Highly potent PDE4 inhibitors with therapeutic potential

Article abstract of DOI:10.1016/j.bmc.2004.06.032

Synthesis and biological evaluation of piperidine derivatives is reported. The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine

Full text of DOI:10.1016/j.bmc.2004.06.032

Bioorganic & Medicinal Chemistry 12 (2004) 4645–4665
Highly potent PDE4 inhibitors with therapeutic potential
Hiroshi Ochiai,a Tazumi Ohtani,a Akiharu Ishida,a Kensuke Kusumi,a Masashi Kato,a
Hiroshi Kohno,a Yoshihiko Odagaki,a Katuya Kishikawa,b Susumu Yamamoto,a
a
Hiroshi Takeda,a Takaaki Obata,a Hisao Nakaia, and Masaaki Toda
*
aMinase Research Institute, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Osaka, Mishima 618-8585, Japan
bDevelopment Planning, Ono Pharmaceutical Co., Ltd, 2-1-5 Doshomachi, Osaka, Chuo-ku 541-8526, Japan
Received 29 May 2004; revised 23 June 2004; accepted 23 June 2004
Available online 20 July 2004
Abstract—The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system
prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Arifloe 1, which is an
orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a–b, 11b–14b,
and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hy-
droxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine
analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy res-
idue with a difluoromethoxy residue led to an increase of in vivo potency. Structure–activity relationships are presented. Single-dose
rat pharmacokinetic data for 11b, 12b, and 17a are also presented.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Arifloe 17 (Fig. 1) has a markedly improved side effect
profile relative to that of the classical PDE4 inhibitor
rolipram. The improved therapeutic index of Ariflo
has been attributed to more selective inhibition of
PDE4 catalytic activity (LPDE4) versus its competi-
tion for the high-affinity[3H]rolipram-binding site
(HPDE4).8,9
The cyclic nucleotide phosphodiesterases (PDEs)1–4 are
enzymes that regulate the cellular levels of cyclic adeno-
sine monophosphate (cAMP) and cyclic guanosine
monophosphate (cGMP) through hydrolysis of these
second messengers. Phosphodiesterase type 4 (PDE4)
is a cAMP-specific PDE expressed by immune and
inflammatory cells, including neutrophils, T-lympho-
cytes, macrophages, and eosinophils. Inhibition of
PDE4 in inflammatory cells influences various specific
responses, such as the production and/or release of pro-
inflammatory mediators, cytokines, and active oxygen
species.5 The potential of PDE4 inhibitors as anti-
inflammatory agents for the treatment of asthma and
rheumatoid arthritis has received considerable atten-
tion.5,6 However, no selective PDE4 inhibitor has been
released yet because the pioneer compounds, typified
by rolipram, had dose-limiting side effects (including
nausea and emesis) that severely restricted their thera-
peutic utility.
A second strategy to decrease side effects is related to the
design of PDE4 subtype selective inhibitors.10–13 Re-
cently, naphthyridine-related PDE4 inhibitors have been
reported to be PDE4D-selective.14 Ariflo is an orally ac-
tive, second-generation PDE4 inhibitor that is also re-
ported to be PDE4D-selective.
The dose-limiting side effects of PDE4 inhibitors, such
as nausea and emesis, are thought to be mediated via
CN
CN
O
O
COOH
N
COX
MeO
1 (Ariflo) ; ClogP = 3.20
MeO
H
Keywords: PDE4 inhibitor; Piperidine analog; Hydroxamic acid;
Difluoromethoxy; Pharmacokinetic data.
2a (X = OH) ; ClogP = 0.07
2b (X = NHOH) ; ClogP = 1.30
*
Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-
9314; e-mail: hi.nakai@ono.co.jp
Figure 1. Molecular design of hydrophilic molecules.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.032

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H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
the central nervous system (CNS).15,16 Accordingly, the
negative charge of Ariflo 1 at a physiological pH might
decrease the occurrence of side effects because of its lim-
ited penetration into the CNS. Based on this hypothesis,
the design of a highly hydrophilic PDE4 inhibitor was
thought to be another possible approach to controlling
side effects because compounds that show good penetra-
tion into the CNS have a relatively high lipophili-
city.17,18
tuted benzyl cyanide 30a–i with a methane sulfonate
29, which was prepared from 27 in two steps, afforded
31a–i, respectively, while acidic deprotection produced
32a–i, respectively. N-Alkylation of 32a–i with ethyl
bromoacetate gave 33a–i, respectively. N-Alkylation
of 32a with triflates, which was derived from methyl
(S)-(ꢀ)-lactate and methyl (R)-(+)-lactate, respectively,
resulted in 33j and 33k, respectively.20 The synthesis
of 33l–n is outlined in Scheme 2A. The key intermedi-
ates 36a–e were prepared by ozonolysis of 35a–e, which
were obtained by dialkylation of appropriately substi-
tuted benzyl cyanides (30a,c, and j–l) with the dichlo-
ride 34. Reductive amination21 of 36e and 36d with
an appropriate amino ester provided 33l–n, respec-
tively.
As shown in Figure 1, focus was placed on the design
and synthesis of a less lipophilic and/or zwitter-ionic
piperidine analog, which was structurally related to Ari-
flo 1 and was expected to show more limited CNS pen-
etration.19 A structure–activity relationship (SAR) study
was started with the expectation that this approach
would provide compounds with an improved therapeu-
tic index. As a result, many analogs were found that
showed increased potency in both in vitro and in vivo
tests. Several of these analogs were estimated to be un-
likely to cause emesis at therapeutic doses based on
the difference between the dose producing the desired
effects and side effects in cross-species and same-species
comparisons (Table 6).
As outlined in Scheme 2B, reductive amination of 36a–b
with an appropriate amino ester afforded 37a–b, respec-
tively. Catalytic hydrogenolysis of 37a–b gave 38a–b,
respectively, while O-alkylation of 38a–b by the appro-
priate method resulted in 33o–w, respectively.
As described in Scheme 2C, reductive amination of 36c
with an appropriate amino ester afforded 39, acidic de-
protection of which provided 40. Then O-alkylation of
40 under appropriate reaction condition, gave 33x–y,
respectively.
This report covers the process of identifying a number of
highly potent PDE4 inhibitors with an improved thera-
peutic potential. SARdata for the piperidine analogs
and rat pharmacokinetic data for selected compounds
are also reported.
The synthesis of 2b–14b and 16b–18b is outlined in
Scheme 3. Alkaline hydrolysis of 33a–k and 33o–w pro-
duced the corresponding carboxylic acids 2a, 10a–14a,
16a–18a, 3a–4a, and 15, 19–26, respectively. Catalytic
hydrogenation of 33l–n and 33x–y afforded the corre-
sponding carboxylic acids 5a–7a and 8a–9a, respec-
tively. Condensation of 2a–14a and 16a–18a with
O-protected hydroxylamine, followed by acidic depro-
tection, resulted in the corresponding hydroxamic acids
2b–14b and 16b–18b, respectively.
2. Chemistry
The synthesis of the compounds 2–26 listed in Tables
1–5 is described in Schemes 1–3. As outlined in Scheme
1, intermediates 32a–i were prepared from a benzyl
cyanide 30.7 Dialkylation of an appropriately substi-
NBoc
H
N
Boc
N
Boc
c
a
b
R1O
R2O
N
HO
OH
HO
OH
MsO
OMs
CN
31a-i
27
(quant)
28
R1O
R2O
(95% in 2 steps)
29
CN
(crude 53% - quant)
HCl
NH
30a-i
d
N
COOEt
30-33
R1O
R2O
R1O
R2O
e
d
g
CN
a
c
e
h
i
CN
32a-i
b
f
33a-i
(90 - 100%)
cPrCH2
Me
R1
R2
cPen
Me
iPr cBu cPen cPen cPen
CHF2
Me
Me
Et
(29 - 61% in 2 steps)
Me
Me Me
Et
iPr
f
33
j
k
R3
N
R4
COOMe
R3
R4
H
Me
H
O
CN
33j-k
(quant)
Me
MeO
Scheme 1. Synthesis of 33a–k. Reagents and conditions: (a) Boc2O, CH2Cl2, 0ꢁC; (b) MsCl, Et3N, CH2Cl2, ꢀ78ꢁC; (c) 30a–i, LiHMDS, THF,
ꢀ78ꢁC; (d) TFA, C6H5SCH3, CH2Cl2, then 4N HCl/EtOAc; (e) K2CO3, BrCH2COOEt, DMF; (f) methyl-S-(ꢀ)-lactate or methyl-R-(+)-lactate,
Tf2O, CH2Cl2, 2,6-lutidine, Et3N.

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4647
R2
N
R3
COOBn
(A)
CHO
CHO
CN
36a-e
R1O
c
R1O
R2O
b
R1O
R2O
a
R1O
R2O
CN
CN
Cl
33l-n
CN
36d-e
MeO
(15 - 25% in 2 steps)
Cl
34
35a-e
(67 - 94%)
30
j
(quant)
33
l
n
m
l
a
k
c
30
a
c
e
cPen
b
d
R1 cPen
Et
35-36
R2
R3
Me CH2 CH2
Me CH2 CH2
MOM
Me
R1
R2
Bn
Me
Bn
Et cPen
Me Me
MOM
Me
R1
R2
Bn
Bn
Et cPen
Me Me
CHF2
CHF2
Me
(B)
N
COR3
N
COR2
N
COR2
c or d
b
R1O
R2O
HO
a
BnO
R1O
CN
36a-b
CN
CN
38a-b
(each 92%)
33o-w
R1O
37a-b
(63% - quant)
(47 and 64% in 2 steps)
33
u
o
p
q
s
v
w
r
t
37-38
a
b
indanyl
cBuCH2 cPrCH2 indanyl
R1
R2
R3
Bu
iBu
cBu
Et
Pr
R1
R2
Me CHF2
OEtOMe
CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2
Me
OEt OMe OMe OMe OMe OMe OMe
OMe
N
OMe
(C)
COOBn
N
COOBn
c or d
N
COOBn
b
R1O
HO
MOMO
MeO
a
CN
33x-y
(each quant)
CN
40
(85 %)
36c
CN
39
MeO
MeO
(65 % in 2 steps)
Compd 33
x
y
R1 cPrCH2 cBu
Scheme 2. (A) Synthesis of 33l–n. Reagents and conditions: (a) 34, LiHMDS, THF, ꢀ78ꢁC; (b) O3, CH2Cl2, then PPh3, ꢀ78ꢁC; (c) NaBH(OAc)3,
AcOH, DCE, or DMF, NH2CR2R3COOBn. (B) Synthesis of 33o–w. Reagents and conditions: (a) NaBH(OAc)3, AcOH, DCE, or DMF,
NH2CH2COR5; (b) H2, 10% Pd/C, MeOH; (c) alcohol, DEAD, PPh3, THF; (d) alkyl halide, K2CO3, DMF. (C) Synthesis of 33x–y. Reagents and
conditions: (a) NaBH(OAc)3, AcOH, DCE, or DMF, NH2C(CH2)2COOBn; (b) 4N HCl/EtOAc, CH2Cl2; (c) alcohol, DEAD, PPh3, THF; (d) alkyl
halide, K2CO3, DMF.
R3
N
R4
COR5
R1O
R2O
a
CN
R3
N
R4
CONHOH
Me Me
R3
N
R
4 H
N
R3
N
R4
COOH
c
33a-k and 33o-w
O
OMe
R1O
R2O
R1O
R2O
O
d
R1O
R2O
CN
CN
2c-14c and 16c-18c
CN
2b-14b and 16b-18b
2a-14a, 15, 16a-18a and 19-26
(25 - 99% in 2 steps)
(54% - quant)
b
R3
N
R4
COOBn
R1O
R2O
CN
33l-n and 33x-y
Scheme 3. Synthesis of 2a–14a, 15, 16a–18a, 19–26, 2b–14b, and 16b–18b. Reagents and conditions: (a) 2N NaOHaq, EtOH; (b) H2, 10% Pd/C,
MeOH; (c) EDC, HOBt, DMF, NH2OC(CH3)2(OCH3); (d) 2N HClaq, MeOH.
3. Results and discussion
inhibitory effect on PDE4 obtained from U937 cells22
(derived from human monocytes). The results of the as-
says were expressed as IC50 values, that is, the test com-
pound concentration that caused 50% inhibition. Test
The series of piperidine analogs listed in Tables 1–5 were
synthesized and biologically evaluated to assess the

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H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
compounds were also evaluated for the inhibitory
effect lipopolysaccharide (LPS)-induced production of
tumor necrosis factor-a (TNF-a) in rats23 and results
were expressed as ID50 values (i.e., the dose that
achieved 50% inhibition relative to the effect of the
vehicle).
To avoid the predicted rapid hydrolysis of 2b, design
and synthesis of a-substituted and a,a-disubstituted
analogs 3b–6b listed in Table 1 was carried out. Com-
pounds 3a–6a were also synthesized and evaluated for
comparison with the corresponding hydroxamic acid
analogs. Introduction of an R-methyl group at the a-
position of 2b gave 3b, which was over 10-fold less po-
tent in vitro and also showed marked loss of in vivo
activity. The corresponding S-isomer 4b exhibited even
lower in vitro activity, but was slightly more potent in
vivo. The R-methyl configuration was found to be supe-
rior to the S-methyl configuration, although introduc-
tion of an alkyl moiety failed to increase the activity
of 2b. Introduction of an a,a-dimethyl moiety into 2b
gave 6b, which showed markedly lower in vitro activity.
Introduction of an a,a-dimethylene moiety, which was
predicted to be smaller than the a,a-dimethyl moiety,
into 2b gave 5b, which also showed lower activity both
in vitro and in vivo. All of the corresponding carboxylic
acid analogs 3a–6a were far less potent in the LPDE4 as-
say than their corresponding hydroxamic acid analogs
3b–6b, respectively. Compounds 3a–4a and 5a–6a
caused less than 50% inhibition at 0.3 and 1lM, respec-
tively. Thus, this modification caused marked reduction
of the activity of the new chemical lead 2b. The enantio-
mers 3b and 4b achieved 26% inhibition and 49% inhibi-
tion, respectively, in the LPS-induced TNF-a
production assay at 0.5h after oral administration, but
showed no effect at 3h after dosing. Accordingly, neither
the inhibitory activity nor the duration of action was not
improved by this modification. Further optimization of
6a–b, which was expected to resist hydrolysis better than
the monomethyl derivatives 3b–4b and exhibit more po-
tent LPDE4 activity than the a,a-dimethyl derivatives
5a–b, was performed as described in Table 2. Replace-
ment of the 3-cyclopentyloxy residue of 6a–b with an
ethoxy residue afforded 7a–b, which showed more
potent and nearly 1.7-fold weaker LPDE4 activity,
respectively. Replacement of the cyclopentyloxy residue
of 6a–b with a cyclopropylmethyloxy residue gave 8a–b,
Table 1 summarized the biological activities of these pip-
eridine analogs. Compound 2a was designed to have less
hydrophobicity than Ariflo 1 and to remove the config-
urational isomerism; it exhibited potent LPDE4 activity,
with an oral dose of 3mg/kg causing 74% inhibition of
LPS-induced TNF-a production in rats. Based on re-
ported information,11 the corresponding hydroxamic
acid analog 2b was extremely potent in the in vitro
LPDE4 assay and also showed an oral ID50 value of
0.04mg/kg in the in vivo TNF-a production assay. Thus,
the hydroxamic acid group, a well known metal chela-
tor, was confirmed to cause a striking increase of inhib-
itory activity.
According to our in vivo evaluation, 2b had no effect on
LPS-induced TNF-a production at 3h after oral admin-
istration (0.1mg/kg), although it showed a very potent
effect at 0.5h. In the early stage of these investigations,
rapid hydrolysis of the hydroxamic acid moiety of 2b
to a carboxylic acid was predicted to be one of the rea-
sons for its short duration of action.
Table 1. Activity profile of piperidine analogs
X
N
O
CN
Me
O
Compound
X
LPDE4a
IC50 (nM)
Inhibition of
TNF-a
production in
ratsb ID50
(mg/kg, po)
2a (Y=OH)
2b (Y=NHOH)
66
0.080
(74%)c
0.043
Table 2. Activity profile of a,a-dimethylene analogs
COY
Me
Me
3a (Y=OH)
3b (Y=NHOH)
>300
0.91
NTe
COY
X
O
N
(26%)d
COY
COY
CN
Me
4a (Y=OH)
4b (Y=NHOH)
>300
5.9
NTe
(49%)d
O
Compound
X=
LPDE4a
Inhibition of TNF-a
IC50 (nM) production in ratsa ID50
(mg/kg, po)
MeMe
COY
5a (Y=OH)
5b (Y=NHOH)
>1000
34
NTe
NTe
7a (Y=OH)
7b (Y=NHOH)
Et-
870
8.0
NTa
(20%)b
6a (Y=OH)
6b (Y=NHOH)
>1000
4.6
NTe
2.0
COY
8a (Y=OH)
8b (Y=NHOH)
>1000
14
NTa
0.87
a Inhibition of PDE4 prepared from U937 cells (a cell line derived from
human monocytes). IC50 represent a mean of n=2.
b ID50 for inhibition of LPS-induced TNF-a production in rats (n=7)
0.5h after oral dosing of a test compound.
c Inhibition% at 3mg/kg, po.
9a (Y=OH)
9b (Y=NHOH)
940
7.6
NTa
5.6
d Inhibition% at 1mg/kg, po.
a See corresponding footnotes from Table 1.
b Inhibition% at 3mg/kg, po.
e Not tested.

