Methyl substitution of 2-aminobicyclo[3.1.0]hexane 2,6-dicarboxylate (LY354740) determines functional activity at metabotropic glutamate receptors: Identification of a subtype selective mGlu2 receptor agonist

Article abstract of DOI:10.1021/jm040222y

LY354740 (1) is a highly potent and selective agonist of metabotropic glutamate (mGlu) receptors 2 and 3. In the present study, we have prepared C3- and C4-methyl-substituted variants of rac-1, compounds 5, 9, and 13. Each of these racemic methyl-substituted analogues displaced specific binding of the mGlu2/3 receptor antagonist ³H-2S-2-amino-2-(1S,2S-2- carboxycycloprop-l-yl)-3-(xanth-9-yl)propanoic acid (³H-LY341495) from membranes expressing mGlu2 or mGlu3 receptor subtypes. Evaluation of the functional effects of this series on second messenger responses in cells expressing human mGlu2 or mGlu3 receptors revealed C3β-methyl analogue 5 to possess antagonist properties at both mGlu2 and mGlu3 receptors while C4β-methyl analogue 9 acts as a full agonist at each of these targets. Unexpectedly, we found that incorporation of a methyl substituent at the C4α-position as in analogue 13 results in a mixed mGlu2 agonist/mGlu3 antagonist pharmacological profile. All of the mGlu2 agonist and mGlu3 antagonist activity of rac-13 was found to reside in its resolved (+)-isomer.

Full text of DOI:10.1021/jm040222y

J. Med. Chem. 2005, 48, 3605-3612
3605
Methyl Substitution of 2-Aminobicyclo[3.1.0]hexane 2,6-Dicarboxylate
(LY354740) Determines Functional Activity at Metabotropic Glutamate
Receptors: Identification of a Subtype Selective mGlu2 Receptor Agonist
Carmen Dominguez,† Lourdes Prieto,† Matthew J. Valli,† Steven M. Massey,† Mark Bures,† Rebecca A. Wright,‡
Bryan G. Johnson,‡ Sherri L. Andis,‡ Ann Kingston,‡ Darryle D. Schoepp,‡ and James A. Monn*,†
Discovery Chemistry and Neuroscience Research Divisions, Eli Lilly and Company, Indianapolis, Indiana 46285
Received December 22, 2004
LY354740 (1) is a highly potent and selective agonist of metabotropic glutamate (mGlu)
receptors 2 and 3. In the present study, we have prepared C3- and C4-methyl-substituted
variants of rac-1, compounds 5, 9, and 13. Each of these racemic methyl-substituted analogues
displaced specific binding of the mGlu2/3 receptor antagonist 3H-2S-2-amino-2-(1S,2S-2-
carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid (3H-LY341495) from membranes expressing
mGlu2 or mGlu3 receptor subtypes. Evaluation of the functional effects of this series on second
messenger responses in cells expressing human mGlu2 or mGlu3 receptors revealed C3â-methyl
analogue 5 to possess antagonist properties at both mGlu2 and mGlu3 receptors while C4â-
methyl analogue 9 acts as a full agonist at each of these targets. Unexpectedly, we found that
incorporation of a methyl substituent at the C4R-position as in analogue 13 results in a mixed
mGlu2 agonist/mGlu3 antagonist pharmacological profile. All of the mGlu2 agonist and mGlu3
antagonist activity of rac-13 was found to reside in its resolved (+)-isomer.
Introduction
(S)-Glutamic acid ((S)-Glu) is the primary excitatory
neurotransmitter in the mammalian central nervous
system (CNS). The neuronal effects of (S)-Glu are
mediated by two heterogeneous families of cell mem-
brane associated receptors: the ion-channel linked, or
Figure 1. Chemical structure of LY354740.
ionotropic glutamate (iGlu), receptors; and the G-
protein-coupled, or metabotropic glutamate (mGlu),
Chemistry
receptors.1,2 The eight known mGlu receptors (mGlu1-
8) are highly heterogeneous with respect to their
structure, function, and localization within the central
nervous system and represent promising targets for
therapeutic intervention in a multiplicity of CNS
disorders.3-10 We have previously reported the identi-
fication of (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-
2,6-dicarboxylate (1, Figure 1), a potent and selective
agonist for mGlu2 and mGlu3 receptors11-13 that has
been found to possess anxiolytic,12,14-19 antipsychotic,20-22
anticonvulsant,23,24 anti-Parkinsonian,25,26 analgesic,27
and neuroprotective28-31 properties in vivo. As part of
our continued interest in this mechanism, we have
explored the effect of substitution of the bicyclic ring
system of 1. In this account, we describe the synthesis
and in vitro pharmacological profile of methyl-substi-
tuted variants of rac-1. Unexpectedly, we have found
that simple methyl substitution at the C3- or C4-
positions of the bicyclo[3.1.0]hexane ring system results
in a spectrum of functional effects, including dual
mGlu2/3 agonist activity, dual mGlu2/3 antagonist
activity, and mixed mGlu2 agonist/mGlu3 antagonist
activity.
