Design, synthesis, and pharmacological characterization of (+)-2- aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740): A potent, selective, and orally active group 2 metabotropic glutamate receptor agonist possessing anticonvulsant and anxiolytic properties

Article abstract of DOI:10.1021/jm9606756

2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (9) was designed as a conformationally constrained analog of glutamic acid. For 9, the key torsion angles (τ₁ and τ₂) which determine the relative positions of the α- amino acid and distal carboxyl functionalities are constrained where τ₁ = 166.9°or 202°and τ₂ = 156°, respectively. We hypothesized that 9 would closely approximate the proposed bioactive conformation of glutamate when acting at group 2 metabotropic glutamate receptors (mGluRs). The racemic target molecule (±)-9, its C2-diastereomer (±)-16, and its enantiomers (+)- 9 (LY354740) and (-)-9 (LY366563) were prepared by an efficient, stereocontrolled, and high-yielding synthesis from 2-cyclopentenone. Our hypothesis that 9 could interact with high affinity and specificity at group 2 mGluRs has been supported by the observation that (±)-9 (EC₅₀ = 0.086 ± 0.025 μM) and its enantiomer (+)-9 (EC₅₀ = 0.055 ± 0.017μM) are highly potent agonists for group 2 mGluRs in the rat cerebral cortical slice preparation (suppression of forskolin-stimulated cAMP formation) possessing no activity at other glutamate receptor sites (iGluR or group 1 mGluR) at concentrations up to 100 μM. Importantly, the mGluR agonist effects of (+)- 9 are evident following oral administration in mice in both the elevated plus maze model of anxiety (ED₅₀ = 0.5 mg/kg) and in the ACPD-induced limbic seizure model (ED₅₀ = 45.6 mg/kg). Thus, (+)-9 is the first orally active group 2 mGluR agonist described thus far and is an important tool for studying the effects of compounds of this class in humans.

Full text of DOI:10.1021/jm9606756

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528
J . Med. Chem. 1997, 40, 528-537
Design , Syn th esis, a n d P h a r m a cologica l Ch a r a cter iza tion of
(+)-2-Am in obicyclo[3.1.0]h exa n e-2,6-d ica r boxylic Acid (LY354740): A P oten t,
Selective, a n d Or a lly Active Gr ou p 2 Meta botr op ic Glu ta m a te Recep tor Agon ist
P ossessin g An ticon vu lsa n t a n d An xiolytic P r op er ties
J ames A. Monn,* Matthew J . Valli, Steven M. Massey, Rebecca A. Wright, Craig R. Salhoff, Bryan G. J ohnson,
Trevor Howe,† Charles A. Alt,‡ Gary A. Rhodes,‡ Roger L. Robey,‡ Kelly R. Griffey,§ J oseph P. Tizzano,§
Mary J . Kallman,§ David R. Helton,§ and Darryle D. Schoepp
Central Nervous System, Process Research and Toxicology Research Divisions, Eli Lilly and Company,
Indianapolis, Indiana 46285, and Lilly Research Centre, Ltd., Windelsham, Surrey, GU20 6PH, U.K.
Received September 27, 1996X
2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (9) was designed as a conformationally
constrained analog of glutamic acid. For 9, the key torsion angles (τ1 and τ2) which determine
the relative positions of the R-amino acid and distal carboxyl functionalities are constrained
where τ1 ) 166.9° or 202° and τ ) 156°, respectively. We hypothesized that 9 would closely
2
approximate the proposed bioactive conformation of glutamate when acting at group 2
metabotropic glutamate receptors (mGluRs). The racemic target molecule (()-9, its C2-
diastereomer (()-16, and its enantiomers (+)-9 (LY354740) and (-)-9 (LY366563) were prepared
by an efficient, stereocontrolled, and high-yielding synthesis from 2-cyclopentenone. Our
hypothesis that 9 could interact with high affinity and specificity at group 2 mGluRs has been
supported by the observation that (()-9 (EC50 ) 0.086 ( 0.025 µM) and its enantiomer (+)-9
(EC50 ) 0.055 ( 0.017 µM) are highly potent agonists for group 2 mGluRs in the rat cerebral
cortical slice preparation (suppression of forskolin-stimulated cAMP formation) possessing no
activity at other glutamate receptor sites (iGluR or group 1 mGluR) at concentrations up to
100 µM. Importantly, the mGluR agonist effects of (+)-9 are evident following oral administra-
tion in mice in both the elevated plus maze model of anxiety (ED50 ) 0.5 mg/kg) and in the
ACPD-induced limbic seizure model (ED50 ) 45.6 mg/kg). Thus, (+)-9 is the first orally active
group 2 mGluR agonist described thus far and is an important tool for studying the effects of
compounds of this class in humans.
In tr od u ction
L-Glutamic acid is the principal excitatory amino acid
(EAA) neurotransmitter in the mammalian central
nervous system.1 Glutamate exerts its effects via
activation of two types of receptors: those which form
ligand-gated cation channels (ionotropic glutamate re-
ceptors, iGluRs) and those which are coupled via G-
proteins to intracellular enzyme systems which influ-
ence the production of second messengers (metabotropic
glutamate receptors, mGluRs). There are currently
eight distinct mGluR proteins (mGluR1-8) which have
been pharmacologically distinguished into three groups
based on amino acid sequence homology, signal trans-
duction mechanisms, and agonist pharmacology.2-6
Group 1 mGluRs (mGluR1 and mGluR5) are posi-
tively coupled to phospholipase C (PLC). When acti-
vated, these mGluRs induce the breakdown of cellular
droxyphenylglycine (2) has been demonstrated to be a
selective agonist for group 1 mGluRs possessing no
effects at group 2 mGluRs, group 3 mGluRs, or
iGluRs.8-10 Group 2 mGluRs (mGluR2 and mGluR3)
are negatively coupled to adenylyl cyclase (AC). Agonist
activation of this receptor class effects the inhibition of
3′,5′-cyclic adenosine monophosphate (c-AMP) forma-
tion. (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG-
1, 3)11,12 and (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)-
glycine (DCG-IV, 4)13,14 are highly potent agonists for
group 2 mGluRs but possess agonist effects at other
glutamate receptor sites at somewhat higher concentra-
tions (3 at group 1 mGluRs, 4 at NMDA receptors). More
phosphoinositides (PI), producing inositol triphosphate
(IP3) and diacylglycerol (DAG) within the target neuron.
