Orally active PDE4 inhibitor with therapeutic potential

Article abstract of DOI:10.1016/j.ejmech.2004.02.010

Based on the promising results obtained by the clinical trial of Ariflo, further optimization of the spatial arrangement of the three pharmacophores (the carboxylic acid moiety, nitrile moiety and 3-cyclopentyloxy-4-methoxyphenyl moiety) in the

Full text of DOI:10.1016/j.ejmech.2004.02.010

European Journal of Medicinal Chemistry 39 (2004) 555–571
www.elsevier.com/locate/ejmech
Original article
Orally active PDE4 inhibitor with therapeutic potential
Hiroshi Ochiai a, Tazumi Ohtani a, Akiharu Ishida a, Katuya Kishikawa b, SusumuYamamoto a,
Hiroshi Takeda a, Takaaki Obata a, Hisao Nakai a,*, Masaaki Toda a
a Medicinal Chemistry Research Laboratories, Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto,
Mishima, Osaka 618-8585, Japan
b Development Planning, Ono Pharmaceutical Co., Ltd., 2-1-5 Doshomachi, Chuo-ku, Osaka 541-8526, Japan
Received 2 September 2003; received in revised form 29 January 2004; accepted 12 February 2004
Available online 28 May 2004
Abstract
Based on the promising results obtained by the clinical trial of Ariflo™, further optimization of the spatial arrangement of the three
pharmacophores (the carboxylic acid moiety, nitrile moiety and 3-cyclopentyloxy-4-methoxyphenyl moiety) in the structure of Ariflo 1 was
attempted using a bicyclo[3·3·0]octane template with more stereochemical diversity than the cyclohexane template of Ariflo 1. Biological
evaluation of the decyanated analogs and further optimization of the cyclopentyloxy moiety of 2a–b were also performed. Among the
compounds tested, 2a, 7a–b and 12a were found to be orally active and were estimated to have therapeutic potential based on cross-species and
same-species comparisons. The structure–activity relationships (SARs) of these compounds were investigated and pharmacokinetic data for
2a and 7b were also obtained by single-dose studies in rats.
© 2004 Elsevier SAS. All rights reserved.
Keywords: PDE4 inhibitor; Bicyclo[3·3·0]octane; Spatial arrangement; Three pharmacophores; Orally active
1. Introduction
Phosphodiesterase type 4 enzyme (PDE4) [1,2] is an en-
zyme involved in the intracellular degradation of cyclic ad-
enosine monophosphate (cAMP) to produce the correspond-
ing 5′-monophosphate, and it has received considerable
attention as a molecular target for the treatment of asthma
and other inflammatory diseases [3,4]. Previous studies in
this field have focused on asthma and chronic obstructive
pulmonary disease (COPD), with numerous inhibitors being
reported that have reached clinical trials [5,6]. However,
clinical use of these pioneer compounds has been limited by
side effects such as nausea, emesis and increased gastric acid
secretion. In order to obtain more useful PDE4 inhibitors
with fewer side effects, two approaches have been attempted.
The first is to design compounds with a reduced affinity for
Fig. 1. Structures of Ariflo, rolipram and new inhibitors.
the so-called high affinity rolipram-binding site (HPDE4) of
PDE4 and a high affinity for its catalytic domain (LPDE4)
[7,8]. Ariflo 1 (Fig. 1) has been reported to be 75-fold more
selective than (R)-rolipram with respect to LPDE4 activity
[9], and has shown efficacy with an improved safety margin
in clinical assessment. The second approach is to design
subtype-selective inhibitors [10–12]. Ariflo 1 is an orally
active second-generation PDE4 inhibitor that is reported to
be PDE4D-selective.
The importance of optimized spatial arrangement of the
carboxylic acid moiety relative to the 3-cyclopentyloxy-4-
methoxyphenyl and nitrile moieties on the cyclohexane ring
is illustrated by comparison of Ariflo 1 (cis-isomer) with its
* Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-9314.
E-mail address: hi.nakai@ono.co.jp (H. Nakai).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.02.010

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H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
Fig. 2. Stereochemical diversity of bicyclo[3·3·0]octane template.
converted to the carboxylic acid analogues 2a and 3, respec-
tively. The stereochemistry of 2a and 3 was determined using
X-ray crystallography, as described in Fig. 6. Details were
also given in Section 5. Condensation of 2a with O-protected
hydroxylamine followed by acidic deprotection provided a
hydroxamic acid 2b. According to the procedure described
above, 24b was converted to 25c and 25d, which were trans-
formed to 4a and 5, respectively.
The synthesis of 6–15 is outlined in Fig. 7. The enol
triflate 30 was prepared from a ketone 29a, which was de-
rived from 3-benzyloxy-4-methoxybenzyl cyanide 26 ac-
cording to the same procedure as described for the prepara-
tion of 23a from 20. Palladium-catalyzed insertion of carbon
monoxide into 30 in the presence of methanol gave 31, after
which catalytic hydrogenation from less hindered side af-
forded a single isomer 32. O-Alkylation of 32 by the conven-
tional method gave 33–42, alkaline hydrolysis of which pro-
duced 6a–15a, respectively. Condensation of 6a–15a with an
O-protected hydroxylamine, followed by acidic deprotec-
tion, yielded the corresponding hydroxamic acids 6b–15b,
respectively. Synthesis of the decyanated analogs 2c and 4b
will be reported elsewhere.
Fig. 3. Stereochemical diversity of cyclohexane template.
trans-isomer [9]. A more restricted spatial arrangement of
these three moieties within their optimized stereochemistry
using another template instead of the cyclohexane ring was
predicted to lead to the discovery of a structurally new inhibi-
tor with improved therapeutic potential. Synthesis and evalu-
ation of a series of bicyclo[3·3·0]octane derivatives was car-
ried out with the expectation of increasing the therapeutic
potential because the bicyclo[3·3·0]octane template provides
more stereochemical diversity (Fig. 2 four stereoisomers
A–D) and it was expected that derivatives based on the
bicyclo[3·3·0]octane template might provide a more optimal
structure than Ariflo 1 and show stronger PDE4 inhibition,
higher LPDE4 selectivity, and better subtype selectivity
compared with the corresponding cyclohexane derivatives
(Fig. 3 two stereoisomers E and F).
3. Results and discussion
Fig.1 shows the configuration of the above-mentioned
three pharmacophores on a new template, the bicyclo[3·3·0]
octane ring, which achieved an increase of activity with an
improved side effect profile.
All four possible isomers 2–5 were synthesized and evalu-
ated, as shown in Table 1. Compounds 2a–b and 3, in which
the aromatic moiety was located outside the concave
bicyclo[3·3·0]octane framework, showed potent inhibition of
PDE4. The LPDE4 inhibitory activity of 2a–b was equipo-
tent with that of Ariflo 1, while that of 3 was 15-fold weaker
than that of 1. Compounds 4 and 5, with the aromatic moiety
located inside the concave framework, showed no LPDE4
inhibitory activity at a concentration of 300 nM.
The carboxylic acid group was preferably oriented in the
syn-direction of the nitrile and located inside the concave
molecule, as illustrated by the greater potency of 2a over 3.
Thus, these results showed that the PDE4 enzyme might
recognize the more accessible aromatic moiety first, while
favoring more hindered carboxylic acid and nitrile groups.
Inhibition of LPS-induced TNF-a production in rats was
evaluated using 2a–b and 3. The in vivo activity of 2a was
fourfold more potent than that of 1, while both 2b and 3 were
less potent than 1. Compound 2a was nearly 10-fold more
potent than 3 (based on ID50 values) for inhibition of LPS-
2. Chemistry
As outlined in Fig. 4, analogues 2–5 were prepared from a
benzylcyanide 20 [9] by the following synthetic pathway.
Dialkylation of 20 with cis-4,5-bis(bromomethy)cyclohex-1-
ene 19, which was prepared from 16 by three steps as de-
scribed in Fig. 4, provided 21 at a good yield. Ozonolysis of
21, followed by oxidation and then esterification, gave the
diester 22. Dieckmann condensation of 22, followed by
demethoxycarbonylation, produced two separable diastere-
oisomers 23a and 23b, which were converted to the ketene-
dithioacetals 24a and 24b, respectively. The stereochemistry
of 23a and 23b was determined based on X-ray crystallogra-
phy (Fig. 5). Details are given in Section 5. Deprotection of
24a yielded two separable isomers 25a and 25b, which were

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
557
Fig. 4. Synthesis of compounds 2–5. (a) LiAlH4, THF, 0 °C; (b) MsCl, Et3N, CH2Cl2, –78 °C; (c) LiBr, DMF, 100 °C; (d) 19, NaHMDS, THF, –78 °C; (e) O3,
CH2Cl2 then PPh3, –78 °C; (f) NaClO2, NaH2PO4, t-BuOH, H2O, 2-methyl-2-butene; (g) CH2N2, Et2O, 0 °C; (h) NaH, DME, reflux; (i) NaCl, DMSO, H2O,
165 °C; (j) 2-trimethylsilyl-1, 3-dithiane, n-BuLi, THF, –78 °C; (k) TFA, 30% H2O2aq, CH3CN, H2O 80 °C, then 2 N NaOHaq 40 °C; (l) CH2N2, Et2O 0 °C;
(m) 2 N KOHaq, THF, MeOH; (n) NH2OC(CH3)2OMe, EDC, HOBt, DMF then MeOH, 1 N HClaq.
induced TNF-a production in rats, as predicted from their in
vitro SAR.
two different sets of SAR for the cyanated and decyanated
analogs.
PDE4 is reported to be inhibited by various hydroxamic
acid derivatives [13,14]. This led us to the synthesis of a
series of hydroxamic acid analogs that were expected to
show increased activity via strong metal-mediated interac-
tions with the enzyme. Conversion of the carboxylic acid
moiety of 2a to a hydroxamic acid afforded 2b, which re-
tained PDE4 inhibitory activity, but the new hydroxamic acid
moiety did not markedly increase the inhibition of PDE4
contrary to our expectations. In addition, the inhibition of
LPS-induced TNF-a production in rats by 2b was markedly
decreased relative to the effect of 2a, probably because of
pharmacodynamic factors such as the low oral availability of
the hydroxamic acid analog.
To assess the influence of the benzylic nitrile moiety on
LPDE4 inhibitory activity, decyanated analogs 2c and 4b
were also synthesized and evaluated. Removal of the ben-
zylic nitrile of 2a produced 2c, with nearly 17-fold reduction
of LPDE4 inhibitory activity, while 2c showed 79% inhibi-
tion of LPS-induced TNF-a production at an oral dose of
3 mg/kg. Although 4a showed less than 50% LPDE4 inhibi-
tory activity at 300 nM, compound 4b without the benzylic
nitrile moiety had an IC50 value of 80 nM and an ID50 value
of 2.2 mg/kg, po. Based on these findings, there seemed to be
The benzylic nitrile moiety seemed to increase the LPDE4
inhibitory activity in conversion from 2c to 2a, but in the
corresponding conversion from 4b to 4a it seemed to have a
deleterious effect. More detailed study of SAR, including the
role played by the benzylic nitrile moiety, is currently under-
way and will be reported in due course.
Further structural optimization of the cyclopentyl moiety
of 2a, which exhibited the most potent activity among the
compounds listed in Table 1, was carried out as shown in
Table 2. Replacement of the cyclopentyl moiety of 2a with
methyl, ethyl and isopropyl groups produced 6a, 7a, and 8a,
respectively, which all showed weaker inhibition of LPDE4.
Replacement of the cyclopentyl moiety of 2a with cyclobutyl
or cyclohexyl moieties afforded 9a and 10a, respectively,
which had fourfold and ninefold less potent LPDE4 inhibi-
tory activity. Introduction of ether oxygen into the cyclo-
hexyl moiety of 10a gave 11a, which showed nearly three-
fold weaker LPDE4 inhibitory activity because of the
presumed lower affinity of the hydrophilic moiety. Replace-
ment of the cyclopentyl moiety of 2a with an 2-isoindanyl
group resulted in 12a, which showed nearly sevenfold stron-
ger LPDE4 inhibitory activity, but was almost fourfold less
potent at inhibiting LPS-induced TNF-a production, presum-

