Prodrugs of 3-(3,4-dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicar boxylic acid (MGS0039): A potent and orally active group II mGluR antagonist with antidepressant-like potential

Article abstract of DOI:10.1016/j.bmc.2006.01.060

3-(3,4-Dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicar boxylic acid 5 (MGS0039) is a highly selective and potent group II metabotropic glutamate receptor (mGluR) antagonist (antagonist activities for mGluR2; IC₅₀ = 20.0 nM, mG

Full text of DOI:10.1016/j.bmc.2006.01.060

Bioorganic & Medicinal Chemistry 14 (2006) 4193–4207
Prodrugs of 3-(3,4-dichlorobenzyloxy)-2-amino-6-
fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039):
A potent and orally active group II mGluR antagonist
with antidepressant-like potential
Akito Yasuhara,* Masato Nakamura, Kazunari Sakagami, Toshiharu Shimazaki,
Ryoko Yoshikawa, Shigeyuki Chaki, Hiroshi Ohta and Atsuro Nakazato
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku,
Saitama-shi, Saitama 331-9530, Japan
Received 5 January 2006; revised 25 January 2006; accepted 26 January 2006
Available online 17 February 2006
Abstract—3-(3,4-Dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid 5 (MGS0039) is a highly selective
and potent group II metabotropic glutamate receptor (mGluR) antagonist (antagonist activities for mGluR2; IC50 = 20.0 nM,
mGluR3; IC50 = 24.0 nM) and is detected in both plasma (492 ng/mL) and brain (13.2 ng/g) at oral administration of 10 ng/mL
[J. Med. Chem. 2004, 47, 4750], but the oral bioavailability of 5 was 10.9%. In order to improve the oral bioavailability of 5, pro-
drugs of 5 were discovered by esterification of carboxyl group on C6-position of bicyclo[3.1.0]hexane ring. Among these compounds,
6-alkyl esters exhibited approximately 10-fold higher concentrations of 5 in the plasma and brain of rats after oral administration
(e.g., ethyl ester of 5; plasma, Cmax = 20.7 1.3 lM) compared to oral administration of 5 (plasma, Cmax = 2.46 0.62 lM). 3-(3,4-
Dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid 6-heptyl ester (7ao), a prodrug of MGS0039,
showed antidepressant-like effects in rat forced swimming test and mouse tail suspension test following oral administration. More-
over, following oral administration of 7ao in mice, high concentrations of MGS0039 were detected in both the brain and plasma,
while 7ao was barely detected. In this paper, we report the synthesis, in vitro metabolic stabilities, and pharmacokinetic profiles of
the prodrugs of 5, and the antidepressant-like effects of 7ao.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
(mGluR4, 6, 7, and 8) are negatively coupled to the
activity of adenylyl cyclase.9–11 Dysfunctional glutama-
tergic systems have been implicated in major psychiatric
disorders such as depression, anxiety, and schizophre-
nia.12 Indeed, the efficacy of group II mGluR agonists
in animal models and in clinical trials suggests that the
agonists may be effective in treating many diseases and
conditions such as schizophrenia,13–15 anxiety,16–19 and
panic disorder,20 while the efficacy of antagonists for
mGluRs has not been clarified in animal models or clin-
ical trials. This might be due to the lack of potent and
selective antagonists for mGluRs.
Glutamate is a major excitatory transmitter in the cen-
tral nervous system, and glutamate receptors are classi-
fied into ionotropic and metabotropic glutamate
receptors.1,2 The metabotropic glutamate receptors
(mGluRs) are coupled to G-protein. The eight mGluRs,
which have been cloned and sequenced, are classified
into three groups (I–III), based on their structure, intra-
cellular signaling mechanisms, and pharmacological
properties.3–9 Group I mGluRs (mGluR1 and mGluR5)
are positively coupled to phospholipase C, while group
II mGluRs (mGluR2 and 3) and group III mGluRs
Our laboratory initiated efforts to identify potent and
selective antagonists for Group II mGluRs, identifying
(1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyl)oxy-
6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid 521
(MGS0039), a potent and selective antagonist for Group
II mGluRs. Moreover, we found that group II mGluR
Keywords: MGS0039; Prodrug; mGluR antagonist; Metabotropic
glutamate receptor; Antidepressant-like effect.
*
Corresponding author. Tel.: +81 48 69 3066; fax: +81 48 652 7254;
e-mail: a.yasuhara@po.rd.taisho.co.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.060

4194
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
antagonists exhibited antidepressant-like potential in
experimental animal models.22
dipeptide carriers, which reports have suggested are
drug delivery systems for low permeability com-
pounds.27 Other ester-type prodrugs more lipophilic
than 5 were sought to increase absorption by passive
transport.28
As shown in Chart 1, a number of antagonists of group
II mGluR have been identified. Compound 1 was found
to be a weak but a selective antagonist for group III
mGluRs.23 Compound 2 was a potent and relatively a
selective antagonist for group II mGluRs. Of the com-
pounds shown in Chart 1, 3–6 bind with very high affin-
ity to rat brain mGluR and are potent functional
antagonists for human mGluRs.21,24–26 We recently
reported on the synthesis, in vitro pharmacological pro-
files, and structure–activity relationships (SARs) of 5, as
well as the pharmacokinetic profiles of several analogues
of 5.21 In particular, 5 (MGS0039) exhibited plasma and
brain concentrations higher than those of (2S)-amino-
2-((1S,2S)-2-carboxycycloprop-1-yl)-3-(9-xanthyl)propi-
onic acid 424,25 (LY341495) upon oral administration of
10 mg/kg to rats, but the oral bioavailability of 5 was
merely 10.9% (4, <5%). The low oral bioavailability of
4 and 5 might be attributable to the low absorption from
the gastrointestinal tract, possibly due to the structural
feature having two carboxyl groups and an amino
group. In contrast, all compounds shown in Chart 1
are modified from glutamic acid and mainly the struc-
tural feature may affect the activity as antagonists for
mGluRs. In an attempt to increase the gastrointestinal
absorption of 5, prodrugs with modification to the car-
boxyl and amino groups in 5 were synthesized, since
prodrugs had potential for good absorption and were
ascribed to biologically active compound 5.
We then examined the antidepressant-like potential of
7ao, a prodrug of MGS0039, using two behavioral des-
pair models of depression.
In this paper, we report on synthesis, in vitro metabolic
stabilities, and pharmacokinetic profiles of prodrugs of
the 5 and 6, as well as the antidepressant-like effects of
7ao.
2. Chemistry
Compound 6, the defluorine analogue of 5, was synthe-
sized from the intermediate 8,29 by the same procedure
for synthesis of 5, as shown in Scheme 1. Etherification
of the 3-hydroxy group in intermediate 8 was performed
using a benzyl trichloroacetimidate, which was prepared
from 1,1,1-trichloroacetonitrile and 3,4-dichlorobenzyl
alcohol in the presence of a catalytic amount of trifluo-
romethanesulfonic acid, yielding 9.30,31
Reduction of the azide group in 9 using the Staudinger
reaction32,33 provided 10 without reductive cleavage of
the benzyl group at C3-position of bicyclo[3.1.0]hexane
ring. Finally, 6 was obtained by hydrolysis of two esters
in 10 with LiOH.
We first designed dipeptides and esters of 5 and/or 6 and
then investigated in vitro metabolic stabilities with liver
S9 fraction and plasma as well as the pharmacokinetic
profiles of the prodrugs. The peptidemimetic prodrugs
of 5 were targeted to increase intestinal absorption via
Scheme 2 depicts the synthesis of dipeptides 7aa and
7ab, 5 coupled with alanine at the amino group of
C2-position and with leucine at the carboxy group of
C6-position in 5.
We reported that reduction of the azide group of 11, an
intermediate for synthesis of 5, provided 12 by the Stau-
dinger reaction.21 The coupling reaction of 12 and a
mixed anhydride of N-Boc-L-alanine with i-butyl chlo-
roformate allowed to proceed to yield 13. Finally, the
protective groups, consisting of ethyl ester and Boc
HOOC
COOH
NH2
H
H2O3P
COOH
NH2
Me
Me
1
CO2Et
2
CO2Et
H
H
a
O
H
H
H
H
N3
N3
H
RO
CO2Bn
COOH
HO CO2Bn
H
9
HOOC
8
COOH
NH2
H NH2
HOOC
CO2Et
CO2H
H
H
H
c
3
4
b
H
H
X
H
NH2
RO CO2Bn
NH2
RO CO2H
COOH
10
6
H
Cl
O
R= 3,4-dichlorobenzyl
X = F: 5
X= H, 6
NH2
HOOC
Scheme 1. Reagents and conditions: (a) 3,4-Cl2-C6H5CH2OC(@NH)
CCl3, TfOH, CHCl3, cyclohexane, rt (b) Me3P, THF, H2O, rt (c) LiOH,
THF, H2O, rt.
Cl
Chart 1. Group II mGluR agonist and antagonists.

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4195
F
F
F
F
H
H
H
H
CO2Et
CO2Et
CO2H
CO2H
d
a
H
N3
H
NH2
H
N3
H
NH2
RO
EtO2C
RO
EtO2C
RO
EtO2C
RO
EtO2C
12
14
11
7ac
a
e
a
f
b
H
F
F
H
H
X
X
H
N
H
H
CO2Et
CO2R1
CO2Et
CO2H
O
H
c
H
H
H
RO
EtO2C
RO
N3
NH2
RO
HO2C
RO
HO2C
NH2
NH2
EtO2C
12
15
7ad-bb
5, 6
b
d
f
F
H
H
N
F
H
F
H
CO2Et
H
CO2Et
X
N
H
CO2H
O
CO2R1
O
H
O
H
RO
EtO2C
e
NH
H
RO
EtO2C
H
NH2
RO
O
N
RO
O
O
H
O
O
16
O
O
NHBoc
13
17
18av-ay, 18ba,1 8bb
R= 3,4-dichlorobenzyl
c
H
N
F
H
Scheme 3. Reagents and conditions: (a) LiOH, THF, H2O, 0 ꢁC; (b)
LiOH, THF, H2O, rt; (c) R1OH, SOCl2. 50–70 ꢁC (d)—(i) allyl
chloroformate, dioxane, satd NaHCO3, rt; (ii) paraformaldehyde,
TsOH, benzene, reflux using Dean–Stark apparatus; (e) R1Cl, NaI,
K2CO3, DMF, 60–80 ꢁC; (f) Pd(PPh3)4, 1,3-dimethylbarbituric acid,
CHCl3, 40 ꢁC.
H
CO2H
F
H
O
H
CO2H
RO
NH2
HO2C
H
RO
NH
7ab
HO2C
O
HCl
H
Me
R= 3,4-dichlorobenzyl
NH2
7aa
at C6-position in 5 or 6 as a single product. Compounds
7av–ay, 7ba, and 7bb required protection of a-amino
acid moiety at C2-position in 5 or 6, since directed ester-
ification of 5 with alkoxycarbonyloxyalkyl, butyroyl-
oxymethyl, and 2-morphorin-4-ylethyl halide under
basic conditions resulted in diesters. Selective a-carbon-
yl-protecting methods with 5-oxazolidinone deriva-
tive34–36 were adopted for syntheses of compounds
7av–ay, 7ba, and 7bb.34–36 Compound 5 was allowed
to react with allyl chloroformate, an amine-protective
agent, in the presence of aqueous sodium bicarbonate
in dioxane to produce the corresponding allyl carba-
mate. Then the reaction of the allyl carbamate with
paraformaldehyde in the presence of p-toluenesulfonic
acid as a catalyst was carried out using the Dean–Stark
apparatus to obtain 17. An esterification of 17, having
the protecting group on a-amino acid moiety, with al-
koxycarbonyloxyalkyl, butyroyloxymethyl, and 2-mor-
phorin-4-ylethyl halide, and excess amount of K2CO3
provided 18av–ay, 18ba, and 18bb. Cleavage of the pro-
tective groups of 18av-ay, 18ba, and 18bb with
Pd(PPh3)4 and barbituric acid37 yielded 7av–ay, 7ba,
and 7bb.
Scheme 2. Reagents and conditions: (a) Me3P, THF, H2O, rt; (b) N-
Boc-L-alanine, N-methylmorphorin, i-Butyl chloroformate, rt; (c) (i)
LiOH, THF, H2O, rt (ii) 4N HCl, rt; (d) LiOH, THF, H2O, 0 ꢁC; (e)
HOBT, EDC, L-leucine Et ester, N-methylmorphorin, 0 ꢁC; (f) LiOH,
THF, H2O, rt.
and ethyl ester, were sequentially removed by hydrolysis
with LiOH and HCl to obtain 7aa. Selective hydrolysis
of ethyl ester at C6-position in 11 with LiOH at 0 ꢁC
provided 14 as a sole product, due to the high reactivity
of ethyl ester for hydrolysis arising from the electroneg-
ativity of the fluoride atom at C6-position in 11. The
coupling reaction of 14 with L-leucine in the presence
of HOBT and EDC provided 15. According to the prep-
aration similar to that for 7aa, reduction of the azide
group of 15 using the Staudinger reaction and hydrolysis
of ethyl ester 16 with basic conditions provided dipep-
tide 7ab.
The synthesis of esters 7ac–bb, monoesters at C2- and
C6-position, from 5 or 6 is shown in Scheme 3.
Compound 7ac, a monoester at C2-position in 5, was
synthesized from 1221 by selective hydrolysis of ethyl es-
ter, according to the preparation of monoester 14, the
first step of synthesis of 7ab. Selective esterification of
5 with alkyl alcohol and benzyl alcohol as a solvent
was performed in the presence of SOCl2 at 50–80 ꢁC
to obtain the corresponding monoesters 7ad–au or 7az
3. Results and discussion
3.1. Metabolic stability study
We first investigated the in vitro metabolism of the pro-
drugs of 5 or 6 using plasma and liver S9 fraction of rat.