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4649
Table 4. Optimization of 4-alkoxy residue
showing no increase and nearly 3-fold weaker LPDE4
activity, respectively. Replacement of the cyclopentyl-
oxy residue of 6a–b with a cyclobutyloxy residue led to
9a–b, which showed stronger and nearly 1.6-fold weaker
LPDE4 activity, respectively. With regard to the effect
on TNF-a production, 8b was more than 2-fold stronger
than 6b while 7b and 9b showed less activity. The dura-
tion of action was also not improved by this modifica-
tion.
COX
N
O
CN
R
O
a
Compound
RLPDE4
Inhibition of
IC50 (nM) TNF-a production
in ratsa ID50
(mg/kg, po)
Therefore, further attempts at optimization of the cyclo-
pentyl moiety of 2a–b were made as shown in Table 3.
Replacement of the cyclopentyl moiety of 2b with n-
alkyl chains, such as methyl and ethyl moieties, resulted
in 10b and 11b, respectively. Compound 10b showed
lower potency in both the in vitro and in vivo tests, while
11b showed 6.5-fold lower in vitro activity, but retained
its in vivo potency. Replacement of the cyclopentyl moi-
ety of 2b with a cyclopropylmethyl moiety gave 12b,
which also had reduced in vitro potency, but retained
its in vivo activity. Replacement of the cyclopentyl moi-
ety of 2b with a branched alkyl moiety, such as an iso-
propyl moiety, gave 13b, which showed a more than
10-fold reduction of in vitro potency and a nearly 2-fold
reduction of in vivo potency. Ring contraction of the cy-
clopentyl moiety of 2b produced a cyclobutyloxy analog
14b, which had nearly 2-fold lower in vitro activity, but
retained its in vivo potency. The corresponding carboxy-
lic acids 10a–14a demonstrated a marked loss of LPDE4
inhibitory activity compared with 2a. Interestingly, the
indan-2-yloxy analog 1524 demonstrated equipotent
16a (X=OH)
16b (X=NHOH)
Et-
2500
6.2
NTa
0.7
17a (X=OH)
17b (X=NHOH)
CHF2-
65
0.051
1.1
0.02
18a (X=OH)
18b (X=NHOH)
i-Propyl- >1000
>30
NTa
NTa
a See corresponding footnotes from Table 1.
inhibition of LPDE4 relative to 2a, but exhibited lower
in vivo activity.
As shown in Table 4, further optimization of the meth-
oxy moiety of 2a–b was carried out. Replacement of the
methoxy moiety of 2b with an ethoxy moiety gave 16b,
which had 75-fold lower LPDE4 inhibitory activity
and nearly 17-fold lower in vivo activity. Replacement
of the methoxy moiety of 2b with an isopropyloxy moi-
ety resulted in 18b, which also showed a marked de-
crease of LPDE4 inhibitory activity. Interestingly,
replacement of the methoxy moiety of 2b with a difluor-
omethoxy25 moiety produced 17b, which was nearly
equipotent with respect to LPDE4 activity and demon-
strated a 2-fold increase of in vivo activity. The corre-
sponding carboxylic acid derivatives 16a and 18a
showed a much lower potency both in vitro and in vivo,
although 17a had nearly the same potency as 2a in both
the LPDE4 assay and the LPS-induced TNF-a produc-
tion assay. As described above, the acceptable modifica-
tions of the methoxy moiety of 2a–b were found to be
much more limited than those of the cyclopentyl moiety,
while the difluoromethoxy moiety was identified as an-
other optimized moiety that could be replaced by a
methoxy. Among the several possible changes in the
physical properties of a molecule that occur when a
hydrogen atom is replaced by a fluorine atom, an in-
crease of at least three factors (steric bulkiness, lipophi-
licity, and metabolic stabilization) would be expected.
As shown in Table 8, these three changes of the physical
properties were estimated from the calculated molal vol-
ume (Mol vol),26 the logD and the duration of action in
vivo, respectively. According to our calculations using
the QMPRPLUS program,27 the molal volume of CHF2
was located between the Me and Et moieties. The logD
values of 2a and 17a were 0.1 and 1.0, respectively. As a
result, the lipophilicity of 17a was found to increase
remarkably with a small change of the molal volume.
Additionally, a longer duration of action was obtained
by this modification of 17a, presumably because of
Table 3. Optimization of 3-alkoxy residue
R
O
N
COX
CN
Me
O
a
Compound
RLPDE4
Inhibition of
IC50 (nM) TNF-a production
in ratsa ID50
(mg/kg, po)
10a (X=OH)
10b (X=NHOH)
Me-
Et-
>100
4.7
NTa
0.23
11a (X=OH)
11b (X=NHOH)
>100
0.52
(0%)b
0.04
12a (X=OH)
12b (X=NHOH)
>100
0.29
(12%)b
0.03
13a (X=OH)
13b (X=NHOH)
i-Propyl-
c-Butyl-
>100
1.0
NTa
0.09
14a (X=OH)
14b (X=NHOH)
>100
0.18
NTa
0.03
15 (X=OH)
67
(29%)c
a See corresponding footnotes from Table 1.
b Inhibition% at 1mg/kg, po.
c Inhibition% at 3mg/kg, po.

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H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
respectively. The 3-npropyloxy analog 20 showed nearly
7-fold lower inhibition of LPDE4 activity than 17a,
while it was slightly more potent in the TNF-a produc-
tion assay. The 3-nbutyloxy analog 21 exhibited nearly
12-fold lower inhibition of LPDE4 activity than 17a,
while it was only slightly less potent in the TNF-a pro-
duction assay. When the 3-ibutyloxy analog 22 was
tested, it showed nearly 5-fold weaker inhibition of
LPDE4 activity than 17a, but showed only slightly lower
potency in the TNF-a production assay. A 3-cyclobutyl-
oxy analog 23 exhibited 7-fold less potent LPDE4 inhib-
itory activity than 17a, while it was equipotent in the
TNF-a production assay. The 3-cyclobutylmethyloxy
analog 24 also showed 7-fold less potent LPDE4 inhib-
itory activity than 17a and was nearly 3-fold less potent
in the TNF-production assay. Finally, the 3-cyclopro-
pylmethyloxy analog 25 exhibited nearly 2-fold weaker
inhibition of LPDE4 activity than 17a, but had a slightly
higher ID50 value in the TNF-a production assay,
and the 3-isoindanyloxy analog 26 showed nearly 3-fold
lower inhibition of LPDE4 activity than 17a.
Table 5. Activity profile of 4-difluoromethoxy analog
COOH
R
O
N
F
CN
F
O
a
Compound RLPDE4
Inhibition of TNF-a
IC50 (nM) production in ratsa ID50
(mg/kg, po)
19
20
21
22
23
Et-
530
490
760
290
450
1.4
0.68
1.3
1.4
1.1
n-Propyl-
n-Butyl-
i-Butyl-
c-Butyl-
460
2.9
24
25
26
150
180
0.72
NTa
a See corresponding footnotes from Table 1.
Further biological evaluation of compounds 2a–b, 11b–
14b, and 17a, which were selected based on the in vitro
LPDE4 assay and in vivo TNF-a production assay, was
carried out as shown in Table 6. Inhibition of slow react-
ing substance of anaphylaxis (SRS-A)-mediated bron-
choconstriction28,29 was investigated in guinea pigs as
a beneficial effect and inhibition of gastric emptying
was assessed in rats30 as an effect. The results were ex-
pressed as ID50 values, that is, the dose that achieved
50% inhibition relative to the vehicle. These compounds
were also evaluated for inhibition of LPS-induced TNF-
a production in human whole blood (HWB)31 to esti-
mate their clinical potential and results were expressed
increased stability due to metabolic changes such as
demethylation.25
Accordingly, further optimization of the 3-alkoxy resi-
due of 4-difluoromethoxyphenyl analogs was carried
out, as illustrated in Table 5. Replacement of the 3-cy-
clopentyloxy residue of the aromatic moiety of 17a with
appropriate alkyl residues afforded 19–26. The 3-ethoxy
analog 19 was nearly 8-fold weaker and slightly less po-
tent than 17a in the in vitro and in vivo evaluations,
Table 6. Further biological evaluation of 1, 2a–b, 11b–14b, and 17a
Compound SRS-A-mediated
bronchoconstrictiona
ID50 (mg/kg, po)
Inhibition of
TNF-a productionb gastric emptyingc TNF-a productiond
ID50 (mg/kg, po) ID50 (mg/kg, po) in HWB IC50 (lM)
Inhibition of
Inhibition of
Ferret emesise
(vomiting/tested)
OVA
challenge (mg/kg, iv)
0.1
0.3
1
3
10
(mg/kg, po)
0.15
0.5
10
(0%)h (74%)i
1 (Ariflo)
2a
2b
4.5
1.7
5.7
NTg
0.3
0.7
0.3
0.8
0.3
4.7
18
5.2
NTg NTg NTg NTg NTg
NTg NTg NTg NTg NTg
NTg
0.3
NTg
NTg
1.1
0.04
0.04
0.03
0.09
0.027
1.1
0.0089
0.0050
0.0021
0.012
0.0027
0.84
0/4
0/4
2/2
1/2
3/5
1/2
0/2
NTg NTg
NTg NTg
11b
12b
0.04
0.3
(57%)f
(77%)f
NTg
NTg 0/2
NTg 0/5
NTg 0/2
NTg 0/2
2/2
NTg
13b
14b
NTg
NTg
4.2
NTg NTg
NTg NTg
17a
NTg NTg NTg 0/6
0/4
a Inhibition of SRS-A-mediated bronchoconstriction and airway microvascular leakage in actively sensitized guinea pigs (n=3–6); OVA challenge 1h
after oral dosing of a test compound.
b See corresponding footnotes from Table 1.
c Inhibition of gastric emptying in rats (n=5).
d Inhibition of LPS-induced TNF-a production in human whole blood. IC50 represent a mean of n=3.
e Vomiting test in fasted ferrets.
f Inhibition% at 0.3mg/kg, po.
g Not tested.
h Inhibition% at 10mg/kg, po.
i Inhibition% at 3mg/kg, po.

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4651
as IC50 values (i.e., the test compound concentration
that caused 50% inhibition relative to the vehicle).
tration versus 0.0131, 0.343, and 14.1lgh/mL after
oral administration. The steady state volume of distri-
bution (Vss) was calculated to be 329, 439, and
286mL/kg, respectively, indicating that these com-
pounds showed moderate distribution to the tissues.
Systemic clearance (CL) was 1770, 2650, and 183mL/
h/kg, respectively. The Cmax values after oral dosing
were 0.0225, 0.267, and 3.61lg/mL, respectively while
the tmax values were 0.50, 1.2, and 1.0h, respectively.
Bioavailability of 11b, 12b, and 17a was 0.2%, 8.7%,
and 68%, respectively.
The effect of these compounds on SRS-A-mediated
bronchoconstriction in actively sensitized guinea pigs
was not always consistent with their inhibition of LPS-
induced TNF-a production in rats, probably because
of differences in pharmacokinetics due to cross-species
comparison. For example, 2a–b, and 12b exhibited
much lower inhibition of SRS-A-mediated bronchocon-
striction in guinea pigs than expected from their effects
on TNF-a production, while 11b was equipotent in each
species.
5. Conclusion
To assess an adverse effect in the same species, inhibition
of gastric emptying by 2b, 11b–14b, and 17a was evalu-
ated in rats. The ID50 values of these compounds for an
effect on gastric emptying were found to be higher than
the values at which TNF-a production was inhibited,
which is one of their beneficial effects.
Based on the hypothesis that the dose-limiting side
effects of PDE4 inhibitors such as nausea and emesis
could be caused by an effect on the CNS, design, and
synthesis of hydrophilic inhibitors, which were predicted
to show limited penetration of the CNS, was carried out.
Several compounds (2b, 11b–14b, and 17a) demon-
strated more potency than Ariflo 1 against SRS-A-medi-
ated bronchoconstriction in actively sensitized guinea
pigs and LPS-induced TNF-a production in rats. In
addition, the potency of 17a relative to 2a was increased
by replacing the methoxy group of 2a with a difluoro-
methoxy group (Table 8). These compounds were also
evaluated for inhibition of gastric emptying in rats and
emesis in ferrets to assess potential side effects. On the
basis of these comparisons, all of the compounds were
estimated to show an improved side effect profile. Be-
cause of a much stronger effect on LPS-induced TNF-
a production in HWB compared with Ariflo 1, these
compounds could show improved potential for clinical
To assess the safety of these compounds in another spe-
cies, a ferret emesis model was used, which is an estab-
lished method for evaluating the side effect profile of
PDE4 inhibitors. Compounds 2b and 11b–13b did not
cause emesis at oral doses up to 0.3mg/kg, while 14b
and 17a did not cause emesis at oral doses up to 1 and
10mg/kg, respectively.
Compounds 2b, 11b–14b, and 17a demonstrated more
potency than Ariflo 1 against SRS-A-mediated broncho-
constriction in actively sensitized guinea pigs and LPS-
induced TNF-a production in rats. Based on their far
stronger effect on LPS-induced TNF-a production in
HWB compared with Ariflo 1, these compounds were
considered to show improved potential for clinical appli-
cation.
Table 8. Increase of the lipophilicity of 17a by replacing the methoxy
moiety of 2a with a difluoromethoxy moiety
4. Pharmacokinetic study
CN
O
Pharmacokinetic data for compounds 11b, 12b, and 17a
were investigated after administration of single doses to
rats (Table 7). Intravenous administration of com-
pounds 11b, 12b, and 17a to rats (3mg/kg, N=3)
resulted in detectable plasma levels (t1/2 =0.49, 0.17,
and 1.6h), respectively, while oral administration of
17a to rats (10mg/kg, N=3) resulted in a t1/2 of 4.4h.
The AUC values of 11b, 12b, and 17a were, respectively
1.78, 1.18, and 20.6lgh/mL after intravenous adminis-
N
COOH
O
R
Compound
CLogP
Mol vol
(cm3/mol)
LogD
(octanol/pH7.4)
2a (R=Me)
0.07
0.51
0.60
0.90
343
350
364
385
0.1
1.0
NT
NT
17a (R=CHF2)
16a (R=Et)
18a (R= i-Pr)
Table 7. Single-dose rat pharmacokinetic data for compounds 11b, 12b, and 17a
Parameter
11b
12b
17a
iv (3mg/kg)
0.49 0.27
po (10mg/kg)
iv (3mg/kg)
0.17 0.02
po (10mg/kg)
iv (3mg/kg)
1.6 0.7
po (3mg/kg)
Cmax (lg/mL)
t1/2 (h)
tmax (h)
0.0225
ND
0.50
0.267 0.118
ND
1.2 1.6
3.61 1.56
4.4 4.7
1.0 0.0
AUC (lgh/mL)
Vss (mL/kg)
CLtotal (mL/h/kg)
%Bioavailabitity
1.78 0.44
329 179
1770 510
0.0131
1.18 0.28
439 178
2650 725
0.343 0.25
20.6 13.1
286 41
183 89
14.1 3.7
0.2
8.7
68.3

4652
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
application. The hydroxamic acid group, a well-known
metal chelator, was again demonstrated to show a
strong interaction with PDE4, as illustrated by data
for 2b, 17b, and other compounds. The configurational
isomerism of Ariflo was abolished without loss of activ-
ity. Pharmacokinetic data for compounds 11b, 12b, and
17a were also obtained in rats.
6.2.3. tert-Butyl 4-cyano-4-[3-(cyclopentyloxy)-4-meth-
oxyphenyl]piperidine-1-carboxylate (31a) (Method A).
To a stirred solution of 30a (2.50g, 10.8mmol) in
THF (150mL) was added LiHMDS (1.0M solution in
THF, 24.0mL, 24.0mmol) at ꢀ78ꢁC under argon
atmosphere. After being stirred at ꢀ78ꢁC for 30min, a
solution of 29 (2.17g, 6.00mmol) in THF (50mL) was
added dropwise. After being stirred at room tempera-
ture for 1.5h, the reaction mixture was poured into
brine, and extracted with EtOAc. The organic layer
was washed with brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (n-hexane/
Et2O, 2/1–1/2–0/1) to give 31a (1.71g, crude 71%) as a
yellow oil: TLC Rf =0.56 (n-hexane/EtOAc, 2/1).
6. Experimental
6.1. General chemical procedures
Analytical samples were homogeneous as confirmed by
thin-layer chromatography (TLC), and yielded spectro-
scopic data consistent with the assigned structures. All
1H NMRspectra were obtained with a Varian Gemi-
ni-200 or MERCURY-300 spectrometer. The chemical
shift values are reported in ppm (d) and coupling con-
stants (J) in Hertz (Hz). Fast atom bombardment
(FAB) and electron ionization (EI) mass spectra were
obtained with a JEOL JMS-DX303HF or JMS-700
spectrometer. Atmospheric pressure chemical ionization
(APCI) mass spectra were determined by a Hitachi M-
1200H spectrometer. IRspectra were measured using a
Perkin–Elmer FTIR1760X or JASCO FTIR-430 spec-
trometer. Elemental analyses were performed with a
Perkin–Elmer PE2400 Series II CHNS/O Analyzer and
are only indicated as the elements within 0.4% of the
theoretical values unless otherwise noted. Column chro-
matography was carried out using silica gel [Merck silica
gel 60 (0.063–0.200mm), Wako Gel C200, Fuji Silysia
FL60D, or Fuji Silysia BW-235]. TLC was also per-
formed on silica gel (Merck TLC plate, silica gel 60
F254).
6.2.4. tert-Butyl 4-cyano-4-[3,4-dimethoxyphenyl]piper-
idine-1-carboxylate (31b). The title compound was pre-
pared from the corresponding benzyl cyanide
according to Method A to give a yellow oil: Yield crude
53%; TLC Rf =0.34 (n-hexane/EtOAc, 2/1).
6.2.5. tert-Butyl 4-cyano-4-[3-ethoxy-4-methoxyphen-
yl]piperidine-1-carboxylate (31c). The title compound
was prepared from the corresponding benzyl cyanide
according to Method A to give a yellow oil: Yield crude
quant; TLC Rf =0.46 (n-hexane/EtOAc, 2/1).
6.2.6. tert-Butyl 4-cyano-4-[3-(cyclopropylmethoxy)-4-
methoxyphenyl]piperidine-1-carboxylate (31d). The title
compound was prepared from the corresponding benzyl
cyanide according to Method A to give a yellow oil:
Yield crude 91%; TLC Rf =0.60 (n-hexane/EtOAc, 2/1).
6.2.7. tert-Butyl 4-cyano-4-[3-(isopropyloxy)-4-methoxy-
phenyl]piperidine-1-carboxylate (31e). The title com-
pound was prepared from the corresponding benzyl
cyanide according to Method A to give a yellow oil:
Yield crude 69%; TLC Rf =0.64 (n-hexane/EtOAc, 2/1).
6.2. Synthesis of compounds 33a–k
6.2.1. tert-Butyl bis(2-hydroxyethyl)carbamate (28). To a
stirred solution of bis(2-hydroxyethyl)amine 27 (20.0g,
190mmol) in CH2Cl2 (200mL) was added a solution
of Boc2O (45.6g, 209mmol) in CH2Cl2 (50mL) at
0ꢁC. After being stirred for 1.5h, the reaction mixture
was concentrated in vacuo. The residue was purified
by column chromatography on silica gel (n-hexane/EtO-
Ac, 2/1–1/2–0/1) to give 28 (41.0g, quant.) as colorless
oil: TLC Rf =0.56 (CHCl3/MeOH, 10/1); 1H NMR
(300MHz, CDCl3) d 3.80 (s, 4H), 3.43 (s, 4H), 3.60–
3.00 (br, 2H), 1.47 (s, 9H).
6.2.8. tert-Butyl 4-cyano-4-[3-(cyclobutyloxy)-4-meth-
oxyphenyl]piperidine-1-carboxylate (31f). The title com-
pound was prepared from the corresponding benzyl
cyanide according to Method A to give a yellow oil:
Yield crude 80%; TLC Rf =0.44 (n-hexane/EtOAc, 2/1).
6.2.9. tert-Butyl 4-cyano-4-[3-(cyclopentyloxy)-4-ethoxy-
phenyl]piperidine-1-carboxylate (31g). The title com-
pound was prepared from the corresponding benzyl
cyanide according to Method A to give a brown oil:
Yield crude 65%; TLC Rf =0.79 (n-hexane/EtOAc, 2/1).
6.2.2. [(tert-Butoxycarbonyl)imino]diethane-2,1-diyl di-
methanesulfonate (29). To a stirred solution of 28
(7.85g, 383.3mmol) and Et3N (16.0mL, 115mmol) in
CH2Cl2 (80mL) was added MsCl (8.89mL, 115mmol)
at ꢀ78ꢁC. After being stirred for 10min, the reaction
mixture was poured into H2O, and extracted with EtO-
Ac. The organic layer was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo to give 29
(13.2g, 36.6mmol, 95% in two steps) as a pale yellow oil:
6.2.10. tert-Butyl 4-cyano-4-[3-(cyclopentyloxy)-4-di-
fluoromethoxyphenyl]piperidine-1-carboxylate (31h). The
title compound was prepared from the corresponding
benzyl cyanide according to Method A to give a
brown oil: Yield crude 77%; TLC Rf =0.76 (n-hexane/
EtOAc, 2/1).
TLC Rf =0.64 (EtOAc); 1H NMR(300MHz, CDCl ) d.
6.2.11. tert-Butyl 4-cyano-4-[3-(cyclopentyloxy)-4-iso-
propoxyphenyl]piperidine-1-carboxylate (31i). The title
compound was prepared from the corresponding benzyl
3
4.40–4.25 (m, 4H), 3.62 (br t, J=5.4Hz, 4H), 3.04 (s,
6H), 1.48 (s, 9H).