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H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
23a
2a
3
Fig. 6. X-ray crystallography of 2a and 3.
The asymmetric unit of crystal of 3 contained two molecules of 3 and a
molecule of isopropanol, while the asymmetric unit of crystal of 2a contai-
ned two molecules of 2a and a molecule of methanol. The solvent molecules
found in both of the units were refined with isotropic temperature parame-
ters, but H atoms were not assigned.
23b
Fig. 5. X-ray crystallography of 23a, 23b.
TNF-a production, was carried out as shown in Table 3.
These compounds were evaluated for inhibition of slow re-
acting substance of anaphylaxis (SRS-A)-mediated bron-
choconstriction [15,16] (a desirable effect) and for inhibition
of gastric emptying [11] (an adverse effect) in rats. The
results were expressed as ID50 values, i.e., the dose that
caused 50% inhibition relative to the vehicle. These com-
pounds were also evaluated for inhibition of LPS-induced
TNF-a production in human whole blood (HWB) [17] to
predict their clinical potential. Results were expressed as
IC50 values, i.e., the test compound concentration that caused
50% inhibition relative to the vehicle.
The potency of these compounds for inhibiting SRS-A-
mediated bronchoconstriction in actively sensitized guinea
pigs was not always consistent with their activity against
LPS-induced TNF-a production in rats, probably because of
differences in pharmacokinetics, the disease models, to the
species studied. Compounds 1, 2a and 7b were evaluated for
their ability to inhibit SRS-A-mediated bronchoconstriction
in guinea pigs challenged by intravenous administration of
OVA (0.15 mg/kg). Compound 2a was nearly twofold less
ably because of pharmacodynamic problems. Replacement
of the cyclopentyl moiety of 2a with a cyclopropylmethyl
group produced 13a, which showed 11-fold weaker LPDE4
inhibitory activity. Replacement of the cyclopentyl moiety of
2a with 4-methylthiobutyl or 3,3,3-trifluoropropyl moieties
afforded 14a and 15a, respectively, which showed fourfold
and eightfold lower LPDE4 inhibitory activity. Both of these
analogs failed to inhibit LPS-induced TNF-a production in
rats at an oral dose of 3 mg/kg. Based on the results of the
LPS-induced TNF-a production assay, the most optimal
structure was 2a among all the tested carboxylic acid ana-
logs. The corresponding hydroxamic acid analogs 6b–15b
were also synthesized and evaluated. Each of these com-
pounds showed stronger LPDE4 inhibitory activity relative
to their corresponding carboxylic acid analogs. However, the
in vivo potency of 12b with the strongest LPDE4 inhibitory
activity did not increase as expected, presumably because of
pharmacodynamic problems. This was also true for the in
vivo potencies of 8b and 14b.
Further evaluation of compounds 2a, 7a–b and 12a,
which were selected on the basis of inhibition of in vivo

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
559
Fig. 7. Synthesis of compounds 6–15. (a) NaHMDS, THF, –78 °C; (b) O3, CH2Cl2 then PPh3, –78 °C; (c) NaClO2, NaH2PO4, t-BuOH, H2O, 2-methyl-2-butene;
(d) CH2N2, Et2O, 0 °C; (e) NaH, DME, reflux; (f) NaCl, DMSO, H2O, 165 °C; (g) LiHMDS, PhNTf2, THF, –78 °C – rt; (h) Pd(OAc)2, Et3N, PPh3, CO, MeOH,
DMF; (i) H2, Pd/C, MeOH; (j) RX, K2CO3, DMF or ROH, RCON=NCOR, PPh3, CH2Cl2; (k) 1 N NaOHaq, MeOH, THF; (l) EDC, HOBt, Et3N,
NH2OC(CH3)2OMe, DMF then 1 N HClaq., MeOH.
potent at inhibiting this type of SRS-A-mediated bronchoc-
onstriction in guinea pigs than Ariflo 1, although 2a was
fourfold stronger for inhibition of LPS-induced TNF-a pro-
duction in rats. Compound 7b showed nearly the same po-
tency as Ariflo 1 in both animal models. Compounds 7a, 7b
and 12a were also evaluated for their ability to inhibit SRS-
A-mediated bronchoconstriction after challenge by a higher
dose of OVA (0.5 mg/kg, iv). Compound 7a failed to inhibit
bronchoconstriction despite a high oral dose of 30 mg/kg
while 7b achieved 49% inhibition at an oral dose of
10 mg/kg. Compound 12a showed 50% inhibition of this
type of SRS-A-mediated bronchoconstriction at an oral dose
of 5.9 mg/kg. Thus, the relative potency of these compounds
against SRS-A-mediated bronchoconstriction in OVA-
sensitized guinea pigs was 12a > 7b, 1 > 2a, 7a.
To assess safety within a single species, inhibition of
gastric emptying by 2a, 7a–b and 12a was evaluated. After
oral dosing, compound 2a caused 73% inhibition of gastric
emptying at 10 mg/kg, while 7a and 7b had ID50 values of
17 and 12 mg/kg, respectively. The oral doses of 7b and 12a
that inhibited gastric emptying were higher than those having
a desirable effect (inhibition of LPS-induced TNF-a produc-
tion in rats). Based on their greater potency than that ofAriflo
1 for blocking LPS-induced TNF-a production in HWB,
7a–b and 12a were estimated to have a greater therapeutic
potential and an improved side effect profile.
tic potential of 2a and Ariflo 1 seemed to be similar. To
evaluate the side effect profile by cross-species comparison,
compounds 2a, 7a–b and 12a were also tested in a ferret
emesis model. All of these compounds did not cause emesis
at oral doses of up to 3 mg/kg, while most caused emesis at an
oral dose of 10 mg/kg.
Pharmacokinetic data for compounds 2a and 7b obtained
after single-dose administration to rats are presented as typi-
cal examples of these derivatives in Table 4. Intravenous
administration of compounds 2a and 7b to rats (3 mg/kg,
n = 3) resulted in increased and decreased T1/2 values of
5.6 and 0.47 h, respectively. The volume of distribution at
steady state (Vss) was calculated to be 1190 and 647 ml/kg,
respectively. Systemic clearance (CL) was 216 and 1953
ml/h/kg, respectively. The area under the concentration vs.
time curve (AUC) was 14.6 and 1.55 µg h/ml, respectively.
Oral administration of 2a and 7b to rats (10 mg/kg, n = 3)
yielded a longer and shorter T1/2 of 8.1 and 4.2 h, respec-
tively. Tmax was 2.8 and 0.50 h, respectively, while the AUC
was 48.4 and 0.296 µg h/ml, respectively. The bioavailability
(BA) of these compounds was calculated to be 99.3% and
5.7%, respectively. Despite its much lower BA (5.7%), 7b
demonstrated a relatively higher efficacy in both of the in
vivo models. A contribution of 7a, which could be produced
by in vivo hydrolysis, was presumed to be involved in this
improved efficacy.
Compound 2a was nearly fourfold more potent for inhibi-
tion of LPS-induced TNF-a production in rats, while it was
nearly twofold less potent and slightly less potent for inhibi-
tion of bronchoconstriction in guinea pigs and inhibition of
TNF-a production in HWB, respectively. Thus, the therapeu-
4. Conclusion
Based on the hypothesis that more rigid spatial fixing of
the three pharmacophores (the carboxylic acid, nitrile, and

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Table 1
aromatic moieties) of Ariflo 1 within their optimized stere-
ochemistry using another template could lead to the discov-
ery of a new inhibitor, the design, synthesis, and evaluation of
bicyclo[3·3·0]octane derivatives showing more stereochemi-
cal diversity than the cyclohexane derivatives was carried
out.Among the compounds tested, compounds 2a, 7a–b, and
12a were estimated to have improved therapeutic potential
with few side effects based on biological data obtained by
both cross-species and same-species comparison. Single-
dose rat pharmacokinetic data for 2a and 7b were also deter-
mined. Synthesis and more detailed SAR will be reported
elsewhere.
MERCURY-300 spectrometer. The chemical shift values are
reported in ppm (d) and coupling constants (J) in Hertz (Hz).
Fast atom bombardment (FAB) and electron ionisation (EI)
mass spectra were obtained with a JEOL JMS-DX303HF or
JMS-700 spectrometer. Atmospheric pressure chemical ioni-
sation (APCI) mass spectra were determined by a Hitachi
M-1200H spectrometer. IR spectra were measured using a
Perkin-Elmer FTIR 1760X or JASCO FTIR-430 spectrom-
eter. 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 chromatography was
carried out using silica gel (Merck silica gel 60 (0.063–
0.200 mm), Wako Gel C200, Fuji Silysia FL60D, or Fuji
Silysia BW-235). TLC was also performed on silica gel
(Merck TLC plate, silica gel 60 F254).
5. Experimental
5.1. Chemistry
5.1.2. Preparation of compounds 23a and 23b
5.1.1. General procedure
Analytical samples were homogeneous as confirmed by
thin-layer chromatography (TLC), and yielded spectroscopic
data consistent with the assigned structures. All H NMR
5.1.2.1. (1R,2S)-Cyclohex-4-ene-1,2-diyldimethanol (17).
To a stirred suspension of LiAlH4 (37 g, 990 mmol) in THF
(1.0 l) was added cis-1,2,3,6-tetrahydrophthalic anhydride
16 (60.0 g, 394 mmol) at 0 °C. After being stirred for 2.5 h,
1
spectra were obtained with a Varian Gemini-200 or

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
561
Table 2
the reaction mixture was quenched with MeOH and poured
into saturated aq Na2SO4. The resulting solid was removed
by filtration and washed with EtOAc. The filtrate was diluted
with brine and extracted with EtOAc. The organic layer was
dried over anhydrous MgSO4 and evaporated to give 17
(55.4 g, 390 mmol, 99%) as a yellow oil: TLC Rf = 0.36
diluted with brine, and extracted with EtOAc. The organic
layer was dried over anhydrous MgSO4, and concentrated in
vacuo to give 18 (18.9 g, quant) as a pale yellow oil: TLC
Rf = 0.18 (n-hexane/EtOAc, 2/1); 1H NMR (300 MHz,
CDCl3) d 5.67 (t, J = 1.5 Hz, 2H), 4.29 (dd, J = 10.2 and
7.2 Hz, 2H), 4.17 (dd, J = 10.2 and 7.2 Hz, 2H), 3.04 (s, 6H),
2.50–2.35 (m, 2H), 2.35–2.15 (m, 2H), 2.10–1.90 (m, 2H).
1
(EtOAc); MS (APCI, Pos. 20 V) m/z = 143 (M + H)+; H
NMR (300 MHz, CDCl3) d 5.62 (brt, 2H), 3.74 (dd,
J = 10.8 and 6.6 Hz, 2H), 3.61 (dd, J = 10.8 and 3.0 Hz, 2H),
2.40–2.00 (m, 8H).
5.1.2.3. (4R,5S)-4,5-Bis(bromomethyl)cyclohexene (19).
The following reaction was carried out under argon atmo-
sphere. To a stirred solution of 18 (18.9 g) in DMF (150 ml)
was added LiBr (17 g, 190 mmol). After being stirred at
100 °C for 2.5 h, the reaction mixture was diluted with
H2O/Et2O. The resulting solid was removed by filtration
through a pad of celite, and washed with Et2O. The filtrate
was washed with H2O and brine, dried over anhydrous
5.1.2.2. ((1R,6S)-6-{[(Methylsulfonyl)oxy]methyl}cyclohex-
3-en-1-yl)methyl methanesulfonate (18). To a stirred solu-
tion of 17 (8.98 g, 63.2 mmol) and Et3N (26 ml, 19 mmol) in
CH2Cl2 (100 ml) was added MsCl (15 ml, 190 mmol) at
–78 °C. After being stirred for 1 h, the reaction mixture was