4196
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
Table 1 shows the transformed ratio from prodrugs to 5
or 6. Following incubation of dipeptides 7aa and 7ab,
compounds with an alanine and a leucine incorporated
at the C2-position and C6-position of bicyclo[3.1.0]hex-
ane ring, no transformation to 5 was observed. The
incubation of 7ac, ethyl ester at the 2-position, with
plasma and S9 fraction of rat also failed to yield 5.
in the high acidity of carboxylic acid at the C6-position
of 5, resulting in ready hydrolysis of the esters using
plasma and liver S9 fractions.
3.2. Plasma concentration profiles and brain penetration
in rats
We investigated the bioavailability of 7aa–ac and 7ae to
determine the suitable chemical modifications (positions
and substitutions) for prodrugs in in vivo studies. Bio-
availability studies of 7aa–ac and 7ae were performed
in fasting Wistar rats, and the pharmacokinetic param-
eters are shown in Table 2.
On the other hand, all compounds of alkyl and benzyl
esters (7ad-as and 7au) at the C6-position of bicy-
clo[3.1.0]hexane ring yielded 5 when incubated with rat
plasma and/or liver S9.
The ratio of hydrolysis of 7az (plasma; 17.7%, S9;
17.8%), an ethyl ester of 6 (defluorine analogue of 5),
was lower than that of 7ae (plasma; 88.7%, S9; 95.9%)
under the same incubation conditions. All of 7av, 7aw,
7ba, and 7bb, of which esters were activated for enzy-
matic hydrolysis and the commonly employed in pro-
drug moieties for acid-derived drugs,38–40 transformed
into biologically active substances 5 in high ratio
(>95%). These findings suggest that the fluorine atom
at the C6-position of bicyclo[3.1.0]hexane ring plays an
important role in the hydrolysis of alkyl esters. We infer
that the high electron-negativity of fluorine atom results
Following intravenous administration of 5 at 1 mg/kg,
the plasma clearance was 176.3 mL/h/kg and the volume
of distribution was 302.8 mL/kg. The values for half-life
(t1/2) and AUCinf were 2.9 h and 15.2 lM h, respectively.
Oral bioavailability studies in fasting rats were per-
formed at 10 mg/kg. Plasma concentration reached max-
imum levels (Cmax) of 2.5 lM at 2.7 h, declining
thereafter with a t1/2 of 2.8 h. The value of AUCinf was
16.6 lM h, and absolute bioavailability was 10.9%. This
low bioavailability is believed to be attributable to low
absorption rather than the first pass effect, since 5 is
stable in rat liver S9 fractions.
Table 1. The ratios of hydrolysis of 7aa–bb to 5 or 6
After oral administration of 7aa–ac and 7ae at a dose of
10 mg/kg, plasma concentrations of 5 reached Cmax of
0.01, 0.3, 1.2, and 20.7 lM at 4.0, 3.3, 4.0, and 1.0 h,
respectively. The values of AUCinf were 0.1, 2.0, 23.8,
and 86.2 lM h, and figures for oral bioavailability deter-
mined by the AUCinf values following intravenous
dosing of 5 were 0.1%, 1.7%, 16.8%, and 66.6%,
respectively.
X
H
COOR1
H
Cl
Cl
O
NH2
R2OOC
7
R1
H
R2
X
Plasma Liver S9
Compared to the three other prodrugs, compound 7ae
exhibited the highest plasma concentration of 5 at the
early sampling time points for both intravenous and oral
administration, with oral bioavailability of biologically
active substance 5 improving from 10.9% to 66.6%.
These findings suggest that 7ae was absorbed rapidly
and hydrolyzed effectively as indicated in in vitro exper-
iments with rat plasma and liver S9 fractions.
aa
Ala
H
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
H
H
H
1.2
0.2
<1.0
<1.0
<1.0
82.6
95.9
100
100
100
100
100
93.1
99
ab Leu
ac
ad Me
H
0.2
87.8
88.7
91.4
88.1
91.2
89.7
62.9
100.0
100.0
NT
ae
af
Et
n-Propyl
ag i-Propyl
ah n-Butyl
ai
aj
i-Butyl
n-Pentyl
The penetration of 5 into the brain relative to that in
plasma was examined in fasting Wistar rats following
oral doses of 10 mg/kg of 5 and 7aa–ac and 7ae. As
shown in Table 3, after oral dosing of 5, the brain levels
were 0.02 and 0.03 lM at 3 and 6 h post-dose, respec-
tively. The corresponding plasma levels were 1.13 and
1.30 lM, and brain to plasma concentration ratios were
0.03 in both time points. Following oral dosing of 7ae,
the increasing of brain levels of 5 was observed. These
levels were 0.30 and 0.25 lM at 3 and 6 h post-dose,
respectively. The corresponding plasma levels were
16.28 and 4.75 lM, and brain/plasma concentration ra-
tios were 0.02 and 0.05, indicating that elevated levels in
the brain were due to higher plasma levels of 5, since
brain/plasma concentration ratios were virtually the
same as dosing of 5. On the other hand, following oral
dose of 7aa–ac, increasing brain levels were not
observed.
ak 3-Methylbutyl
al n-Hexyl
am 4-Methylpentyl
an Cyclohexyl
ao n-Heptyl
99.1
100
98
95.6
NT
NT
ap 5-Methylhexyl
aq Cyclohexylmethyl
94.6
99.1
94.9
90.6
NT
100
100
100
NT
95.4
17.8
97.7
100
33.7
NT
NT
ar
as
at
n-Octyl
6-Methylheptyl
n-Decyl
NT
au Benzyl
av
95.6
98.2
96.7
NT
CH(Me)OCOOEt
aw CH(Me)OCOO-cyclohexyl
ax CH2OCO(CH2)2Me
ay 2-Morphorin-4-ylethyl
54.9
17.7
100.0
100.0
az
ba CH(Me)OCOOEt
Et
bb CH(Me)OCOO-cyclohexyl
NT, not tested.

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4197
Table 2. Pharmacokinetic parameters of 5 after intravenous (1 mg/kg) and oral dosing (10 mg/kg) of prodrugs (7aa–ae) to Wistar rats
Dosed compound
Route
Dose (lmol/kg)
Tmax (h)
Cmax (lM)
t1/2 (h)
AUCinf (lM h)
F (%)
7aa
7ab
7ac
7ae
iv
iv
iv
iv
2.1
2.0
2.5
2.3
1.2 0.8
1.7 2.1
2.0 0.0
0.03 0.0
0.03 0.02
0.04 0.01
0.4 0.10
40.2 6.96
11.5 12.9
8.7 4.6
4.0 0.8
1.6 0.2
0.3 0.2
0.1 0.0
3.5 0.7
19.5 1.5
7aa
7ab
7ac
7ae
po
po
po
po
20.6
20.4
24.6
22.6
4.0 0.0
3.3 1.2
4.0 0.0
1.0 0.0
0.01 0.00
0.3 0.2
1.2 0.6
5.4 1.2
3.5 1.3
12.4 4.0
2.0 0.4
0.1 0.1
2.0 0.8
0.1
1.7
23.8 10.9
86.2 6.9
16.8
66.6
20.7 1.3
Results are expressed as the mean SD of three animals.
Table 3. Brain penetration of 5 after oral dosing of 5 and its prodrug (10 mg/kg) to fasting Wistar rats
Time (h)
3
Dosed compound
Dose (lmol/kg)
Plasma (lM)
Brain (lM)
Brain/plasma
5
26.4
20.6
20.4
24.6
22.6
1.13 0.08
0.01 0.00
0.41 0.30
1.62 0.42
16.28 0.95
0.02 0.01
0.00 0.00
0.00 0.00
0.02 0.00
0.30 0.03
0.03 0.01
0.00 0.00
0.00 0.00
0.01 0.00
0.02 0.00
7aa
7ab
7ac
7ae
6
5
26.4
20.6
20.4
24.6
22.6
1.30 0.91
0.01 0.00
0.23 0.11
1.17 0.53
4.75 0.99
0.03 0.02
0.00 0.00
0.00 0.01
0.03 0.01
0.25 0.01
0.03 0.01
0.00 0.00
0.01 0.03
0.03 0.00
0.05 0.01
7aa
7ab
7ac
7ae
Results are expressed as means SD of three animals.
In the studies above, 7ae showed the highest improve-
ment in oral bioavailability, and this result well corre-
sponded with the fact of efficient enzymatic hydrolysis
in the in vitro study. Moreover, an approximately 10-
fold improvement was obtained in brain levels of 5. In
contrast, no improvement in oral bioavailability or
brain levels was observed following oral dosing of
7aa–ac. These results are consistent with the resistance
to enzymatic hydrolysis observed in the in vitro study.
Based on these pharmacokinetic profiles of four
prodrugs and in vitro experiments, it may be difficult
to improve the oral bioavailability of 5 via esters at
the C2-position and derivatives of 5 modified with
amino acids. Thus, we focused our examination on
esters on the C6-position of bicyclo[3.1.0]hexane ring.
In cases of 7av and 7aw, bioavailabilities based on plas-
ma concentration of 5 were 12.2% and 12.7%, respec-
tively. Compound 7ay, a substituted alkyl ester, was
administered at a dose of 10 mg/kg to fasting rats. Plas-
ma concentrations of 5 reached peak levels at 2.3 h with
the Cmax value of 0.5 lM. Bioavailability was a mere
2.0%. Compounds 7au–aw and 7ax completely trans-
formed into 5 in in vitro experiments, and large non-en-
zymatic degradations were observed. These products,
which are alkoxycarbonylethyl esters, did not show im-
proved bioavailability for 5, in spite of ensuring stability
in dosing solution. These findings suggest that resistance
to chemical degradation as well as high hydrolysis rate
in liver S9 and plasma are required to improve bioavail-
ability of 5.
Oral bioavailability studies of alkyl esters 7ad–bb were
performed in rats at doses of 10 mg/kg. The pharmaco-
kinetic parameters are shown in Table 4.
We next investigated 7az–bb, prodrugs of 6 (defluorine
analogue of 5).
We examined the pharmacokinetic profiles of 6, which
exhibited high affinity for mGluR2 (Ki = 2.54 nM)
and potent antagonist activity for mGluR2
(IC50 = 34.2 nM) as well as 5.41 Following intravenous
administration of 1 mg/kg 6 to non-fasting rats, plasma
levels appeared to decline biphasically with t1/2 of 4.3 h
and total plasma clearance of 258.4 mL/h/kg. The vol-
ume of distribution was estimated at 446.4 mL/kg, and
the AUC value was 10.8 lM h. Following oral admin-
istration of 10 mg/kg 6 to fasting rats, plasma concen-
trations reached a Cmax of 0.4 lM at 4.7 h, declining
thereafter with a t1/2 of 3.8 h. The AUCinf value was
3.8 lM h, and absolute bioavailability was 3.6%. We
also investigated the prodrugs of 6 to improve the oral
bioavailability of 6 based on the successful results with
prodrugs for 5.
Compounds 7ad–at, alkyl esters, were administered at a
dose of 10 mg/kg to fasting rats. Plasma concentrations
of 5 reached peak levels within 0.5–1.5 h. Bioavailabili-
ties based on plasma concentration of 5, for 7ad, 7ag,
and 7ai, were >70% with Cmax values of 22.9–28.9 lM.
The next highest values for bioavailability (50.7–67.4%)
were obtained in 7af, 7ah, 7aj, 7am, 7an, and 7aq with
Cmax values of 15.5–24.8 lM. Brain levels were 0.19–
0.24 lM at 6 h. All alkyl esters were completely hydro-
lyzed by liver S9 fractions as well as plasma in rats with
improved bioavailability and increase in brain levels.
In the case of 7au, a benzyl ester, plasma concentrations
of 5 reached the maximum levels at 1.3 h with a Cmax
value of 13.2 lM and bioavailability was 41.5%.

