H), 1.91–2.09 (m, 5H), 2.17–2.24 (m, 2H), 2.31 (s, 3H),
3.20–3.90 (series of m, total 11H, including s, 3H, at d 3.33),
4.07–4.13 (m, 2H), 4.23–4.26 (m, 1H), 6.72–6.76 (m, 1H), 7.08–
7.15 (m, 2H), 7.21–7.23 (m, 1H), 7.41 (broad s, 1H), 8.04–8.07
(m, 1H); MS (ESI), m/z 586 [M+H]+.
oral exposure (AUC1 = 1253 ng h/mL at 0.5 mg/kg) and bioavail-
ability (F = 54%).
4. Conclusion
In this study, we explored the effects of substituents at the C-3
or the C-5 position in the pyrrolidine ring of 2-(phenylamino)-7-
fluorobenzoxazolyl derivative 3 on in vitro activity and physico-
chemical properties. Consequently, we made it clear that introduc-
tion of substituents into the C-5 position was effective to maintain
high activity in the presence of 3% HSA while adjusting lipophilic
and hydrophilic balance of compound suitable for oral administra-
tion regimen, resulting in the identification of zwitterionic com-
pound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine
moiety, which demonstrated excellent efficacy at oral dosing
(12.5 mg/kg) in a murine asthma model as well as favorable aque-
Compound 6b–6i, 6k, and 6m–6n were prepared according to
general procedure A.
5.3. Ethyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(3R)-methoxy-
(2R)-pyrrolidinylmethoxy]cyclohexanecarboxylate (6b)
Yield 100%. A brown viscous oil. 1H NMR (CDCl3) d 1.19–1.27
(m, 5H), 1.39–1.49 (m, 2H), 1.82–2.28 (series of m, total 7H), 2.30
(s, 3H), 3.16–3.29 (m, 1H), 3.41 and 3.42 (each s, total 3H), 3.44–
3.78 (series of m, total 5H), 3.90–4.00 (m, 2H), 4.07–4.15 (m, 2H),
4.28–4.32 (m, 1H), 6.72–6.77 (m, 1H), 7.05–7.24 (m, 4H), 8.07–
8.09 (m, 1H); MS (ESI), m/z 586 [M+H]+.
ous solubility (JP1, 89 lg/mL; JP2, 462 lg/mL). Furthermore, this
compound showed good oral bioavailability (F = 54%) in monkeys.
5. Experimental
5.1. General
5.4. Methyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5R)-
methoxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6c)
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-
Yield 99%. A pale yellow oil. 1H NMR (CDCl3) d 1.20–1.33 (m,
2H), 1.40–1.53 (m, 2H), 1.86–2.11 (m, 8H), 2.23–2.27 (m, 1H),
2.30 (s, 3H), 3.19–3.28 (m, 1H), 3.34–3.71 (m, total 10H), 3.83–3.97
Table 4
Pharmacokinetic properties of 7n in monkeys (n = 3)
F
a(%)
p.o. (0.5 mg/kg)
iv (0.5 mg/kg)
b
c
d
f
AUC1 (ng h/mL)
Cmax (ng/mL)
T1/2 (h)
AUC1 (ng h/mL)
2324
CLe (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
54
1253
511
1.7
3.8
0.125
6.5
a
b
c
d
e
f
Oral bioavailability.
Pharmacokinetic area under curve.
Pharmacokinetic maximum concentration.
Plasma half-life.
Pharmacokinetic clearance.
Volume of distribution.

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
49
(m, 2H), 4.19–4.25 (m, 2H), 6.72–6.76 (m, 1H), 7.05–7.35 (m,
5.10. Methyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5R)-methyl-(2S)-
4H), 8.05 (dd, J = 2.4, 10.7 Hz, 1 H); MS (ESI), m/z 586 [M+H]+.
pyrrolidinylmethoxy]cyclohexanecarboxylate (6i)
5.5. Methyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5S)-
methoxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6d)
Yield 88%. A pale yellow solid. IR (ATR) 2941, 1730, 1637, 1610,
1575 cmꢁ1 1H NMR (CDCl3) d 1.11–1.63 (series of m, total 8H),
;
1.88–2.31 (series of m, total 11H) 3.12–3.26 (m, total 1H), 3.34–
3.47 (m, total 1H), 3.62–3.92 (series of m, total 6H, including s, to-
tal 3 H, at d 3.63 and 3.67), 4.16–4.26 (series of m, total 2H), 6.74
(dt, J = 2.8 and 8.0 Hz, 1H), 7.10–7.16 (series of m, total 2H), 7.21
and 7.23 (each d, J = 4.4 Hz, total 2H), 8.06–8.10 (m, 1H); MS
(ESI), m/z 556 [M+H]+; Anal. Calcd for C30H35F2N3O5ꢀ0.75H2O: C,
63.31; H, 6.46; N, 7.38. Found: C, 63.40; H, 6.27; N, 7.22.
Yield 87%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.08–
2.30 (m, 13H), 2.31 (s, 3H), 3.09–3.48 (m, 6H, including each s, total
3H, at d 3.30 and 3.38), 3.49–3.70 (m, 5H, including each s, total 3H,
at d 3.63 and 3.67), 3.73–3.85 (m, 1H), 3.87–3.96 (m, 1H), 4.08–
4.29 (m, 2H), 6.70–6.79 (m, 1H), 7.09–7.17 (m, 2H), 7.20–7.25
(m, 1H), 8.04–8.12 (m, 1H); MS (ESI), m/z 586 [M+H]+.
5.11. Methyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5S)-
methylthiomethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6k)
5.6. Methyl trans-4-[1-[[7-fluoro-2-(2-methylphenylamino)-6-
benzoxazolyl]acetyl]-(5S)-methoxymethyl-(2S)-pyrrolidinyl
methoxy]cyclohexanecarboxylate (6e)
Yield 100%. A pale yellow amorphous solid. 1H NMR (CDCl3) d
1.10–1.55 (series of m, total 4H), 1.89–2.17 (m, total 10H, including
s, 3H, at d 2.13), 2.18–2.35 (m, total 5H, including each s, total 3H,
at d 2.30 and 2.31), 2.53–3.45 (series of m, total 3H), 3.54 (m, 1H),
3.59–3.70 (m, 4H, including s, 3H, at d 3.67), 3.80 (d, J = 16.0 Hz,
1H), 3.88 (d, J = 16.0 Hz, 1H), 4.10-4.29 (m, 2H), 6.74 (m, 1H),
7.06–7.29 (m, 4H), 8.07 (br d, J = 10.8 Hz, 1H); MS (ESI), m/z 602
[M+H]+.
Yield 91%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.06–
1.54 (m, 4H), 1.80–2.10 (m, 7H), 2.11–2.42 (m, 5H, including each
s, total 3H, at d 2.35 and 2.36), 3.09–3.47 (m, 6H, including each s,
total 3H, at d 3.30 and 3.38), 3.48–3.70 (m, 5H, including each s, to-
tal 3H, at d 3.63 and 3.66), 3.72–3.84 (m, 1H), 3.85–3.95 (m, 1H),
4.05–4.29 (m, 2H), 7.04–7.16 (m, 2H), 7.17–7.36 (m, 4H), 8.05–
8.12 (m, 1H); MS (ESI), m/z 568 [M+H]+.
5.7. Methyl trans-4-[(5S)-ethoxymethyl-1-[[7-fluoro-2-(5-
fluoro-2-methylphenylamino)-6-benzoxazolyl]acetyl]-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (6f)
5.12. Methyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5S)-
dimethylanimomethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6m)
Yield 80%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.13
and 1.22 (each t, J = 6.8 Hz, total 3H), 1.11–1.53 (series of m, 4H),
1.85–2.08 (m, 7H), 2.15–2.32 (m, 2H), 2.32 (s, 3H), 3.11–3.64 (ser-
ies of m, 7H), 3.63 and 3.67 (each s, total 3H), 3.77–3.96 (series of
m, 2H), 4.11–4.25 (m, 2H), 6.73–6.77 (m, 1H), 7.13–7.16 (m, 2H),
7.22–7.25 (m, 2H), 8.09 and 8.12 (each t, J = 3.2 Hz, total 1H); MS
(ESI), m/z 600 [M+H]+.
Yield 73%. A colorless oil. 1H NMR (CDCl3) d 1.09–1.57 (m, 4H),
1.88–2.71 (series of m, total 20H, including each s, 3H at d 2.28 and
6H at d 2.31,), 3.09–3.31 (m, 1H), 3.35–3.96 (series of m, total 7H,
including each s, total 3H, at d 3.63 and 3.67), 4.01–4.28 (m, 2H),
6.70–6.79 (m, 1H), 7.06–7.32 (m, 4H), 8.03–8.13 (m, 1H); MS
(ESI), m/z 599 [M+H]+.
5.13. Ethyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5S)-
[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6n)
5.8. Methyl trans-4-[(5S)-ethoxymethyl-1-[[7-fluoro-2-(2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (6g)
Yield 92%. A pale yellow solid. IR (ATR) 2941, 1732, 1635,
1576 cmꢁ1; 1H NMR (CDCl3) d 1.13 and 1.22 (each t, J = 7.1 Hz, total
3H), 1.26 (m, 2H), 1.44 (m, 2H), 1.87–2.08 (series of m, total 7H),
2.22 (m, 2H), 2.35 (m, 3H), 3.18 (m, 1H), 3.38 (m, 2H), 3.51 (m,
3H), 3.63 and 3.66 (each s, total 3 H), 3.64 (m, 1H), 3.76–3.94 (m,
2H), 4.22 (m, 2H), 6.92 (m, 1H), 7.08 (t, J = 7.2 Hz, 1H), 7.12 (d,
J = 7.2 Hz, 1H), 7.22 (m, 2H), 7.33 (d, J = 7.2 Hz, 1H), 8.10 (d,
J = 8.0 Hz, 1H); MS (ESI), m/z 582 [M+H]+; Anal. Calcd for
Yield 81%. A yellow oil. 1H NMR (CDCl3) d 1.13–1.52 (m, 7H),
1.83–2.27 (m, 5H), 2.31 (s, 3H), 2.53–2.61 (m, 1H), 2.62 (s, 3H),
2.75–2.84 (m, 1H), 3.06–3.31 (m, 1H), 3.34–3.51 (m, 3H), 3.52 (s,
3H), 3.64–4.00 (m, 2H), 4.03–4.32 (m, 3H), 4.04–4.04 (m, 4H),
6.74 (td, J = 2.5 and 8.2 Hz, 1H), 7.11–7.25 (m, 4H), 8.04–8.13 (m,
1H); MS (ESI), m/z 629 [M+H]+.
5.14. General procedure B: preparation of methyl trans-4-[1-[[7-
fluoro-2-(5-fluoro-2-methylphenylamino)-6-benzoxazolyl]
acetyl]-(5S)-fluoromethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6j)
C
32H40FN3O6: C, 66.08; H, 6.93; N, 7.22. Found: C, 65.94; H, 6.92;
N, 7.03.
5.9. Methyl trans-4-[(5S)-acetoxymethyl-1-[[7-fluoro-2-(5-
fluoro-2-methylphenylamino)-6-benzoxazolyl]acetyl]-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (6h)
Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-fluoromethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate
(29f)
(280 mg,
Yield 67%. A pale yellow amorphous solid. 1H NMR (CDCl3) d
1.06–1.55 (series of m, total 4H), 1.57–2.41 (series of m, total
15H, including each s, each 3H, at d 2.02 and 2.11), 3.06–4.53 (ser-
ies of m, total 12H, including s, 3H, at d 3.67), 6.75 (m, 1H), 7.02–
7.39 (series of m, total 3H), 8.08 (m, 1H).
0.750 mmol) was taken up in 4 N HCl–dioxane (10 mL). The mix-
ture was stirred for 3 h and concentrated under reduced pressure
to give methyl trans-4-[(5S)-fluoromethyl-(2S)-pyrrolidinylmeth-
oxy]cyclohexanecarboxylate hydrochloride (5h) as a colorless so-
lid. Compound 5h was taken up in DMF (10 mL) and 4b (239 mg,

50
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
0.75 mmol), Et3N (552
l
l, 3.75 mmol), EDCꢀHCl (216 mg,
ica gel with CHCl3–MeOH (30:1, v/v) to give the title compound
(193.0 mg, 100%) as a brown oil. 1H NMR (CDCl3) d 1.18–1.35 (m,
2H), 1,39–1.69 (m, 2H), 1.90–2.40 (series of m, total 12H, including
s, 3H, at d 2.33), 2.78 (dd, J = 10.0 and 14.0 Hz, 1H), 3.01 (s, 3H),
3.26 (m, 1H), 3.47 (dd, J = 6.8 and 9.6 Hz, 1H), 3.54 (dd, J = 6.0
and 9.6 Hz, 1H), 3.59–3.77 (m, 4H, including s, 3H, at d 3.67),
3.83 (d, J = 15.6 Hz, 1H), 3.92 (d, J = 15.6 Hz, 1H), 4.21 (dd, J = 6.4
and 12.4 Hz, 1H), 4.42 (m, 1H), 6.77 (m, 1H), 7.07 (dd, J = 6.8 and
6.8 Hz, 1H), 7.16 (dd, J = 6.8 and 6.8 Hz, 1H), 7.21–7.32 (m, 1H),
7.93–8.12 (m, 1H); MS (ESI), m/z 634 [M+H]+.
1.13 mmol), and HOBt (153 mg, 1.13 mmol) were added. After
72 h stirring, the mixture was diluted with EtOAc (300 mL), fol-
lowed by washing with brine (2 ꢂ 100 mL), drying over MgSO4,
and concentration under reduced pressure. The residue was chro-
matographed on silica gel with CHCl3–EtOAc (4:1, v/v) as eluent
to give the title compound (370 mg, 86% for two steps) as a pale
yellow foam. 1H NMR (CDCl3) d 1.11–2.30 (series of m, total
16H), 3.09–4.75 (series of m, total 12H), 6.70–6.75 (m, 1H), 6.98
(m, 1H), 7.08–7.15 (m, 2H), 7.21–7.24 (m, 1H), 8.07–8.10 (m,
1H); MS (ESI), m/z 574 [M+H]+.
Compound 6o–6q were prepared from 31c–31e according to
general procedure B.