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4653
cyanide according to Method A to give a yellow oil:
Yield crude 83%; TLC Rf =0.76 (n-hexane/EtOAc, 1/1).
AcOH, 10/1/0.2); MS (APCI, Pos. 40V) m/z=275
(M+H)+; 1H NMR(300MHz, CDCl 3) d 10.40–9.60
(br, 2H), 7.10–7.00 (m, 2H), 6.90 (d, J=9.0Hz, 1H),
4.58 (sept, J=6.0Hz, 1H), 3.86 (s, 3H), 3.80–3.60 (m,
2H), 3.45–3.35 (m, 2H), 2.80–2.60 (m, 2H), 2.35–2.20
(m, 2H), 1.38 (d, J=6.0Hz, 6H).
6.2.12.
4-[3-Cyclopentyloxy)-4-methoxyphenyl]piper-
idine-4-carbonitrile hydrochloride (32a) (Method B). To
a stirred solution of 31a (1.71g) in CH2Cl2 (5mL) were
added PhSMe (5.0mL) and TFA (3.0mL). After being
stirred at room temperature for 1h, the reaction mixture
was poured into H2O, and extracted with CH2Cl2. The
organic layer was dried over MgSO4 and concentrated
in vacuo. After addition of 4N HCl/EtOAc (1.4mL),
the resulting mixture was evaporated. The residue was
triturated with Et2O to give 32a (971mg, 2.89mmol,
48% in two steps) as a white powder: TLC Rf =0.33
(CHCl3/MeOH, 10/1); MS (MALDI, Pos.) m/z=301
6.2.17. 4-[3-Cyclobutyloxy-4-methoxyphenyl]piperidine-
4-carbonitrile hydrochloride (32f). The title compound
was prepared from the corresponding tert-butylcarbo-
nate according to Method B to give a white powder:
Yield 60% in two steps; TLC Rf =0.31 (CHCl3/MeOH,
9/1); MS (APCI, Pos. 20V) m/z=287 (M+H)+; 1H
NMR(300MHz, CDCl ) d 10.20–9.80 (m, 2H), 7.10–
3
7.00 (m, 1H), 6.90–6.85 (m, 2H), 4.70 (quintet,
J=7.2Hz, 1H), 3.88 (s, 3H), 3.75–3.65 (m, 2H), 3.45–
3.30 (m, 2H), 2.78–2.60 (m, 2H), 2.60–2.50 (m, 2H),
2.35–2.15 (m, 4H), 1.95–1.60 (m, 2H).
(M+H)+; 1H NMR(300MHz, CDCl ) d 10.02 (br s,
3
2H), 7.10–7.00 (m, 2H), 6.88 (d, J=9.0Hz, 1H), 4.82
(m, 1H), 3.86 (s, 3H), 3.80–3.60 (m, 2H), 3.50–3.30 (m,
2H), 2.80–2.60 (m, 2H), 2.40–2.20 (m, 2H), 2.10–1.80
(m, 6H), 1.80–1.60 (m, 2H).
6.2.18. 4-[3-Cyclopentyloxy-4-ethoxyphenyl]piperidine-4-
carbonitrile hydrochloride (32g). The title compound was
prepared from the corresponding tert-butylcarbonate
according to Method B to give a white powder: Yield
45% in two steps; TLC Rf =0.31 (CHCl3/MeOH, 10/1);
MS (APCI, Pos. 20V) m/z=315 (M+H)+; 1H NMR
(300MHz, CDCl3) d 9.99 (br s, 2H), 7.05–6.95 (m,
2H), 6.88 (d, J=8.4Hz, 1H), 4.81 (m, 1H), 4.06 (q,
J=7.2Hz, 2H), 3.75–3.60 (m, 2H), 3.50–3.30 (m, 2H),
2.80–2.60 (m, 2H), 2.35–2.20 (m, 2H), 2.00–1.70 (m,
6H), 1.70–1.50 (m, 2H), 1.43 (t, J=7.2Hz, 3H).
6.2.13. 4-[3,4-Dimethoxyphenyl]piperidine-4-carbonitrile
hydrochloride (32b). The title compound was prepared
from the corresponding tert-butylcarbonate according
to Method B to give a white powder: Yield 34% in
two steps; TLC Rf =0.23 (CHCl3/MeOH/AcOH, 10/1/
0.2); MS (APCI, Pos. 40V) m/z=247 (M+H)+; 1H
NMR(300MHz, CDCl ) d 10.00 (br s, 2H), 7.08 (dd,
3
J=8.4, 2.4Hz, 1H), 7.05 (d, J=2.4Hz, 1H), 6.89 (d,
J=8.4Hz, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.75–3.65
(m, 2H), 3.50–3.30 (m, 2H), 2.80–2.65 (m, 2H), 2.35–
2.20 (m, 2H).
6.2.19.
4-[3-Cyclopentyloxy-4-difluoromethoxyphen-
yl]piperidine-4-carbonitrile hydrochloride (32h). The title
compound was prepared from the corresponding tert-
butylcarbonate according to Method B to give a white
powder: Yield 34% in two steps; TLC Rf =0.42
6.2.14. 4-[3-Ethoxy-4-methoxyphenyl]piperidine-4-carbo-
nitrile hydrochloride (32c). The title compound was pre-
pared from the corresponding tert-butylcarbonate
according to Method B to give a white powder: Yield
45% in two steps; TLC Rf =0.30 (CHCl3/MeOH, 9/1);
MS (APCI, Pos. 20V) m/z=261 (M+H)+; 1H NMR
(300MHz, CDCl3) d 10.05–9.95 (m, 2H), 7.08–7.04 (m,
2H), 6.90 (d, J=8.4Hz, 1H), 4.15 (q, J=6.9Hz, 2H),
3.88 (s, 3H), 3.75–3.65 (m, 2H), 3.45–3.35 (m, 2H),
2.71 (br t, J=12.6Hz, 2H), 2.27 (br d, J=14.1Hz,
2H), 1.48 (t, J=6.9Hz, 3H).
(CHCl3/MeOH/AcOH, 10/1/0.2); MS (APCI, Pos.
1
40V) m/z=275 (M+H)+; H NMR(300MHz, CDCl )
3
d 10.40–9.60 (br, 2H), 7.10–7.00 (m, 2H), 6.90 (d,
J=9.0Hz, 1H), 4.58 (sept, J=6.0Hz, 1H), 3.86 (s, 3H),
3.80–3.60 (m, 2H), 3.45–3.35 (m, 2H), 2.80–2.60 (m,
2H), 2.35–2.20 (m, 2H), 1.38 (d, J=6.0Hz, 6H).
6.2.20.
4-[3-Cyclopentyloxy-4-isopropoxyphenyl]piper-
idine-4-carbonitrile hydrochloride (32i). The title com-
pound was prepared from the corresponding tert-
butylcarbonate according to Method B to give a white
powder: Yield 32% in two steps; TLC Rf =0.35
(CHCl3/MeOH, 9/1); MS (APCI, Pos. 20V) m/z=329
(M+H)+; 1H NMR(300MHz, CDCl 3) d 10.20–9.85
(m, 2H), 7.27–6.90 (m, 3H), 4.83–4.75 (m, 1H), 4.45
(sept, J=6.0Hz, 1H), 3.75–3.63 (m, 2H), 3.45–3.30 (m,
2H), 2.75–2.60 (m, 2H), 2.35–2.22 (m, 2H), 1.95–1.75
(m, 6H), 1.70–1.40 (m, 2H), 1.33 (d, J=6.0Hz, 6H).
6.2.15. 4-[3-Cyclopropylmethoxy-4-methoxyphenyl]pip-
eridine-4-carbonitrile hydrochloride (32d). The title com-
pound was prepared from the corresponding tert-
butylcarbonate according to Method B to give a white
powder: Yield 61% in two steps; TLC Rf =0.42
(CHCl3/MeOH/AcOH, 10/1/0.2); MS (APCI, Pos.
1
40V) m/z=287 (M+H)+; H NMR(300MHz, CDCl )
3
d 10.50–9.00 (br, 2H), 7.10–7.00 (m, 2H), 6.90 (d,
J=8.7Hz, 1H), 3.90 (d, J=6.3Hz, 2H), 3.89 (s, 3H),
3.75–3.65 (m, 2H), 3.45–3.30 (m, 2H), 2.75–2.60 (m,
2H), 2.35–2.20 (m, 2H), 1.34 (m, 1H), 0.75–0.55 (m,
2H), 0.45–0.30 (m, 2H).
6.2.21. Ethyl {4-cyano-4-[3-(cyclopentyloxy)-4-methoxy-
phenyl]piperidin-1-yl}acetate (33a) (Method C). To a stir-
red solution of 32a (300mg, 0.893mmol) in DMF
(4.0mL) were added K2CO3 (246mg, 1.79mmol) and
BrCH2COOEt (0.15mL, 1.3mmol). After being stirred
at room temperature for 20h, the reaction mixture was
diluted with EtOAc and washed with H2O and then
brine. The organic layer was dried over MgSO4 and
6.2.16. 4-[3-Isopropyloxy-4-methoxyphenyl]piperidine-4-
carbonitrile hydrochloride (32e). The title compound
was prepared from the corresponding tert-butylcarbo-
nate according to Method B to give a white powder:
Yield 29% in two steps; TLC Rf =0.42 (CHCl3/MeOH/

4654
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel (n-hexane/Et2O, 2/1–
1/1–0/1) to give 33a (341mg, 0.883mmol, 99%) as a col-
orless oil: TLC Rf =0.36 (n-hexane/EtOAc, 2/1); 1H
2H), 4.67 (quint, J=7.2Hz, 1H), 4.22 (q, J=7.2Hz,
2H), 3.87 (s, 3H), 3.31 (s, 2H), 3.12–3.05 (m, 2H),
2.73–2.60 (m, 2H), 2.55–2.43 (m, 2H), 2.33–2.00 (m,
6H), 1.93–1.78 (m, 1H), 1.76–1.60 (m, 1H), 1.30 (t,
J=7.2Hz, 3H).
NMR(300MHz, CDCl ) d 7.10–6.95 (m, 2H), 6.86 (d,
3
J=8.8Hz, 1H), 4.79 (m, 1H), 4.22 (q, J=7.0Hz, 2H),
3.85 (s, 3H), 3.31 (s, 2H), 3.15–3.00 (m, 2H), 2.75–2.55
(m, 2H), 2.30–2.05 (m, 2H), 2.20–2.00 (m, 2H), 2.00–
1.80 (m, 6H), 1.80–1.50 (m, 2H), 1.30 (t, J=7.0Hz, 3H).
6.2.27. Ethyl {4-cyano-4-[3-(cyclopentyloxy)-4-ethoxy-
phenyl]piperidin-1-yl}acetate (33g). The title compound
was prepared from the corresponding piperidine accord-
ing to Method C to give a pale yellow oil: Yield 96%;
TLC Rf =0.80 (CHCl3/MeOH, 10/1); 1H NMR
(300MHz, CDCl3) d 7.05–6.95 (m, 2H), 6.86 (d,
J=9.0Hz, 1H), 4.78 (m, 1H), 4.22 (q, J=7.2Hz, 2H),
4.06 (q, J=7.2Hz, 2H), 3.30 (s, 2H), 3.10–3.00 (m,
2H), 2.75–2.60 (m, 2H), 2.25–2.10 (m, 2H), 2.15–2.00
(m, 2H), 1.95–1.75 (m, 6H), 1.70–1.55 (m, 2H), 1.42 (t,
J=7.2Hz, 3H), 1.30 (t, J=7.2Hz, 3H).
6.2.22. Ethyl [4-cyano-4-(3,4-dimethoxyphenyl)piperidin-
1-yl]acetate (33b). The title compound was prepared
from the corresponding piperidine according to Method
C to give a pale yellow oil: Yield 97%; TLC Rf =0.33 (n-
hexane/EtOAc, 2/1); 1H NMR(300MHz, CDCl 3) d 7.06
(dd, J=8.4, 2.4Hz, 1H), 6.99 (d, J=2.4Hz, 1H), 6.87 (d,
J=8.4Hz, 1H), 4.22 (q, J=7.4Hz, 2H), 3.90 (s, 3H),
3.89 (s, 3H), 3.31 (s, 2H), 3.15–3.00 (m, 2H), 2.75–2.60
(m, 2H), 2.30–2.15 (m, 2H), 2.15–2.00 (m, 2H), 1.30 (t,
J=7.4Hz, 3H).
6.2.28. Ethyl {4-cyano-4-[3-(cyclopentyloxy)-4-(difluoro-
methoxy)phenyl]piperidin-1-yl}acetate (33h). The title
compound was prepared from the corresponding piper-
idine according to Method C to give a pale yellow oil:
Yield 94%; TLC Rf =0.20 (n-hexane/EtOAc, 2/1); 1H
NMR(300MHz, CDCl 3) d 7.16 (d, J=8.4Hz, 1H),
7.10 (d, J=2.4Hz, 1H), 7.03 (dd, J=8.4, 2.4Hz, 1H),
6.54 (t, J=75.3Hz, 1H), 4.83 (m, 1H), 4.22 (q,
J=7.2Hz, 2H), 3.31 (s, 2H), 3.20–3.00 (m, 2H), 2.80–
2.60 (m, 2H), 2.30–2.10 (m, 2H), 2.15–2.00 (m, 2H),
2.00–1.70 (m, 6H), 1.70–1.55 (m, 2H), 1.30 (t,
J=7.2Hz, 3H).
6.2.23. Ethyl [4-cyano-4-(3-ethoxy-4-methoxyphenyl)pip-
eridin-1-yl]acetate (33c). The title compound was pre-
pared from the corresponding piperidine according to
Method C to give a pale yellow oil: Yield 100%; TLC
Rf =0.50 (EtOAc); 1H NMR(300MHz, CDCl 3) d
7.30–6.95 (m, 2H), 6.87 (d, J=8.4Hz, 1H), 4.22 (q,
J=7.2Hz, 2H), 4.10 (q, J=7.2Hz, 2H), 3.88 (s, 3H),
3.31 (s, 2H), 3.13–3.05 (m, 2H), 2.73–2.60 (m, 2H),
2.26–2.15 (m, 2H), 2.12–2.05 (m, 2H), 1.47 (t,
J=7.2Hz, 3H), 1.30 (t, J=7.2Hz, 3H).
6.2.29. Ethyl {4-cyano-4-[3-(cyclopentyloxy)-4-isoprop-
oxyphenyl]piperidin-1-yl}acetate (33i). The title com-
pound was prepared from the corresponding
piperidine according to Method C to give a pale yellow
6.2.24. Ethyl {4-cyano-4-[3-(cyclopropylmethoxy)-4-
methoxyphenyl]piperidin-1-yl}acetate (33d). The title
compound was prepared from the corresponding piper-
idine according to Method C to give a pale yellow oil:
Yield 93%; TLC Rf =0.36 (n-hexane/EtOAc, 2/1); 1H
NMR(300MHz, CDCl 3) d 7.05 (dd, J=8.7, 2.7Hz,
1H), 6.99 (d, J=2.7Hz, 1H), 6.87 (d, J=8.7Hz, 1H),
4.22 (q, J=7.2Hz, 2H), 3.88 (s, 3H), 3.86 (d,
J=6.9Hz, 2H), 3.30 (s, 2H), 3.15–3.00 (m, 2H), 2.75–
2.60 (m, 2H), 2.25–2.10 (m, 2H), 2.15–2.00 (m, 2H),
1.30 (t, J=7.2Hz, 3H), 1.40–1.20 (m, 1H), 0.70–0.60
(m, 2H), 0.40–0.30 (m, 2H).
1
oil: Yield 90%; TLC Rf =0.95 (CHCl3/MeOH, 9/1); H
NMR(300MHz, CDCl ) d 7.01–6.97 (m, 2H), 6.89 (d,
3
J=7.8Hz, 1H), 4.80–4.74 (m, 1H), 4.42 (sept,
J=6.0Hz, 1H), 4.22 (q, J=7.0Hz, 2H), 3.30 (s, 2H),
3.12–3.02 (m, 2H), 2.72–2.61 (m, 2H), 2.26–2.14 (m,
2H), 2.12–2.05 (m, 2H), 1.90–1.75 (m, 2H), 1.65–1.50
(m, 6H), 1.31 (d, J=6.0Hz, 6H), 1.28 (t, J=7.0Hz,
3H).
6.2.30. Methyl (2R)-2-{4-cyano-4-[3-(cyclopentyloxy)-4-
methoxyphenyl]piperidin-1-yl}propanoate (33j). The fol-
lowing reaction was carried out under argon atmos-
phere. To a stirred solution of methyl-(S)-(ꢀ)-lactate
(0.34mL, 3.56mmol) in CH2Cl2 (3.0mL) were added
Tf2O (0.66mL, 3.93mmol) and 2,6-lutidine (0.46mL,
3.93mmol) at 0ꢁC. After being stirred at room temper-
ature for 30min, a solution of 32a (400mg, 1.19mmol)
in CH2Cl2 (2.5mL) and Et3N (0.36mL, 2.38mmol) were
added to the reaction mixture. After being stirred at
room temperature for 18h, the reaction mixture was
poured into H2O, extracted with EtOAc. The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography on
silica gel (n-hexane/EtOAc, 3/1) to give 33j (492mg,
6.2.25. Ethyl [4-cyano-4-(3-isopropoxy-4-methoxyphen-
yl)piperidin-1-yl]acetate (33e). The title compound was
prepared from the corresponding piperidine according
to Method C to give a pale yellow oil: Yield 93%;
TLC Rf =0.34 (n-hexane/EtOAc, 2/1); 1H NMR
(300MHz, CDCl3) d 7.10–7.00 (m, 2H), 6.87 (d,
J=8.4Hz, 1H), 4.54 (sept, J=6.0Hz, 1H), 4.22 (q,
J=7.2Hz, 2H), 3.85 (s, 3H), 3.30 (s, 2H), 3.20–3.00
(m, 2H), 2.75–2.60 (m, 2H), 2.25–2.10 (m, 2H), 2.15–
2.00 (m, 2H), 1.37 (d, J=6.0Hz, 6H), 1.30 (t,
J=7.2Hz, 3H).
6.2.26. Ethyl {4-cyano-4-[3-(cyclobutyloxy)-4-methoxy-
phenyl]piperidin-1-yl}acetate (33f). The title compound
was prepared from the corresponding piperidine accord-
ing to Method C to give a pale yellow oil: Yield 99%;
TLC Rf =0.95 (CHCl3/MeOH, 9/1); 1H NMR
(300MHz, CDCl3) d 7.02–7.00 (m, 1H), 6.90–6.80 (m,
quant.) as a colorless oil: TLC Rf =0.90 (CHCl3/MeOH,
1
9/1); H NMR(300MHz, CDCl ) d 7.05–6.97 (m, 2H),
3
6.87–6.83 (m, 1H), 4.85–4.75 (m, 1H), 3.84 (s, 3H),
3.76 (s, 3H), 3.39 (q, J=7.0Hz, 1H), 3.10–2.95 (m,