The rac-C3â-methyl analogue 5 was prepared by the
method outlined in Scheme 1. Alkylation of the lithium
enolate of 212,32 with methyl iodide at -78 °C afforded
C3â-methyl ketone 3. This was converted to a mixture
of C2-spiro-5′-hydantoins by the Bucherer-Bergs method,
and the desired diastereomer 4 was isolated directly
from the reaction mixture as a precipitate. Exhaustive
hydrolysis of 4 followed by ion exchange chromatogra-
phy then provided the desired zwitterion rac-5. The rac-
C4R- and C4â-methyl-substituted analogues of 1 were
prepared from the common R,â-unsaturated ketone
intermediate 6 (Scheme 2). Dimethyl cuprate addition
to 6 yielded the C4â-methyl analogue 7 that was
converted to the desired amino acid precursor 8 by the
Strecker reaction and in situ acylation followed by
chromatographic separation of the C2-acetamidonitrile
diastereomers. Hydrolysis of 8 in refluxing aqueous HCl
then afforded rac-9. Alternatively, 7 was converted to
the TMS-enol ether and oxidized to the C4-methyl-
substituted unsaturated ketones 10 by the Saegusa
method.33 Hydrogenation of 10 (H2/Pd-C) afforded
exclusively the C4R-methyl bicyclic ketone 11. Conver-
sion to the desired C2-spiro-5′-hydantoin 12 was achieved
in low overall yield via the Bucherer-Bergs reaction
followed by crystallization from EtOH. Base-mediated
hydrolysis of 12 provided the desired C4R-methyl bicy-
clic amino acid rac-13. The individual enantiomers of
13 were obtained in an analogous manner from indi-
* To whom correspondence should be addressed. Telephone: (317)
276-9101. Fax: (317) 277-7600. E-mail: Monn@Lilly.com.
† Discovery Chemistry.
‡ Neuroscience Research.
10.1021/jm040222y CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/22/2005

3606 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Dominguez et al.
Scheme 1
Scheme 2
vidual enantiomers of 11, obtained via chiral chroma-
tography (Scheme 3). Assignment of relative stereo-
chemistry for methyl-substituted derivatives 5, 9, and
13 was based on analysis of proton-proton nuclear
Overhauser effects (NOEs) in hydantoins 4 and 12 and
acetamido nitrile 8. Key NOEs are depicted in Figure
2.
Biochemical Methods
Test compounds were evaluated for their ability to
displace 3H-2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-
yl)-3-(xanth-9-yl)propanoic acid from membranes ex-
pressing individual recombinant human mGlu2 and
mGlu3 receptor subtypes.34,35 The Ki values were cal-
culated from the IC50 values employing the Cheng-
Prusoff equation36 and represent the mean of at least
three separate experiments. Test compounds were also
evaluated for their ability to influence the production
of second messengers in “RGT” cells. RGT cells are
nonneuronal AV12-664 cells coexpressing both GLAST
(a recombinant glutamate transporter to minimize
constituent glutamate activity) and a recombinant hu-
man group I (mGlu1a or mGlu5a), group II (mGlu2 or
mGlu3), or group III (mGlu4a, mGlu6, mGlu7a or
mGlu8) receptor employing methods previously de-
scribed.11,37
Figure 2. Key NOEs observed for methyl-substituted amino
acid precursors.

Subtype Selective mGlu2 Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3607
Scheme 3
Table 1. Effects of Bicyclic Amino Acids 1, 5, 9, and 13 at Recombinant Human mGlu2 and mGlu3 receptors in Vitro
displacement of 3H-341495 binding to membranes
functional (cAMP) responses in mGlu
expressing recombinant mGlu2 or mGlu3a Ki (µM)
receptor-expressing cellsb EC50 (µM) or IC50 (µM)
compd
mGlu2
mGlu3
mGlu2
mGlu3
1
0.074 ( 0.009
0.983 ( 0.035
0.635 ( 0.097
0.409 ( 0.025
0.165 ( 0.003
>100
0.093 ( 0.003
0.146 ( 0.056
0.511 ( 0.020
0.570 ( 0.049
0.302 ( 0.048
>100
0.010 ( 0.002
1.75 ( 0.62
0.045 ( 0.005
0.397 ( 0.039
0.161 ( 0.019
>100
0.038 ( 0.003
9.83 ( 0.37
0.034 ( 0.004
2.94 ( 0.20
1.05 ( 0.23
>100
5
9
13
(+)-13
(-)-13
a For experimental details, see refs 34 and 35. b For experimental details, see ref 11. Roman values are EC50 values, and italic values
are IC50 values. At concentrations up to 100 µM, no agonist or antagonist activity was observed for 5, 9, or 13 in cells expressing mGlu1a,
mGlu5a, mGlu4a, mGlu7a, or mGlu8 receptors.
Molecular Modeling
0.025 µM, respectively (Table). Comparable affinity of
these compounds for 3H-antagonist labeled mGlu3
receptors was also observed, with Ki values of 0.146 (
0.056, 0.511 ( 0.020, and 0.570 ( 0.049 µM for ana-
logues 5, 9, and 13, respectively. Affinity for both mGlu2
and mGlu3 receptors was generally diminished relative
to the unsubstituted parent amino acid 1. Evaluation
of the functional effects of this series on cAMP responses
in RGT cells (Table) identified the C3â-methyl analogue
5 as a full antagonist at both mGlu2 and mGlu3
receptors (IC50 ) 1.75 ( 0.62 and 9.83 ( 0.37 µM,
respectively). In contrast, C4â-methyl analogue 9 was
found to possess full agonist activity at each of these
targets with potency (EC50 ) 0.045 ( 0.005 µM at
mGlu2 and 0.034 ( 0.004 µM at mGlu3) comparable to
1. Methyl substitution at the C4R-position as in ana-
logue 13 resulted in mixed mGlu2 agonist (EC50 ) 0.397
( 0.039 µM)/mGlu3 antagonist (IC50 ) 2.94 ( 0.20 µM)
activity. The mGlu activity in rac-13 was found to reside
exclusively in the (+)-isomer (for (+)-13: mGlu2, Ki )
0.165 µM, EC50 ) 0.161 ( 0.019 µM; mGlu3, Ki ) 0.302
µM, IC50 ) 1.05 ( 0.23 µM). The other enantiomer, (-)-
13, was devoid of mGlu2 and mGlu3 activity at concen-
trations up to 100 µM. For concentrations up to 100 µM,
none of the racemic or nonracemic methyl-substituted
analogues displayed either agonist or antagonist activity
in cells expressing recombinant human mGlu1a, mGlu5a,
mGlu4a, mGlu7a, or mGlu8 receptors.