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H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
Table 3
Biological profile of 1, 2a, 7a, 7b and 12a
Table 4
Single-dose pharmacokinetic data for compounds 2a and 7b
MgSO4, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (n-hexane) to give
19 (6.09 g, 22.9 mmol, 36% in two steps) as a pale yellow oil:
TLC Rf = 0.76 (n-hexane/EtOAc, 8/1); 1H NMR (300 MHz,
CDCl3) d 5.63 (t, J = 1.2 Hz, 2H), 3.41 (dd, J = 10.2 and
5.4 Hz, 2H), 3.32 (dd, J = 10.2 and 8.4 Hz, 2H), 2.45–2.30
(m, 2H), 2.35–2.20 (m, 2H), 2.15–2.00 (m, 2H).
5.1.2.4. A mixture (21) of (2r,3aR,7aS)-2-[3-(cyclopentyl-
oxy)-4-methoxyphenyl]-2,3,3a,4,7,7a-hexahydro-1H-indene-
2-carbonitrile and (2s,3aR,7aS)-2-[3-(cyclopentyloxy)-4-
methoxyphenyl]-2,3,3a,4,7,7a-hexahydro-1H-indene-2-carbo-
nitrile. The following reaction was carried out under argon
atmosphere. To a stirred solution of 2-(3-cyclopentyloxy-4-
methoxyphenyl)acetonitrile 20 (1.3 g, 5.4 mmol) in THF

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
563
(20 ml) was added NaHMDS (1.0 M solution in THF, 12 ml,
12 mmol) at –78 °C. The reaction mixture was stirred at
–78 °C for 20 min, and a solution of 19 (799 mg, 3.00 mmol)
in THF (10 ml) was added dropwise. After being stirred at
room temperature for 2 h, the resulting mixture was diluted
with brine, and extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous MgSO4, and con-
centrated in vacuo. The residue was purified by column
chromatography on silica gel (n-hexane/EtOAc, 10/1) to give
an isomeric mixture 21 (725 mg, 2.15 mmol, 72%; major:mi-
nor = 3:1) as a pale yellow oil: TLC Rf = 0.64 (n-
stirred solution of 22 (2.66 g, 6.20 mmol) in DME (50 ml)
was added NaH (60% in mineral oil, 1.5 g, 37 mmol). After
heating under reflux for 1.5 h, the reaction mixture was
neutralized with 2 N aq HCl (19 ml), diluted with H2O, and
extracted with Et2O. The organic layer was dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was
used for the next reaction without further purification: TLC
Rf = 0.61 and 0.53 (n-hexane/EtOAc, 2/1).
To a stirred solution of the above-described residue in
DMSO (50 ml) and H2O (5.0 ml) was added NaCl (3.0 g).
After being stirred at 165 °C for 1 h, the reaction mixture was
diluted with H2O, and extracted with n-hexane/EtOAc. The
organic layer was dried over anhydrous MgSO4, concen-
trated in vacuo, and purified by column chromatography on
silica gel (n-hexane/EtOAc, 4/1) to give 23a (1.00 g,
2.95 mmol, 48% in two steps) and 23b (469 mg, 1.38 mmol,
22% in two steps). Compound 23a was obtained as a pale
yellow powder: TLC Rf = 0.29 (n-hexane/EtOAc, 2/1); MS
1
hexane/EtOAc, 4/1); H NMR (300 MHz, CDCl3) d 7.00–
6.90 (m, 2H), 6.85–6.80 (m, 1H), 5.83 & 5.75 (m, 2H), 4.81
(m, 1H), 3.84 & 3.84 (s, 3H), 2.70–2.20 (m, 8H), 2.20–2.00
(m, 2H), 2.00–1.70 (m, 6H), 1.70–1.50 (m, 2H).
5.1.2.5. An isomeric mixture (22) of dimethyl 2,2′-{(1R,
2S,4s)-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]
1
(EI, Pos.) m/z = 339 (M)+; H NMR (300 MHz, CDCl3) d
cyclopentane-1,2-diyl}diacetate and dimethyl 2,2′-{(1R
2S,4r)-4-cyano-4-[3-(cyclopentyloxy)-4-methoxy-phenyl]
,
6.95 (d, J = 2.1 Hz, 1H), 6.92 (dd, J = 8.4, 2.1 Hz, 1H), 6.84
(d, J = 8.4 Hz, 1H), 4.81 (m, 1H), 3.85 (s, 3H), 3.00–2.85 (m,
2H), 2.75–2.55 (m, 4H), 2.40–2.20 (m, 4H), 2.00–1.75 (m,
6H), 1.75–1.55 (m, 2H). Compound 23b was obtained as a
pale yellow powder: TLC Rf = 0.36 (n-hexane/EtOAc, 2/1);
MS (EI, Pos.) m/z = 339 (M)+; 1H NMR (300 MHz, CDCl3)
d 6.95–6.90 (m, 2H), 6.84 (d, J = 9.0 Hz, 1H), 4.79 (m, 1H),
3.85 (s, 3H), 3.30–3.15 (m, 2H), 2.85 (dd, J = 13.2, 7.5 Hz,
2H), 2.68 (dd, J = 19.5, 9.9 Hz, 2H), 2.17 (dd, J = 19.5,
3.9 Hz, 2H), 2.00–1.80 (m, 8H), 1.70–1.55 (m, 2H).
cyclopentane-1,2-diyl}diacetate. To a stirred solution of 21
(2.78 g, 8.25 mmol) in CH2Cl2 (50 ml) was bubbled ozone
(O2 containing ca 3% O3) at –78 °C for 1 h. To the resulting
mixture was added Ph3P (3.2 g, 12 mmol), and stirring was
continued at –78 °C for 30 min. After being stirred at room
temperature for 1 h, the resulting mixture was concentrated
in vacuo. The residue was used for the next reaction without
further purification. TLC Rf
=
0.50 (n-hexane/
EtOAc, 1/2).
To a stirred solution of the above-described residue in
t-BuOH (50 ml)/2-methyl-2-butene (5.2 ml)/H2O (10 ml)
were added NaClO2 (3.0 g, 33 mmol) and NaH2PO4 (2.4 g,
20 mmol).After being stirred at room temperature for 3 h, the
reaction mixture was diluted with CH2Cl2 and extracted with
2 N aq NaOH. The aqueous layer was acidified with 2 N aq
HCl, and then extracted with EtOAc. The organic layer was
dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was used for the next reaction without further
purification: TLC Rf = 0.39 (EtOAc).
A solution of the above-described residue in Et2O (60 ml)
was treated with CH2N2/Et2O at 0 °C until the evolution of
gas subsided. The resulting solution was quenched with
AcOH and then evaporated. The residue was purified by
column chromatography on silica gel (n-hexane/EtOAc, 2/1)
to give an isomeric mixture 22 (2.66 g, 6.20 mmol, 75% in
three steps; mixture rate was not clear.) as a pale yellow oil:
TLC Rf = 0.35 (n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20V)
m/z = 430 (M + H)+, 362, 330; 1H NMR (300 MHz, CDCl3)
d 7.00–6.90 (m, 2H), 6.85–6.80 (m, 1H), 4.81 (m, 1H), 3.84
(s, 3H), 3.70 & 3.70 (s, 6H), 3.10–2.60 (m, 2H), 2.70–2.20
(m, 8H), 2.00–1.75 (m, 6H), 1.80–1.50 (m, 2H).
5.1.3. Preparation of compounds 25a–25d
5.1.3.1. (2s,3aR,6aS)-2-[3-(Cyclopentyloxy)-4-methoxyphe-
nyl]-5-(1,3-dithian-2-ylidene)octahydropentalene-2-carbo-
nitrile (24a). The following reaction was carried out under
argon atmosphere. To a stirred solution of 2-trimethylsilyl-1,
3-dithiane (2.2 g, 12 mmol) in THF (10 ml) was added
n-BuLi (1.53 M solution in n-hexane, 7.5 ml, 12 mmol) at
0 °C, and then the reaction mixture was stirred at 0 °C for
10 min. To this solution was added a solution of 23a (1.30 g,
3.83 mmol) in THF (5.0 ml) at –78 °C. After being stirred at
0 °C for 30 min, the reaction mixture was quenched with
brine, and extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous MgSO4, and con-
centrated in vacuo. The residue was purified by column
chromatography on silica gel (n-hexane/EtOAc, 8/1–6/1) to
give 24a (633 mg, 1.44 mmol, 37%) as a pale yellow amor-
phous solid and to recover 23a (700 mg, 2.06 mmol, 54%):
TLC Rf = 0.54 (n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20V)
1
m/z = 442 (M + H)+; H NMR (300 MHz, CDCl3) d 6.95–
6.85 (m, 2H), 6.82 (d, J = 8.0 Hz, 1H), 4.80 (m, 1H), 3.84 (s,
3H), 3.00–2.85 (m, 2H), 2.90–2.75 (m, 2H), 2.80–2.40 (m,
6H), 2.40–2.00 (m, 4H), 2.00–1.70 (m, 8H), 1.80–1.50 (m,
2H).
5.1.2.6.
(2s,3aR,6aS)-2-[3-(Cyclopentyloxy)-4-methoxy-
(23a)
phenyl]-5-oxooctahydropentalene-2-carbonitrile
and (2r,3aR,6aS)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-
5-oxooctahydropentalene-2-carbonitrile (23b). The follow-
ing reaction was carried out under argon atmosphere. To a
5.1.3.2. (2r,3aR,6aS)-2-[3-(Cyclopentyloxy)-4-methoxyphe-
nyl]-5-(1,3-dithian-2-ylidene)octahydropentalene-2-carbo-