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4655
1
2H), 2.85–2.68 (m, 2H), 2.15–2.05 (m, 4H), 2.00–1.75
(m, 6H), 1.65–1.45 (m, 2H), 1.35 (d, J=7.0Hz, 3H).
20V) m/z=260 (M+H)+; H NMR(300MHz, CDCl )
3
d 7.21 (d, J=2.1Hz, 1H), 7.12 (dd, J=8.4, 2.1Hz, 1H),
6.87 (d, J=8.4Hz, 1H), 5.85–5.78 (m, 2H), 5.22 (s,
2H), 3.88 (s, 3H), 3.52 (s, 3H), 3.32–3.23 (m, 2H),
2.97–2.88 (m, 2H).
6.2.31. Methyl (2S)-2-{4-cyano-4-[3-(cyclopentyloxy)-4-
methoxyphenyl]piperidin-1-yl}propanoate (33k). The title
compound was prepared according to the same proce-
dures as described for the preparation of 33k from 32a
using methyl-(R)-(+)-lactate instead of methyl-(S)-(ꢀ)-
6.3.5. 1-[3-Ethoxy-4-methoxyphenyl]cyclopent-3-ene-1-
carbonitrile (35d). The title compound was prepared
from the corresponding benzylcyanide according to
Method D to give a pale yellow oil: Yield 67%; TLC
Rf =0.35 (n-hexane/EtOAc, 5/1); 1H NMR(300MHz,
CDCl3) d 7.00 (dd, J=8.4, 2.1Hz, 1H), 6.96 (d,
J=2.1Hz, 1H), 6.84 (d, J=8.4Hz, 1H), 5.86–5.78 (m,
2H), 4.11 (q, J=6.9Hz, 2H), 3.87 (s, 3H), 3.34–3.22
(m, 2H), 2.99–2.87 (m, 2H), 1.47 (t, J=6.9Hz, 3H).
lactate to give a colorless oil: TLC Rf =0.90 (CHCl3/
1
MeOH, 9/1); H NMR(300MHz, CDCl ) d 7.05–6.97
3
(m, 2H), 6.87–6.83 (m, 1H), 4.85–4.75 (m, 1H), 3.84 (s,
3H), 3.76 (s, 3H), 3.39 (q, J=7.0Hz, 1H), 3.10–2.95
(m, 2H), 2.85–2.68 (m, 2H), 2.15–2.05 (m, 4H), 2.00–
1.75 (m, 6H), 1.65–1.45 (m, 2H), 1.35 (d, J=7.0Hz, 3H).
6.3. Synthesis of compounds 33l–n
6.3.6. Benzyl 2-{4-cyano-4-[3-(cyclopentyloxy)-4-meth-
oxyphenyl]piperidin-1-yl}-2-methylpropanoate
6.3.1. 1-[3-Cyclopentyloxy)-4-methoxyphenyl]cyclopent-
3-ene-1-carbonitrile (35e) (Method D). The following
reaction was carried out under argon atmosphere. To
a stirred solution of 30i (4.00g, 17.3mmol) in THF
(75mL) was added LiHMDS (1.0M solution in THF,
40.4mL, 40.4mmol) at ꢀ78ꢁC. After stirring for 1h,
cis-2-butenyl-1,4-dichloride (1.20mL, 11.5mmol) was
added to the reaction mixture at ꢀ78ꢁC. After being
stirred for 1.5h, the reaction mixture was poured into
saturated aqueous NH4Cl, and extracted with EtOAc.
The organic layer was washed with H2O, brine, dried
over anhydrous MgSO4, and concentrated in vacuo.
The residue was purified by column chromatography
on silica gel (n-hexane/EtOAc, 8/1) to give 35e (3.05g,
(33l)
(Method E). To a solution of 35e (460mg, 1.63mmol)
in CH2Cl2 (10mL) was bubbled ozone (O2 containing
ca. 3% O3) at ꢀ78ꢁC for 25min. To the reaction mixture
was added Ph3P (513mg, 1.96mmol), and the reaction
mixture was stirred at ꢀ78ꢁC for 30min. After being
stirred at room temperature for 1h, the reaction mixture
was concentrated in vacuo to give 36e (1.27g, quant.) as
a yellow amorphous solid. The product was used for the
next reaction without further purification.
To a stirred solution of 36e (1.27g) and benzyl-2-amino-
2-methylpropanoate
(374mg,
1.63mmol)
in
ClCH2CH2Cl (10mL) were added NaBH(OAc)3
(1.03g, 4.88mmol) and AcOH (0.56mL, 9.77mmol).
After being stirred at room temperature for 3h, the reac-
tion mixture was diluted with EtOAc. The mixture was
washed with saturated aqueous NaHCO3 and brine.
The organic layer was dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (n-hexane/EtO-
Ac, 4/1) to give 33l (196mg, 0.412mmol, 25%) as a pale
yellow oil: TLC Rf =0.62 (n-hexane/EtOAc, 2/1); 1H
NMR(300MHz, CDCl 3) d 7.45–7.30 (m, 5H), 7.05–
6.95 (m, 2H), 6.85 (d, J=9.0Hz, 1H), 5.19 (s, 2H),
4.80 (m, 1H), 3.84 (s, 3H), 3.05–2.95 (m, 2H), 2.80–
2.60 (m, 2H), 2.10–2.00 (m, 4H), 2.05–1.80 (m, 6H),
1.80–1.50 (m, 2H), 1.38 (s, 6H).
10.8mmol, 94%) as a yellow oil: TLC Rf =0.39 (n-hex-
1
ane/EtOAc, 2/1); H NMR(300MHz, CDCl ) d 7.00–
3
6.95 (m, 2H), 6.82 (d, J=8.7Hz, 1H), 5.82 (s, 2H),
4.78 (m, 1H), 3.84 (s, 3H), 3.35–3.20 (m, 2H), 3.00–
2.85 (m, 2H), 2.00–1.75 (m, 6H), 1.70–1.55 (m, 2H).
6.3.2. 1-[3-Benzyloxy-4-methoxyphenyl]cyclopent-3-ene-
1-carbonitrile (35a). The title compound was prepared
from the corresponding benzylcyanide according to
Method D to give a white powder: Yield 93%; TLC
Rf =0.70 (n-hexane/EtOAc, 2/1); 1H NMR(300MHz,
CDCl3) d 7.45–7.20 (m, 5H), 7.02 (dd, J=8.4, 2.1Hz,
1H), 6.94 (d, J=2.1Hz, 1H), 6.85 (d, J=8.4Hz, 1H),
5.76 (s, 2H), 5.15 (s, 2H), 3.88 (s, 3H), 3.15–3.30 (m,
2H), 2.90–2.75 (m, 2H).
6.3.7. Benzyl 1-{4-cyano-4-[3-(cyclopentyloxy)-4-metho-
xyphenyl]piperidin-1-yl}cyclopropanecarboxylate (33m).
The title compound was prepared from the correspond-
ing cyclopentene according to Method E to give a pale
6.3.3.
1-[3-Benzyloxy-4-difluoromethoxyphenyl]cyclo-
pent-3-ene-1-carbonitrile (35b). The title compound was
prepared from the corresponding benzylcyanide accord-
ing to Method D to give a pale yellow powder: Yield
79%; TLC Rf =0.62 (n-hexane/EtOAc, 3/1); MS (APCI,
Pos. 20V) m/z=342 (M+H)+; 1H NMR(300MHz,
CDCl3) d 7.45–7.31 (m, 5H), 7.16 (d, J=8.4Hz, 1H),
7.12 (d, J=2.1Hz, 1H), 7.02 (dd, J=8.4, 2.1Hz, 1H),
6.57 (t, J=75.0Hz, 1H), 5.83–5.77 (m, 2H), 5.15 (s,
2H), 3.33–3.24 (m, 2H), 2.91–2.83 (m, 2H).
yellow oil: Yield 20% in two steps; TLC Rf =0.50 (n-hex-
1
ane/EtOAc, 2/1); H NMR(300MHz, CDCl ) d 7.50–
3
7.30 (m, 5H), 7.00 (d, J=2.1Hz, 1H), 6.98 (dd,
J=9.0, 2.1Hz, 1H), 6.84 (d, J=9.0Hz, 1H), 5.19 (s,
2H), 4.80 (m, 1H), 3.84 (s, 3H), 3.65–3.50 (m, 2H),
3.00–2.90 (m, 2H), 2.10–2.00 (m, 2H), 2.00–1.75 (m,
8H), 1.70–1.55 (m, 2H), 1.40–1.35 (m, 2H), 1.00–0.95
(m, 2H).
6.3.4. 1-[3-Methoxymethoxy-4-methoxyphenyl]cyclopent-
3-ene-1-carbonitrile (35c). The title compound was pre-
pared from the corresponding benzylcyanide according
to Method D to give a pale yellow oil: Yield 84%;
TLC Rf =0.42 (n-hexane/EtOAc, 2/1); MS (APCI, Pos.
6.3.8. Benzyl 1-[4-cyano-4-(3-ethoxy-4-methoxyphe-
nyl)piperidin-1-yl]cyclopropanecarboxylate (33n). The
title compound was prepared from the corresponding
cyclopentene according to Method E to give a pale

4656
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
yellow oil: Yield 15% in two steps; TLC Rf =0.48 (n-hex-
ane/EtOAc, 3/1); MS (APCI, Pos. 20V) m/z=435
J=12.0Hz, 2H), 2.66 (td, J=12.0, 2.7Hz, 2H), 2.16
(td, J=13.5, 3.9Hz, 2H), 2.12–2.03 (m, 3H).
1
(M+H)+; H NMR(300MHz, CDCl ) d 7.45–7.28 (m,
3
5H), 7.02–6.97 (m, 2H), 6.85 (d, J=9.0Hz, 1H), 5.20
(s, 2H), 4.12 (q, J=7.0Hz, 2H), 3.87 (s, 3H), 3.62–3.51
(m, 2H), 3.00–2.91 (m, 2H), 2.08–2.00 (m, 2H), 1.90–
1.79 (m, 2H), 1.47 (t, J=7.0Hz, 3H), 1.38–1.34 (m,
2H), 0.99–0.94 (m, 2H).
6.4.5. Ethyl {4-cyano-4-[3-(2,3-dihydro-1H-inden-2-yl-
oxy)-4-methoxyphenyl]piperidin-1-yl}acetate (33o)
(Method G). To a stirred solution of 38a (642mg,
2.02mmol) in THF (10mL) were added PPh3 (794mg,
3.03mmol),
diethylazodicarboxylate
(0.60mL,
3.03mmol) and 2-indanol (406mg, 3.03mmol). After
being stirred at room temperature for 15h, the reaction
mixture was purified by column chromatography on sil-
ica gel (n-hexane/EtOAc, 2/1) to give 33o (671mg,
1.54mmol, 77%) as a yellow oil: TLC Rf =0.62 (n-hex-
6.4. Synthesis of compounds 33o–w
6.4.1. Ethyl {4-[3-(benzyloxy)-4-methoxyphenyl]-4-cyano-
piperidin-1-yl}acetate (37a). The title compound was
prepared from the corresponding cyclopentene accord-
ing to Method E to give a pale yellow oil: Yield 47%
ane/EtOAc, 1/2); MS (APCI, Pos. 20V) m/z=435
1
(M+H)+; H NMR(300MHz, CDCl ) d 7.26–7.16 (m,
3
4H), 7.09–7.05 (m, 2H), 6.90–6.86 (m, 1H), 5.20 (m,
1H), 4.22 (q, J=7.2Hz, 2H), 3.82 (s, 3H), 3.44–3.35
(m, 2H), 3.31 (s, 2H), 3.27–3.19 (m, 2H), 3.14–3.05 (m,
2H), 2.73–2.63 (m, 2H), 2.26–2.16 (m, 2H), 2.15–2.06
(m, 2H), 1.30 (t, J=7.2Hz, 3H).
1
in two steps; TLC Rf =0.31 (n-hexane/EtOAc, 2/1); H
NMR(300MHz, CDCl 3) d 7.50–7.40 (m, 2H), 7.45–
7.25 (m, 3H), 7.10–7.00 (m, 2H), 6.89 (d, J=8.7Hz,
1H), 5.15 (s, 2H), 4.22 (q, J=7.2Hz, 2H), 3.88 (s, 3H),
3.30 (s, 2H), 3.15–3.00 (m, 2H), 2.70–2.55 (m, 2H),
2.20–2.05 (m, 2H), 2.15–1.95 (m, 2H), 1.30 (t,
J=7.2Hz, 3H).
6.4.6. Methyl {4-cyano-4-[3-(cyclobutyloxy)-4-(difluoro-
methoxy)phenyl]piperidin-1-yl}acetate (33t). The title
compound was prepared from the corresponding phenol
and alcohol according to Method G to give an orange
oil: Yield 77%; TLC Rf =0.54 (toluene/EtOAc, 1/1);
MS (APCI, Pos. 20V) m/z=395 (M+H)+; 1H NMR
(300MHz, CDCl3) d 7.17 (d, J=8.1Hz, 1H), 7.02 (dd,
J=8.1, 2.1Hz, 1H), 6.97 (d, J=2.1Hz, 1H), 6.58 (t,
J=75.0Hz, 1H), 4.69 (m, 1H), 3.76 (s, 3H), 3.33 (s,
2H), 3.09 (dt, J=12.3, 2.7Hz, 2H), 2.66 (td, J=12.3,
2.7Hz, 2H), 2.54–2.43 (m, 2H), 2.29–2.13 (m, 4H),
2.11–2.02 (m, 2H), 1.89 (m, 1H), 1.72 (m, 1H).
6.4.2. Methyl {4-[3-(benzyloxy)-4-(difluoromethoxy)phen-
yl]-4-cyanopiperidin-1-yl}acetate (37b). The title com-
pound was prepared from the corresponding
cyclopentene according to Method E to give a brown
oil: Yield 64% in two steps; TLC Rf =0.31 (n-hexane/Et-
OAc, 1/1); MS (APCI, Pos. 20V) m/z=431 (M+H)+; 1H
NMR(300MHz, CDCl ) d 7.42–7.30 (m, 5H), 7.20 (d,
3
J=8.1Hz, 1H), 7.18 (d, J=2.4Hz, 1H), 7.07 (dd,
J=8.1, 2.4Hz, 1H), 6.58 (t, J=75.0Hz, 1H), 5.15 (s,
2H), 3.76 (s, 3H), 3.32 (s, 2H), 3.08 (dt, J=12.0,
2.7Hz, 2H), 2.66 (td, J=12.0, 2.7Hz, 2H), 2.18 (td,
J=12.0, 3.9Hz, 2H), 2.09–2.01 (m, 2H).
6.4.7. Methyl {4-cyano-4-[4-(difluoromethoxy)-3-(2,3-di-
hydro-1H-inden-2-yloxy)phenyl]piperidin-1-yl}acetate
(33w). The title compound was prepared from the corre-
sponding phenol and alcohol according to Method G to
6.4.3. Ethyl [4-cyano-4-(3-hydroxy-4-methoxyphenyl)pip-
eridin-1-yl]acetate (38a) (Method F). A solution of 37a
(892mg, 2.18mmol) in EtOH (12mL) was hydrogenated
under atmospheric pressure of H2 gas in the presence of
10% Pd/C (100mg) for 2.5h. The catalyst was removed
by filtration through a pad of Celite, and washed with
MeOH. The filtrates were concentrated in vacuo to give
38a (642mg, 2.02mmol, 92%) as a white powder: TLC
give a pale yellow oil: Yield 63%; TLC Rf =0.29 (n-hex-
1
ane/EtOAc, 1/1); H NMR(300MHz, CDCl ) d 7.27–
3
7.16 (m, 6H), 7.07 (dd, J=8.1, 2.4Hz, 1H), 6.38 (t,
J=75.3Hz, 1H), 5.26–5.20 (m, 1H), 3.76 (s, 3H), 3.41
(dd, J=16.5, 6.3Hz, 2H), 3.34 (s, 2H), 3.20 (dd,
J=16.5, 3.3Hz, 2H), 3.11 (d, J=12.0Hz, 2H), 2.69 (dt,
J=12.0, 2.4Hz, 2H), 2.28–2.19 (m, 2H), 2.14–2.04 (m,
2H).
Rf =0.43 (n-hexane/EtOAc, 1/2); MS (APCI, Neg.
1
20V) m/z=317 (MꢀH)ꢀ; H NMR(300MHz, CDCl )
3
d 7.06 (d, J=2.4Hz, 1H), 7.00 (dd, J=8.1, 2.4Hz, 1H),
6.85 (d, J=8.1Hz, 1H), 5.66 (br, 1H), 4.22 (q,
J=7.2Hz, 2H), 3.90 (s, 3H), 3.30 (s, 2H), 3.11–3.03
(m, 2H), 2.72–2.62 (m, 2H), 2.20–2.04 (m, 4H), 1.30 (t,
J=7.2Hz, 3H).
6.4.8. Methyl {4-cyano-4-[4-(difluoromethoxy)-3-ethoxy-
phenyl]piperidin-1-yl}acetate (33p) (Method H). To a stir-
red solution of 38b (904mg, 2.66mmol) in DMF (10mL)
were added EtI (0.32mL, 4.0mmol) and K2CO3
(919mg, 6.65mmol). After being stirred at 60ꢁC for
3h, the reaction mixture was poured into ice H2O and
extracted with EtOAc. The organic layer was washed
with brine, dried over anhydrous MgSO4, concentrated
in vacuo, and purified by silica gel column chromatogra-
phy (n-hexane/EtOAc, 1/1–2/1) to give 33p (951mg,
2.58mmol, 97%) as a pale yellow oil: TLC Rf =0.33 (n-
hexane/EtOAc, 1/2); 1H NMR(300MHz, CDCl 3) d
7.17 (d, J=8.4Hz, 1H), 7.09 (d, J=2.1Hz, 1H), 7.04
(dd, J=8.4, 2.1Hz, 1H), 6.57 (t, J=75.3Hz, 1H), 4.11
(q, J=6.9Hz, 2H), 3.75 (s, 3H), 3.32 (s, 2H), 3.08 (d,
6.4.4. Methyl {4-cyano-4-[4-(difluoromethoxy)-3-hy-
droxyphenyl]piperidin-1-yl}acetate (38b). The title com-
pound was prepared from the corresponding benzyl
ether according to Method F to give an ivory powder:
Yield 92%; TLC Rf =0.55 (CHCl3/MeOH, 9/1); MS
(APCI, Pos. 20V) m/z=341 (M+H)+; 1H NMR
(300MHz, CDCl3) d 7.16 (d, J=2.4Hz, 1H), 7.13 (d,
J=8.4Hz, 1H), 7.02 (dd, J=8.4, 2.4Hz, 1H), 6.54 (t,
J=73.5Hz, 1H), 3.76 (s, 3H), 3.32 (s, 2H), 3.07 (d,