The crystal structure coordinates of rat mGlu1 ligand-
binding domain with glutamate bound38 were obtained
from the Protein Data Bank (PDB code 1ewk).39 The
molecular modeling program Insight200040 was used to
manipulate the protein structures studied. The protein
sequence of rat mGlu1 ligand-binding domain was
aligned with sequences of human mGlu2 and mGlu3
using the Align123 utility in Insight2000. Homology
models for each of the human mGlu ligand binding
domains were built by replacing the appropriate rat
mGlu1 residue with the corresponding residue present
in the human form. The models were energy-minimized
using the Discover module in Insight2000. By use of the
rat mGlu1 crystal structure as a guide, glutamate was
manually docked into each homology model. Additional
energy minimization was then performed on each
complex. The three-dimensional structures of 5, 9, and
13 were generated using Concord.41 Compounds 5, 9,
and 13 were then manually docked into the glutamate
binding site, followed by energy minimization.
Results
Racemic methyl-substituted analogues 5, 9, and 13
displaced specific binding of the mGlu2/3 receptor
antagonist 3H-2S-2-amino-2-(1S,2S-2-carboxycycloprop-
1-yl)-3-(xanth-9-yl)propanoic acid from membranes ex-
pressing recombinant human mGlu2 receptors with Ki
values of 0.983 ( 0.035, 0.635 ( 0.097, and 0.409 (

3608 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Dominguez et al.
Figure 4. Proposed binding mode for 9 (cyan) in glutamate
binding pocket of mGlu2 (green). The methyl group of 9
interacts with a tyrosine residue (Y216, magenta) and the
methylene groups of an arginine residue (R271, magenta).
Compare this to the proposed binding mode for compound 5
in Figure 3. In this case the methyl interacts with the same
tyrosine residue (Y216) and an additional tyrosine residue,
Y144.
Figure 3. Proposed binding mode for 5 (cyan) in the glutamate
binding pocket of mGlu2 (green). The methyl group of 5 fits
into a small hydrophobic pocket comprising two tyrosine
residues (Y144 and Y216 shown in magenta).
Discussion
As part of our ongoing efforts to identify novel
pharmacological agents to selectively modulate the
function of metabotropic glutamate receptor subtypes,
we substituted the C3- and C4-positions in the bicyclic
ring system of the potent and selective mGlu2/3 receptor
agonist 1 with a methyl group. Methyl substitution at
the C3â-position (5) resulted in an order of magnitude
loss of affinity at mGlu2 but no significant loss in
affinity at mGlu3 compared to the unsubstituted parent.
Moreover, 5 was found to possess antagonist activity
at each of these targets, perhaps suggesting that this
modification interferes with critical conformational
changes in mGlu2 and mGlu3 that are required for
receptor signaling. In contrast, while receptor affinity
was decreased relative to 1 for C4â-methyl analogue 9,
functional agonist activity at both mGlu2 and mGlu3
receptors was fully maintained in this molecule. Finally,
as in the case of â-methyl-substituted analogues 5 and
9, affinity of C4R-methyl-substituted analogue 13 at
both mGlu2 and mGlu3 was diminished compared to
the unsubstituted parent. However, 13 unexpectedly
produced opposite functional effects in the mGlu2- and
mGlu3-expressing cell lines, acting as an agonist at
mGlu2 and an antagonist at mGlu3. These functional
effects were maintained with an approximately 2-fold
increase in potency for the resolved enantiomer (+)-13.
By use of the crystal structure of glutamate bound to
rat mGlu138 as a guide, binding modes for 5, 9, and 13
to the ligand-binding pockets of both mGlu2 and mGlu3
were manually generated (Figures 3-6). The proposed
binding modes result in a hydrogen-bonding network
very similar to that described for the glutamate-mGlu1
crystal structure (see ref 38 and figures therein for
details). The methyl group of 5 (mixed mGlu2/mGlu3
antagonist) fits into a hydrophobic region defined by two
tyrosine residues (Y144 and Y216) as depicted in Figure
3. Y144 and Y216 arise from different lobes of the
glutamate binding site, and mutational studies have
shown that each is important for ligand binding to both
mGlu2 and mGlu3.41 It is intriguing to speculate that
the methyl group of 5, by virtue of being situated
between these residues, might impede the closure of the
Figure 5. Proposed binding mode for 13 (cyan) in the
glutamate binding pocket of mGlu2 (green). In this binding
mode the methyl group of 13 is colliding with tyrosine Y216
and glycine G296 (with the R-carbon hydrogens shown in gray).
