Synthesis, in vitro pharmacology, and structure-activity relationships of 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as mGluR2 antagonists

Article abstract of DOI:10.1016/j.bmc.2005.12.061

Chemical modification of the bicyclo[3.1.0]hexane ring C-3 position led to the discovery of 3-alkoxy-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, 3-benzylthio-, and 3-benzylamino-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives, metabotropic glutamate receptor 2 (mGluR2) antagonists. In particular, 3-(3,4-dichlorobenzyloxy)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ae), (1R,2S,5R,6R)-2-amino-3-(3,4-dichlorobenzylthio)-6-fluorobicyclo[3.1.0]h exane-2,6-carboxylic acid (15at), and (1R,2S,5R,6R)-2-amino-3-(N-(3,4-dichlorobenzylamino))-6-fluorobicyclo[3. 1.0]hexane-2,6-carboxylic (15ba) showed high affinity for the mGluR2 receptor (15ae: K_i = 2.51 nM, 15at: K_i = 1.96 nM, and 15ba: K_i = 3.29 nM) and potent antagonist activity for mGluR2 (15ae; IC₅₀ = 34.21 nM, 15at; IC₅₀ = 13.34 nM, and 15ba; IC₅₀ = 35.96 nM). No significant agonist activity for mGluR2 was observed with 15ae, 15at, or 15ba. This paper reports on the synthesis, in vitro pharmacological profile, and structure-activity relationships (SARs) of 3-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid.

Full text of DOI:10.1016/j.bmc.2005.12.061

Bioorganic & Medicinal Chemistry 14 (2006) 3405–3420
Synthesis, in vitro pharmacology, and structure–activity
relationships of 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid
derivatives as mGluR2 antagonists
Akito Yasuhara,* Kazunari Sakagami, Ryoko Yoshikawa, Shigeyuki Chaki,
Masato Nakamura and Atsuro Nakazato
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku,
Saitama-shi, Saitama 331-9530, Japan
Received 24 November 2005; revised 28 December 2005; accepted 29 December 2005
Available online 20 January 2006
Abstract—Chemical modification of the bicyclo[3.1.0]hexane ring C-3 position led to the discovery of 3-alkoxy-2-aminobicy-
clo[3.1.0]hexane-2,6-dicarboxylic acid, 3-benzylthio-, and 3-benzylamino-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid derivatives, metabotropic glutamate receptor 2 (mGluR2) antagonists. In particular, 3-(3,4-dichlorobenzyloxy)-2-aminobicy-
clo[3.1.0]hexane-2,6-dicarboxylic acid (15ae), (1R,2S,5R,6R)-2-amino-3-(3,4-dichlorobenzylthio)-6-fluorobicyclo[3.1.0]hexane-2,6-
carboxylic acid (15at), and (1R,2S,5R,6R)-2-amino-3-(N-(3,4-dichlorobenzylamino))-6-fluorobicyclo[3.1.0]hexane-2,6-carboxylic
(15ba) showed high affinity for the mGluR2 receptor (15ae: Ki = 2.51 nM, 15at: Ki = 1.96 nM, and 15ba: Ki = 3.29 nM) and potent
antagonist activity for mGluR2 (15ae; IC50 = 34.21 nM, 15at; IC50 = 13.34 nM, and 15ba; IC50 = 35.96 nM). No significant agonist
activity for mGluR2 was observed with 15ae, 15at, or 15ba. This paper reports on the synthesis, in vitro pharmacological profile,
and structure–activity relationships (SARs) of 3-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
aptically and negatively coupled to the activity of ade-
nylcyclase.3,4 mGluRs have been implicated in the
pathology of major psychiatric disorders such as depres-
sion, anxiety, and schizophrenia11 due to their critical
role as modulators of synaptic transmission, ion channel
activity, and synaptic plasticity.9 Indeed, the efficacy of
group II mGluR agonists in animal models and in clin-
ical trials suggests that their inhibition of the glutama-
tergic system may make agonists of the group II
mGluRs effective in the treatment of many diseases
and conditions, including schizophrenia,12–14 anxi-
ety,15–18 and panic disorders.19
L-Glutamate, a major excitatory neurotransmitter in the
mammalian central nervous system, is involved in sever-
al physiological and pathological conditions, exerting its
effects through either ionotropic glutamate receptors
(iGluRs), in which the receptors have an ion channel
structure, or metabotropic glutamate receptors
(mGluRs), which are members of the GPCR family C
and characterized by a large extracellular amino-termi-
nal agonist binding site.1–4 mGluRs are classified as
one of eight subtypes organized into three groups based
on sequence homology, signal transduction mecha-
nisms, and pharmacological properties.3,5–10 Group I
mGluRs (mGluR1 and 5) are positively coupled to
phospholipase C. Their activation produces phosphoin-
ositide turn over and diacylglycerol within target neu-
rons. Both group II (mGluR2 and 3) and group III
mGluRs (mGluR4 and mGluR 6–8) are located presyn-
In contrast, the efficacy of antagonists for mGluRs has
not been clarified in animal models or clinical trials. This
may be due to the lack of potent and selective antago-
nists of mGluRs. Our laboratory initiated efforts to
identify potent and selective antagonists for group II
mGluRs, reporting that 3-alkoxy-2-amino-6-fluorobicy-
clo[3.1.0]hexane-2,6-dicarboxylic acid derivatives 2 are
potent and selective antagonists for group II mGluRs,
exhibiting good absorption and blood–brain barrier
(BBB) penetration.20 Compounds 2 were identified
through chemical modification of 1,12 a typical group
Keywords: mGluR2 antagonist; 2-Aminobicyclo[3.1.0]hexane-2,6-di-
carboxylic acids; Synthesis; Structure–activity relationship.
*
Corresponding author. Tel.: +81 486693029; fax: +81 486527254;
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.2005.12.061

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A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
II mGluR agonist. Compound 1, a compound incorpo-
rating a fluorine atom at the C-6 position of 4 reported
by Kew et al.21 strongly inhibited cAMP formation with
the same EC50 value as 4, exhibiting better oral activity
than the original compound 4. Of the compounds which
we have reported, 2-amino-3-(3,4-dichlorobenzyloxy)-6-
ested in chemical modifications of 2, focusing on the
incorporation of an S-arylthio, S-benzylthio (S-ana-
logue) and N-benzylamino group (N-analogue) on the
C-3 position of group II mGluR agonists 2. These trans-
formations may expand the structural diversity of the
compounds, since the nitrogen atom is capable of hav-
ing another substituent, and the sulfur atom has defer-
ent oxidation states (Chart 1).
fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid
3
(MGS0039) was the most potent group II mGluR antag-
onist with respect to affinity, selectivity, oral absorption,
and BBB penetration, exhibiting dose-dependent antide-
pressant-like effects in the rat forced swim test and
mouse tail suspension test. In a previous paper,20 we
reported that incorporating an hydroxyl or alkoxy
group at the C-3 position of group II mGluR agonist
1 produced a group II mGluR antagonist, despite the
size of the alkoxy group. Our interest in chemical mod-
ification of 2 to identify group II mGluR antagonists fo-
cused on the defluorine compounds of 2. The fluorine
atom at a-position of the carboxyl group enhances the
acidity of the carboxyl group at the C-6 position on
the bicyclo[3.1.0]hexane ring of 2. We assumed that
the defluorination of 2 would dramatically affect the
acidity of the compounds and their resulting binding
affinity and compound absorption. We were also inter-
Here, we focus on the defluorine compound at the C-6
position of the bicyclo[3.1.0]hexane ring of 2, S-ana-
logues, and N-analogues. We discuss the synthesis, in vi-
tro
pharmacological
profile,
structure–activity
relationships of 3-alkoxy-2-aminobicyclo[3.1.0]hexane-
2,6-dicarboxylic acid, and pharmacokinetic profiles of
15ae, 15at, and 15ba.
2. Chemistry
Scheme 1 illustrates the synthesis of the key intermedi-
ate 9. Treating the optically pure compound 522 with
lithium bis(trimethylsilyl)amide and N-phenylbis(triflu-
oromethanesulfonimide) produced the corresponding
enol triflate. The a,b-unsaturated carbonyl compound
6 was obtained from the enol triflate through a palla-
dium-catalyzed reaction with carbon monoxide in the
presence of ethanol. We used the method of transfor-
mation from a,b-unsaturated carbonyl compound into
b-hydroxy a-amino acid reported by Shao and Good-
man,23 while the key intermediate 9 was synthesized
from 6. Oxidation of the double bond in 6 with
OsO4 and N-methylmorphorine N-oxide (NMO)
resulted in diol 7. In the reaction, the oxidant
approaches the double bond at the opposite side of
the fused cyclopropane ring, resulting in 7 as the sin-
gle product. The reaction of 7 with SOCl2 followed by
oxidation with NaIO4 and RuCl3 provided a cyclic
sulfate 8. Regioselective nucleophilic displacement by
NaN3 at the C-2 position of 8 proceeded24,25 due to
the high ability of the cyclic sulfate as a leaving
group, followed by hydrolysis, providing the key inter-
mediate 9.
Antagonists
Agonists
F
F
H
H
CO2H
CO2H
H
NH2
H
NH2
RO
HO2C
CO2H
Cl
H
Cl
CO2H
F
H
CO2H
H
H
NH2
NH2
HO2C
O
CO2H
3 (MGS0039)
4
Chart 1. Group II mGluR agonists and antagonists.
H
H
H
CO2Et
CO2Et
CO2Et
b
a
c
H
H
H
HO
CO2Et
HO
O
CO2Et
6
5
7
H
CO2Et
H
CO2Et
d
H
H
O
CO2Et
O
HO
EtO2C
S
N3
O
O
9
8
Scheme 1. Reagents and conditions: (a) i—HMDS, n-BuLi, Tf2NPh, THF, ꢀ60 ꢁC; ii—CO, Pd(OAc)2, (i-Pr)2NEt, PPh3, EtOH, rt; (b) 4% OsO4,
50% NMO, MeCN, rt; (c) SOCl2, Et3N, CH2Cl2, 4 ꢁC; (d) i—NaN3, DMF, 50 ꢁC; ii—H2SO4, Et2O, H2O, rt.

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3407
With the key intermediates 9 and 1026 in hand, we ad-
dressed the synthesis of 3-alkoxy-2-aminobicy-
clo[3.1.0]hexane-2,6-dicarboxylic acid 15aa–am, as
shown in Scheme 2. The 3-hydroxy groups in the key
intermediates 9 and 10 were etherized with benzyl tri-
chloroacetimidates and a catalytic amount of trifluo-
romethanesulfonic acid to yield 11ad, 11ae, and 11ag
(R1 = Et), and 12aa–ac, 12af, and 12ah–am
(R1 = Bn).27,28 The benzyl trichloroacetimidates were
prepared from 1,1,1-trichloroacetonitrile and the corre-
sponding alcohol (R2OH). For the reaction of 10 with
1-(3,4-dichlorophenyl)ethanol, the mixture of two dia-
stereomers 12al and 12am was separated by silica gel
chromatography.
Scheme 3 shows the synthesis of 15an–ax, bicy-
clo[3.1.0]hexane ring system having aryl-, benzyl-, and
alkylthio groups. Compounds 15an–ax were synthesized
from 16.20
Compound 16 was treated with anhydride and pyridine
to yield a key intermediate 17 to introduce a thioether
moiety. Nucleophilic substitution reaction of 17 with
various sodium benzyl and alkyl thiorates was allowed
to proceed to render the corresponding thioethers
(18an–ax). A Staudinger reaction29,30 was then per-
formed by trimethylphosphine in the presence of a small
amount of water in THF to obtain 19an–ax. Finally,
hydrolysis of the ester groups of C-3 and C-6 positions
in 19an–ax with lithium hydroxide provided 15an–ax.
Reduction of compounds 11ad, 11ae, 11ag, 12aa–ac,
12af, and 12ah–am using the Staudinger reaction29,30
provided compounds 13ad, 13ae, 13ag, 14aa–ac, 14af,
and 14ah–am without reductive cleavage of the benzyl
group at the C-3 position. Finally, 2,6-dicarboxylic acid
15aa–am were obtained by hydrolysis of the esters 13ad,
13ae, 13ag, 14aa–ac, 14af, and 14ah–am with LiOH.
Scheme 4 illustrates the preparation of 15ay and 15az,
sulfinyl and sulfonyl derivatives. m-CPBA was used for
oxidation of the thioether moiety, since the oxidation
state could be controlled by the amount of m-CPBA.
Oxidation of 18at with 1.2 and 2.5 equiv of m-CPBA
provided 20 and 22 in 84% and 91% yields, respectively.
Compound 20 was transformed into 15ay through
reduction by the Staudinger reaction and hydrolysis by
lithium hydroxide. After 23 was obtained by the same
reduction from 20, cyclization reaction of 23 occurred
to give 24 instead of 15az under the hydrolysis condi-
tions using lithium hydroxide. The cyclization reaction
may be attributable to the high acidity of the proton
at the benzyl position in 23. Thus, we attempted hydro-
lysis of 23 under acid conditions to avoid the cyclization
reaction. This produced a satisfactory yield of 15az,
after which no 23 was detected.
Scheme 5 shows the synthesis of 15ba, 15bb, and 15bc
compounds having an amino group and having an
amide moiety at the C-3 position of the bicy-
clo[3.1.0]hexane ring. Reduction of the azide group in
16 and the protection of the generated amino group with
a Boc group were performed first, since the synthesis of
15ba and 15bb required distinguishing between the two
amino groups at the C-3 and C-2 positions of 15ba
and 15bb. Reduction of azide group of 16 by the Stau-
dinger reaction was allowed to proceed to give 25. Treat-
ment of Boc2O and sodium bicarbonate in THF
provided 26, after which compound 26 was treated with
Scheme 2. Reagents and conditions: (a) R2OC(@NH)CCl3, TfOH,
CHCl3, cyclohexane, rt; (b) Me3P, THF, H2O, rt; (c) LiOH–H2O,
THF, H2O, rt.
Scheme 3. Reagents and conditions: (a) Tf2O, pyridine, CH2Cl2, 0 ꢁC; (b) R2SH, EtONa, DMF, rt; (c) Me3P, THF, H2O, rt; (d) LiOH–H2O, THF,
H2O, rt.