Group 1 mGluRs are activated by (1S,3R)-1-amino-
cyclopentane-1,3-dicarboxylic acid (1), although this
compound produces group 2 mGluR agonist effects at
similar concentrations.7 More recently, 3,5-dihy-
* Corresponding author: Dr. J ames A. Monn, Eli Lilly and Com-
pany, Lilly Corporate Center, mail drop code 0510, Indianapolis, IN
46285. Telephone: (317) 276-9101. FAX: (317) 277-1125. E-mail:
Monn@Lilly.com.
† Lilly Research Centre, Ltd.
‡ Process Research, Eli Lilly and Company.
§ Toxicology Research Divisions, Eli Lilly and Company.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00675-9 CCC: $14.00 © 1997 American Chemical Society

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mGluR Agonist Effects of LY354740
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 529
Ta ble 1. Torsion Angle Measurements and CHARMm Energy Calculations for Various Conformations of Compounds 3 and 9
conformer of 3a conformer of 9b
A (flap up) B (flap down)
measurement
a
b
c
d
e
f
τ1 angle (deg)c
τ2 angle (deg)c
35
140.9
6.64
2.30
651.328
12.82
85
153
217
274
141.3
5.09
0.74
651.192
11.44
332
166.9
155.9
13.02
0
862.407
8.77
200.2
157.0
15.60
2.59
862.410
8.77
136.6
7.47
3.12
150.0
3.35
0
642.168
8.70
146.3
6.97
2.63
143.9
7.99
3.64
d
absolute CHARMm (energy (kcal/mol-1
)
relative CHARMm energy (kcal/mol-1
4-31G nuclear repulsion energy (Eh)e
4-31G dipole (debye)f
)
d
a
d
See Figure 1 for graphical representation. b See Figure 2 for Newman projection representations. c See ref 24 for definition. CHARMm
force field, refs 25 and 26.
selective (although less potent) group 2 mGluR agonists
include (2S,1′S,2′R,3′R)-2-(2-carboxy-3-(methoxymeth-
yl)cyclopropyl)glycine (cis-MCG-I, 5)15 and (2R,4R)-4-
aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC, 6).16,17
Group 3 mGluRs (mGluR4, mGluR7, and mGluR8) are
also negatively coupled to AC, but can be distiguished
from the group 2 class both by protein sequence homol-
ogy and agonist pharmacology. Group 3 mGluRs are
selectively activated by 2-amino-4-phosphonobutyrate
(AP4, 7) and L-serine O-phosphate (L-SOP, 8).18-22 As
part of our research program directed toward the
preparation of highly potent and selective ligands for
mGluRs, we have focused on the identification of highly
potent, selective, and systemically active group 2 mGluR
agonists and antagonists.
Molecu la r Mod elin g
Because of the high degree of potency and good
24
F igu r e 1. Effect of rotation about τ1 of 3 on the calculated
selectivity evinced by 3 toward group 2 mGluRs, this
partially constrained glutamic acid analog has been the
subject of inquiry regarding the preferred conformation
of glutamate when interacting at these receptor sites.23
In this study, a computer-assisted comparison of the
low-energy conformers of 1 and 3 was made, from which
the likely bioactive conformation of glutamate for group
2 mGluR protein-agonist interaction was inferred.
Specifically, the group 2 mGluR-active conformation of
glutamate was hypothesized to be one in which the τ1
and τ2 angles24 approximate 177° and -174°, respec-
tively (e.g. a nearly fully extended glutamate backbone
conformation). This hypothesis is further supported by
the observation that 6, a highly selective group 2 mGluR
agonist, preferentially adopts a fully extended glutamate
conformation in the vacuum phase.17 We have ourselves
examined the effect of rotation about the C2-C3 bond
of 3 on the CHARMm energy25,26 for this molecule
(Figure 1, Table 1) and have arrived at a similar
conclusionsthe lowest energy conformer of 3 exhibits
values for τ1 and τ2 of 153° and 150°, respectively
(conformer “c”, Figure 1, Table 1). However, two other
low-energy conformations of 3 (conformers “a” and “e”,
Figure 1, Table 1) cannot be ruled out as possible group
2 mGluR-active forms of 3 as these would be expected
to be energetically accessible at physiologically relevant
temperatures.
CHARMm potential energy. See molecular modeling section
for details.
F igu r e 2. Numbering system for 9 and Newman projections
of calculated low-energy conformations. Reader is viewing 9
down the axis of the C2-C1 carbon-carbon bond.
F igu r e 3. Thermal elipsoid representation of (+)-9. The
non-H atom elipsoids enclose the 50% probability limits.
To more fully test the hypothesis that a fully extended
glutamate backbone is required for optimal group 2
mGluR protein-ligand interaction, we designed bicyclic
amino acid 9 in which the glutamic acid skeleton is
incorporated into a fused bicyclo[3.1.0]hexane nucleus
(Figure 2). For compound 9, two low-energy conforma-
tions (envelope flap down and up, Figure 2) were
observed which differed by only 2.59 kcal/mol (Table 1).
For each of these conformers, the τ1 angle24 is close to
180° (166.9° for conformer A, 202° for conformer B) and
the τ2 angle24 is constrained at 156° in conformer A and
at 157° in conformer B. In close agreement with these
calculated values, the τ1 and τ2 angles observed in the
X-ray crystal structure of (+)-9 (Figure 3) were deter-
mined to be 171.38° and 150.60°, respectively.

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530 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Monn et al.
Sch em e 1
a
(a) Ethyl (dimethylsufuranylidene)acetate, benzene, 50 °C; (b) ethyl (dimethylsufuranylidene)acetate, toluene, room temperature; (c)
ethyl (dimethylsulfonium)acetate bromide, DBU, toluene, room temperature; (d) (NH4)2CO3, KCN, EtOH, H2O, 50 °C; (e) (NH4)2CO3,
KCN, EtOH, H2O, 35 °C; (f) NaOH, H2O, reflux; (g) EtOH, SOCl2, reflux; (h) NaOH, H2O, room temperature; (i) ion-exchange
chromatography.
Ch em istr y
related amino diethyl esters 14 and 15 (75:25 isolated
ratio), with 14 being isolated in 66% overall yield from
10. Alkaline hydrolysis of 14 followed by ion exchange
chromatography then yielded the target racemic amino
acid (()-9, while saponification of 15 afforded (()-16.