two lobes of the glutamate binding site, a conformational
change required to elicit an agonist response.38 Incor-
poration of the methyl group at the adjacent C4â-
position, as in 9 (mixed mGlu2/mGlu3 agonist, Figure
4), results in the methyl substituent interacting with
Y216 and the methylene groups of arginine R271, each
of which arise from the same lobe of the glutamate
binding site. These interactions apparently do not
preclude the ability of either mGlu2 or mGlu3 receptors
to adopt a fully closed (agonist) conformation. The
proposed binding mode for 13, which displays agonist
activity at mGlu2 and antagonist activity at mGlu3, is
shown in Figures 5 and 6. The change in stereochemical
configuration of the methyl group in 13 compared to 9
leads to steric interactions with Y216 and glycine G296
in both mGlu2 (Figure 5) and mGlu3 (Figure 6) recep-
tors. Energy minimization of 13 bound to each receptor
results in an approximately 0.5 Å shift in several
residues in the binding site, including Y216, G296, and
leucine L300, shown in orange in the upper-left portion
of Figure 6. L300 is interesting in that it represents the

Subtype Selective mGlu2 Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3609
a VG 70SE or Varian MAT 731 instrument. Preparative HPLC
was performed with the Waters Prep LC2000 apparatus using
dual silica gel PrepPAK-500 cartridges. Solvent systems
employed are given in parentheses for each example. Cation-
exchange chromatography was performed with Dowex 50X8-
100 ion-exchange resin and anion-exchange chromatography
with Bio-Rad AG1-X8 anion-exchange resin (hydroxide form).
Compounds for which satisfactory combustion analyses could
be obtained are identified as such within the Experimental
Section, and a table is provided with detailed elemental
analyses as Supporting Information. For compound 3, analyti-
cal HPLC data (normal phase conditions) are included in the
experimental part as an indicator of purity (column, Kromasil
Si 60, 250 mm × 4.6 mm, 5 µm; isocratic mode, hexane/IPA
5%; flow rate, 1 mL/min; detection with UV-vis diode array
detector, 210-400 nM).
(1SR,3RS,5RS,6SR)-Ethyl-2-oxo-3-methylbicyclo[3.1.0]-
hexane 6-Carboxylate (3). To a solution of 2 (317 mg, 1.88
mmol) in anhydrous THF (30 mL) at -78 °C and under argon
was added a 1 M solution of lithium bis-trimethylsilylamide
in THF (1.9 mL, 1.88 mL). The reaction mixture was stirred
for 45 min at this temperature, and then this solution was
cannuled into a solution of methyl iodide (0.35 mL, 5.6 mmol)
in THF (10 mL) at -78 °C. The reaction mixture was stirred
for 1 h at -78 °C and then overnight at room temperature,
quenched with saturated ammonium chloride solution (20 mL),
and extracted with CH2Cl2 (3 × 50 mL). The combined organic
phases were dried over MgSO4, filtered, and evaporated to
dryness. Purification of the crude by flash chromatography
(hexane/ethyl acetate 4:1) afforded 3 (0.24 g, 1.32 mmol) as
Figure 6. Proposed binding mode for 13 (cyan) in the
glutamate binding pocket of mGlu3 (green). Energy minimiza-
tion of this complex causes movement in several residues,
including glutamine Q306. This glutamine is a leucine residue
in mGlu2 (L300, orange) and represents the only residue that
is different among the residues in the proximity of the
glutamate binding site of mGlu2 vs mGlu3. Movement of this
key residue may give rise to different conformational changes
in the two receptors, which may account for the difference in
functional activities observed for 13 at mGlu2 vs mGlu3.
1
an oil in 70% yield. FDMS: M+ ) 182. H NMR (CDCl3), δ:
only nonidentical amino acid close to the ligand-binding
pocket of mGlu2 and mGlu3 (L300 in the former and
glutamine Q306 in the latter, also shown in Figure 6).
Recent work has demonstrated that the mutation
Q306A in mGlu3 results in an approximate 87% loss in
agonist (3H-DCG-IV) binding compared to the wild-type
receptor whereas the analogous mutation in mGlu2
(L300A) had a slightly less pronounced effect (67% loss
4.0 (q, 2H, J ) 7.1 Hz), 2.32-2.1 (m, 3H), 1.98-1.85 (m, 2H),
1.75-1.6 (m, 1H), 1.15 (t, 3H, J ) 7.1 Hz), 0.9 (d, 3H, J ) 6.9
Hz). 13C NMR (CDCl3), δ: 212, 170.26, 60.95, 35.77, 35.21,
31.46, 27.19, 27.07, 13.95, 13.79. Compound was checked on
analytical HPLC for quality control. Purity of the desired
compound (under normal phase conditions) was >98%.
(1SR,2SR,3SR,5RS,6SR)-Ethyl-2-spiro-5-hydantoin-3-
methylbicyclo[3.1.0]hexane 6-Carboxylate (4). To a solu-
tion of 3 (500 mg, 2.7 mmol) in ethanol (1.3 mL) and water
(3.3 mL) was added potassium cyanide (195 mg, 3 mmol) and
ammonium carbonate (782 mg, 8.1 mmol). The mixture was
heated at 55 °C overnight. After the mixture was cooled to
room temperature, the precipitated material was filtered and
washed with EtOH-H2O to afford 4 (0.27 g, 1.1 mmol) in 40%
yield. FDMS: M+ ) 252. 1H NMR (DMSO-d6), δ: 10.65 (s, 1H),
7.9 (s, 1H), 4.05 (q, 2H, J ) 7.1 Hz), 2.05-1.6 (m, 5H), 1.2 (t,
3H, J ) 7.1 Hz), 0.8 (d, 3H, J ) 5.9). 13C NMR (DMSO-d6), δ:
175.86, 172.02, 156.75, 71.86, 60.40, 35.76, 33.12, 32.00, 25.97,
20.48, 14.27, 12.18. Anal. (C12H16N2O4) C, H, N.