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A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
Scheme 4. Reagents and conditions: (a) m-CPBA (1.2 equiv), CH2Cl2, rt; (b) Me3P, THF, H2O, rt; (c) LiOH–H2O, THF, H2O, rt; (d) m-CPBA
(2.5 equiv), CH2Cl2, rt; (e) 60% H2SO4, 130 ꢁC.
Scheme 5. Reagents and conditions: (a) Me3P, THF, H2O, rt; (b) Boc2O, satd NaHCO3, THF, rt; (c) Tf2O, pyridine, CH2Cl2, 0 ꢁC; (d) NaN3, DMF,
rt; (e) H2, Pd/C, EtOH, rt; (f) 3,4-dichlorobenzyl bromide, pyridine, CHCl3, rt; (g) MeI, K2CO3, DMF, rt; (h) 4 M HCl/AcOEt, 0 ꢁC; (i) LiOH–H2O,
THF, H2O, rt; (j) 3,4-dichlorobenzoyl chloride, rt.

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3409
trifluoromethanesulfonyl anhydride and pyridine to ob-
3. Results and discussion
tain 27. Transformation from 27 into 28 was achieved
by nucleophilic substitution with sodium azide, followed
by hydrogenation of the introduced azide group with 5%
Pd/C as a catalyst. The N-mono-substituted compound
30 was produced via benzylation of an amino group at
the C-3 position of 29 with 3,4-dichlorobenzyl bromide
and pyridine in chloroform. The N-di-substituted com-
pound 31 was synthesized from 30 by methyl iodide
and K2CO3 in DMF. Following removal of the Boc
group in 30 and 31, hydrolysis of 14ba and 14bb with
lithium hydroxide provided 15ba and 15bb.
3.1. Pharmacological profile studies
The binding affinity of 15aa–bc for mGluR2 was
evaluated by [3H]-(1S,2S,3S,5R,6S)-2-amino-3-fluoro-
bicyclio[3.1.0]hexane-2,6-dicarboxylic
MGS0008) binding using CHO cells stably expressing
mGluR2.12 Table 1 gives the Ki values obtained for
mGluR2.
acid
([3H]-
The agonist activity of 15ae, 15aj, 15an, 15at, and 15ay–
ba was evaluated by measuring the agonist-dependent
inhibition of forskolin-induced cyclic AMP (cAMP) for-
mation in mGluR2 expressing cells. The antagonist
activities of the compounds were measured with 30 lM
glutamic acid. Table 2 shows the agonist and antagonist
activities of the compounds.
The treatment of 29, the intermediate of synthesis of
15ba and 15bb, with 3,4-dichlorobenzoyl chloride and
pyridine in chloroform, gave 32. The removal of the
Boc group and hydrolysis of 32 in the same manner
from 30 to 15ba provided 15bc.
Table 1. Binding affinity for mGluR2
X
H
CO2H
H
R
Y
CO2H
H2N
15
X
Y
R
Affinity Ki (nM)
aa
ab
H
H
H
H
H
H
H
H
H
H
H
H
H
F
O
O
O
O
O
O
O
O
O
O
O
O
O
S
2-Naphtyl-CH2
2-Ph-PhCH2
4-Cl-PhCH2
4.02 (2.53)a
9.27 (4.07)a
8.46 (3.17)a
8.17 (2.94)a
2.51 (2.38)a
4.84 (2.27)a
7.59 (3.57)a
6.05
ac
ad
3-NC-PhCH2
3,4-Cl2-PhCH2
3,4-F2-PhCH2
3-Cl-4-F-PhCH2
Ph2CH
ae
af
ag
ah
ai
(4-Cl-Ph)2CH
(4-F-Ph)2CH
(3,4-Cl2-Ph)CH
8.65
2.24
aj
ak
8.60
5.02
al
am
(R )-3,4-Cl2-Ph(Me)CH
*
(S )-3,4-Cl2-Ph(Me)CH
*
Thiophen-2-yl-CH2
2-Ph-PhCH2
3.86
6.37
an
ao
F
S
3.77
3.77
ap
aq
F
S
4-MeO-PhCH2
4-F-PhCH2
F
S
2.13
21.90
ar
F
S
4-t-Bu-PhCH2
3-CF3-PhCH2
3,4-Cl2-PhCH2
3-Cl-2,6-F2-PhCH2
Ph(Me)CH
as
F
S
5.15
1.96
at
au
F
S
F
S
4.92
5.70
6.37 (3.75)a
3.69
11.28
av
aw
F
S
F
S
(4-F-Ph)2CH
3,4-Cl2-Ph–
ax
ay
F
S
F
SO
SO2
NH
N-Me
N
3,4-Cl2-PhCH2
3,4-Cl2-PhCH2
3,4-Cl2-PhCH2
3,4-Cl2-PhCH2
3,4-Cl2-PhCO
az
ba
F
14.61
3.29
F
bb
bc
F
6.98
168.01
2.61
F
3 (Ref. 20)
LY341495
(Ref. 31)
2.90
a The figure within parentheses is the Ki value of the corresponding fluorinated compound 2.20

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A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
Table 2. Antagonist and agonist activities
15au at the benzene ring slightly reduced mGluR2 affin-
ity (Ki = 4.92 nM).
15 Antagonist activity IC50 (nM) Agonist activity EC50 (nM)
ae
aj
34.21
38.87
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
We then examined the introduction of alkyl and phenyl
groups at the benzyl position. The Ki value of the
1-bis(4-fluorophenyl)methyl compound 15aw for
mGluR2 was 6.37 nM, lower than that of the corre-
sponding O-benzyl compound (Ki = 3.75.20) The bind-
ing affinity of 15av was evaluated as a mixture of two
unseparated diasteromers by column chromatography
(Ki = 5.70 nM).
an 100.69
13.34
55.19
at
ay
az 689.07
ba 35.96
At first, to examine the effects of defluorination of 2 hav-
ing the substituent/s on benzyl group at the C-3 position
of the bicyclo[3.1.0]hexane ring on affinity for mGluR2,
we investigated some corresponding defluorinated com-
pounds to 2, for which high mGluR2 affinities had been
reported in a previous paper.20 Compounds 15aa–af
showed Ki values of 4.02 nM (2.53 nM), 9.27 (4.07),
8.46 (3.17), 8.17 (2.94), 2.51 (2.38), 4.84 (2.27), and
7.59 (3.57), respectively (the figure within parentheses
is the Ki value of the corresponding fluorinated com-
pound 220). Compounds 15aa–ad, 15af, and 15ag
showed between 1.6- and 2.8-fold lower affinity than
the corresponding 2, but 15ae showed almost the same
affinity for mGluR2. Compound 15ae was the corre-
sponding defluorinated compound of MGS0039, the
most potent compound in the series of fluorinated com-
pounds at the C-6 position. We then examined the intro-
duction of a methyl and substituted phenyl group on the
methylene of the benzyl group at the C-3 position of the
bicyclo[3.1.0]hexane ring. Compounds 15ai and 15ak–am
also tended to show lower affinities for mGluR2 com-
pared to the corresponding 2, but 15ai showed only high
affinity.
In general, incorporating S-benzyl compounds increased
binding affinities for mGluR2 compared to O-benzyl
compounds. The 3,4-dichlorobenzyl group also showed
marked effectiveness in the S-benzyl compounds.
Previously, we reported20 on the introduction of an O-
benzyl group by benzylation with trichloroacetimidate
of the corresponding benzyl alcohol, preventing synthe-
sis of the O-aryl compounds. Thus, we were interested in
the binding affinity for mGluR2 of O-aryl compound.
Introduction of the S-benzyl group was performed by
nucleophilic displacement with the corresponding ben-
zylmercaptan under basic conditions, and the synthesis
of S-aryl compound succeeded. We reported that S-ben-
zyl compounds generally showed good binding affinity
compared to O-benzyl compounds. For this reason, we
examined S-aryl compounds, and 15ax showed high
binding affinity for mGluR2. However, the Ki value of
15ax was slightly lower than those of 15ae and 15at.
As the next step in chemical modification, we investigat-
ed the transformation of benzyl thioether moiety of
15at. We synthesized a sulfinyl compound (15ay) and
sulfonyl compound (15az), each having a sulfur atom
of two deferent oxidation states. Their Ki values were
lower than those of 15at (15ay: Ki = 11.28 nM, 15ba:
Ki = 14.61 nM).
These findings suggest a fluorine atom must be intro-
duced to increase binding affinity, and introducing a
3,4-dichlorobenzyl group and bis(4-fluorophenyl)methyl
group at the C-3 position is particularly effective in
increasing binding affinity for mGluR2. Compounds
15ae and 15aj with high binding affinities for mGluR2
also showed high antagonist activity (15ae:
IC50 = 34.21 nM, 15aj: IC50 = 38.87 nM), but no signifi-
cant agonist activity (ED50 > 100,000 nM).
Our final chemical modification focused on incorporat-
ing the N-alkyl group at the C-3 position of group II
mGluR antagonists 2. Compound 15ba, an N-benzyl
compound corresponding to 3, exhibited high binding
affinities (Ki = 3.29 nM) for mGluR2, identical to 3.
Compound 15bb, N,N-disubstituted compound, exhibit-
ed 2.5-fold lower mGluR2 affinity than 15ba. Com-
pound 15bc, a benzoyl compound, exhibited much
lower affinity for mGluR2 (Ki = 168.01 nM). These find-
ings suggest that the oxygen atom of ether bond in 3 can
be replaced by a sulfur or nitrogen atom to produce
nearly the same binding affinity for mGluR2, while
increase of the size of around sulfur or nitrogen atom
reduced mGluR2 binding affinity.
We next examined chemical modifications, focusing on
incorporating S-analogues and N-analogues of the
group II mGluR antagonist 2.
We began by investigating chemical modification of the
substituent on the benzene ring of benzyl group at the C-
3 position of the bicyclo[3.1.0]hexane ring. Introducing
single substituents, a phenyl group (15ao), methoxy
group (15ap), fluorine atom (15aq), and trifluoromethyl
group (15as), resulted in good affinity for mGluR2.
Compounds 15ao, 15ap, and 15as showed binding affin-
ities higher than those of the corresponding O-benzyl
compounds 2. The Ki value of 15ar having a bulky alkyl
group was lower than those of other single substituted
compounds. In the S-benzyl compounds, 15at with a
3,4-dichlorobenzyl group also showed marked binding
affinity for mGluR2. Introduction of three halogen
atoms, two fluorine atoms, and a chlorine atom in
Finally, we performed evaluations of plasma levels for
the three compounds 15ae, 15at, and 15ba. All three
showed high affinity for mGluR2, while retaining a
3,4-dichlorobenzyl group.
Table 3 gives plasma levels and pharmacokinetic param-
eters for 15ae, 15at, and 15ba following oral administra-
tion to rats at a dose of 10 mg/kg.