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4657
J=12.0Hz, 2H), 2.66 (dt, J=12.0, 2.7Hz, 2H), 2.26–
2.16 (m, 2H), 2.10–2.05 (m, 2H), 1.46 (t, J=6.9Hz, 3H).
1H), 3.88 (d, J=6.9Hz, 2H), 3.76 (s, 3H), 3.32 (s, 2H),
3.08 (dt, J=12.0, 2.7Hz, 2H), 2.66 (td, J=12.0, 2.7Hz,
2H), 2.27–2.15 (m, 2H), 2.12–2.05 (m, 2H), 1.28 (m,
1H), 0.69–0.63 (m, 2H), 0.40–0.33 (m, 2H).
6.4.9. Methyl {4-cyano-4-[4-(difluoromethoxy)-3-prop-
oxyphenyl]piperidin-1-yl}acetate (33q). The title com-
pound was prepared from the corresponding alkyl
halide and 38b according to Method H to give a pale
brown powder: Yield 99%; TLC Rf =0.56 (n-hexane/Et-
OAc, 1/1); 1H NMR(300MHz, CDCl 3) d 7.18 (d,
J=8.1Hz, 1H), 7.10 (d, J=2.4Hz, 1H), 7.05 (dd,
J=8.1, 2.4Hz, 1H), 6.57 (t, J=75.0Hz, 1H), 3.99 (t,
J=7.0Hz, 2H), 3.76 (s, 3H), 3.33 (s, 2H), 3.15–3.00
(m, 2H), 2.75–2.60 (m, 2H), 2.30–2.15 (m, 2H), 2.15–
2.00 (m, 2H), 1.86 (sext, J=7.0Hz, 2H), 1.06 (t,
J=7.0Hz, 3H).
6.5. Synthesis of compounds 33x–y
6.5.1. Benzyl 1-{4-cyano-4-[4-methoxy-3-(methoxymetho-
xy)phenyl]piperidin-1-yl}cyclopropanecarboxylate (39).
The title compound was prepared from the correspond-
ing cyclopentene according to Method E to give a beige
solid: Yield 65% in two steps; TLC Rf =0.45 (n-hexane/
EtOAc, 2/1); MS (APCI, Pos. 20V) m/z=451 (M+H)+;
1H NMR(300MHz, CDCl ) d 7.45–7.29 (m, 5H), 7.22
3
(d, J=2.4Hz, 1H), 7.14 (dd, J=8.4, 2.4Hz, 1H), 6.88
(d, J=8.4Hz, 1H), 5.23 (s, 2H), 5.19 (s, 2H), 3.88 (s,
3H), 3.61–3.50 (m, 2H), 3.53 (s, 3H), 3.00–2.92 (m,
2H), 2.08–2.00 (m, 2H), 1.89–1.78 (m, 2H), 1.38–1.34
(m, 2H), 1.00–0.95 (m, 2H).
6.4.10. Methyl {4-[3-butoxy-4-(difluoromethoxy)phenyl]-
4-cyanopiperidin-1-yl}acetate (33r). The title compound
was prepared from the corresponding alkyl
halide and 38b according to Method H to give a pale
brown oil: Yield 100%; TLC Rf =0.57 (n-hexane/EtOAc,
1/1); MS (APCI, Pos. 20V) m/z=397 (M+H)+; 1H NMR
(300MHz, CDCl3) d 7.17 (d, J=8.5Hz, 1H), 7.09 (d,
J=2.2Hz, 1H), 7.05 (dd, J=8.5, 2.2Hz, 1H), 6.56 (t,
J=75.0Hz, 1H), 4.03 (t, J=6.5Hz, 2H), 3.76 (s, 3H),
3.33 (s, 2H), 3.13–3.05 (m, 2H), 2.72–2.62 (m, 2H),
2.27–2.17 (m, 2H), 2.11–2.04 (m, 2H), 1.86–1.76 (m,
2H), 1.60–1.45 (m, 2H), 0.99 (t, J=7.4Hz, 3H).
6.5.2. Benzyl 1-[4-cyano-4-(3-hydroxy-4-methoxyphen-
yl)piperidin-1-yl]cyclopropanecarboxylate hydrochloride
(40). To a stirred solution of 39 (1.80g, 3.99mmol) in
CH2Cl2 (10mL) was added 4N HCl/EtOAc (10mL).
After being stirred at room temperature for 1h, the reac-
tion mixture was concentrated in vacuo. The residue was
triturated with EtOAc to give 40 (1.51g, 3.41mmol,
85%) as a pale beige powder: TLC Rf =0.38 (n-hexane/
EtOAc, 2/1); MS (APCI, Pos. 20V) m/z=407 (M+H)+;
1H NMR(300MHz, CDCl ) d 7.42–7.32 (m, 5H), 7.19
3
(d, J=2.7Hz, 1H), 7.07 (dd, J=8.4, 2.7Hz, 1H), 6.86
(d, J=8.4Hz, 1H), 5.90–5.83 (br s, 1H), 5.22 (s, 2H),
4.50–4.36 (m, 2H), 3.89 (s, 3H), 3.56–3.47 (m, 2H),
3.27–3.09 (m, 2H), 2.30–2.23 (m, 2H), 2.22–2.12 (m,
2H), 1.72–1.65 (m, 2H), 1.70–1.50 (br, 1H).
6.4.11. Methyl {4-cyano-4-[4-(difluoromethoxy)-3-isobut-
oxyphenyl]piperidin-1-yl}acetate (33s). The title com-
pound was prepared from the corresponding alkyl
halide and 38b according to Method H to give a pale
brown powder: Yield 90%; TLC Rf =0.40 (n-hexane/Et-
OAc, 1/1); 1H NMR(300MHz, CDCl 3) d 7.18 (d,
J=7.8Hz, 1H), 7.10–7.00 (m, 2H), 6.56 (t, J=75.3Hz,
1H), 3.78 (d, J=6.6Hz, 2H), 3.76 (s, 3H), 3.33 (s, 2H),
3.20–3.00 (m, 2H), 2.80–2.60 (m, 2H), 2.30–2.15 (m,
2H), 2.25–2.00 (m, 1H), 2.20–2.00 (m, 2H), 1.05 (d,
J=6.6Hz, 6H).
6.5.3. Benzyl 1-{4-cyano-4-[3-(cyclopropylmethoxy)-
4-methoxyphenyl]piperidin-1-yl}cyclopropanecarboxylate
(33x). The title compound was prepared from the corre-
sponding alkyl haride and 40 according to Method H to
give a colorless oil: Yield quant; TLC Rf =0.57 (n-hex-
ane/EtOAc, 2/1); MS (APCI, Pos. 20V) m/z=461
1
(M+H)+; H NMR(300MHz, CDCl ) d 7.45–7.28 (m,
3
6.4.12. Methyl {4-cyano-4-[3-(cyclobutylmethoxy)-4-(di-
fluoromethoxy)phenyl]piperidin-1-yl}acetate (33u). The
title compound was prepared from the corresponding
alkyl halide and 38b according to Method H to give a pale
5H), 7.02–6.96 (m, 2H), 6.87–6.82 (m, 1H), 5.19 (s,
2H), 3.88–3.84 (m, 5H), 3.61–3.49 (m, 2H), 3.00–2.90
(m, 2H), 2.08–1.99 (m, 2H), 1.90–1.77 (m, 2H), 1.38–
1.32 (m, 2H), 0.98–0.96 (m, 2H), 0.69–0.61 (m, 2H),
0.40–0.33 (m, 3H).
brown oil: Yield 84%; TLC Rf =0.84 (CHCl3/MeOH, 9/
1
1); H NMR(300MHz, CDCl ) d 7.17 (d, J=8.4Hz,
3
1H), 7.09 (d, J=2.1Hz, 1H), 7.05 (dd, J=8.4, 2.1Hz,
1H), 6.57 (t, J=75.3Hz, 1H), 3.99 (d, J=6.6Hz, 2H),
3.76 (s, 3H), 3.33 (s, 2H), 3.09 (dt, J=11.7, 2.4Hz,
2H), 2.81 (m, 1H), 2.67 (td, J=11.7, 2.4Hz, 2H), 2.28-
1.82 (m, 10H).
6.5.4. Benzyl 1-{4-cyano-4-[3-(cyclobutyloxy)-4-methoxy-
phenyl]piperidin-1-yl}cyclopropanecarboxylate (33y). The
title compound was prepared from the corresponding
alcohol and 40 according to Method G to give a white
powder: Yield quant; TLC Rf =0.52 (n-hexane/EtOAc,
2/1); MS (APCI, Pos. 20V) m/z=461 (M+H)+; 1H
NMR(300MHz, CDCl 3) d 7.45–7.29 (m, 5H), 6.97
(dd, J=8.4, 2.4Hz, 1H), 6.87 (d, J=2.4Hz, 1H), 6.84
(d, J=8.4Hz, 1H), 5.19 (s, 2H), 4.69 (quint, J=7.0Hz,
1H), 3.86 (s, 3H), 3.61–3.50 (m, 2H), 3.00–2.90 (m,
2H), 2.55–2.43 (m, 2H), 2.33–2.18 (m, 2H), 2.08–1.99
(m, 2H), 1.89–1.60 (m, 4H), 1.38–1.34 (m, 2H), 0.99–
0.94 (m, 2H).
6.4.13. Methyl {4-cyano-4-[3-(cyclopropylmethoxy)-4-(di-
fluoromethoxy)phenyl]piperidin-1-yl}acetate (33v). The
title compound was prepared from the corresponding
alkyl halide and 38b according to Method H to give a
pale yellow oil: Yield 93%; TLC Rf =0.80 (CHCl3/
1
MeOH, 9/1); H NMR(300MHz, CDCl 3) d 7.18 (d,
J=8.1Hz, 1H), 7.09–7.03 (m, 2H), 6.63 (t, J=75.6Hz,

4658
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
6.6. Synthesis of compounds 2a–14a, 15, 16a–18a, 19–26,
2b–14b, and 16b–18b
(300MHz, DMSO-d6) d 7.10–7.00 (m, 2H), 6.97 (d,
J=9.3Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 4.00–3.00
(br, 1H), 3.23 (s, 2H), 3.05–2.95 (m, 2H), 2.65–2.50
(m, 2H), 2.20–1.95 (m, 4H); Anal. Found C16H20N2O4-
2H2O (C, H, N).
6.6.1. {4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphen-
yl]piperidin-1-yl}acetic acid (2a) (Method I). To a stirred
solution of 33a (330mg, 0.855mmol) in EtOH (5.0mL)
was added 2N NaOH (0.86mL, 1.71mmol). After being
stirred at room temperature for 35min, the reaction
mixture was acidified with 2N HCl (0.86mL,
1.71mmol), and concentrated in vacuo. The residue
was purified by column chromatography on silica gel
(CHCl3/MeOH/H2O, 10/2/0.1) to give 2a (278mg,
0.77mmol, 91%) as a white powder: TLC Rf =0.22
(CHCl3/MeOH/AcOH, 10/1/0.2); IR(KBr) 3446, 2959,
2870, 2233, 1721, 1631, 1517, 1444, 1417, 1361, 1260,
6.6.5. {4-Cyano-4-[3-ethoxy-4-methoxyphenyl]piperidin-
1-yl}acetic acid (11a). The title compound was prepared
from the corresponding ester according to Method I to
give a white powder: Yield quant; TLC Rf =0.30
(CHCl3/MeOH, 9/1); MS (MALDI, Pos.) m/z=319
(M+H)+; IR(KBr) 3436, 2982, 2242, 1637, 1520, 1400,
1
1264, 1176, 1152, 1025, 907, 635; H NMR(300MHz,
CDCl3) d 7.10–7.05 (m, 2H), 6.95–6.85 (m, 1H), 4.15
(q, J=6.9Hz, 2H), 3.88 (s, 3H), 3.50–3.40 (m, 4H),
3.10–2.95 (m, 2H), 2.60–2.40 (m, 3H), 2.25–2.15 (m,
2H), 1.49 (t, J=6.9Hz, 3H); Anal. Found
C17H22N2O43/4H2O (C, H, N).
1169, 1151, 1088, 1025, 985; MS (MALDI, Pos.) m/
1
z=359 (M+H)+; H NMR(300MHz, CDCl ) d 7.10–
3
7.00 (m, 2H), 6.88 (d, J=9.0Hz, 1H), 4.83 (m, 1H),
4.30–4.00 (br, 1H), 3.85 (s, 3H), 3.56 (br d, J=12.6Hz,
2H), 3.46 (s, 2H), 2.99 (br t, J=12.6Hz, 2H), 2.51 (br
t, J=12.6Hz, 2H), 2.19 (br d, J=12.6Hz, 2H), 2.05–
1.75 (m, 6H), 1.70–1.55 (m, 2H); Anal. Found
C20H26N2O43/7H2O (C, H, N); HRMS (EI) calcd for
C20H26N2O4 358.1893. Found 358.1901.
6.6.6. {4-Cyano-4-[3-(cyclopropylmethoxy)-4-methoxy-
phenyl]piperidin-1-yl}acetic acid (12a). The title com-
pound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
74%; TLC Rf =0.59 (CHCl3/MeOH/AcOH, 10/2/1);
MS (MALDI, Pos.) m/z=345 (M+H)+, 367 (M+Na)+,
299; IR(KBr) 3436, 3082, 3004, 2932, 2837, 2237,
1637, 1522, 1462, 1398, 1356, 1287, 1249, 1176, 1153,
1142, 1090, 1026, 1013, 966; 1H NMR(300MHz,
DMSO-d6) d 7.05–6.95 (m, 3H), 3.83 (d, J=6.9Hz,
2H), 3.76 (s, 3H), 3.60–2.90 (br, 1H), 3.27 (s, 2H),
3.05–2.95 (m, 2H), 2.70–2.50 (m, 2H), 2.20–1.95 (m,
4H), 1.20 (m, 1H), 0.65–0.55 (m, 2H), 0.40–0.25 (m,
2H); Anal. Found C19H24N2O4ÆH2O (C, H, N).
6.6.2. (2R)-2-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxy-
phenyl]piperidin-1-yl}propanoic acid (3a). The title com-
pound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
54%; TLC Rf =0.20 (CHCl3/MeOH, 9/1); MS (MALDI,
Pos.) m/z=373 (M+H)+, 395 (M+Na)+, 411 (M+K)+;
IR(KBr) 3453, 2958, 2348, 2232, 1617, 1519, 1449,
1
1365, 1261, 1150, 1023; H NMR(300MHz, CDCl ) d
3
7.10–7.00 (m, 2H), 6.90–6.85 (m, 1H), 4.88–4.80 (m,
1H), 3.85 (s, 3H), 3.50 (br q, J=7.0Hz, 1H), 3.28–3.04
(m, 3H), 3.00–2.90 (m, 1H), 2.50–2.15 (m, 5H), 2.05–
6.6.7. [4-Cyano-4-(3-isopropoxy-4-methoxyphenyl)piper-
idin-1-yl]acetic acid (13a). The title compound was pre-
pared from the corresponding ester according to
Method I to give a white powder: Yield quant; TLC
Rf =0.10 (EtOAc); MS (MALDI, Pos.) m/z=333
(M+H)+; IR(KBr) 3650, 3401, 2980, 2838, 2550, 2232,
1638, 1518, 1446, 1400, 1371, 1331, 1263, 1178, 1155,
1104; 1H NMR(300MHz, CDCl 3) d 7.15–7.00 (m,
2H), 6.95–6.85 (m, 1H), 4.59 (sept, J=6.0Hz, 1H),
3.86 (s, 3H), 3.60–3.50 (m, 2H), 3.46 (s, 2H), 3.05–2.93
(m, 2H), 2.85–2.60 (m, 1H), 2.60–2.40 (m, 2H), 2.24–
2.12 (m, 2H), 1.37 (d, J=6.0Hz, 6H); Anal. Found
C18H24N2O4Æ2/3H2O (C, H, N).
1.80 (m, 6H), 1.70–1.55 (m, 2H), 1.48 (br d, J=7.0Hz,
30
D
Anal. Found C21H28N2O42/3H2O (C, H, N).
3H); Optical rotation ½aŠ þ 10:69 (c 0.305, DMSO);
6.6.3. (2S)-2-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxy-
phenyl]piperidin-1-yl}propanoic acid (4a). The title com-
pound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
91%; TLC Rf =0.20 (CHCl3/MeOH, 9/1); MS (MALDI,
Pos.) m/z=373 (M+H)+, 395 (M+Na)+; (KBr) 3443,
2959, 2370, 2231, 1616, 1519, 1449, 1366, 1262, 1151,
1023.; 1H NMR(300MHz, CDCl 3) d 7.10–7.00 (m,
2H), 6.90–6.80 (m, 1H), 4.86–4.58 (m, 1H), 3.84 (s,
3H), 3.54–3.44 (m, 1H), 3.34–3.20 (m, 2H), 3.14–3.02
(m, 1H), 3.00–2.86 (m, 1H), 2.50–1.75 (m, 11H), 1.75–
6.6.8.
{4-Cyano-4-[3-(cyclobutyloxy)-4-methoxyphen-
yl]piperidin-1-yl}acetic acid (14a). The title compound
was prepared from the corresponding ester according
to Method I to give a white powder: Yield 60%; TLC
Rf =0.10 (EtOAc); MS (MALDI, Pos.) m/z=345
(M+H)+; IR(KBr) 3449, 2940, 2856, 2369, 2242, 1637,
1521, 1403, 1265, 1152, 1078; 1H NMR(300MHz,
CDCl3) d 7.10–7.00 (m, 1H), 6.95–6.85 (m, 2H), 4.71
(quint, J=7.5Hz, 1H), 3.87 (s, 3H), 3.70–3.40 (m, 2H),
3.49 (s, 2H), 3.10–2.95 (m, 2H), 2.70–2.00 (m, 9H),
2.00–1.80 (m, 1H), 1.80–1.60(m, 1H); Anal. Found
C19H24N2O4Æ2/3H2O (C, H, N).
1.55 (m, 2H), 1.45 (br d, J=7.0Hz, 3H); Optical rota-
30
D
C21H28N2O44/3H2O (C, H, N).
tion ½aŠ ꢀ 10:40 (c 0.245, DMSO); Anal. Found
6.6.4.
[4-Cyano-4-(3,4-dimethoxyphenyl)piperidin-1-
yl]acetic acid (10a). The title compound was prepared
from the corresponding ester according to Method I to
give a white powder: Yield 94%; TLC Rf =0.38
(CHCl3/MeOH/AcOH, 10/2/1); MS (MALDI, Pos.) m/
z=305 (M+H)+, 259; IR(KBr) 3494, 3006, 2963,
2840, 2594, 2241, 1614, 1520, 1443, 1412, 1381, 1335,
1265, 1243, 1201, 1174, 1151, 1090, 1025; 1H NMR
6.6.9. {4-Cyano-4-[3-(2,3-dihydro-1H-inden-2-yloxy)-4-
methoxyphenyl]piperidin-1-yl}acetic acid (15). The title