Optical resolution of the 12 was achieved as depicted
in Scheme 2. Alkaline hydrolysis of the C6-ester
functionality of 12 afforded carboxylic acid 17 in 95%
yield. Treatment of 17 with either (R)- or (S)-1-
phenylethylamine in acetone:H2O (1.6:1) at room tem-
perature followed by filtration afforded the diastereo-
merically pure salts 18 (from (R)-1-phenylethylamine)
and 19 (from (S)-1-phenylethylamine), which were
subsequently converted to the free carboxylic acids (-)-
17 and (+)-17, respectively.30 Alkaline hydrolysis of (-)-
17 afforded (+)-9, while acid hydrolysis of (+)-17
provided (-)-9. The structure of (+)-9 was confirmed
by single-crystal X-ray analysis (Figure 3).28
The preparation of (()-9 and its racemic C2-dia-
stereoisomer (()-16 is depicted in Scheme 1. Carboxy-
cyclopropanation of 2-cyclopentenone utilizing ethyl (di-
methylsulfuranylidene)acetate (EDSA)27 under stand-
ard conditions (80 °C, benzene) afforded a 69:31 ratio
of cycloadducts 10 and 11 in a combined yield of 64%
(41% isolated yield of 10). We found that the dia-
stereoselectivity of this reaction could be dramatically
enhanced (10:11 ) 97:3) by simply performing the
cycloaddition at room temperature either with isolated
EDSA as above or with EDSA generated in situ with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Thus, treat-
ment of ethyl dimethylsulfonium bromide with DBU in
toluene at room temperature followed by addition of
2-cyclopentenone afforded the highly enriched mixture
of diastereomers from which 10 was isolated in 68%
yield after chromatography. The structure of 10 was
confirmed by single-crystal X-ray analysis.28 Ketone 10
was then subjected to hydantoin formation under
Bucherer-Bergs conditions ((H4N)2CO3, KCN, EtOH,
H2O, 50-60 °C), yielding an inseparable mixture of 12
and 13 (78:22 ratio based on analytical reverse phase
HPLC analysis). Lowering the reaction temperature to
30-35 °C resulted in a significant enhancement in the
observed diastereoselectivity, affording a mixture of 12
and 13 (87:13 ratio). From this mixture 12 could be
isolated by selective crystallization in 73% yield.29
Alternatively, exhaustive hydrolysis of the 78:22 mix-
ture of 12 and 13 followed by esterification and chro-
matographic separation afforded the diastereomerically
Molecu la r Mod elin g
All molecular mechanics calculations and conforma-
tional searches were carried out under CHARMm 23.1
with a continuum dielectric constant of 80 and in the
fully protonated form. Charges were assigned using
CHARMm templates, smoothed over all atoms, and a
unit positive charge of +1 applied. Both 3 and 9 were
initially minimized and subjected to conformational
searches in order to determine the optimal orientations
of the polar functional groups. Protonated NH3+ groups
were used to avoid complicating symmetry problems
which would arise from using an unprotonated NH2

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mGluR Agonist Effects of LY354740
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 531
Sch em e 2
a
(a) NaOH, H2O, room temperature; (b) (R)-(+)-1-phenylethylamine, acetone, H2O; (c) (S)-(-)-1-phenylethylamine, acetone, H2O; (d)
HCl, H2O; (e) NaOH, H2O, reflux, then precipitation at pH ) 3; (f) 48% HBr, reflux, then precipitation at pH ) 3.
group. The conformational searches on 9 were 10°
incremental grid scans about the C2-C2′ and C6-C6′
axes (see Figure 2). The torsions were fixed at each
increment, and 250 steps of ABNR minimization were
performed. The open chain molecule 3 presented a more
complex conformational searching problem since the
range of conformations is increased by the freely rotat-
ing τ1 angle. In order to be as thorough as possible on
these searches, combinations of grid scans were exe-
cuted around the C1-C1′, C3-C3′ and C1-C2 bonds
(see Figure 1) using the same protocol as outlined for
9. From the best minima determined at this stage,
further one-bond conformational searches were carried
out around τ1 at 1° intervals with 250 steps ABNR
performed at each increment. These searches were
carried out in two ways, the first in which the the τ1
angle was held fixed at each 1° increment and the
second in which τ1 was allowed to fully relax from each
1° increment.
within 1 min following injection; behaviors lasted ap-
proximately 20 min.
Au tom a ted Eleva ted P lu s-Ma ze Assa y. Male NIH
Swiss mice (20-35 g) were obtained from Harlan
Sprague-Dawley (Cumberland, IN) and acclimated at
least 3 days before study onset. Mice were housed at
23 ( 2 °C (relative humidity 30-70%) and given food
and water ad libitum. The photoperiod was 12 h of light
and 12 h of dark, with dark onset at approximately 1800
h. All experiments were carried out in accordance with
USDA Animal Care and Use Guidelines. Construction
of the elevated plus-maze was based on a design
validated for mice.37 The entire maze was made of clear
Plexiglas. The maze possesses two open arms (30 × 5
× 0.25 cm) and two enclosed arms (30 × 5 × 15 cm).
The floor of each maze arm was corrugated to provide
texture. The arms extended from a central platform and
were angled at 90° from each other. The maze was
elevated to a height of 45 cm above the floor and
illuminated by red light. Individual infrared photocells
were mounted along each arm of the maze to monitor
closed arm, open arm, or nosepoke activity. Mice were
individually placed on the closed platform of the maze,
and the number of closed arm, open arm, and nosepoke
counts were recorded and used as a measure of arm
entries and time spent on various sections of the maze
over a 5-min test period. All compounds were admin-
istered to the mice orally 30 min before evaluation in
the maze. Elevated plus maze data were evaluated by
an analysis of variance followed by a Tukey’s standard-
ized range test for post hoc comparisons of group means.
Statistical analyses were performed using Statistical
Analysis Systems (SAS Institute, Inc., 1985).