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3411
Table 3. Pharmacokinetic parameters of 15ae, at, and ba following oral administration to rats
Compound
15ae
15ata
15baa
3 (Ref. 20)
LY341495 (Ref. 31)
10
0.5
64
2.64
Dose (mg/kg)
Tmax (h)
3
10
30
5.3 2.3
10
10
10
6
3.3 1.2
81.9 23.1
4.0 0.4
702.5 75.6
—
4.7 3.1
147.0 39.5
4.0 0.5
3.33 1.15
116 51.0
—
3.33 1.15
96.7 8.62
—
Cmax (ng/mL)
t1/2 (h)
519.3 49.9
3.2 0.4
492
2.3
6810
—
AUCinf (ng h/mL)
AUC0–6h (ng h/mL)
1385.1 57.4
—
5919.1 585.3
—
—
—
284
—
500 213
455 28.9
Each value represents the mean SD of three animals.
a t1/2 and AUCinf could not be estimated for limited sampling points.
Following oral administration of 15ae, maximum plasma
concentrations (Cmax) of 81.9, 147.0, and 519.3 ng/mL
were reached at 3.3–5.3 h at dose of 3, 10, and 30 mg/kg,
respectively. Cmax values and corresponding exposure,
as measured by AUCinf, increased less than dose, pro-
portionally. Plasma levels of 15at and 15ba following
oral administration were also evaluated at 10 mg/kg in
rats. The Cmax levels of these compounds were 116
and 96.7 ng/mL, somewhat lower than that of 15ae,
and the corresponding AUC0–6h values were 500 and
455 ng h/mL, respectively.
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 obtained using a Varian Gemini 2000 (200 MHz),
Varian Unity Inova 300 (300 MHz) or JEOL Lamb-
da500 (500 MHz). Chemical shifts are reported in parts
per million relative to TMS or sodium 3-trimethylsilyl-
propionate-2,2,3,3-d4 (TMSP) as an internal standard.
Mass spectra (MS) were obtained on a JEOL JMS-
SX102 or Micromass Platform LC. Optical rotations
were determined with a JASCO DIP-360 polarimeter.
Elemental analyses were performed on a Perkin-Elmer
2400.
At oral administration, all compounds, which were stud-
ied at 10 mg/kg (po) in rat, exhibited the higher plasma
concentrations than those for the known group II mGluR
antagonist (2S)-amino-2-((1S,2S)-2-carboxycycloprop-
1-yl)-3-(9-xanthyl)propionic acid (LY341495).31,32
5.1.1.
(1S,5R,6S)-6-Bicyclo[3.1.0]hex-2-ene-2,6-dicar-
4. Conclusions
boxylic acid diethyl ester (6). To a mixture of HMDS
(9.0 mL, 42.8 mmol) and THF (46 mL) was added BuLi
(2.66 M hexane solution, 16.1 mL, 42.8 mmol) under
nitrogen atmosphere at ꢀ70 to ꢀ68 ꢁC. After the mix-
ture was stirred for 0.5 h, a THF (23 mL) solution of 6
(6.00 g, 35.7 mmol) was added to the mixture at ꢀ70
to ꢀ60 ꢁC and stirred for 1 h. A THF (700 mL) solution
of N-phenylbis(trifluoromethanesulfonimide) (213 g,
0.597 mol) at ꢀ70 to ꢀ60 ꢁC was added to the mixture
and stirred for 2 h. The mixture was stirred for 2 h, after
removing the dry ice–acetone bath. The reaction mixture
was diluted with ether. The ethereal solution was
washed with saturated NaHCO3 (3·) and brine, dried
with Na2SO4, concentrated under reduced pressure,
and chromatographed (hexane/AcOEt = 20:1) to get a
colorless oil (12.68 g). A mixture of the obtained color-
less oil (12.68 g), diisopropylethylamine (7.3 mL,
42.2 mmol), triphenylphosphine (663 mg, 2.53 mmol),
ethanol (68 mL), DMF (67.8 mL), and Pd(OAc)2
(285 mg, 1.27 mmol) was stirred for 18 h at room tem-
perature under carbon monoxide atmosphere. The mix-
ture was diluted with 1 N HCl and extracted with ether
(3·). The combined ethereal layer was washed with sat-
urated NaHCO3 (4·) and saturated brine, dried with
Na2SO4, evaporated, and chromatographed (hexane/
AcOEt = 20:1) to yield 7 (5.86 g, 73%) as a colorless
In this paper, we have reported the syntheses and SARs.
Overall, S-analogues showed good mGluR2 binding
affinity compared to O-benzyl group, while introducing
a bulky alkyl group in benzene moiety and increase of
the size of around sulfur or nitrogen atom did not im-
prove the binding affinity. The fluorine atom and the
3,4-dichlorobenzyl group (15ae, 15at, and 15ba) played
important roles in increasing binding affinity. Of the
compounds discussed, 15ae, 15aj, 15an, 15at, and
15ay–ba also showed antagonist activity. In particular,
15at with a 3,4-dichlorobenzyl group showed the highest
mGluR2 affinity among known group II mGluR antag-
onists, and the presence of a 3,4-dichlorobenzyl group
and fluorine atom at the 6 position of bicyclo[3.1.0]hex-
ane ring effectively increased affinity for mGluR2
receptor.
Preliminarily examined at a dose of 10 mg/kg (po) in
rats, compounds 15ae, 15at, and 15ba exhibited plasma
concentrations higher than that of known group II
mGluR antagonist (LY341495), but the plasma concen-
trations of those compounds did not reach the plasma
concentration of 3. Given the high binding affinity for
mGluR2, those compounds have the potency of group
II mGluR2 antagonists, although they require further
improvements in plasma concentrations. We believe
studies of SARs and the resulting pharmacokinetic pro-
files will provide new opportunities to explore and im-
prove upon new group II mGluR2 antagonists.
1
oil. H NMR (300 MHz, CDCl3, TMS) d 1.26 (3H, t,
J = 7.1 Hz), 1.31 (3H, t, J = 6.8 Hz), 2.22–2.30 (1H,
m), 2.59–2.91 (4H, m), 4.08–4.29 (4H, m), 6.53 (1H, s);
26
MS (ESI, Pos) m/z 247 (M++Na); ½aꢁ +145.5 (c 0.95,
D
CHCl3).

3412
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
5.1.2.
(1S,5R,6S)-2,3-Dihydroxybicyclo[3.1.0]hexane-
5.1.5. General method for the synthesis of 15aa–ac and
15ae–ak (method A)
2,6-dicarboxylic acid diethyl ester (7). A mixture of 7
(5.86 g, 26.1 mmol), MeCN (150 mL), H2O (54 mL),
50% aqueous NMO (10.8 mL, 52.2 mol), and 4% aque-
ous OsO4 (8.3 mL, 1.31 mmol) was stirred for 1 h at
room temperature. NaHSO3 was added to the reaction
mixture with ice-cooling stirred for 30 min. The mixture
was filtered using Celite Pad, and the filtrate was extract-
ed with AcOEt (2·). The combined AcOEt layer was
washed with saturated brine, dried with Na2SO4, evapo-
rated, and chromatographed (hexane/AcOEt = 2:1) to
yield 7 (5.43 g, 81%) as a white solid. 1H NMR
(200 MHz, CDCl3, TMS) d 1.25 (3H, t, J = 7.3 Hz),
1.33 (3H, t, J = 7.0 Hz), 1.73–2.16 (4H, m), 2.27–2.45
(2H, m), 3.83 (1H, s), 4.00–4.15 (1H, m), 4.12 (2H, q,
5.1.5.1. (1S,2R,3R,5R,6S)-2-Azido-3-(3,4-dichloroben-
zyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid diethyl
ester (11ae). A THF (153 mL) solution of 3,4-dichlorob-
enzyl alcohol (200 g, 1.13 mol) was added to a THF
(227 mL) suspension of NaH (60%, 4.52 g, 113 mmol)
and stirred for 0.5 h at room temperature. Trichloro-
acetonitrile (113 mL, 1.13 mol) was added to the mix-
ture with ice-cooling and stirred for 1.5 h at room
temperature. Pentane (150 mL) and methanol (3.7 mL)
were added to the reaction mixture. After stirring for
30 min at room temperature, a formed precipitate was
filtered off. The filtrate was concentrated under reduced
pressure to yield crude 3,4-dichlorobenzyl-2,2,2-trichlo-
roacetoimidate (358 g) as a brown viscous liquid.
J = 7.0 Hz), 4.21–4.41 (2H, m); MS (ESI, Pos) m/z 281
27
D
(M++Na); ½aꢁ ꢀ89.7 (c 0.32, CHCl3).
Trifluoromethanesulfonic acid (161 lL) was added to
the mixture of 3,4-dichlorobenzyl-2,2,2-trichloroace-
toimidate (1.47 g, 4.56 mmol), 9 (860 mg, 3.04 mol),
CHCl3 (3.6 mL), and cyclohexane (7.2 mL) under nitro-
gen atmosphere. After the mixture was stirred for 1 h at
room temperature, a formed precipitate was filtered off.
Saturated NaHCO3 was added to the filtrate with ice-
cooling. The mixture was extracted with CHCl3 (2·).
The CHCl3 layer was washed with saturated brine, dried
with Na2SO4, concentrated under reduced pressure, and
chromatographed (hexane/AcOEt = 15:1) to yield 11ae
5.1.3. (1S,2S,3R,5R,6S)-3,3-Dioxo-tetrahydro-2,4-dioxa-
acid
3k6-thiacyclopropa[a]pentalene-1,1b-dicarboxylic
diethyl ester (8). SOCl2 (2.47 mL) was added to a mix-
ture of 7 (5.86 g, 22.7 mmol), CH2Cl2 (82 mL), and
Et3N (6.96 mL, 49.9 mmol) with ice-cooling and the
mixture was stirred for 15 min. H2O was added to the
reaction mixture, and CH2Cl2 layer was separated.
The CH2Cl2 layer was washed with H2O and saturated
brine, dried with MgSO4, and evaporated. The residue
was dissolved with CCl4 (42 mL), MeCN (42 mL), and
H2O (50 mL). NaIO4 (6.31 g, 29.5 mol) and RuCl3ÆH2O
(50 mg) were added to the solution with ice-cooling and
stirred for 2 h. The mixture was filtered using Celite Pad,
and the filtrate was partitioned into organic and aque-
ous layers, and the aqueous layer was extracted with
ether. The combined organic layer was washed with sat-
urated brine, dried with MgSO4, evaporated, and chro-
matographed (hexane/AcOEt = 4:1) to yield 8 (6.37 g,
88%) as a white solid. 1H NMR (200 MHz, CDCl3,
1
(440 mg, 33%) as a colorless oil. H NMR (200 MHz,
CDCl3, TMS) d 1.27 (3H, t, J = 7.0 Hz), 1.32 (3H, t,
J = 7.5 Hz), 1.75 (1H, t, J = 3.1 Hz), 2.03–2.38 (4H,
m), 3.54 (1H, dd, J = 7.47 Hz), 4.14 (4H, q,
J = 7.2 Hz), 4.23–4.46 (3H, m), 4.58 (1H, d,
J = 12.3 Hz), 7.09 (1H, dd, J = 8.4, 2.0 Hz), 7.36 (1H,
d, J = 2.0 Hz), 7.40 (1H, d, J = 8.4 Hz); MS (ESI, Pos)
24
m/z; 464 (M++Na); ½aꢁ +2.5 (c 0.70, CHCl3).
D
TMS)
d
1.26 (3H, t, J = 7.3 Hz), 1.37 (3H, t,
5.1.5.2. (1S,2R,3R,5R,6S)-2-Amino-3-(3,4-dichloro-
benzyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic
J = 7.3 Hz), 1.71 (1H, dd, J = 3.5, 3.5 Hz), 2.26–2.38
(1H, m), 2.56–2.68 (3H, m), 4.14 (2H, q, J = 7.3 Hz),
4.29–4.47 (2H, m), 5.35 (1H, dd, J = 6.2, 6.2 Hz); MS
acid
diethyl ester (13ae). A mixture of 11ae (540 mg,
1.22 mmol), Me3P (1 M THF solution, 1.34 mL), THF
(18 mL), and H2O (1.4 mL) was stirred for 22 h at room
temperature. 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 chromatographed (hexane/AcOEt = 3:1–1:1) to
yield 13ae (160 mg, 39%) as a pale yellow oil. 1H
NMR (200 MHz, CDCl3, TMS) d 1.25 (3H, d,
J = 7.5 Hz), 1.30 (3H, d, J = 7.47 Hz), 1.72 (1H, dd,
J = 3.1, 3.1 Hz), 1.97–2.37 (4H, m), 3.46 (1H, dd,
J = 8.6, 7.3 Hz), 4.06–4.54 (4H, m), 4.41 (1H, d,
J = 12.3 Hz), 4.51 (1H, d, J = 12.3 Hz), 7.08 (1H, dd,
J = 8.4, 1.8 Hz), 7.35 (1H, d, J = 1.8 Hz), 7.39 (1H, d,
24
D
(ESI, Pos) m/z 343 (M++Na); ½aꢁ ꢀ35.7 (c 0.27,
CHCl3).
5.1.4.
(1S,2S,3R,5R,6S)-2-Azido-3-hydroxybicyclo-
[3.1.0]hexane-2,6-dicarboxylic acid diethyl ester (9). A
mixture of 8 (6.37 g, 19.9 mmol), DMF (78 mL), H2O
(8 mL), and NaN3 (2.33 g, 35 mmol) was stirred for
1 h at 50 ꢁC. The reaction mixture was concentrated
under reduced pressure. The residue was dissolved with
ether (400 mL) and H2O (11 mL). 20% H2SO4 (31 mL)
was added dropwise to the mixture with ice-cooling
and vigorously stirred for 20 h at room temperature.
The organic layer of the mixture was separated,
washed with saturated brine, dried with MgSO4, evap-
orated, and chromatographed (hexane/AcOEt = 4:1) to
yield 9 (88.5 g, 91%) as a colorless oil. 1H NMR
(200 MHz, CDCl3, TMS) d 1.27 (3H, t, J = 7.3 Hz),
1.38 (3H, t, J = 7.3 Hz), 1.83 (1H, dd, J = 3.1,
3.1 Hz), 1.99–2.46 (4H, m), 3.73–3.90 (1H, m), 4.14
J = 8.4 Hz); MS (ESI, Pos) m/z 438 (M++Na);
23
D
½aꢁ +12.6 (c 0.33, CHCl3).
5.1.5.3. (1S,2R,3R,5R,6S)-2-Amino-3-(3,4-dichloro-
benzyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic
acid
(15ae). A mixture of 13ae (160 mg, 0.384 mmol), THF
(4 mL), H2O (2 mL), and LiOHÆH2O (48 mg,
1.15 mmol) was stirred for five days at room tempera-
ture. The reaction mixture was acidified with 1 N HCl
(2H, q, J = 7.0 Hz), 4.36 (2H, q, J = 7.0 Hz); MS
27
(ESI, Pos) m/z; 306 (M++Na); ½aꢁ ꢀ49.1 (c 0.22,
D
CHCl3).