5.19. General procedure C: preparation of trans-4-[1-[[7-fluoro-
2-(5-fluoro-2-methylphenylamino)-6-benzoxazolyl]acetyl]-(3S)-
methoxy-(2R)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid
(7a)
5.15. Ethyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-methyl
phenylamino)-6-benzoxazolyl]acetyl]-(5S)-(1-isoxazolidinyl
methyl)-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (6o)
To a stirred solution of 6a (323 mg, 0.55 mmol) in THF (4 mL)
was added 0.5 N NaOH (3.5 mL, 1.75 mmol) and the reaction mix-
ture was stirred at room temperature for 20 h. The mixture was
acidified with 1 N HCl and extracted with EtOAc. The extract
was washed with brine, dried over Na2SO4, and evaporated. The
residue was purified by flash column chromatography with
CHCl3–MeOH (9:1, v/v) as eluent to give the title compound
(193 mg, 63%) as a colorless amorphous solid. IR (ATR) 2937,
2864, 1720, 1701, 1639, 1610, 1577, 1550, 1502, 1450, 1281,
For ethyl trans-4-[(5S)-(1-isoxazolidinylmethyl)-(2S)-pyrrolidi-
nylmethoxy]cyclohexanecarboxylate dihydrochloride (5l);
A
brown oil. MS (ESI), m/z 341 [M+H]+.
For 6o; Yield 63% (two steps). A colorless oil. 1H NMR (CDCl3) d
1.06–1.56 (m, 8H), 1.85–2.37 (series of m, total 14H), 2.88 and 2.96
(each s, total 3H), 3.09–3.60 (m, 2H), 3.66–4.02 (m, 4H), 4.04–4.40
(m, 4H), 6.74 (td, J = 2.5 and 8.1 Hz, 1H), 6.98–7.24 (m, 3H), 7.40 (s,
1H), 8.00–8.09 (m, 2H); MS (ESI), m/z 641 [M+H]+.
1201, 1068, 802 cmꢁ1 1H NMR (DMSO-d6) d 1.10–1.40 (series of
;
m, total 4H), 1.84–2.18 (series of m, total 7H), 2.30 (s, 3H),
3.17–3.65 (series of m, total 8H, including s, 3H, at d 3.23),
3.71–4.15 (series of m, total 4H), 6.86–6.91 (m, 1H), 7.07–7.10
(m, 1H), 7.20–7.27 (m, 2H), 7.91–7.94 (m, 1H), 10.04 (broad s,
1H), 12.04 (broad s, 1H); MS (ESI), m/z 558 [M+H]+; Anal. Calcd
for C29H33F2N3O6: C, 62.47; H, 5.97; N, 7.54; F, 6.81. Found: C,
62.27; H, 6.12; N, 7.30; F, 6.65.
5.16. Ethyl trans-4-[1-[[7-fluoro-2-[[5-fluoro-2-methyl
phenyl]amino]-6-benzoxazolyl]acetyl]-(5S)-(4-morpholinyl)
methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (6p)
Yield 80% (two steps). A colorless oil. 1H NMR (CDCl3) d 1.12–
1.55 (m, 7H), 1.90–2.75 (series of m, total 16H), 3.15–4.22 (series
of m, total 15H), 6.72–6.77 (m, 1H), 7.13–7.27 (m, 3H), 8.09–8.12
(m, 1H).
Compound 7b–7q were prepared according to general proce-
dure C.
5.17. Ethyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-methylphenyl
amino)-6-benzoxazolyl]acetyl]-(5S)-(3-methoxyazetidinyl
methyl)-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (6q)
5.20. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(3R)-methoxy-(2R)-pyrrolidinyl
methoxy]cyclohexanecarboxylic acid (7b)
For ethyl trans-4-[(5S)-(3-methoxyazetidinylmethyl)-(2S)-pyr-
rolidinylmethoxy]cyclohexanecarboxylate dihydrochloride (5n);
1H NMR (DMSO-d6) d 1.13–1.29 (m, 5H, including t, J = 7.0 Hz,
3H, at d 1.17), 1.30–1.44 (m, 2H), 1.50–1.72 (m, 2H), 1.86–1.96
(m, 2H), 1.98–2.11 (m, 3H), 2.12–2.21 (m, 1H), 2.22–2.31 (m,
1H), 3.25 (s, 3H), 3.29–3.93 (m, 8H), 3.94–4.10 (m, 3H, including
q, J = 7.0 Hz, 2H, at d 4.04), 4.11–4.56 (m, 3H), 8.99–9.33 (m, 1H),
9.42–9.72 (m, 1H), 10.83–11.18 (m, 1H); MS (ESI), m/z 355 [M+H]+.
For 6q; Yield 56% (two steps). A yellow oil. 1H NMR (CDCl3) d
1.08–1.55 (m, 8H), 1.83–2.67 (series of m, total 10H), 2.77–4.40
(series of m, total 20H), 6.74 (t, J = 8.2 Hz, 1H), 7.06–7.16 (m, 2H),
7.20 and 7.21 (each t, J = 2.2 Hz, total 1H), 7.41 (s, 1H), 8.00–8.07
(m, 1H).
Yield 69%. A colorless amorphous solid. 1H NMR (DMSO-d6) d
1.15–1.40 (m, 4H), 1.76–2.17 (series of m, total 7H), 2.29 (s,
3H), 3.12–4.39 (series of m, total 12H, including s, 3H, at d
3.31), 6.87–6.91 (m, 1H), 7.00–7.10 (m, 1H), 7.19–7.28 (m, 2H),
7.91–7.94 (m, 1H), 10.01 (broad s, 1H), 12.02 (broad s, 1H); MS
(ESI), m/z 558 [M+H]+; Anal. Calcd for C29H33F2N3O6ꢀ0.25H2O: C,
61.97; H, 6.01; N, 7.48; F, 6.76. Found: C, 62.11; H, 5.96; N,
7.44; F, 6.73.
5.21. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5R)-methoxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7c)
5.18. Methyl trans-4-[1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(5S)-
methanesulfonylmethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (6l)
Yield 78%. A colorless solid. IR (ATR) 2935, 2861, 1724, 1701,
1639, 1610, 1577, 1502, 1450, 1348, 1280, 1201, 1105, 1068,
999, 802 cmꢁ1 1H NMR (DMSO-d6) d 1.13–1.36 (m, 4H), 1.75–
;
1.94 (m, 8H), 2.14 (m, 1H), 2.28 (s, 3H), 3.15–3.53 (m, 8H), 3.86–
3.88 (m, 2H), 3.90–4.02 (m, 1H), 4.21–4.23 (m, 1H), 6.86–6.89
(m, 1H), 7.02–7.04 (m, 1H), 7.18–7.26 (m, 2H), 7.90–7.93 (m,
1H); MS (ESI), m/z 572 [M+H]+; Anal. Calcd for C30H35F2N3O6: C,
63.04; H, 6.17; N, 7.35; F, 6.65. Found: C, 63.01; H, 6.35; N, 6.96;
F, 6.28.
To a stirred solution of 6k (154.9 mg, 0.257 mmol) in CH2Cl2
(3.0 mL) was added mCPBA (133.3 mg, 0.772 mmol) at 0 °C. After
stirring for 1.5 h at 0 °C, the reaction mixture was diluted with
CHCl3–MeOH (10:1, v/v), washed with satd NaHCO3 aq, dried over
Na2SO4, and evaporated. The residue was chromatographed on sil-

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
51
5.22. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-methoxymethyl-(2S)-pyrrolidinyl
methoxy]cyclohexanecarboxylic acid (7d)
(broad s, 1H); MS (ESI), m/z 558 [M+H]+; Anal. Calcd for
C29H33F2N3O6ꢀ0.5H2O: C, 61.48; H, 6.05; F, 6.71; N, 7.42. Found:
C, 61.59; H, 5.98; F, 6.68; N, 7.20.
Yield 94%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.06–
1.58 (m, 4H), 1.82–2.11 (m, 7H), 2.11–2.38 (m, 5H, including each
s, total 3H, at d 2.29 and 2.31), 3.06–3.28 (m, 1H), 3.28–3.48 (m, 5H,
including each s, total 3H, at d 3.30 and 3.39), 3.49–3.65 (m, 2H),
3.71–3.87 (m, 1H), 3.87–3.98 (m, 1H), 4.09–4.30 (m, 2H), 6.72–
6.80 (m, 1H), 7.09–7.22 (m, 3H), 7.79–7.94 (m, 1H); MS (ESI), m/z
572 [M+H]+; Anal. Calcd for C30H35F2N3O6: C, 63.04; H, 6.17; N,
7.35. Found C, 63.29; H, 6.32; N, 7.35.
5.27. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5R)-methyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (7i)
Yield 89%.
A colorless solid. IR (ATR) 2933, 1639, 1610,
1577 cmꢁ1 1H NMR (CDCl3) d 1.15–1.61 (series of m, total 8H),
;
1.95–2.29 (series of m, total 11H), 3.14–3.23 (m, 1 H), 3.36–3.92
(series of m, total 4H), 4.14–4.26 (series of m, total 2H), 6.75 (td,
J = 2.4 and 8.4 Hz, 1H), 7.10–7.16 (series of m, total 4H), 7.75 and
7.78 (each dd, J = 2.8 and 10.8 Hz, total 1 H); MS (ESI), m/z 542
[M+H]+; HRMS (ESI) Calcd for C29H33F2N3O5+H: 542.24665. Found:
542.24704.
5.23. trans-4-[1-[[7-Fluoro-2-(2-methylphenylamino)-6-
benzoxazolyl]acetyl]-(5S)-methoxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7e)
Yield 93%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.06–
1.57 (m, 4H), 1.77–2.41 (series of m, total 12H, including each s, to-
tal 3H, at d 2.34 and 2.35), 3.06–3.46 (m, 6H, including each s, total
3H, at d 3.29 and 3.37), 3.47–3.63 (m, 2H), 3.69–3.83 (m, 1H), 3.85–
3.97 (m, 1H), 4.07–4.29 (m, 2H), 7.05–7.17 (m, 3H), 7.18–7.32 (m,
2H), 7.82 and 7.87 (each d, J = 7.8 Hz, total 1H); MS (ESI), m/z 554
[M+H]+; Anal. Calcd for C30H36FN3O6ꢀ0.5H2O: C, 64.56; H, 6.59; N,
7.53. Found: C, 64.64; H, 6.60; N, 7.35.
5.28. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-fluoromethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7j)
Yield 53%. A colorless amorphous solid. IR (ATR) 2939, 2862,
1701, 1639, 1610, 1577, 1502, 1452, 1411, 1280, 1201, 1105,
1068, 1004, 802 cmꢁ1 1H NMR (DMSO-d6) d 1.10–1.39 (m, 4H),
;
1.78–2.18 (m, 9H), 2.30 (s, 3H), 3.08–4.57 (series of m, total 9H),
6.89 (dt, J = 2.7 and 8.3 Hz, 1 H), 7.10 (t, J = 7.8 Hz, 1H), 7.20–7.28
(m, 2H), 7.93 (dd, J = 2.2 and 11.5 Hz, 1H), 10.04 (broad s, 1H),
12.05 (broad s, 1H); MS (ESI), m/z 560 [M+H]+; Anal. Calcd for
5.24. trans-4-[(5S)-Ethoxymethyl-1-[[7-fluoro-2-(5-fluoro-2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7f)
C29H32F3N3O5: C, 62.25; H, 5.76; N, 7.51. Found: C, 62.16; H,
Yield 100%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.22
and 1.31 (each t, J = 6.8 Hz, total 3H), 1.18–1.69 (series of m, total
4H), 1.93–2.16 (m, 8H), 2.25–2.34 (m, 2H), 2.40 and 2.43 (each s,
total 3H), 3.17–3.35 (series of m, total 1H), 3.44–3.73 (series of
m, total 6H), 3.83–4.08 (series of m, total 2H), 4.19–4.34 (m, 2H),
6.84 (td, J = 2.8 and 8.4 Hz, 1H), 7.20–7.30 (series of m, total 3H),
7.92 and 8.01 (each dd, J = 2.8 and 10.8 Hz, total 1H); MS (ESI),
m/z 586 [M+H]+; Anal. Calcd for C31H37F2N3O6ꢀ0.5H2O: C, 63.09;
H, 6.40; N, 7.12. Found: C, 62.89; H, 6.38; N, 7.03.
5.97; N, 7.24.
5.29. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-methylthiomethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7k)
Yield 90%. A colorless amorphous solid. IR (ATR) 2937, 2862,
1701, 1639, 1610, 1577, 1502, 1452, 1423, 1280, 1203, 1105,
1068, 802 cmꢁ1 1H NMR (CDCl3) d 1.07–1.57 (m, 4H), 1.80–2.45
;
(series of m, total 15H, including s, 3H, at d 2.14 and each s, total
3H, at d 2.30 and 2.31), 2.50–3.95 (series of m, total 7H), 4.06–
4.35 (m, 2H), 6.75 (m, 1H), 7.05–7.35 (m, 3H), 7.80–8.00 (m, 1H);
MS (ESI), m/z 588 [M+H]+; Anal. Calcd for C30H35F2N3O5Sꢀ0.75H2O:
C, 59.94; H, 6.12; F, 6.32; N, 6.99; S, 5.33. Found C, 59.83; H, 5.79; F,
6.39; N, 6.93, S, 5.62.
5.25. trans-4-[(5S)-Ethoxymethyl-1-[[7-fluoro-2-(2-
methylphenylamino)-6-benzoxazolyl]acetyl]-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7g)
Yield 89%. A colorless solid. IR (ATR) 1699, 1637, 1577 cmꢁ1; 1H
NMR (CDCl3) d 1.23 and 1.22 (each t, J = 6.8 Hz, total 3H), 1.25 (m,
2H), 1.42 (m, 2H), 1.84–2.07 (series of m, total 7H), 2.22 (m, 2H),
2.34 and 2.35 (each s, total 3H), 3.10–3.22 (m, 1H), 3.34–3.64 (ser-
ies of m, total 6H), 3.85 (m, 2H), 4.18 (m, 2H), 7.09 (t, J = 7.2 Hz,
1H), 7.15 (m, 2H), 7.22 (m, 1H), 7.27 (s, 1H), 7.29 (m, 1H), 7.87
and 7.96 (each d, J = 8.0 Hz, total 1H); MS (ESI), m/z 568 [M+H]+;
Anal. Calcd for C31H38FN3O6ꢀ0.25H2O: C, 65.59; H, 6.75; N, 7.40.
Found: C, 65.21; H, 6.78; N, 7.21.