4198
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
Table 4. Pharmacokinetic parameters and brain levels of 5 and 6 after oral dosing (10 mg/kg) of 7ad–bb to male Sprague–Dawley or Wistar rats
Dosed compound
Dose (lmol/kg)
Tmax (h)
Cmax (lM)
t1/2 (h)
AUCinf (lM h)
Fa (%)
Brain at 6 h (lM)
7ad
7af
7ag
7ah
7ai
23.3
21.9
21.9
21.2
21.2
22.3
22.3
21.6
21.6
20.1
21.0
21.0
21.0
19.6
20.4
21.6
19.3
21.4
20.2
18.2
1.0 0.0
1.0 0.0
1.0 0.0
1.3 0.6
0.8 0.3
1.3 0.6
1.3 0.6
1.0 0.0
0.8 0.3
1.0 0.0
1.0 0.0
1.3 0.6
0.5 0.0
1.3 0.6
1.0 0.0
0.7 0.3
0.8 0.3
1.3 0.6
1.0 0.0
1.0 0.0
28.9 7.2
24.8 6.2
22.9 2.1
17.0 0.5
24.1 6.6
23.9 4.0
17.8 5.4
20.8 5.2
18.3 5.0
16.6 3.1
14.9 3.7
12.9 0.9
17.3 1.3
15.5 3.2
17.7 4.2
15.0 1.2
14.5 0.9
13.2 2.4
5.3 3.1
1.4 0.2
1.6 0.1
2.0 0.8
1.8 0.1
1.5 0.3
1.1 0.1
1.4 0.4
1.2 0.3
2.0 0.9
1.7 0.3
1.2 0.2
2.0 0.1
1.6 0.5
1.5 0.6
1.2 0.2
1.6 0.4
1.1 0.1
1.4 0.1
1.2 0.1
1.0 0.1
NT
90.0 18.1
80.6 12.8
88.3 6.4
63.7 1.8
86.0 13.6
72.0 9.7
54.4 18.8
54.9 4.6
59.9 10.2
59.9 15.6
45.4 14.9
105.4 27.4
45.1 6.0
61.5 20.3
48.6 8.3
42.9 7.3
43.2 3.8
48.3 11.2
13.5 7.4
12.6 1.3
70.6
67.4
73.9
54.9
74.1
59.1
44.7
46.5
50.7
54.5
39.6
73.0b
39.4
57.5
43.6
36.3
41.0
41.5
12.2
12.7
0.20 0.01
0.23 0.03
0.25 0.03
0.24 0.03
0.26 0.08
0.23 0.05
0.20 0.05
0.21 0.01
0.19 0.01
0.22 0.05
0.19 0.01
7aj
7ak
7al
7am
7an
7ao
(7ao
7ap
7aq
7ar
7as
7at
7au
7av
7aw
7ax
7ay
7az
7ba
7bb
5
0.15 0.04
0.19 0.01
0.19 0.01
0.18 0.04
0.18 0.21
0.19 0.03
0.07 0.03
0.06 0.00
4.2 0.7
17.7
23.5
21.0
18.9
26.4
27.8
2.3 1.5
2.0 0.0
1.7 0.6
1.3 0.6
2.7 1.2
4.7 3.1
0.5 0.1
3.7 0.2
2.8 0.9
1.9 1.4
2.5 0.6
0.4 0.1
1.1 0.5
1.9 0.6
1.2 0.3
1.8 1.1
2.8 0.1
4.0 0.5
1.9 0.4
18.5 1.9
9.8 2.2
6.8 4.3
16.6 6.1
3.8 0.2
2.0
20.2
11.9
9.2
0.00 0.00
0.09 0.02
0.07 0.01
0.05 0.04
0.03 0.02
0.01 0.00
10.9
3.6
6
Results are expressed as means SD of three animals. 7ad–7bb: Sprague–Dawley rats; 5 and 6: Wistar rats; NT, not tested.
a F = (AUCinf/dose)/(AUCinf, iv/dose, iv) · 100.
b The oral bioavailability was evaluated by adding the sampling points up to 24 h.
After oral administration of 10 mg/kg 7az–bb to fasting
rats, the plasma concentration of 6 for each prodrugs
reached the maximum levels of 3.7, 2.8, and 1.9 lM at
2.0, 1.7, and 1.3 h, respectively. The corresponding AU-
Cinf values were 18.5, 9.8, and 6.8 lM h, and bioavaila-
bilities of the active form were 20.2%, 11.9%, and 9.2%,
respectively. Plasma concentrations of 6 reached peak
levels of 4.8, 0.0, and 0.0 lM at 0.5, 0.8, and 0.8 h,
respectively.
prodrug 7ad–at of 5. In contrast, 7ba and 7bb were sta-
ble against chemical degradation and were completely
hydrolyzed by liver S9 fractions. However, the stability
did not improve bioavailability. These findings suggest
the need for further investigation of the pH range for
chemical stability, stability in the absorption phase (in
intestinal microsomes and/or S9 fractions), penetration
into absorption sites, and other related issues.
Finally, we confirmed that 7ao was not a ligand for
mGluR2. The affinity of 7ao was evaluated by [3H]-
(1S,2S,3S,5R,6S)-2-amino-3-fluorobicyclo[3.1.0]hexane-
2,6-dicarboxylic acid binding using CHO cells stably
expressing mGluR2.13 Compound 7ao did not exhibit
significant affinity for mGluR2 (7ao: Ki = >1000 nM,
5: Ki = 2.38 nM21). These findings suggest that 7ao is
not a ligand for mGluR2 and acts as a prodrug of 5.
Compound 7ae, an ethyl ester of 5, was almost com-
pletely hydrolyzed by rat liver S9 fractions and plasma,
but 7az, an ethyl ester of 6, was stable in liver S9 frac-
tions and plasma. Moreover, 7ae exhibited increased
the bioavailability of 5 to 66.6% in rats, while 7az in-
creased the bioavailability only to 20%. Based on the im-
proved bioavailability in ester-type prodrugs related to
the hydrolysis rate of prodrugs in liver S9 fractions
and plasma, the fluoride atom in 7ad–ay appears to play
an important role in hydrolysis. Ester-type prodrugs im-
proved the bioavailability of 5 having fluoride atom at
the C6-position of the bicyclo[3.1.0]hexane ring.
3.3. Antidepressant-like effects of the prodrug (7ao) of 5
We examined the antidepressant potential of 7ao, a pro-
drug of 5, in two despair behavioral models of
depression.
In the case of 7av and 7aw, which are alkoxycarbonyl-
oxyethyl esters of 5, we observed non-enzymatic degra-
dation with 40–80%, although they were completely
transformed to 5 in liver S9 fractions. We had assumed
that 7av and 7aw might have been transformed into 5 in
the gastrointestinal tract due to low stabilities for chem-
ical degradation and the hydrolysis has given the low
bioavailability of 5 comparing with the alkyl ester-type
3.4. Forced swimming test in rats
Oral administration of 7ao dose-dependently and signif-
icantly reduced immobility time with the lowest active
dose of 3 mg/kg (Fig. 1a). According to the behavioral
scoring method reported by Detke et al.,42 7ao signifi-
cantly increased swimming behavior without changing

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4199
a
200
180
160
140
120
100
80
40
b
vehicle
1mg/kg
3mg/kg
10mg/kg
**
**
35
30
25
20
15
10
5
*
*
*
*
60
40
20
0
0
climbing
swimming Immobility
vehicle
1
3
10
7ao (mg/kg, p.o.)
Figure 1. Effect of 7ao in forced swimming test in rats. The effect of 7ao was evaluated by both the method (a) duration of immobility and by the
time-sampling technique (b) described by Detke et al. (1995). Data represent means SEM (n = 8). *P < 0.05, **P < 0.01 versus vehicle-treated
group (Dunnett’s test).
Table 5. Mean plasma and brain levels (lM) of 5 and 7ao after oral
dosing of 7ao to male ICR mice at a dose of 1, 3, and 10 mg/kg
climbing behavior (Fig. 1b), similar to the findings for 5
and fluvoxamine.21
Dose (mg/kg)
5
7ao
Brain
3.5. Tail suspension test in mice
Plasma
Brain
Plasma
1
3
0.52 0.35 0.04 0.02 0.00 0.00 0.00 0.00
1.03 0.31 0.10 0.02 0.00 0.00 0.00 0.00
5.51 2.79 0.36 0.13 0.01 0.01 0.00 0.00
Compound 7ao showed dose-dependent and significant
antidepressant-like effects on the mouse tail suspension
test following oral administration (Fig. 2).
10
Results are expressed as means SD of 15 animals.
After 10 min test session, mice were sacrificed, and con-
centrations of 5 and 7ao in the brain and plasma were
measured. High concentrations of 5 were detected in
both the brain and plasma, while 7ao was barely detect-
ed (Table 5).
Oral administration of 7ao exhibited significant antide-
pressant-like effects in two behavior despair models,
the forced swimming test and the tail suspension test,
both of which are indicative of antidepressant-like po-
tential in humans. Compound 7ao showed antidepres-
sant-like effects at doses, which did not affect
locomotor activity, indicating that antidepressant-like
effects are not ascribed to altered locomotion.
3.6. Effect on spontaneous locomotor activity
Compound 7ao did not affect spontaneous locomotor
activity in rats at 3 and 10 mg/kg, po, while 7ao signifi-
cantly increased locomotor activity at 30 mg/kg, po
(Fig. 3). In contrast, oral administration of 7ao did not
affect spontaneous locomotor activity up to 30 mg/kg
in mice (Fig. 3).
Moreover, following oral administration of 7ao in mice,
a large amount of 5 was detected in both brain and plas-
ma (at rats more than 10-fold that of the IC50 value),
while 7ao was barely detected.
These results indicate that 7ao, a prodrug of 5, shows
antidepressant-like effects in rodent models of depres-
sion following oral administration, and that these effects
are attributable to 5.
300
250
200
**
**
150
4. Conclusion
100
50
0
This paper presented in vitro metabolic stabilities and
pharmacokinetic profiles for prodrugs of 3-(3,4-dichlo-
robenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,
6-dicarboxylic acid 5 (MGS0039) and 3-(3,4-dichlo-
robenzyloxy)-2-aminobicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid 6 and antidepressant-like effects of 7ao. Of
the prodrugs presented, the alkyl esters at the C6-posi-
tion of the bicyclo[3.1.0]hexane ring exhibited approxi-
mately 10-fold higher concentrations of MGS0039 in
plasma and brain, while most alkyl esters were not
vehicle
1
3
10
7ao (mg/kg, p.o.)
Figure 2. Effect of 7ao in tail suspension test in mice. Compound 7ao
was administered po 2 h prior to the test. Data represent mean-
s
test).
SEM (n = 15). **P < 0.01 versus vehicle-treated group (Dunnett’s