P h a r m a cologica l Meth od s
Bioch em ist r y. Radioligand binding to NMDA,
AMPA, kainate, and metabotropic glutamate receptors
was performed utilizing [3H]CGS19755, [3H]AMPA,
[3H]kainate, and [3H]glutamate as the radioligands,
respectively.31-34 Measurement of cyclic adenosine
monophosphate (cAMP) and tritiated inositol mono-
phosphates (3H-IP) levels in rat cerebral cortical slices
was performed as previously described.16,35
Mou se Lim bic Seizu r e Assa y. Evaluation of the
effects of compound (+)-9 in NIH Swiss mice in the
presence and absence of 1 was performed as previously
described.36 Briefly, male NIH Swiss mice (20-25 g,
Charles River Labs., Inc., Portage, MI) were physically
restrained to allow unilateral intracerebral (ic) injec-
tions, which were made using a 10 µL Hamilton micro-
syringe. The entry site was 2 mm lateral from bregma,
with the syringe placed parallel to the midline angled
45° posterior and the injection was made to a depth of
3.7 mm. Animals were pretreated (20 min) with either
of sterile water (5 µL, ic), (+)-9, or (-)-9 at the doses
indicated. Animals were observed for 30 min following
administration of 1S,3R-ACPD. Limbic seizures in
treated mice were characterized by the presence of at
least one episode of clonic forelimb contractions followed
by hindlimb rearing to a praying stance, then loss of
balance, and falling. The onset of these behaviors was
Resu lts
In Vitr o EAA Ra d ioliga n d Bin d in g. Compounds
3 (Tocris Neuramin, Bristol, U.K.), (()-9, (+)-9, (-)-9,
and (()-16 were initially evaluated for their ability to
displace [3H]CGS19755 (10 nM),31 [3H]AMPA (5 nM),32
[3H]kainate (5 nM),33 and ACPD-sensitive [3H]glutamate
binding (10 nM)34 in rat forebrain membranes (Table
2). Compounds 3 and 9 (racemate and enantiomers)
were ineffective in displacing [3H]CGS19755, [3H]-
AMPA, or [3H]kainate binding at concentrations up to
100 µM. Compound 16 evinced some displacement of
[3H]CGS19755 binding, but only at high concentrations
(IC50 ) 62.4 µM). In contrast, 3, (()-9, and (+)-9

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532 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Monn et al.
Ta ble 2. Glutamate Receptor Radioligand Displacement and Second Messenger Responses Produced by Conformationally
Constrained Glutamic Acid Analogs
IC50 (µM)
EC50 (µM)
ACPD-sensitive
3H]glutamatea
inhibition of forskolin-
stimulation of
compd
[3H]CGS19755b
[3H]AMPAc
[3H]KAd
stimulated cAMPe
3H]IP formationf
3
0.454 ( 0.038
0.318 ( 0.091
>100
0.180 ( 0.052
>100
>100
>100
62.4g
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.1 ( 0.29
0.086 ( 0.025
NTh
0.055 ( 0.017
>300
25.4 ( 0.2
NTh
(()-9
(()-16
(+)-9
(-)-9
NTh
>300
NTh
a
b
d
g
See ref 34. See ref 31. c See ref 32. See ref 33. e See ref 16. f See ref 35. Single analysis (triplicate analysis at 1, 10, and 100 µM).
h
Not tested.
F igu r e 4. Comparison of 3 (open squares), (()-9 (open triangles), (+)-9 (closed circles), and (-)-9 (closed triangles) on forskolin
(30 µM)-stimulated cAMP formation in adult rat cerebral cortical slices (A) and on [3H]phosphoinositide hydrolysis in neonatal
rat cerebral cortical slices (B). *p < 0.05 when compared to the basal value.
potently displaced ACPD-sensitive [3H]glutamate bind-
ing, wtih IC50 values of 0.454 ( 0.038, 0.318 ( 0.091,
and 0.180 ( 0.052 µM, respectively (N ) 3). Compounds
16 and (-)-9 were each ineffective in displacing ACPD-
sensitive [3H]glutamate binding at concentrations up to
100 µM.
In Vitr o m Glu R F u n ction a l Resp on ses. Com-
pounds 3, (()-9, (+)-9 and (-)-9 were then evaluated
in adult rat cerebral cortical slices for their ability to
influence forskolin-stimulated cAMP formation (Figure
4A)16 or basal [3H]IP formation (compounds 3 and (+)-9
only, Figure 4B)35. As shown in Figure 4A, 3, (()-9, and
(+)-9 are effective in decreasing forskolin-stimulated
cAMP formation in the adult rat cerebral cortex, with
EC50 values of 1.1 ( 0.29, 0.086 ( 0.025,and 0.055 (
0.017 µM for 3, (()-9 and (+)-9, respectively. The
F igu r e 5. Anticonvulsant effect of (+)-9 on limbic seizures
agonist effect of (+)-9 is highly stereospecific, as its
enantiomer, (-)-9, evinced an agonist effect only at high
concentrations (EC50 ) 22 ( 3.4 µM) which may be
attributed to the presence of a small amount (ca. 0.25%)
of (+)-9 enantiomeric contamination. Moreover, while
3 is effective in stimulating the formation of [3H]IP
formation in the adult rat cerebral cortex (EC50 ) 25.4
( 0.2 µM), (+)-9 (up to 300 µM) does not stimulate PI
hydrolysis above basal levels in this tissue (Figure 4B).
These results are summarized in Table 2.
produced by 1 (400 nmol, ic) in NIH Swiss mice.36 Compound
(+)-9 was administered by either intraperatoneal (solid bar)
or oral (hashed bar) route to NIH Swiss mice 30 min prior to
the administration of 1.
decrease in the number of animals exhibiting limbic
seizures over the 15 min observation period (ED50 ) 17
mg/kg, ip, Figure 5, Table 3). The anticonvulsant effect
of (+)-9 in this assay was also observed following oral
administration (30 min pretreatment time, ED50 ) 45.6
mg/kg, po, Figure 5, Table 3). This effect was stereo-
specific, as (-)-9 (up to 100 mg/kg, ip) was ineffective
in blocking ACPD-induced limbic seizures (Table 3).
Eleva ted P lu s Ma ze. Oral administration of (+)-9
(0.3-10 mg/kg) resulted in dose-related increases in
open arm activity with significance at 1, 3, and 10 mg/
kg and an oral ED50 of 0.5 mg/kg (Figure 6, Table 3).