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4659
compound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
76%; TLC Rf =0.68 (CHCl3/MeOH/AcOH, 30/2/1);
MS (APCI, Pos. 20V) m/z=407 (M+H)+; IR(KBr)
3653, 3392, 2956, 2836, 2234, 1633, 1521, 1481, 1441,
1421, 1401, 1357, 1322, 1268, 1248, 1198, 1174, 1138,
1018, 992, 977; 1H NMR(300MHz, DMSO- d6) d
7.28–7.23 (m, 2H), 7.19–7.13 (m, 2H), 7.10 (d,
J=1.8Hz, 1H), 7.06 (dd, J=8.6, 1.8Hz, 1H), 6.98 (d,
J=8.6Hz, 1H), 5.28 (m, 1H), 3.69 (s, 3H), 3.80–2.60
(br, 1H), 3.39–3.30 (m, 2H), 3.25 (s, 2H), 3.16–2.98
(m, 4H), 2.66–2.53 (m, 2H), 2.15–1.97 (m, 4H); Anal.
Found C24H26N2O4ÆH2O (C, H, N); HRMS (EI) calcd
for C24H26N2O4 406.1893. Found 406.1888.
Rf =0.15 (CHCl3/MeOH/AcOH, 9/1/0.1); MS (APCI,
Neg. 40V) m/z=353 (MꢀH)ꢀ; IR(KBr) 3442, 2984,
2235, 1631, 1519, 1487, 1422, 1349, 1300, 1274, 1226,
1
1177, 1110, 1029, 970; H NMR(300MHz, DMSO- d6)
d 7.22–7.19 (m, 2H), 7.09 (dd, J=8.4, 2.1Hz, 1H), 7.06
(t, J=74.4Hz, 1H), 4.13 (q, J=6.9Hz, 2H), 4.00–3.00
(br, 1H), 3.21 (s, 2H), 3.01 (d, J=12.0Hz, 2H), 2.57
(dt, J=11.7, 3.0Hz, 2H), 2.13–1.99 (m, 4H), 1.32 (t,
J=6.9Hz, 3H); HRMS (EI) calcd for C17H20F2N2O4
354.1391. Found 354.1383.
6.6.14. {4-Cyano-4-[4-(difluoromethoxy)-3-propoxyphen-
yl]piperidin-1-yl}acetic acid (20). The title compound was
prepared from the corresponding ester according to
Method I to give a white powder: Yield 60%; TLC
Rf =0.60 (CHCl3/MeOH/AcOH, 10/2/1); MS (MALDI,
Pos.) m/z=369 (M+H)+; IR(KBr) 3438, 2967, 2237,
1631, 1511, 1473, 1421, 1390, 1344, 1297, 1273, 1226,
1179, 1113, 1072, 1033, 978; 1H NMR(300MHz,
DMSO-d6) d 12.40–11.00 (br, 1H), 7.30–7.15 (m, 2H),
7.11 (dd, J=8.7, 2.7Hz, 1H), 7.05 (t, J=74.4Hz, 1H),
4.04 (t, J=6.6Hz, 2H), 3.23 (s, 2H), 3.10–2.95 (m,
2H), 2.70–2.50 (m, 2H), 2.20–2.00 (m, 4H), 1.74 (sext,
J=6.6Hz, 2H), 0.98 (t, J=6.6Hz, 3H); Anal. Found
C18H22F2N2O4 (C, H, N).
6.6.10.
{4-Cyano-4-[3-(cyclopentyloxy)-4-ethoxyphen-
yl]piperidin-1-yl}acetic acid (16a). The title compound
was prepared from the corresponding ester according
to Method I to give a white powder: Yield 82%; TLC
Rf =0.39 (CHCl3/MeOH/AcOH, 10/1/0.2); MS (APCI,
Neg. 40V) m/z=371 (MꢀH)ꢀ; IR(KBr) 3453, 2968,
2874, 2236, 1640, 1520, 1485, 1450, 1418, 1374, 1352,
1333, 1297, 1262, 1244, 1154, 1125, 1089, 1039, 1007,
1
989; H NMR(300MHz, DMSO- d3) d 7.05–6.90 (m,
3H), 4.83 (m, 1H), 4.00 (q, J=6.9Hz, 2H), 4.00–3.00
(br, 1H), 3.23 (s, 2H), 3.05–2.95 (m, 2H), 2.65–2.50
(m, 2H), 2.15–1.90 (m, 4H), 1.95–1.80 (m, 2H), 1.80–
1.60 (m, 4H), 1.65–1.50 (m, 2H), 1.29 (t, J=6.9Hz,
3H); Anal. Found C21H28N2O4Æ2/3H2O (C, H, N).
6.6.15. {4-[3-Butoxy-4-(difluoromethoxy)phenyl]-4-cyano-
piperidin-1-yl}acetic acid (21). The title compound was
prepared from the corresponding ester according to
Method I to give a pale brown powder: Yield 78%;
TLC Rf =0.56 (CHCl3/MeOH/AcOH, 10/2/1); MS
(APCI, Neg. 20V) m/z=381 (MꢀH)ꢀ; IR(KBr) 3451,
2987, 2981, 2879, 2696, 2547, 2236, 1611, 1521, 1423,
6.6.11. {4-Cyano-4-[3-(cyclopentyloxy)-4-(difluorometh-
oxy)phenyl]piperidin-1-yl}acetic acid (17a). The title
compound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
quant; TLC Rf =0.35 (CHCl3/MeOH/AcOH, 10/1/0.2);
MS (APCI, Neg. 40V) m/z=393 (MꢀH)ꢀ; IR(KBr)
3427, 2965, 2875, 2239, 1611, 1516, 1420, 1271, 1226,
1126, 1041, 988; 1H NMR(300MHz, DMSO- d3) d
7.25–7.15 (m, 2H), 7.09 (dd, J=8.1, 2.1Hz, 1H), 7.01
(t, J=75.0Hz, 1H), 4.98 (m, 1H), 3.60–3.00 (br, 1H),
3.26 (s, 2H), 3.10–2.95 (m, 2H), 2.70–2.50 (m, 2H),
2.20–2.00 (m, 4H), 2.00–1.80 (m, 2H), 1.80–1.60 (m,
4H), 1.65–1.50 (m, 2H); Anal. Found C20H24F2N2O4
(C, H, N).
1298,
1124,
1034;
1H
NMR(300MHz,
CDCl3+DMSO-d6) d 7.17 (d, J=8.1Hz, 1H), 7.09 (dd,
J=8.1, 2.1Hz, 1H), 7.04 (d, J=2.1Hz, 1H), 6.57 (t,
J=75.2Hz, 1H), 4.03 (t, J=6.5Hz, 2H), 3.57 (br, 1H),
3.29 (s, 2H), 3.20–3.10 (m, 2H), 2.72–2.61 (m, 2H),
2.30–2.19 (m, 2H), 2.13–2.05 (m, 2H), 1.86–1.76 (m,
2H), 1.58–1.44 (m, 2H), 0.99 (t, J=7.5Hz, 3H); Anal.
Found C19H24F2N2O4 (C, H, N).
6.6.16. {4-Cyano-4-[4-(difluoromethoxy)-3-isobutoxyphen-
yl]piperidin-1-yl}acetic acid (22). The title compound was
prepared from the corresponding ester according to
Method I to give a white powder: Yield 74%; TLC
Rf =0.63 (CHCl3/MeOH/AcOH, 10/2/1); MS (MALDI,
Pos.) m/z=83 (M+H)+, 405 (M+Na)+; IR(KBr) 3071,
2965, 2235, 1640, 1518, 1473, 1420, 1407, 1389, 1370,
1338, 1316. 1290, 1271, 1256, 1227, 1177, 1106, 1046,
1028, 963; 1H NMR(300MHz, DMSO- d6) d 12.20–
10.80 (br, 1H), 7.25–7.20 (m, 2H), 7.11 (dd, J=8.7,
2.4Hz, 1H), 7.04 (t, J=74.4Hz, 1H), 3.86 (d,
J=6.3Hz, 2H), 3.23 (s, 2H), 3.10–2.95 (m, 2H), 2.65–
2.50 (m, 2H), 2.20–2.00 (m, 5H), 0.98 (d, J=6.6Hz,
6H); Anal. Found C19H24F2N2O4ÆH2O (C, H, N).
6.6.12. {4-Cyano-4-[3-(cyclopentyloxy)-4-isopropoxyphen-
yl]piperidin-1-yl}acetic acid (18a). The title compound
was prepared from the corresponding ester according
to Method I to give a white powder: Yield 69%; TLC
Rf =0.20 (CHCl3/MeOH, 9/1); MS (MALDI, Pos.) m/
z=387 (M+H)+, 409 (M+Na)+; IR(KBr) 3433, 2970,
2882, 2232, 1632, 1513, 1404, 1370, 1265, 1172, 1136,
1109; 1H NMR(300MHz, CDCl
) d 7.04 (d,
3
J=2.1Hz, 1H), 7.00 (dd, J=8.4, 2.1Hz, 1H), 6.91 (d,
J=8.4Hz, 1H), 4.84–4.78 (m, 1H), 4.44 (sept,
J=6.0Hz, 1H), 3.48–3.34 (m, 4H), 3.02–2.90 (m, 2H),
2.48–2.30 (m, 2H), 2.20–1.75 (m, 9H), 1.70–1.60 (m,
2H), 1.32 (d, J=6.0Hz, 6H).
6.6.17. {4-Cyano-4-[3-(cyclobutyloxy)-4-(difluorometh-
oxy)phenyl]piperidin-1-yl}acetic acid (23). The title com-
pound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
89%; TLC Rf =0.67 (CHCl3/MeOH, 3/1); MS (MALDI,
Pos.) m/z=381 (M+H)+, 403 (M+Na)+; IR(KBr) 3430,
6.6.13. {4-Cyano-4-[4-(difluoromethoxy)-3-ethoxyphen-
yl]piperidin-1-yl}acetic acid (19). The title compound
was prepared from the corresponding ester according
to Method I to give a white powder: Yield 88%; TLC

4660
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
2239, 1636, 1516, 1408, 1371, 1318, 1275, 1111, 1074,
1050; 1H NMR(300MHz, DMSO- d6) d 7.21 (d,
J=8.1Hz, 1H), 7.10 (dd, J=8.1, 2.1Hz, 1H), 7.08 (t,
J=74.4Hz, 1H), 7.06 (d, J=2.1Hz, 1H), 4.85 (m, 1H),
4.25–2.60 (br s, 1H), 3.24 (s, 2H), 3.01 (br d,
J=12.0Hz, 2H), 2.58 (br t, J=12.0Hz, 2H), 2.48–
2.34 (m, 2H), 2.16–1.94 (m, 6H), 1.77 (m, 1H), 1.62
(m, 1H); Anal. Found C19H22F2N2O4Æ1/4H2O (C, H,
N).
MeOH. The filtrates were concentrated in vacuo. The
residue was purified by column chromatography on sil-
ica gel (CHCl3/MeOH/H2O, 10/2/0.1) to give 5a (140mg,
0.363mmol, 96 %) as a white powder: TLC Rf =0.34
(CHCl3/MeOH, 10/1); MS (MALDI, Pos.) m/z=387
(M+H)+, 409 (M+Na)+; IR(KBr) 3571, 3422, 2955,
2853, 2685, 2606, 2238, 1626, 1591, 1519, 1497, 1466,
1420, 1406, 1389, 1365, 1351, 1286, 1257, 1211, 1200,
1170, 1149, 1123, 1084, 1063, 1019, 988; 1H NMR
(300MHz, DMSO-d6) d 7.05–6.90 (m, 3H), 4.84 (m,
1H), 3.74 (s, 3H), 3.80–3.00 (br, 1H), 3.15–3.00 (m,
2H), 2.65–2.50 (m, 2H), 2.20–2.05 (m, 2H), 2.10–1.95
(m, 2H), 2.00–1.80 (m, 2H), 1.80–1.60 (m, 4H), 1.65–
1.55 (m, 2H), 1.25 (s, 6H); HRMS (FAB) calcd for
C22H31N2O4 387.2284. Found 387.2289.
6.6.18. {4-Cyano-4-[3-(cyclobutylmethoxy)-4-(difluoro-
methoxy)phenyl]piperidin-1-yl}acetic acid (24). The title
compound was prepared from the corresponding ester
according to Method I to give a white powder: Yield
98%; TLC Rf =0.53 (CHCl3/MeOH, 3/1); MS (MALDI,
Pos.) m/z=395 (M+H)+, 417 (M+Na)+; IR(KBr) 2976,
2229, 1638, 1518, 1408, 1385, 1368, 1338, 1316, 1271,
1258, 1100, 1047; 1H NMR(300MHz, DMSO- d6) d
7.25 (d, J=2.4Hz, 1H), 7.21 (d, J=8.4Hz, 1H), 7.11
(dd, J=8.4, 2.4Hz, 1H), 7.03 (t, J=74.4Hz, 1H), 4.06
(d, J=6.6Hz, 2H), 4.00–2.80 (br s, 1H), 3.24 (s, 2H),
3.02 (br d, J=12.0Hz, 2H), 2.72 (m, 1H), 2.59 (td,
J=12.0, 2.7Hz, 2H), 2.17–1.95 (m, 6H), 2.00–1.75 (m,
4H); HRMS (EI) calcd for C20H24F2N2O4 394.1704.
Found 394.1715.
6.6.22. 1-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphen-
yl]piperidin-1-yl}cyclopropanecarboxylic acid (6a). The
title compound was prepared from the corresponding
benzyl ester according to Method J to give a white pow-
der: Yield 93%; TLC Rf =0.45 (CHCl3/MeOH, 10/1);
MS (MALDI, Pos.) m/z=385 (M+H)+, 407 (M+Na)+;
IR(KBr) 3440, 2956, 2870, 2236, 1725, 1605, 1519,
1445, 1418, 1364, 1259, 1170, 1148, 1082, 1027, 996;
1H NMR(300MHz, DMSO- d6) d 12.29 (br s, 1H),
7.05–6.90 (m, 3H), 4.82 (m, 1H), 3.73 (s, 3H), 3.45–
3.30 (m, 2H), 2.95–2.85 (m, 2H), 2.10–1.95 (m, 2H),
2.00–1.60 (m, 8H), 1.65–1.50 (m, 2H), 1.25–1.10 (m,
2H), 0.95–0.80 (m, 2H); HRMS (EI) calcd for
C22H28N2O4 384.2046. Found 384.2032.
6.6.19. {4-Cyano-4-[3-(cyclopropylmethoxy)-4-(difluoro-
methoxy)phenyl]piperidin-1-yl}acetic acid hydrochloride
(25). The title compound was prepared from the corre-
sponding ester according to Method I to give a white
powder: Yield 74%; TLC Rf =0.61 (CHCl3/MeOH, 2/
1); MS (MALDI, Pos.) m/z=381 (M+H)+, 403
(M+Na)+; IR(KBr) 3482, 2943, 2672, 2239, 1750,
1521, 1407, 1297, 1237, 1187, 1132, 1051; 1H NMR
(300MHz, DMSO-d6) d 7.21–7.14 (m, 2H), 7.02 (t,
J=74.4Hz, 1H), 7.01 (dd, J=8.7, 2.1Hz, 1H), 4.12 (s,
2H), 3.95–2.95 (br s, 2H), 3.85 (d, J=6.9Hz, 2H), 3.58
(br d, J=12.0Hz, 2H), 3.23 (br t, J=12.0Hz, 2H),
2.54–2.31 (m, 4H), 1.14 (m, 1H), 0.47 (m, 2H), 0.24
(m, 2H); HRMS (EI) calcd for C19H22F2N2O4
380.1548. Found 380.1550.
6.6.23. 1-[4-Cyano-4-(3-ethoxy-4-methoxyphenyl)piper-
idin-1-yl]cyclopropanecarboxylic acid (7a). The title com-
pound was prepared from the corresponding benzyl
ester according to Method J to give a white powder:
Yield 68%; TLC Rf =0.38 (CHCl3/MeOH, 9/1); MS
(FAB, Pos.) m/z=345 (M+H)+; IR(KBr) 3443, 3025,
2930, 2230, 1732, 1621, 1522, 1494, 1451, 1372,
1
1255, 1151, 1022, 923; H NMR(300MHz, DMSO- d6)
d 12.45–12.15 (br, 1H), 7.03–6.93 (m, 3H), 4.04 (q,
J=6.9Hz, 2H), 3.75 (s, 3H), 3.45–3.35 (m, 2H),
2.95–2.86 (m, 2H), 2.09–1.98 (m, 2H), 1.86–1.72
(m, 2H), 1.32 (t, J=6.9Hz, 3H), 1.21–1.16 (m, 2H),
0.92–0.86 (m, 2H); Anal. Found C19H24N2O4 (C,
H, N).
6.6.20. {4-Cyano-4-[4-(difluoromethoxy)-3-(2,3-dihydro-
1H-inden-2-yloxy)phenyl]piperidin-1-yl}acetic acid (26).
The title compound was prepared from the correspond-
ing ester according to Method I to give a white powder:
Yield 86%; TLC Rf =0.24 (CHCl3/MeOH/AcOH, 9/1/
0.1); MS (APCI, Neg. 40V) m/z=441 (MꢀH)ꢀ; IR
(KBr) 3132, 2237, 1637, 1515, 1407, 1388, 1367, 1311,
6.6.24. 1-{4-Cyano-4-[3-(cyclopropylmethoxy)-4-metho-
xyphenyl]piperidin-1-yl}cyclopropanecarboxylic
acid
1
(8a). The title compound was prepared from the corre-
sponding benzyl ester according to Method J to give a
white powder: Yield 99%; TLC Rf =0.35 (n-hexane/EtO-
Ac, 1/1); MS (APCI, Pos. 20V) m/z=371 (M+H)+; IR
(KBr) 3448, 3012, 2959, 2942, 2910, 2864, 2236, 1700,
1638, 1607, 1519, 1473, 1442, 1420, 1403, 1335, 1280,
1252, 1182, 1169, 1151, 1125, 1017, 965; 1H NMR
(300MHz, DMSO-d6) d 12.5–12.0 (br, 1H), 7.04–6.93
(m, 3H), 3.82 (d, J=7.2Hz, 2H), 3.77 (s, 3H), 3.46–
3.36 (m, 2H), 2.94–2.86 (m, 2H), 2.07–1.97 (m, 2H),
1.85–1.71 (m, 2H), 1.26–1.14 (m, 3H), 0.91–0.85
(m, 2H), 0.60–0.53 (m, 2H), 0.35–0.28 (m, 2H);
HRMS (EI) calcd for C21H26N2O4 370.1893. Found
370.1873.
1271, 1221, 1177, 1118, 1035, 960; H NMR(300MHz,
DMSO-d6) d 7.32–7.14 (m, 7H), 6.91 (t, J=74.4Hz,
1H), 5.43–5.37 (m, 1H), 4.00–2.60 (br, 1H), 3.39 (dd,
J=16.8, 6.0Hz, 2H), 3.24 (s, 2H), 3.07–3.00 (m, 4H),
2.60 (dt, J=11.7, 3.0Hz, 2H), 2.17–2.03 (m, 4H); Anal.
Found C24H24F2N2O4Æ1/2H2O (C, H, N).
6.6.21. 2-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphen-
yl]piperidin-1-yl}-2-methylpropanoic acid (5a) (Method
J). A solution of 33l (180mg, 0.378mmol) in MeOH
(4.0mL) and THF (4.0mL) was hydrogenated under
atmospheric pressure of H2 gas in the presence of 10%
Pd/C (20mg) for 1.5h. The catalyst was removed by fil-
tration through a pad of Celite, and washed with