4200
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
Rats
Mice
10000
8000
6000
4000
2000
0
30000
25000
20000
15000
10000
5000
0
*
vehicle
3
10
30
vehicle
3
10
30
7ao (mg/kg, p.o.)
7ao (mg/kg, p.o.)
Figure 3. Effect of 7ao on spontaneous locomotor activity in rats and mice. Compound 7ao was administered po 2 h prior to the test. Data represent
means SEM (n = 8 for rats, 10 for mice). *P < 0.05 versus vehicle-treated group (Dunnett’s test).
detected. In contrast, the prodrugs of 6 exhibited poor
pharmacokinetic profiles. These findings indicate that
the fluorine atom at the C6-position of the bicy-
clo[3.1.0]hexane ring plays an important role in improv-
ing pharmacokinetic profiles, especially for hydrolysis of
alkyl ester-type prodrugs.
for 30 min at room temperature, the formed precipitate
was filtered off. The filtrate was concentrated under re-
duced pressure to yield crude 3,4-dichlorobenzyl-2,2,2-
trichloroacetoimidate (2.38 g) as a brown viscous liquid.
Trifluoromethane sulfonic acid (360 lL) was added to
the mixture of 3,4-dichlorobenzyl-2,2,2-trichloroace-
toimidate (6.77 g, 21.1 mmol), 8 (5.10 g, 14.0 mol),
CH2Cl2 (18 mL), and cyclohexane (36 mL) under a
nitrogen atmosphere. The mixture was then stirred for
1 h at room temperature. Trifluoromethane sulfonic
acid (360 lL) was added to the mixture, which was stir-
red for 2 h, after which the precipitate was filtered off.
Saturated NaHCO3 was added to the filtrate with ice-
cooling. The mixture was extracted with CHCl3, and
the CHCl3 layer was washed with saturated brine, dried
with Na2SO4, concentrated under reduced pressure, and
chromatographed (hexane/AcOEt 15:1) to yield 9
Compound 7ao, a prodrug of MGS0039, showed antide-
pressant-like effects in rat forced swimming test and
mouse tail suspension test following oral administration,
effects attributable to MGS0039.
5. Experimental
5.1. Chemistry
Melting points were determined on a Yanaco MP-500D
melting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were ob-
tained using a Varian Gemini 2000 (200 MHz), or
Varian Unity Inova 300 (300 MHz), JEOL Alpha500
or JEOL Lambda500. Chemical shifts are reported in
parts per million relative to tetramethylsilane (TMS)
1
(2.36 g, 32%) as a colorless oil. H NMR (200 M Hz,
CDCl3, TMS) d 1.26 (3H, t, J = 7.0 Hz), 1.75 (1H, t,
J = 3.1 Hz), 2.02–2.36 (4H, m), 3.55 (1H, dd, J = 8.8,
7.5 Hz), 4.13 (2H, q, J = 7.0 Hz), 4.34 (1H, d,
J = 12.3 Hz), 4.51 (1H, d, J = 12.3 Hz), 5.22 (1H, d,
J = 11.9 Hz), 5.34 (1H, d, J = 11.9 Hz), 7.01 (1H, dd,
or
sodium
3-trimethylsilylpropionate-2,2,3,3-D4
J = 8.1, 2.0 Hz), 7.22–7.47 (7H, m); MS (ion spray)
26
(Positive) m/z; 526 (M+Na)+; ½aꢀ +7.83 (c 3.5, CHCl3).
(TMSP) as an internal standard. Mass spectra (MS)
were obtained on a JEOL JMS-SX102 (FAB) or Micro-
mass Platform LC (Ion Spray). Optical rotations were
determined with a JASCO DIP-360 polarimeter and
are reported at the sodium D-line (589 nm). Elemental
analyses were performed using a Perkin-Elmer 2400. Sil-
ica gel (C-200, 100–200 mesh (Wako Pure Chemical))
was used for column chromatography, using the solvent
systems (volume ratios) indicated below.
D
5.1.2. (1S,2R,3R,5R,6S)-2-Amino-3-(3,4-dichlorobenzyl-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 2-benzyl
6-ethyl ester (10). A mixture of 9 (2.31 g, 4.42 mmol),
Me3P (1 M THF solution, 4.86 mL), THF (64 mL),
and H2O (6.4 mL) was stirred for 12 h at room temper-
ature. Ether was added to the reaction mixture, and the
organic layer of the mixture was separated. The organic
layer was washed with saturated NaHCO3 and saturated
brine, dried with Na2SO4, evaporated, and chromato-
graphed (hexane/AcOEt 4:1–1:1) to yield 10 (1.51 g,
5.1.1. (1S,2R,3R,5R,6S)-2-Azido-3-(3,4-dichlorobenzyl-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 2-benzyl
6-ethyl ester (9). A THF (153 mL) solution of 3,4-dichlo-
robenzyl alcohol (1.04 g, 9.62 mmol) was added to a
Et2O (2 mL) suspension of NaH (60%, 38 mg,
0.962 mmol) and stirred for 0.5 h at room temperature.
Trichloroacetonitrile (0.96 mL, 9.62 mmol) was added
to the mixture with ice-cooling and stirred for 1.5 h at
room temperature. Pentane (1 mL) and methanol
(37 lL) were added to the reaction mixture. After stirring
1
69%) as a pale yellow oil. H NMR (200 MHz, TMS,
CDCl3) d 1.24 (3H, t, J = 7.3 Hz), 1.73 (1H, t,
J = 3.1 Hz,), 1.95–2.33 (4H, m), 3.47 (1H, dd, J = 8.8,
7.0 Hz), 4.10 (4H, q, J = 7.3 Hz), 4.37 (1H, d,
J = 12.3 Hz), 4.46 (1H, d, J = 12.3 Hz), 5.18 (1H, d,
J = 12.3 Hz), 5.30 (1H, d, J = 12.3 Hz), 7.01 (1H, dd,
J = 8.4, 2.2 Hz), 7.29–7.38 (7H, m); MS (ion spray)
27
D
(Positive) m/z 501 (M++Na); ½aꢀ +15.9 (c 5.7, CHCl3).

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4201
5.1.3. (1S,2R,3R,5R,6S)-2-Amino-3-(3,4-dichlorobenzyl-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (6).
white powder. Mp > 260 (decomp.); 1H NMR
(300 MHz, CD3OD, TMS) d 1.49 (3H, d, J = 6.8 Hz),
2.07–2.18 (1H, m), 2.21–2.38 (1H, m), 2.44–2.56 (1H,
m), 2.75–2.83 (1H, m), 4.02–4.22 (2H, m), 4.50 (1H, d,
J = 11.3 Hz), 4.71 (1H, d, J = 11.3 Hz), 7.29 (1H, d,
A
mixture of 10 (1.48 g, 2.98 mmol), THF (27 mL), H2O
(13.5 mL), and LiOH Æ H2O (345 mg, 8.2 mmol) was
stirred for 4 days at room temperature. The reaction
mixture was acidified with 1 N HCl and stirred for 1 h
at room temperature. The solution was chromato-
graphed on AG50W-X8 cation-exchange resin (Bio-
Rad) (H2O–50% aqueous THF–10% aqueous pyridine)
J = 7.8 Hz), 7.50–7.58 (2H, m); MS (ion spray) (Nega-
26
D
tive) m/z 447 (Mꢁ1)ꢁ; ½aꢀ ꢁ56.7 (c = 0.22, MeOH);
Anal. Calcd for C18H19Cl2FNO6ÆHCl: C, 44.51; H,
4.15; N, 5.77. Found: C, 44.45; H, 4.12; N, 5.76.
to yield
6 (980 mg, 88%) as a white powder.
Mp > 250 ꢁC (decomp.); 1H NMR (300 MHz, D2O,
TMSP) d 1.61 (1H, t, J = 3.0 Hz), 1.97–2.41 (4H, m),
3.78 (1H, dd, J = 8.5, 7.9 Hz), 4.50 (1H, d,
J = 12.1 Hz), 4.55 (1H, d, J = 12.1 Hz), 7.27–7.31 (1H,
5.1.6. (1R,2R,3R,5R,6R)-2-Azido-3-(3,4-dichlorobenzyl-
oxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid
2-ethyl ester (14). One molar LiOH solution (2.23 mL)
was added to a mixture of 11 (932 mg, 2.02 mmol),
THF (16 mL), and H2O (8 mL) with ice-cooling and
stirred for 30 min. The mixture was acidified (pH 1) with
1 M HCl and extracted with AcOEt. The organic layer
was washed with saturated brine, dried with Na2SO4,
and evaporated to yield 14 (901 mg) as a brown colorless
m, J = 12.1 Hz), 7.53–7.58 (2H, m); MS (ion spray)
28
(Negative) m/z 358 (Mꢁꢁ1); ½aꢀ +5.1 (c 2.0, 1 N
D
NaOH).
5.1.4. (1R,2R,3R,5R,6R)-2-((2S)-2-tert-Butoxycarbon-
ylaminopropionylamino)-3-(3,4-dichlorobenzyloxy)-6- flu-
orobicyclo[3.1.0]hexane-2,6-dicarboxylic acid diethylester
(13). N-Methylmorpholin (184 mL, 1.67 mmol) and i-
butyl chloroformate (218 lL, 1.67 mmol) were added
to a CH2Cl2 solution of N-Boc-L-alanine (316 mg,
1.67 mmol) at ꢁ14 ꢁC under a nitrogen atmosphere.
After dropwise addition of a CH2Cl2 solution of 1221
(691 mg, 1.59 mmol) to the mixture at room tempera-
ture, the mixture was stirred for 0.5 h. The reaction
mixture was washed with 1 M HCl, dried with Na2SO4,
evaporated, and chromatographed (hexane/AcOEt 2:1)
1
oil. H NMR (200 MHz, CDCl3, TMS) d 1.31 (3H, t,
J = 7.0 Hz), 2.31–2.56 (4H, m), 3.90–4.05 (2H, m),
4.18–4.49 (3H, m), 4.65 (1H, d, J = 12.3 Hz), 7.10 (1H,
dd, J = 8.1, 2.0 Hz), 7.35–7.44 (2H, m).
5.1.7. (1R,2R,3R,5R,6R)-2-Azido-3-(3,4-dichlorobenzyl-
oxy)-6-((1S)-1-ethoxycarbonyl-3-methylbutylcarbamoyl)-
6-fluorobicyclo[3.1.0]hexane-2-carboxylic acid ethyl ester
(15). HOBT Æ H2O (378 mg, 2.47 mmol) and EDC Æ HCl
(455 mg, 2.37 mmol) were added to a DMF (8.5 mL)
solution of 14 (854 mg, 1.98 mmol), L-leucine ethyl ester
(464 mg, 2.37 mmol), and N-methylmorphrin (594 mL,
2.37 mmol) with ice-cooling. The mixture was stirred
for 12 h at room temperature and diluted with AcOEt.
The mixture was washed with 1 M HCl and saturated
brine, dried with Na2SO4, evaporated, and chromato-
graphed (hexane/AcOEt 8:1) to yield 15 (998 mg, 91%)
1
to yield 13 (902 mg, 94%) as a colorless oil. H NMR
(200 MHz, CDCl3, TMS) d 1.25 (3H, t, J = 7.1 Hz),
1.28 (3H, t, J = 7.1 Hz), 1.34 (3H, d, J = 7.0 Hz), 1.39
(9H, s), 2.18–2.31 (1H, m), 2.32–2.54 (2H, m), 3.08
(1H, dd, J = 2.9 Hz, 7.9 Hz), 3.86–4.04 (1H, m), 4.06–
4.16 (5H, m), 4.42 (1H, d, J = 11.6 Hz), 4.65 (1H, d,
J = 11.6 Hz), 4.76–4.96 (1H, m), 7.06–7.24 (1H, m),
7.12 (1H, dd, J = 2.0 Hz, 8.1 Hz), 7.39 (1H, d,
1
as a colorless oil. H NMR (200 MHz, CDCl3, TMS)
d 0.96 (6H, d, J = 5.5 Hz), 1.29 (3H, t, J = 7.0 Hz),
1.30 (3H, t, J = 7.0 Hz), 1.52–1.80 (3H, m), 2.26–2.57
(4H, m), 3.86–4.02 (1H, m), 4.22 (2H, q, J = 7.0 Hz),
4.10–4.38 (2H, m), 4.42 (1H, d, J = 12.2 Hz), 4.50–4.66
(1H, m), 4.65 (1H, d, J = 12.2 Hz), 6.79 (1H, d,
J = 8.1 Hz), 7.11 (1H, dd, J = 8.1, 2.0 Hz), 7.38 (1H, d,
J = 2.0 Hz), 7.40 (1H, d, J = 8.1 Hz); MS (ion spray)
(Negative) m/z 630 (Mꢁ1)ꢁ; ½aꢀ
24
D
ꢁ33.6 (c 0.42,
CHCl3).
5.1.5. (1R,2R,3R,5R,6R)-2-((2S)-2-Aminopropionylami-
no)-3-(3,4-dichlorobenzyloxy)-6-fluoro-bicyclo[3. 1.0]hex-
ane-2,6-dicarboxylic acid (7aa). A mixture of 13 (459 mg,
0.751 mmol), 2.5 M aqueous LiOH (6.0 mL, 15 mmol),
and H2O (6 mL) was stirred for 2 days at room temper-
ature. The mixture was extracted three times with
AcOEt, and the AcOEt layer was dried with Na2SO4
and concentrated under reduced pressure to obtain a
colorless oil (470 mg). The oil was used for the next reac-
tion without further purification, despite containing an
unidentified impurity. A mixture of the oil (375 mg),
LiOH Æ H2O (135 mg, 3.21 mmol), and H2O (7.5 mL)
was stirred for 8 days at room temperature. The mixture
was washed 10 times with AcOEt, acidified (pH 2) with
1 M HCl, and extracted with AcOEt. The AcOEt layer
was washed with brine, dried with Na2SO4, and concen-
trated. Four normal solution of HCl/AcOEt (4.6 mL)
was added to the residue, and the mixture was stirred
for 15 h at room temperature. The obtained precipitate
was filtrated, washed with AcOEt (5 mL), and dried un-
der reduced pressure to yield 7aa (138 mg, 61%) as a
J = 2.0 Hz), 7.40 (1H, d, J = 8.1 Hz); MS (ion spray)
28
(Negative) m/z 571 (Mꢁ1)ꢁ; ½aꢀ ꢁ20.0 (c 0.39, CHCl3).
D
5.1.8. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyl-
oxy)-6-((1S)-1-ethoxycarbonyl-3-methylbutylcarbamoyl)-
6-fluorobicyclo[3.1.0]hexane-2-carboxylic Acid Ethyl
Ester (16). A mixture of 15 (996 mg, 1.74 mmol), 1 M
Me3P/THF (1.91 mL, 1.91 mmol), THF (25 mL), and
H2O (2.5 mL) was stirred for 5 h at room temperature.
Et2O and saturated NaHCO3 were added to the mixture
and stirred for 1 h at room temperature. The ethereal
layer was separated and washed with saturated brine,
dried with Na2SO4, and evaporated in vacuo. Silica gel
and CHCl3 were added to the obtained residue. The
CHCl3 was evaporated under reduced pressure, allowed
to stand for 2 days, and chromatographed (hexane/
AcOEt 3:2–1:1) to yield 16 (784 mg, 82%) as a colorless
1
oil. H NMR (200 MHz, CDCl3, TMS) d 0.96 (6H, d,
J = 5.9 Hz), 1.29 (6H, t, J = 7.1 Hz), 1.52–1.77 (3H, m),
1.85 (2 H, s), 2.10–2.28 (2H, m), 2.36–2.48 (2H, m),