5.30. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-methanesulfonylmethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7l)
Yield 98%. A colorless amorphous solid. IR (ATR) 2933, 1722,
1639, 1610, 1577, 1504, 1452, 1408, 1298, 1201, 1128, 1105,
1068, 960 cmꢁ1 1H NMR (CDCl3) d 1.18–1.38 (m, 2H), 1.40–1.60
;
(m, 2H), 1.95–2.18 (m, 6H), 2.18–2.40 (series of m, total 6H, includ-
ing s, 3H, at d 2.32), 2.77 (dd, J = 10.4 and 14.0 Hz, 1H), 3.01 (s, 3H),
3.27 (m, 1H), 3.48 (dd, J = 6.4 and 9.2 Hz, 1H), 3.54 (dd, J = 5.6 and
9.2 Hz, 1H), 3.68 (d, J = 14.0 Hz, 1H), 3.84 (d, J = 16.0 Hz, 1H), 3.90
(d, J = 16.0 Hz, 1H), 4.21 (dd, J = 6.4 and 12.4 Hz, 1H), 4.42 (m,
1H), 6.77 (m, 1H), 7.09 (dd, J = 7.6 and 7.6 Hz, 1H), 7.16 (dd,
J = 7.6 and 7.6 Hz, 1H), 7.19–7.30 (m, 1H), 7.98 (m, 1H); MS (ESI),
m/z 620 [M+H]+; Anal. Calcd for C30H35F2N3O7Sꢀ0.25H2O: C,
57.73; H, 5.73; F, 6.09; N, 6.73; S, 5.14. Found: C, 57.63; H, 5.64;
F, 5.87; N, 6.51; S, 5.04.
5.26. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-hydroxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7h)
Yield 60%. A colorless amorphous solid. IR (ATR) 2939, 2864,
1703, 1639, 1610, 1577, 1502, 1452, 1280, 1203, 1068, 804 cmꢁ1
;
1H NMR (DMSO-d6) d 1.07–1.46 (m, 4H), 1.79–2.21 (m, 9H), 2.32
(s, 3H), 3.06–4.99 (series of m, total 9H), 6.78 (m, 1H), 7.06 and
7.08 (each d, J = 8.0 Hz, total 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.19 (d,
J = 7.6 Hz, 1H), 7.89 and 7.92 (each s, total 1H), 8.11 (s, 1H), 9.87

52
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
5.31. trans-4-[(5S)-Dimethylanimomethyl-1-[[7-fluoro-2-(5-
fluoro-2-methylphenylamino)-6-benzoxazolyl]acetyl]-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7m)
5.36. Methyl 1-benzyloxycarbonyl-(3S)-hydroxy-(2S)-
pyrrolidinecarboxylate (9)
To a cooled (0 °C), stirred solution of SOCl2 (27.0 mL, 0.37 mol)
Yield 62%. A pale yellow amorphous solid. IR (ATR) 2937,
in MeOH (200 mL) was added trans-3-hydroxy-L-proline (8)
2864, 1712, 1637, 1577, 1452 cmꢁ1
;
1H NMR (DMSO-d6)
d
(16.2 g, 0.12 mol) and the reaction mixture was stirred at 0 °C to
room temperature for 2 h. After removal of the solvent, the crude
solid was triturated with Et2O to give methyl (3S)-hydroxy-(2S)-
pyrrolidinecarboxylate hydrochloride (22.4 g) as a colorless solid.
To a stirred suspension of methyl (3S)-hydroxy-(2S)-pyrrolidin-
ecarboxylate hydrochloride (22.4 g, 0.12 mol) and Et3N (42.9 mL,
0.31 mol) in MeCN–H2O (400 mL, 1:1, v/v) was added ZCl
(22.9 mL, 0.16 mol) and the reaction mixture was stirred at room
temperature for 20 h. The mixture was diluted with H2O and ex-
tracted with EtOAc. The extract was washed with brine, dried over
Na2SO4, and evaporated. The residue was purified by column chro-
matography on silica gel with hexane–EtOAc (4:1 to 1:1, v/v) as
eluent to give the title compound (33.2 g, 96%) as a colorless oil.
1H NMR (CDCl3) d 1.91–1.99 (m, 1H), 2.08–2.24 (m, 2H), 3.65–
3.73 (m, 2H), 3.76 (s, 3H), 4.29 and 4.37 (each m, total 1H), 4.48
(m, 1H), 5.03–5.21 (m, 2H), 7.28–7.37 (m, 5H); MS (ESI), m/z 280
[M+H]+.
1.04–1.45 (m, 4H), 1.79–2.26 (m, 8H), 2.30 (s, 3H), 2.43–2.87
(m, 9H), 2.91–3.66 (m, 5H), 3.86–4.06 (m, 1H), 4.17–4.36 (m,
1H), 6.85–6.98 (m, 1H), 7.06–7.18 (m, 1H), 7.18–7.36 (m, 2H),
7.86–7.99 (m, 1H), 10.05 (s, 1H), 12.02 (broad s, 1H); MS (ESI),
m/z 585 [M+H]+; HRMS (ESI) Calcd for C31H38F2N4O5+H:
585.28885. Found: 585.28695.
5.32. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-
(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7n)
Yield 99%. A colorless amorphous solid. IR (ATR) 2937, 2862,
1701, 1639, 1610, 1577, 1550 cmꢁ1 1H NMR (CDCl3) d 1.03–1.58
;
(m, 4H), 1.82–2.47 (m, 12H), 2.52–2.68 (m, 4H), 2.72–2.85 (m,
1H), 3.32–4.07 (m, 1H), 3.39–4.02 (m, 7H), 4.08–4.32 (m, 2H),
6.72–6.80 (m, 1H), 7.07–7.25 (m, 3H), 7.75–7.91 (m, 1H); MS
(ESI), m/z 601 [M+H]+; Anal. Calcd for C31H38F2N4O6ꢀ0.5H2O: C,
61.07; H, 6.45; F, 6.23; N, 9.19. Found: C, 61.15; H, 6.46; F, 6.23;
N, 8.99.
5.37. 1-Benzyloxycarbonyl-(2R)-tert-butyldimethylsilyl
oxymethyl-(3S)-hydroxypyrrolidine (10)
5.33. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-(1-isoxazolidinylmethyl)-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7o)
To a cooled (0 °C), stirred solution of 9 (32.3 g, 0.116 mol) in tol-
uene (330 mL) was added NaBH4 (10.8 g, 0.289 mol) and MeOH
(70.3 mL, 1.74 mol). After being stirred at room temperature for
4 h, the mixture was quenched by H2O and extracted with EtOAc.
The extract was washed with brine, dried over Na2SO4, and evapo-
rated to give 1-benzyloxycarbonyl-(3S)-hydroxy-(2R)-pyrrolidine-
methanol (25.9 g, 89%) as a colorless viscous oil. MS (ESI), m/z
252 [M+H]+.
To a cooled (0 °C), stirred solution of 1-benzyloxycarbonyl-(3S)-
hydroxy-(2R)-pyrrolidinemethanol (26.6 g, 0.106 mol) and Et3N
(22.1 mL, 0.159 mol) in CH2Cl2 (400 mL) was added TBDMSCl
(17.5 g, 0.116 mol) and the reaction mixture was stirred at room
temperature for 2 days. The mixture was diluted with H2O and ex-
tracted with EtOAc. The extract was washed with brine, dried over
Na2SO4, and evaporated. The residue was purified by column chro-
matography on silica gel with hexane–EtOAc (4:1 to 2:1, v/v) as
eluent to give the title compound (34.7 g, 90%) as a colorless solid.
1H NMR (CDCl3) d ꢁ0.04 and ꢁ0.02 (each s, total 3H), 0.03 and 0.05
(each s, total 3H), 0.84 and 0.88 (each s, total 9H), 1.74–1.89 (m,
2H), 2.09–2.19 (m, 1H), 3.38–3.92 (series of m, total 5H), 4.40 (m,
1H), 5.06–5.20 (m, 2H), 7.28–7.36 (m, 5H); MS (ESI), m/z 366
[M+H]+.
Yield 67%. A colorless amorphous solid. 1H NMR (CDCl3) d 1.03–
1.60 (m, 4H), 1.87–2.36 (m, 17H), 2.89 and 2.96 (each s, total 3H),
3.33–3.71 (m, 2H), 3.74–4.02 (m, 4H), 4.08–4.39 (m, 2H), 6.76 (tt,
J = 2.3 and 8.1 Hz, 1H), 7.18–7.08 (m, 3H), 7.84–7.74 (m, 1H); MS
(ESI), m/z 613 [M+H]+; HRMS (ESI) Calcd for C32H38F2N4O6+H:
613.28377. Found: 613.28085.
5.34. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-(4-morpholinyl)methyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7p)
Yield 69%. A colorless solid. IR (ATR) 3224, 1716, 1637, 1610,
1577, 1452 cmꢁ1 1H NMR (DMSO-d6) d 1.10–1.40 (m, 4H), 1.75–
;
2.28 (m, 9H), 2.30 (s, 3H), 2.35–2.60 (m, 4H), 3.10–4.20 (m, 13H),
6.87–6.90 (m, 1H), 7.09–7.28 (m, 3H), 7.29 (dd, J = 2.7 and
11.3 Hz, 1H); MS (ESI), m/z 627 [M+H]+; Anal. Calcd for
C
33H40F2N4O6ꢀ0.7H2O: C, 62.00; H, 6.53; N, 8.76. Found: C, 61.79;
H, 6.25; N, 8.72.
5.38. 1-Benzyloxycarbonyl-(3S)-methoxy-(2R)-pyrrolidine
methanol (11)
5.35. trans-4-[1-[[7-Fluoro-2-(5-fluoro-2-methylphenylamino)-
6-benzoxazolyl]acetyl]-(5S)-(3-methoxyazetidinylmethyl)-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylic acid (7q)
To a cooled (0 °C), stirred solution of 10 (2.60 g, 7.11 mmol) and
NaH (0.34 g, 14.2 mmol) in DMF (10 mL) was added MeI (0.53 mL,
8.51 mmol) and the reaction mixture was stirred at room temper-
ature for 13 h. The mixture was quenched by H2O and extracted
with EtOAc. The extract was washed with brine, dried over Na2SO4,
and evaporated. The residue was purified by column chromatogra-
phy on silica gel with hexane–EtOAc (6:1, v/v) as eluent to give 1-
benzyloxycarbonyl-(2R)-tert-butyldimethylsilyloxymethyl-(3S)-
methoxypyrrolidine (2.73 g, 100%) as a colorless oil. 1H NMR
(CDCl3) d 0.05 (s, 6H), 0.90 and 0.93 (each s, total 9H), 2.02–2.08
(m, 2H), 3.35–3.37 (each s, total 3H), 3.41–3.64 (m, 3H), 3.75–
3.98 (m, 3H), 5.08–5.27 (m, 2H), 7.32–7.40 (m, 5H).
Yield 74%. A colorless amorphous solid. IR (ATR) 2933, 2858,
1712, 1637, 1610, 1576, 1552 cmꢁ1 1H NMR (CDCl3) d 1.09–1.34
;
(m, 2H), 1.34–1.55 (m, 2H), 1.81–2.14 (m, 8H), 2.15–2.35 (m, 5H,
including s, total 3H, at d 2.27 and 2.30), 2.54–2.74 (m, 1H),
2.95–3.29 (m, 6H, including s, total 3H, at d 3.14 and 3.26), 3.33–
3.69 (m, 3H), 3.76–3.96 (m, 3H), 3.96–4.21 (m, 3H), 6.70–6.78
(m, 1H), 7.03–7.23 (m, 3H), 7.85 and 7.95 (each dd, J = 2.6 and
10.8 Hz, total 1H); MS (ESI), m/z 627 [M+H]+; Anal. Calcd for
C
33H40F2N4O6ꢀH2O: C, 61.48; H, 6.57; F, 5.89; N, 8.69. Found: C,
61.44; H, 6.53; F, 5.65; N, 8.30.

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
53
To a cooled (0 °C), stirred solution of 1-benzyloxycarbonyl-(2R)-
tert-butyldimethylsilyloxymethyl-(3S)-methoxypyrrolidine
(2.73 g, 7.19 mmol) in THF (15 mL) was added tetrabutylammo-
nium fluoride (TBAF) (1 M solution in THF, 14.4 mL, 14.4 mmol)
and the reaction mixture was stirred at room temperature for
18 h. The mixture was concentrated to a small volume, diluted
with 1 N HCl, and extracted with EtOAc. The extract was washed
with brine, dried over Na2SO4, and evaporated. The residue was
purified by column chromatography on silica gel with CHCl3–
MeOH (20:1, v/v) as eluent to give the title compound (2.01 g,
100%) as a colorless oil. 1H NMR (CDCl3) d 1.98 (m, 2H), 3.34 (s,
3H), 3.41–3.74 (m, 5H), 3.88 and 4.01 (each m, total 1H), 5.14 (s,
2H), 7.30–7.36 (m, 5H); MS (ESI), m/z 266 [M+H]+.
purified by flash column chromatography with hexane–EtOAc (2:1,
v/v) as eluent to give the title compound (cis/trans = ca. 1/1,
896.0 mg, 54%) as a colorless oil. MS (ESI), m/z 372 [M+H]+.
5.41. Ethyl trans-4-[1-tert-butoxycarbonyl-(3S)-methoxy-(2R)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (14)
To a stirred solution of 13 (437 mg, 1.18 mmol) in THF (4 mL)
was added 1 N NaOH (3.5 mL, 3.50 mmol) and the reaction mixture
was stirred at room temperature for 4 days. The mixture was acid-
ified with 1 N HCl and extracted with EtOAc. The extract was
washed with brine, dried over Na2SO4, and evaporated to give
4-[1-tert-butoxycarbonyl-(3S)-methoxy-(2R)-pyrrolidinylmethoxy]
cyclohexanecarboxylic acid (413 mg, 98%) as a pale yellow oil.
A mixture of 4-[1-tert-butoxycarbonyl-(3S)-methoxy-(2R)-pyr-
5.39. Methyl 4-[(3S)-methoxy-(2R)-pyrrolidinyl
methoxy]benzoate (12)
rolidinylmethoxy]cyclohexanecarboxylic
acid
(413 mg,
1.16 mmol), EtI (940 l, 11.8 mmol), and K2CO3 (325 mg,
l
To
a
stirred solution of 11 (1.90 g, 7.16 mmol), methyl
2.35 mmol) in DMF (10 mL) was stirred at 70 °C for 17 h. After
being cooled to room temperature, the mixture was acidified with
1 N HCl, and extracted with EtOAc. The extract was washed with
brine, dried over Na2SO4, and evaporated. The residue was purified
by flash column chromatography with hexane–EtOAc (5:1, v/v) as
eluent to give the title compound (142 mg, 31%) as a colorless oil.