3
in H-DCG-IV binding).42 The functional consequence
of these mutations (e.g., evaluation of agonist/antagonist
activity) was not reported. On the basis of our observed
activity of 13 as an agonist at mGlu2 and an antagonist
at mGlu3, we speculate that while a slight movement
of L300 side chain in mGlu2 has no effect on the ability
of the mGlu2 glutamate binding site to adopt an agonist
conformation, a comparable displacement of the ω-a-
mide functionality in Q306 in mGlu3 may preclude this
conformational change, resulting in the observed an-
tagonist effect.
(1SR,2SR,3SR,5RS,6SR)-2-Amino-3-methylbicyclo[3.1.0]-
hexane 2,6-Dicarboxylic Acid (5). A mixture 4 (250 mg, 0.99
mmol) in 12 N HCl (10 mL) was refluxed overnight. The
resulting solution was evaporated to dryness, yielding a white
solid. Compound 5 (78 mg, 0.39 mmol) was isolated in 40%
yield as a zwitterion after ion exchange chromatography on
Dowex 50X8 50-100 mesh using 10% solution of pyridine in
Summary
We have found that substitution at the C3- or C4-
positions of 1 by a methyl group results in generally
diminished mGlu2/3 receptor affinity and agonist, an-
tagonist, or mixed agonist/antagonist activity in func-
tional assays depending on methyl group regiochemistry
and stereochemistry. Importantly, C4R-methyl-substi-
tuted analogue 13 is the first example of a glutamate
site agonist capable of functionally differentiating be-
tween mGlu2 and mGlu3 receptors. Owing to this
unique pharmacological profile, 13 may be a useful tool
for studying mGlu2 receptor activation in vivo.
1
water as the eluent. FDMS: M+ ) 199. H NMR (D2O-Pyr-
d5), δ: 2.0-1.7 (m, 6H), 0.9 (d, 3H). 13C NMR (D2O-Pyr-d5),
δ: 179.95, 173.37, 70.08 36.46, 33.69, 31.79, 26.44, 23.75,
12.18. Anal. (C9H13NO4) C, H, N.
(1SR,5RS,6SR)-Ethyl-2-oxobicyclo[3.1.0]hex-3-ene 6-Car-
boxylate (6). Iodotrimethylsilane (75 g, 375 mmol) was added
dropwise to a 0 °C solution of 2 (42 g, 250 mmol) and Et3N (75
g, 750 mmol) in anhydrous CH2Cl2 (1 L). The resulting reaction
mixture was allowed to warm to ambient temperature as it
was stirred overnight. The reaction mixture was washed three
times with aqueous NH4Cl and brine, dried over MgSO4, and
concentrated to yield the crude silyl enol ether. This was
dissolved in anhydrous CH3CN (600 mL), treated in one
portion with Pd(OAc)2 (61.7 g, 275 mmol), and stirred at
ambient temperature overnight. The reaction mixture was
diluted with Et20 (600 mL) and filtered through a pad of Celite.
The filtrate was concentrated to yield the crude product that
Experimental Section
Melting points were obtained using a Thomas-Hoover capil-
liary melting point apparatus and are uncorrected. 1H and 13
C
NMR spectra were obtained at 300.15 and 75.48 MHz,
respectively, with TMS as an internal standard. Field desorp-
tion mass spectrometry (FDMS) was performed using either

3610 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Dominguez et al.
was purified by HPLC (10% EtOAc/hexanes to 50% EtOAc/
hexanes), affording 6 (38.22 g, 230 mmol, 92% yield). Mp )
75-77 °C. FDMS: M+ ) 166. 1H NMR (CDCl3) δ: 7.61 (dd, J
) 5.5 and 2.4 Hz, 1H), 5.73 (d, J ) 5.5 Hz, 1H), 4.15 (q, J )
7.1 Hz, 2H), 2.97-2.94 (m, 1H), 2.64-2.61 (m, 1H), 2.27 (t, J
) 2.7 Hz, 1H), 1.27 (t, J ) 7.1 Hz, 3H). 13C NMR (CDCl3) δ:
203.24, 167.92, 159.66, 129.60, 61.32, 45.82, 30.01, 28.91,
14.11. Anal. (C9H10O3) C, H.
(1SR,4SR,5RS,6SR)-Ethyl-2-oxo-4-methylbicyclo[3.1.0]-
hexane 6-Carboxylate (7). Methyllithium (71 mL of a 1.4
M solution in Et2O, 100 mmol) was added to a mixture of
copper(I) iodide (9.5 g, 50 mmol) in anhydrous Et2O (10 mL)
at 0 °C. The solution was stirred for 30 min at 0 °C and cooled
to -78 °C. Compound 6 (1.6 g, 10 mmol) in Et2O (25 mL) was
added dropwise. Upon complete addition, the mixture was
stirred for another hour at -20 °C for 1 h and then was
quenched with saturated ammonium chloride solution and
extracted with Et2O. The combined organic phases were dried
over MgSO4, filtered, and evaporated to dryness. Purification
of the crude product by flash chromatography (hexane/ethyl
acetate 4:1) gave 7 (1.52 g, 8.3 mmol, 83% yield). FDMS: M+
) 182. 1H NMR (CDCL3), δ: 4.1 (q, 2H, J ) 7.15 Hz), 2.5 (m,
1H), 2.35-2.19 (m, 3H), 2.1 (t, 1H, J ) 2.60 Hz), 1.65 (d, 1H,
J ) 18.60 Hz), 1.19 (t, 3H, J ) 7.15 Hz), 1.10 (d, 3H, J ) 6.90
Hz). 13C NMR (CDCl3), δ: 211.13, 170.16, 61.13, 40.45, 36.09,
34.96, 29.93, 26.92, 21.80, 14.03. Anal. (C10H14O3) C, H.