4202
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
24
spray) (Negative) m/z 466 (Mꢁ1)ꢁ; ½aꢀ +5.3 (c 0.34,
3.69–3.87 (1H, m), 4.21 (2H, q, J = 7.1 Hz), 4.15–4.36
(2H, m), 4.45 (1H, d, J = 12.1 Hz), 4.64 (1H, d,
J = 12.1 Hz), 4.55–4.69 (1H, m), 6.77 (1H, dd, J = 8.0,
3.4 Hz), 7.10 (1H, dd, J = 8.4, 1.8 Hz), 7.38 (1H, d,
D
MeOH); Anal. Calcd for C22H20Cl2FNO5: C, 52.35;
H, 4.19; N, 2.77. Found: C, 52.10; H, 4.40; N, 2.75.
J = 1.8 Hz), 7.39 (1H, d, J = 8.4 Hz); MS (ion spray)
5.1.12. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid 6-methyl ester (7ad). A mixture of 5 (114 mg,
0.301 mmol), SOCl2 (88 lL, 1.21 mmol), and MeOH
(1.1 mL) was stirred for 1 h at 50 ꢁC. After cooling,
MeOH was evaporated under reduced pressure. Diiso-
propyl ether (IPE) was added to the residue, and the ob-
tained precipitate was filtered to yield 7ad (102 mg, 79%)
as a white powder. Mp > 190 ꢁC (decomp.); H NMR
(200 MHz, CD3OD, TMS) d 2.37–2.63 (4H, m), 3.81
(3H, s), 4.03–4.18 (1H, m), 4.55 (2H, s), 7.26 (1H, dd,
J = 8.3, 1.8 Hz), 7.48 (1H d, J = 8.3 Hz), 7.53 (1H, d,
22
(Negative) m/z 545 (Mꢁ1)ꢁ; ½aꢀ +2.4 (c 0.65, CHCl3).
D
5.1.9. (1R,2R,3R,5R,6R)-2-Amino-6-((1S)-1-carboxy-3-
methylbutylcarbamoyl)-3-(3,4-dichlorobenzyloxy)-6-fluo-
robicyclo[3.1.0]hexane-2-carboxylic acid (7ab). A mix-
ture of 16 (400 mg, 0.731 mmol), LiOHÆH2O (76.7 mg,
1.83 mmol), THF (8.4 mL), and H2O (4.2 mL) was stir-
red for 24 h at room temperature. The mixture was
diluted with H2O, acidified (pH 1) with 1 M HCl, and
chromatographed on AG50W-X8 cation-exchange resin
(Bio-Rad) (H2O–50% aqueous THF–10% aqueous pyri-
dine) to yield 7ab (250 mg, 59%) as a white powder.
1
J = 1.8 Hz); MS (ion spray) (Negative) m/z 390
30
(Mꢁ1)ꢁ; ½aꢀ minus13.2 (c = 0.64, MeOH); Anal. Calcd
1
Mp > 190 ꢁC. H NMR (300 MHz, CD3OD, TMS) d
D
0.89–0.94 (6H, m), 1.62–1.71 (3H, m), 2.37–2.39 (2H,
m), 2.44–2.49 (1H, m), 2.55 (1H, dd, J = 14.0, 7.9 Hz),
4.10–4.14 (1H, m), 4.29–4.32 (1H, m), 4.54 (1H, d,
J = 11.6 Hz), 4.61 (1H, d, J = 11.6 Hz), 5.30 (1H, d,
for C16H16Cl2FNO5ÆHClÆ1.5H2O: C, 42.17; H, 4.42; N,
3.07. Found: C, 42.42; H, 4.46; N, 3.01.
5.1.13. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid ethyl ester (7ae). Mp > 158 ꢁC (decomp.); 1H NMR
(300 MHz, CD3OD, TMS) d 1.29 (3H, t, J = 6.9 Hz),
2.34–2.50 (2H, m), 2.52–2.68 (2H, m), 4.05–4.18 (1H,
m), 4.20–4.34 (1H, m), 4.48–4.63 (1H, m), 7.15–7.20
J = 8.4 Hz), 7.55 (1H, d, J = 8.4 Hz), 7.56 (1H, s); MS
25
D
(ion spray) (Negative) m/z 489 (Mꢁ1)ꢁ; ½aꢀ ꢁ7.6
(c 0.46, MeOH); Anal. Calcd for C21H25Cl2FNO6Æ
1.2H2O:C, 49.17; H, 5.38; N, 5.46. Found: C, 49.34;
H, 5.66; N, 5.52.
(1H, m), 7.47 (1H, d, J = 7.3 Hz), 7.52 (1H, s); MS
29
D
5.1.10. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid 2-ethyl ester (7ac). A mixture of 17 (150 mg,
0.354 mmol), LiOHÆH2O (17.8 mg, 0.425 mmol), THF
(3.5 mL), and (H2O (1.7 mL) was stirred for 2 h with
ice-cooling. The mixture was acidified with 1 M HCl
(0.45 mL), diluted with H2O, and chromatographed on
AG50W-X8 cation-exchange resin (Bio-Rad) (H2O–
50% aqueous THF–10% aqueous pyridine) to yield
7ac21 (107 mg, 75%) as a white powder. Mp > 223 ꢁC;
1H NMR (300 MHz, CD3OD, TMS) d 1.31 (3H, t,
J = 7.3 Hz), 2.26 (1H, dd, J = 7.9, 2.4 Hz), 2.30–2.33
(1H, m), 2.44–2.49 (1H, m), 2.55 (1H, dd, J = 13.4,
7.3 Hz), 4.08–4.12 (1H, m), 4.29–4.42 (2H, m), 4.51
(1H, d, J = 12.8 Hz), 4.54 (1H, d, J = 12.8 Hz), 7.22
(ion spray) (Negative) m/z 404 (Mꢁ1)ꢁ; ½aꢀ ꢁ8.1
(c = 0.24, MeOH); Anal. Calcd for C17H18Cl2FNO5Æ
HClÆ0.5H2O: C, 45.20; H, 4.46; N, 3.10. Found: C;
45.34, H, 4.51; N, 2.99.
5.1.14. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid ethyl ester propyl ester (7af). Mp > 149 ꢁC (de-
1
comp.); H NMR (200 MHz, CD3OD, TMS) d 0.92–
1.00 (3H, m), 1.69 (2H, sextet, J = 6.8 Hz), 2.37–2.65
(4H, m), 4.02–4.12 (1H, m), 4.17 (2H, t, J = 6.6 Hz),
4.51 (1H, d, J = 11.9 Hz,), 4.59 (1H, d, J = 11.9 Hz),
7.27 (2H, dd, J = 8.4, 1.8 Hz), 7.48 (1H, d, J = 8.4 Hz),
7.54 (1H, d, J = 1.8 Hz); MS (ion spray) (Negative)
28
D
m/z 418 (Mꢁ1)ꢁ; ½aꢀ +7.8 (c 0.35, MeOH) as the salt
(1H, d, J = 8.5 Hz), 7.49 (1H, d, J = 8.5 Hz), 7.50 (1H,
free form; Anal. Calcd for C18H20Cl2FNO5: C, 51.44;
H, 4.80; N, 3.33. Found: C, 51.40; H, 4.82; N, 3.30.
28
D
s); MS (ion spray) (Negative) m/z 404 (Mꢁ1)ꢁ; ½aꢀ
ꢁ24.3 (c 0.46, MeOH); Anal. Calcd for
C21H25Cl2FNO6Æ0.4H2O: C, 49.82; H, 4.53; N, 3.42.
Found: C, 49.74; H, 4.65; N, 3.34.
5.1.15. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid iso-propyl ester (7ag). Mp > 161 ꢁC (decomp.); H
1
5.1.11. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid 6-benzyl ester (7au). A mixture of 5 (400 mg,
1.06 mmol), SOCl2 (0.31 mL, 4.26 mmol), and benzyl
alcohol (6 mL) was stirred for 2.5 h at 50 ꢁC. After
cooling, benzyl alcohol was evaporated under reduced
pressure. The residue was chromatographed on
reverse-phase silica gel (Wakogelꢂ50C18, H2O–30%
MeCN) to yield 7au (240 mg, 48%). Mp > 193 ꢁC
(decomp.); 1H NMR (200 MHz, CD3OD, TMS) d
2.30–2.61 (4H, m), 3.95–4.05 (1H, m), 4.46 (1H, d,
J = 11.8 Hz), 4.58 (1H, d, J = 11.8 Hz), 5.23 (2H, s),
7.28 (1H, dd, J = 8.2, 1.9 Hz), 7.33–7.41 (5H, m), 7.45
(1H, d, J = 8.2 Hz), 7.53 (1H, d, J = 1.9 Hz); MS (ion
NMR (500 MHz, CD3OD, TMS) d 1.28 (6H, d,
J = 5.8 Hz), 2.38–2.44 (2H, m), 2.54–2.62 (2H, m),
4.07–4.12 (1H, m), 4.53 (1H, d, J = 11.6 Hz), 4.57 (1H,
d, J = 11.6 Hz), 5.07–5.12 (1H, m), 7.26 (1H, dd,
J = 1.8, 8.6 Hz), 7.48 (1H, d, J = 8.6 Hz), 7.52 (1H, d,
J = 1.8 Hz); MS (ion spray) (Negative) m/z 418
28
(Mꢁ1)ꢁ; ½aꢀ +10.4 (c 0.25, MeOH) as the salt free
D
form; Anal. Calcd for C18H20Cl2FNO5ÆHCl: C, 47.34;
H, 4.63; N, 3.07. Found: C, 47.41; H, 4.90; N, 3.11.
5.1.16. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid butyl ester (7ah). Mp > 146 ꢁC (decomp.); 1H
NMR (500 MHz, CD3OD, TMS) d 0.95 (3H, t,