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4661
6.6.25. 1-{4-Cyano-4-[3-(cyclobutyloxy)-4-methoxyphen-
yl]piperidin-1-yl}cyclopropanecarboxylic acid (9a). The
title compound was prepared from the corresponding
benzyl ester according to Method J to give a white pow-
der: Yield 65%; TLC Rf =0.36 (CHCl3/MeOH, 19/1);
MS (APCI, Pos. 20V) m/z=371 (M+H)+; IR(KBr)
3445, 2940, 2236, 1715, 1605, 1520, 1443, 1419, 1357,
1266, 1174, 1150, 1078, 1050, 1023, 984; 1H NMR
(300MHz, DMSO-d6) d 12.5–12.1 (br, 1H), 7.01–6.93
(m, 2H), 6.84 (d, J=1.8Hz, 1H), 4.73 (quint,
J=7.5Hz, 1H), 3.75 (s, 3H), 3.44–3.36 (m, 2H), 2.95–
2.86 (m, 2H), 2.46–2.33 (m, 2H), 2.11–1.96 (m, 4H),
1.83–1.55 (m, 4H), 1.21–1.15 (m, 2H), 0.92–0.86 (m,
2H); HRMS (EI) calcd for C21H26N2O4 370.1893.
Found 370.1882.
1H), 3.08–2.98 (m, 2H), 2.90–2.75 (m, 1H), 2.75–2.60
(m, 1H), 2.15–2.00 (m, 4H), 1.95–1.65 (m, 6H), 1.55–
1.45 (m, 2H), 1.43 (br d, J=6.6Hz, 3H); Optical rota-
30
tion ½aŠ þ 8:8 (c 0.37, DMSO); Anal. Found
D
C21H29N3O46/5HClÆ1/5H2NOH (C, H, N).
6.6.28.
(2S)-2-{4-Cyano-4-[3-(cyclopentyloxy)-4-meth-
oxyphenyl]piperidin-1-yl}-N-hydroxypropanamide hydro-
chloride (4b). The title compound was prepared from the
corresponding carboxylic acid according to Method K
to give a white powder: Yield 44% in two steps; TLC
Rf =0.45 (CHCl3/MeOH, 9/1); MS (MALDI, Pos.) m/
z=388 (M+H)+; IR(KBr) 3370, 3167, 2940, 2714,
2232, 1685, 1519, 1465, 1443, 1420, 1382, 1356, 1259,
1
1179, 1149, 1130, 1106, 1016; H NMR(300MHz, py-
ridine-d5+CDCl3) d 7.11 (d, J=2.1Hz, 1H), 7.02 (dd,
J=2.1, 8.7Hz, 1H), 6.88 (d, J=8.7Hz, 1H), 6.80–6.20
(m, 3H), 4.80–4.72 (m, 1H), 3.72 (s, 3H), 3.40 (br q,
J=6.9Hz, 1H), 3.14–3.02 (m, 2H), 3.00–2.88 (m, 1H),
2.82–2.70 (m, 1H), 2.20–2.05 (m, 4H), 1.95–1.65 (m,
6.6.26. 2-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphen-
yl]piperidin-1-yl}-N-hydroxyacetamide
hydrochloride
(2b) (Method K). To stirred solution of 2a
a
(239mg, 0.668mmol) in DMF (4.0mL) were added
EDC (192mg, 1.00mmol), HOBt (135mg, 1.00mmol)
6H), 1.55–1.45 (m, 2H), 1.41 (d, J=6.9Hz, 3H); Optical
30
D
and
(1-methoxy-1-methyethyl)oxyamine
(351mg,
rotation ½aŠ ꢀ 8:7 (c 0.15, DMSO); Anal. Found
3.34mmol). After being stirred at room temperature
for 3h, the mixture was poured into H2O, and extracted
with EtOAc. The organic layer was washed with H2O,
dried over MgSO4, and concentrated in vacuo. The res-
idue was purified by column chromatography on silica
gel (n-hexane/EtOAc, 1/1–0/1) to give 2c (289mg,
0.649mmol, 97%) as a pale yellow oil: TLC Rf =0.26
(EtOAc); 1H NMR(300MHz, CDCl 3) d 8.94 (br s,
1H), 7.05–6.90 (m, 2H), 6.87 (d, J=8.4Hz, 1H),
4.81 (m, 1H), 3.86 (s, 3H), 3.36 (s, 3H), 3.23 (s, 2H),
3.10–3.00 (m, 2H), 2.80–2.65 (m, 2H), 2.20–2.00
(m, 2H), 2.10–1.75 (m, 8H), 1.70–1.55 (m, 2H), 1.46 (s,
6H).
C21H29N3O46/5HClÆ1/5H2 NOH (C, H, N).
6.6.29. 2-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphen-
yl]piperidin-1-yl}-N-hydroxy-2-methylpropanamide hydro-
chloride (5b). The title compound was prepared from the
corresponding carboxylic acid according to Method K
to give a white powder: Yield 62% in two steps; TLC
Rf =0.38 (CHCl3/MeOH, 10/1); MS (FAB, Pos.) m/
z=402 (M+H)+; IR(KBr) 3423, 3235, 2959, 2871,
2709, 2564, 2237, 1732, 1677, 1604, 1590, 1524, 1465,
1446, 1374, 1328, 1307, 1274, 1248, 1218, 1151, 1123,
1063, 1028, 993; 1H NMR(300MHz, pyridine-
d5+CDCl3) d 7.14 (d, J=2.1Hz, 1H), 6.99 (dd, J=8.4,
2.1Hz, 1H), 6.91 (d, J=8.4Hz, 1H), 5.95 (br s, 3H),
4.72 (m, 1H), 3.73 (s, 3H), 3.10–3.00 (m, 2H), 2.80–
2.65 (m, 2H), 2.20–2.00 (m, 4H), 2.00–1.70 (m, 6H),
1.60–1.40 (m, 2H), 1.39 (s, 6H); Anal. Found
C22H31N3O4ÆHClÆH2O (C, H, N).
To a stirred solution of 2c (280mg, 0.629mmol) in
MeOH (3.0mL) was added 2N HCl (0.35mL,
0.692mmol). After being stirred at room temperature
for 1h, the reaction mixture was concentrated in vacuo.
The residue was triturated with i-Pr2O/MeOH to give 2b
(189mg, 0.462mmol, 73%) as a white powder: TLC
Rf =0.38 (CHCl3/MeOH, 10/1); MS (MALDI, Pos.) m/
z=374 (M+H)+; 1H NMR(300MHz, pyridine-
d5+CDCl3) d 7.09 (d, J=2.4Hz, 1H), 6.99 (dd, J=8.4,
2.4Hz, 1H), 6.88 (d, J=8.4Hz, 1H), 6.08 (br s, 3H),
4.75 (m, 1H), 3.72 (s, 3H), 3.30 (s, 2H), 3.02 (br d,
J=14.4Hz, 2H), 2.75–2.60 (m, 2H), 2.20–1.95 (m, 4H),
2.00–1.65 (m, 6H), 1.60–1.40 (m, 2H); Anal. Found
C20H26N2O4Æ3/7H2O (C, H, N).
6.6.30. 1-{4-Cyano-4-[3-(cyclopentyloxy)-4-methoxyphen-
yl]piperidin-1-yl}-N-hydroxycyclopropanecarboxamide hy-
drochloride (6b). The title compound was prepared from
the corresponding carboxylic acid according to Method
K to give a white powder: Yield 84% in two steps; TLC
Rf =0.45 (CHCl3/MeOH, 10/1); MS (MALDI, Pos.)
m/z=400 (M+H)+, 422 (M+Na)+; IR(KBr) 3437,
3198, 2960, 2871, 2642, 2600, 2553, 2240, 1731, 1662,
1519, 1444, 1419, 1361, 1334, 1305, 1257, 1171, 1151,
1
1133, 1055, 1027, 993; H NMR(300MHz, pyridine-
6.6.27. (2R)-2-{4-Cyano-4-[3-(cyclopentyloxy)-4-meth-
oxyphenyl]piperidin-1-yl}-N-hydroxypropanamide hydro-
chloride (3b). The title compound was prepared from the
corresponding carboxylic acid according to Method K
to give a white powder: Yield 75% in two steps; TLC
Rf =0.45 (CHCl3/MeOH, 9/1); MS (MALDI, Pos.) m/
z=388 (M+H)+; IR(KBr) 3371, 3164, 2939, 2674,
2232, 1665, 1519, 1465, 1443, 1420, 1382, 1355, 1259,
1179, 1149, 1130, 1106, 1067, 1016; 1H NMR
(300MHz, pyridine-d5+CDCl3) d 7.08–7.00 (m, 1H),
7.00–6.95 (m, 1H), 6.85 (d, J=8.7Hz, 1H), 5.95–5.50
(m, 3H), 4.80–4.72 (m, 1H), 3.73 (s, 3H), 3.38–3.25 (m,
d5+CDCl3) d 7.12 (d, J=2.4Hz, 1H), 6.99 (dd, J=8.4,
2.4Hz, 1H), 6.87 (d, J=8.4Hz, 1H), 6.90–6.00 (br,
3H), 4.75 (m, 1H), 3.73 (s, 3H), 3.00–2.90 (m, 2H),
2.90–2.70 (m, 2H), 2.20–2.00 (m, 4H), 2.00–1.60 (m,
6H), 1.60–1.40 (m, 2H), 1.35–1.25 (m, 2H), 1.10–1.00
(m, 2H); Anal. Found C22H29N3O4ÆHClÆH2O (C, H, N).
6.6.31. 1-[4-Cyano-4-(3-ethoxy-4-methoxyphenyl)piper-
idin-1-yl]-N-hydroxycyclopropanecarboxamide hydrochlo-
ride (7b). The title compound was prepared from the
corresponding carboxylic acid according to Method K

