Rational design and enantioselective synthesis of (1R,4S,5R,6S)-3- azabicyclo[3.3.0]octane-4,6-dicarboxylic acid - A novel inhibitor at human glutamate transporter subtypes 1, 2, and 3

Article abstract of DOI:10.1021/jm0508336

The natural product kainic acid is used as template for the rational design of a novel conformationally restricted (S)-glutamic acid (Glu) analogue, (1R,45,5R,6S)-3-azabicyclo[3.3.0]octane-4,6-dicarboxylic acid (1a), The target structure 1a was synthesize

Full text of DOI:10.1021/jm0508336

172
J. Med. Chem. 2006, 49, 172-178
Rational Design and Enantioselective Synthesis of
(1R,4S,5R,6S)-3-Azabicyclo[3.3.0]octane-4,6-dicarboxylic Acid s A Novel Inhibitor at Human
Glutamate Transporter Subtypes 1, 2, and 3
Lennart Bunch,* Birgitte Nielsen, Anders A. Jensen, and Hans Bra¨uner-Osborne
Department of Medicinal Chemistry, The Danish UniVersity of Pharmaceutical Sciences, UniVersitetsparken 2, DK-2100 Copenhagen, Denmark
ReceiVed August 22, 2005
The natural product kainic acid is used as template for the rational design of a novel conformationally
restricted (S)-glutamic acid (Glu) analogue, (1R,4S,5R,6S)-3-azabicyclo[3.3.0]octane-4,6-dicarboxylic acid
(1a). The target structure 1a was synthesized from commercially available (S)-pyroglutaminol, in an
enantioselective fashion, in 14 steps. Pharmacological characterization of 1a at human glutamate transporter
subtypes 1, 2, and 3 yielded Ki values of 127, 52, and 46 µM, respectively. Furthermore, binding studies at
native ionotropic Glu (iGlu) receptors revealed low affinity for R-amino-3-hydroxy-5-methyl-4-isoxazolepro-
pionate (AMPA)-preferring iGlu receptors (IC50 > 100 µM), whereas affinities for the KAIN-preferring
iGlu receptors and the N-methyl-D-aspartate (NMDA)-preferring group of iGlu receptors were in the low
micromolar range (IC50 ) 14 and 2.9 µM, respectively). At metabotropic Glu receptors (mGluR), EC50
values for 1a were >1000 µM for mGluR1 and 4, representing group I and III, respectively, and ∼1000
µM (agonist) for mGluR2, representing group II.
Introduction
The design and synthesis of novel rigid carbo- or heterocyclic
skeletons that mimic the biologically active conformations of
the major excitatory neurotransmitter (S)-glutamate (Glu) in the
central nervous system (CNS) continues to be an important topic
of research. In the healthy CNS, activation of Glu receptors is
involved in important neurophysiological processes such as
Figure 1. Rationally designed novel EAAT inhibitors 1a and 2.
memory and learning, motor functions, and neural plasticity and
development.1 However, under conditions of metabolic stress
and oxygen deprivation, Glu is a neurotoxic agent. Thus, it is
believed that neurodegenerative diseases such as Alzheimer’s
disease, Huntington’s disease, amyotrophic lateral sclerosis
(ALS), epilepsy, and cerebral stroke may be directly related to
disordered glutamatergic neurotransmission originating from
dysfunction of either the Glu receptors, ionotropic receptors
(iGluR), or metabotropic receptors (mGluR) or the Glu reuptake
system, excitatory amino acid transporters (EAATs).1
To this date, several conformationally restricted Glu ana-
logues, such as simple three- to six-membered carbocyclic
analogues, substituted norprolines,2 pyrrolidines and piperidines,
as well as highly rigid analogues based on the bicyclo[1.1.1]-
pentane,3 spiro[2.2]pentane,4 bicyclo[2.1.1]hexane,5 bicyclo-
[3.1.0]hexane,6 bicyclo[2.2.1]heptane,7 the 7-azanorbonane,8 or
the 2-azanorbonane9 skeletons, have been designed and syn-
thesized. Subsequent pharmacological characterization of these
Glu analogues has provided important information about the
binding mode of Glu to Glu receptors and transporters, as well
as the structural requirements for Glu receptor agonist/antagonist
activity, Glu transporter substrate/inhibitor activity, and subtype
selectivity. Considering the essential role played by the EAATs
in the maintenance of synaptic Glu concentrations below
neurotoxic levels, surprisingly little attention has been paid to
the Glu transporters as potential drug targets in the treatment
of the plethora of neurotoxic and neurodegenerative disorders
mentioned above.10,11 Recently, it has been suggested that the
EAATs may exhibit reverse-transport of Glu under ischemic
conditions and that selective EAAT inhibitors may prevent
this.12 However, at present only a few selective EAAT inhibitors
have been described, none of which, apart from dihydrokainic
acid (DHK), displaying significant EAAT subtype selectivity.13
In this paper we describe the rational design and enantioselective
synthesis of a novel conformationally restricted Glu analogue
1a (Figure 1), whose chemical structure holds the small, highly
rigid heterocyclic skeleton, 3-azabicyclo[3.3.0]octane. The
compound is characterized pharmacologically at human glutamate
transporters EAAT1, EAAT2, and EAAT3, and iGlu receptors.