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4203
J = 7.9 Hz), 1.40 (2H, sextet, J = 7.9 Hz), 1.65 (2H,
quintet, J = 7.9 Hz), 2.38–2.44 (2H, m), 2.53–2.62
(2H, m), 4.06–4.10 (1H, m), 4.22 (2H, t, J = 6.7 Hz),
4.52 (1H, d, J = 11.6 Hz), 4.58 (1H, d, J = 11.6 Hz),
7.26 (1H, dd, J = 1.8, 7.9 Hz), 7.47 (1H, d,
5.1.21. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-Dichloroben-
zyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid ethyl
ester (7az). Mp > 188 ꢁC (decomp.); 1H NMR
(300 MHz, CD3OD, TMS)
d
1.24 (3H, t,
J = 7.1 Hz), 1.97–2.01 (1H, m), 2.15–2.51 (4H, m),
3.76–3.80 (1H, m), 4.13 (2H, q, J = 7.2 Hz), 4.50
(2H, s), 7.24 (1H, dd, J = 8.2, 1.9 Hz), 7.48 (1H, d,
J = 7.9 Hz) 7.53 (1H, d, J = 1.8 Hz); MS (ion spray)
+10.4 (c 0.25,
28
D
(Negative) m/z 432 (Mꢁ1)ꢁ; ½aꢀ
MeOH) as the salt free form; Anal. Calcd for
C19H22Cl2FNO5Æ0.5HCl: C, 51.48; H, 5.23; N, 3.16.
Found: C, 51.27; H, 5.20; N, 3.06.
J = 8.2 Hz), 7.51 (1H, d, J = 1.9 Hz); MS (ion spray)
30
D
(Negative) m/z 386 (Mꢁ1)ꢁ; ½aꢀ
ꢁ1.4 (c 0.13,
MeOH); Anal. Calcd for C17H19Cl2NO5Æ0.9HCl: C,
48.49; H, 4.76; N, 3.33. Found: C, 48.77; H, 4.91;
N, 3.31.
5.1.17. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid 3-methylbutyl ester (7ai). Mp > 158 ꢁC (decomp.);
1H NMR (300 MHz, CD3OD, TMS) d 0.95 (6H, d,
J = 6.7 Hz), 1.90–2.03 (1H, m), 2.35–2.66 (4H, m), 4.00
(2H, d, J = 6.5 Hz), 4.03–4.20 (1H, m), 4.52 (1H, d,
J = 10.7 Hz), 4.58 (1H, d, J = 10.7 Hz), 7.26 (1H, dd,
J = 8.2, 1.9 Hz), 7.47 (1H, d, J = 8.2 Hz), 7.53 (1H, d,
5.1.22. Typical procedure 2
5.1.22.1. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlorob-
enzyloxy)-6-fluorobicyclo[3.1.0]- hexane-2,6-dicarboxylic
acid 6-(3-methyl)butyl ester (7ak). A mixture of 5
(50 mg, 0.132 mmol), SOCl2 (35 mL, 0.481 mmol),
and 3-methylbutan-1-ol (1 mL) was stirred for 2.5 h
at 60 ꢁC. After cooling, the mixture was concentrated
under reduced pressure. Propylene oxide (0.5 mL) and
EtOH (0.5 mL) were added to the residue, and the
mixture was refluxed for 45 min. The obtained precip-
itate was filtered and washed with Et2O, IPE, and
hexane to yield 7ak (43 mg, 73%) as a white powder.
Mp > 235 ꢁC (decomp.); 1H NMR (300 MHz,
CD3OD, TMS) d 0.93 (6H, d, J = 6.5 Hz), 1.52–1.59
(2H, m), 1.65–1.74 (1H, m), 2.31–2.64 (4H, m),
3.97–4.05 (1H, m), 4.24 (2H, t, J = 6.7 Hz), 4.47
(1H, d, J = 12.1 Hz), 4.59 (1H, d, J = 12.1 Hz), 7.28
(1H, dd, J = 8.2, 2.0 Hz), 7.45 (1H, d, J = 8.2 Hz),
J = 1.9 Hz); MS (ion spray) (Negative) m/z 432
30
(Mꢁ1)ꢁ; ½aꢀ ꢁ7.9 (c 0.67, MeOH); Anal. Calcd for
D
C22H26Cl2FNO5ÆHCl: C, 51.73; H, 5.33; N, 2.74. Found:
C, 52.13; H, 5.61; N, 2.70.
5.1.18. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid pentyl ester (7aj). Mp > 148 ꢁC (decomp.); 1H
NMR (300 MHz, CD3OD, TMS) d 0.93 (3H, t,
J = 7.3 Hz), 1.34–1.37 (2H, m), 1.65–1.71 (2H, m),
2.39–2.45 (2H, m), 2.55–2.60 (2H, m), 4.08–4.12 (1H,
m), 4.03 (2H, t, J = 6.7 Hz), 4.53 (1H, d, J = 12.2 Hz),
4.58 (1H, d, J = 12.2 Hz), 7.26 (1H, dd, J = 8.6,
1.8 Hz), 7.48 (1H, d, J = 8.6 Hz), 7.53 (1H, d,
7.54 (1H, d, J = 2.0 Hz); MS (ion spray) (Negative)
30
m/z 446 (Mꢁ1)ꢁ; ½aꢀ +9.2 (c 0.47, MeOH); Anal.
D
J = 1.8 Hz); MS (ion spray) (Negative) m/z 446
Calcd for C20H24Cl2FNO5: C, 53.58; H, 5.40; N,
3.12. Found: C, 53.55; H, 5.36; N, 3.14.
30
(Mꢁ1)ꢁ; ½aꢀ ꢁ8.6 (c 0.59, MeOH); Anal. Calcd for
D
C20H24Cl2FNO5: C, 53.58; H, 5.40; N, 3.12. Found: C,
53.27; H, 5.42; N, 3.08.
5.1.22.2. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-Dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid hexyl ester (7al). Mp > 200 ꢁC (decomp.); 1H
NMR (300 MHz, CD3OD, TMS) d 0.91 (3H, t,
J = 6.7 Hz), 1.26–1.43 (6H, m), 1.60–1.72 (2 H, m),
2.31–2.67 (4H, m), 3.97–4.06 (1H, m), 4.19 (2H, t,
J = 6.6 Hz), 4.47 (1H, d, J = 12.0 Hz), 4.59 (1H, d,
J = 12.0 Hz), 7.28 (1H, dd, J = 8.2, 1.7 Hz), 7.45
5.1.19. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
1
acid cyclohexyl ester (7an). Mp > 167 ꢁC (decomp.); H
NMR (300 MHz, CD3OD, TMS) d 1.27–1.89 (10H,
m), 2.32–2.64 (4H, m), 3.95–4.08 (1H, m) 4.47 (1H, d,
J = 12.0 Hz), 4.59 (1H, d, J = 12.0 Hz), 7.28 (1H, dd,
J = 8.2, 1.9 Hz), 7.45 (1H, d, J = 8.24 Hz), 7.54 (1H, d,
(1H, d, J = 8.2 Hz), 7.54 (1H, d, J = 1.7 Hz); MS
27
D
J = 1.86 Hz); MS (ion spray) (Negative) m/z 458
(ion spray) (Negative) m/z 460 (Mꢁ1)ꢁ; ½aꢀ +5.0
30
(Mꢁ1)ꢁ; ½aꢀ ꢁ5.3 (c 0.11, MeOH); Anal. Calcd for
(c = 0.57, MeOH); Anal. Calcd for C21H26Cl2FNO5:
C, 54.55; H, 5.67; N, 3.03. Found: C, 54.59; H,
5.68; N, 3.06.
D
C22H26Cl2FNO5ÆHCl: C, 54.79; H, 5.26; N, 3.04. Found:
C, 54.69; H, 5.30; N, 3.05.
5.1.20. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichloroben-
zyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid cyclohexylmethyl ester (7aq). Mp > 153 ꢁC (de-
5.1.22.3. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid 4-methylpentyl ester (7am). Mp > 198 ꢁC
(decomp.); 1H NMR (300 MHz, CD3OD, TMS) d
0.91 (6H, d, J = 6.7 Hz), 1.19–1.31 (2H, m), 1.49–
1.72 (3H, m), 2.32–2.66 (4H, m), 3.98–4.70 (1H, m),
4.18 (1H, t, J = 6.6 Hz), 4.48 (1H, d, J = 11.8 Hz),
4.60 (1H, d, J = 11.8 Hz), 7.29 (1H, dd, J = 8.2,
2.1 Hz), 7.46 (1H, d, J = 8.2 Hz), 7.55 (1H, d,
1
comp.); H NMR (500 MHz, CD3OD, TMS) d 0.97–
1.05 (2H, m), 1.16–1.33 (3H, m), 1.63–1.77 (6H, m),
2.40–2.46 (2H, m), 2.58–2.60 (2H, m), 4.03 (2H, d,
J = 6.7 Hz), 4.09–4.13 (1H, m), 4.54 (1H, d,
J = 11.6 Hz), 4.58 (1H, d, J = 11.6 Hz), 7.26 (1H, dd,
J = 1.8, 8.6 Hz), 7.48 (1H, d, J = 8.6 Hz), 7.52 (1H, d,
J = 1.8 Hz); MS (ion spray) (Negative) m/z 472
J = 2.1 Hz); MS (ion spray) (Positive) m/z 462
30
(Mꢁ1)ꢁ; ½aꢀ ꢁ6.9 (c 0.47, MeOH); Anal. Calcd for
28
(M+1)+; ½aꢀ +7.0 (c 0.41, MeOH); Anal. Calcd for
D
D
C17H18Cl2FNO5ÆHCl: C, 46.12; H, 4.33; N, 3.16. Found:
C, 46.07; H, 4.61; N, 3.22.
C21H26Cl2FNO5: C, 54.55; H, 5.67; N, 3.03. Found:
C, 54.52; H, 5.68; N, 3.00.