564
H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
nitrile (24b). Compound 24b was prepared from 23b in 42%
yield according to the same procedure as described for the
preparation of 24a from 23a. Pale yellow amorphous solid:
TLC Rf = 0.69 (n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20V)
Rf = 0.61 (n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20 V)
m/z = 384 (M + H)+; H NMR (300 MHz, CDCl3) d 7.00–
1
6.90 (m, 2H), 6.84 (d, J = 9.0 Hz, 1H), 4.81 (m, 1H), 3.84 (s,
3H), 3.68 (s, 3H), 3.05–2.90 (m, 3H), 2.70–2.55 (m, 2H),
2.30–2.15 (m, 2H), 2.05–1.60 (m, 12H). Compound 25c was
obtained (35% in two steps) as a pale yellow oil: TLC
Rf = 0.58 (n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20 V)
1
m/z = 442 (M + H)+; H NMR (300 MHz, CDCl3) d 6.95–
6.90 (m, 2H), 6.83 (d, J = 9.0 Hz, 1H), 4.79 (m, 1H), 3.84 (s,
3H), 3.10–2.95 (m, 2H), 2.95–2.75 (m, 4H), 2.75–2.55 (m,
4H), 2.47 (dd, J = 18.3 and 2.1 Hz, 2H), 2.20–2.10 (m, 2H),
2.00–1.80 (m, 6H), 1.80–1.65 (m, 2H), 1.70–1.55 (m, 2H).
1
m/z = 384 (M + H)+; H NMR (300 MHz, CDCl3) d 6.95–
6.85 (m, 2H), 6.83 (d, J = 8.4 Hz, 1H), 4.79 (m, 1H), 3.84 (s,
3H), 3.69 (s, 3H), 3.15–3.00 (m, 2H), 2.81 (m, 1H), 2.70–
2.60 (m, 2H), 2.05–1.75 (m, 12H), 1.70–1.55 (m, 2H).
5.1.3.3. Methyl (2r,3aR,5r,6aS)-5-cyano-5-[3-(cyclopentyl-
oxy)-4-methoxyphenyl]octahydropentalene-2-carboxylate
(25b) and methyl (2s,3aR,5r,6aS)-5-cyano-5-[3-(cyclo-
pentyloxy)-4-methoxyphenyl]octahydropentalene-2-carbo-
xylate (25a). To a stirred solution of 24a (548 mg,
1.24 mmol) in MeCN/H2O (4/1, 10 ml) were added TFA
(0.38 ml) and 30% aq H2O2 (2.0 ml). After being stirred at
80 °C for 30 min, the reaction mixture was cooled to 40 °C.
To this solution was added 2 N aq NaOH (19 ml). After being
stirred at 40 °C for 20 min, the reaction mixture was neutral-
ized with 2 N aq HCl (16 ml), and then extracted with
CH2Cl2. The organic layer was dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (CHCl3/MeOH 1/0–
20/1) to give a mixture of 2a and 3: TLC Rf = 0.52
(CHCl3/MeOH, 10/1).
A solution of a mixture of 2a and 3 in Et2O/THF (5/2,
7.0 ml) was treated with CH2N2/Et2O at 0 °C until the
evolution of gas subsided. The resulting solution was
quenched with AcOH, and then concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(n-hexane/EtOAc, 6/1–4/1) to give 25b (80.4 mg,
0.210 mmol, 18% in two steps) and 25a (108 mg,
0.282 mmol, 25% in two steps). Compound 25b was ob-
tained as a pale yellow oil: TLC Rf = 0.65 (n-hexane/EtOAc,
2/1); MS (APCI, Pos. 40 V) m/z = 384 (M + H)+, 352, 316;
1H NMR (300 MHz, CDCl3) d 6.92 (d, J = 1.8 Hz, 1H), 6.90
(dd, J = 7.5, 1.8 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 4.79 (m,
1H), 3.84 (s, 3H), 3.69 (s, 3H), 2.79 (m, 1H), 2.80–2.55 (m,
4H), 2.05–1.80 (m, 12H), 1.70–1.55 (m, 2H). Compound 25a
was obtained as a pale yellow oil: TLC Rf = 0.57 (n-
hexane/EtOAc, 2/1); MS (APCI, Pos. 40 V) m/z = 384 (M +
H)+, 352, 316, 284; 1H NMR (300 MHz, CDCl3) d 6.93 (d,
J = 1.8 Hz, 1H), 6.91 (dd, J = 8.4, 1.8 Hz, 1H), 6.82 (d,
J = 8.4 Hz, 1H), 4.79 (m, 1H), 3.84 (s, 3H), 3.70 (s, 3H), 2.80
(m, 1H), 2.80–2.55 (m, 2H), 2.60–2.40 (m, 2H), 2.35–2.20
(m, 4H), 2.00–1.75(m, 8H), 1.70–1.50 (m, 2H).
5.1.4. General procedure for the preparation of
compounds 2a, 3, 4a and 5
The following compounds 2a, 3, 4a and 5 were prepared
from the corresponding ester derivatives according to the
same procedures as described for the preparation of 2a from
25a.
5.1.4.1. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclopentyloxy)-4-
methoxyphenyl]octahydropentalene-2-carboxylic acid (2a).
To a stirred solution of 25a (96.7 mg, 0.252 mmol) in
MeOH/THF (2/1, 1.5 ml) was added 1 N aq KOH (0.50 ml).
After being stirred at room temperature for 4 h, the reaction
mixture was extracted with EtOAc. The aqueous layer was
acidified with 2 N aq HCl (0.50 ml), and extracted with
EtOAc. The organic layer was dried over anhydrous MgSO4,
and concentrated in vacuo. The residual solid was triturated
with a mixture of n-hexane/Et2O (9/1) to give 2a (69.8 mg,
0.189 mmol, 75%) as a white powder: TLC Rf = 0.39
(CHCl31/MeOH, 10/1); MS (APCI, Neg. 40.V) m/z = 368 (M
– H)–; H NMR (300 MHz, CDCl3) d 6.95–6.85 (m, 2H),
6.82 (d, J = 7.8 Hz, 1H), 4.79 (m, 1H), 3.84 (s, 3H), 2.84 (m,
1H), 2.90–2.60 (m, 2H), 2.55–2.40 (m, 2H), 2.40–2.20 (m,
4H), 2.10–1.75 (m, 9H), 1.80–1.55 (m, 2H); IR (KBr) 2960,
2872, 2232, 1701, 1591, 1517, 1468, 1443, 1415, 1343,
1252, 1150, 1026, 992; Anal. Found: C22H27NO4 (C, H, N).
5.1.4.2. (2r,3aR,5r,6aS)-5-Cyano-5-[3-(cyclopentyloxy)-4-
methoxyphenyl]octahydropentalene-2-carboxylic acid (3).
Compound 3 was obtained (91%) as a white powder: TLC
Rf = 0.39 (CHCl3/MeOH, 10/1); MS (APCI, Neg. 40 V)
1
m/z = 368 (M – H)–; H NMR (300 MHz, CDCl3) d 6.95–
6.85 (m, 2H), 6.82 (d, J = 8.1 Hz, 1H), 4.79 (m, 1H), 3.84 (s,
3H), 3.03 (m, 1H), 2.80–2.65 (m, 2H), 2.70–2.55 (m, 2H),
2.10–1.90 (m, 4H), 1.95–1.80 (m, 9H), 1.70–1.55 (m, 2H);
IR (KBr) 2959, 2872, 2232, 1736, 1701, 1590, 1517, 1467,
1445, 1415, 1343, 1292, 1249, 1233, 1169, 1151, 1138,
1027, 990; Anal. Found: C22H27NO4·0.25H2O (C, H, N).
5.1.3.4. Methyl (2s,3aR,5s,6aS)-5-cyano-5-[3-(cyclopentyl-
oxy)-4-methoxyphenyl]octahydropentalene-2-carboxylate
(25c) and methyl (2r,3aR,5s,6aS)-5-cyano-5-[3-(cyclo-
pentyloxy)-4-methoxyphenyl]octahydropentalene-2-carbo-
xylate (25d). Compounds 25c and 25d were prepared from
24 according to the same procedures as described for the
preparation of 25a and 25b from 24a. Compound 25d was
obtained (10% in two steps) as a white powder: TLC
5.1.4.3. (2s,3aR,5s,6aS)-5-Cyano-5-[3-(cyclopentyloxy)-4-
methoxyphenyl]octahydropentalene-2-carboxylic acid (4a).
Compound 4a was obtained (83%) as a white powder: TLC
Rf = 0.56 (CHCl3/MeOH, 10/1); MS (APCI, Neg. 40 V)
m/z = 368 (M – H)–; 1H NMR (300 MHz, CDCl3) d 6.93 (d,
J = 2.1 Hz, 1H), 6.91 (dd, J = 8.1, 2.1 Hz, 1H), 6.83 (d,

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
565
J = 8.1 Hz, 1H), 4.80 (m, 1H), 3.84 (s, 3H), 3.20–3.00 (m,
2H), 2.85 (m, 1H), 2.75–2.60 (m, 2H), 2.05–1.75 (m, 12H),
1.70–1.55 (m, 3H); IR (KBr) 2960, 2871, 2228, 1131, 1702,
1590, 1517, 1464, 1442, 1418, 1265, 1166, 1145, 1027, 855;
Anal. Found C22H27NO4 (C, H, N).
Pale yellow oil: TLC Rf = 0.61 (n-hexane/EtOAc, 3/1); MS
(APCI, Pos. 20 V) m/z = 360 (M + H)+. 1H NMR (200 MHz,
CDCl3) d 7.48–7.26 (m, 5H), 7.04 & 7.00 (dd, J = 8.4, 2.4 Hz,
1H), 6.93 (d, J = 2.4 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 5.18 &
5.16 (s, 2H), 3.89 & 3.88 (s, 3H), 2.70–2.45 (m, 1H), 2.45–
1.93 (m, 8H), 1.93–1.70 (m, 1H).
5.1.4.4. (2r,3aR,5s,6aS)-5-Cyano-5-[3-(cyclopentyloxy)-4-
methoxyphenyl]octahydropentalene-2-carboxylic acid (5).
Compound 5 was obtained (64%) as a white powder: TLC
Rf = 0.56 (CHCl3/MeOH, 10/1); MS (APCI, Neg. 60 V)
5.1.6.2. An isomeric mixture (28) of dimethyl 2,2′-
{(1R,2S,4s)-4-[3-(benzyloxy)-4-methoxyphenyl]-4-cyano-
cyclopentane-1,2-diyl}diacetate and dimethyl 2,2′-
{(1R,2S,4r)-4-[3-(benzyloxy)-4-methoxyphenyl]-4-cyano-
cyclopentane-1,2-diyl}diacetate. An isomeric mixture 28
was prepared from 27 in 78% yield in three steps according
to the same procedures as described for the preparation of 22
from 21. Pale yellow oil: TLC Rf = 0.38 (n-hexane/EtOAc,
2/1); MS (APCI, Pos. 20 V) m/z = 452 (M + H)+; 1H NMR
(200 MHz, CDCl3) d 7.49–7.26 (m, 5H), 7.00 (m, 1H), 6.93
(d, J = 2.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 5.18 & 5.16 (s,
2H), 3.89 (s, 3H), 3.70 & 3.70 (s, 6H), 3.10–2.58 (m, 2H),
2.80–2.14 & 1.84–1.72 (m, 8H).
1
m/z = 368 (M – H)–; H NMR (300 MHz, CDCl3) d 7.00–
6.90 (m, 2H), 6.83 (d, J = 9.0 Hz, 1H), 4.80 (m, 1H), 3.84 (s,
3H), 3.10–2.95 (m, 3H), 2.70–2.60 (m, 2H), 2.40–2.20 (m,
2H), 2.00–1.60 (m, 10H), 1.70–1.55 (m, 3H); IR (KBr) 2960,
2871, 2228, 1698, 1602, 1589, 1517, 1462, 1442, 1418,
1266, 1166, 1145, 855; Anal. Found: C22H27NO4 (C, H, N).
5.1.5. Preparation of compound 2b
5.1.5.1. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclopentyloxy)-4-
methoxyphenyl]-N-hydroxyoctahydropentalene-2-carboxa-
mide (2b). To a stirred solution of 2a (490 mg, 1.33 mmol) in
DMF (10 ml) were added EDC·HCl (380 mg, 2.0 mmol),
HOBt (270 mg, 2.0 mmol) and (1-methoxy-1-
methyethyl)oxyamine (700 mg, 6.7 mmol). After being
stirred at room temperature for 2 h, 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 residue was purified by short column chromatog-
raphy on silica gel (n-hexane/EtOAc, 2/3-0/1) to give an
amide (453 mg) as a white amorphous solid: TLC Rf = 0.55
(CHCl3/MeOH, 10/1).
To a stirred solution of an amide (453 mg) in MeOH
(8.0 ml) was added 2 N aq HCl (49 µl, 0.098 mmol, 0.1 eq).
After being stirred at room temperature for 2 h, the reaction
mixture was concentrated in vacuo. The residue was tritu-
rated with i-Pr2O/MeOH to give 2b (258 mg, 0.673 mmol,
two steps 52%) as a white powder: TLC Rf = 0.50
(CHCl31/MeOH, 10/1); MS (APCI, Neg. 40 V) m/z = 383 (M
– H)–; H NMR (300 MHz, CDCl3) d 9.40–8.50 (br, 2H),
6.95–6.85 (m, 2H), 6.82 (d, J = 8.7 Hz, 1H), 4.79 (m, 1H),
3.84 (s, 3H), 2.80–2.50 (m, 3H), 2.50–2.10 (m, 6H), 2.05–
1.70 (m, 8H), 1.70–1.50 (m, 2H); IR (KBr) 3344, 2952, 2870,
2221, 1670, 1590, 1513, 1441, 1417, 1358, 1321, 1301,
1260, 1163, 1140, 1028, 989; Anal. Found:
C22H28N2O4·0.25H2O (C, H, N).
5.1.6.3. (2s,3aR,6aS)-2-[3-(Benzyloxy)-4-methoxyphenyl]-
5-oxooctahydropentalene-2-carbonitrile (29a) and (2r,3aR,
6aS)-2-[3-(benzyloxy)-4-methoxyphenyl]-5-oxooctahydro-
pentalene-2-carbonitrile (29b). An isomeric mixture con-
sisting of 29a and 29b was prepared from 28 according to the
same procedures as described for the preparation of an iso-
meric mixture consisting of 23a–b from 22 and purified by
column chromatography on silica gel to afford 29a and 29b.
Compound 29a was obtained (48% in two steps) as a white
powder: TLC Rf = 0.44 (n-hexane/EtOAc, 1/1); MS (APCI,
Pos. 20 V) m/z = 394 (M + MeOH + H)+, 362 (M + H)+; 1H
NMR (300 MHz, CDCl3) d 7.46–7.28 (m, 5H), 6.98 (dd,
J = 8.4, 2.1 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.87 (d,
J = 2.1 Hz, 1H), 5.19 (s, 2H), 3.90 (s, 3H), 2.80–2.65 (m, 2H),
2.62–2.48 (m, 4H), 2.30–2.18 (m, 4H). Compound 29b was
obtained as a white powder: TLC Rf = 0.53 (n-
1
hexane/EtOAc, 1/1); H NMR (300 MHz, CDCl3) d 7.46–
7.29 (m, 5H), 6.98 (dd, J = 8.5 and 2.3 Hz, 1H), 6.88 (d,
J = 8.5 Hz, 1H), 6.87 (d, J = 2.3 Hz, 1H), 5.20 (s, 2H), 3.90 (s,
3H), 2.78–2.64 (m, 2H), 2.62–2.48 (m, 4H), 2.30–2.18 (m,
4H).
5.1.6.4. An enantiomeric mixture (30) of ((3aS,5S,6aS)-
5-[3-(benzyloxy)-4-methoxyphenyl]-5-cyano-1,3a,4,5,6,6a-
hexahydropentalen-2-yl trifluoromethanesulfonate and (3aR
,
5R,6aR)-5-[3-(benzyloxy)-4-methoxyphenyl]-5-cyano-1,3a,
4,5,6,6a-hexahydropentalen-2-yl trifluoromethanesulfonate.
The following reaction was carried out under argon atmo-
sphere. To a stirred solution of 29a (11.4 g, 31.4 mmol) in
THF (300 ml) was added LiHMDS (1.0 M solution in THF,
38 ml, 38 mmol) at –78 °C. After being stirred at –78 °C for
45 min, Tf2NPh (12 g, 35 mmol) was added. After being
stirred at 0 °C for additional 1 h, the reaction mixture was
diluted with brine, and extracted with EtOAc. The organic
layer was washed with brine, dried over anhydrous MgSO4,
5.1.6. Preparation of compound 32
5.1.6.1. An isomeric mixture (27) of (2r,3aR,7aS)-2-
[3-(benzyloxy)-4-methoxyphenyl]-2,3,3a,4,7,7a-hexahydro-
1H-indene-2-carbonitrile and (2s,3aR,7aS)-2-[3-(benzyl-
oxy)-4-methoxyphenyl]-2,3,3a,4,7,7a-hexahydro-1H-indene-
2-carbonitrile. An isomeric mixture 27 (major:minor = 3:1)
was prepared from 26 in 92% yield according to the same
procedures as described for the preparation of 21 from 20.