1H NMR (CDCl3) d 1.22–1.26 (m, total 5H, including t, J = 7.1 Hz, 3H,
at d 1.25), 1.40–1.50 (m, total 11H, including s, 9H, at d 1.46), 1.98–
2.07 (m, 6H), 2.24–2.28 (m, 1H), 3.20–3.26 (m, 1H), 3.32–3.50 (ser-
ies of m, total 6H, including s, 3H, at d 3.32), 3.60–3.68 (m, 1H),
3.76–3.92 (m, 2H), 4.11 (q, J = 7.1 Hz, 2H).
4-hydroxybenzoate (1.20 g, 7.89 mmol) and Ph3P (2.25 g,
8.58 mmol) in THF (20 mL) was added diisopropyl azodicarboxyl-
ate (DIAD) (1.69 mL, 8.58 mmol) and the reaction mixture was re-
fluxed for 12 h. After the mixture was cooled to the room
temperature, the solvent was evaporated. The residue was purified
by column chromatography on silica gel with hexane–EtOAc (1:1,
v/v) as eluent to give methyl 4-[1-benzyloxycarbonyl-(3S)-meth-
oxy-(2R)-pyrrolidinylmethoxy]benzoate as a crude product. MS
(ESI), m/z 400 [M+H]+.
A stirred solution of the crude product in MeOH (50 mL) was
hydrogenated over 10% Pd/C (1.50 g) for 20 h. The mixture was fil-
tered to remove the catalyst, and the solvent was evaporated. The
residue was purified by column chromatography on silica gel with
CHCl3–MeOH (30:1, v/v) as eluent to give the title compound
(1.39 g, 73% for two steps) as a brown oil. 1H NMR (CDCl3) d
1.87–2.01 (m, 2H), 3.03–3.18 (m, 2H), 3.35 (s, 3H), 3.43–3.47 (m,
1H), 3.79–3.82 (m, 1H), 3.89 (s, 3H), 3.95–4.02 (m, 2H), 6.91–
6.93 (m, 2H), 7.97–8.00 (m, 2H).
5.42. Ethyl trans-4-[(3S)-methoxy-(2R)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (5a)
To a stirred solution of 14 (142 mg, 0.37 mmol) in CH2Cl2 was
added TFA (3 mL) and the reaction mixture was stirred at room
temperature for 1 h. The mixture was made basic with satd NaH-
CO3 aq and extracted with CHCl3. The extract was washed with
brine, dried over Na2SO4, and evaporated to give the title com-
pound (97 mg, 92%) as a pale yellow oil. 1H NMR (CDCl3) d 1.20–
1.30 (m, total 5H, including t, J = 7.3 Hz, 3H, at d 1.24), 1.41–1.51
(m, 2H), 1.78–1.92 (m, 2H), 1.98–2.08 (m, 4H), 2.20–2.28 (m,
1H), 2.99–3.05 (m, 2H), 3.13–3.27 (m, 2H), 3.31 (s, 3H), 3.41–3.50
(m, 2H), 3.64–3.67 (m, 1H), 4.11 (q, J = 7.3 Hz, 2H).
5.40. Methyl 4-[1-tert-butoxycarbonyl-(3S)-methoxy-(2R)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (13)
A stirred solution of 12 (1.39 g, 5.24 mmol) in MeOH/AcOH
(22 mL, 10:1, v/v) was hydrogenated at 10 atm over Rh/Al2O3
(0.55 g) for 2 days. The mixture was filtered to remove the catalyst
and the solvent was evaporated. The residue was dissolved in
CHCl3, washed with satd NaHCO3 aq, brine, dried over Na2SO4,
and evaporated to give methyl 4-[(3S)-methoxy-(2R)-pyrrolidinyl-
methoxy]cyclohexanecarboxylate (1.42 g) as a crude product.
A mixture of the crude product (1.42 g), (Boc)2O (1.26 g,
5.77 mmol), and DMAP (0.06 g, 0.49 mmol) in MeCN (20 mL) was
stirred at room temperature for 20 h. After removal of the solvent,
the residue was purified by column chromatography on silica gel
with hexane–EtOAc (4:1 to 3:1, v/v) as eluent to give methyl 4-
[1-tert-butoxycarbonyl-(3S)-methoxy-(2R)-pyrrolidinylmeth-
oxy]cyclohexanecarboxylate (cis-rich, 1.67 g, 86% for 2 steps) as a
pale yellow oil. MS (ESI), m/z 372 [M+H]+.
To a stirred solution of the cis-rich product (1.67 g, 4.50 mmol)
in MeOH (15 mL) was added NaOMe (0.73 g, 13.5 mmol) and the
reaction mixture was heated under reflux for 24 h. After being
cooled to the room temperature, the mixture was concentrated
to a small volume. The mixture was acidified with 1 N HCl, and ex-
tracted with EtOAc. The extract was washed with brine, dried over
Na2SO4, and evaporated to give the crude product.
5.43. 1-Benzyloxycarbonyl-(2R)-tert-butyldimethylsilyl
oxymethyl-3-pyrrolidinone (15)
To a cooled (ꢁ78 °C), stirred solution of (COCl)2 (17.9 mL,
0.205 mol) and DMSO (21.8 mL, 0.307 mol) in CH2Cl2 (250 mL)
was added 10 (37.5 g, 0.102 mol) in CH2Cl2 (100 mL). After the
mixture was stirred at -78 °C for 1.5 h, Et3N (71.4 mL, 0.512 mol)
was added and the reaction mixture was stirred at ꢁ78 °C to room
temperature for 13 h. The mixture was poured into ice-H2O and ex-
tracted with CHCl3. The extract was washed with brine, dried over
Na2SO4, and evaporated. The residue was purified by column chro-
matography on silica gel with hexane–EtOAc (10:1 to 5:1, v/v) as
eluent to give the title compound (37.8 g, 100%) as a light reddish
brown oil. 1H NMR (CDCl3) d ꢁ0.05 and ꢁ0.02 (each s, total 6H),
0.82 (s, 9H), 2.45–2.62 (m, 2H), 3.67–3.74 (m, 1H), 3.88–4.19 (ser-
ies of m, total 4H), 5.14–5.25 (m, 2H), 7.32–7.38 (m, 5H).
5.44. 1-Benzyloxycarbonyl-(2R)-tert-butyldimethylsilyl
oxymethyl-(3R)-hydroxypyrrolidine (16)
To a stirred solution of the crude product in MeOH–benzene
(20 mL, 1:4, v/v) was added TMSCHN2 (2.0 M solution in hexane,
1.10 mL, 2.20 mmol) and the reaction mixture was stirred at room
temperature for 2 h. After removal of the solvent, the residue was
To a cooled (ꢁ10 °C), stirred solution of 15 (37.8 g, 0.104 mol) in
MeOH (300 mL) was added NaBH4 (4.73 g, 0.125 mol) and the reac-
tion mixture was stirred at ꢁ10 °C to room temperature for 7 h.

54
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
After being concentrated to a small volume, the mixture was
quenched by H2O and extracted with EtOAc. The extract was
washed with brine, dried over Na2SO4, and evaporated. The residue
was purified by column chromatography on silica gel with hexane–
EtOAc (5:1 to 3:1, v/v) as eluent to give the title compound (39.0 g,
100%) as a pale yellow oil. 1H NMR (CDCl3) d ꢁ0.02 (s, 3H), 0.05 and
0.08 (each s, total 3H), 0.85 and 0.88 (each s, total 9H), 1.94–2.08
(m, 2H), 3.32–3.34 and 3.48–3.52 (each m, total 3H), 3.85–4.13
(series of m, total 3H), 4.46–4.50 (m, 1H), 5.05–5.20 (m, 2H),
7.32–7.36 (m, 5H).
1.80–1.88 (m, 2H), 1.98–2.05 (m, 2H), 2.30 (m, 1H), 3.23–3.54 (ser-
ies of m, total 8H, including s, 3H, at d 3.41), 3.89–3.93 (m, 2H),
4.11 (q, J = 7.1 Hz, 2H).
5.48. Ethyl trans-4-[1-(tert-butoxycarbonyl)-(3R)-methoxy-
(2R)-pyrrolidinylmethoxy]cyclohexanecarboxylate (20)
A
mixture of 19 (1.77 g, 4.59 mmol) and NaOEt (0.94 g,
13.8 mmol) in EtOH (15 mL) was refluxed under N2 atmosphere
for 16 h. After being cooled to room temperature, the mixture
was acidified with 1 N HCl and extracted with EtOAc. The extract
was washed with brine, dried over Na2SO4, and evaporated. The
residue was dissolved in DMF (15 mL). To the solution was added
K2CO3 (1.58 g, 11.4 mmol) and EtI (0.69 mL, 8.63 mmol), and the
reaction mixture was stirred at 60 °C for 2 h. After being cooled
to room temperature, the mixture was acidified with 1 N HCl and
extracted with EtOAc. The extract was washed with brine, dried
over Na2SO4, and evaporated. The residue was purified by flash col-
umn chromatography with hexane–EtOAc (5:1, v/v) as eluent to
give the title compound (551 mg, 31%) as a colorless oil. 1H NMR
(CDCl3) d 1.20–1.30 (series of m, total 5H, including t, J = 7.1 Hz,
3H, at d 1.24), 1.40–1.50 (m, total 11H, including s, 9H, at d 1.46),
1.97–2.08 (m, 6H), 2.21–2.27 (m, 1H), 3.20–3.42 (m, total 6H,
including s, 3H, at d 3.40), 3.57–3.69 (m, 2H), 3.89–3.95 (m, 2H),
4.11 (q, J = 7.1 Hz, 2H).
5.45. 1-Benzyloxycarbonyl-(3R)-methoxy-(2R)-
pyrrolidinemethanol (17)
The title compound was prepared in a procedure similar to that
described for 11, starting from 16 via 1-benzyloxycarbonyl-(2R)-
tert-butyldimethylsilyloxymethyl-(3R)-methoxypyrrolidine.
For 1-benzyloxycarbonyl-(2R)-tert-butyldimethylsilyloxymeth-
yl-(3R)-methoxypyrrolidine; Yield 85%. A colorless oil. 1H NMR
(CDCl3) d ꢁ0.02 to 0.08 (m, total 6H), 0.87 and 0.90 (each s, total
9H), 2.04 (m, 2H), 3.38–3.51 (m, total 5H, including s, 3H, at d
3.39), 3.76 (m, 1H), 3.88–3.92 (m, 3H), 5.10–5.13 (m, 2H), 7.29–
7.36 (m, 5H).
For 17; Yield 100%. A colorless oil. 1H NMR (CDCl3) d 1.65 (s, 1H),
1.96–2.14 (m, 2H), 3.37 and 3.41 (each s, total 3H), 3.50–3.53 (m,
2H), 3.80–4.08 (m, 4H), 5.11–5.18 (m, 2H), 7.31–7.37 (m, 5H).
5.49. Ethyl trans-4-[(3R)-methoxy-(2R)-
5.46. Ethyl 4-[(3R)-methoxy-(2R)-pyrrolidinylmethoxy]
pyrrolidinylmethoxy]cyclohexanecarboxylate (5b)
benzoate (18)
The title compound was prepared in a procedure similar to
that described for 5a, starting from 20. Yield 95%. A pale yellow
oil. 1H NMR (CDCl3) d 1.20–1.30 (series of m, total 5H, including
t, J = 7.1 Hz, 3H, at d 1.24), 1.41–1.51 (m, 2H), 1.89–2.12 (m, 6H),
2.20–2.28 (m, 1H), 2.68 (broad s, 1H), 2.88–2.94 (m, 1H), 3.11–
3.32 (m, total 6H, including s, 3H, at d 3.29), 3.52–3.56 (m,
1H), 3.67–3.71 (m, 1H), 3.82–3.85 (m, 1H), 4.11 (q, J = 7.1 Hz,
2H).
To a stirred solution of 17 (2.17 g, 8.18 mmol), ethyl 4-hydroxy-
benzoate (1.36 g, 8.18 mmol) and Ph3P (2.57 g, 9.80 mmol) in THF
(50 mL) was added DIAD (2.42 mL, 12.3 mmol) and the reaction
mixture was heated under reflux for 7 h. After being cooled to
room temperature, the solvent was evaporated. The residue was
purified by column chromatography on silica gel with hexane–
EtOAc (3:1, v/v) as eluent to give ethyl 4-[1-benzyloxycarbonyl-
(3R)-methoxy-(2R)-pyrrolidinylmethoxy]benzote (5.71 g) as
a
crude product. A stirred solution of the crude product (5.17 g) in
MeOH (100 mL) was hydrogenated over 20% Pd(OH)2/C (1.03 g)
for 22 h. The mixture was filtered to remove the catalyst and the
solvent was evaporated. The residue was purified by column chro-
matography on silica gel with CHCl3–MeOH (60:1 to 10:1, v/v) as
eluent to give the title compound (1.81 g, 79% for two steps) as a
colorless oil. 1H NMR (CDCl3) d 1.38 (t, J = 7.1 Hz, 3H), 1.93–1.98
(m, 2H), 2.18 (broad s, 1H), 2.92–2.99 (m, 1H), 3.14–3.18 (m, 1H),
3.30 (s, 3H), 3.38–3.45 (m, 1H), 3.93–3.96 (m, 1H), 4.05–4.09 (m,
1H), 4.21–4.25 (m, 1H), 4.34 (q, J = 7.1 Hz, 2H), 6.94–6.96 (m,
2H), 7.97–7.99 (m, 2H).
5.50. (2S)-Benzyloxymethyl-1-tert-butoxycarbonyl-(5R)-
methoxymethylpyrrolidine (22)
To a cooled (0 °C), stirred solution of (2S)-benzyloxymethyl-1-
tert-butoxycarbonyl-(5R)-hydroxymethylpyrrolidine
(190 mg, 0.59 mmol) and NaH (28 mg, 1.17 mmol) in DMF (5 mL)
was added MeI (44 l, 0.71 mmol) and the reaction mixture was
(21)
l
stirred at room temperature for two days. The mixture was
quenched by H2O, extracted with EtOAc, washed with brine, dried
over Na2SO4, and evaporated. The residue was purified by column
chromatography on silica gel with hexane–EtOAc (5:1, v/v) as elu-
ent to give the title compound (183 mg, 92%) as a colorless oil. 1H
NMR (CDCl3) d 1.44 (s, 9H), 1.87–1.99 (m, 4H), 3.28–3.34 (m, total
5H, including s, 3H, at d 3.31), 3.49 (m, 1H), 3.51–3.61 (m, 1H), 3.96
(m, 2H), 4.52 (s, 2H), 7.25–7.33 (m, 5H).