4204
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
27
z 466 (Mꢁ1)ꢁ; ½aꢀ +10.5 (c 0.52, MeOH); Anal. Calcd
5.1.22.4. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid heptyl ester (7ao). Mp > 214 ꢁC (decomp.); H
D
for C25H34Cl2FNO5: C, 57.92; H, 6.61, N, 2.70. Found:
C, 57.85; H, 6.60; N, 2.72.
1
NMR (300 MHz, CD3OD, TMS) d 0.90 (3H, t,
J = 6.5 Hz), 1.26–1.42 (8H, m), 1.59–1.72 (2H, m),
2.31–2.40 (2H, m), 2.48 (1H, dd, J = 13.3, 7.4 Hz),
2.50–2.66 (1H, m), 3.98–4.03 (1H, m), 4.19 (2H, t,
J = 6.5 Hz), 4.47 (1H, d, J = 12.0 Hz), 4.59 (1H, d,
J = 12.0 Hz), 7.28 (1H, dd, J = 8.2 Hz), 7.45 (1H, d,
5.1.22.9. (10R,20R,30R,50R,60R)-30-(3,4-Dichloroben-
zyloxy)-60-fluoro-3-allyloxycarbonyl-5-oxo-oxazinone-4-
spiro-20-bicyclo[3.1.0]hexane-60-carboxylic acid (17). A
mixture of 5 (1.55g, 3.97 mmol), allyl chloroformate
(957 mg, 7.94 mmol), saturated NaHCO3 (13 mL), and
dioxane (5.3 mL) was stirred for 18 h at room tempera-
ture. The mixture was diluted with H2O (5 mL) and
washed with AcOEt. The aqueous layer was acidified
(pH 1) with 1 N HCl and extracted with AcOEt. The
AcOEt layer was washed with H2O, dried with Na2SO4,
and evaporated under reduced pressure to obtain a
white foam. A mixture of the white foam, paraformalde-
hyde (503 mg), TsOHÆH2O (36 mg, 0.189 mmol), and
benzene was refluxed for 6 h using Dean–Stark appara-
tus. After cooling, the mixture was diluted with AcOEt,
washed with H2O, dried with Na2SO4, and evaporated
under reduced pressure to yield 17 (1.67 g, 89%) as a
white foam. 1H NMR (300 MHz, CDCl3, TMS) d
2.07–2.54 (m, 4H), 4.17–4.24 (1H, m), 4.39 (1H, d,
J = 12.3 Hz), 4.52 (1H, d, J = 12.3 Hz), 4.63 (2H, d,
J = 6.2 Hz), 5.23 (1H, d, J = 4.4 Hz), 5.28–5.54 (2H,
m), 5.53 (1H, d, J = 4.5 Hz), 5.85–5.98 (1H, m), 7.07
(1H, dd, J = 8.2, 1.9 Hz), 7.32 (1H, d, J = 1.9 Hz), 7.41
(1H, d, J = 8.2 Hz); MS (ion spray) (Negative) m/z 472
(Mꢁ1).
5.1.22.10. (10R,20R,30R,50R,60R)-30-(3,4-Dichloroben-
zyloxy)-60-fluoro-3-allyloxycarbonyl-5-oxo-oxazinone-4-
spiro-20-bicyclo[3.1.0]hexane-60-carboxylic acid 1-ethoxy-
carbonyloxyethyl ester (18av). A mixture of 17 (250 mg,
0.527 mmol), 1-chloroethyl ethyl carbonate (193 mg,
1.27 mmol), NaI (190 mg, 1.27 mmol), K2CO3 (87 mg,
0.633 mmol), and DMF was stirred for 3 h at 75 ꢁC.
After the mixture was stirred for 12 h at room tempera-
ture, AcOEt was added to the mixture. The AcOEt layer
was washed with H2O and saturated brine, dried with
Na2SO4, evaporated, and chromatographed (hexane/
AcOEt 5:1) to yield 18av as a pale yellow oil.
J = 8.2 Hz), 7.54 (1H, d, J = 2.0 Hz); MS (ion spray)
27
(Positive) m/z 476 (M+1)+; ½aꢀ +8.2 (c 0.49, MeOH);
D
Anal. Calcd for C22H28Cl2FNO5: C, 55.47; H, 5.92; N,
2.94. Found: C, 55.37; H, 5.90; N, 2.76.
5.1.22.5. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid 4-methylpentyl ester (7ap). Mp > 209 ꢁC
1
(decomp.); H NMR (300 MHz, CD3OD, TMS) d 0.90
(6H, d, J = 6.5 Hz), 1.17–1.71 (7H, m), 2.32–2.40 (2H,
m), 2.49 (1H, dd, J = 13.2, 7.3 Hz), 2.55–2.66 (1H, m),
3.98–4.07 (1H, m), 4.20 (2H, t, J = 6.5 Hz), 4.48 (1H,
d, J = 12.0 Hz), 4.60 (1H, d, J = 12.0 Hz), 7.29 (1H,
dd, J = 8.2, 1.9 Hz), 7.46 (1H, d, J = 8.2 Hz), 7.55 (1H,
d, J = 1.9 Hz); MS (ion spray) (Positive) m/z 476
30
(M+1)+; ½aꢀ +4.9 (c 0.48, MeOH); Anal. Calcd for
D
C22H28Cl2FNO5: C; 55.47; H, 5.92; N, 2.94. Found: C,
55.30; H, 5.99; N, 2.92.
5.1.22.6. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
1
ylic acid octyl ester (7ar). Mp > 203 ꢁC (decomp.); H
NMR (300 MHz, CD3OD, TMS) d 0.90 (3H, t,
J = 6.6 Hz), 1.25–1.42 (10H, m), 1.60–1.71 (2H, m),
2.31–2.40 (2H, m), 2.48 (1H, dd, J = 13.4, 7.6 Hz),
2.5–2.7 (1H, m), 4.05–4.06 (1H, m), 4.19 (2H, t,
J = 6.6 Hz), 4.47 (1H, d, J = 12.0 Hz), 4.59 (1H, d,
J = 12.0 Hz), 7.28 (1H, dd, J = 8.2, 1.9 Hz), 7.45 (1H,
d, J = 8.2 Hz), 1.9 (1H, d, J = 1.9 Hz); MS (ion spray)
30
(Positive) m/z 490 (M+1)+; ½aꢀ +6.3 (c 0.52, MeOH);
D
Anal. Calcd for C23H30Cl2FNO5: C, 56.33; H, 6.17; N,
2.86. Found: C, 56.11; H, 6.18; N, 2.86.
5.1.22.7. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid 6-methylheptyl ester (7as). Mp > 193 ꢁC
5.1.22.11. (1R,2R,3R,5R,6R)2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-carboxylic
acid 1-ethoxycarbonyloxyethyl ester (7av). A mixture of
18av (168 mg, 0.285 mmol), Pd(PPh3)4 (10 mg,
8.5 mmol), 1,3-dimethylbarbituric acid (133 mg,
0.854 mmol), and CHCl3 (2.9 mL) was stirred for 1.5 h
at 40 ꢁC under nitrogen atmosphere. The mixture was
concentrated under reduced pressure. AcOEt was added
to the residue and stirred for 1 h at room temperature.
After the mixture was stirred for 1 h with ice cooling,
the obtained precipitate was filtrated off, after which
the filtrate was evaporated under reduced pressure.
The obtained residue was purified using reverse-phase
silica gel (Wakogelꢂ50C18, H2O–40% MeCN) to yield
7av (118 mg, 84%). A white powder. Mp ꢂ126 ꢁC (de-
1
(decomp.); H NMR (300 MHz, CD3OD, TMS) d 0.89
(6H, d, J = 6.7 Hz), 1.15–1.39 (6H, m), 1.47–1.71 (3H,
m), 2.33–2.65 (4H, m), 4.02 (1H, m), 4.19 (2H, t,
J = 6.5 Hz), 4.48 (1H, d, J = 12.1 Hz), 4.60 (1H, d,
J = 12.1 Hz), 7.29 (1H, dd, J = 8.2, 2.0 Hz), 7.46 (1H,
d, J = 8.2 Hz), 7.54 (1H, d, J = 2.0 Hz); MS (ion spray)
30
(Negative) m/z 488 (Mꢁ1)ꢁ; ½aꢀ +4.7 (c 0.53, MeOH);
D
Anal. Calcd for C23H30Cl2FNO5: C, 56.33; H, 6.17; N,
2.86. Found: C, 56.09; H, 6.04; N, 2.87.
5.1.22.8. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
1
ylic acid decyl ester (7at). Mp > 196 ꢁC (decomp.); H
1
NMR (300 MHz, CD3OD, TMS) d 2.30–2.61 (4H, m),
3.95–4.05 (1H, m), 4.46 (1H, d, J = 11.8 Hz), 4.58 (1H,
d, J = 11.8 Hz), 5.23 (2H, s), 7.28 (1H, dd, J = 8.2,
1.9 Hz), 7.33–7.41 (5H, m), 7.45 (1H, d, J = 8.2 Hz),
7.53 (1H, d, J = 1.9 Hz); MS (ion spray) (Negative) m/
comp.); H NMR (200 MHz, CD3OD, TMS) d 2.37–
2.63 (4H, m), 3.81 (3H, s), 4.03–4.18 (1H, m), 4.55
(2H, s), 7.26 (1H, dd, J = 8.3, 1.8 Hz), 7.48 (1H d,
J = 8.3 Hz), 7.53 (1H, d, J = 1.8 Hz); MS (ion spray)
(Negative) m/z 390 (Mꢁ1); Anal. Calcd for

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4205
C16H16Cl2F2NO5ÆHCl Æ 0.5H2O: C, 47.73; H, 4.61; N,
2.78. Found: C, 47.73; H, 4.47; N, 2.81.
(ion spray) (Negative) m/z 528 (Mꢁ1)ꢁ; Anal. Calcd
for C24H29Cl2NO8ÆH2O: C, 53.63; H, 5.42; N, 2.7.
Found: C, 53.44; H, 5.61; N, 2.60.
5.1.22.12. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid 1-cyclohexyloxycarbonyloxyethyl ester (7aw).
Mp > 133 ꢁC (decomp.); H NMR (300 MHz, CD3OD,
TMS) d 1.21–1.61 (8H, m), 1.68–1.96 (4H, m), 2.30–2.66
(4H, m), 3.98–4.07 (1H, m), 4.47 (1H, d, J = 12.0 Hz),
4.57–4.67 (2H, m), 6.76 (1H, q, J = 5.3 Hz), 7.28 (1H,
dd, J = 8.3, 2.0 Hz), 7.45 (1H, d, J = 8.3 Hz), 7.54 (1H,
d, J = 2.0 Hz); MS (ion spray) (Negative) m/z 458
(Mꢁ1)ꢁ; Anal. Calcd for C24H28Cl2FNO8Æ0.2H2O: C,
52.22; H, 5.19; N, 2.54. Found: C, 52.03; H, 5.17; N,
2.46.
5.2. Metabolic stability study
1
Liver S9 fractions (1 mg protein/mL) from rat (Gentest)
were incubated with 3lM prodrugs in the presence of an
NADPH generating system (125 lg/mL NADP+,
2.5 mM MgCl2, and 1.92 mM glucose-6-phosphate) in
0.255 M phosphate buffer containing 0.575% (w/v)
KCl (pH 7.4) for 60 min at 37 ꢁC. All experiments were
performed in triplicate. After incubation, a 2-fold vol-
ume of DMSO was added to incubation medium, and
the tube was vortexed and centrifuged at 11,200g
(4 ꢁC) for 10 min. The resulting supernatant was ana-
lyzed with a LC–MS/MS system.
5.1.22.13. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid butanoyloxymethyl ester (7ax). Mp > 152 ꢁC
Rat plasma were spiked with prodrugs at concentrations
of 1000 ng/mL (approximately 2.3 lM) and then incu-
bated for 2 h at 37 ꢁC. 200 lL of I.S. working solution
(250 ng/mL) with methanol or acetonitrile was added
to 50 lL aliquot of plasma sample, and the tube was
vortexed and centrifuged at 11,200g (4 ꢁC) for 10 min.
The resulting supernatant was analyzed by a LC–MS/
MS system.
1
(decomp.); H NMR (300 MHz, CD3OD, TMS) d 0.95
(3H, t, J = 7.4 Hz), 1.64 (2H, sextet, J = 7.4 Hz), 2.31–
2.97 (6H, m), 3.97–4.06 (1H, m), 4.47 (1H, d,
J = 12.1 Hz), 4.59 (2H, d, J = 12.1 Hz), 5.83 (2H, s),
7.28 (1H, dd, J = 8.2, 2.0 Hz), 7.45 (1H, d, J = 8.2 Hz),
7.54 (1H, d, J = 2.0 Hz); MS (ion spray) (Negative)
30
m/z 476 (Mꢁ1)ꢁ; ½aꢀ +10.5 (c = 0.53, MeOH); Anal.
D
Calcd for C20H22Cl2FNO7Æ0.7H2O: C, 48.93; H, 4.80;
N, 2.85. Found: C, 48.97; H, 4.77; N, 2.85.
5.2.1. Pharmacokinetics. Animals. The experiments used
7-week-old Male Wistar or Sprague–Dawley rats
(Charles River, Japan). All animals in these experi-
ments were used following acclimation for at least 4
days before entry into a study. The rats were given ac-
cess to water and administered a standard laboratory
diet (MF, Oriental Yeast Co, Japan) ad libitum during
acclimation. The environmental parameters during
breeding were maintained at relative humidity
50 20% and temperature 23 3 ꢁC. The animals
were fasted overnight (about 18 h) before and 4 h after
dosing when studies were conducted in the fasting
state. Drinking water was made freely available at all
times.
5.1.22.14. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid 2-(morphrin-4-yl)ethyl ester (7ay). Mp >
1
190 ꢁC (decomp.); H NMR (300 MHz, D2O, TMSP)
d 2.47–1.58 (4H, m), 3.33–3.41 (4H, m), 3.52–3.58
(2H, m), 3.92–4.13 (5H, m), 4.48–4.64 (4H, m), 7.29
(1H, dd, J = 8.2, 1.9 Hz), 7.53–756 (2H, m); MS (ion
30
D
spray) (Negative) m/z 489 (Mꢁ1)ꢁ; ½aꢀ +3.5 (c 0.31,
MeOH); Anal. Calcd for C21H25Cl2FN2O6Æ2HCl Æ H2O:
C, 43.32; H, 5.02; N, 4.81. Found: C, 43.48; H, 4.87;
N, 4.88.
5.1.22.15. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)bicyclo[3.1.0]hexane-2,6-carboxylic acid 1-
ethoxycarbonyloxyethyl ester (7ba). Mp > 194 ꢁC
(decomp.); 1H NMR (300 MHz, CD3OD, TMS) d
1.22–1.32 (3H, m), 1.42–1.50 (3H, m), 1.87–1.94 (1H,
m), 2.11–2.27 (2H, m), 2.32–2.41 (2H, m), 3.61–3.70
(1H, m), 4.11–4.23 (2H, m), 4.44 (1H, d, J = 12.0 Hz),
4.51 (1H, d, J = 12.0 Hz), 6.63–6.72 (1H, m), 7.22–7.30
(1H, m), 7.45 (1H, d, J = 8.24 Hz), 7.53 (1H, s); MS
(ion spray) (Negative) m/z 474 (Mꢁ1)ꢁ; Anal. Calcd
for C20H23Cl2NO8ÆH2O: C, 49.50; H, 4.98; N, 2.89.
Found: C, 49.46; H, 4.88; N, 2.85.
5.2.2. Plasma concentrations. In the case of rats, 0.2–
0.3 mL blood samples were taken from the tail vein
using a Multivette containing EDTA, or from the jugu-
lar vein using a syringe, and then transferred to a Mul-
tivette at 0.5, 1, 2, 4 and 6 h post-dosing. For mice, after
tail suspension test, animals were immediately eutha-
nized under anesthesia by ether, and blood samples were
taken via the femoral vein with tube (containing
EDTA). Plasma was separated by centrifugation
(11,200g, 4 ꢁC, 2–3 min). In the case of prodrugs, plasma
was immediately mixed with 5 N HCl (plasma/5 N HCl
50:1) to avoid enzymatic degradation in plasma. The
plasma samples were immediately frozen with liquid
N2 and stored at ꢁ80 ꢁC until bioanalysis.
5.1.22.16. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlo-
robenzyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid
1-cyclohexyloxycarbonyloxyethyl
ester
(7bb).
1
Mp > 198 ꢁC (decomp.); H NMR (300 MHz, CD3OD,
TMS) d 1.30–1.98 (14H, m), 2.11–2.27 (2H, m), 2.31–
2.42 (2H, m), 3.59–3.71 (1H, m), 4.44 (1H, d,
J = 12.1 Hz), 4.51 (1H, d, J = 12.1 Hz), 4.54–4.66 (1H,
m), 6.61–6.72 (1H, m), 7.26 (1H, dd, J = 8.2, 1.7 Hz),
7.45 (1H, d, J = 8.2 Hz), 7.53 (1H, d, J = 1.7 Hz); MS
To a 50 lL aliquot of a plasma sample, 200 lL of
I.S. working solution (250 ng/mL) with methanol or
acetonitrile was added, and the tube was vortexed
and centrifuged at 11,200g (4 ꢁC) for 10 min. The
resulting supernatant was analyzed by a LC–MS/MS
system.