Cl. The organic phase was dried (MgSO4) and concentrated to
dryness. The residue was dissolved in CH3CN, cooled to 5 °C,
and treated with Pd(OAc)2 (11.0 g, 49 mmol) in one portion.
The reaction was allowed to continue at room temperature
overnight, then was diluted with Et2O, filtered through a pad
of silica gel and Celite, and concentrated to dryness yielding
10 (7.1 g, 35.4 mmol, 88% yield). The product solidified to a
low-melting solid on standing, mp ) 50-52 °C. FDMS: M+
)
1
180. H NMR (CDCl3) δ: 5.41 (s, 1H), 4.15 (q, J ) 7 Hz, 2H),
2.80-2.75 (m, 1H), 2.60-2.55 (m, 1H), 2.22 (t, J ) 2.5 Hz, 1H),
2.18 (s, 3H), 1.25 (t, J ) 7 Hz, 3H). Anal. (C10H12O3.0.05CH2-
Cl2) C, H.
(1SR,4RS,5RS,6SR)-Ethyl-2-oxo-4-methylbicyclo[3.1.0]-
hexane 6-Carboxylate (11). To a solution of 10 (5.55 g, 30.8
mmol) in EtOH (100 mL) was added 5% Pd-C (0.6 g). The
mixture was subjected to H2(g) at 40 psi for 1 h. The mixture
was diluted with Et2O and filtered through Celite. The solvent
was evaporated, and the crude product was purified by HPLC
(hexanes-ethyl acetate 3:1), providing 11 (5.08 g, 28 mmol,
1
91%). FDMS: M+ ) 182. H NMR (CDCl3) δ: 4.18-4.08 (m,
2H), 2.70-2.60 (m, 1H), 2.51-2.48 (m, 1H), 2.28-2.25 (m, 1H),
2.18 (ddd, J ) 18.4, 8.8, and 1.0 Hz, 1H), 2.03 (dd, J ) 3.3
and 2.5 Hz), 1.61 (ddd, J ) 18.4, 9.5, and 1.0 Hz, 1H), 1.26 (t,
J ) 7 Hz, 3 H), 1.15 (d, J ) 7 Hz, 3H). 13C NMR (CDCl3) δ:
210.8, 170.47, 61.22, 39.56, 37.17, 34.79, 29.56, 23.76, 18.07,
14.09. Anal. (C10H14O3) C, H.
(1SR,2SR,4RS,5RS,6SR)-Ethyl-2-spiro-5-hydantoin-4-
methylbicyclo[3.1.0]hexane 6-Carboxylate (12). To a solu-
tion of 11 (1.82 g, 10 mmol) in ethanol (20 mL) and water (10
mL) was added KCN (1.0 g, 15 mmol) and (NH4)2CO3 (2.0 g,
25 mmol) and the mixture was heated at 50 °C for 48 h. The
reaction mixture was diluted with H2O (30 mL) and cooled in
an ice bath, and the resulting solids were filtered to yield 0.5
g of crude product. This was recrystallized from EtOH to yield
a single diastereomer 12 (0.18 g, 0.71 mmol, 7.1%). Mp ) 222-
224 °C. FDMS: M+ ) 252. 1H NMR (pyridine-d5), δ: 12.47 (s,
1H), 9.58 (s, 1H), 4.08 (q, 2H, J ) 7.1 Hz), 2.97 (ddd, J ) 10.5,
6.8, and 3.8 Hz, 1H), 2.45 (dd, J ) 6.3 and 3.2 Hz, 1H), 2.25
(ddd, J ) 6.3, 3.2, and 3.2 Hz, 1H), 2.16 (dd, J ) 3.2 and 3.2
Hz, 1H), 2.11 (dd, J ) 13.7 and 7.9 Hz, 1H), 1.23 (dd, J ) 13.7
and 10.5 Hz, 1H), 1.08 (t, J ) 7.1 Hz, 3H), 0.89 (d, J ) 6.8 Hz,
3H). 13C NMR (pyridine-d5), δ: 178.65, 172.33, 157.97, 70.17,
60.74, 38.43, 34.56, 33.85, 33.39, 18.92, 18.92, 14.20. Anal.
(C12H16N2O4) C, H, N.
(1SR,2SR,4RS,5RS,6SR)-2-Amino-4-methylbicyclo[3.1.0]-
hexane 2,6-Dicarboxylic Acid (13). A mixture of 12 (0.15
g, 0.6 mmol) and 1 N NaOH solution (15 mL) was heated under
reflux for 5 days. The pH was adjusted to 1 by addition of 1 N
HCl, and the resulting solution was evaporated to dryness,
yielding a white solid. The solids were redissolved at pH 12,
the solids were removed by filtration, and the filtrate was
applied to an anion exchange column (AG1-X8, acetate form).
Amino acid 13 (0.067 g, 0.34 mmol, 56%) was isolated following
elution with 3 N AcOH. Mp >270 °C (dec). FDMS: M+ (+H)
) 200. 1H NMR (D2O, KOD), δ: 2.25-2.10 (m, 1H), 1.70-1.43
(m, 3H), 1.22 (br.s, 1H), 0.7-0.67 (m, 3H), 0.52-0.43 (m, 1H).