566
H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
and concentrated in vacuo to give a mixture 30 (29.1 g) as a
yellow oil: TLC Rf = 0.53 (n-hexane/EtOAc, 2/1); 1H NMR
(300 MHz, CDCl3) d 7.45–7.20 (m, 5H), 6.94 (dd, J = 8.3,
2.3 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 2.3 Hz, 1H),
5.62 (m, 1H), 5.18 (s, 2H), 3.89 (s, 3H), 3.27–3.15 (m, 1H),
2.90–2.79 (m, 1H), 2.74–2.56 (m, 1H), 2.56–2.40 (m, 2H),
2.30–2.10 (m, 3H).
powder: TLC Rf = 0.29 (n-hexane/EtOAc, 2/1); MS (APCI,
Pos.20V) m/z = 330 (M + H)+; 1H NMR (300 MHz, CDCl3)
d 6.95–6.90 (m, 2H), 6.85–6.81 (m, 1H), 3.91 (s, 3H), 3.88 (s,
3H), 3.70 (s, 3H), 2.87–2.74 (m, 1H), 2.74–2.62 (m, 2H),
2.52–2.44 (m, 2H), 2.34–2.20 (m, 4H), 1.96–1.84 (m, 2H).
5.1.7.2. Methyl (2s,3aR,5r,6aS)-5-cyano-5-(3-isopropoxy-4-
methoxyphenyl)octahydropentalene-2-carboxylate (35). To
a stirred solution of 32 (0.10 g, 0.32 mmol) in CH2Cl2
(4.0 ml) were added i-PrOH (30 µl, 0.38 mmol), PPh3
(0.12 g, 0.48 mmol) and ADDP (0.12 g, 0.48 mmol). After
being stirred at room temperature overnight, the reaction
mixture was poured into n-hexane and the precipitates were
removed by filtration. The filtrates and washings were com-
bined and concentrated in vacuo. The resulting residue was
purified by column chromatography on silica gel (n-
hexane/EtOAc, 3/1) to give 35 (0.11 g, 0.31 mmol, 98%) as a
pale yellow oil: TLC Rf = 0.38 (n-hexane/EtOAc, 2/1); MS
(APCI, Pos. 20 V) m/z = 358 (M + H)+; 1H NMR (300 MHz,
CDCl3) d 6.96–6.92 (m, 2H), 6.85–6.82 (m, 1H), 4.54 (sep-
tet, J = 6.0 Hz, 1H), 3.85 (s, 3H), 3.70 (s, 3H), 2.87–2.74 (m,
1H), 2.74–2.60 (m, 2H), 2.51–2.42 (m, 2H), 2.32–2.20 (m,
4H), 1.95–1.83 (m, 2H), 1.37 (d, J = 6.0 Hz, 6H).
5.1.6.5. An enantiomeric mixture (31) of methyl
(3aS,5S,6aS)-5-[3-(benzyloxy)-4-methoxyphenyl]-5-cyano-
1,3a,4,5,6,6a-hexahydropentalene-2-carboxylate and me-
thyl (3aR,5R,6aR)-5-[3-(benzyloxy)-4-methoxyphenyl]-5-
cyano-1,3a,4,5,6,6a-hexahydropentalene-2-carboxylate. To
a stirred solution of 30 (29.1 g) in DMF/MeOH (6/1, 350 ml)
were added PPh3 (820 mg, 3.1 mmol), Pd(OAc)2 (350 mg,
1.6 mmol) and Et3N (8.8 ml, 63 mmol). After being stirred at
room temperature overnight under CO atmosphere, the reac-
tion mixture was diluted with H2O and extracted with
EtOAc/n-hexane. The organic layer was washed with H2O,
dried over MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (n-
hexane/EtOAc, 4/1) to give an enantiomeric mixture 31
(8.82 g, 21.9 mmol, 70% in two steps) as a yellow oil: TLC
Rf = 0.46 (n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20 V)
1
m/z = 404 (M + H)+; H NMR (200 MHz, CDCl3) d 7.47–
5.1.7.3. Methyl (2s,3aR,5r,6aS)-5-cyano-5-[3-(2,3-dihydro-
1H-inden-2-yloxy)-4-methoxyphenyl]octahydropentalene-
2-carboxylate (39). To a stirred solution of 32 (0.10 g,
0.32 mmol) in THF (3.0 ml) were added indan-2-ol (64 mg,
0.48 mmol), PPh3 (125 mg, 0.48 mmol) and DIPAD (0.10 ml,
0.51 mmol). After being stirred at room temperature over-
night, the reaction mixture was concentrated in vacuo. The
resulting residue was purified by column chromatography on
silica gel (n-hexane/EtOAc, 2/1) to give 39 (192 mg, quant)
as a colorless oil: TLC Rf = 0.65 (n-hexane/EtOAc, 1/1); MS
(APCI, Pos. 20 V) m/z = 432 (M + H)+; 1H NMR (300 MHz,
CDCl3) d 7.28–7.16 (m, 4H), 6.99–6.95 (m, 2H), 6.87–6.83
(m, 1H), 5.25–5.17 (m, 1H), 3.81 (s, 3H), 3.70 (s, 3H), 3.38
(dd, J = 16.7, 6.6 Hz, 2H), 3.23 (dd, J = 16.7, 3.9 Hz, 2H),
2.87–2.61 (m, 3H), 2.51–2.43 (m, 2H), 2.34–2.21 (m, 4H),
1.96–1.84 (m, 2H).
7.26 (m, 5H), 6.96 (dd, J = 8.4 and 2.2 Hz, 1H), 6.88 (d,
J = 2.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.64 (m, 1H), 5.18
(s, 2H), 3.89 (s, 3H), 3.75 (s, 3H), 3.44–3.25 (m, 1H), 2.90–
2.35 (m, 5H), 2.27–2.09 (m, 2H).
5.1.6.6. Methyl (2s,3aR,5r,6aS)-5-cyano-5-(3-hydroxy-4-
methoxyphenyl)octahydropentalene-2-carboxylate (32). A
solution of 31 (9.36 g, 23.2 mmol) in MeOH (230 ml) was
hydrogenated under atmospheric pressure of H2 gas in the
presence of 10% Pd/C (1.0 g) for 3 days. The catalyst was
removed by filtration through a pad of celite, and washed
with MeOH. The filtrate was concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(n-hexane/EtOAc, 3/1) to give 32 (1.93 g, 6.12 mmol, 26%)
as a white powder: TLC Rf = 0.48 (n-hexane/EtOAc, 1/1);
1
MS (APCI, Neg. 20 V) m/z = 314 (M – H)–; H NMR
(300 MHz, CDCl3) d 6.98–6.80 (m, 3H), 5.63 (s, 1H), 3.90 (s,
3H), 3.69 (s, 3H), 2.89–2.58 (m, 3H), 2.52–2.40 (m, 2H),
2.32–2.17 (m, 4H), 1.97–1.79 (m, 2H).
5.1.7.4. Methyl (2s,3aR,5r,6aS)-5-cyano-5-(3-ethoxy-4-me-
thoxy-phenyl)octahydropentalene-2-carboxylate (34). Com-
pound 34 was prepared from 32 in quantitative yield accord-
ing to the same procedures as described for the preparation of
33 from 32. White powder: TLC Rf = 0.40 (n-hexane/EtOAc,
2/1); MS (APCI, Pos. 20 V) m/z = 344 (M + H)+; 1H NMR
(300 MHz, CDCl3) d 6.94–6.89 (m, 2H), 6.85–6.81 (m, 1H),
4.12 (q, J = 7.0 Hz, 2H), 3.87 (s, 3H), 3.70 (s, 3H), 2.86–2.74
(m, 1H), 2.74–2.61 (m, 2H), 2.51–2.43 (m, 2H), 2.32–2.20
(m, 4H), 1.95–1.83 (m, 2H), 1.47 (t, J = 7.0 Hz, 3H).
5.1.7. Preparation of compounds 33 and 42
5.1.7.1. Methyl (2s,3aR,5r,6aS)-5-cyano-5-(3,4-dimethoxy-
phenyl)octahydropentalene-2-carboxylate (33). To a stirred
solution of 32 (0.10 g, 0.32 mmol) in DMF (4 ml) were added
K2CO3 (0.13 g, 0.95 mmol, 3.0 eq) and MeI (60 µl,
0.95 mmol). After being stirred at room temperature for 2 h,
the reaction mixture was poured into H2O, and diluted with
EtOAc. The organic layer was washed with H2O, brine, and
dried over anhydrous MgSO4. The resulting solution was
concentrated in vacuo to give 33 (0.11 g, quant) as a white
5.1.7.5. Methyl (2s,3aR,5r,6aS)-5-cyano-5-[3-(cyclobutyl-
oxy)-4-methoxyphenyl]octahydropentalene-2-carboxylate
(36). Compound 36 was prepared from 32 in 53% yield
according to the same procedures as described for the prepa-