5.47. Ethyl 4-[1-(tert-butoxycarbonyl)-(3R)-methoxy-(2R)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (19)
A mixture of 18 (1.81 g, 6.48 mmol) in MeOH–TFA (31 mL, 30:1,
v/v) was hydrogenated over 5% Rh/Al2O3 (0.54 g) at 10 atm H2 for
15 h. The mixture was filtered to remove the catalyst, and the sol-
vent was evaporated. The residue was dissolved in MeCN–satd
NaHCO3 aq (20 mL, 1:1, v/v). (Boc)2O (1.70 g, 7.79 mmol) was
added to the mixture, and the mixture was stirred at room temper-
ature for 2 days. The mixture was acidified with 1 N HCl and ex-
tracted with EtOAc. The extract was washed with brine, dried
over Na2SO4, and evaporated. The residue was purified by column
chromatography on silica gel with CHCl3–MeOH (60:1, v/v) as elu-
ent to give the title compound (1.77 g, 71%) as a colorless oil. 1H
NMR (CDCl3) d 1.21–1.29 (m, total 5H, including t, J = 7.1 Hz, 3H,
at d 1.24) 1.45–1.66 (m, total 13H, including s, 9H, at d 1.46),
5.51. 1-tert-Butoxycarbonyl-(5R)-methoxymethyl-(2S)-
pyrrolidinemethanol (23)
A stirred solution of 22 (183 mg, 0.55 mmol) in MeOH (10 mL)
was hydrogenated over 10% Pd/C (40 mg) for 15 h. The mixture
was filtered to remove the catalyst and the solvent was evaporated.
The residue was purified by column chromatography on silica gel
with hexane–EtOAc (2:1, v/v) as eluent to give the title compound
(116 mg, 87%) as a colorless oil. 1H NMR (CDCl3) d 1.48 (s, 9H),
1.87–2.01 (m, 4H), 3.36 (s, 3H), 3.38–3.39 (m, 1H), 3.45–3.51 (m,
1H), 3.78–3.99 (m, 3H), 4.66–4.69 (m, 1H).

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
55
5.52. Methyl 4-[(5R)-methoxymethyl-(2S)-pyrrolidinylmethoxy]
benzoate (24)
To a stirred solution of methyl trans-4-[1-tert-butoxycarbonyl-
(5R)-methoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohexanecar-
boxylate (321 mg, 0.83 mmol) in CH2Cl2 (3 mL) was added TFA
(3 mL) and the reaction mixture was stirred at room temperature
for 1 h. The mixture was made basic with satd NaHCO3 aq and ex-
tracted with CHCl3. The extract was washed with brine, dried over
Na2SO4, and evaporated to give the title compound (240 mg, 100%)
as a pale yellow oil. 1H NMR (CDCl3) d 1.21–1.33 (m, 2H), 1.41–1.50
(m, 2H), 1.80–1.86 (m, 3H), 1.99–2.09 (m, 5H), 2.23–2.30 (m, 1H),
3.18–3.26 (m, 1H), 3.29–3.44 (m, total 8H, including s, 3H, at d
3.36), 3.49–3.52 (m, 1H), 3.66 (s, 3H).
To a stirred solution of 23 (950 mg, 3.87 mmol), methyl 4-
hydroxybenzoate (648 mg, 4.26 mmol) and Ph3P (1.22 g,
4.65 mmol) in THF (20 mL) was added DIAD (1.14 mL, 5.79 mmol)
and the reaction mixture was heated under reflux for 10 h. After
being cooled to the room temperature, the solvent was evaporated.
The residue was purified by column chromatography on silica gel
with hexane–EtOAc (6:1, v/v) as eluent to give methyl 4-[1-tert-
butoxycarbonyl-(5R)-methoxymethyl-(2S)-pyrrolidinylmethoxy]
benzoate (1.62 g) as a crude product.
To a stirred solution of the crude product (1.62 g) in CH2Cl2
(15 mL) was added TFA (15 mL) and the reaction mixture was stir-
red at room temperature for 17 h. The mixture was made basic
with satd NaHCO3 aq and extracted with CHCl3. The extract was
washed with brine, dried over Na2SO4, and evaporated. The residue
was purified by column chromatography on silica gel with CHCl3–
MeOH (20:1, v/v) as eluent to give the title compound (1.03 g, 95%
for 2 steps) as a pale yellow oil. 1H NMR (CDCl3) d 1.49–2.01 (m,
5H), 3.27–3.33 (m, 1H), 3.37–3.44 (m, total 5H, including s, 3H,
at d 3.37), 3.53–3.59 (m, 1H), 3.87–4.00 (m, total 5H, including s,
3 H, at d 3.88), 6.09 (d, J = 8.8 Hz, 2 H), 7.97 (d, J = 8.8 Hz, 2 H).
5.54. (2S)-Benzyloxymethyl-1-tert-butoxycarbonyl-(5S)-
methoxymethylpyrrolidine (26a)
The title compound was prepared in a procedure similar to that
described for 22, starting from (2S)-benzyloxymethyl-1-tert-
butoxycarbonyl-(5S)-hydroxymethylpyrrolidine (25a). Yield 74%.
A colorless oil. 1H NMR (CDCl3) d 1.40 and 1.47 (s, total 9H),
1.86–2.02 (m, 4H), 3.20–3.68 (series of m, 4H), 3.34 (s, 3H), 3.86–
4.00 (m, 2H), 4.46–4.58 (m, 2H), 7.27–7.34 (m, 5H).
5.55. 1-tert-Butoxycarbonyl-(2S)-hydroxymethyl-(5S)-
methoxymethylpyrrolidine (27a)
5.53. Methyl trans-4-[(5R)-methoxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5c)
The title compound was prepared in a procedure similar to that
described for 23, starting from 26a. Yield 100%. A colorless oil. 1H
NMR (CDCl3) d 1.49 (s, 9H), 1.57–1.70 (m, 1H), 1.87–1.98 (m,
2H), 2.01–2.14 (m, 1H), 3.17–4.02 (series of m, 6H), 3.35 (s, 3H).
A stirred solution of 24 (1.03 g, 3.69 mmol) in MeOH–TFA
(21 mL, 20:1, v/v) was hydrogenated over Rh/Al2O3 (0.50 g) at
10 atm H2 for 1 day. The mixture was filtered to remove the cata-
lyst and the solvent was evaporated to give methyl 4-[(5R)-
methoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxyl-
ate TFA salt (1.15 g) as a crude product.
5.56. Methyl 4-[1-tert-butoxycarbonyl-(5S)-methoxymethyl-
(2S)-pyrrolidinylmethoxy]benzoate (28a)
To a stirred solution of 27a (865 mg, 3.53 mmol), triphenyl-
To a stirred solution of the crude product (1.15 g) in 1,4-diox-
ane-satd NaHCO3 aq (30 mL, 1:1, v/v) was added (Boc)2O (0.81 g,
3.71 mmol) and the reaction mixture was stirred at room temper-
ature for 2 h. The mixture was acidified with 1 N HCl, and extracted
with EtOAc. The extract was washed with brine, dried over Na2SO4,
and evaporated. The residue was purified by column chromatogra-
phy on silica gel with hexane–EtOAc (5:1, v/v) as eluent to give
methyl 4-[1-tert-butoxycarbonyl-(5R)-methoxymethyl-(2S)-pyrro-
lidinylmethoxy]cyclohexanecarboxylate (1.32 g, 93% for 2 steps) as
a colorless oil. 1H NMR (CDCl3) d 1.46 (s, 9H), 1.64–1.67 (m, 2H),
1.80–1.98 (m, 6H), 2.10–2.34 (m, 5H), 3.32–3.39 (m, total 5H,
including s, 3H, at d 3.35), 3.46–3.63 (m, 3H), 3.67 (s, 3H), 3.91
(m, 2H).
To a stirred solution of methyl 4-[1-tert-butoxycarbonyl-(5R)-
methoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarbox-
ylate (1.32 g, 3.42 mmol) in MeOH (10 mL) was added NaOMe
(0.55 g, 10.2 mmol) and the reaction mixture was heated under re-
flux for 20 h. After being cooled to the room temperature, the mix-
ture was concentrated to a small volume. The mixture was diluted
with H2O, and extracted with EtOAc. The extract was washed with
brine, dried over Na2SO4, and evaporated to give the crude product.
To a stirred solution of the crude product in MeOH–benzene
phosphine (1.11 g, 4.23 mmol) and methyl 4-hydroxybenzoate
(536 mg, 3.53 mmol) was added DIAD (803 ll, 3.88 mmol) at room
temperature, and the resulting mixture was stirred at 60 °C for 2 h.
The mixture was concentrated in vacuo, and the residue was chro-
matographed on silica gel with hexane–EtOAc (3:1, v/v) as eluent
to give the title compound (1.13 g, 84%) as a colorless oil. 1H
NMR (CDCl3) d 1.48 (s, 9H), 1.94–2.14 (m, 4H), 3.26–4.27 (series
of m, 6H), 3.36 (s, 3H), 3.88 and 3.89 (each s, total 3H), 6.93 and
6.96 (each d, J = 8.8 Hz, total 2H), 7.96 and 7.99 (each d,
J = 4.0 Hz, total 2H).
5.57. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-
methoxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (29a)
A mixture of 28a (1.02 g, 2.69 mmol) and 5% Rh/Al2O3 (500 mg)
in EtOH–AcOH (10:1, v/v, 55 mL) was stirred under hydrogen
atmosphere (8 atm) at room temperature for 2.5 h. The mixture
was filtered, and the filtrate was concentrated in vacuo. The resi-
due was chromatographed on silica gel with hexane–EtOAc (3:1,
v/v) as eluent to give methyl 4-[1-tert-butoxycarbonyl-(5S)-
methoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxyl-
ate (771 mg, 74%) as a colorless oil. A mixture of the oil (770 mg,
1.99 mmol) in MeOH was added NaOMe (324 mg, 5.99 mmol) at
room temperature, and the resulting mixture was stirred under re-
flux overnight. After being cooled to room temperature, the mix-
ture was added to 1 N HCl and concentrated in vacuo, then
extracted with CHCl3–MeOH (10:1). The extract was dried over
Na2SO4, then concentrated in vacuo to give a crude colorless oil.
To a stirred solution of the oil in benzene–MeOH (10:1, 22 mL)
was added TMSCHN2 (2.0 M in hexane, 0.50 mL, 0.25 mmol) at
room temperature, and the resulting mixture was stirred for an
(10 mL, 1:4, v/v) was added TMSCHN2 (2.0 M solution, 680 ll,
1.36 mmol) and the reaction mixture was stirred at room temper-
ature for 3 h. After the mixture was evaporated, the residue was
purified by flash column chromatography with hexane–EtOAc
(7:1, v/v) as eluent to give methyl trans-4-[1-tert-butoxycar-
bonyl-(5R)-methoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohex-
anecarboxylate (321 mg, 24%) as a pale yellow oil. 1H NMR (CDCl3)
d 1.22–1.33 (m, 2H), 1.41–1.51 (m, total 11H, including s, 9H, at d
1.46), 1.87–2.07 (m, 8H), 2.24–2.29 (m, 1H), 3.17–3.63 (m, total 8H,
including s, 3H, at d 3.35), 3.66 (s, 3H), 3.87–3.97 (m, 2H).

56
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
hour. The mixture was concentrated in vacuo, and the residue was
purified by flash chromatography with hexanae–EtOAc (5:1, v/v) as
eluent to give the title compound (178 mg, 23%) as a colorless oil.
1H NMR (CDCl3) d 1.15–1.33 (m, 2H), 1.36–1.54 (m, 11H, including
s, 9H, at d 1.47), 1.78–2.10 (m, 8H), 2.19–2.33 (m, 1H), 3.12–3.50
(m, 7H, including s, 3H, at d 3.34), 3.51–3.62 (m, 1H), 3.65 and
3.66 (each s, total 3H), 3.75–3.97 (m, 2H); MS (ESI), m/z 386
[M+H]+.
ate (4.96 g, 22.7 mmol) in CH2Cl2 (100 mL) was added Et3N
(4.32 mL, 31.0 mmol) at room temperature, and the resulting
mixture was stirred for 3.5 h. The mixture was concentrated in
vacuo, and the residue was chromatographed on silica gel with
hexane–EtOAc (3:1) as eluent to give methyl 4-[1-tert-butoxycar-
baonyl-(5S)-ethoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohex-
anecarboxylate (7.45 g, 90%) as a colorless oil. To a mixture of
methyl 4-[1-tert-butoxycarbaonyl-(5S)-ethoxymethyl-(2S)-pyrro-
lidinylmethoxy]cyclohexanecarboxylate (7.45 g, 18.7 mmol) and
NaH (60% in mineral oil, 895 mg, 22.4 mmol) in DMF (100 mL)
was added MeOH (1.51 mL, 37.3 mmol) at 0 °C, and the resulting
mixture was stirred at room temperature for 1 h. The mixture
was poured into 1 N HCl, then extracted with Et2O. The extract
was washed with water and brine, dried over Na2SO4, then concen-
trated in vacuo to give a crude colorless oil. To a stirred solution of
the oil in benzene–MeOH (6:1, 60 mL) was added trimethylsilyl
diazomethane (2.0 M in hexane, 1.87 mL, 3.73 mmol) at room tem-
perature, and the resulting mixture was stirred for 1 h. The mixture
was concentrated in vacuo, and the residue was purified by flash
chromatography with hexane–EtOAc (5:1) as eluent to give the ti-
tle compound (1.37 g, 18%) as a colorless oil. 1H NMR (CDCl3) d
1.12–1.31 (m, 5H), 1.37–1.52 (m, 11H, including s, 9H, at d 1.46),
1.81–2.10 (m, 8H), 2.19–2.31 (m, 1H), 3.14–3.69 (m, 10H, including
each s, total 3H, at d 3.65 and 3.66), 3.75–3.97 (m, 2H).
5.58. Methyl trans-4-[(5S)-methoxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5d)
The title compound was prepared in a procedure similar to that
described for 5a, starting from 29a. Yield 100%. A yellow oil. 1H
NMR (CDCl3) d 1.18–1.31 (m, 2H), 1.38–1.52 (m, 2H), 1.81–1.95
(m, 4H), 1.96–2.12 (m, 4H), 2.21–2.32 (m, 1H), 3.17–3.47 (m,
10H), 3.66 (s, 3H); MS (ESI), m/z 286 [M+H]+.