13C NMR (D2O, KOD) δ: 180.97, 180.16, 64.26, 40.17, 34.39,
31.46, 31.33, 19.04, 14.47. Anal. (C9H13NO4) C, H, N.
Separation of the Enantiomers of rac-11. The individual
enantiomers of rac-11 were obtained by chiral column chro-
matography (Chiralpak AD 8 cm × 28 cm column; 500 mg of
racemate loaded each run; elution with 100% CH3OH at 300
mL/min; detection at 220 nm). Retention times for (+)-11 and
(-)-11 were 6.22 and 10.07 min, respectively. From 8.5 g (46.6
mmol) of rac-11 was obtained 3.56 g (19.5 mmol, 42% yield,
>98.8% ee) of (+)-11 and 3.66 g (20.0 mmol, 43% yield, 98.8%
ee) of (-)-11. Optical rotations: (+)-11, RD 78.5 (c 1.07, CH3-
OH); (-)-11, Rd -83.3 (c 1.08, CH3OH).
(+)-Ethyl-2-spiro-5-hydantoin-4-methylbicyclo[3.1.0]-
hexane 6-Carboxylate ((+)-12). To a solution of (+)-11 (3.20
g, 17.6 mmol) in ethanol (15 mL) and water (15 mL) was added
KCN (1.71 g, 26.3 mmol) and (NH4)2CO3 (4.12 g, 52.8 mmol),
and the mixture was heated at 50 °C overnight and then at
room temperature for an additional 24 h. The resulting solids
(1SR,2SR,4SR,5RS,6SR)-Ethyl-2-acetamido-2-cyano-4-
methylbicyclo[3.1.0]hexane 6-Carboxylate (8). A hetero-
geneous mixture of alumina (14 g, Merck, type 90 for column
chromatography, neutral, activity I) and ammonium chloride
(26 mmol) in acetonitrile (50 mL) was ultrasonically irradiated
for 30 min. Then, a solution of 7 (2.19 mmol) in acetonitrile (5
mL) was added, and after sonication for an addition 2 h, 2.19
mmol of KCN was added. The mixture was sonicated over-
night, and then the alumina was filtered off and the filtrate
was concentrated to dryness to give a mixture of diastereo-
meric aminonitriles. To a solution of this mixture (1.25 mmol)
in dry CH2Cl2 at 0 °C was added ethyl diisopropylamine (1.37
mmol), and the resultant mixture was stirred for 15 min.
Acetyl chloride (1.37 mmol) was added, and the mixture was
stirred at ambient temperature for 5 h. The reaction was
quenched with water and extracted with CH2Cl2. The com-
bined organic extracts were dried over MgSO4 and evaporated
to dryness. Desired diastereomer 8 (0.16 g, 0.66 mmol) was
isolated in 30% yield from the mixture by column chromatog-
raphy (hexane/ethyl acetate 1:1), using 230-400 mesh silica
1
gel (Merck). FDMS: M+ ) 250. H NMR (CDCl3), δ: 6.15 (s,
1H), 4.1 (q, 2H, J ) 7.1 Hz), 2.7 (dd, 1H, J ) 2.8 and 6.2 Hz),
2.55 (d, 1H, J ) 15 Hz), 2.45 (m, 1H), 2.15-1.95 (m, 5H), 1.62
(t, 1H, J ) 3.5 Hz), 1.55 (dd, 1H, J ) 7.8 and 15 Hz), 1.25 (m,
6H). 13C NMR (CDCl3), δ: 171.16, 169.97, 121.09, 61.28, 55.05,
42.26, 34.94, 34.62, 34.29, 23.03, 22.04, 21.15, 14.25. Anal.
(C13H18N2O3) C, H,N.
(1SR,2SR,4SR,5RS,6SR)-2-Amino-4-methylbicyclo[3.1.0]-
hexane 2,6-Dicarboxylic Acid (rac-9). A mixture of 8 (0.8
mmol) and 5 N HCl solution (10 mL) was heated under reflux
overnight. The resulting solution was evaporated to dryness,
yielding a white solid. Amino acid 9 (0.05 g, 0.25 mmol) was
isolated in 31% yield as a zwitterion after ion exchange
chromatography on Dowex 50X8 50-100 mesh using 10%
pyridine-water as eluent. Mp > 300 °C. FDMS: M+ ) 199.
1H NMR (D2O, Pyr-d5), δ: 2.2 (dd, 1H, J ) 3.1 and 6.2 Hz),
1.94 (m, 1H), 1.8-1.6 (m, 3H), 1.52 (t, 1H, J ) 3.1 Hz), 0.9 (d,
3H, J ) 7.2 Hz). 13C NMR (D2O, KOD) δ: 181.32, 179.68, 67.05,
41.58, 35.02, 34.77, 33.61, 25.56, 20.24. Anal. (C9H13NO4) C,
H, N.