Closed arm activity counts were not significantly altered
Mou se Lim bic Seizu r e Mod el. When compound 1
is administered (400 nmol, ic) to NIH Swiss mice, a
characteristic limbic seizure is subsequently observed
which persists for the entire 20 min observation period
(Figure 5).36 Direct ic administration of (+)-9 (12.5-200
nmol, ic) did not result in limbic seizure activity on its
own. Intraperatoneal (ip) administration of (+)-9 (30
min prior to direct injection of 1) produced a dose-related

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mGluR Agonist Effects of LY354740
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 533
Ta ble 3. Effect of (+)-9 or (-)-9 in the Mouse Limbic Seizure
investigated (+)-9 in vivo so as to study the physiological
and behavioral consequences of activation of this class
of mGluRs.
Modela and in the Automated Elevated plus Maze Assayb
compd
assay
route
ED50 (mg/kg)
(+)-9
limbic seizure
limbic seizure
elevated plus maze
ip
po
po
17
45.6
0.5
A substantial body of evidence now exists which
suggests that a number of mGluRs, in particular
mGluR2, mGluR4, and mGluR7, are presynaptically
localized and function as inhibitory autoreceptors which
regulate neurotransmitter release in the CNS.5 In
support of the hypothesis that mGluR2 may serve in
this capacity, the mixed group 1 mGluR/group 2 mGluR
agonist 1 depresses excitatory neuronal transmission
in a number of brain regions by a presynaptic mecha-
nism.5 Previous studies have shown that direct intra-
thalamic administration of 1 to mice produces (by a
group 1 mGluR mechanism) limbic seizures that are
blocked by either direct or systemic administration of
selective group 2 mGluR agonists, such as 6.17 How-
ever, while 6 is an extremely useful tool for studying
group 2 mGluR function in vitro, its utility is limited
by its relatively low (µM) agonist potency. We thus
examined the more potent group 2 mGluR agonist (+)-9
for its effects on ACPD-induced seizures in mice. Like
6, systemic administration of (+)-9 produced a dose
dependent decrease in the number of mice exhibiting
limbic seizures in this model (ED50 ) 17 mg/kg, ip).
Importantly, the anticonvulsant activity of (+)-9 was
maintained following oral administration (ED50 ) 45.6
mg/kg, po). These data provide further evidence for the
lack of group 1 mGluR agonist (seizure-producing)
activity in (+)-9, and the anticonvulsant activity in (+)-9
is consistent with its selective effects on decreasing
cAMP formation (group 2 mGluR activity).
(-)-9
limbic seizure
elevated plus maze
ip
ip
>100
>100
a
b
See ref 36. See ref 37.
F igu r e 6. The effect of (+)-9 on automated elevated plus maze
following oral administration in NIH Swiss mice. Nosepoke
counts represent incomplete movements (head only) into the
open arm from the closed arm. Open arm counts represent
activity in the open (sideless) arms of the maze. Closed arm
counts represent activity in the enclosed arms of the maze.
(+)-9 was administered 30 min before testing on the maze.
Values represent mean ( SE of activity counts in each zone
during a 5 min test period. *Significantly different from control,
p < 0.05.
The elevated plus maze is a widely used test of
anxiety which has been validated for both rats and mice
and is sensitive to both anxiolytic and anxiogenic
drugs.37,40,41 The test procedure is based upon the
rodent’s natural aversion to heights and open spaces.
Clinically proven anxiolytics such as diazepam (Valium)
and buspirone (Buspar) are effective at increasing open
arm activity (reducing fear) in the elevated plus maze.41,42
However, all clinically available anxiolytics are re-
stricted in use by their adverse side effect profile,
dependence producing properties43,44 or limited ef-
ficacy.45,46 The benzodiazepines are postulated to pro-
duce their anxiolytic effects by suppression of excitatory
pathways within the limbic system.47 Agonists acting
at group 2 or group 3 mGluRs have been shown to
attenuate excitatory synaptic transmission within cer-
tain limbic pathways including the amygdala,48 as well
as the perforant49 and mossey fiber50 pathways of the
hippocampus. The ability of (+)-9 to suppress ACPD-
induced limbic seizures is consistent with such a mech-
anism. With this in mind, we profiled the effects of
(+)-9 on the murine automated evaluated plus maze
model of anxiety. When examined for its effects in this
assay, (+)-9 (0.3-10 mg/kg, po) was highly efficacious
in increasing open arm activity while not affecting
closed arm activity in mice (ED50 ) 0.5 mg/kg, po),
indicative of anxiolytic effects. This degree of oral
efficacy compares favorably with that observed for
diazepam.41 Furthermore, the lack of effect of (+)-9 at
any dose on closed arm activity suggests that this
compound is neither enhancing nor diminishing loco-
motor activity at these doses. The effect of (+)-9 was
stereospecific, as the mGluR inactive enantiomer (-)-9
at any dose of (+)-9. The anxiolytic effect of (+)-9 was
stereospecific, as (-)-9 (10 mg/kg) had no effect on open
or closed arm activity (Table 3).
Discu ssion
As part of our ongoing program aimed at delineating
the structural and conformational aspects of agonist and
antagonist interactions with excitatory amino acid
receptors, we designed and prepared the conformation-
ally constrained glutamic acid analog (()-9 and its
constituent enantiomers (+)-9 and (-)-9 in which the
two torsion angles (τ1 and τ2) which define the geometric
relationship between the R-amino acid and distal car-
boxylic acid functionalities are constrained in an ex-
tended arrangement. Evaluation of the ability of these
analogs to displace radioligand binding from iGluRs and
mGluRs demonstrated that (()-9 potently displaces
ACPD-sensitive [3H]glutamate binding (an indication of
group 2 mGluR affinity)38,39 from rat forebrain homo-
genates and that this activity is evinced exclusively by
the (+)-enantiomer. Furthermore, (+)-9 is devoid of
binding affinity for the glutamate recognition site on
the NMDA, AMPA, and kainate receptors. In rat
cerebral cortical slices, both (()-9 and (+)-9 potently
activate mGluRs negatively coupled to AC, while dem-
onstrating no agonist activity for stimulation of PI
hydrolysis in this tissue. Thus, as a result of restricting
the conformation of glutamic acid by its incorporation
into the bicyclo[3.1.0]hexane framework, we have si-
multaneously achieved a high degree of potency and
selectivity for (+)-9 at mGluRs negatively coupled to AC,
presumably group 2 mGluRs.39 We have therefore

+
+
534 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Monn et al.