Rational Design of 1a. It has been suggested by us14 and
others10,15,16 that the inhibitory binding conformation at EAAT1-3
is similar to the folded Glu conformation also observed for iGluR
agonist activity,17 while EAAT substrates bind in an extended
Glu conformation, however, different from the conformation
observed for mGluR agonist activation.18 The natural product
kainic acid (KAIN), as well as its close structural analogue
DHK, are both selective inhibitors at the EAAT2 subtype (Ki
) 60 and 31 µM, respectively) (Table 1). However, the C-4-
dihydro KAIN/DHK-analogue CPAA19 is a non-subtype-selec-
tive EAAT inhibitor with 4-8-fold lower potency at EAAT1,
-2, and -3 (Ki > 250 µM) (Table 1). To investigate the role of
the C-4 substituent in these observations and with the aim of
designing a small rigid skeleton that could potentially be a non-
subtype-selective EAAT1-3 inhibitor, we performed a super-
imposition study of the low-energy folded conformations of
KAIN, DHK, and CPAA (Figure 2). As expected, the isopro-
pylene group of KAIN and the isopropyl group of DHK align
* To whom correspondence should be addressed. Phone: +45 35306244.
Fax: +45 35306040. E-mail lebu@dfuni.dk.
10.1021/jm0508336 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/02/2005

3-Azabicyclo[3.3.0]octane-4,6-dicarboxylic Acid
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 173
Table 1. Reported EAAT Inhibitors Kainic Acid (KAIN),
Dihydrokainic Acid (DHK), and 2-Carboxy-3-pyrrolidineacidic Acid
(CPAA)
Ki (µM)
EAAT1
EAAT2
EAAT3
KAINa
DHKa
>3000
>3000
>250
60
31
>250
>3000
>3000
>250
Figure 3. Superimposition by fitting the ammonium nitrogen and the
two carboxylate groups of calculated low-energy, folded conformations
of dihydrokainic acid (DHK) (type code), 1a (magenta), and 2 (yellow).
CPAAb
a Data taken from ref 21. b Data taken from ref 19.
Scheme 1
Figure 2. Superimposition by fitting the ammonium nitrogen and the
two carboxylate groups of calculated low-energy, folded conformations
of dihydrokainic acid (DHK) (type code), kainic acid (KAIN) (green),
and 2-carboxy-3-pyrrolidineacetic acid (CPAA) (orange).
1a, conformationally restricted bicyclic Glu analogue 2 (Figure
1), previously synthesized in our group,9 also fulfills the before
mentioned structural requirements and exerts the same ambiguity
with respect to the total volume occupied (Figure 3). Thus, both
compounds 1a and 2 are candidates as novel non-subtype-
selective EAAT1-3 inhibitors.
well; thus, unfavorable van der Waals interactions of these
substituents must be responsible for the observed lack of potency
at EAAT1 and EAAT3. On the other hand, the C-4-dihydro
analogue, CPAA, is a nonselective EAAT inhibitor. This can
be understood from the fact that no structural features of this
compound project into the area in space occupied by the
isopropylene or the isopropyl groups. However, from this
superimposition study it is not clear to us why CPAA is at least
4-fold less potent than KAIN and at least 8-fold less potent
than DHK. We speculate that the observed 4-8-fold decreased
potency of CPAA is solely due to the amplified desolvation
and entropic energies of CPAA compared with KAIN/DHK.
We also take note of a reported KAIN analogue that restricts
the orientation of the isopropylene group.20 However, in a
binding study on native rat neurons, the compound exhibited
significantly less affinity (10-20-fold) than KAIN itself, and
no data as to its characteristics as an EAAT inhibitor were
presented.
On the basis of the modeling considerations described above,
we predicted that truncation of the isopropylene group in KAIN,
linking the remaining methyl group to the R-carbon of the acetic
acid moiety, would offer a new small rigidified Glu analogue
that is a potential non-subtype-selective EAAT inhibitor. The
compound, (1R,4S,5R,6S)-3-azabicyclo[3.3.0]octane-4,6-dicar-
boxylic acid (1a) (Figure 1) was subjected to a stochastic
conformational search, which revealed that the low-energy
conformation of 1a mimics the folded Glu conformation very
well and that the area probing selectivity for EAAT2 is not
affected (Figure 3). However, methylene groups C7H2 and C8H2
in 1a do reside in a volume not occupied by KAIN/DHK. This
could result in unfavorable van der Waals interactions and thus
decreased or abolished potency. With this ambiguity in mind,
we predicted 1a to be a candidate as a novel nonselective
EAAT1-3 inhibitor. In addition to newly designed structure
Retrosynthetic Analysis of 1a. The 4,6-disubstituted 3-aza-
bicyclo[3.3.0] heterocarbon skeleton is an unexplored target
structure in synthetic organic chemistry. Given the importance
of stereochemistry in medicinal chemistry, we sought to follow
an eantioselective route to 1a. Thus, a retrosynthetic analysis
of 1a (Scheme 1) suggests the synthesis of suitably protected
diol 3, which may be obtained from key intermediate 4 by
hydroboration of the alkene from the less sterically hindered
face. The identification of a pyroglutaminol moiety in 4 suggests
its synthesis from the reaction of readily available enantiopure
protected 3,4-didehydropyroglutaminol 6,22 with the cuprate
formed from vinyl bromide 7. This reaction sets the desired
absolute stereochemistry at C5 in 5, which again allows for
cyclization into the cis fused ring system in 4 via generation of
the enolate. The reaction may be explored as a one-pot
annulation reaction (LG ) Cl), but since 6 has been shown to
be sensitive in cuprate addition reactions and the corresponding
vinyllithio anion of vinyl bromide 7 (LG ) Cl) is unstable at
temperatures above -50 °C,23 the reaction may necessarily be
carried out in two steps (LG being a masked leaving group,
e.g. O-PMB) with isolation and appropriate functional group
transformation of 5.