(1SR,5RS,6SR)-Ethyl-2-oxo-4-methylbicyclo[3.1.0]hex-
3-ene 6-Carboxylate (10). A solution of 7 (8.15 g, 44.7 mmol)
and Et3N (10.86 g, 107 mmol) in CH2Cl2 at 0 °C was treated
dropwise with TMSI (10.74 g, 53.7 mmol) at a rate that
maintained the internal temperature below 5 °C. Upon
complete addition, the mixture was allowed to stir at this
temperature for 2 h. The reaction mixture was diluted with
Et2O and washed with a saturated aqueous solution of NH4-

Subtype Selective mGlu2 Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3611
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Metabotropic glutamate receptors: potential drug targets for the
treatment of schizophrenia. Curr. Drug Targets 2002, 1, 261-
281.
were filtered and washed with EtOH and CH2Cl2 to yield (+)-
12 (1.25 g, 4.96 mmol, 28%). FDMS: M+ ) 252. 1H NMR
(pyridine-d5), δ: 12.43 (s, 1H), 9.53 (s, 1H), 4.08 (q, 2H, J )
7.1 Hz), 2.98 (ddd, J ) 10.5, 6.8, and 3.8 Hz, 1H), 2.45 (dd, J
) 6.3 and 3.2 Hz, 1H), 2.26 (ddd, J ) 6.3, 3.2, and 3.2 Hz,
1H), 2.16 (dd, J ) 3.2 and 3.2 Hz, 1H), 2.12 (dd, J ) 13.7 and
7.9 Hz, 1H), 1.23 (dd, J ) 13.7 and 10.5 Hz, 1H), 1.10 (t, J )
7.1 Hz, 3H), 0.89 (d, J ) 6.8 Hz, 3H). 13C NMR (pyridine-d5),
δ: 178.66, 172.33, 158.00, 70.20, 60.75, 38.46, 34.57, 33.87,
33.41, 18.95, 17.01, 14.22. RD 17.82 (c 1.01, CH3OH). Anal.
(C12H16N2O4‚0.4CH2Cl2) C, H, N.
(-)-Ethyl-2-spiro-5-hydantoin-4-methylbicyclo[3.1.0]-
hexane 6-Carboxylate ((-)-12). To a solution of (-)-11 (3.20
g, 17.6 mmol) in ethanol (15 mL) and water (15 mL) was added
KCN (1.71 g, 26.3 mmol) and (NH4)2CO3 (4.12 g, 52.8 mmol),
and the mixture was heated at 50 °C overnight, then at room
temperature for an additional 24 h. The resulting solids were
filtered and washed with EtOH to yield (-)-12 (1.62 g, 6.4
mmol, 36%). FDMS: M+ ) 252. 1H NMR (pyridine-d5), δ:
12.43 (s, 1H), 9.53 (s, 1H), 4.08 (q, 2H, J ) 7.1 Hz), 2.98 (ddd,
J ) 10.5, 6.8, and 3.8 Hz, 1H), 2.45 (dd, J ) 6.3 and 3.2 Hz,
1H), 2.26 (ddd, J ) 6.3, 3.2, and 3.2 Hz, 1H), 2.16 (dd, J ) 3.2
and 3.2 Hz, 1H), 2.12 (dd, J ) 13.7 and 7.9 Hz, 1H), 1.23 (dd,
J ) 13.7 and 10.5 Hz, 1H), 1.10 (t, J ) 7.1 Hz, 3H), 0.89 (d, J
) 6.8 Hz, 3H). 13C NMR (pyridine-d5), δ: 178.66, 172.33,
158.00, 70.20, 60.75, 38.46, 34.57, 33.87, 33.41, 18.95, 17.01,
14.22. RD -23.16 (c 0.95, CH3OH). Anal. (C12H16N2O4‚0.1H2O)
C, H, N.
(+)-2-Amino-4-methylbicyclo[3.1.0]hexane 2,6-Dicar-
boxylic Acid ((+)-13). A mixture of (+)-12 (1.40 g, 5.5 mmol)
and 2 N NaOH solution (28 mL) was heated under reflux for
24 h. The pH was adjusted to 7 by addition of 1 N HCl, and
the resulting solution was evaporated to dryness, yielding a
white solid. The solids were redissolved at pH 12 and applied
to an anion exchange column (AG1-X8, OH form). Amino acid
(+)-13 (0.60 g, 3.0 mmol, 55%) was isolated following elution
with 3 N AcOH. Mp >250 °C (dec). FDMS: M+ (+1) ) 200.
1H NMR (D2O, KOD), δ: 2.60-2.45 (m, 1H), 2.05-1.80 (m,
3H), 1.58-1.52 (m, 1H), 1.00 (t, J ) 7 Hz, 3H), 0.78 (dd, J )
13.7 and 10.5 Hz, 1H). RD 53.61 (c 0.97, 1 N HCl). Anal. (C9H13-
NO4‚0.1AcOH) C, H, N.
(-)-2-Amino-4-methylbicyclo[3.1.0]hexane 2,6-Dicar-
boxylic Acid ((-)-13). A mixture of (-)-12 (1.80 g, 7.1 mmol)
and 2 N NaOH solution (36 mL) was heated under reflux for
24 h. The pH was adjusted to 7 by addition of 1 N HCl, and
the resulting solution was evaporated to dryness, yielding a
white solid. The solids were redissolved at pH 12 and applied
to an anion exchange column (AG1-X8, OH form). Amino acid
(-)-13 (1.10 g, 5.6 mmol, 78%) was isolated following elution
with 3 N AcOH. Mp >250 °C (dec). FDMS: M+ (+1) ) 200.
1H NMR (D2O, KOD), δ: 2.60-2.45 (m, 1H), 2.05-1.80 (m,
3H), 1.58-1.52 (m, 1H), 1.00 (t, J ) 7 Hz, 3H), 0.78 (dd, J )
13.7 and 10.5 Hz, 1H). RD -54.74 (c 0.95, 1 N HCl). Anal.
(C9H13NO4‚0.1AcOH) C, H, N.
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Supporting Information Available: Results from com-
bustion analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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