(CH3), 22.49 (CH2), 26.53 (CH), 29.24 (CH), 31.93 (CH2), 35.81
(CH), 61.28 (CH2), 170.47 (CO2Et), 211.80 (CdO). Anal. Calcd
for C9H12O3: C, H. 11: FDMS M+ ) 168; 1H NMR (CDCl3) δ
1.30 (t, J ) 7 Hz, 3H), 2.05-2.15 (m, 1H), 2.20-2.50 (m, 6H),
4.15 (q, J ) 7 Hz, 2 H); 13C NMR (CDCl3) δ 14.50 (CH3), 20.35
(CH2), 28.99 (CH), 30.24 (CH), 34.56 (CH), 38.30 (CH2), 61.52
(CH2), 170.18 (CO2Et), 213.78 (CdO). Anal. (C9H12O3) C, H.
(1SR,5RS,6SR)-Eth yl 2-Oxobicyclo[3.1.0]h exa n e-6-ca r -
boxyla te (10) by in Situ Gen er a tion of EDSA. A suspen-
sion of ethyl (dimethylsulfonio)acetate bromide (45.5 g, 198.6
mmol)27 in toluene (350 mL) was stirred at room temperature
as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added in one
portion. The resulting mixture was stirred at room temper-
ature for 1 h. 2-Cyclopenten-1-one (19.57 g, 238 mmol) was
then added, and the reaction mixture was allowed to stir at
room temperature overnight. The mixture was poured into a
suspension of 1 N HCl in brine, extracted with Et2O, dried
over MgSO4, and concentrated under reduced pressure. Capil-
liary GC analysis revealed the presence of 10 and 11 in a ratio
of 98:2. Chromatography (HPLC: 10% EtOAc/hexanes to 50%
EtOAc/hexanes) afforded pure cycloadduct 10 which was
identical in every respect to that which was isolated in the
preceding example.
(1SR,2SR,5RS,6SR)-Diet h yl 2-Am in ob icyclo[3.1.0]-
h exa n e-2,6-d ica r boxyla te (14) a n d (1SR,2RS,5RS,6SR)-
Dieth yl 2-Am in obicyclo[3.1.0]h exa n e-2,6-d ica r boxyla te
(15). To an ethanolic solution (200 mL) of 10 (22.81 g, 135.6
mmol) was added an aqueous solution (200 mL) of KCN (9.71
g, 149.2 mmol) and (H4N)2CO3 (21.2 g, 271.2 mmol) at room
temperature. The reaction mixture was then stirred at 50 °C
overnight to produce a mixture of 12 and 13. The reaction
mixture was cooled to room temperature, NaOH (16.2 g, 407
mmol) was added, and the resulting solution was refluxed for
48 h. The reaction mixture was then cooled to 0 °C, acidified
to pH 1 with concentrated HCl, and concentrated to dryness.
The residue was reconstituted in EtOH and treated at 0 °C
with thionyl chloride (80.6 g, 678 mmol), and the resulting
reaction mixture was refluxed for 48 h. After the reaction
mixture was concentrated to dryness under reduced pressure,
the products were partitioned between 1 N NaOH and Et2O.
The organic phase was separated, and the aqueous one was
extracted with Et2O (3 × 100 mL). The combined organic
phases were dried over K2CO3 and concentrated under reduced
pressure to afford the mixture of 14 and 15 (24.65 g, 102 mmol,
75:25 GC ratio) in 75% combined yield from 10.
was ineffective in producing an effect on either open or
closed arm activity at doses up to 10 mg/kg (data not
shown). These data support the hypothesis that sup-
pression of excitatory neurotransmission in limbic struc-
tures in the brain, possibly through the inhibition of
excitatory neurotransmitter release, can produce anxio-
lytic effects in rodents. Further evaluation of this
important pharmacological tool is currently underway
and will be reported in due course.
Su m m a r y
Compound 9 was designed as a constrained glutamic
acid analog which closely mimics the proposed bioactive
conformation of this neurotransmitter when acting at
group 2 mGluRs. This hypothesis has been supported
by the observation that (()-9 and (+)-9 are exceptionally
potent agonists at group 2 mGluRs, possessing no
activity at other glutamate receptors at concentrations
several orders of magnitude greater than that required
to suppress forskolin-stimulated cAMP formation in rat
cerebral cortical slices. Importantly, the mGluR agonist
effects of (+)-9 are stereospecific and apparent following
oral administration in mice in the elevated plus maze
model of anxiety and in the ACPD-induced limbic
seizure assay. Thus, (+)-9 is the first orally active group
2 mGluR agonist described thus far and may be an
important tool for studying the effects of compounds of
this class in humans.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were obtained using
a Thomas-Hoover capilliary melting point apparatus and are
uncorrected. Analytical gas chromatography (GC) was per-
formed on a Hewlett-Packard 5890 series II gas chromato-
graph utillizing an Ultra 2 (cross-linked 5% Ph Me silicone)
capilliary column (25 m × 0.32 mm × 0.52 µm film thickness).
1H and 13C NMR spectra were obtained at 300.15 and 75.48
MHz, respectively, with TMS as an internal standard. Field
desorption mass spectroscopy (FDMS) was performed using
either a VG 70SE or Varian MAT 731 instrument. Optical
rotations were obtained using the Perkin-Elmer 241 polarim-
eter and are reported at the sodium D line (589 nm), unless
otherwise noted. 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. Preparative centrifugal thin
layer chromatography (PC-TLC) was performed on a Harrison
Model 7924A chromatotron using Analtech silica gel GF rotors.
The solvent system employed is indicated in the particular
example. Cation-exchange chromatography was performed
with Dowex 50 × 8-100 ion-exchange resin and anion-
exchange chromatography with Bio-Rad AG 1-X8 anion-
exchange resin (acetate form converted to hydroxide form).
Chiral HPLC was performed on a Chriobotic 4.6 × 250 mm
column utilizing 20% THF/0.1% TFA/water (0.6 mL/min) as
the eluent system at a solvent flow rate of 0.6 mL/min.
Detection was performed at λ ) 230 nm.