5.59. (2S)-Benzyloxymethyl-1-tert-butoxycarbonyl-(5S)-
ethoxymethylpyrrolidine (26b)
The title compound was prepared in a procedure similar to that
described for 22, starting from 25a using EtI. Yield 90%. A colorless
oil. 1H NMR (CDCl3) d 1.16 and 1.18 (each t, J = 7.3 Hz, total 3H),
1.40 and 1.47 (each s, total 9H), 1.84–2.12 (m, 4H), 3.16–3.73 (m,
6H), 3.80–4.05 (m, 2H), 4.42–4.62 (m, 2H), 7.23–7.38 (m, 5H);
MS (ESI), m/z 350 [M+H]+.
5.63. Methyl trans-4-[(5S)-ethoxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5e)
5.60. 1-tert-Butoxycarbonyl-(5S)-ethoxymethyl-(2S)-
hydroxymethylpyrrolidine (27b)
The title compound was prepared in a procedure similar to that
described for 5a, starting from 29b. Yield 97%. A pale yellow oil. 1H
NMR (CDCl3) d 1.15–1.32 (m, 5H, including t, J = 6.9 Hz, 3H, at d
1.19), 1.37–1.54 (m, 2H), 1.83–2.15 (m, 8H), 2.20–2.32 (m, 1H),
3.17–3.27 (m, 1H), 3.27–3.44 (m, 6H), 3.49 (q, J = 6.9 Hz, 2H),
3.66 (s, 3H); MS (ESI), m/z 300 [M+H]+.
The title compound was prepared in a procedure similar to that
described for 23, starting from 26b. Yield 97%. A colorless oil. 1H
NMR (CDCl3) d 1.18 (t, 3H), 1.48 (s, 9H), 1.49–2.18 (m, 4H), 3.24–
3.87 (m, 6H), 3.88–4.30 (m, 2H).
5.64. Methyl 4-[(5S)-benzyloxymethyl-1-tert-butoxycarbonyl-
(2S)-pyrrolidinylmethoxy]benzoate (28c)
5.61. Methyl 4-[1-tert-butoxycarbonyl-(5S)-ethoxymethyl-(2S)-
pyrrolidinylmethoxy]benzoate (28b)
The title compound was prepared in a procedure similar to that
described for 28a, starting from 25a. Yield 80%. A clear oil. 1H NMR
(CDCl3) d 1.40 and 1.48 (each s, total 9H), 1.92–2.23 (m, 4H), 3.31–
3.71 (series of m, total 2H), 3.80–3.96 (m, total 4H, including each
s, total 3H, at d 3.88 and 3.89), 3.96–4.28 (series of m, total 3H),
4.45–4.60 (m, 2H), 6.95 (m, 2H), 7.23–7.39 (m, 5H), 7.97 (m, 2H).
The title compound was prepared in a procedure similar to that
described for 28a, starting from 27b. Yield 84%. A colorless oil. 1H
NMR (CDCl3) d 1.13–1.24 (m, 3H), 1.47 (s, 9H), 1.90–2.23 (m,
4H), 3.23–3.68 (m, 4H), 3.79–4.31 (m, 7H), 6.88–7.02 (m, 2H),
7.92–8.06 (m, 2H); MS (ESI), m/z 394 [M+H]+.
5.62. Methyl trans-4-[1-tert-butoxycarbaonyl-(5S)-
ethoxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (29b)
5.65. Methyl 4-[1-tert-butoxycarbonyl-(5S)-hydroxymethyl-
(2S)-pyrrolidinylmethoxy]benzoate (28d)
The title compound was prepared in a procedure similar to that
described for 23, starting from 28c. Yield 91%. A colorless foam. 1H
NMR (CDCl3) d 1.48 (s, 9H), 1.57–1.74 (m, 2H), 1.97–2.25 (m, 2H),
3.53–3.78 (m, 2H), 3.84–3.97 (m, 4H, including s, 3H, at d 3.89),
4.01–4.28 (m, 3H), 6.93 (m, 2H), 7.98 (m, 2H); MS (ESI), m/z 366
[M+H]+.
To a stirred solution of 28b (9.10 g, 23.13 mmol) in CH2Cl2
(100 mL) was added TFA (50 mL) at room temperature, and the
resulting mixture was stirred for 20 min. The mixture was concen-
trated in vacuo and poured into satd NaHCO3 aq The mixture was
extracted with CHCl3–MeOH (10:1), and the extract was dried over
Na2SO4, then concentrated in vacuo to give methyl 4-[(5S)-ethoxy-
methyl-(2S)-pyrrolidinylmethoxy]benzoate (6.24 g, 92%) as a pale
yellow oil. A mixture of methyl 4-[(5S)-ethoxymethyl-(2S)-pyrro-
lidinylmethoxy]benzoate (6.24 g, 21.3 mmol) and 5% Rh/AlO3
(1.41 g) in MeOH–AcOH (7:1, 80 mL) was stirred under hydrogen
atmosphere (11 atm) at room temperature overnight. The mixture
was filtered, and the filtrate was concentrated in vacuo to give
methyl 4-[(5S)-ethoxymethyl-(2S)-pyrrolidinylmethoxy]cyclohex-
anecarboxylate (6.19 g, 97%) as a colorless oil. To a stirred mixture
of methyl 4-[(5S)-ethoxymethyl-(2S)-pyrrolidinylmethoxy]cyclo-
hexanecarboxylate (6.19 g, 20.7 mmol) and di-tert-butyl dicarbon-
5.66. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-
hydroxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (29c)
The title compound was prepared in a procedure similar to that
described for 29a, starting from 28d. Yield 18% (three steps). A pale
yellow oil. 1H NMR (CDCl3) d 1.17–2.40 (series of m, total 22H,
including s, 9H, at d 1.48), 3.14–4.28 (series of m, total 10H, includ-
ing each s, total 3H, at d 3.66 and 3.67).

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
57
5.67. Methyl trans-4-[(5S)-acetoxymethyl-(2S)-pyrrolidinyl
methoxy]cyclohexanecarboxylate (5f)
10 mL). To this solution was added TMSCHN2 (2.0 M in hexane,
600 l, 1.2 mmol), and the resulting mixture was stirred at room
temperature for 16 h. To the reaction mixture was added AcOH
(200 l, 0.12 mmol) and concentrated in vacuo. The residue was
l
To a stirred solution of 29c (280.6 mg, 0.755 mmol) in pyridine
(6.0 mL) was added (AcO)2O (3.0 mL) at room temperature. After
being stirred for 2.5 h, the reaction was quenched with EtOH at
room temperature. The mixture was diluted with EtOAc, washed
with 1 N HCl, dried over Na2SO4, and concentrated to give methyl
trans-4-[(5S)-acetoxymethyl-1-tert-butoxycarbonyl-(2S)-pyrrolidi-
nylmethoxy]cyclohexanecarboxylate (29d). To a stirred solution of
29d in CH2Cl2 (6.0 mL) was added TFA (6.0 mL) at 0 °C. The mixture
was stirred for 2 h at room temperature, concentrated, and neu-
tralized with satd NaHCO3 aq The mixture was extracted with
CHCl3–MeOH (10:1, v/v), dried over Na2SO4 and evaporated to give
the title compound (100%) as a pale yellow oil, which was used to
the subsequent reaction without further purification. 1H NMR
(CDCl3) d 1.17–1.57 (series of m, total 5H), 1.61–2.21 (series of
m, total 10H, including s, 3H, at d 2.07), 2.26 (m, 1H), 3.16–3.40
(series of m, total 2H), 3.40–3.55 (m, 2H), 3.66 and 3.67 (each s, to-
tal 3H), 3.89 (dd, J = 7.6 and 10.8 Hz, 1H), 4.03 (dd, J = 4.8 and
10.8 Hz, 1H), 4.14 and 4.49 (each m, total 1H); MS (ESI), m/z 314
[M+H]+.
l
purified by flash column chromatography with hexane–EtOAc
(5:1, v/v) as eluent to give methyl trans-4-[1-tert-butoxycar-
bonyl-(5R)-methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarbox-
ylate (29e) (120 mg, 27% for 2 steps) as a pale yellow oil.
To a stirred solution of 29e (234 mg, 0.66 mmol) in CH2Cl2
(5 mL) was added TFA (1 mL), and the mixture was stirred at room
temperature for 3 h. After the reaction mixture was concentrated
in vacuo, the residue was treated with satd NaHCO3 aq and ex-
tracted with CHCl3–MeOH (5:1, v/v). The extract was dried over
MgSO4, and concentrated to give the title compound (168 mg,
quant.) as a pale yellow oil. 1H NMR (CDCl3) d 1.13 (d, J = 6.0 Hz,
3H), 1.27 (m, 3H), 1.39-1.48 (series of m, total 3H), 1.88–2.09 (ser-
ies of m, total 7H), 2.27 (tt, J = 4.0 and 12.0 Hz, 1H), 3.23 (m, 2H),
3.32–3.42 (series of m, total 3H), 3.66 (s, 3H); MS (ESI), m/z 256
[M+H]+.
5.70. (2S)-Benzyloxymethyl-1-tert-butoxycarbonyl-(5S)-
methansulfonyloxymethylpyrrolidine (25b)
5.68. Methyl 4-[1-tert-butoxycarbonyl-(5R)-methyl-(2S)-
To a stirred solution of 25a (2.0 g, 6.22 mmol) in CH2Cl2 (10 mL)
pyrrolidinylmethoxy]benzoate (28e)
was added Et3N (1.7 mL, 12.4 mmol) and MsCl (722 ll, 9.33 mmol)
at 0 °C. After being stirred at the same temperature for 1 h, the
mixture was quenched with H2O (50 mL) and extracted with CHCl3
(200 mL). The extract was washed with brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was chro-
matographed on silica gel with CHCl3–EtOAc (4:1) as eluent to give
the title compound (2.54 g, quant.) as a yellow oil. 1H NMR (CDCl3)
d 1.40–1.48 (m, 9H), 1.87–2.21 (m, 4H), 2.99–3.02 (m, 3H), 3.36–
4.57 (series of m, total 8H), 7.26–7.36 (m, 5H); MS (ESI), m/z 400
[M+H]+.
The title compound was prepared in a procedure similar to that
described for 28a, starting from 27c. Yield 81%. A colorless oil. 1H
NMR (CDCl3) d 1.16 and 1.20 (each d, J = 6.4 Hz, total 3H), 1.47 (s,
9H), 1.55 (m, 1H), 2.01 (m, 1H), 2.12 (m, 2H), 3.80–3.95 (series of
m, total 2H), 3.87 (s, 3H), 4.11–4.26 (series of m, total 2H), 6.95
(m, 2H), 7.96 (d, J = 8.8 Hz, 2H).
5.69. Methyl trans-4-[(5R)-methyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5g)
5.71. (2S)-Benzyloxymethyl-1-tert-butoxycarobnyl-(5S)-
fluoromethylpyrrolidine (26c)
To a stirred solution of 28e (471 mg, 1.37 mmol) in CH2Cl2
(6 mL) was added TFA (1 mL) at room temperature. After being
stirred for 3 h at room temperature, the reaction mixture was con-
centrated in vacuo to give methyl 4-[(5R)-methyl-(2S)-pyrrolidi-
nylmethoxy]benzoate as a yellow oil. The oil was dissolved in
MeOH (12 mL) and hydrogenated at 20 atm over Rh/Al2O3
(250 mg) for 5 h. After removal of the catalyst by filtration, the fil-
trate was concentrated in vacuo to give methyl 4-[(5R)-methyl-
(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (550 mg,
quant.) as a pale brown solid. To a solution of this solid in
MeCN–H2O (1:1, v/v, 12 mL) was added Boc2O (299 mg,
To a stirred solution of 25b (499 mg, 1.25 mmol) in THF (22 mL)
was added TBAF (1.0 M in THF, 12.5 mL, 12.5 mmol). After being
refluxed for 8 h, the reaction mixture was poured into H2O and ex-
tracted with EtOAc. The extract was dried over MgSO4 and concen-
trated under reduced pressure. The residue was chromatographed
on silica gel with hexane–EtOAc (4:1) as eluent to give the title
compound (273 mg, 68%) as a colorless oil. 1H NMR (CDCl3) d
1.40 and 1.46 (each s, total 9H), 1.87–2.18 (m, 4H), 3.33–3.68 (m,
2H), 3.90–4.04 (m, 2H), 4.23–4.62 (m, 4H), 7.31 (m, 5H); MS
(ESI), m/z 324 [M+H]+.
1.37 mmol) and Et3N (420 ll, 3.01 mmol), and the mixture was
stirred at room temperature for 14 h. The reaction mixture was
concentrated to a small volume. The residue was partitioned be-
tween 1 N HCl and EtOAc. The phases were separated and the
aqueous phase was extracted with EtOAc. The combined extracts
were washed with brine, dried over Na2SO4, and concentrated in
vacuo to give methyl 4-[1-tert-butoxycarbonyl-(5R)-methyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (440 mg, 90%) as a
pale yellow oil. 1H NMR (CDCl3) d 1.07–1.34 (m, 4H), 1.38–1.58
(m, 12H, including s, 9H, at d 1.46), 1.58–1.74 (m, 2H), 1.74–2.23
(m, 6H), 2.25–2.45 (m, 1H), 3.14–3.62 (m, 3H), 3.67 (s, 3H), 3.77–
4.06 (m, 2H).
The resulting oil was dissolved in MeOH (40 mL). To this solu-
tion was added NaOMe (200 mg, 3.70 mmol) under N2, and the
mixture was refluxed for 18 h. After being cooled to room temper-
ature, the resulting mixture was concentrated, acidified with 1 N
HCl at 0 °C, and extracted with EtOAc. The extract was washed
with brine, dried over Na2SO4, and concentrated in vacuo to give
5.72. 1-tert-Butoxycarbonyl-(5S)-fluoromethyl-(2S)-
hydroxymethylpyrrolidine (27d)
The title compound was prepared in a procedure similar to that
described for 23, starting from 26c. Yield 83%. A colorless oil. 1H
NMR (CDCl3) d 1.48 (s, 9H), 1.93–2.11 (m, 4H), 3.41–4.51 (series
of m, 6H); MS (ESI), m/z 234 [M+H]+.