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
567
ration of 35 from 32. Colorless oil: TLC Rf = 0.40 (n-
hexane/EtOAc, 2/1); MS (APCI, Pos. 20 V) m/z = 370 (M +
H)+; 1H NMR (200 MHz, CDCl3) d 6.91 (dd, J = 8.4, 2.2 Hz,
1H), 6.82 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 4.68
(quintet, J = 7.0 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 2.90–2.60
(m, 3H), 2.56–2.36 (m, 4H), 2.36–2.18 (m, 6H), 1.98–1.66
(m, 4H).
5.1.7.10. Methyl (2s,3aR,5r,6aS)-5-cyano-5-[4-methoxy-3-
(3,3,3-trifluoropropoxy)phenyl]octahydropentalene-2-carbo-
xylate (42). Compound 42 was prepared from 32 in 58%
yield according to the same procedures as described for the
preparation of 35 from 32. Colorless oil: TLC Rf = 0.46
(n-hexane/EtOAc, 2/1); MS (APCI, Pos. 20 V) m/z = 412 (M
1
+ H)+; H NMR (300 MHz, CDCl3) d 7.01 (dd, J = 8.4,
2.4 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H),
4.25 (t, J = 6.8 Hz, 2H), 3.86 (s, 3H), 3.70 (s, 3H), 2.87–2.60
(m, 5H), 2.50–2.42 (m, 2H), 2.33–2.20 (m, 4H), 1.96–1.84
(m, 2H).
5.1.7.6. Methyl (2s,3aR,5r,6aS)-5-cyano-5-[3-(cyclohexyl-
oxy)-4-methoxyphenyl]octahydropentalene-2-carboxylate
(37). Compound 37 was prepared from 32 in 60% yield
according to the same procedures as described for the prepa-
ration of 35 from 32. Colorless oil: TLC Rf = 0.54 (n-
hexane/EtOAc, 2/1); MS (APCI, Pos. 20 V) m/z = 398 (M +
5.1.8. General procedure for the preparation of
compounds 6a–15a
The following compounds 6a–15a were prepared from the
corresponding ester derivatives 33–42, respectively, accord-
ing to the same procedures as described for the preparation of
6a from 33.
1
H)+; H NMR (200 MHz, CDCl3) d 6.97–6.91 (m, 2H),
6.86–6.81 (m, 1H), 4.26–4.10 (m, 1H), 3.84 (s, 3H), 3.70 (s,
3H), 2.90–2.56 (m, 3H), 2.53–2.40 (m, 2H), 2.33–2.18 (m,
4H), 2.08–1.68 (m, 6H), 1.68–1.42 (m, 2H), 1.42–1.22 (m,
4H).
5.1.8.1. (2s,3aR,5r,6aS)-5-Cyano-5-(3,4-dimethoxyphenyl)
octahydropentalene-2-carboxylic acid (6a). To a stirred so-
lution of 33 (0.11 g, 0.32 mmol) in MeOH/THF (1/1, 4.0 ml)
was added 1 N aq NaOH (1.6 ml). After being stirred at room
temperature for 1 h, the reaction mixture was acidified with
1 N aq HCl, and extracted with EtOAc. The organic layer was
washed with H2O, brine, dried over anhydrous MgSO4 and
concentrated in vacuo. The residual solid was triturated with
n-hexane/EtOAc to give 6a (0.10 g, 0.32 mmol, 99%) as a
white powder: TLC Rf = 0.53 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 314 (M – H)–; 1H NMR (300 MHz,
CDCl3) d 6.96–6.91 (m, 2H), 6.85–6.81 (m, 1H), 3.91 (s,
3H), 3.88 (s, 3H), 2.91–2.78 (m, 1H), 2.78–2.65 (m, 2H),
2.52–2.44 (m, 2H), 2.36–2.25 (m, 4H), 2.02–1.90 (m, 2H),
1.60 (br, 1H); IR (KBr) 3449, 2941, 2879, 2838, 2227, 1690,
1589, 1521, 1465, 1414, 1291, 1256, 1214, 1148, 1024, 873;
Anal. Found: C18H21NO4 (C, H, N).
5.1.7.7. Methyl (2s,3aR,5r,6aS)-5-cyano-5-[4-methoxy-3-
(tetrahydro-2H-pyran-4-yloxy)phenyl]octahydropentalene-
2-carboxylate (38). Compound 38 was prepared from 32 in
79% yield according to the same procedures as described for
the preparation of 35 from 32. Colorless oil: TLC Rf = 0.39
(n-hexane/EtOAc, 1/1); MS (APCI, Pos. 20 V) m/z = 400 (M
1
+ H)+; H NMR (200 MHz, CDCl3) d 7.03–6.96 (m, 2H),
6.89–6.83 (m, 1H), 4.50–4.34 (m, 1H), 4.08–3.95 (m, 2H),
3.85 (s, 3H), 3.70 (s, 3H), 3.60–3.48 (m, 2H), 2.89–2.58 (m,
3H), 2.51–2.39 (m, 2H), 2.33–2.18 (m, 4H), 2.08–1.76 (m,
6H).
5.1.7.8. Methyl (2s,3aR,5r,6aS)-5-cyano-5-[3-(cyclopropyl-
methoxy)-4-methoxyphenyl]octahydropentalene-2-carboxy-
late (40). Compound 40 was prepared from 32 in quantitative
yield according to the same procedures as described for the
preparation of 33 from 32. White powder: TLC Rf = 0.70
(n-hexane/EtOAc, 1/1); MS (APCI, Pos. 20 V) m/z = 370 (M
5.1.8.2. (2s,3aR,5r,6aS)-5-Cyano-5-(3-ethoxy-4-methoxy-
phenyl)octahydropentalene-2-carboxylic acid (7a). Com-
pound 7a was obtained (quant) from 34 as a white powder:
TLC Rf = 0.53 (CHCl31/MeOH, 10/1); MS (APCI, Neg. 20V)
m/z = 328 (M – H)–; H NMR (300 MHz, CDCl3) d 6.94–
6.90 (m, 2H), 6.85–6.81 (m, 1H), 4.12 (q, J = 7.1 Hz, 2H),
3.87 (s, 3H), 2.90–2.78 (m, 1H), 2.78–2.64 (m, 2H), 2.51–
2.43 (m, 2H), 2.35–2.25 (m, 4H), 2.00–1.89 (m, 2H), 1.55
(br, 1H), 1.48 (t, J = 7.1 Hz, 3H); IR (KBr) 3450, 2927, 2216,
1690, 1590, 1522, 1466, 1445, 1420, 1326, 1296, 1251,
1215, 1165, 1147, 1040, 929; Anal. Found: C19H23NO4 (C,
H, N).
1
+ H)+; H NMR (300 MHz, CDCl3) d 6.94–6.90 (m, 2H),
6.85–6.80 (m, 1H), 3.88–3.84 (m, 5H), 3.70 (s, 3H), 2.85–
2.74 (m, 1H), 2.74–2.60 (m, 2H), 2.55–2.45 (m, 2H), 2.36–
2.19 (m, 4H), 1.96–1.82 (m, 2H), 1.40–1.22 (m, 1H), 0.70–
0.60 (m, 2H), 0.41–0.35 (m, 2H).
5.1.7.9. Methyl (2s,3aR,5r,6aS)-5-cyano-5-{4-methoxy-3-
[4-(methylsulfanyl)butoxy]phenyl}octahydropentalene-2-
carboxylate (41). Compound 41 was prepared from 32 in
quantitative yield according to the same procedures as de-
scribed for the preparation of 35 from 32. Colorless oil: TLC
Rf = 0.61 (n-hexane/EtOAc, 1/1); MS (APCI, Pos. 20 V)
5.1.8.3. (2s,3aR,5r,6aS)-5-Cyano-5-(3-isopropoxy-4-metho-
xy-phenyl)octahydropentalene-2-carboxylic acid (8a).
Compound 8a was obtained (98%) from 35 as a white pow-
der: TLC Rf = 0.53 (CHCl3/MeOH, 10/1); MS (APCI, Neg.
1
m/z = 418 (M + H)+; H NMR (300 MHz, CDCl3) d 6.94–
6.90 (m, 2H), 6.85–6.81 (m, 1H), 4.05 (t, J = 6.3 Hz, 2H),
3.85 (s, 3H), 3.70 (s, 3H), 2.86–2.74 (m, 1H), 2.74–2.62 (m,
2H), 2.59 (t, J = 7.2 Hz, 2H), 2.51–2.43 (m, 2H), 2.32–2.20
(m, 4H), 2.11 (s, 3H), 2.01–1.75 (m, 4H), 1.73–1.66 (m, 2H).
1
20 V) m/z = 342 (M – H)–; H NMR (300 MHz, CDCl3) d
6.96–6.92 (m, 2H), 6.86–6.82 (m, 1H), 4.54 (septet,