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3413
and stirred for 1 h at room temperature. The solution
was chromatographed on AG50W-X8 cation-exchange
resin (Bio-Rad) (H2O–50% aqueous THF–10% aqueous
pyridine) to yield 15ae (66 mg, 49%) 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,
5.1.5.8. (1S,2R,3R,5R,6S)-2-Amino-3-(3-chloro-4-flu-
orobenzyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid
(15ag). Starting from 9 and 3-chloro-4-fluorobenzyl
alcohol, 15ag was obtained as a white powder.
Mp > 270 ꢁC (decomp.). 1H NMR (500 MHz, D2O,
TMSP) d 1.60–1.65 (1H, m), 1.96–2.21 (3H, m), 2.36–
2.43 (1H, m), 3.81 (1H, t, J = 8.1 Hz), 4.64 (2H, s),
7.17–7.24 (1H, m), 7.35–7.40 (1H, m), 7.46–7.52 (1H,
23
D
m, J = 12.1 Hz), 7.53–7.58 (2H, m); MS (ESI, Nega)
m); MS (ESI, Nega) m/z 342 (Mꢀꢀ1); ½aꢁ +3.6 (c
28
m/z 358 (Mꢀꢀ1); ½aꢁ +5.1 (c 2.00, 1 N NaOH); Anal.
0.12, 1 N NaOH); Anal. Calcd for C15H15F2NO5Æ0.1-
H2O: C, 54.75; H, 4.66; N, 4.26. Found: C, 54.61; H,
4.61; N, 4.22.
D
Calcd for C15H15Cl2NO5: C, 50.02; H, 4.20; N, 3.89.
Found: C, 49.99; H, 4.21; N, 3.80.
5.1.5.9. (1S,2R,3R,5R,6S)-2-Amino-3-(diphenylmeth-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ah).
Starting from 10 and diphenylmethyl alcohol, 15ah
was obtained as a white powder. Mp > 228 (decomp.).
1H NMR (300 MHz, D2O, TMSP) d 1.49 (1H, dd,
J = 2.8, 2.8 Hz), 1.93–2.05 (2H, m), 2.14–2.33 (2H, m),
3.75 (1H, dd, J = 7.8, 7.8 Hz), 5.55 (1H, s), 7.30–7.48
5.1.5.4.
methyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic
(15aa). Starting from 10 and 2-naphthenylmethyl alco-
(1S,2R,3R,5R,6S)-2-Amino-3-(2-naphthyl-
acid
hol, 15aa was obtained as
a
white powder.
Mp > 213 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMSP) d 1.57–1.63 (1H, m), 1.95–2.06 (2H, m), 2.14–
2.25 (1H, m), 2.33–2.43 (1H, m), 3.83 (1H, dd, J = 8.9,
8.9 Hz), 4.68–4.80 (2H, m), 7.53–7.64 (3H, m), 7.88–
(10H, m); MS (ESI, Nega) m/z 366 (Mꢀꢀ1);
29
½aꢁ ꢀ14.3 (c 0.38, 1 N NaOH); Anal. Calcd for
8.01 (4H, m); MS (ESI, Nega) m/z 340 (Mꢀꢀ1);
D
28
½aꢁ +24.1 (c 0.26, 1 N NaOH); Anal. Calcd for
C21H21NO5Æ0.7H2O: C, 66.37; H, 5.94; N, 3.69. Found:
C, 66.53; H, 5.84; N, 3.83.
D
C19H19NO5Æ0.2H2O: C, 66.15; H, 5.67; N, 4.06. Found:
C, 66.16; H, 5.58; N, 3.84.
5.1.5.10. (1S,2R,3R,5R,6S)-2-Amino-3-(bis(4-chloro-
phenyl)methoxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15ai). Starting from 10 and bis(4-chlorophe-
nyl)methyl alcohol, 15ai was obtained as a white pow-
der. Mp > 222 ꢁC (decomp.). 1H NMR (300 MHz,
D2O, TMSP) d 1.45–1.53 (1H, m), 1.91–2.05 (2H, m),
2.15–2.26 (2H, m), 3.73 (1H, dd, J = 8.1, 8.1 Hz), 5.53
5.1.5.5. (1S,2R,3R,5R,6S)-2-Amino-3-(2-phenylben-
zyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ab).
Starting from 10 and 2-phenylbenzyl alcohol, 15ab was
obtained as a white powder. Mp > 205 ꢁC (decomp.).
1H NMR (300 MHz, D2O, TMSP) d 1.53–1.56 (1H,
m), 1.90–2.21 (4H, m), 3.56 (1H, dd, J = 7.5, 7.5 Hz),
4.43 (1H, d, J = 10.9 Hz), 4.50 (1H, d, J = 10.9 Hz),
(1H, s), 7.31–7.45 (8H, m); MS (ESI, Nega) m/z 434
28
(Mꢀꢀ1); ½aꢁ ꢀ14.3 (c 0.38, 1 N NaOH); Anal. Calcd
7.39–7.57 (9H, m); MS (ESI, Nega) m/z 366 (Mꢀꢀ1);
D
26
½aꢁ ꢀ12.9 (c 0.17, 1 N NaOH); Anal. Calcd for
for C21H19Cl2NO5Æ0.8H2O: C, 55.96; H, 4.61; N, 3.11.
Found: C, 56.20; H, 4.89; N, 2.95.
D
C21H21NO5Æ0.75H2O: C, 66.22; H, 5.95; N, 3.68. Found:
C, 66.25; H, 5.78; N, 3.68.
5.1.5.11. (1S,2R,3R,5R,6S)-2-Amino-3-(bis(4-fluoro-
phenyl)methoxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15aj). Starting from 10 and bis(4-fluorophe-
nyl)methyl alcohol, 15aj was obtained as a white powder.
Mp > 295 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMSP) d 1.49 (1H, dd, J = 3.8, 3.8 Hz), 1.88–2.04 (2H,
m), 2.17–2.24 (2H, m), 3.73 (1H, dd, J = 8.4, 8.4 Hz),
5.1.5.6. (1S,2R,3R,5R,6S)-2-Amino-3-(4-chloroben-
zyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ac).
Starting from 10 and 4-chlorobenzyl alcohol, 15ac was
obtained as a white powder. Mp > 205 ꢁC (decomp.).
1H NMR (300 MHz, D2O, TMSP) d 1.61 (1H, dd,
J = 3.3, 3.3 Hz), 1.99–2.05 (2H, m), 2.11–2.20 (1H, m),
2.34–2.40 (1H, m), 3.78 (1H, dd, J = 7.6, 7.6 Hz), 4.78
(2H, s), 7.35 (2H, d, J = 8.5 Hz), 7.43 (2H, d,
5.54 (1H, s), 7.09–7.17 (2H, m), 7.35–7.42 (2H, m); MS
25
(ESI, Nega) m/z 402 (Mꢀꢀ1); ½aꢁ ꢀ12.8 (c 0.18, 1 N
D
NaOH); Anal. Calcd for C21H19F2NO5Æ1.5H2O: C,
58.60; H, 5.15; N, 3.25. Found: C, 58.78; H, 4.85; N, 3.28.
J = 8.5 Hz); MS (ESI, Nega) m/z 324 (Mꢀꢀ1);
27
½aꢁ +5.1 (c 0.58, 1 N NaOH); Anal. Calcd for
D
C15H16ClNO5Æ0.1H2O: C, 55.00; H, 4.99; N, 4.28.
Found: C, 54.81; H, 4.91; N, 4.27.
5.1.5.12.
(1S,2R,3R,5R,6S)-2-Amino-3-(bis(3,4-
dichlorophenyl)methoxy)bicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid (15ak). Starting from 10 and bis(4-fluor-
ophenyl)methyl alcohol, 15ak was obtained as a white
5.1.5.7. (1S,2R,3R,5R,6S)-2-Amino-3-(3,4-difluoro-
benzyloxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic
1
acid
(15af). Starting from 10 and 3,4-difluorobenzyl alcohol,
15af was obtained as a white powder. Mp > 298 ꢁC (de-
powder. Mp > 295 ꢁC (decomp.). H NMR (300 MHz,
D2O, TMSP) d 1.56–1.69 (1H, m), 2.02–2.48 (4H, m),
3.49–3.72 (1H, m), 5.39–5.58 (1H, m), 7.20–7.69 (6H,
1
24
m); MS (ESI, Nega) m/z 502 (Mꢀꢀ1); ½aꢁ ꢀ3.43 (c
comp.). H NMR (300 MHz, D2O, TMSP) d 1.60–1.64
(1H, m), 1.98–2.08 (2H, m), 2.11–2.20 (1H, m), 2.34–
2.41 (1H, m), 3.78 (1H, dd, J = 7.3, 7.3 Hz), 4.51 (2H,
D
0.34, 1 N NaOH).
s), 7.11–7.32 (3H, m); MS (ESI, Nega) m/z 326 (MꢀꢀH);
5.1.6. Method B
26
½aꢁ ꢀ3.1 (c 0.37, 1 N NaOH); Anal. Calcd for
5.1.6.1. (1S,2R,3R,5R,6S)-2-Azido-3-(2-cyanobenzyl-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid diethyl
ester (11ad). A THF (1.6 mL) solution of 3-cyanobenzyl
D
C15H15ClNO5Æ0.3H2O: C, 51.60; H, 4.50; N, 4.01.
Found: C, 51.56; H, 4.36; N, 3.90.

3414
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
alcohol (1.0 g, 7.51 mmol) was added to a THF (1.1 mL)
suspension of NaH (60%, 30 mg, 0.75 mmol) and stirred
for 0.5 h at room temperature. Trichloroacetonitrile
(0.75 mL, 7.51 mol) was added to the mixture with ice-
cooling and stirred for 2 h at room temperature. Pentane
(1.3 mL) and methanol (27 lL) were added to the reac-
tion mixture. After stirring for 15 min at room tempera-
ture, a formed precipitate was filtered off. The filtrate
was concentrated under reduced pressure to yield crude
3-cyanobenzyl-2,2,2-trichloroacetoimidate (2.02 g) as a
brown viscous liquid.
30% MeCN) to yield 15ad (46 mg, 5%) as a white pow-
der. Mp > 230 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMSP) d 1.57–1.66 (1H, m), 1.94–2.45 (4H, m), 3.75–
3.88 (1H, m), 4.60 (2H, s), 7.64 (4H, m); MS (ESI, Nega)
28
m/z 315 (Mꢀꢀ1); ½aꢁ ꢀ1.1 (c 0.20, 1 N NaOH).
D
5.1.7. Method C
5.1.7.1. (1S,2R,3R,5R,6S)-2-Azido-3-((R )-1-(3,4-
dichlorophenyl)ethoxy)bicyclo[3.1.0]hexane-2,6-dicarbox-
*
ylic acid diethyl ester (15al) and (1S,2R,3R,5R,6S)-2-
azido-3-((S )-1-(3,4-dichlorophenyl)ethoxy)bicyclo[3.1.0]-
*
hexane-2,6-dicarboxylic acid diethyl ester (15am). A
THF (0.3 mL) solution of 1-(3,4-dichlorophenyl)ethanol
(500 mg, 2.62 mol) was added to a THF (0.3 mL) sus-
pension of NaH (60%, 10 mg, 0.25 mmol) and stirred
for 0.5 h at room temperature. Trichloroacetonitrile
(0.26 mL, 2.62 mol) was added to the mixture with ice-
cooling and stirred for 2 h at room temperature. Pentane
(0.46 mL) and methanol (9.3 lL) were added to the reac-
tion mixture. After stirring for 10 min at room tempera-
ture, a formed precipitate was filtered off. The filtrate
was concentrated under reduced pressure to yield crude
1-(3,4-dichlorophenyl)ethyl-2,2,2-trichloroacetoimidate
(860 mg) as a brown viscous liquid.
Trifluoromethanesulfonic acid (50 lL) was added to the
mixture of 3-cyanobenzyl-2,2,2-trichloroacetoimidate
(471 mg, 1.70 mmol), 9 (320 mg, 1.13 mmol), CHCl3
(1.4 mL), and cyclohexane (2.8 mL) under nitrogen
atmosphere, and stirred for 1 h. Then trifluoromethane-
sulfonic acid (50 lL) was added to the reaction mixture.
After the mixture was stirred for 2 h at room tempera-
ture, a formed precipitate was filtered off. Saturated
NaHCO3 was added to the filtrate with ice-cooling.
The mixture was extracted with CHCl3 (2·). The CHCl3
layer was washed with saturated brine, dried with
Na2SO4, concentrated under reduced pressure, and
chromatographed (hexane/AcOEt = 10:1) to yield 11ad
(210 mg, 46%) as
a
pale yellow oil. 1H NMR
Trifluoromethanesulfonic acid (14 lL) was added to the
mixture of 1-(3,4-dichlorophenyl)ethyl-2,2,2-trichloro-
acetoimidate (548 mg, 1.64 mmol), 10 (375 mg,
1.09 mol), CH2Cl2 (1.4 mL), and cyclohexane (2.8 mL)
under nitrogen atmosphere. After the mixture was stir-
red for 1 h at room temperature, a formed precipitate
was filtered off. Saturated NaHCO3 was added to the fil-
trate with ice-cooling. The mixture was extracted with
CHCl3 (2·). The CHCl3 layer was washed with saturat-
ed brine, dried with Na2SO4, and concentrated under re-
duced pressure. The residue was purified with silica gel
column chromatography to yield 15al (196 mg, 38%)
and 15am (238 mg, 46%).
(200 MHz, CDCl3, TMS) d 1.27 (3H, t, J = 7.5 Hz),
1.31 (3H, t, J = 7.0 Hz), 1.76 (1H, t, J = 3.1 Hz), 3.55
(1H, dd, J = 8.6, 7.3 Hz), 4.14 (2H, q, J = 7.5 Hz),
4.23–4.41 (2H, m), 4.46 (1H, d, J = 12.3 Hz), 4.61 (2H,
s), 4.66 (1H, d, J = 12.3 Hz), 7.40–7.71 (4H, m); MS
(ESI, Pos) m/z 421 (M++Na).
5.1.6.2. (1S,2R,3R,5R,6S)-2-Amino-3-(2-cyanobenzyl-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid diethyl
ester (13ad). A mixture of 11ad (210 mg, 0.525 mmol),
Me3P (1 M THF solution, 0.58 mL), THF (7 mL), and
H2O (0.6 mL) was stirred for 20 h at room temperature.
Ether was added to the reaction mixture, and the organ-
ic 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 = 1:1–1:2) to yield 13ad
Compound 15al: Rf = 0.72 (hexane/AcOEt = 3:1, TLC;
60F254). 1H NMR (300 MHz, D2O, TMSP) d 1.14–
1.29 (6H, m), 1.63 (1H, dd, J = 2.6, 2.6 Hz), 1.90–2.29
(4H, m), 3.33 (1H, dd, J = 8.1, 8.1 Hz), 4.09 (2H, q,
J = 7.2 Hz), 4.58 (1H, q, J = 6.2 Hz), 5.25 (1H, d,
J = 11.9 Hz), 5.42 (1H, d, J = 11.9 Hz), 6.98-7.06 (1H,
1
(59 mg, 30%) as a colorless oil. H NMR (200 MHz,
CDCl3, TMS) d 1.25 (3H, t, J = 7.0 Hz), 1.30 (3H, t,
J = 7.0 Hz), 1.73 (1H, dd, J = 3.1, 3.1 Hz), 1.98–2.37
(4H, m), 3.48 (1H, dd, J = 8.8, 7.5 Hz), 4.11 (2H, q,
J = 7.0 Hz), 4.27 (2H, m), 4.48 (1H, d, J = 12.3 Hz),
m), 7.24–7.49 (7H, m); MS (ESI, Pos) m/z 540
26
(M++Na); ½aꢁ +78.5 (c 3.2, CHCl3).
D
4.59 (1H, d, J = 12.3 Hz), 7.37–7.59 (4H, m); MS (ESI,
Compound 15am: Rf = 0.65 (hexane/AcOEt = 3:1, TLC;
60F254), H NMR (300 MHz, D2O, TMSP) d 1.18–1.29
(6H, m), 1.65 (1H, dd, J = 3.3, 3.3 Hz), 2.01–2.35 (4H,
m), 3.44 (1H, dd, J = 7.9, 7.9 Hz), 4.10 (2H, q,
J = 7.3 Hz), 4.32 (1H, q, J = 6.6 Hz), 5.16 (1H, d,
J = 12.3 Hz), 5.33 (1H, d, J = 12.3 Hz), 7.03–7.11 (1H,
24
D
1
Pos) m/z 395 (M++Na); ½aꢁ +2.5 (c 0.71, CHCl3); Anal.
Calcd for C16H16N2O5Æ0.3H2O: C, 59.73; H, 5.20; N,
8.71. Found: C, 59.75; H, 5.15; N, 8.59.
5.1.6.3. (1S,2R,3R,5R,6S)-2-Amino-3-(2-cyanobenzyl-
oxy)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ad). A
mixture of 13ad (96 mg, 0.258 mmol), THF (4 mL),
H2O (2 mL), and LiOHÆH2O (27 mg, 0.643 mmol) was
stirred for five days at room temperature. HCl (1 N)
was added to the reaction mixture 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)
and reverse-phase silica gel (Wakogelꢂ50C18, H2O–
m), 7.30–7.44 (7H, m); MS (ESI, Pos) m/z 540
27
(M++Na); ½aꢁ ꢀ15.5 (c 3.2, CHCl3).
D
5.1.7.2.
(1S,2R,3R,5R,6S)-2-Amino-3-((R )-1-(3,4-
*
dichlorophenyl)ethoxy)bicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid diethyl ester (14al). A mixture of 12al (189 mg,
0.360 mmol), Me3P (1 M THF solution, 0.4 mL), THF
(5.4 mL), and H2O (0.5 mL) was stirred for 13 h at room
temperature. Ether was added to the reaction mixture,