Results and Discussion
The enantioselective synthesis of 1a (Scheme 2) commenced
with the conversion of the alcohol group in 3-bromo-3-buten-
1-ol (available from Sigma-Aldrich) to its corresponding
chloride, 8a. Copper(I)-mediated conjugate addition to 6 has

174 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Bunch et al.
Scheme 2a
a Reagents and conditions: (a) for 8a, NCS, PPh3, DCM, rt, 20 h (49%); for 8b, PMB-TCA, La(OTf)3, toluene, rt, 5 min (77%); (b) 2.5 equiv of 8a or
8b, t-BuLi, CuCN, TMSCl, THF, -78 to -50 °C, then 6, -50 °C, 1 h (9a, 24-38%; 9b, 86%); (c) DDQ, H2O, DCM, rt, 1 h (90%); (d) PhSO2Cl, Et3N,
DMAP, DCM, rt, 4 h (92%); (e) 1.03 equiv of KHMDS, THF, -78 °C to room temperature, 1 h, then rt, 1 h (90%); (f) 1.03 equiv LHMDS, THF, -78 to
0 °C, 18 h (92%); (g) 3% RhCl(PPh3)3, CatBH, THF, rt, 1 h, then 4 equiv of BH3‚THF, rt, 20 h, then 35% H2O2, NaOH, rt, 30 min (one-pot 85%, 80% de);
(h) TBAF, rt, 30 min (97%); (i) RuCl3, NaIO4, H2O, MeCN, EtOAc, rt, 1 h (77%); (j) 20 equiv of HCl/EtOAc, rt, 1-2 h (83%, 84% de); (k) Recrystallization
from glacial AcOH (70%, 99% ee, 96% de).
previously been investigated by us.24 However our optimized
protocol for this specific reaction failed on larger scales (2
Table 2. Pharmacological Characterization of Conformationally
Restricted Glu Analogues 1a and 2 at Human EAAT1-3
mmol) giving inconsistent yields (24-38%) of the desired
product 9a. We believe this observation is due to problems with
metal-halogen exchange of 8a. Even though 9a was readily
cyclized into key intermediate 10 in high yield, we decided to
use a more traditional synthetic pathway by the use of protecting
group chemistry. The p-methoxybenzyl (PMB) group was
chosen because of its possibly selective removal and following
conversion of the alcohol to a leaving group. Thus, 8b was
prepared by the use of a very mild method for the introduction
of a PMB group,25 and using our protocol, conjugate addition
of its corresponding cuprate to 6 gave 9b in high yield (86%).
Selective removal of the PMB group followed by conversion
of the free alcohol 9c to its corresponding phenylsulfonic ester
9d allowed for cyclization into key intermediate 10 in high yield.
Subsequent hydroboration of the methylidene group in com-
pound 10 gave a high degree of facial selectivity (ratio 9:1) by
the use of Wilkinson’s catalyst/catecholborane.9 In one pot, the
endo-carbonyl group was subsequently reduced by the addition
of borane to give alcohols 11a/11b (85% yield). Cleavage of
the O-TBS silyl ether by treatment with tetrabutylammonium
fluoride gave diols 12a/12b in 97%, which were oxidized to
their corresponding diacids 13a/13b via the modified Sharpless
procedure26 (RuCl3/NaIO4). Removal of the BOC group with
HCl(g)/EtOAc gave the target compound 1a/1b as their HCl
salts in 83% yield, de ) 84%. Recrystallization of this crude
diastereomeric mixture from glacial AcOH gave 1a, in 70%
yield, de ) 96%.
The conformationally restricted Glu analogues 1a, synthesized
as described in Scheme 2, and 2, synthesized as described earlier
by us,9 were characterized pharmacologically at the glutamate
transporter subtypes EAAT1, EAAT2, and EAAT3, using a
FLIPR membrane potential (FMP) assay (see Experimental
Section for details). The assay was performed essentially as
described previously,21 and results are summarized in Table 2.
Compound 1a was found to be an inhibitor at the three EAAT
subtypes tested here, with modest potency at EAAT1 (Ki ) 127
µM) and midrange potencies, comparable with KAIN and DHK,
at EAAT2 and EAAT3 (Ki ) 52 and 46 µM, respectively). On
the other hand, compound 2 was found to be inactive as substrate
Ki (µM)
EAAT1
EAAT2
EAAT3
1a
2
127
52
46
>400a
>400a
>400a
a Also inactive when tested as a substrate.
Table 3. Pharmacological Characterization of Conformationally
Restricted Glu Analogues 1a and 2 at iGlu receptors
IC50 (µM)
Ki (µM):
[3H]AMPA
[3H]KAIN
[3H]CGP39653
1a
2a
>100
>300
14
>160
2.9
>300
a Data taken from ref 9.
and inhibitor at EAAT1, EAAT2 and EAAT3 with Km and Ki
values >400 µM. Furthermore, 1a was characterized in binding
studies at native ionotropic Glu (iGlu) receptors, and results
are summarized in Table 3. The novel Glu analogue 1a was
inactive at R-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
(AMPA)-preferring iGlu receptors (IC50 >100 µM), while
affinities for the KAIN-preferring iGlu receptors and the
N-methyl-D-aspartate (NMDA)-preferring group of iGlu recep-
tors were in the low micromolar range (IC50 ) 14 and 2.9 µM,
respectively). Pharmacological characterization of 1a in a
functional assay27 at the metabotropic Glu receptor subtypes
mGluR1, mGluR2, and mGluR4, representing groups I, II, and
III, respectively, gave EC50 values of >1000 µM for mGluR1
and -4, and ∼1000 µM (agonist) for mGluR2.