(1SR,2SR,5RS,6SR)-Diet h yl 2-Am in ob icyclo[3.1.0]-
h exa n e-2,6-d ica r boxyla te (14). To a stirred solution of the
preceding mixture (20.71 g, 85.8 mmol) in EtOAc (200 mL)
was added a solution of oxalic acid (15.46 g, 171.7 mmol) in
EtOH (50 mL). The resulting reaction mixture was stirred at
room temperature for 1 h. Additional EtOH (50 mL) was then
added, and the mixture was allowed to stir at room temper-
ature overnight. The resulting salt (5.8 g, 17.5 mmol) was
removed by filtration. The filtrate, which was now enriched
to a 10:1 ratio (GC) favoring diastereomer 14, was concentrated
to dryness under reduced pressure, partitioned with 1 N
NaOH, extracted with Et2O, washed with brine, dried over
K2CO3, and concentrated. Chromatography (HPLC: CH2Cl2/
5% NH4OH:MeOH, 97:3) afforded 14 (13.48 g, 55.9 mmol) in
1
66% isolated yield: FDMS M+ (+H) ) 242; H NMR (CDCl3)
δ 1.10-1.21 (m, 1H), 1.26 (t, J ) 7 Hz, 3H), 1.34 (t, J ) 7 Hz,
3H), 1.75 (t, J ) 2 Hz, 1H), 1.80-2.20 (m, 7H), 4.12 (q, J ) 7
Hz, 2H), 4.21 (q, J ) 7 Hz, 2H); 13C NMR (CDCl3) δ 14.17
(CH3), 14.23 (CH3), 21.17 (CH), 27.23 (CH2), 29.23 (CH), 34.07
(CH2), 37.24 (CH), 60.59 (CH2), 61.38 (CH2), 65.64 (C), 173.04
(CdO), 175.61 (CdO). Anal. (C12H19NO4) C, H, N.
(1SR,5RS,6SR)-Eth yl 2-Oxobicyclo[3.1.0]h exa n e-6-ca r -
boxyla te (10) a n d (1SR,5RS,6RS)-Eth yl 2-Oxobicyclo-
[3.1.0]h exa n e-6-ca r boxyla te (11). The procedure of Payne27
was followed. A solution of 2-cyclopentenone (19.0 g, 231.9
mmol) and ethyl (dimethylsufuranylidene)acetate (17.2 g,
115.9 mmol) in benzene (150 mL) was heated at 50 °C for 6 h
and then allowed to stir at room temperature overnight. The
reaction mixture (GC: 69:31 ratio of 10:11) was concentrated
to dryness and then subjected to preparative HPLC (10%
EtOAc in hexane eluent), affording 10 (7.95 g, 47.3 mmol) in
41% yield and 11 (1.76 g, 10.5 mmol) in 9.0% yield. 10:
(1SR,2RS,5RS,6SR)-Diet h yl 2-Am in ob icyclo[3.1.0]-
h exa n e-2,6-d ica r boxyla te (15). The oxalic acid salt from
above (GC: 63.4:36.6 ratio of 15:14) was converted to the
corresponding free base and subjected to preparative chroma-
tography (HPLC: CH2Cl2/5% NH4OH:MeOH, 97:3) to yield an
additional 1.93 g (8.0 mmol) of 14 (total yield of 14 including
that obtained above ) 15.41 g, 63.9 mmol, 74.4%) and 3.65 g
1
1
crystallized on standing, mp 42-44 °C; FDMS M+ ) 168; H
(15.1 mmol) of 15 (17.6%): FDMS M+ (+H) ) 242; H NMR
NMR (CDCl3) δ 1.30 (t, J ) 7 Hz, 3H), 2.0-2.4 (m, 6H), 2.48-
(CDCl3) δ 1.06 (t, J ) 7 Hz, 3H), 1.08 (t, J ) 7 H, 3H), 1.31
(dd, J ) 13.7, 8.2 Hz, 1H),1.53-1.65 (m 3H), 1.71-1.78 (m,
2.55 (m, 1H), 4.15 (q, J ) 7 Hz, 2 H); 13C NMR (CDCl3) δ 14.19

+
+
mGluR Agonist Effects of LY354740
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 535
3H), 1.83-1.93 (m, 2H), 3.88 (q, J ) 7 Hz, 2H), 3.93-4.04 (m,
2H); 13C NMR (CDCl3) δ 14.01 (CH3), 14.08 (CH3), 22.27 (CH),
25.70 (CH2), 27.13 (CH), 32.96 (CH2), 37.43 (CH), 60.38 (CH2),
61.18 (CH2), 64.84 (C), 172.64 (CdO), 174.96 (CdO). Anal.
(C12H19NO4) C, H, N.
allowed to cool slowly to room temperature. The product
crystallized almost immediately. The reaction mixture was
stirred at room temperature for several hours and filtered. The
cake was washed with acetone (20 mL) and dried to afford 19
(7.64 g, 23.1 mmol) in 46% yield: mp 218-224 °C; H NMR
(DMSO-d6) δ 1.30 (m, 4H), 1.69 (m, 5H), 1.93 (m, 1H), 4.09 (q,
1
(1SR,2SR,5RS,6SR)-2-Am in obicyclo[3.1.0]h exa n e-2,6-
d ica r boxyla te [(()-9]. The diethyl ester 14 (3.1 g, 12.8
mmol) was stirred at room temperature in a 1:1 mixture of 2
N NaOH/THF (50 mL total volume) overnight. The THF was
removed under reduced pressure, and the subsequent aqueous
solution was adjusted to pH 11. Ion exchange chromatography
(Bio-Rad AG1-X8 anion-exchange: elute with 50% HOAc-
H2O) afforded (()-9 (2.12 g, 11.4 mmol) in 89% yield: mp >250
°C dec; FDMS M+ (+H) ) 186; 1H NMR (D2O + KOD) δ 1.1-
1.35 (m, 1H), 1.55 (m, 1H), 1.8-2.15 (m, 5H); 13C NMR (D2O
+ KOD) δ 24.71 (CH), 27.39 (CH2), 28.73 (CH), 32.90 (CH2),
34.70 (CH), 67.61 (C), 180.00 (CdO), 181.99 (CdO). Anal.
(C8H11NO4) C, H, N.
1H), 6.70 (s, 2H, NH2), 7.28 (m, 5H), 7.94 (s, 1H, NH); [R]D
)
32.73° (c ) 1.02, H2O) Anal. (C17H21N3O4‚1/2H2O) C, H, N.