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3415
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
chromatographed (hexane/AcOEt = 2:1–1:1) to yield
14al (150 mg, 85%) as a colorless oil. 1H NMR
(200 MHz, CDCl3, TMS) d 1.21 (3H, t, J = 7.2 Hz),
1.23 (3H, d, J = 7.2 Hz), 1.59–1.62 (1H, m), 1.82–2.25
(4H, m), 3.22 (1H, dd, J = 8.6, 7.3 Hz), 3.72–3.78 (1H,
m), 4.07 (2H, q, J = 7.2 Hz), 5.15 (1H, d, J = 12.3 Hz),
5.32 (1H, d, J = 12.3 Hz), 7.06 (1H, d, J = 8.4 Hz),
formed precipitate was filtered off. The filtrate was con-
centrated under reduced pressure and chromatographed
(hexane/AcOEt = 5:1) to yield 17 (166 mg, 96%) as a col-
orless oil. H NMR (300 MHz, CDCl3, TMS): d 1.35
(3H, t, J = 7.0 Hz), 1.38 (3H, t, J = 7.0 Hz), 2.35–2.50
1
(2H, m), 2.62–2.86 (2H, m), 4.31 (2H, q, J = 7.0 Hz),
4.27–4.55 (2H, m), 4.94–5.10 (1H, m). MS (FAB, Pos)
26
m/z 434 (M++1); ½aꢁ ꢀ41.4 (c 1.1, CHCl3).
D
5.1.9. Method D
7.30–7.41 (7H, m); (ESI, Pos) m/z 622 (M++Na);
5.1.9.1. (1R,2S,3R,5R,6R)-2-Azido-3-(3,4-dichlo-
robenzylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxyl-
ic acid diethyl ester (18at). Sodium (29 mg, 1.28 mmol)
was dissolved to EtOH (1.28 mL). 3,4-dichlorobenzyl
mercaptan (226 mg, 1.28 mmol) was added to the solu-
tion, stirred for 5 min, and evaporated under reduced
pressure. A DMF solution (0.46 mL) of 17 (231 mg,
0.533 mmol) was added to the DMF solution (4.6 mL)
of the residue and stirred for 30 min at room tempera-
ture. Et2O was added to the reaction mixture, and the
ethereal layer was washed with H2O and saturated
brine, dried with Na2SO4, concentrated under reduced
pressure, and chromatographed (hexane/AcOEt = 8:1)
to yield 18at (147 mg, 58%) as a colorless oil. 1H
28
D
½aꢁ +50.8 (c 1.19, CHCl3).
5.1.7.3.
(1S,2R,3R,5R,6S)-2-Amino-3-((S )-1-(3,4-
*
dichlorophenyl)ethoxy)bicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid diethyl ester (14am). Starting from 12am, 14am
was obtained as an oil. Yield 89%. 1H NMR (200 MHz,
CDCl3, TMS) d 1.21 (3H, t, J = 7.2 Hz), 1.23 (3H, d,
J = 7.2 Hz), 1.59–1.62 (1H, m), 1.82–2.25 (4H, m), 3.22
(1H, dd, J = 8.6, 7.3 Hz), 3.72–3.78 (1H, m), 4.07 (2H,
q, J = 7.2 Hz), 5.15 (1H, d, J = 12.3 Hz), 5.32 (1H, d,
J = 12.3 Hz), 7.06 (1H, d, J = 8.4 Hz), 7.30–7.41 (7H,
29
m); (ESI, Pos) m/z 622 (M++Na); ½aꢁ ꢀ39.1 (c 1.7,
D
CHCl3)
NMR (300 MHz, CDCl3, TMS)
d 1.34 (3H, t,
5.1.7.4.
(1S,2R,3R,5R,6S)-2-Amino-3-((R )-1-(3,4-
J = 7.0 Hz), 1.38 (3H, t, J = 7.0 Hz), 2.20–2.49 (4H,
m), 2.99–3.13 (1H, m), 3.68 (1H, d, J = 13.6 Hz), 3.84
(1H, d, J = 13.6 Hz), 4.22–4.51 (4H, m), 7.16 (1H, dd,
*
dichlorophenyl)ethoxy)bicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid (15al). A mixture of 14al (146 mg, 0.30 mmol),
THF (4.5 mL), H2O (0.5 mL), and LiOHÆH2O (37 mg,
0.88 mmol) was stirred for four days at room tempera-
ture. HCl (1 N) was added to the reaction mixture and
stirred for 10 min at room temperature. The solution
was chromatographed on AG50W-X8 cation-exchange
resin (Bio-Rad) (H2O–50% aqueous THF–10% aqueous
pyridine) to yield 15al (87 mg, 77%) as a white powder.
Mp > 277 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMSP) d 1.36 (3H, d, J = 6.1 Hz), 1.47–1.51 (1H, m),
1.87–2.11 (4H, m), 3.67 (1H, dd, J = 8.1 Hz), 4.54 (1H,
q, J = 6.1 Hz), 7.26 (1H, d, J = 8.5 Hz), 7.52 (1H, s),
J = 8.1, 2.0 Hz), 7.34–7.46 (m, 2H); MS (ESI, Pos) m/z
24
D
498 (M++Na); ½aꢁ +129.9 (c 0.48, CHCl3).
5.1.9.2. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyl-
sulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (19at). A mixture of 18at (72 mg,
0.151 mmol), Me3P (1 M THF solution, 166 lL), THF
(2.2 mL), and H2O (220 lL) was stirred for 4 h at room
temperature. Ether was added to the reaction mixture,
and the organic layer was separated. The organic layer
was washed with saturated NaHCO3 and saturated
brine, dried with Na2SO4, evaporated, and chromato-
graphed (hexane/AcOEt = 3:1–2:1) to yield 29at
7.55 (1H, d, J = 8.5 Hz); MS (ESI, Nega) m/z 372
28
(Mꢀꢀ1); ½aꢁ +88.4 (c 0.40, 1 N NaOH); Anal. Calcd
D
for C16H17Cl2NO5Æ0.2H2O: C, 50.86; H, 4.64; N, 3.71.
Found: C, 50.81; H, 4.58; N, 3.66.
(43 mg, 63%) as a
pale yellow oil. 1H NMR
(300 MHz, CDCl3, TMS): d 1.31 (3H, t, J = 7.2 Hz),
1.35 (3H, t, J = 7.2 Hz), 2.08–2.15 (1H, m), 2.24–2.40
(3H, m), 2.86–2.93 (1H, m), 3.73 (1H, d, J = 13.4 Hz),
3.88 (1H, d, J = 13.4 Hz), 4.21–4.37 (4H, m), 7.15 (1H,
dd, J = 8.2, 2.2 Hz), 7.36 (1H, d, J = 8.2 Hz), 7.42 (1H,
5.1.7.5.
(1S,2R,3R,5R,6S)-2-Amino-3-((S )-1-(3,4-
*
dichlorophenyl)ethoxy)bicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid (15am). Starting from 14am, 15am was obtained
as an oil. Yield 13%. Mp > 239 ꢁC (decomp.). 1H NMR
(300 MHz, D2O, TMSP) d 1.36 (3H, d, J = 6.5 Hz),
1.46–1.50 (1H, m), 1.95–2.04 (2H, m), 3.49 (1H, dd,
J = 7.9, 7.9 Hz), 4.51 (2H, q, J = 6.5 Hz), 7.30 (1H, d,
d, J = 2.2 Hz); MS (ESI, Pos) m/z 472 (M++Na);
23
D
½aꢁ +94.4 (c 0.25, CHCl3).
5.1.9.3. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyl-
sulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15at). A mixture of 19at (41 mg, 91 lmol), THF
(0.8 mL), H2O (0.4 mL), and LiOHÆH2O (10 mg,
0.24 mmol) was stirred for three days at room tempera-
ture. LiOHÆH2O (2 mg, 0.05 mmol) was added to the reac-
tion mixture and stirred for 2.5 days. The reaction mixture
was acidified with 1 N HCl and stirred for 1 h at room
temperature. The solution was chromatographed on
AG50W-X8 cation-exchange resin (Bio-Rad) (H2O–
50% aqueous THF–10% aqueous pyridine) to yield
15at (25 mg, 69%) as a white powder. Mp > 220 ꢁC (de-
J = 8.4 Hz), 7.55 (1H, s), 7.57 (1H, d, J = 8.4 Hz); MS
28
D
(ESI, Nega) m/z 372 (Mꢀꢀ1); ½aꢁ ꢀ64.0 (c 0.24, 1 N
NaOH); HR-MS Calcd for C16H16Cl2NO5: 372.0406.
Found: 372.0424.
5.1.8.
(1R,2R,3R,5R,6R)-2-Azido-6-fluoro-3-trifluoro-
methanesulfonyloxybicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (17). Tf2O (78 lL, 0.47 mmol) was add-
ed to a mixture of 16 (120 mg, 0.40 mmol), CH2Cl2
(4 mL), and pyridine (48 lL, 0.59 mmol) with ice-cool-
ing under nitrogen atmosphere and stirred for 1 h. After
Et2O (30 mL) was added to the reaction mixture, a
1
comp.). H NMR (300 MHz, D2O, TMSP) d 2.17–2.48