5.73. Methyl 4-[1-tert-butoxycarbonyl-(5S)-fluoromethyl-(2S)-
pyrrolidnylmethyl]benzoate (28f)
The title compound was prepared in a procedure similar to that
described for 28a, starting from 27d. Yield 92%. A colorless oil. 1H
NMR (CDCl3) d 1.47 and 1.48 (each s, total 9H), 1.99–2.26 (m, 4H),
3.88–4.72 (series of m, 9H), 6.91–6.97 (m, 2H), 7.96–7.99 (m, 2H);
MS (ESI), m/z 368 [M+H]+.
a
yellow oil, which was dissolved in benzene–MeOH (4:1,

58
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
5.74. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-fluoromethyl-
(2S)-pyrrolidinylmethyl]cyclohexanecarboxylate (29f)
total 2H), 3.22 (m, 1H), 3.26–3.48 (m, 4H), 3.66 (s, 3H); MS (ESI),
m/z 302 [M+H]+.
The title compound was prepared in a procedure similar to that
described for 29a, starting from 28f via methyl 4-[1-(tert-
butoxycarbonyl)-(5S)-fluoromethyl-(2S)-pyrrolidinylmethyl]
cyclohexanecarboxylate.
For methyl 4-[1-(tert-butoxycarbonyl)-(5S)-fluoromethyl-(2S)-
pyrrolidinylmethyl]cyclohexanecarboxylate; Yield 54%. A colorless
oil. 1H NMR (CDCl3) d 1.11–2.41 (m, 22H), 3.13–3.73 (m, 6H), 3.79–
4.07 (m, 2H), 4.21–4.44 (m, 1H), 4.45–4.66 (m, 1H); MS (ESI), m/z
374 [M+H]+.
5.78. Ethyl 4-[(5S)-benzyloxymethyl-1-tert-butoxycarbonyl-
(2S)-pyrrolidinylmethoxy]benzoate (28g)
The title compound was prepared in a procedure similar to that
described for 28a, starting from 25a using ethyl 4-hydroxybenzo-
ate. Yield 74%. A colorless oil.
5.79. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-
benzyloxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (29i)
For 29f; Yield 41% (two steps). A pale yellow oil. 1H NMR
(CDCl3) d 1.10–1.30 (m, 2H), 1.34–1.52 (m, 11H, including s, 9H,
at d 1.44), 1.79–2.30 (m, 9H), 3.11–3.23 (m, 1H), 3.24–3.49 (m,
1H), 3.50–3.69 (m, 4H, including each s, total 3H, at d 3.63 and
3.64), 3.78–4.03 (m, 2H), 4.17–4.40 (m, 1H), 4.42–4.62 (m, 1H);
MS (ESI), m/z 374 [M+H]+.
A mixture of 28g (54 g, 115 mmol) and Pd(OH)2 (7.0 g) in EtOH–
AcOH (5:1, 600 mL) was stirred under hydrogen atmosphere
(1 atm) at room temperature for 5 days. The mixture was filtered
to remove the catalyst and the solvent was evaporated to give
ethyl 4-[1-tert-butoxycarbonyl-(5S)-hydroxymethyl-(2S)-pyrrolid-
inylmethoxy]benzoate (28h), which was used to the subsequent
reaction without further purification. To a stirred solution of 28h
(43.6 g, 115 mmol) in CH2Cl2 (150 mL) was added TFA (200 mL)
at 0 °C. After being stirred at room temperature for 40 min, the
mixture was concentrated. A mixture of the residue and 5% Rh/
Al2O3 (10 g) in EtOH–AcOH–TFA (25:5:1, v/v, 620 mL) was stirred
under hydrogen atmosphere (10 atm) at room temperature over-
night. The mixture was filtered, and the filtrate was concentrated
in vacuo. To a stirred solution of the residue in CH2Cl2 (500 mL)
was added Et3N (64 mL, 460 mmol) and Boc2O (27.6 g, 127 mmol)
at 0 °C. After the mixture was stirred at room temperature for
75 min, Et3N (100 mL, 719 mmol) was added to the mixture, which
was stirred for 75 min. The solution was concentrated and the res-
idue was chromatographed on silica gel with hexane–EtOAc (4:1 to
1:3, v/v) to give ethyl 4-[1-tert-butoxycarbonyl-(5S)-hydroxy-
methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate (30 g,
68% for 4 steps) as a light blue oil. 1H NMR (CDCl3) d 1.20–1.29
(m, 3H), 1.37–1.71 (m, 13H, including s, 9H, at d 1.47), 1.73–2.55
(m, 9H), 3.12–3.37 (m, 1H), 3.38–3.48 (m, 1H), 3.48–4.08 (m,
5H), 4.08–4.17 (m, 2H), 4.19–4.40 (m, 1H).
To a stirred solution of ethyl 4-[1-tert-butoxycarbonyl-(5S)-
hydroxymethyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylate
(30 g, 77.9 mmol) and benzyl bromide (18.6 mL, 155.8 mmol) in
DMF (300 mL) was added NaH (4.7 g, 116.9 mol) portionwise at
0 °C. After being stirred for 3 h, the mixture was diluted with
H2O, and extracted with EtOAc. The extract was washed with brine,
dried over Na2SO4, and concentrated. The residue was chromato-
graphed on silica gel with hexane–EtOAc (7:1 to 4:1, v/v) to give
ethyl 4-[(5S)-benzyloxymethyl-1-tert-butoxycarbonyl-(2S)-pyrro-
lidinylmethoxy]cyclohexanecarboxylate (30 g, 81%) as a colorless
oil. To a stirred solution of the oil (30 g, 63.2 mmol) in DMF
(300 mL) was added NaH (5.56 g, 139.0 mmol) and EtOH
(7.36 mL, 126.4 mmol) at 0 °C. After the mixture was stirred at
0 °C for 40 min, H2O (1.36 mL, 75.8 mmol) was added to the mix-
ture, which was stirred at room temperature overnight. To the
mixture was added H2O (1.36 mL, 63.2 mmol), K2CO3 (26.2 g,
189.6 mmol) and EtI (15.2 mL, 189.6 mmol), the resulting mixture
was stirred at 70 °C for 30 min. The mixture was diluted with H2O
and EtOAc and extracted with EtOAc. The extract was washed with
brine, dried over Na2SO4, and concentrated. The residue was puri-
fied by flash column chromatography with hexane–EtOAc (8:1, v/
v) as eluent to give the title compound (16 g, 53% for two steps)
as a yellow oil. 1H NMR (CDCl3) d 1.16–1.30 (m, 5H), 1.33–1.54
(m, 11H), 1.84–2.11 (m, 8H), 2.18–2.30 (m, 1H), 3.14–3.32 (m,
2H), 3.35–3.50 (m, 1H), 3.54–3.71 (m, 2H), 3.76–4.03 (m, 2H),
4.06–4.16 (m, 2H), 4.44–4.60 (m, 2H), 7.22–7.37 (m, 5H).
5.75. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-
methanesulfonyloxymethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (29g)
The title compound was prepared in a procedure similar to that
described for 25b, starting from 29c. Yield 73%. A clear oil. 1H NMR
(CDCl3) d 1.12–1.34 (m, 2H), 1.36–1.55 (m, total 11H, including
each s, total 9H, at d 1.47 and 1.49), 1.60–2.38 (series of m, total
9H), 3.00 (s, 3H), 3.19 (m, 1H), 3.31–3.65 (series of m, total 2H),
3.66 (s, 3H), 3.81–4.38 (series of m, total 4H); MS (ESI), m/z 450
[M+H]+.
5.76. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-
methylthiomethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (29h)
To a stirred solution of 29g (270.5 mg, 0.602 mmol) in DMF
(5.5 mL) was added sodium thiomethoxide (126.5 mg,
1.805 mmol). After being stirred for 4.5 h at 60 °C, the mixture
was diluted with EtOAc, washed with 1 N HCl, dried over Na2SO4,
and concentrated. The residue was dissolved in benzene–MeOH
(10:1, v/v, 5.5 mL), and to the solution was added TMSCHN2
(0.30 mL, 2.0 M in hexane, 0.602 mmol) at 0 °C. The mixture was
stirred for 30 min at room temperature, and then the reaction
was quenched with AcOH at 0 °C. The solution was concentrated
and the residue was chromatographed on silica gel with hexane–
EtOAc (2:1, v/v) to give the title compound (202.4 mg, 84%) as a
clear oil. 1H NMR (CDCl3) d 1.15–1.33 (m, 2H), 1.37–1.55 (m, total
11H, including each s, total 9H, at d 1.46 and 1.47), 1.85–2.10 (m,
total 7H), 2.13 and 2.16 (each s, total 3H), 2.20–2.39 (m, 2H),
2.79 and 2.95 (each d, J = 12.4 Hz, total 1H), 3.15–3.70 (series of
m, total 7H, including each s, total 3H, at d 3.65 and 3.66), 3.84
(m, 1H), 3.93 (m, 1H); MS (ESI), m/z 402 [M+H]+.
5.77. Methyl trans-4-[(5S)-methylthiomethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5i)
To a stirred solution of 29h (202.4 mg, 0.504 mmol) in CH2Cl2
(4 mL) was added TFA (2 mL) at 0 °C. After being stirred at room
temperature for 2 h, the mixture was concentrated, diluted with
CHCl3 and neutralized with sat NaHCO3 aq The resulting mixture
was extracted with CHCl3–MeOH (10:1, v/v), and the organic layer
was dried over Na2SO4, and concentrated to give the title com-
pound (165.6 mg, quant.) as a pale yellow oil, which was used to
the subsequent reaction without further purification. 1H NMR
(CDCl3) d 1.18–1.33 (m, 2H), 1.37–1.56 (m, 4H), 1.87–2.31 (series
of m, total 11H, including s, 3H, at d 2.12), 2.54 and 2.55 (each s,

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
59
5.80. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-
hydroxymethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (29j)
as a colorless solid. 1H NMR (DMSO-d6) d 1.12–1.29 (m, 2H), 1.30–
1.45 (m, 2H), 1.48–1.77 (m, 2H), 1.81–2.11 (m, 5H), 2.12–2.36 (m,
2H), 2.84 (s, 6H), 3.22–3.44 (m, 2H), 3.47–3.72 (m, 6H, including s,
3H, at d 3.59), 3.73–3.89 (m, 1H), 3.94–4.12 (m, 1H), 8.76–9.28 (m,
1H), 9.61–9.87 (m, 1H), 10.65–10.89 (m, 1H); MS (ESI), m/z 299
[M+H]+.
A mixture of 29i (16 g, 33.7 mmol) and Pd(OH)2 (2.0 g) in EtOH–
AcOH (4:1, 250 mL) was stirred under hydrogen atmosphere
(1 atm) at room temperature overnight. The mixture was filtered
to remove the catalyst and the filtrate was evaporated. The residue
was purified by flash column chromatography with hexane–EtOAc
(3:1, v/v) as eluent to give the title compound (13 g, 100%) as col-
orless oil. 1H NMR (CDCl3) d 1.17–1.31 (m, 5H, including t,
J = 7.2 Hz, 3H, at d 1.25,), 1.39–1.72 (m, 11H, including s, 9H, at d
1.48), 1.75–2.19 (m, 8H), 2.19–2.31 (m, 1H), 3.13–3.25 (m, 1H),
3.28–3.76 (m, 4H), 3.77–4.36 (m, 4H, including q, J = 7.2 Hz, 2H,
at d 4.12).
5.83. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-
[methoxy(methyl)amino]methyl-(2S)-pyrrolidinyl
methoxy]cyclohexanecarboxylate (31b)
5.83.1. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-formyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (30b)
The title compound was prepared in a procedure similar to that
described for 30a, starting from 29j. A colorless oil. 1H NMR (CDCl3)
d 1.15–1.30 (m, 5H), 1.36–1.52 (m, 11H, including s, total 9H, at d
1.42 and 1.49), 1.80–2.11 (m, 8H), 2.15–2.32 (m, 1H), 3.15–3.28 (m,
1H), 3.35–3.66 (m, 2H), 3.95–4.31 (m, 4H), 9.52 and 9.58 (each d,
J = 2.1 Hz, total 1H).
5.81. General procedure D: preparation of methyl trans-4-[1-
tert-butoxycarbonyl-(5S)-dimethylaminomethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (31a)
5.81.1. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-formyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (30a)
5.83.2. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-
[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (31b)
To a stirred solution of 29c (3.56 g, 9.58 mmol) and trichloroi-
socyanuric acid (2.45 g, 10.5 mmol) in CH2Cl2 (50 mL) was added
2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) (0.15 g, 0.96 mmol)
at 0 °C. After being stirred at room temperature for 30 min, the
reaction mixture was filtered through a Celite pad. The filtrate
was made basic with satd NaHCO3 aq and extracted with CHCl3.
The extract was washed with brine, dried over Na2SO4 and concen-
trated in vacuo. The residue was chromatographed on silica gel
with hexane–EtOAc (2:1) to give the title compound (2.88 g, 81%)
as a colorless oil. 1H NMR (CDCl3) d 1.17–1.30 (m, 2H), 1.36–1.52
(m, 11H, including s, toral 9H, at d 1.42 and 1.49), 1.83–2.09 (m,
8H), 2.20–2.33 (m, 1H), 3.16–3.27 (m, 1H), 3.36–3.74 (m, 5H,
including s, total 3H, at d 3.66 and 3.67), 3.98–4.31 (m, 2H), 9.52
and 9.59 (each d, J = 2.1 Hz, total 1H).
Yield 67% (two steps). A colorless oil. 1H NMR (CDCl3) d 1.16–
1.32 (m, 5H), 1.39–1.52 (m, 12H), 1.89–2.51 (m, 9H), 2.57 and
2.59 (each s, total 3H), 2.81–3.41 (m, 3H), 3.46 and 3.47 (each s, to-
tal 3H), 3.54–4.01 (m, 3H), 4.11 (q, J = 7.1 Hz, 2H); MS (ESI), m/z
429 [M+H]+.
5.84. Ethyl trans-4-[(5S)-[methoxy(methyl)amino]methyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate (5k)
The title compound was prepared in a procedure similar to that
described for 5a, starting from 31b. Yield 99%. A brown oil. 1H NMR
(CDCl3) d 1.20–1.31 (m, 5H), 1.37–1.52 (m, 3H), 1.54–2.34 (m, 9H),
2.52–2.56 (m, 2H), 2.56 (s, 3H), 3.18–3.48 (m, 5H), 3.52 (s, 3H), 4.11
(q, J = 7.1 Hz, 2H); MS (ESI), m/z 329 [M+H]+.