From the pharmacological data presented above for 1a, we
can conclude that the two methylene groups C7H2 and C8H2
are not in critical conflict with parts of the glutamate transporter
proteins EAAT1, EAAT2, and EAAT3. Thus, this volume can
be viewed as allowed in the future design of EAAT inhibitors.
Furthermore, as predicted in the design phase, 1a is characterized
as a non-subtype-selective EAAT inhibitor. The fact that the
highly rigid heterocyclic skeleton locks the Glu backbone in
the folded Glu conformation underlines that this conformation
indeed is the biologically active one for EAAT inhibitors.

3-Azabicyclo[3.3.0]octane-4,6-dicarboxylic Acid
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 175
THF (2 mL) was added and the reaction mixture allowed to warm
to -50 °C (clear solution). After 5 min, the flask was recooled to
-78 °C and 6 (655 mg, 2.0 mmol) dissolved in dry THF (2.0 mL)
was added, followed by TMSCl (0.65 mL, 5.0 mmol). The reaction
mixture was allowed to warm to -50 °C and stirred at this
temperature for 1 h. The reaction was quenched with saturated NH4-
Cl and extracted with EtOAc. The organic layer was washed with
brine, dried (Na2SO4), and concentrated. Purification of the crude
product by flash chromatography (heptane/EtOAc 6:1, Rf ) 0.23)
gave 9a, as a colorless oil (321 mg, 24-38%): [R]20589 ) -51.91
(c ) 1.0, CHCl3); 1H NMR δ 4.96 (br s, 1H), 4.92 (br s, 1H), 3.98
(m, 1H), 3.84 (dd, 1H, J ) 10 & 5 Hz), 3.74 (dd, 1H, J ) 10 &
3 Hz), 3.64 (t, 2H, J ) 7 Hz), 2.94 (dd, 1H, J ) 17 & 8 Hz), 2.82
(br d, 1H, J ) 10 Hz), 2.51 (br t, 2H, J ) 7 Hz), 2.34 (dd, 1H, J
) 17 & 2 Hz), 1.51 (s, 9H), 0.88 (s, 9H), 0.06 (s, 3H), 0.04 (s,
3H); 13C NMR δ 173.94, 150.21, 146.76, 112.02, 83.54, 64.22,
64.21, 42.75, 38.80, 38.16, 38.10, 28.60, 26.36, 18.72, -4.93. Anal.
(C20H36ClNO4Si) C, H, N.
The question remains as to why activity at EAAT1-3 is not
observed for 2-azanorborane analogue 2. We propose that the
origin for this observation is the development of disfavored van
der Waals interactions between 2 and the EAAT protein. In
detail, C7 in 2 overlaps well with C4 in KAIN, and C6 in 2 is
included in the acceptable volume defined by C7 and C8 of 1a.
However, these two positive conclusions for 2 do not combine.
In this context, it is important to point out that C1 in 1a is not
overlapping with C7 in 2. Thus, we suggest that the lack of
EAAT activity for 2 can be ascribed to the compound’s inability
to maneuver in the EAAT protein because of the strict relative
position in space of C1 and C6.
Conclusions
In conclusion, we have carried out the rational design and
enantioselective synthesis of a novel conformationally restricted
Glu analogue 1a. The enantioselective synthesis was carried
out in 14 steps from commercially available (S)-pyroglutaminol.
As predicted in the design phase, 1a was characterized as a
non-subtype-selective EAAT inhibitor (EAAT1, EAAT2, and
EAAT3). Furthermore, we have characterized the 2-azanorbor-
nane analogue 2 previously synthesized in our group. However,
against our prediction, this compound was not found to be a
ligand at EAAT1-3. To meet the requirements from the
scientific community for subtype-selective EAAT inhibitors, we
advocate for the design and synthesis of substituted analogues
of 1a. This work is currently ongoing in our group.
(4R,5S)-1-tert-Butoxycarbonyl-5-(tert-butyldimethylsilyloxy-
methyl)-4-(4-(4-methoxybenzyloxy)but-1-en-2-yl)pyrrolidin-2-one
(9b). To a solution of 8b (2.72 g, 10.0 mmol) in dry THF (34 mL)
at -78 °C was added t-BuLi (11.8 mL, 1.7 M in hexanes, 20.0
mmol). After stirring for 15 min, a slurry of CuCN (450 mg, 5.0
mmol) in dry THF (4 mL) was added and the reaction mixture
allowed to warm to -50 °C (clear solution). After 5 min, the flask
was recooled to -78 °C and 6 (1.31 g, 4.0 mmol) dissolved in dry
THF (4.0 mL) was added followed by TMSCl (1.30 mL, 10.0
mmol). The reaction mixture was allowed to warm to -50 °C and
stirred at this temperature for 1 h. The reaction was quenched with
saturated NH4Cl and extracted with EtOAc. The organic layer was
washed with brine, dried (Na2SO4), and concentrated. Purification
of the crude product by flash chromatography (heptane/EtOAc 4:1,
Experimental Section
Rf ) 0.20) gave 9b, as a colorless oil (1.78 g, 86%): [R]20
)
Chemistry. All reagents were obtained from commercial sup-
pliers and used without further purification. THF was distilled from
sodium/benzophenone. NMR (300 MHz) spectra were recorded in
CDCl3 using CHCl3 as reference, unless otherwise noted. Melting
points are uncorrected. Merck silica gel (35-70 mesh) was used
for flash chromatography.