568
H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
J = 6.0 Hz, 1H), 3.85 (s, 3H), 2.90–2.78 (m, 1H), 2.77–2.64
(m, 2H), 2.50–2.42 (m, 2H), 2.34–2.24 (m, 4H), 2.00–1.88
(m, 2H), 1.60 (br, 1H), 1.3 7 (d, J = 6.0 Hz, 6H); IR (KBr)
2973, 2220, 1705, 1590, 1518, 1466, 1442, 1417, 1384,
1372, 1330, 1292, 1257, 1218, 1162, 1146, 1112, 1031, 947;
Anal. Found: C20H25NO4 (C, H, N).
IR (KBr) 3271, 3071, 3042, 3000, 2958, 2906, 2875, 2836,
2231, 1740, 1705, 1594, 1515, 1465, 1441, 1417, 1402,
1352, 1332, 1254, 1207, 1173, 1152, 1081, 1020, 953; Anal.
Found: C26H27NO4 (C, H, N).
5.1.8.8. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclopropylmethoxy)-
4-methoxyphenyl]octahydropentalene-2-carboxylic
acid
(13a). Compound 13a was obtained (70%) from 40 as a
white powder: TLC Rf = 0.40 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 354 (M – H)–; 1H NMR (300 MHz,
CDCl3) d 6.95–6.90 (m, 2H), 6.83 (d, J = 9.0 Hz, 1H), 3.87 (s,
3H), 3.87 (d, J = 6.9 Hz, 2H), 2.90–2.76 (m, 1H), 2.76–2.64
(m, 2H), 2.52–2.40 (m, 2H), 2.36–2.24 (m, 4H), 2.00–1.85
(m, 2H), 1.62 (brs, 1H), 1.42–1.22 (m, 1H), 0.70–0.62 (m,
2H), 0.42–0.34 (m, 2H); IR (KBr) 3225, 3000, 2969, 2898,
2841, 2244, 2034, 1738, 1591, 1519, 1470, 1448, 1407,
1323, 1285, 1246, 1203, 1188, 1170; Anal. Found:
C21H25NO4·0.5H2O (C, H, N).
5.1.8.4. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclobutyloxy)-4-
methoxyphenyl]octahydropentalene-2-carboxylic acid (9a).
Compound 9a was obtained (97%) from 36 as a white pow-
der: TLC Rf = 0.57 (CHCl3/MeOH, 10/1); MS (APCI, Neg.
1
20 V) m/z = 354 (M – H)–; H NMR (300 MHz, CDCl3) d
6.90 (dd, J = 8.3, 2.2 Hz, 1H), 6.82 (d, J = 8.3 Hz, 1H), 6.80
(d, J = 2.2 Hz, 1H), 4.68 (quintet, J = 7.5 Hz, 1H), 3.86 (s,
3H), 2.91–2.78 (m, 1H), 2.78–2.64 (m, 2H), 2.54–2.41 (m,
4H), 2.36–2.19 (m, 6H), 2.00–1.81 (m, 3H), 1.78–1.62 (m,
2H); IR (KBr) 3325, 2963, 2876, 2230, 1733, 1592, 1519,
1466, 1446, 1421, 1400, 1357, 1308, 1260, 1244, 1194,
1148, 1073, 1019, 945; Anal. Found: C21H25NO4 (C, H, N).
5.1.8.9. (2s,3aR,5r,6aS)-5-Cyano-5-{4-methoxy-3-[4-(methyl-
sulfanyl)butoxy]phenyl}octahydropentalene-2-carboxylic acid
(14a). Compound 14a was obtained (86%) from 41 as a
white powder: TLC Rf = 0.48 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 402 (M – H)–; 1H NMR (300 MHz,
CDCl3) d 6.95–6.91 (m, 2H), 6.85–6.81 (m, 1H), 4.06 (t,
J = 6.3 Hz, 2H), 3.86 (s, 3H), 2.91–2.78 (m, 1H), 2.78–2.65
(m, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.51–2.43 (m, 2H), 2.35–
2.25 (m, 4H), 2.12 (s, 3H), 2.01–1.88 (m, 4H), 1.88–1.75 (m,
2H), 1.61 (br, 1H); IR (KBr) 3436, 2963, 2838, 2218, 1695,
1637, 1589, 1519, 1465, 1420, 1331, 1295, 1260, 1212,
1163, 1146, 1060, 1022, 936; Anal. Found: C22H29NO4S (C,
H, N, S).
5.1.8.5. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclohexyloxy)-4-
methoxyphenyl]octahydropentalene-2-carboxylic
acid
(10a). Compound 10a was obtained (90%) from 37 as a
white powder: TLC Rf = 0.68 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 382 (M – H)–; 1H NMR (300 MHz,
CDCl3) d 6.97–6.92 (m, 2H), 6.86–6.82 (m, 1H), 4.24–4.14
(m, 1H), 3.84 (s, 3H), 2.90–2.78 (m, 1H), 2.76–2.64 (m, 2H),
2.50–2.42 (m, 2H), 2.34–2.24 (m, 4H), 2.05–1.88 (m, 4H),
1.88–1.78 (m, 2H), 1.63–1.49 (m, 3H), 1.42–1.25 (m, 4H);
IR (KBr) 3431, 2936, 2858, 2225, 1701, 1604, 1586, 1517,
1438, 1420, 1297, 1254, 1219, 1145, 1043, 1022, 959; Anal.
Found: C23H29NO4·0.25H2O (C, H, N).
5.1.8.10. (2s,3aR,5r,6aS)-5-Cyano-5-[4-methoxy-3-(3,3,3-
trifluoropropoxy)phenyl]octahydropentalene-2-carboxylic
acid (15a). Compound 15a was obtained (79%) from 42 as a
white powder: TLC Rf = 0.68 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 369 (M – H)–; 1H NMR (300 MHz,
CDCl3) d 7.01 (dd, J = 8.4, 2.1 Hz, 1H), 6.95 (d, J = 2.1 Hz,
1H), 6.87 (d, J = 8.4 Hz, 1H), 4.26 (t, J = 6.9 Hz, 2H), 3.86 (s,
3H), 2.91–2.78 (m, 1H), 2.78–2.60 (m, 4H), 2.50–2.41 (m,
2H), 2.36–2.25 (m, 4 H), 2.01–1.90 (m, 2H), 1.90–1.40 (br,
1H); IR (KBr) 2964, 2887, 2226, 1698, 1603, 1588, 1521,
1462, 1441, 1414, 1349, 1327, 1290, 1246, 1219, 1196,
1156, 1143, 1042, 1024, 1004, 905; Anal. Found:
C20H22F3NO4 (C, H, N).
5.1.8.6. (2s,3aR,5r,6aS)-5-Cyano-5-[4-methoxy-3-(tetrahy-
dro-2H-pyran-4-yloxy)phenyl]octahydropentalene-2-carbo-
xylic acid (11a). Compound 11a was obtained (94%) from
38 as a white powder: TLC Rf = 0.49 (CHCl3/MeOH, 10/1);
1
MS (APCI, Neg. 20 V) m/z = 384 (M – H)–; H NMR
(300 MHz, CDCl3) d 7.02–6.97 (m, 2H), 6.88–6.84 (m, 1H),
4.47–4.38 (m, 1H), 4.06–3.98 (m, 2H), 3.85 (s, 3H), 3.59–
3.50 (m, 2H), 2.90–2.78 (m, 1H), 2.78–2.64 (m, 2H), 2.49–
2.40 (m, 2H), 2.34–2.25 (m, 4H), 2.05–1.91 (m, 4H), 1.91–
1.77 (m, 2H), 1.56 (br, 1H); IR (KBr) 3435, 2958, 2217, 1732,
1708, 1590, 1519, 1417, 1303, 1259, 1146, 1067, 1051, 1028,
853; Anal. Found: C22H27NO5·0.25H2O (C, H, N).
5.1.8.7.
(2s,3aR,5r,6aS)-5-Cyano-5-[3-(2,3-dihydro-1H-
5.1.9. General procedure for the preparation of
compounds 6b and 15b
The following compounds 6b–15b were prepared from
the corresponding carboxylic acid derivatives 6a–15a, re-
spectively, according to the same procedures as described for
the preparation of 2b from 2a.
inden-2-yloxy)-4-methoxyphenyl]octahydropentalene-2-car-
boxylic acid (12a). Compound 12a was obtained (90%) from
39 as a white powder: TLC Rf = 0.52 (CHCl3/MeOH, 10/1);
1
MS (APCI, Neg. 20 V) m/z = 416 (M – H)–; H NMR
(300 MHz, CDCl3) d 7.27–7.16 (m, 4H), 6.99–6.95 (m, 2H),
6.87–6.83 (m, 1H), 5.25–5.17 (m, 1H), 3.81 (s, 3H), 3.38 (dd,
J = 16.5, 6.5 Hz, 2H), 3.21 (dd, J = 16.5, 3.9 Hz, 2H),
2.91–2.78 (m, 1H), 2.78–2.65 (m, 2H), 2.51–2.43 (m, 2H),
2.36–2.25 (m, 4H), 2.01–1.89 (m, 2H), 1.80–1.30 (br, 1H);
5.1.9.1. (2s,3aR,5r,6aS)-5-Cyano-5-(3,4-dimethoxyphenyl)-
N-hydroxyoctahydropentalene-2-carboxamide (6b). Com-
pound 6b was obtained (49%) from 6a as a white powder:

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
569
TLC Rf = 0.50 (CHCl3/MeOH, 10/1); MS (FAB, Pos.)
m/z = 331 (M + H)+; H NMR (300 MHz, DMSO-d6) d
1.83 (m, 2H), 1.77–1.64 (m, 4H), 1.55–1 .22 (m, 6H); IR
(KBr) 3423, 2937, 2860, 2232, 1655, 1509, 1446, 1415,
1259, 1145, 1020, 855; Anal. Found: C23H30N2O4·0.25H2O
(C, H, N).
1
10.34 & 9.93 (br, 1H), 9.00 & 8.68 (br, 1H), 7.00–6.91 (m,
3H), 3.77 (s, 3H), 3.74 (s, 3H), 2.64–2.37 (m, 5H), 2.28–2.18
(m, 2H), 2.02–1.92 (m, 2H), 1.77–1.66 (m, 2H); IR (KBr)
3403, 3222, 3019, 2943, 2835, 2220, 1683, 1653, 1592,
1518, 1457, 1412, 1329, 1257, 1146, 1062, 1023, 859;
5.1.9.6. (2s,3aR,5r,6aS)-5-Cyano-N-hydroxy-5-[4-methoxy-
3-(tetrahydro-2H-pyran-4-yloxy)phenyl]octahydropentalene-
2-carboxamide (11b). Compound 11b was obtained (63%)
from 11a as a white powder: TLC Rf = 0.30 (CHCl3/MeOH,
10/1); MS (APCI, Neg. 20 V) m/z = 399 (M – H)-; 1H NMR
(300 MHz, DMSO-d6) d 10.34 & 9.75 (s, 1H), 8.99 & 8.67 (s,
1H), 7.06–6.95 (m, 3H), 4.57–4.46 (m, 1H), 3.88–3.80 (m,
2H), 3.75 (s, 3H), 3.49–3.40 (m, 2H), 2.57–2.37 (m, 5H),
2.30–2.18 (m, 2H), 2.02–1.85 (m, 4H), 1.77–1 .66 (m, 2H),
1.64–1.50 (m, 2H); IR (KBr) 3250, 2956, 2231, 1659, 1515,
1467, 1416, 1362, 1258, 1184, 1146, 1088, 1031, 989;
HRMS (FAB) calcd for C18H23N2O4 (M
+ H)+
m/z = 331.1658, found 331.1636.
5.1.9.2.
2s,3aR,5r,6aS)-5-Cyano-5-(3-ethoxy-4-methoxy-
phenyl)-N-hydroxyoctahydropentalene-2-carboxamide (7b).
Compound 7b was obtained (30%) from 7a as a white pow-
der: TLC Rf = 0.43 (CHCl3/MeOH, 10/1); MS (FAB, Pos.)
1
m/z = 345 (M + H)+; H NMR (300 MHz, DMSO-d6) d
10.35 & 9.98 (br, 1H), 10.17 & 9.75 (br, 1H), 7.00–6.91 (m,
3H), 4.03 (q, J = 6.9 Hz, 2H), 3.74 (s, 3H), 2.68–2.36 (m,
5H), 2.26–2.17 (m, 2H), 2.02–1.92 (m, 2H), 1.77–1.65 (m,
2H), 1.31 (t, J = 6.9 Hz, 3H); IR (KBr) 3192, 2976, 2220,
1655, 1523, 1465, 1446, 1419, 1391, 1329, 1258, 1146,
1041, 1025, 930; Anal. Found: C19H24N2O4 (C, H, N).
HRMS (FAB) calcd for C22H29N2O5 (M
+ H)+
m/z = 401.2076, found 401.2101.
5.1.9.7.
(2s,3aR,5r,6aS)-5-Cyano-5-[3-(2,3-dihydro-1H-
inden-2-yloxy)-4-methoxyphenyl]-N-hydroxyoctahydropen-
talene-2-carboxamide (12b). Compound 12b was obtained
(34%) from 12a as a white powder: TLC Rf = 0.31
(CHCl3/MeOH, 10/1); MS (FAB, Pos.) m/z = 433 (M + H)+;
1H NMR (300 MHz, DMSO-d6) d 10.34 (s, 1H), 8.67 (s, 1H),
7.28–7.24 (m, 2H), 7.19–7.14 (m, 2H), 7.04–6.98 (m, 2H),
6.94 (d, J = 8.4 Hz, 1H), 5.30–5.22 (m, 1H), 3.69 (s, 3H),
3.38–3.30 (m, 2H), 3.01 (dd, J = 17.1, 2.1 Hz, 2H), 2.65–2.36
(m, 5H), 2.28–2.21 (m, 2H), 2.04–1.94 (m, 2H), 1.79–1.67
(m, 2H); IR (KBr) 3390, 3239, 2960, 2904, 2836, 2218,
1655, 1591, 1515, 1460, 1442, 1416, 1355, 1330, 1251,
1208, 1150, 1119, 1020, 952; Anal. Found:
C26H28N2O4·0.25H2O (C, H, N).
5.1.9.3. (2s,3aR,5r,6aS)-5-Cyano-N-hydroxy-5-(3-isopro-
poxy-4-methoxyphenyl)octahydropentalene-2-carboxamide
(8b). Compound 8b was obtained (43%) from 8a as a white
powder: TLC Rf = 0.45 (CHCl3/MeOH, 10/1); MS (APCI,
1
Neg. 20 V) m/z = 357 (M – H)–; H NMR (300 MHz,
DMSO-d6) d 10.34 & 9.75 (br, 1H), 8.99 & 8.68 (br, 1H),
7.00–6.92 (m, 3H), 4.57 (septet, J = 6.0 Hz, 1H), 3.73 (s, 3H),
2.64–2.37 (m, 5H), 2.24–2.17 (m, 2H), 2.02–1.92 (m, 2H),
1.77–1.65 (m, 2H), 1.23 (d, J = 6.0 Hz, 6H); IR (KBr) 3434,
3185, 2973, 2233, 1627, 1520, 1466, 1418, 1376, 1256,
1223, 1176, 1148, 1110, 1020, 989; Anal. Found:
C20H26N2O4·0.5H2O (C, H, N).
5.1.9.8. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclopropylmetho-
xy)-4-methoxyphenyl]-N-hydroxyoctahydropentalene-2-car-
boxamide (13b). Compound 13b was obtained (28%) from
13a as a white powder: TLC Rf = 0.40 (CHCl3/MeOH, 9/1);
5.1.9.4. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclobutyloxy)-4-
methoxyphenyl]-N-hydroxyoctahydropentalene-2-carboxa-
mide (9b). Compound 9b was obtained (57%) from 9a as a
white powder: TLC Rf = 0.27 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 369 (M – H)–; 1H NMR (300 MHz,
DMSO-d6) d 10.34 (d, J = 1.5 Hz, 1H), 8.68 (d, J = 1.5 Hz,
1H), 6.98–6.91 (m, 2H), 6.83–6.79 (m, 1H), 4.70 (quintet,
J = 7.7 Hz, 1H), 3.74 (s, 3H), 2.62–2.33 (m, 7H), 2.24–2.18
(m, 2H), 2.10–1.92 (m, 4H), 1.82–1.54 (m, 4H); IR (KBr)
3234, 2939, 2231, 1627, 1519, 1465, 1443, 1417, 1259,
1147, 1077, 1023, 959; HRMS (FAB) calcd for C21H27N2O4
(M + H)+ m/z = 371.1971, found 371.1988.
1
MS (APCI, Neg. 20 V) m/z = 369 (M – H)–; H NMR
(300 MHz, DMSO-d6) d 10.34 (s, 1H), 8.68 (s, 1H), 7.02–
6.94 (m, 3H), 3.81 (d, J = 6.9 Hz, 2H), 3.75 (s, 3H), 2.62–
2.38 (m, 5H), 2.28–2.14 (m, 2H), 2.02–1.90 (m, 2H), 1.78–
1.62 (m, 2H), 1.28–1.12 (m, 1H), 0.60–0.52 (m, 2H), 0.36–
0.26 (m, 2H); IR (KBr) 3213, 2935, 2359, 2232, 1619, 1519,
1465, 1407, 1258, 1145, 1021; Anal. Found:
C21H26N2O4·0.25H2O (C, H, N).
5.1.9.9. (2s,3aR,5r,6aS)-5-Cyano-N-hydroxy-5-{4-methoxy-
3-[4-(methylsulfanyl)butoxy]phenyl}octahydropentalene-2-
carboxamide (14b). Compound 14b was obtained (55%)
from 14a as a white powder: TLC Rf = 0.38 (CHCl3/MeOH,
5.1.9.5. (2s,3aR,5r,6aS)-5-Cyano-5-[3-(cyclohexyloxy)-4-
methoxyphenyl]-N-hydroxyoctahydropentalene-2-carboxa-
mide (10b). Compound 10b was obtained (76%) from 10a as
a white powder: TLC Rf = 0.38 (CHCl3/MeOH, 10/1); MS
(APCI, Neg. 20 V) m/z = 397 (M – H)–; 1H NMR (300 MHz,
DMSO-d6) d 10.34 & 9.75 (s, 1H), 8.99 & 8.68 (s, 1H),
7.00–6.92 (m, 3H), 4.35–4.24 (m, 1H), 3.74 (s, 3H), 2.64–
2.36 (m, 5H), 2.25–2.16 (m, 2H), 2.03–1.92 (m, 2H), 1.90–
1
10/1); MS (FAB, Pos.) m/z = 419 (M + H)+; H NMR
(300 MHz, DMSO-d6) d 10.34 (s, 1H), 8.68 (s, 1H), 6.99–
6.91 (m, 3H), 3.99 (t, J = 6.2 Hz, 2H), 3.74 (s, 3H), 2.60–2.38
(m, 7H), 2.24–2.18 (m, 2H), 2.03 (s, 3H), 2.05–1.92 (m, 2H),
1.83–1.63 (m, 6H); IR (KBr) 3435, 3188, 3013, 2955, 1836,