5.81.2. Methyl trans-4-[1-tert-butoxycarbonyl-(5S)-
dimethylaminomethyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (31a)
5.85. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-(1-
isoxazolidinylmethyl)-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (31c)
To a stirred mixture of 30a (545 mg, 1.48 mmol) and Me2NH
(2.0 M in THF, 0.74 mL, 1.48 mmol) in THF (30 mL) was added NaB-
H(OAc)3 (941 mg, 4.44 mmol) at room temperature. After being
stirred for 2.5 h, the reaction mixture was poured into satd NaHCO3
aq and extracted with EtOAc. The extract was washed with brine,
dried over Na2SO4 and condensed in vacuo. The residue was chro-
matographed on silica gel with CHCl3–MeOH (20:1) to give the title
compound (470 mg, 80%) as a colorless oil. 1H NMR (CDCl3) d 1.18–
1.32 (m, 3H), 1.39–1.42 (m, 1H), 1.46 and 1.47 (each s, total 9H),
1.58 (m, 1H), 1.83–2.19 (m, 10H), 2.25 (s, 3H), 2.28 (s, 3H), 3.17–
3.24 (m, 1H), 3.32 and 3.36 (each t, J = 8.8 Hz, total 1H), 3.58 (dd,
J = 2.7 and 8.6 Hz, 1H), 3.65 and 3.66 (each s, total 3H), 3.71–3.91
(m, 2H); MS (ESI), m/z 399 [M+H]+.
Yield 39% (two steps). A colorless oil. 1H NMR (CDCl3) d 1.11–
1.34 (m, 6H), 1.35–1.62 (m, 12H), 1.75–2.14 (m, 8H), 2.15–2.32
(m, 3H), 2.79–3.13 (m, 2H), 3.14–3.42 (m, 2H), 3.49–3.72 (m,
1H), 3.73–4.06 (m, 4H), 4.11 (q, J = 7.1 Hz, 2H); MS (ESI), m/z 441
[M+H]+.
5.86. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-(4-
morpholinyl)methyl-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (31d)
Yield 77% (two steps). A yellow oil. 1H NMR (CDCl3) d 1.22–1.26
(m, 5H), 1.40–1.46 (m, 11H), 1.85–2.62 (m, 13H), 3.20–3.90 (m,
11H), 4.10–4.13 (m, 2H); MS (ESI), m/z 455 [M+H]+.
Compound 31b–31e were prepared according to general proce-
dure D.
5.87. Ethyl trans-4-[1-tert-butoxycarbonyl-(5S)-(3-
methoxyazetidinylmethyl)-(2S)-pyrrolidinylmethoxy]
cyclohexanecarboxylate (31e)
5.82. Methyl trans-4-[(5S)-dimethylaminomethyl-(2S)-
pyrrolidinylmethoxy]cyclohexanecarboxylate dihydrochloride
(5j)
Yield 85%. A yellow oil. 1H NMR (CDCl3) d 1.09–1.31 (m, 6H),
1.42–1.51 (m, 11H), 1.74–2.30 (m, 9H), 2.35–2.78 (m, 1H), 2.88–
3.27 (m, 5H), 3.28–3.93 (m, 7H), 3.97–4.06 (m, 1H), 4.06–4.16
(m, 2H); MS (ESI), m/z 455 [M+H]+.
A solution of 31a (470 mg, 1.18 mmol) in 4 N HCl–dioxane
(20 mL) was stirred at room temperature for 12 h. The reaction
mixture was concentrated under reduced pressure. The residue
was washed with Ether to give the title compound (373 mg, 85%)

60
M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
5.88. Determination of aqueous solution solubility
1 nM). For assays conducted in the presence of human serum albu-
min, 50 l of compound at various concentrations and an equal
l
JP1 and JP2 were used as solvents for solubility measurements.
Two milligrams of one of the compounds was accurately weighed
into a disposable glass tube equipped with a fitted cap, and 1 mL of
one of the solvents was added. The glass tube was capped, soni-
cated for 5 min, and shaken vigorously for 30 s at 5-min intervals
volume of 2 nM Eu-labeled 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 incubated for
60 min at room temperature and the wells were washed 4 times
with 300 ll of chilled Wash Buffer. Finally, 100 ll of the enhance-
for 30 min. The sample solutions were filtered through a 0.45
l
m
ment 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 Research
fluorometer, model 1234-001; PerkinElmer Inc.). The concentra-
tion of compound required for 50% inhibition in the assay was
determined.
membrane filter (Millex-HV; Millipore Corp.). The filtrate was di-
luted if necessary, and the resulting solutions were assayed by
High-performance liquid chromatography (HPLC) system (Waters
Alliance 2690; Waters Corp.). The concentration of the solution
was calculated from the calibration curve for the appropriate
compound.
5.91. Murine bronchial inflammatory model
5.89. Distribution coefficient (logD)
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,
500 lg protein of Ascaris suum extract (LSL Co., Ltd) in 0.2 mL saline
containing 4.5 mg aluminum hydroxide was injected intraperito-
neally. On day 22, the mice were challenged intratracheally under
anesthesia with 300 lg (30 ll) protein of Ascaris suum extract. In
the negative control group, sensitized mice were challenged with
saline instead of the antigen.
The same volume of 1-octanol and JP2 (pH 6.8) were mixed and
shaken for 30 min at room temperature. The resulting solution was
allowed to stand for 24 h at room temperature; this process
yielded 1-octanol-saturated JP2 solution layer (SJP2) and a JP2-sat-
urated 1-octanol layer (Soct). Two milligrams of one of the com-
pounds was accurately weighed into a disposable glass tube. Ten
milliliters of the Soct was added and the compound was dissolved.
After filtering the sample solution (Millex-LH; Millipore Corp.),
3 mL of the resulting solution was mixed with 3 mL of SJP2. This
mixture was shaken for 30 min and then centrifuged for 10 min
at 3000 rpm. The upper and lower fractions of solutions were col-
lected separately as the Soct phase and SJP2 phase. One part of the
Soct phase was diluted with 25 parts of an acetonitrile–water
(1:1, v/v) solution, and then loaded onto HPLC system to determine
the concentration of the compound. The SJP2 phase was diluted
with 2 parts of the acetonitrile–water (1:1, v/v) solution, and then
loaded onto HPLC system to determine the concentration of the
compound. The octanol-JP2 partition coefficient (log D) was calcu-
lated from the following equation:
5.92. Effect on eosinophil infiltration
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 (for 7c, 7f, and 7g) and 5 or 12.5 mg/kg (for 7n). Forty-eight
hours after antigen challenge, the mice were sacrificed and BALF
was collected using tracheal polyethylene cannula with
2 ꢂ 0.5 mL Hanks’ balanced salt solution. The cells in the BALF
were counted in a particle analyzer CDA-500 (Sysmex Corp.). Cyto-
centrifuged 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.
log D ðpH 6:8Þ ¼ log ½ðpeak area of Soct sample
ꢂ 25Þ=ðpeak area of SJP2 sample ꢂ 2Þꢃ
5.90. VLA-4/VCAM-1 binding assay
A human VLA-4-expressing cell line, 4B4, was established at
5.93. Effect on hyper-responsiveness
Pharmacopeia Inc., by transfecting both the
a4 gene and b1 gene
of VLA-4 into CHO-K1 cells. The 4B4 cells were maintained in
Ham’s F-12 medium (Sigma Corp.) supplemented with 10% (v/v)
fetal calf serum (REHATUIN Fetal Bovine Serum, Serologicals
Compound 7n, which was dissolved in 0.5% MC containing
0.03% Tween 80, was orally administered 15 min before and 8,
24, and 32 h after the antigen challenge at a dose of 2 or
12.5 mg/kg. The bronchial hyper-responsiveness in each mouse
was estimated from the increase in lung resistance by acetylcho-
line chloride (ACh; Sigma Corp.) injection at 48 h after the antigen
challenge. Ten minutes before the start of the measurement, the
mice were anesthetized by an intraperitoneal injection of pento-
barbital at a dose of 100 mg/kg. The trachea was cannulated and
connected to a rodent ventilator (Mini Vent type 845; Hugo Sachs
Electronik-Harvard Apparatus) with an in-line pressure transducer
(TRD-4510; Buxco Electronics, Inc.) that was coupled to a pulmon-
ary mechanics analyzer (Bio-System XA; Buxco Electronics, Inc.).
The flows were determined by measuring the differential pressure
(TRD-5100; Buxco Electronics, Inc.) across eight layers of 400-mesh
wire cloth covering a 1.3-cm hole in a plethysmograph box (Plyan-
M; Buxco Electronics, Inc.). The mice were placed in the plethys-
mograph box and then ventilated at 140 strokes/min with a stroke
Corp.), 100 U/mL penicillin (Invitrogen Corp.), 100
lg/mL strepto-
mycin (Invitrogen Corp.), 2 mM -glutamine (Invitrogen Corp.)
L
and 1 mg/mL G-418 (Geneticin, Invitrogen Corp.). A Eu-labeling Re-
agent (PerkinElmer Inc.) was used to label the human VCAM-1/Fc
chimera (R&D Systems Inc.). The Eu-labeled protein was purified
with a PD-10 column (Amersham Biosciences KK.) and stored at
ꢁ80 °C until use. All assays were performed in duplicate. In prepa-
ration 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-culture plate
(Costar Inc.). The plates were incubated at 37 °C in a 5% CO2 atmo-
sphere for 2 days. Prior to the assay, the medium was discarded
and each well was washed twice with 300
(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-labeled human VCAM-1/Fc
chimera diluted with the Wash Buffer (final concentration:
ll of chilled Wash Buffer
l
l
volume of 150 ll. After establishing a stable baseline of lung resis-

M. Setoguchi et al. / Bioorg. Med. Chem. 21 (2013) 42–61
61
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.
tance, ACh, dissolved in saline was cumulatively administered (25,
50, and 100 ( g/mL)/kg) via the tail vein, and the changes in lung
resistance were monitored.
l
7. (a) Podolsky, D. K. N. Engl. 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. Pulm. 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. Engl. 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. Engl. 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.
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.
12. Setoguchi, M.; Iimura, S.; Sugimoto, Y.; Yoneda, Y.; Chiba, J.; Watanabe, T.;
Muro, F.; Iigo, Y.; Takayama, G.; Yokoyama, M.; Taira, T.; Aonuma, M.; Takashi,
T.; Nakayama, A.; Machinaga, N. Bioorg. Med. Chem. 2012, 20, 1201.
13. (a) Alelyunas, Y. W.; Empfield, J. R.; McCarthy, D.; Spreen, R. C.; Bui, K.; Pelosi-
Kilby, L.; Shen, C. Bioorg. Med. Chem. Lett. 2010, 20, 7312; (b) Ishikawa, M.;
Hashimoto, Y. J. Med. Chem. 2011, 54, 1539.
14. (a) Chiba, J.; Takayama, G.; Takashi, T.; Yokoyama, M.; Nakayama, A.; Baldwin,
J. J.; McDonald, E.; Moriarty, K. J.; Sarko, C. R.; Saionz, K. W.; Swanson, R.;
Hussain, Z.; Wong, A.; Machinaga, N. Bioorg. Med. Chem. 2006, 14, 2725; (b)
Chiba, J.; Iimura, S.; Yoneda, Y.; Sugimoto, Y.; Horiuchi, T.; Muro, F.; Ochiai, Y.;
Ogasawara, T.; Tsubokawa, M.; Iigou, Y.; Takayama, G.; Taira, T.; Takata, Y.;
Yokoyama, M.; Takashi, T.; Nakayama, A.; Machinaga, N. Chem. Pharm. Bull.
2006, 54, 1515.
15. (a) Katoh, T.; Nagata, Y.; Kobayashi, Y.; Arai, K.; Minami, J.; Terashima, S.
Tetrahedron Lett. 1993, 34, 5743; (b) Katoh, T.; Nagata, Y.; Kobayashi, Y.; Arai,
K.; Minami, J.; Terashima, S. Tetrahedron 1994, 50, 6221.
5.94. Pharmacokinetic studies on monkeys
Female cynomolgus monkeys (3.5–4 kg, HAMRI Co.) were used.
The animals were fasted for 18 h prior to dosing. Each group con-
sisted in three animals. Compound 7n was suspended in 0.5% (w/
v) MC for oral dosing (0.5 mg/kg) or dissolved in saline with
3 equiv NaOH for intravenous dosing (0.5 mg/kg). Blood samples
(1 mL) were collected after 0.08, 0.25 (for iv), 0.25 (for p.o.), 0.5,
1, 2, 4, 8, and 10 (for p.o.) h. After the 4 h sampling, the animals
were provided with food. These analytical samples were left to
stand at room temperature, followed by centrifugation at
15,000 rpm for 10 min at 4 °C. The plasma fractions were subse-
quently stored in a ꢁ20 °C freezer until being analyzed. The con-
centrations of the compounds were determined by an LC/MS/MS
method, comprised of an Alliance 2695 HPLC (Waters), Symmetry
Shield RP8, 2.1 ꢂ 50 mm, 3.5
lm column (Waters), and TSQ-700
(Thermo Electron, Waltham, MA). The mobile phase consisted of
10 mM HCOONH4 in water/methanol; the gradient condition was
90/10 to 10/90. The plasma concentrations versus the time data
were analyzed by non-compartmental approaches using the Win-
Nonlin software program (version 1.13.1 Pharsight, Mountain
View, CA).
All the animal experiments were conducted with the approval
of the Animal Experiment Ethics Committee of Daiichi Pharmaceu-
tical Co., Ltd.
References and notes
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.
16. (a) Takano, S.; Ogasawara, K., Iwabuchi, Y.; Moriya, M. JP03220175.; (b)
Takano, S.; Moriya, M.; Iwabuchi, Y.; Ogasawara, K. Tetrahedron Lett. 1989, 30,
3805.
17. Siegrist, R.; Zürcher, M.; Baumgartner, C.; Seiler, P.; Diederich, F.; Daum, S.;
Fischer, G.; Klein, C.; Dangl, M.; Schwaiger, M. Helv. Chim. Acta 2007, 90, 217.
18. 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.
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.
4. (a) Barthel, S. R.; Johansson, M. W.; McNamee, D. M.; Mosher, D. F. J. Leukoc.
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. 1817, 2007, 56; (b) Carter, R. A.; Wicks, I. P. Arthritis Rheum. 2001, 44,
985; (c) Seiffge, D. J. Rheumatol. 1996, 23, 2086.