2-Bromo-4-chlorobut-1-ene (8a). To a solution of triphenylphos-
phine (9.45 g, 36 mmol) in dry dichloromethane (100 mL) was
added 3-bromo-3-buten-1-ol (3.0 mL, 30 mmol). The flask was
then cooled to 0 °C and a solution of N-chlorosuccinimide (4.80 g,
36 mmol) in dry dichloromethane (50 mL) was added dropwise
via a funnel. The reaction was allowed to warm to room temperature
and stirring continued overnight. The organic layer was concentrated
on a rotary evaporator (50 °C, 1 atm) and freezing cold diethyl
ether was added. The slurry was filtered and the filtrate concentrated
on a rotary evaporator (50 °C, 1 atm). The crude product was
distilled (50 °C, 20 mmHg) to give 8a, as a clear oil (2.5 g, 49%):
1H NMR δ 5.74-5.70 (m, 1H), 5.56 (d, 1H, J ) 2 Hz), 3.69 (br
t, 2H, J ) 7 Hz), 2.84 (br t, 2H, J ) 7 Hz); 13C NMR δ 129.12,
119.71, 44.11, 41.84. Anal. (C4H6BrCl) C, H.
1-((3-Bromobut-3-enyloxy)methyl)-4-methoxybenzene (8b).
To a solution of 3-bromo-3-buten-1-ol (760 mg, 5.0 mmol) and
p-methoxybenzyl trichloroacetimidate28 (2.1 g, 7.5 mmol) in dry
toluene (25 mL) was added La(OTf)3 (147 mg, 0.25 mmol). The
reaction mixture was stirred for 5 min and then filtered on silica
gel. The silica gel cake was washed with toluene (3 × 25 mL),
and the collective organic layers were concentrated. The crude
product was purified using flash chromatography (heptane/EtOAc
19:1, Rf ) 0.18) to give 8b, as a colorless oil (1.04 g, 77%): 1H
NMR δ 7.26 (d, 2H, J ) 9 Hz), 6.88 (d, 2H, J ) 9 Hz), 5.67 (dd,
1H, J ) 3 & 2 Hz), 5.48 (d, 1H, J ) 2 Hz), 4.48 (s, 2H), 3.81 (s,
3H), 3.64 (t, 2H, J ) 6 Hz), 2.71 (br t, 2H, J ) 6 Hz); 13C NMR
δ 159.32, 130.92, 130.36, 129.49, 118.57, 113.98, 73.01, 67.67,
55.59, 42.05. Anal. (C12H15BrO2) C, H.
589
1
-40.70 (c ) 1.0, CHCl3); H NMR δ 7.22 (d, 2H, J ) 10 Hz),
6.85 (d, 2H, J ) 9 Hz), 4.87 (br s, 1H), 4.84 (br s, 1H), 4.42 (s,
2H), 3.97 (m, 1H), 3.87 (dd, 1H, J ) 10 & 4 Hz), 3.78 (s, 3H),
3.66 (dd, 1H, J ) 10 & 2 Hz), 3.56 (t, 2H, J ) 7 Hz), 2.90 (dd,
1H, J ) 17 & 9 Hz), 2.80 (br d, 1H, J ) 10 Hz), 2.38-2.30 (m,
3H), 1.50 (s, 9H), 0.87 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H); 13C
NMR δ 173.87, 158.97, 149.81, 147.50, 130.05, 129.15, 113.65,
110.46, 82.80, 72.65, 68.36, 63.88, 63.82, 55.24, 38.75, 37.93,
34.98, 28.12, 25.88, 18.23, -5.43, -5.44. Anal. (C28H45NO6Si) C,
H, N.
(4R,5S)-1-tert-Butoxycarbonyl-5-(tert-butyldimethylsilyloxy-
methyl)-4-(4-hydroxybut-1-en-2-yl)pyrrolidin-2-one (9c). To a
solution of 9b (1.78 g, 3.43 mmol) in dichloromethane (40 mL)
and H2O (2 mL) was added DDQ (1.56 g, 6.86 mmol). The reaction
mixture was stirred at room temperature for 1 h and then quenched
with saturated NaHCO3. The aqueous phase was extracted with
dichloromethane, and the collective organic layers were washed
with brine, dried (Na2SO4), and concentrated. Purification of the
crude product by flash chromatography (heptane/EtOAc 3:2, Rf )
0.25) gave 9c, as a colorless oil (1.23 g, 90%): [R]20589 ) -50.37
(c ) 1.0, CHCl3); 1H NMR δ 4.90 (br s, 1H), 4.86 (br s, 1H), 3.94
(m, 1H), 3.82 (dd, 1H, J ) 10 & 5 Hz), 3.72 (t, 2H, J ) 6 Hz),
3.68 (dd, 1H, J ) 10 & 2 Hz), 2.88 (dd, 1H, J ) 17 & 9 Hz), 2.79
(br d, 1H, J ) 9 Hz), 2.36-2.24 (m, 3H), 1.48 (s, 9H), 0.83 (s,
9H), 0.01 (s, 3H), 0.0 (s, 3H); 13C NMR δ 174.03, 150.09, 147.20,
111.41, 83.30, 64.14, 64.10, 60.84, 38.79, 38.07, 38.03, 28.44,
26.18, 18.55, -5.10. Anal. (C20H37NO5Si) C, H, N.