570
H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
2233, 1626, 1520, 1467, 1421, 1388, 1257, 1224, 1149,
1022, 989; Anal. Found: C22H30N2O4S (C, H, N, S).
and was stored at –20 °C 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 solution was prepared by adding
3H-cAMP (300,000 dpm (5000 Bq)/assay) and 100 µmol/l
cAMP solution to 100 mmol/l Tris–HCl (pH 8.0) containing
5 mmol/l ethylene glycol-bis (b-aminoethyl ether) and
N,N,N′,N′-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 30 min at 30 °C.Assays were performed in duplicate
at 3–4 different concentrations of each test compound, and
the IC50 values were determined.
5.1.9.10. (2s,3aR,5r,6aS)-5-Cyano-N-hydroxy-5-[4-metho-
xy-3-(3,3,3-trifluoropropoxy)phenyl]octahydropentalene-2-
carboxamide (15b). Compound 15b was obtained (44%)
from 15a as a white powder: TLC Rf = 0.35 (CHCl3/MeOH,
1
10/1); MS (FAB, Pos.) m/z = 413 (M + H)+; H NMR
(300 MHz, DMSO-d6) d 10.33 (s, 1H), 8.66 (s, 1H), 7.05–
7.01 (m, 2H), 6.99–6.95 (m, 1H), 4.21 (t, J = 6.2 Hz, 2H),
3.75 (s, 3H), 2.85–2.69 (m, 2H), 2.64–2.38 (m, 5H), 2.28–
2.18 (m, 2H), 2.02–1.93 (m, 2H), 1.78–1.66 (m, 2H); IR
(KBr) 3193, 2961, 2234, 1627, 1521, 1467, 1420, 1395,
1258, 1223, 1150, 1071, 1022, 854; HRMS (FAB) calcd for
C20H24F3N2O4 (M + H)+ m/z = 413.1688, found 413.1678.
5.3.2. Inhibition of LPS-induced plasma TNF-␣ production
in rats
Male Crj:CD(SD)IGS rats aged 6 weeks (n = 7) were
fasted overnight, and the test compounds (0.01–
0.1 mg/10 ml/kg) were administered orally at 1 h before
i.v.injection of 1 µg/kg of LPS (Escherichia coli Serotype
055 B5). The plasma TNF-a level was measured with a
commercially available ELISA kit (R&D Systems) at 90 min
after LPS challenge. The percent inhibition (the dosage re-
quired to inhibit plasma TNF-a production by 50%) was
determined by the following formula:
5.2. X-ray crystallography
All diffraction data were obtained using a Rigaku AFC5R
4-circle diffractometer with graphite monochromate Cu-Ka
(k = 1.54187 Å) radiation and an RU-200 X-ray generator.
Data collection was carried out at room temperature. Correc-
tion was done for Lorentz polarization absorption and decay.
The software package teXsan [18] was used for analysis and
for drawing figures. The positions of non-H atoms were
easily determined using the program SHELXS86 [19], while
the positions of the H atoms were deduced from the non-H
atom coordinations and confirmed by Fourier synthesis.
Then the non-H atoms were refined with anisotropic tem-
perature parameters, and H atoms were refined w ith isotro-
pic parameters.
% Inhibition = 100 − (C − S) ⁄ (L − S) × 100
where C is the plasma TNF-a concentration in LPS-treated
animals pretreated with a test compound, L is the plasma
TNF-a concentration in LPS-treated animals pretreated with
saline, S is the plasma TNF-a concentration in saline-treated
animals pretreated with saline.
5.2.1. Summary of the X-ray crystallographic studies
5.3.3. SRS-A-mediated bronchoconstriction in guinea pigs
Male Hartley guinea pigs aged 7 weeks (n = 5) were
actively sensitized by intraperitoneal administration of 1 mg
of ovalbumin (OVA) containing 5 × 109 killed Bordetella
pertussis organisms on day 0. On day 14, the bronchocon-
strictor response was measured using a modified version of
the method of Konzett and Rössler. Bronchoconstriction was
induced by an i.v. injection of OVA (0.15–0.5 mg/kg). Sen-
sitized animals were treated with both a cyclooxygenase
inhibitor (indomethacin at 5 mg/kg i.v., 3 min before OVA)
and an antihistamine (pyrilamine at 1 mg/kg i.v., 1 min
before OVA) to ensure that endogenous SRS-A was solely
responsible for bronchoconstriction. Test compounds were
administered orally at 1 h before antigen challenge. Bron-
choconstrictor response was measured for 15 min and the
result was represented as the area under the curve (AUC
0–15 min).
Compounds
23b
23a
3
2a
Lattice parameter a(Å) 10.357(4) 8.248(6)
b(Å) 15.537(3) 22.814(6)
c(Å) 6.248(2) 10.107(7)
␣(°) 95.52(2)
11.14(2)
7.561(1)
9.98(1)
92.9(1)
114.8(1)
86.8(1)
2108(5)
P-1
11.135(6)
21.07(1)
9.962(4)
92.05(4)
14.95(3)
87.20(5)
2116(1)
P-1
b(°) 104.46(2) 105.53(6)
c(°) 106.76(2)
Volume (Å3)
Space group
No. observed
reflections
(I>3.00r(I))
No. variables
R-factor
916.7(5)
P-1
1832(2)
P21/a
2314
1758
2309
2091
226
226
509
511
0.063
0.049
0.055
0.085
0.066
0.076
0.099
0.105
Rw
R = R(|Fobs| – |Fcalc||)/R|Fobs
|
Rw = [(R w(|Fobs| – |Fcalc|)2/R w Fobs2)]1/2
5.3. Pharmacology
5.3.4. Gastric emptying in rats
5.3.1. Assay of human PDE4 activity
Male Sprague–Dawley rats were fasted overnight and
were orally administered test compounds or 0.5w/v% meth-
ylcellulose (10 ml/kg). In addition, 0.05 mg/ml of phenol red
solution was orally administered in a volume of 1.5 ml at
The method of Reeves et al. [20] was modified to isolate
phosphodiesterase type 4 isozyme (PDE4). The enzyme was
prepared from U937 cells derived from human monocytes,

H. Ochiai et al. / European Journal of Medicinal Chemistry 39 (2004) 555–571
571
20 min 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 (75 mg/kg, i.p.), and then the
stomach was isolated without leakage of phenol red. The
stomach was cut open and the phenol red solution was
drained into a beaker containing 100 ml of 0.1 N NaOH. Part
of the solution was filtrated (pore size: 0.45 µm) and the
absorbance at 546 nm was measured to determine the amount
of dye remaining in the stomach. Then the gastric emptying
rate was calculated by the following formula:
7b). Blood samples were taken from the jugular vein into a
heparinized syringe at 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 h after
oral administration.
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100 × (0.75 − concentration of dye in the stomach) ⁄ 0.75.
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0.05 mg/ml, which was achieved by adding 1.5 ml of phenol
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gastric emptying by the test compounds was calculated by
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solution of the test compound (10 µl) dissolved in DMSO
was added to 180 µl of whole blood, and the mixture was
pre-incubated for 30 min at 37 °C. Then 10 µl of 2 µg/ml of
LPS was added and incubated for 6 h at 37 °C, after which the
plasma TNF-a concentration was measured with a human
TNF-a ELISA kit (DIACLONE). Assays were performed in
duplicate at 3–4 different concentrations of each test com-
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5.3.6. Ferret emetic study
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night and test compounds were administered orally. Their
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5.3.7. Pharmacokinetic study
Male Sprague–Dawley (SD) rats weighing 280 20 g
were fasted for 20 h before and during the experiment. Test
compounds suspended in 0.5% methylcellulose were admin-
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