4206
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
5.2.3. Brain concentrations. After blood sampling at 6 h,
animals were decapitated at the specified time. The brain
(cerebrum) was immediately excised and weighed, and
dura mater removed with soft paper. Each tissue was
added 4-fold distilled water and homogenized.
5.3.4. Tail suspension test in mice. Tail suspension tests
were performed by the method described by Steru
et al., with modification. Mice were suspended by the
tail using adhesive tape from a metal rod fixed 45 cm
above the surface of a table in a sound-isolated room.
The mouse was positioned at least 15 cm away from
the nearest object. Test sessions were videotaped for
10 min, and the immobility time was determined by an
observer. Mice were considered immobile only when
they hung passively and completely motionless. Com-
pound 8ao was administered po 2 h prior to the test.
To a 50 lL aliquot of a plasma or tissue homogenate
sample, 200 lL of I.S. working solution (250 ng/mL)
with methanol or acetonitrile was added, and the tube
was vortexed and centrifuged at 11,200g (4 ꢁC) for
10 min. The resulting supernatant was analyzed by s
LC–MS/MS system.
5.3.5. Spontaneous locomotor activity. Spontaneous
locomotor activity was determined as reported.22 Ani-
mals were individually housed in transparent acrylic
cages (for rats, 45 · 28.5 · 29.5 cm; for mice, 30 cm
diameter, 30 cm height), and spontaneous locomotor
activity was recorded every 5 min for 60 min, using a
SCANET apparatus (Neuroscience Inc., Japan) placed
in a sound-proof box. Compound 8ao was administered
po 2 h before the start of measurement.
5.3. Antidepressant-like effects
5.3.1. Animals. Male ICR mice (25–35 g, Charles River,
Yokohama, Japan) were used for tail suspension test
and locomotor activity. All of these animals were
maintained under a 12 h light/dark cycle (light on
7:00 AM) in a temperature- and humidity-controlled
holding room. Food and water were available
ad libitum.
5.3.6. Statistical analysis. Data from in vivo experiments
were analyzed by one-way ANOVA. Significant differ-
ences between groups were determined by Dunnett’s
test.
5.3.2. Ethics. All studies were reviewed by the Taisho
Pharmaceutical Co., Ltd. Animal Care Committee and
met the Japanese Experimental Animal Research Asso-
ciation standards, as defined in The Guidelines for
Animal Experiments (1987).
Acknowledgments
5.3.3. Forced swimming test in rats. Forced swimming
tests were performed by the method previously report-
ed, and the effect of the compound was evaluated by
measuring the period of immobility and by a time-sam-
pling technique. A time-sampling technique was used
to score several types of behavior (immobility, swim-
ming, and climbing) as described by Derke et al.42
Swimming sessions were conducted by placing rats in
cylinders containing 25 ꢁC water at a depth of 30 cm,
sufficient to prevent rats from keeping their heads
above water by standing on the bottom. Two swim-
ming sessions were conducted; an initial 15 min pretest
between 10:00 AM and 4:00 PM, followed 24 h later by
a 5 min test. Compound 8a was administered po during
the period between these two sessions (24 and 2 h prior
to the test). Following both swimming sessions, the rats
were removed from the cylinders, placed in a heated
cage for 15 min, and then returned to their home cages.
Test sessions were videotaped from the front of the cyl-
inders for later scoring. The water in the cylinders was
changed after every trial. A time-sampling technique
was used to score behavior during a single viewing:
At the end of each 5-s period during the test session,
the scorer, who remained unaware of the drug admin-
istered, classified the rat’s behavior into one the follow-
ing three categories: (1) immobility-floating in the
water without struggling and making only movements
necessary to keep its head above water; (2) swim-
ming-making active swimming motions between quad-
rants of the cylinder, beyond those necessary merely
to maintain head above water and moving around in
the cylinder; and (3) climbing movements with fore-
paws in and out of the water, usually directed against
the walls of the cylinder.
The authors thank Professor Sigetada Nakanishi at
Department of Biological Sciences, Kyoto University
Faculty of Medicine, for provision of CHO cell line sta-
bly expressing rat mGluR2.
References and notes
1. Collingridge, G. L.; Lester, R. A. Pharmacol. Rev. 1989,
40, 143.
2. Monaghan, D. T.; Bridges, R. J.; Cotman, C. W. Annu.
Rev. Pharmcol. Toxicol. 1989, 29, 365.
3. Nakanishi, S. Science 1992, 258, 597.
4. Pin, J. P.; Duvoision, R. Neuropharmacology 1995, 34, 1.
5. Schoepp, D. D.; Conn, P. J. Trends Pharmacol. Sci. 1993,
14, 13.
6. Bockaert, J.; Pin, J.; Fagni, L. Fundam. Clin. Pharmcol.
1993, 7, 473.
7. Nakanishi, S.; Masu, M. Annu. Rev. Biophys. Biomol.
Struct. 1994, 23, 319.
8. Hollmann, N.; Heinemann, S. Annu. Rev. Neurosci. 1994,
17, 31.
9. Conn, P. J.; Pin, J. P. Annu. Rev. Pharmcol. Toxicol. 1997,
37, 205.
10. Schoepp, D. D.; Johnson, B. G.; Monn, J. A.
J. Neurochem. 1992, 58, 1184.
11. Nakajima, Y.; Iwakabe, H.; Akazawa, C.; Nawa, H.;
Shigemoto, R.; Mizuno, N.; Nalanishi, S. J. Biol. Chem.
1993, 268, 11868.
12. Meldrum, B. S. J. Nutr. 2000, 130, 1007S–1015S.
13. Nakazato, A.; Kumagai, T.; Sakagami, K.; Yoshikawa,
R.; Suzuki, Y.; Chaki, S.; Ito, H.; Taguchi, T.; Nakanishi,
S.; Okuyama, S. J. Med. Chem. 2000, 43, 4893.
14. Cartmell, J.; Monn, J. A.; Schoepp, D. D. Psychophar-
macology 2000, 148, 423.

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 4193–4207
4207
15. Moghaddam, B.; Adams, B. W. Science 1998, 281, 1349.
16. Grillon, C.; Cordova, J.; Levine, L. R.; Morgan, C. A.
Psychopharmacology 2003, 168, 446.
27. Fei, Y. J.; Kanai, Y.; Nussberger, S.; Ganapathy, V.;
Leibach, F. H.; Romero, M. F.; Singh, S. K.; Boron, W.
F.; Hediger, M. A. Nature 1994, 563.
17. Tizzano, J. P.; Griffey, K. I.; Schoepp, D. D. Pharmacol.
Biochem. Behav. 2002, 73, 367.
28. Taylor, M. D. Adv. Drug Delivery Rev. 1996, 19, 131.
29. Adam, G; Huguenin-Virchaus, P. N.; Mutel, V.; Stadler,
H.; Woltering, T. J. DE Patent 19941675, 2000; . Chem.
Abstr. 2000, 132, 166520.
30. Wessel, H, P.; Iversen, T.; Bundle, D. R. J. Chem. Soc.,
Perkin Trans. 1 1985, 2247.
31. Widmer, U. Synthesis 1987, 568.
18. Helton, D. R.; Tizzano, J. P.; Monn, J. A.; Schoepp, D.
D.; Kallman, M. J. J. Pharmcol. Exp. Ther. 1998, 284, 651.
19. Monn, J. A.; Valli, M. J.; Massey, S. M.; Wright, R. A.;
Salhoff, C. R.; Johnson, B. G.; Howe, T.; Alt, C. A.;
Rhodes, G. A.; Robey, R. L.; Griffey, K. R.; Tizzano, J.
P.; Kallman, M. J.; Helton, D. R.; Schoepp, D. D. J. Med.
Chem. 1997, 40, 528.
32. Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett.
1983, 24, 763.
20. Shekhar, A.; Keim, S. R. Neuropharmacology 2000, 39,
1139.
33. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F.
Teterahedron 1981, 37, 437.
21. Nakazato, A.; Sakagami, K.; Yasuhara, A.; Ohta, H.;
Yoshikawa, R.; Itoh, M.; Nakamura, M.; Chaki, S.
J. Med. Chem. 2004, 47, 4570.
34. Itoh, M. Chem. Phram. Bull. 1969, 17, 1679.
35. Paleo, M. R.; Sardina, F. J. Tetrahedron Lett. 1996, 37,
3403.
22. Chaki, S.; Yoshikawa, R.; Hirota, S.; Shimazaki, T.;
Maeda, M.; Kawashima, N.; Yoshimizu, T.; Yasuhara,
A.; Sakagami, K.; Okuyama, S.; Nakanishi, S.; Nakazato,
A. Neuropharmcology 2004, 46, 457.
23. Jane, D. E.; Jones, P. L. St. J.; Pook, P. C.; Tse, H. W.;
Watkins, J. C. Br. J. Pharmacol. 1994, 112, 809.
24. Ornstein, P. L.; Bleisch, T. J.; Arnold, M. B.; Wright, R.
A.; Johnson, B. G.; Schoepp, D. D. J. Med. Chem. 1998,
41, 346.
25. Ornstein, P. L.; Bleisch, T. J.; Arnold, M. B.; Kennedy, J.
H.; Wright, R. A.; Hohnson, B. G.; Tizzano, J. P.; Helton,
D. R.; Kallman, M. J.; Schoepp, D. D.; Herin, M. J. Med.
Chem. 1998, 41, 358.
26. Pellicciari, R.; Marinozzi, M.; Natalini, B.; Costantino,
G.; Luneia, R.; Giorgi, G.; Moroni, F.; Thomsen, C.
J. Med. Chem. 1996, 39, 2259.
36. Walter, M. W.; Adlington, R. M.; Baldwin, J. E.; Chuhan,
J.; Schofield, C. J. Tetrahedron Lett. 1995, 36, 7761.
37. Kunz, H.; Ma¨rz, J. Synlett 1992, 591.
38. Bundgaard In A Textbook of Drug Design and Develop-
ment; Krogsgaard-Larsen, P., Bundgaard, H., Eds.; Har-
wood Academic: Langhorne, 1991; pp 113–191.
39. Jeffrey, P. K.; Valentino, J. S. Adv. Drug Delivery Rev.
1996, 19, 287.
40. Freeman, S.; Ross, K. C.. In Ellis, G. P., Luscombe, D.
K., Eds.; Progress in Medicinal Chemistry; Elsevier
Science, BV: Amsterdam, 1997; vol. 34, pp 111–147.
41. Yasuhara, A.; Sakagami, K.; Yoshikawa, R.; Chaki, S.;
Nakamura, M.; Nakazato, A. Bioorg. Med. Chem. in
press, doi:10.1016/j.bmc.2005.12.061.
42. Detke, M. J.; Rickels, M.; Lucki, I. Psychopharmacology
1995, 121, 66.