(4R,5S)-1-tert-Butoxycarbonyl-5-(tert-butyldimethylsilyloxy-
methyl)-4-(4-(phenylsulfonyloxy)but-1-en-2-yl)pyrrolidin-2-one
(9d). To a solution of 9c (1.23 g, 3.09 mmol), N,N-(dimethylamino)-
pyridine (38 mg, 0.31 mmol), and triethylamine (0.85 mL, 6.17
mmol) in dry dichloromethane (20 mL) at 0 °C was added dropwise
phenylsulfonyl chloride (0.57 mL, 4.32 mmol). The ice bath was
removed and the reaction mixture allowed to stir at room temper-
ature for 4 h. Dichloromethane (100 mL) was added and the organic
phase washed with aqueous HCl (25 mL, 1 M), saturated NaHCO3
(25 mL), brine; dried (Na2SO4); and then concentrated. Purification
of the crude product by flash chromatography (heptane/EtOAc 2:1,
(4R,5S)-1-tert-Butoxycarbonyl-5-(tert-butyldimethylsilyloxy-
methyl)-4-(4-chlorobut-1-en-2-yl)pyrrolidin-2-one (9a). To a
solution of 8a (850 mg, 5.0 mmol) in dry THF (16 mL) at -78 °C
was added t-BuLi (5.88 mL, 1.7 M in hexanes, 10.0 mmol). After
stirring for 15 min, a slurry of CuCN (225 mg, 2.50 mmol) in dry

176 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Rf ) 0.28) gave 9d, as a colorless oil (1.54 g, 92%): [R]20
Bunch et al.
)
(1R,4S,5R,6S)-N-tert-Butoxycarbonyl-3-azabicyclo[3.3.0]octane-
4,6-dicarboxylic Acid (13a) and (1R,4S,5R,6R)-N-tert-Butoxy-
carbonyl-3-azabicyclo[3.3.0]octane-4,6-dicarboxylic Acid (13b).
To 12a/12b (ratio 9:1) (353 mg, 1.30 mmol) dissolved in MeCN
(10.0 mL) and EtOAc (10.0 mL) was added a solution of RuCl3·
H2O (5.4 mg, 0.026 mmol) and NaIO4 (2.28 g, 10.66 mmol) in
H2O (15.0 mL). The reaction mixture was stirred for 1 h and then
filtered on filter paper, and the filter cake was washed with EtOAc.
The aqueous phase was extracted with EtOAc, and the collective
organic layers were washed with brine, dried (Na2SO4), and
concentrated. Purification of the crude product by flash chroma-
tography (dichloromethane:MeOH:AcOH 100:8:2, Rf ) 0.23) gave
589
-32.54 (c ) 1.0, CHCl3); 1H NMR δ 7.87 (m, 2H), 7.64 (m, 1H),
7.54 (m, 2H), 4.86 (br s, 1H), 4.78 (br s, 1H), 4.16 (br t, 2H, J )
7 Hz), 3.90 (m, 1H), 3.84 (dd, 1H, J ) 10 & 4 Hz), 3.68 (dd, 1H,
J ) 10 & 2 Hz), 2.87 (dd, 1H, J ) 17 & 9 Hz), 2.72 (br d, 1H, J
) 9 Hz), 2.39 (dt, 2H, J ) 7 & 2 Hz), 2.23 (dd, 1H, J ) 17 & 2
Hz), 1.51 (s, 9H), 0.87 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H); 13C NMR
δ 173.83, 150.17, 145.32, 136.22, 134.24, 129.65, 128.19, 112.30,
83.58, 68.73, 64.27, 64.12, 39.03, 38.09, 34.29, 28.58, 26.35, 18.71,
-4.95. Anal. (C26H41NO7SSi) C, H, N.
(1R,4S,5R)-N-tert-Butoxycarbonyl-3-aza-4-(tert-butyldimeth-
ylsilyloxymethyl)-6-methylidene-2-oxobicyclo[3.3.0]octane (10).
To a solution of 9d (632 mg, 1.17 mmol) in dry THF (23 mL) at
-78 °C was added LHMDS (1.21 mL, 1.21 mmol, 1 M in hexanes).
The reaction mixture was stirred for 30 min, warmed to -50 °C,
and left overnight, reaching 10 °C as the end temperature. The
reaction was quenched with saturated NH4Cl and extracted with
EtOAc. The organic layer was washed with brine, dried (Na2SO4),
and concentrated. Purification of the crude product by flash
chromatography (heptane/EtOAc 9:1, Rf ) 0.24) gave 10, as a white
13a/13b (ratio 9:1), as a white foam (300 mg, 77%): [R]24
)
589
-63.67 (c ) 0.5, CHCl3); 1H NMR (major diastereomer 13a, two
1
1
rotamers) δ 10.40 (br s, 2H), 4.44 (br s, /2H), 4.32 (br s, /2H),
3.90-3.50 (br m, 2H), 3.20 (br s, 2H), 2.94 (br s, 1H), 2.10 (br s,
3H), 1.76 (br s, 1H), 1.61 (br s, 5H), 1.57 (br s, 4H); 13C NMR
(major diastereomer 13a, two rotamers) δ 178.90, 177.72, 176.35,
155.56, 154.48, 81.64, 81.27, 61.63, 61.11, 53.63, 53.27, 52.03,
50.46, 48.85, 43.12, 42.54, 31.64, 31.38, 28.87, 28.70, 27.59, 27.19.