4662
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
to give a pale yellow powder: Yield 89% in two steps;
TLC Rf =0.42 (CHCl3/MeOH, 9/1); MS (FAB, Pos.)
m/z=360 (M+H)+; IR(KBr) 3439, 3138, 2980, 2907,
2385, 1656, 1523, 1446, 1419, 1386, 1264, 1251, 1173,
6.6.35. 2-[4-Cyano-4-(3-ethoxy-4-methoxyphenyl)piper-
idin-1-yl]-N-hydroxyacetamide hydrochloride (11b). The
title compound was prepared from the corresponding
carboxylic acid according to Method K to give a white
powder: Yield 64% in two steps; TLC Rf=0.15 (EtOAc);
MS (APCI, Neg. 20V) m/z=332 (MꢀH)ꢀ; IR(KBr)
3421, 3107, 2987, 2839, 2242, 1698, 1608, 1525, 1457,
1423, 1396, 1366, 1340, 1318, 1262, 1250, 1177, 1158,
1138, 1084, 1034; 1H NMR(300MHz, pyridine-
d5+CDCl3) d 8.30–7.00 (m, 5H), 6.93–6.87 (m, 1H),
3.95 (q, J=6.9Hz, 2H), 3.74 (s, 3H), 3.34 (s, 2H), 3.10–
3.00 (m, 2H), 2.75–2.60 (m, 2H), 2.15–1.95 (m, 4H),
1.33 (t, J=6.9Hz, 3H); Anal. Found C17H23N3O4ÆH-
ClÆ1/4H2O (C, H, N).
1
1151, 1129, 1043, 1025, 961; H NMR(300MHz, pyri-
dine-d5+CDCl3)
d
8.00–7.20 (br, 3H), 7.09 (d,
J=1.8Hz, 1H), 7.04 (dd, J=8.4, 1.8Hz, 1H), 6.89 (d,
J=8.4Hz, 1H), 3.91 (q, J=6.9Hz, 2H), 3.74 (s, 3H),
2.99–2.79 (m, 4H), 2.19–2.10 (m, 4H), 1.37–1.27 (m,
5H), 1.09–1.03 (m, 2H); Anal. Found C19H25N3O4ÆHCl
(C, H, N).
6.6.32. 1-{4-Cyano-4-[3-(cyclopropylmethoxy)-4-meth-
oxyphenyl]piperidin-1-yl}-N-hydroxycyclopropanecarbox-
amide hydrochloride (8b). The title compound was
prepared from the corresponding carboxylic acid
according to Method K to give a white powder: Yield
91% in two steps; TLC Rf =0.60 (CHCl3/MeOH, 9/1);
MS (APCI, Pos. 20V) m/z=386 (M+H)+; IR(KBr)
3432, 3127, 3005, 2872, 2632, 2593, 2538, 2411, 2389,
2238, 1654, 1605, 1592, 1521, 1467, 1444, 1421, 1409,
1360, 1331, 1251, 1176, 1153, 1135, 1051, 1024, 1010,
6.6.36. 2-{4-Cyano-4-[3-(cyclopropylmethoxy)-4-meth-
hydro-
oxyphenyl]piperidin-1-yl}-N-hydroxyacetamide
chloride (12b). The title compound was prepared from
the corresponding carboxylic acid according to Method
K to give a white powder: Yield 52% in two steps; TLC
Rf =0.31 (CHCl3/MeOH, 10/1); MS (MALDI, Pos.) m/
z=360 (M+H)+; IR(KBr) 3126, 3002, 2239, 1699,
1607, 1593, 1521, 1466, 1423, 1407, 1363, 1340, 1314,
1254, 1201, 1177, 1153, 1139, 1093, 1014, 966; 1H
NMR(300MHz, pyridine- d5+CDCl3) d 8.80–7.50 (br,
3H), 7.08 (d, J=2.4Hz, 1H), 7.02 (dd, J=8.4, 2.4Hz,
1H), 6.87 (d, J=8.4Hz, 1H), 3.83 (d, J=6.9Hz, 2H),
3.73 (s, 3H), 3.30 (s, 2H), 3.10–2.90 (m, 2H), 2.70–2.55
(m, 2H), 2.15–1.95 (m, 4H), 1.26 (m, 1H), 0.55–0.45
(m, 2H), 0.35–0.25 (m, 2H); Anal. Found
C19H25N3O4ÆHCl (C, H, N).
1
965; H NMR(300MHz, pyridine- d5+CDCl3) d 8.60–
6.80 (br, 3H), 7.15 (d, J=2.0Hz, 1H), 7.05 (dd, J=9.0,
2.0Hz, 1H), 6.91 (d, J=9.0Hz, 1H), 3.81 (d, J=6.9Hz,
2H), 3.73 (s, 3H), 2.99–2.79 (m, 4H), 2.20–2.02 (m,
4H), 1.37–1.31 (m, 2H), 1.31–1.20 (m, 1H), 1.08–1.03
(m, 2H), 0.55–0.47 (m, 2H), 0.32–0.26 (m, 2H); Anal.
Found C21H27N3O4ÆHCl (C, H, N).
6.6.33. 1-{4-Cyano-4-[3-(cyclobutyloxy)-4-methoxyphe-
nyl]piperidin-1-yl}-N-hydroxycyclopropanecarboxamide
hydrochloride (9b). The title compound was prepared
from the corresponding carboxylic acid according to
Method K to give a white powder: Yield 99% in two
steps; TLC Rf =0.64 (CHCl3/MeOH, 9/1); MS (APCI,
Pos.20V) m/z=386 (M+H)+; IR(KBr) 3450, 3099,
2983, 2878, 2632, 2590, 2505, 2412, 2233, 1650, 1593,
1521, 1464, 1448, 1419, 1384, 1352, 1262, 1249, 1189,
1171, 1151, 1134, 1080, 1053, 1042, 1025, 978; 1H
NMR(300MHz, pyridine- d5+CDCl3) d 8.00–7.10
(br, 3H), 7.04 (d, J=2.1Hz, 1H), 7.00 (dd, J=8.4,
2.1Hz, 1H), 6.90 (d, J=8.4Hz, 1H), 4.62 (quint,
J=7.5Hz, 1H), 3.75 (s, 3H), 3.00–2.80 (m, 4H), 2.42–
2.30 (m, 2H), 2.23–2.02 (m, 6H), 1.74–1.60 (m, 1H),
1.58–1.40 (m, 1H), 1.38–1.32 (m, 2H), 1.09–1.03 (m,
2H); HRMS (FAB) calcd for C21H28N3O4 386.2080.
Found 386.2072.
6.6.37. 2-[4-Cyano-4-(3-isopropoxy-4-methoxyphenyl)pip-
eridin-1-yl]-N-hydroxyacetamide hydrochloride (13b).
The title compound was prepared from the correspond-
ing carboxylic acid according to Method K to give a
white powder: Yield 25% in two steps; TLC Rf =0.22
(EtOAc); MS (APCI, Neg. 20V) m/z=346 (MꢀH)ꢀ;
IR(KBr) 3431, 3153, 2976, 2232, 1686, 1518, 1445,
1419, 1264, 1175, 1152, 1108, 1026; 1H NMR
(300MHz, pyridine-d5+CDCl3) d 7.10 (d, J=2.1Hz,
1H), 7.02 (dd, J=6.0, 2.1Hz, 1H), 6.88 (d, J=6.0Hz,
1H), 6.25–5.50 (m, 3H), 4.51 (sept, J=6.0Hz, 1H),
3.73 (s, 3H), 3.28 (s, 2H), 3.05–2.95 (m, 2H), 2.75–2.55
(m, 2H), 2.15–1.95 (m, 4H), 1.28 (d, J=6.0Hz, 6H);
Anal. Found C18H25N3O4ÆHClÆH2O (C, H, N).
6.6.38. 2-{4-Cyano-4-[3-(cyclobutyloxy)-4-methoxyphen-
yl]piperidin-1-yl}-N-hydroxyacetamide
6.6.34. 2-[4-Cyano-4-(3,4-dimethoxyphenyl)piperidin-1-
yl]-N-hydroxyacetamide hydrochloride (10b). The title
compound was prepared from the corresponding carb-
oxylic acid according to Method K to give a white pow-
der: Yield 42% in two steps; TLC Rf =0.21 (CHCl3/
MeOH, 10/1); MS (MALDI, Pos.) m/z=320 (M+H)+;
IR(KBr) 3431, 3120, 2999, 2959, 2841, 2244, 1701,
1609, 1523, 1469, 1445, 1415, 1365, 1340, 1320, 1263,
1249, 1161, 1140, 1091, 1023, 969; 1H NMR
(300MHz, pyridine-d5+CDCl3) d 7.05–6.95 (m, 2H),
6.85 (d, J=9.0Hz, 1H), 6.80–6.00 (br, 3H), 3.75 (s,
6H), 3.28 (s, 2H), 3.05–2.95 (m, 2H), 2.70–2.55 (m,
2H), 2.20–1.90 (m, 4H); HRMS (FAB) calcd for
C16H22N3O4 320.1610. Found 320.1627.
hydrochloride
(14b). The title compound was prepared from the corre-
sponding carboxylic acid according to Method K to
give a white powder: Yield 58% in two steps; TLC
Rf =0.20 (EtOAc); MS (MALDI, Pos.) m/z=360
(M+H)+; IR(KBr) 3393, 3171, 2939, 2654, 2571,
2242, 1697, 1521, 1443, 1420, 1355, 1261, 1175, 1152,
1137, 1074, 1024; 1H NMR(300MHz, pyridine-
d5+CDCl3) d 7.05–7.00 (m, 2H), 6.95–6.88 (m, 1H),
6.80–6.20 (m, 3H), 4.65 (quint, J=6.9Hz, 1H), 3.75
(s, 3H), 3.36 (s, 2H), 3.10–3.00 (m, 2H), 2.75–2.65
(m, 2H), 2.40–2.30 (m, 2H), 2.20–1.90 (m, 6H),
1.75–1.40 (m, 2H); Anal. Found C19H25N3O4ÆHCl (C,
H, N).

H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
4663
6.6.39. 2-{4-Cyano-4-[3-(cyclopentyloxy)-4-ethoxyphen-
yl]piperidin-1-yl}-N-hydroxyacetamide hydrochloride
tetraacetic acid. The substrate solution was mixed with
the enzyme solution containing a test compound dis-
solved in dimethylsulfoxide (DMSO), and incubation
was done for 30min at 30ꢁC. Assays were performed
in duplicate at three to four different concentrations of
each test compound, and the IC50 values were deter-
mined.
(16b). The title compound was prepared from the corre-
sponding carboxylic acid according to Method K to give
a white powder: Yield 58% in two steps; TLC Rf =0.36
(CHCl3/MeOH, 10/1); MS (FAB, Pos.) m/z=388
(M+H)+; IR(KBr) 3293, 3120, 2972, 2659, 2610, 2565,
2254, 1694, 1604, 1516, 1477, 1451, 1420, 1361, 1317,
1262, 1184, 1129, 1092, 1046, 975; 1H NMR
(300MHz, pyridine-d5+CDCl3) d 8.10–7.20 (br, 3H),
7.10 (d, J=2.1Hz, 1H), 7.00 (dd, J=8.4, 2.1Hz, 1H),
6.90 (d, J=8.4Hz, 1H), 4.76 (m, 1H), 3.97 (q,
J=6.9Hz, 2H), 3.31 (s, 2H), 3.10–2.95 (m, 2H), 2.75–
2.60 (m, 2H), 2.20–1.95 (m, 4H), 2.00–1.65 (m, 6H),
1.60–1.40 (m, 2H), 1.31 (t, J=6.9Hz, 3H); Anal. Found
C21H29N3O4ÆHCl (C, H, N).
6.8. Inhibition of LPS-induced plasma TNF-a production
in rats
Male Crj:CD(SD)IGS rats aged 6weeks (n=7) were
fasted overnight, and the test compounds (0.01–0.1mg/
10mL/kg) were administered orally at 1h before intrave-
nous injection of 1lg/kg of LPS (Escherichia coli Sero-
type 055 B5). The plasma TNF-a level was measured
with a commercially available ELISA kit (R&D Sys-
tems) at 90min after LPS challenge. The percent inhibi-
tion (the dosage required to inhibit plasma TNF-a
production by 50%) was determined by the following
formula:
6.6.40. 2-{4-Cyano-4-[3-(cyclopentyloxy)-4-(difluorometh-
oxy)phenyl]piperidin-1-yl}-N-hydroxyacetamide hydrochlo-
ride (17b). The title compound was prepared from the
corresponding carboxylic acid according to Method K
to give a white powder: Yield 81% in two steps; TLC
Rf =0.36 (CHCl3/MeOH, 10/1); MS (FAB, Pos.) m/
z=410 (M+H)+; IR(KBr) 3296, 3119, 2973, 2659,
2612, 2568, 2253, 1692, 1606, 1508, 1479, 1452, 1420,
1385, 1317, 1291, 1267, 1236, 1183, 1147, 1107, 1056,
%Inhibition ¼ 100 ꢀ ðC ꢀ SÞ=ðL ꢀ SÞ ꢁ 100
C: Plasma TNF-a concentration in LPS-treated animals
pretreated with a test compound. L: Plasma TNF-a con-
centration in LPS-treated animals pretreated with saline.
S: Plasma TNF-a concentration in saline-treated ani-
mals pretreated with saline.
1
1003, 976; H NMR(300MHz, pyridine- d5+CDCl3) d
7.23 (d, J=8.4Hz, 1H), 7.18 (d, J=1.8Hz, 1H), 6.97
(dd, J=8.4, 1.8Hz, 1H), 6.97 (t, J=75.0Hz, 1H), 6.60–
5.60 (br, 3H), 4.74 (m, 1H), 3.31 (s, 2H), 3.10–3.00 (m,
2H), 2.70–2.60 (m, 2H), 2.20–2.00 (m, 4H), 1.85–1.60
(m, 6H), 1.60–1.40 (m, 2H); Anal. Found
C20H25F2N3O4ÆHCl (C, H, N).
6.9. SRS-A mediated bronchoconstriction in guinea pigs
Male Hartley guinea pigs aged 7weeks (n=5) were
actively sensitized by intraperitoneal administration of
1mg of ovalbumin (OVA) containing 5·109 killed
Bordetella pertussis organisms on day 0. On day 14,
the bronchoconstrictor response was measured using a
modified version of the method of Konzett and Ro¨ssler.
Bronchoconstriction was induced by an intravenous
injection of OVA (0.15–0.5mg/kg). Sensitized animals
were treated with both a cyclooxygenase inhibitor (indo-
methacin at 5mg/kg i.v., 3min before OVA) and an anti-
histamine (pyrilamine at 1mg/kg i.v., 1min before OVA)
to ensure that endogenous SRS-A was solely responsible
for bronchoconstriction. Test compounds were adminis-
tered orally at 1h before antigen challenge. Bronchocon-
strictor response was measured for 15min and the result
was represented as the area under the curve (AUC 0–
15min).
6.6.41. 2-{4-Cyano-4-[3-(cyclopentyloxy)-4-isopropoxy-
phenyl]piperidin-1-yl}-N-hydroxyacetamide hydrochloride
(18b). The title compound was prepared from the corre-
sponding carboxylic acid according to Method K to give
a white powder: Yield 71% in two steps; TLC Rf =0.40
(CHCl3/MeOH, 9/1); MS (MALDI, Pos.) m/z=402
(M+H)+; IR(KBr) 3291, 3118, 2971, 2658, 2561, 2253,
1693, 1601, 1513, 1451, 1418, 1370, 1319, 1262, 1183,
1130, 1101, 1045; 1H NMR(300MHz, pyridine-
d5+CDCl3) d 7.15–7.10 (m, 1H), 7.05–6.90 (m, 2H),
5.80–5.35 (m, 3H), 4.78–4.72 (m, 1H), 4.46 (sept,
J=6.0Hz, 1H), 3.28 (s, 2H), 3.04–2.96 (m, 2H), 2.68–
2.58 (m, 2H), 2.15–1.95 (m, 4H), 1.95–1.65 (m, 6H),
1.58–1.45 (m, 2H), 1.28 (d, J=6.0Hz, 6H); Anal. Found
C22H31N3O4ÆHClÆ1/3H2O (C, H, N).
6.10. Gastric emptying in rats
6.7. Assay of human PDE4 activity
Male Sprague-Dawley rats were fasted overnight and
were orally administered test compounds or 0.5w/v%
methylcellulose (10mL/kg). In addition, 0.05mg/mL of
phenol red solution was orally administered in a volume
of 1.5mL at twenty minutes after dosing with the test
compounds. Forty minutes after administration of the
test compounds, both the cardia and pylorus of the
stomach were ligated under anesthesia with sodium
pentobarbital (75mg/kg, i.p.), and then the stomach
was isolated without leakage of phenol red. The stom-
ach was cut open and the phenol red solution was
drained into a beaker containing 100mL of 0.1N
The method of Reeves et al.32 was modified to isolate
phosphodiesterase type 4 isozyme (PDE4). The enzyme
was prepared from U937 cells derived from human
monocytes, and was stored at ꢀ20ꢁC after preparation.
Measurement of PDE4 activity was performed using this
stored enzyme after it was diluted with distilled water
containing bovine serum albumin. The substrate solu-
3
tion was prepared by adding H–cAMP (300,000dpm
(5000Bq)/assay) and 100lmol/L cAMP solution to
100mmol/L Tris–HCl (pH8.0) containing 5mmol/L eth-
ylene glycol-bis (b-aminoethyl ether) and N,N,N0,N0-

4664
H. Ochiai et al. / Bioorg. Med. Chem. 12 (2004) 4645–4665
2. Torphy, T. J. Am. J. Respir. Crit. Care Med. 1998, 157,
351.
3. Essayan, D. M. J. Allergy Clin. Immunol. 2001, 108, 671.
4. Soderling, S. H.; Beavo, J. A. Curr. Opin. Cell Biol. 2000,
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5. Teixeira, M. M.; Gristwood, R. W.; Cooper, N.; Helle-
well, P. G. Trends Pharmacol. Sci. 1997, 18, 164.
6. Dyke, H. J.; Montana, J. G. Exp. Opin. Invest. Drugs
2002, 11, 1.
7. Christensen, S. B.; Guider, A.; Forster, C. F.; Gleason, J.
G.; Bender, P. E.; Karponski, J. M.; Dewolf, W. E.;
Barnette, M. S.; Underwood, D. C.; Griswold, D. E.;
Cieslinski, L. B.; Burman, M.; Bochnowicz, S.; Osborn, R.
R.; Manning, C. D.; Grous, M.; Hillegas, L. M.; Bartus, J.
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Torphy, T. J. J. Med. Chem. 1998, 41, 821.
NaOH. Part of the solution was filtrated (pore size:
0.45lm) and the absorbance at 546nm was measured
to determine the amount of dye remaining in the stom-
ach. Then the gastric emptying rate was calculated by
the following formula:
Gastric emptying rate ¼ 100 ꢁ ð0:75 ꢀ concentration of
dye in the stomachÞ=0:75
A value of 0.75lg/mL was equal to a concentration of
0.05mg/mL, which was achieved by adding 1.5mL of
phenol red to 100mL of 0.1N NaOH. The 50% inhibi-
tion rate for gastric emptying by the test compounds
was calculated by defining gastric emptying after vehicle
administration as 100%.
8. Schneider, H. H.; Schmiechen, R.; Brezinski, M.; Seidler,
J. Eur. J. Pharmacol. 1986, 127, 105.
6.11. Inhibitory activity on LPS-induced TNF-a produc-
tion in human whole blood
9. Duplantier, A. J.; Biggers, M. S.; Chambers, R. J.; Cheng,
J. B.; Cooper, K.; Damon, D. B.; Eggler, J. F.; Kraus, K.
G.; Marfat, A.; Masamune, H.; Pillar, J. S.; Shirley, J. T.;
Umland, J. P.; Watson, J. W. J. Med. Chem. 1996, 39, 120.
10. Hughes, B.; Owens, R.; Perry, M.; Warvellow, G.; Allen,
R. Drug Discov. Today 1997, 2, 89.
11. Kleinman, E. F.; Campbell, E.; Giordano, L. A.; Cohan,
V. L.; Jenkinson, T. H.; Cheng, J. B.; Shirley, J. T.;
Pettipher, E. R.; Salter, E. D.; Hibbs, T. A.; Dicapua, F.
M.; Bordner, J. J. Med. Chem. 1998, 41, 266.
12. Freyne, E. J.; Diels, G. S.; Matesanz-Ballesteros, M. E.;
Diaz-Martinez, A. WO 9950262, 1999.
13. Hersperger, R.; Bray-French, K.; Mazzoni, L.; Muller, T.
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15. Giembycz, M. A. Expert Opin. Invest. Drugs 2001, 10,
1361.
Under the supervision of a physician, blood was col-
lected into a heparinized tube (final concentration:
10U/mL heparin sodium) from a forearm vein in three
healthy male donors. A solution of the test compound
(10lL) dissolved in DMSO was added to 180lL of
whole blood, and the mixture was pre-incubated for
30min at 37ꢁC. Then 10lL of 2lg/mL of LPS was
added and incubated for 6h at 37ꢁC, after which the
plasma TNF-a concentration was measured with a
human TNF-a ELISA kit (DIACLONE). Assays were
performed in duplicate at three to four different
concentrations of each test compound, and the IC50 val-
ues were determined.
16. Dyke, H. J.; Montana, J. G. Expert Opin. Invest. Drugs
1999, 8, 1301.
6.12. Ferret emetic study
17. Ajay; Bemis, G. W.; Murcko, M. A. J. Med. Chem. 1999,
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Male ferrets (weighting about 1.2kg) were fasted over-
night and test compounds were administered orally.
Their behavior was observed throughout a 1h period
after gavage. Results were expressed as the number of
animals that vomited relative to the animals tested.
20. Mickelson, J. W.; Belonga, K. L.; Jacobsen, E. J. J. Org.
Chem. 1995, 60, 4177.
21. Verardo, G.; Giumanini, A. G.; Favret, G.; Strazzolini, P.
Synthesis 1991, 6, 447.
6.13. Pharmacokinetic study
22. Torphy, T. J.; Zhou, H.; Cieslinski, L. B. J. Pharmacol.
Exp. Ther. 1992, 263, 1195.
Pharmacokinetic parameters were determined in rats
after intravenous (3mg/mL/kg, iv) or oral (10mg/5mL/
kg, po) administration. Rats (n=3) were given the test
compound intravenously in 20% HP-d-CD solution, or
orally in 0.5% methylcelluslose suspension. Blood sam-
ples were collected from jugular veins into a heparinized
syringe at 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0h after dosing.
Plasma samples were analyzed after extraction of the
test compounds by simple liquid–liquid extraction. LC/
MS/MS analysis was performed with a Quattro II
(Micromass Co., Ltd). HPLC was carried out using
a HP1100 (Agilent Co., Ltd) apparatus equipped with
YMC-Pack Pro C18 (2.1·150mm, YMC Corporation).
23. Tracey, K. J.; Cerami, A. Annu. Rev. Med. 1994, 45, 491.
24. Cheng, J. B.; Cooper, K.; Duplantier, A. J.; Eggler, J. F.;
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