3416
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
(4H, m), 3.04–3.13 (1H, m), 3.80 (1H, d, J = 14.9 Hz), 3.85
(1H, d, J = 14.9 Hz), 7.31 (1H, d, J = 8.1 Hz), 7.53 (1H, d,
J = 8.1 Hz), 7.59 (1H, s); MS (ESI, Nega) m/z 392
0.2H2O: C, 59.26; H, 6.39; N, 3.64. Found: C, 59.40;
H, 6.34; N, 3.61.
30
(Mꢀꢀ1); ½aꢁ +47.5 (c 0.41, 1 N NaOH); Anal. Calcd
5.1.9.9. (1R,2S,3R,5R,6R)-2-Amino-3-(3-trifluorometh-
ylbenzylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid (15as). Starting from 17 and 3-trifluoro-
methylbenzyl mercaptan, 15as was obtained as a white
D
for C15H14Cl2NO4S: C, 45.70; H, 3.58; N, 3.55. Found:
C, 45.49; H, 3.60; N, 3.47.
1
powder. Mp > 271 ꢁC (decomp.). H NMR (300 MHz,
5.1.9.4.
(1R,2S,3R,5R,6R)-2-Amino-3-(thiophen-2-
D2O, TMSP): d 2.16–2.47 (4H, m), 3.04–3.15 (1H, m),
3.89 (1H, d, J = 13.2 Hz), 3.94 (1H, d, J = 13.2 Hz),
ylmethylsulfanyl)-6- fluorobicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid (15an). Starting from 17 and 2-thienylmeth-
anethiol, 15an was obtained as a white powder.
Mp > 230 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMS) d 2.16–2.32 (2H, m), 2.41–2.50 (2H, m), 3.06–
3.18 (1H, m), 4.07 (2H, s), 6.97–7.10 (2H, m), 7.35–
7.50–7.78 (4H, m); MS (ESI, Nega) m/z 392 (Mꢀꢀ1);
24
½aꢁ +38.9 (c 0.36, 1 N NaOH); Anal. Calcd for
D
C16H15F4NO4S: C, 48.85; H, 3.84; N, 3.56. Found: C,
48.75; H, 3.87; N, 3.42.
7.43 (1H, m); MS (ESI, Nega) m/z 330 (Mꢀꢀ1);
25
½aꢁ +38.7 (c 0.35, 1 N NaOH); Anal. Calcd for
5.1.9.10. (1R,2S,3R,5R,6R)-2-Amino-3-(2,6-difluoro-3-
chlorobenzylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-di-
carboxylic acid (15au). Starting from 17 and 2,6-diflu-
oro-4-chlorobenzyl mercaptan, 15au was obtained as a
white powder. Mp > 234 ꢁC (decomp.). 1H NMR
(300 MHz, D2O, TMSP): d 2.09–2.62 (4H, m), 3.11–
3.40 (1H, m), 3.79–4.11 (2H, m), 6.87–7.23 (1H, m),
D
C13H14FNO4S2: C, 47.12; H, 4.26; N, 4.24. Found: C,
47.00; H, 4.30; N, 4.19.
5.1.9.5. (1R,2S,3R,5R,6R)-2-Amino-3-(2-phenylbenz-
ylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15ao). Starting from 17 and 2-phenylbenzyl mer-
7.32–7.61 (1H, m); MS (ESI, Nega) m/z 394 (Mꢀꢀ1);
captan, 15ao was obtained as
a white powder.
24
½aꢁ +66.9 (c 0.23, 1 N NaOH); Anal. Calcd for
Mp > 230 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMS) d 2.08–2.35 (4H, m), 2.87–3.00 (1H, m), 3.78–
4.01 (2H, m), 7.26–7.62 (9H, m); MS (ESI, Nega) m/z
D
C15H13ClF3NO4S: C, 45.52; H, 3.31; N, 3.54. Found:
C, 45.47; H, 3.40; N, 3.27.
24
440 (Mꢀꢀ1); ½aꢁ +49.3 (c 0.21, 1 N NaOH); Anal.
D
Calcd for C21H20FNO4SÆ0.5H2O: C, 61.45; H, 5.16; N,
3.41. Found: C, 61.46; H, 5.08; N, 3.44.
5.1.9.11. (1R,2S,3R,5R,6R)-2-Amino-3-(2-phenylethyl-
sulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15av). Starting from 17 and 1-phenylethanethiol,
15av was a mixture of diasteromers obtained as a
white powder. Mp > 307 ꢁC (decomp.). 1H NMR
(300 MHz, D2O, TMSP): d 1.48–1.62 (3H, m), 1.82–
1.94 (1H, m), 2.05–2.32 (2H, m), 2.43–2.71 (1H, m),
2.83–3.03 (1H, m), 4.04–4.24 (1H, m), 7.27–7.52
(5H, m); MS (ESI, Nega) m/z 338 (Mꢀꢀ1); Anal.
Calcd for C16H18FNO4SÆ0.1H2O: C, 56.33; H, 5.38;
N, 4.11. Found: C, 56.23; H, 5.44; N, 4.21.
5.1.9.6. (1R,2S,3R,5R,6R)-2-Amino-3-(4-methoxybenz-
ylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15ap). Starting from 17 and 4-methoxylbenzyl
mercaptan, 15ap was obtained as a white powder.
Mp > 240 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMS) d 2.16–2.42 (4H, m), 3.03–3.13 (1H, m), 3.77–
3.87 (5H, m), 7.00 (2H, d, J = 8.7 Hz), 7.35 (2H, d,
J = 8.7 Hz); MS (ESI, Nega) m/z 440 (Mꢀꢀ1);
26
½aꢁ +31.0 (c 0.48, 1 N NaOH); Anal. Calcd for
D
C16H18NO5S2: C, 54.07; H, 5.11; N, 3.94. Found: C,
53.83; H, 5.12; N, 3.92.
5.1.9.12. (1R,2S,3R,5R,6R)-2-Amino-3-(1-bis(4-fluor-
ophenyl)methylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-
dicarboxylic acid (15aw). Starting from 17 and bis(4-flu-
orophenyl)methanethiol, 15aw was obtained as a white
5.1.9.7. (1R,2S,3R,5R,6R)-2-Amino-3-(4-fluorobenzyl-
sulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15aq). Starting from 17 and 4-fluorobenzyl mercap-
tan, 15aq was obtained as a white powder. Mp > 243 ꢁC
1
powder. Mp > 234 ꢁC (decomp.). H NMR (300 MHz,
D2O, TMSP): d 2.06–2.60 (4H, m), 2.91–3.08 (1H, m),
5.43 (1H, s), 6.99–7.25 (2H, m), 7.39–7.65 (4H, m);
24
D
MS (ESI, Nega) m/z 436 (Mꢀꢀ1); ½aꢁ +15.4 (c 0.36,
1
(decomp.). H NMR (300 MHz, D2O, TMSP) d 2.13–
1 N NaOH); Anal. Calcd for C21H18F3NO4SÆ0.5H2O:
C, 56.50; H, 4.29; N, 3.14. Found: C, 56.54; H, 4.38;
N, 3.11.
2.54 (4H, m), 2.97–3.19 (1H, m), 3.84 (2H, s), 7.03–7.29
(2H, m), 7.33–7.55 (2H, m); MS (ESI, Nega) m/z 342
25
D
(Mꢀꢀ1); ½aꢁ +25.4 (c 0.20, 1 N NaOH); Anal. Calcd
for C15H13Cl2FNO5S2Æ0.1H2O: C, 52.20; H, 4.44; N,
4.06. Found: C, 52.00; H, 4.48; N, 4.06.
5.1.9.13. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichloro-
phenylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid (15ax). Starting from 17 and 3,4-dichlorobenzyl
mercaptan, 15ax was obtained as a white powder.
Mp > 234 ꢁC (decomp.). 1H NMR (500 MHz, D2O,
TMSP): d 1.60 (1H, dd, J = 3.1, 3.1 Hz), 1.93–1.96 (1H,
m), 2.03–2.05 (1H, m), 2.15–2.19 (2H, m), 2.69 (1H, dd,
J = 8.5, 9.7 Hz), 3.77 (1H, d, J = 14.0 Hz), 3.81 (1H, d,
J = 14.0 Hz), 7.31 (1H, dd, J = 8.5, 1.8 Hz), 7.53 (1H,
d, J = 8.5 Hz), 7.58 (1H, d, J = 1.8 Hz); MS (ESI, Nega)
5.1.9.8.
(1R,2S,3R,5R,6R)-2-Amino-3-(4-tert-butyl-
benzylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid (15ar). Starting from 16 and 4-tert-butylbenzyl
mercaptan, 15ar was obtained as a white powder.
Mp > 271 ꢁC (decomp.). 1H NMR (300 MHz, D2O,
TMSP): d 1.31 (9H, s), 2.20–2.47 (4H, m), 2.06–3.20
(1H, m), 3.83 (2H, s), 7.32–7.40 (2H, m), 7.46–7.56
26
(2H, m); MS (ESI, Nega) m/z 380 (Mꢀꢀ1); ½aꢁ ꢀ1.1
D
m/z 374 (Mꢀꢀ1); ½aꢁ28 +22.0 (c 0.033, 1 N NaOH); Anal.
(c 0.63, 1 N NaOH); Anal. Calcd for C19H24Cl2FNO4SÆ
D

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3417
Calcd for C14H12Cl2FNO4S: C, 44.22; H, 3.18; N, 3.68.
Found: C, 44.45; H, 3.34; N, 3.59.
0.414 mmol), and CH2Cl2 (3.94 mL) with ice-cooling,
the mixture was stirred for 3 h. Saturated NaHCO3
was added to the mixture, and the organic layer was sep-
arated. The organic layer was washed with saturated
NaHCO3 and saturated brine, dried with Na2SO4, evap-
orated, and chromatographed (hexane/AcOEt = 5:1) to
5.1.10. (1R,2S,3R,5R,6R)-2-Azido-3-(3,4-dichlorophenyl-
sulfinyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid
diethyl ester (20). After m-CPBA (32 mg, 0.12 mmol) was
added to a mixture of 18at (73 mg, 0.153 mmol) and
CH2Cl2 (1.46 mL) in dry ice bath, the mixture was stir-
red for 1 h. The mixture was stirred for 3.5 h with ice-
cooling and stirred at room temperature for 11 h. After
m-CPBA (15 mg, 0.06 mmol) was added to the reaction
mixture in dry ice bath, the mixture was stirred for 1 h.
Then the mixture was stirred for 4 h with ice-cooling.
Saturated NaHCO3 was added to the reaction mixture,
and the organic layer was separated. The organic layer
was washed with saturated brine, dried over with
Na2SO4, evaporated, and chromatographed (CHCl3/
EtOH = 4:1–2:1) to yield 20 (63 mg, 84%) as a colorless
1
yield 22 (193 mg, 91%) as a pale yellow oil. H NMR
(300 MHz, CDCl3, TMS): d 1.36 (3H, t, J = 7.0 Hz),
1.39 (3H, t, J = 7.0 Hz), 2.33–2.58 (3H, m), 2.86–3.05
(1H, m), 3.53 (1H, dd, J = 11.2, 8.1 Hz), 4.24–4.46
(6H, m), 7.28 (1H, dd, J = 8.4, 2.2 Hz), 7.44–7.56 (2H,
29
m); MS (ESI, Pos) m/z 530 (M++Na); ½aꢁ +7.9 (c
D
0.70, CHCl3).
5.1.14. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichlorophen-
ylsulfonyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (23). In a manner similar to the prep-
aration of 21, 23 (346 mg, 97%) was obtained from 22
(377 mg, 0.742 mmol) as a colorless oil. Mp > 160 ꢁC
1
oil. H NMR (300 MHz, CDCl3, TMS): d 1.36 (3H, t,
1
J = 7.0 Hz), 1.38 (3H, t, J = 7.0 Hz), 2.33 (1H, dd,
J = 14.1, 8.4 Hz), 2.43–2.61 (2H, m), 2.80–2.97 (1H,
m), 3.11–3.24 (1H, m), 3.79 (1H, d, J = 13.2 Hz), 4.09
(1H, d, J = 13.2 Hz), 4.25–4.43 (4H, m), 7.17 (1H, dd,
(decomp.). H NMR (200 MHz, D2O, TMSP): d 1.34
(3H, t, J = 7.0 Hz), 1.36 (3H, t, J = 7.0 Hz), 2.28–2.42
(3H, m), 2.83–3.01 (1H, m), 3.41–3.53 (1H, m), 4.23–
4.37 (6H, m), 7.28 (1H, dd, J = 8.4, 1.8 Hz), 7.46
J = 8.4, 2.2 Hz), 7.40–7.50 (2H, m); MS (ESI, Pos) m/z
(1H, d, J = 8.4 Hz), 7.55 (1H, d, J = 1.8 Hz); MS
28
514 (M++Na); ½aꢁ +36.0 (c 0.49, CHCl3).
29
(ESI, Pos) m/z 482 (M++1); ½aꢁ +24.0 (c 0.86,
D
D
CHCl3).
5.1.11. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichlorophen-
ylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (21). A mixture of 20 (61 mg,
0.124 mmol), Me3P (1 M THF solution, 136 lL), THF
(1.7 mL), and H2O (170 lL) was stirred for 2 h at room
temperature. Ether was added to the reaction mixture,
and the organic layer was separated. The organic layer
was washed with saturated NaHCO3 and saturated
brine, dried with Na2SO4, evaporated, and chromato-
graphed (CHCl3/EtOH = 50:1) to get residue. The resi-
due was purified with column chromatography
(hexane/AcOEt = 2:5) to yield 21 (41 mg, 71%) as a pale
5.1.15. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichlorophen-
ylsulfonyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15az). A mixture of 23 (108 mg, 0.224 mmol) and
60% H2SO4 (1.08 mL) was heated at 130 ꢁC for three
days using a sealed tube. After cooling, the mixture
was neutralized with 5 N NaOH and stirred for 1 h at
room temperature. The solution was chromatographed
on AG50W-X8 cation-exchange resin (Bio-Rad)
(H2O–50% aqueous THF–10% aqueous pyridine) to
yield 15az (76 mg, 80%) as
a
white powder.
1
1
Mp > 230 ꢁC (decomp.). H NMR (300 MHz, D2O): d
2.33–2.45 (3H, m), 2.82–2.94 (1H, m), 3.98 (1H, dd,
J = 10.1, 9.5 Hz), 4.55 (1H, d, J = 15.3 Hz), 4.60 (1H,
d, J = 15.3 Hz), 7.37 (1H, d, J = 8.6 Hz), 7.63 (1H, d,
yellow oil. H NMR (300 MHz, CDCl3, TMS): d 1.34
(3H, t, J = 7.0 Hz), 1.35 (3H, t, J = 7.0 Hz), 2.30–2.43
(3H, m), 2.78–3.12 (2H, m), 3.80 (1H, d, J = 13.2 Hz),
4.19–4.36 (5H, m), 7.17 (1H, dd, J = 8.4, 2.2 Hz), 7.44
J = 8.3 Hz), 7.64 (1H, s); MS (ESI, Nega) m/z 424
(1H, d, J = 8.4 Hz), 7.44 (1H, d, J = 2.2 Hz); MS (ESI,
25
D
(Mꢀꢀ1); ½aꢁ ꢀ5.1 (c 0.72, 1 N NaOH); Anal. Calcd
29
Pos) m/z 488 (M++Na); ½aꢁ +59.1 (c 0.32, CHCl3).
D
for C15H14Cl2FNO6S: C, 42.27; H, 3.31; N, 3.29. Found:
C, 42.60; H, 3.44; N, 3.22.
5.1.12. (1R,2S,3R,5R,6R)-2-Amino-3-(3,4-dichlorophen-
ylsulfanyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid (15ay). In a manner similar to the preparation of
15at, 15ay (17 mg, 52%) was obtained from 21 (38 mg,
81.5 lmol) as a white powder. Mp > 160 ꢁC (de-
comp.). 1H NMR (300 MHz, D2O, TMSP): d 2.16–
2.29 (2H, m), 2.44–2.49 (2H, m), 2.77–2.88 (2H, m),
3.44–3.53 (1H, m), 4.05 (1H, d, J = 13.1 Hz), 4.26 (1
H. d, J = 13.1 Hz), 7.29 (1H, d, J = 8.5 Hz), 7.56
5.1.16. (1R,2R,3S,5R,6R)-2-Amino-6-fluoro-3-hydroxybi-
cyclo[3.1.0]hexane-2,6-dicarboxylic acid diethyl ester (25).
A mixture of 16 (245 mg, 0.813 mmol), 1M Me3P/THF
(0.89 mL), THF (7.0 mL), and H2O (0.7 mL) was stir-
red for 12 h at room temperature. Et2O and saturated
NaHCO3 were added to the mixture, and the resultant
mixture was stirred for 1 h. The aqueous layer was sep-
arated and extracted with CHCl3. The combined
organic layer was washed with saturated brine, dried
with Na2SO4, evaporated, and chromatographed (hex-
ane/AcOEt = 50:1) to yield 25 (163 mg, 73%) as a col-
(1H, s), 7.60 (1H, d, J = 8.5 Hz); MS (ESI, Nega)
25
m/z 408 (Mꢀꢀ1). ½aꢁ +79.7 (c 0.30, 1 N NaOH);
D
HR MS calcd for C15H13Cl2FO5S: 407.9876. Found:
407.9893.
1
orless oil. H NMR (200 MHz, CDCl3, TMS): d 1.32
(6H, t, J = 7.3 Hz), 2.07–2.23 (2H, m), 2.41 (1H, dd,
J = 8.1, 3.3 Hz), 2.71–2.91 (1H, m), 4.10–4.41 (5H,
5.1.13. (1R,2S,3R,5R,6R)-2-Azido-3-(3,4-dichlorophenyl-
sulfonyl)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (22). After m-CPBA (274 mg,
1.03 mmol) was added to a mixture of 18at (197 mg,
26
m); MS (ESI, Nega) m/z 276 (M++1); ½aꢁ +2.8 (c
D
1.5, CHCl3).