Anal. (C14H21NO6) C, H, N: calcd, 56.18, 7.07, 4.68; found, 54.66,
6.88, 4.32.
solid (410 mg, 92%): mp ) 65-66 °C; [R]20 ) -164.75 (c )
589
1.0, CHCl3); 1H NMR δ 5.06 (d, 1H, J ) 2 Hz), 5.01 (d, 1H, J )
2 Hz), 3.99 (m, 1H), 3.92 (dd, 1H, J ) 10 & 4 Hz), 3.78 (dd, 1H,
J ) 10 & 2 Hz), 3.16 (br t, 1H, J ) 8 Hz), 3.00 (br d, 1H, J ) 8
Hz), 2.42-2.18 (m, 3H), 1.97-1.80 (m, 1H), 1.52 (s, 9H), 0.88 (s,
9H), 0.07 (s, 3H), 0.05 (s, 3H); 13C NMR δ 176.65, 154.73, 149.68,
107.72, 82.78, 65.79, 64.28, 49.10, 43.52, 32.28, 28.94, 28.13,
25.88, 18.22, -5.39. Anal. (C20H35NO4Si) C, H, N.
(1R,4S,5R,6S)-3-Azabicyclo[3.3.0]octane-4,6-dicarboxylic Acid
(1a) and (1R,4S,5R,6R)-3-Azabicyclo[3.3.0]octane-4,6-dicarbox-
ylic Acid (1b). To 13a/13b (ratio 9:1) (300 mg, 1.0 mmol) in EtOAc
(15 mL) at 0 °C was added HCl(g)/EtOAc (2 mL, 12 mmol, 6 M).
The reaction mixture was stirred at room temperature for 1 h and
then concentrated. The solid was triturated with freezing cold diethyl
ether to give the HCl salt of 1a/1b (ratio 92:8), as a white solid
(202 mg, 83%). Recrystallization from glacial acetic acid gave 1a,
as white crystals (141 mg, 60%, diastereomeric ratio 98:2, ee >
99%): TLC (butanol/EtOAc/AcOH/H2O 1:1:1:1) Rf ) 0.57; mp
) 186-188 °C; [R]25589 ) +14.40 (c ) 0.25, H2O); 1H NMR (D2O)
δ 4.25 (d, 1H, J ) 7 Hz), 3.64 (q, 1H, J ) 11 & 7 Hz), 3.25 (q,
1H, J ) 16 & 8 Hz), 3.14-3.00 (m, 3H), 2.05-1.75 (m, 3H), 1.72-
1.60 (m, 1H); 13C NMR (D2O) δ 177.77, 171.99, 62.07, 51.23,
48.77, 47.73, 42.58, 29.49, 28.16. Anal. (C9H14ClNO4) C, H, N:
calcd, 45.87, 5.99, 5.94; found, 45.35, 5.68, 4.96. Characteristic
1H NMR of 1b (D2O) δ 4.17 (d, 1H, J ) 7 Hz).
Molecular Modeling Study of Glu Analogues. The modeling
study was performed using the software package MOE (Molecular
Operating Environment, v2004.03, Chemical Computing Group,
2004) using the build-in mmff94x force field and the GB/SA
continuum solvent model. Each compound was submitted to a
stochastic conformational search, and with respect to its global
minimum returned (∆G in kcal/mol), conformations above +7 kcal/
mol were discarded. For all compounds, the γ-carboxylate group
was protonated prior to execution of the conformational search, as
this gave a larger and thus more reliable number of output
conformations. Superimpositions of ligands were carried out using
the built-in function in MOE, by fitting the ammonium group and
the two carboxylate groups.
(1R,4S,5R,6S)-N-tert-Butoxycarbonyl-3-aza-4-(tert-butyldi-
methylsilyloxymethyl)-6-(hydroxymethyl)bicyclo[3.3.0]octane (11a)
and (1R,4S,5R,6R)-N-tert-Butoxycarbonyl-3-aza-4-(tert-butyldi-
methylsilyloxymethyl)-6-(hydroxymethyl)bicyclo[3.3.0]octane
(11b). To a solution of 10 (740 mg, 1.94 mmol) in dry THF (8
mL) was added RhCl(PPh3)3 (56 mg, 0.058 mmol) dissolved in
dry THF (14 mL) and the reaction mixture stirred for 5 min. A
solution of catecholborane (3.82 mL, 3.82 mmol, 1 M in THF)
was added and the reaction mixture stirred for 1 h. A solution of
borane (7.64 mL, 7.64 mmol, 1 M in THF) was the added and
stirring continued for 20 h. The flask was then cooled to 0 °C and
H2O (1.0 mL) was added carefully followed by NaOH (15.7 mL,
2 N) and H2O2 (4.85 mL, 35 w/w%). The reaction mixture was
then stirred at room temperature for 1 h and quenched with saturated
NaHCO3. The aqueous phase was extracted with EtOAc, and the
collective organic layers were washed with brine, dried (Na2SO4),
and concentrated. Purification of the crude product by flash
chromatography (heptane/diethyl ether 2:3, Rf ) 0.23) gave 11a/
11b (ratio 9:1), as a colorless oil (639 mg, 85%): [R]20589 ) -76.87
1
(c ) 0.5, CHCl3); H NMR (two diastereomers) δ 4.00-3.20 (m,
7H), 2.69 (br s, 2H), 2.30 (m, 1H), 1.92 (m, 1H), 1.64-1.40 (m,
2H), 1.46 (s, 9H), 1.20 (m, 1H), 1.93 (s, 9H), 0.13 (s, 6H); 13C
NMR (two diastereomers) δ 154.26, 79.84, 65.94, 65.31, 63.93,
57.46, 53.87, 53.23, 49.84, 48.94, 47.58, 43.10, 42.23, 32.40, 29.00,
26.50, 27.88, 19.02, -4.92. Anal. (C20H39NO4Si) C, H, N.
Determination of Diastereomeric Excess/Diastereomeric Ra-
tio. Chiral HPLC was performed using a Sumichiral OA-5000
column (4.6 × 150 mm, Sumika Chemical Analysis Service). The
column was eluted at 1.0 mL/min with an aqueous solution of
ammonium acetate (10 mM) containing copper(II) acetate (0.1 mM),
adjusted to pH 4.7, and 2-propanol (9:1 v/v). The column was
connected to a TSP HPLC system consisting of a P2000 pump, an
AS3000 autoinjector equipped with a column oven (60 °C), and
an SM5000 PDA detector. For data handling, TSP PC1000 software
was used. On the basis of peak areas at 240 nm, the diastereomeric
excess/diastereomeric ratio were determined.
Pharmacological Characterization of 1a at iGluR. Rat brain
membrane preparations used in the receptor binding experiments
were prepared according to the method described by Ransom and
Stec.29 Affinity for AMPA,30 KAIN,31 and NMDA32 receptor sites
was determined using 5 nM [3H]AMPA, 5 nM [3H]KAIN, and 2
nM [3H]CGP 39653 with some modifications previously de-
scribed.22
(1R,4S,5R,6S)-N-tert-Butoxycarbonyl-3-aza-4,6-bis(hydroxy-
methyl)bicyclo[3.3.0]octane (12a) and (1R,4S,5R,6R)-N-tert-
Butoxycarbonyl-3-aza-4,6-bis(hydroxymethyl)bicyclo[3.3.0]-
octane (12b). To a solution of 11a/11b (ratio 9:1) (642 mg, 1.66
mmol) in dry THF (14 mL) was added tetrabutylammonium fluoride
(2.66 mL, 2.66 mmol, 1 M in THF). The reaction mixture was
stirred for 30 min then quenched with half-saturated NaHCO3. The
aqueous phase was extracted with EtOAc, and the collective organic
layers were washed with brine, dried (Na2SO4), and concentrated.
Purification of the crude product by flash chromatography (EtOAc,
Rf ) 0.20) gave 12a/12b (ratio 9:1), as a colorless oil (435 mg,
97%): [R]20
) -99.43 (c ) 0.52, CHCl3); 1H NMR (two
589
diastereomers) δ 3.90-3.30 (m, 7H), 2.60 (br s, 2H), 2.33 (m, 1H),
1.92 (m, 1H), 1.63 (m, 1H), 1.45 (s, 9H), 1.40-1.20 (m, 2H); 13
C
NMR (two diastereomers) δ 156.05, 80.47, 65.76, 64.84, 63.64,
58.60, 57.48, 53.47, 52.95, 50.34, 49.60, 46.93, 42.78, 31.40, 28.97,
27.32. Anal. (C14H25NO4) C, H, N: calcd, 61.97, 9.29, 5.16; found,
59.51, 9.29, 4.69.

3-Azabicyclo[3.3.0]octane-4,6-dicarboxylic Acid
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 177
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J. Org. Chem. 1999, 64 (6), 2050-2056.
Pharmacological Characterization of 1a and 2 at Human
EAATs. The pharmacological properties of 1a and 2 at human
EAAT1, EAAT2, and EAAT3 were determined in the FLIPR
membrane potential (FMP) assay. The construction of human
embryonic kidney 293 (HEK293) cell lines stably expressing human
EAAT1, EAAT2, and EAAT3 has been reported previously, and
the pharmacological characterization was performed essentially as
described here.21 Briefly, cells were split into poly-D-lysine-coated
black-walled clear-bottom 96-well plates in Dulbecco’s modified
Eagle medium supplemented with penicillin (100 U/mL), strepto-
mycin (100 µg/mL), 10% dialyzed fetal bovine serum, and 1 mg/
mL G-418. Then 16-24 h later the medium was aspirated, and the
cells were washed with 100 µL of Krebs buffer (140 mM NaCl/
4.7 mM KCl/2.5 mM CaCl2/1.2 mM MgCl2/11 mM HEPES/10 mM
D-glucose, pH 7.4). Then 50 µL of Krebs buffer was added to each
well (in the characterization of nonsubstrate inhibitors, the inhibitors
were added to this buffer). Krebs buffer (50 µL) supplemented with
FMP assay dye was then added to each well, and the plate was
incubated at 37 °C for 30 min. The plate was assayed at 30 °C in
a NOVOstar plate reader measuring emission at 560 nm caused by
excitation at 530 nm before and up to 1 min after addition of 25
µL of substrate solution. The experiments were performed in
triplicate at least three times for each compound. For the charac-
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substrate concentration. IC50 values were converted to Ki values
by the use of the Cheng-Prusoff equation.33
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Acknowledgment. We would like to thank Mie Kusk, for
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Council, the Lundbeck Foundation, and the Novo Nordisk
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Supporting Information Available: Combustion analysis data.
This material is available free of charge via the Internet at http://
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JM0508336