3418
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
5.1.17. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonylamino)-
6-fluoro-3-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (26). A mixture of 25 (160 mg,
0.581 mmol), Boc2O (152 mg, 0.697 mmol), saturated
NaHCO3 (0.8 mL), and THF (0.8 mL) was stirred for
4 h at room temperature. The mixture was extracted
with AcOEt, and the organic layer was washed with sat-
urated brine, dried with Na2SO4, evaporated, and chro-
matographed (hexane/AcOEt = 2:1) to yield 26 (214 mg,
0.150 mmol), and CHCl3 (0.25 mL) with ice-cooling,
the mixture was stirred for 19 h at room temperature.
H2O was added to the mixture, and the organic layer
was separated. The aqueous layer was extracted with
CHCl3. The combined organic layer was washed with
saturated brine, dried with Na2SO4, and evaporated to
yield 30 (31 mg, 43%) as a colorless oil. 1H NMR
(200 MHz, CDCl3, TMS): d 1.23–1.34 (6H, m), 1.44
(9H, s), 2.03–2.26 (2H, m), 2.43 (1H, dd, J = 13.0,
7.3 Hz), 2.83–2.93 (1H, m), 3.02–3.15 (1H, m), 3.71
(1H, d, J = 13.2 Hz), 3.80 (1H, d, J = 13.2 Hz), 4.12–
4.39 (4H, m), 4.82 (1H, s), 7.11 (1H, dd, J = 8.1,
1
98%) as a colorless oil. H NMR (300 MHz, CDCl3,
TMS): d 1.29 (3H, t, J = 7.0 Hz), 1.30 (3H, t,
J = 7.0 Hz), 1.44 (9H, s), 2.20–2.48 (3H, m), 2.77–2.98
(2H, m), 4.07–4.48 (5H, m), 5.57 (1H, s); MS (ESI,
2.0 Hz), 7.33–7.45 (2H, m); MS (ESI, Nega) m/z 531
22
D
27
D
Nega) m/z 398 (M++Na); ½aꢁ ꢀ14.0 (c 0.90, CHCl3).
(Mꢀꢀ1); ½aꢁ ꢀ15.1 (c 0.52, CHCl3).
5.1.18. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonylamino)-
6-fluoro-3-trifluoromethanesulfonyloxybicyclo[3.1.0]hex-
ane-2,6-dicarboxylic acid diethyl ester (27). In a manner
similar to the preparation of 17 from 16, 27 (84 mg,
71%) was obtained from 26 (150 mg, 0.296 mmol) as a
colorless oil. 1H NMR (200 MHz, CDCl3, TMS): d
1.25–1.41 (6H, m), 1.44 (9H, s), 2.13–2.26 (1H, m),
2.40–2.57(2H, m), 2.97–3.20 (1H, m), 4.14–4.47 (4H,
5.1.22. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonylamino)-
3-(N-3,4-chlorobenzyl-N-methylamino)-6-fluorobicyclo-
[3.1.0]hexane-2,6-dicarboxylic acid diethyl ester (31). A
mixture of 30 (136 mg, 0.255 mmol), MeI (64 mL,
1.02 mmol), K2CO3 (71 mg, 0.510 mmol), and DMF
was stirred for three days at room temperature. Satu-
rated Na2S2O3 was added to the mixture and extracted
with AcOEt. The AcOEt layer was washed with satu-
rated brine, dried with Na2SO4, evaporated, and
chromatographed (hexane/AcOEt = 5:1) to yield 31
m), 5.32 (1H, s), 5.99 (1H, d, J = 8.4 Hz); MS (ESI,
28
Nega) m/z 506 (Mꢀꢀ1); ½aꢁ +79.8 (c 0.50, CHCl3).
D
1
(126 mg, 90%) as a colorless oil. H NMR (200 MHz,
5.1.19. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonylamino)-
3-azido-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid
diethyl ester (28). A mixture of 27 (150 mg, 0.296 mmol),
NaN3 (29 mg, 0.443 mmol), and DMF (1,5 mL) was
stirred for 4 h at room temperature and stirred for 26 h
at 30 ꢁC. Et2O was added to the mixture, and the ethereal
layer was washed with H2O and saturated brine, dried
with Na2SO4, evaporated, and chromatographed
(hexane/AcOEt = 5:1–3:1) to yield 28 (84 mg, 71%) as a
colorless oil. 1H NMR (200 MHz, CDCl3, TMS): d
1.29 (3H, t, J = 7.0 Hz), 1.33 (3H, t, J = 7.0 Hz), 1.45
(9H, s), 2.21–2.56 (3H, m), 2.92 (1H, dd, J = 7.7,
CDCl3, TMS) d 1.28 (3H, t, J = 7.0 Hz), 1.29 (3H, t,
J = 7.0 Hz), 1.43 (9H, s), 2.11 (3H, s), 2.16–2.58 (3H,
m), 2.80–3.07 (2H, m), 3.29 (1H, d, J = 13.6 Hz,),
3.78 (1H, d, J = 13.6 Hz), 4.05–4.43 (4H, m), 4.86
(1H, s), 7.08 (1H, dd, J = 8.4, 1.8 Hz), 7.31–7.41 (2H,
25
m); MS (ESI, Pos) m/z 547 (M++1); ½aꢁ ꢀ51.9 (c
D
0.50, CHCl3).
5.1.23.
5.1.23.1. (1R,2R,3S,5R,6R)-2-Amino-3-(N-3,4-chloro-
benzylamino)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid diethyl ester (14ba). A mixture of 30 (28 mg,
52.5 lmol) and 4 M HCl/AcOEt was stirred for 6 h with
ice-cooling. Saturated NaHCO3 was added to the mix-
ture, and the organic layer was separated. The aqueous
layer was extracted with AcOEt. The combined AcOEt
layer was washed with saturated brine, dried with
Na2SO4, and evaporated to yield 14ba (21 mg, 91%)
as a pale yellow oil. 1H NMR (200 MHz, CDCl3,
2.4 Hz), 3.78–3.88 (1H, m), 4.17–4.41 (4H, m), 5.01
26
D
(1H, s); MS (ESI, Pos) m/z 423 (M++1); ½aꢁ +0.79 (c
1.4, CHCl3).
5.1.20. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonylamino)-
3-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid
diethyl ester (29). A mixture of 28 (78 mg, 0.195 mmol),
5% Pd/C (8 mg), and EtOH (0.8 mL) was stirred for
12 h under hydrogen atmosphere at room temperature.
The mixture was filtered using Celite pad. The filtrate
was concentrated under reduced pressure. The residue
was purified with silica gel column chromatography (hex-
ane/AcOEt = 50:1–20:1) to give 29 (44 mg, 60%) as a col-
TMS) d 1.31 (3H, t, J = 7.0 Hz), 1.34 (3H, t,
J = 6.2 Hz), 2.03–2.28 (3H, m), 2.35–2.51 (1H, m),
2.94–3.08 (1H, m), 3.77 (2H, s), 4.16–4.40 (4H, m),
7.12 (1H, d, J = 7.9 Hz), 7.35 (1H, d, J = 7.9 Hz), 7.40
24
D
(1H, s); MS (ESI, Pos) m/z 433 (M++1); ½aꢁ ꢀ8.4 (c
1
0.56, CHCl3).
orless solid. H NMR (200 MHz, CDCl3, TMS): d 1.30
(3H, t, J = 7.0 Hz), 1.32 (3H, t, J = 7.0 Hz), 1.44 (9H, s),
2.06–2.27 (2H, m), 2.40–2.55 (1H, m), 2.61–2.72 (1H,
m), 3.28–3.47 (1H, m), 4.17–4.41 (4H, m), 5.05 (1H, s);
5.1.23.2. (1R,2R,3S,5R,6R)-2-Amino-3-(N-3,4-chloroben-
zyl-N-methylamino)-6-fluorobicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid diethyl ester (14bb). Starting from 31, 14bb was
obtained as a colorless oil. 1H NMR (200 MHz, CDCl3,
27
MS (ESI, Pos) m/z 397 (M++Na); ½aꢁ ꢀ14.2 (c 1.4,
D
CHCl3).
TMS):
d 1.33 (3H, t, J = 7.0 Hz), 1.35 (3H, t,
5.1.21. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonylamino)-
3-(N-3,4-chlorobenzylamino)-6-fluorobicyclo[3.1.0]hexane-
2,6-dicarboxylic acid diethyl ester (30). After 3,4-dichlo-
robenzyl bromide (36 mg, 0.150 mmol) was added to a
mixture of 29 (51 mg, 0.136 mmol), pyridine (12 lL,
J = 7.0 Hz), 2.06 (3H, s), 2.03–2.21 (1H, m), 2.23–2.60
(3H, m), 2.68–2.84 (1H, m), 3.22 (1H, d, J = 14.1 Hz),
3.97 (1H, d, J = 14.1 Hz), 4.18–4.32 (4H, m), 7.07 (1H,
dd, J = 8.1, 2.0 Hz), 7.30–7.39 (2H, m); MS (ESI, Pos)
23
m/z 447 (M++1); ½aꢁ ꢀ24.9 (c 0.84, CHCl3).
D

A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
3419
5.1.24.
5.1.24.1. (1R,2R,3S,5R,6R)-2-amino-3-(N-3,4-chloro-
5.2.
benzylamino)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic (15ba). In a manner similar to the preparation of
15ay, 15ba (17 mg, 71%) was obtained from 14ba
(28 mg, 64.6 lmol) as a white powder. Mp > 190 ꢁC
(decomp.). 1H NMR (300 MHz, D2O, TMSP): d
2.31–2.77 (4H, m), 3.59–3.74 (1H, m), 4.06 (1H, d,
J = 13.5 Hz), 4.15 (1H, m, J = 13.5 Hz), 7.35 (1H, d,
5.2.1. Pharmacology. Cell culture: CHO cell line stably
expressing rat mGluR2 was cultured in DMEM supple-
mented with 10% dialyzed fetal bovine serum, 2 mM
glutamine, 1% proline, 1 mM sodium pyruvate, 1 mM
succinic acid, 50 U/mL penicillin, and 50 lg/mL strepto-
mycin. Cells were maintained at 37 ꢁC in a humidified
atmosphere of 5% CO2 in air.
J = 7.77 Hz), 7.58–7.64 (2H, m); MS (ESI, Nega) m/z
27
375 (Mꢀꢀ1); ½aꢁ ꢀ14.6 (c 0.29, 1 N NaOH); Anal.
5.2.1.1. [3H]-(1S,2S,3S,5R,6S)-2-Amino-3-fluorobi-
cyclo[3.1.0]hexane-2,6-dicarboxylic acid ([3H]MGS0008)
binding. The binding of [3H]MGS0008 (34 Ci/mmol)
was performed according to the method previously
described.11 CHO cells stably expressing mGluR2
were collected by centrifugation at 1000 rpm for
5 min. The cells were homogenized with 50 mM
Tris–HCl buffer (pH 7.4) and centrifuged at 48,000g
for 20 min at 4 ꢁC. The pellet was suspended in
50 mM Tris–HCl buffer (pH 7.4), and incubated at
37 ꢁC for 15 min, after which the pellet was washed
twice with 50 mM Tris–HCl buffer (pH 7.4) by resus-
pension and recentrifugation. The pellet obtained was
then suspended in 50 mM Tris–HCl buffer (pH 7.4)
containing 2 mM MgCl2 and served as a crude mem-
brane preparation.
D
Calcd for C15H13Cl2FN2O4ÆH2O: C, 45.59; H, 4.08;
N, 7.09. Found: C, 45.67; H, 4.29; N, 6.99.
5.1.24.2. (1R,2R,3S,5R,6R)-2-Amino-3-(N-3,4-chloro-
benzyl-N-methylamino)-6-fluorobicyclo[3.1.0]hexane-2,6-
dicarboxylic acid (15bb). Starting from 14bb, 15bb was
obtained as a white powder. Mp > 164 ꢁC (decomp.).
1H NMR (300 MHz, D2O, TMSP): d 2.31–2.41 (1H,
m), 2.45–2.53 (1H, m), 2.64 (3H, s), 2.73–2.82 (2H,
m), 3.72–3.82 (1H, m), 4.01 (1H, d, J = 13.4 Hz), 4.27
(1H, d, J = 13.4 Hz), 7.35–7.41 (1H, m), 7.61–7.69
24
(2H, m); MS (ESI, Nega) m/z 389 (Mꢀꢀ1); ½aꢁ ꢀ35.2
D
(c 0.51, 1 N NaOH). HR MS calcd for
C16H16Cl2FN2O4: 389.0483. Found: 389.0471.
5.1.25.
For a typical binding experiment, the reaction was ini-
tiated by incubating 0.5 mL of the crude membrane
preparation with [3H]MGS0008. The reaction mixture
was incubated for 1 h at 25 ꢁC. The reaction was termi-
nated by rapid filtration through Whatman GF/C glass
fiber filters presoaked with 0.3% polyethyleneimine,
after which the filters were washed three times with
3 mL of ice-cold 50 mM HEPES buffer (pH 7.4), using
a multicell harvester M-48 (Brandel Biomedical Re-
search and Development Laboratories, Inc, Gaithers-
burg, MD, USA). Aquazol-2 scintillator (Perkin-
Elmer, Wellesley, MA, USA) (10 mL) was added, and
filter-bound radioactivity was counted in a liquid scin-
tillation spectrometer (LS6000TA, Beckman Instru-
ments Inc, Fullerton, CA, USA). Nonspecific binding
was determined in the presence of 10 lM LY354740.
5.1.25.1. (1R,2R,3S,5R,6R)-2-(tert-Butoxycarbonyl-
amino)-3-(N-3,4-chlorobenzoylamino)-6-fluorobicyclo[3.1.0]-
hexane-2,6-dicarboxylic acid diethyl ester (32). A mixture of
29 (17 mg, 45.4 lmol), 3,4-dichlorobenzoyl chloride
(14 mg, 68.1 lmol), pyridine (7.3 lL, 90.8 lmol), and
CHCl3 (0.17 mL) was stirred for 3 h at room temperature.
The mixture was concentrated under reduced pressure and
chromatographed (CHCl3/AcOEt = 100:1) to yield 3
(21 mg, 84%) as a colorless oil. 1H NMR (200 MHz,
CDCl3, TMS): d 1.19 (3H, t, J = 7.0 Hz), 1.31 (3H, t,
J = 7.3 Hz), 1.41 (9H, s), 2.21–2.64 (3H, m), 2.82–2.91
(1H, m), 4.07–4.37 (4H, m), 4.58-4.75 (1H, m), 6.20 (1H,
s), 6.39–6.50 (1H, m), 7.46–7.57 (2H, m), 7.80–7.85 (1H,
23
m); MS (ESI, Nega) m/z 545 (Mꢀꢀ1). ½aꢁ +12.1 (c 0.95,
D
CHCl3).
5.1.25.2. (1R,2R,3S,5R,6R)-2-Amino-3-(3,4-chloroben-
zoylamino)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid diethyl ester (14bc). Starting from 32, 14bc was ob-
In the competition binding assay, the reaction was car-
ried out using 3.0 nM [3H]MGS0008. The concentration
of the test compound that caused 50% inhibition of
specific binding of [3H]MGS0008 (IC50 value) was
determined from each concentration–response curve.
1
tained as a colorless oil. H NMR (200 MHz, CDCl3,
TMS) d 1.30 (3H, t, J = 6.8 Hz), 1.33 (3H, t, J =
7.0 Hz), 2.09–2.43 (3H, m), 2.53–2.38 (1H, m),
4.19–4.38 (4H, m), 4.52–4.71 (1H, m), 7.48–7.55 (2H,
m), 7.81–7.91 (1H, m).
5.2.1.2. Measurements of cAMP formation. Agonist
activities for mGluR2 were evaluated by measuring an
agonist-dependent inhibition of forskolin-induced cyclic
AMP formation in mGluR2 expressing CHO cells.
Briefly, CHO cells11 expressing mGluR2 were seeded
in a 96-well plate at a density of 1.26 · 104 cells per well
and grown for two days. The medium was changed to
fresh medium without 2 mM glutamine, and the cells
were incubated for 4–5 h. The cells were pre-incubated
with PBS containing 1 mM 3-isobutyl-1-methylxanthine
(IBMX) (PBS-IBMX) for 20 min at 37 ꢁC. The reaction
was started by replacing the medium with fresh PBS-
IBMX containing 10 lM forskolin and various concen-
5.1.25.3. (1R,2R,3S,5R,6R)-2-Amino-3-(3,4-chloro-
benzoylamino)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarbox-
ylic acid (15bc). Starting from 14bc, 15bc was obtained
1
as a white powder. Mp > 210 ꢁC (decomp.). H NMR
(300 MHz, D2O, TMSP): d 2.33–2.42 (2H, m), 2.57–
2.67 (2H, m), 4.46–4.55 (1H, m), 7.58–7.68 (2H, m),
7.87–7.90 (1H, m); MS (ESI, Nega) m/z 389 (Mꢀꢀ1);
28
½aꢁ +6.0 (c 0.34, 1 N NaOH); Anal. Calcd for
D
C15H13Cl2FN2O5Æ0.3H2O: C, 45.43; H, 3.46, N; 7.06.
Found: C, 45.58; H, 3.73; N, 6.91.

3420
A. Yasuhara et al. / Bioorg. Med. Chem. 14 (2006) 3405–3420
9. Nakanishi, S.; Masu, M. Annu. Rev. Biophys. Biomol.
Struct. 1994, 23, 319–348.
10. Hollmann, N.; Heinemann, S. Annu. Rev. Neurosci. 1994,
17, 31–108.
11. Meldrum, B. S. J. Nutr. 2000, 130, 1007S–1015S.
12. 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, 4893–4909.
13. Cartmell, J.; Monn, J. A.; Schoepp, D. D. Psychophar-
macology 2000, 148, 423–429.
trations of test agents. After incubation for 15 min at
37 ꢁC, the reaction was terminated by adding ice-cold
100% ethanol and allowed to settle on ice for 40 min.
The supernatants were evaporated and cAMP levels
were determined by cAMP enzyme immunoassay
(EIA) system (Amersham). Antagonist activities of com-
pounds were measured under 30 lM glutamic acid con-
dition, and the test compound was added to the cells
20 min before addition of glutamic acid.
14. Moghaddam, B.; Adams, B. W. Science 1998, 281, 1349–
1352.
5.3.
15. Grillon, C.; Cordova, J.; Levine, L. R.; Morgan, C. A.
Psychopharmacology 2003, 168, 446–454.
16. Tizzano, J. P.; Griffey, K.; Schoepp, D. D. Pharmacol.,
Biochem. Behav. 2002, 73, 367–374.
17. Helton, D. R.; Tizzano, J. P.; Monn, J. A.; Schoep, D. D.;
Kallman, M. J. J. Pharmacol. Exp. Ther. 1998, 284, 651–
660.
18. 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. A.; Schoepp, D. D.;
J. Med. Chem. 1997, 40, 528–537.
19. Shekhar, A.; Keim, S. R. Neuropharmacology 2000, 39,
1139–1146.
20. Nakazato, A.; Sakagami, K.; Yasuhara, A.; Ohta, H.;
Yoshikawa, R.; Itoh, M.; Nakamura, M.; Chaki, S.
J. Med. Chem. 2004, 47, 4570–4587.
21. Kew, J. N. C.; Ducarre, J.-M.; Woltering, T.; Adam, G.;
Mutel, V.; Kemp, J. A. Abstract, 29th Annual Meeting of
Society of Neuroscience, Miami, Oct 23–28, 1999.
22. Monn, J. A.; Valli, M. J.; Massey, S. M.; Hansen, M. M.;
Kress, T. J.; Wepsiec, J. P.; Harkness, A. R.; Grutsch, J.
L.; Wright, R. A.; Johnson, B. G.; Andis, S. L.; Kingston,
A.; Tomlinson, R.; Lewis, R.; Griffey, K. R.; Tizzano, J.
P.; Schoepp, D. D. J. Med. Chem. 1999, 42, 1027–1040.
23. Shao, H.; Goodman, M. J. Org. Chem. 1996, 61, 2582–
2583.
5.3.1. Pharmacokinetics evaluation. Each compounds
was administered to male Wistar or Sprague–Dawley
rats (Charles River, Japan) orally. Blood was collected
from the tail vein, and plasma was separated by centri-
fugation (11,200g, 4 ꢁC, 3 min) and stored at ꢀ80 ꢁC
until bioanalysis. Deproteinized supernatant with meth-
anol of plasma samples was analyzed by liquid chroma-
tography with mass detection on an Agilent ZORBAX
SB-C18 column (5 lm, 50 · 2.1 mm). The analyte was
eluted with linear gradient mobile phase by increasing
acetonitrile concentration in 0.1% acetic acid from 5%
to 95% over 4 min at a flow rate of 250 lL/min. Tandem
mass spectrometric detection was carried out using Tur-
boIonSpray (AB/MDS Sciex API3000) in positive ion
mode. The lower limits of quantitation (LLOQ) were
1 ng/mL.
Acknowledgments
The authors thank Professor Sigetada Nakanishi at
Department of Biological Sciences, Kyoto University
Faculty of Medicine, for provision of CHO cell line
stably expressing rat mGluR2.
24. Lohray, B. B. Synthesis 1992, 1035–1052.
25. Gao, Y.; Sharpless, K. B. J. Am. Chem. Soc. 1988, 110,
7538–7539.
26. Adam, G.; Huguenin-Virchaus, P. N.; Mutel, V.; Stadler,
H.; Woltering, T. J. DE Patent 19941675, 2000; Chem.
Abstr. 2000, 132, 166520.
27. Wessel, H. P.; Iversen, T.; Bundle, D. R. J. Chem. Soc.,
Perkin Trans. 1 1985, 2247–2250.
28. Widmer, U. Synthesis 1987, 568–570.
References and notes
1. Colinrige, G. L.; Lester, R. A. Pharmacol. Rev. 1989, 40,
143–210.
2. Monaghan, D. T.; Bridges, R. J.; Cotman, C. W. Annu.
Rev. Pharmacol. Toxicol. 1989, 29, 365–402.
3. Conn, P. J.; Pin, J.-P. Annu. Rev. Pharmacol. Toxicol.
1997, 37, 205–237.
´
29. Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett.
1983, 24, 763–764.
30. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F.
Tetrahedron 1981, 37, 437–472.
4. Schoepp, D. D.; Jane, D. E.; Monn, J. A. Neuropharma-
cology 1999, 38, 1431–1476.
31. Ornstein, P. L.; Bleisch, T. J.; Arnold, M. B.; Wright, R.
A.; Johnson, B. G.; Schoepp, D. D. J. Med. Chem. 1998,
41, 346–357.
5. Nakanishi, S. Science 1992, 258, 597–603.
6. Pin, J. P.; Duvoision, R. J. Neurochem. 1992, 58, 1184–
1186.
32. 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. J. Med. Chem.
1998, 41, 358–378.
7. Schoepp, D. D.; Conn, P. J. Trends Pharmacol. Sci. 1993,
14, 13–20.
8. Bockaert, J.; Pin, J.; Fagni, L. Fundam. Clin. Pharmacol.
1993, 7, 473–485.