(-)-Bicyclo[3.1.0]h exa n e-6-ca r b oxyla t e-2-sp ir o-5′-h y-
d a n toin [(-)-17]. A mixture of 18 (0.74 g, 2.2 mmol) and
water (10 mL) was stirred at 25 °C while the pH was adjusted
to 1.0 using 1 N HCl. The reaction mixture was stirred for 1
h, and the product was collected by filtration and dried to give
(-)-17 (0.35 g, 1.7 mmol) in 76% yield: mp 310 °C dec; FDMS
1
M+ ) 210; H NMR (DMSO-d6) δ 1.28 (m, 1H), 1.68 (m, 2H),
1.85 (m, 3H), 7.91 (s, 1H, NH), 10.54 (s, 1H, NH), 12.16 (s,
1H, CO2H); [R]D ) -24.22° (c ) 1.0, MeOH). Anal. (C9H10N2O4)
C, H, N. Chiral HPLC (see general methods section) indicated
that the product was 99.9% ee (retention time ) 9.61 min)
(+)-Bicyclo[3.1.0]h exa n e-6-ca r b oxyla t e-2-sp ir o-5′-h y-
d a n toin [(+)-17]. A mixture of 19 (5.10 g, 15.4 mmol) and
water (51 mL) was stirred at room temperature while the pH
was adjusted to 1.0 using 1 N HCl. The reaction mixture was
stirred for 30 min. The product was collected by filtration and
dried to give (+)-17 (2.55 g, 12.1 mmol) in 79% yield: mp 310
°C dec; 1H NMR (DMSO-d6) δ 1.28 (m, 1H), 1.68 (m, 2H), 1.85
(m, 3H), 7.91 (s, 1H, NH), 10.54 (s, 1H, NH), 12.16 (s, 1H,
CO2H); [R]D ) 25.25° (c ) 1.01, MeOH). Anal. (C9H10N2O4)
C, H, N. Chiral HPLC (see general methods section) indicated
that the product was 99.9% ee (retention time ) 8.95 min).
(+)-2-Am in obicyclo[3.1.0]h exa n e-2,6-d ica r boxylic Acid
[(+)-9]. A mixture consisting of (-)-17 (184 g, 876.2 mmol)
and 3 N NaOH (1750 mL) was heated at reflux until the
reaction was judged complete by HPLC. After 28 h, the
solution was cooled to room temperature and filtered through
glass paper to remove trace amounts of insoluble material. The
pH of the solution was adjusted to 3.0 using concentrated HCl.
The reaction mixture was stirred 1 h at room temperature and
2 h at 0 °C. The precipitated product was collected by
filtration, washed with cold water (170 mL), and dried to afford
(+)-9 (152.5 g, 750.5 mmol) in 86% yield: mp 271 °C; FDMS
M+ (+H) ) 186; 1H NMR (trifluoroacetic acid-d) δ 1.75 (m,
1H), 2.13 (m, 2H), 2.40 (m, 3H), 2.57 (m, 1H); 13C NMR (D2O
+ KOD) δ 24.82 (CH), 27.57 (CH2), 29.29 (CH), 31.66 (CH2),
(1SR,2RS,5RS,6SR)-2-Am in obicyclo[3.1.0]h exa n e-2,6-
d ica r boxyla te (16). The diethyl ester 15 (1.0 g, 4.1 mmol)
was stirred at room temperature in a 1:1 mixture of 1 N NaOH/
THF (30 mL total volume) overnight. The THF was removed
under reduced pressure, and the subsequent aqueous solution
was adjusted to pH 11. Ion exchange chromatography (Bio-
Rad AG1-X8 anion-exchange: elute with 3 N AcOH) afforded
(()-16 (0.66 g, 3.6 mmol) in 87% yield, mp >260 °C dec; FDMS
1
M+(+H) ) 186; H NMR (D2O + KOD) δ 1.35-1.45 (m, 2H),
1.60-1.75 (m, 3H), 1.80-2.00 (m, 2H); 13C NMR (D2O + KOD)
δ 24.61 (CH), 25.02 (CH2), 25.52 (CH), 31.73 (CH2), 36.53 (CH),
65.99 (C), 176.52 (CdO), 180.80 (CdO). Anal. (C8H11NO4) C,
H, N.
(1SR,2SR,5RS,6SR)-Eth yl Bicyclo[3.1.0]h exa n e-6-ca r -
boxyla te-2-sp ir o-5′-h yd a n toin (12). A mixture of 10 (5.05
g, 30.0 mmol), KCN (2.15 g, 33.0 mmol), and (NH4)2CO3 (5.77
g, 73.9 mmol) in a mixture of EtOH (30 mL) and H2O (12 mL)
was stirred at 35 °C for 15 h. The reaction mixture was cooled
to 0 °C, and H2O (33 mL) was added to the mixture. After 2
h at 0 °C, the precipitate was isolated by filtration and dried
to give 12 (5.23 g, 22.0 mmol) in 73% yield: mp 219-221 °C;
FDMS M+ ) 238; 1H NMR (DMSO-d6) δ 1.14 (t, J ) 7 Hz,
3H), 1.30 (m, 1H), 1.82 (m, 5H), 1.97 (m, 1H), 4.02 (q, J ) 7
Hz, 2H), 7.89 (s, 1H, NH) 10.57 (s, 1H, NH); 13C NMR (DMSO-
d6) δ 14.01 (CH3), 20.02 (CH), 25.58 (CH2), 27.37 (CH), 29.45
(CH2), 32.95 (CH), 60.15 (CH2), 68.61 (C), 156.14 (CdO), 171.75
(CdO), 177.43 (CdO). Anal. (C11H14N2O4) C, H, N.
(1SR,2SR,5RS,6SR)-Bicyclo[3.1.0]h exan e-6-car boxylate-
2-sp ir o-5′-h yd a n toin (17). A mixture of 12 (16.32 g, 68.6
mmol) and 2 N NaOH (137 mL) was stirred at room temper-
ature for 1 h. Concentrated HCl was added to adjust the pH
to 1.0. The resulting precipitate was isolated by filtration and
dried to give 17 (13.70 g, 65.2 mmol) in 95% yield: mp 278-
280 °C; FDMS M+ ) 210; 1H NMR (DMSO-d6) δ 1.28 (m, 1H),
1.68 (m, 2H), 1.85 (m, 3H), 7.91 (s, 1H, NH), 10.54 (s, 1H, NH),
12.16 (s, 1H, CO2H); 13C NMR (DMSO-d6) δ 20.10 (CH), 25.59
(CH2), 27.01 (CH), 29.55 (CH2), 32.71 (CH), 68.62 (C), 156.15
(CdO), 173.35 (CdO), 177.50 (CdO). Anal. (C9H10N2O4) C,
H, N.
(R)-(+)-1-P h en yleth yla m in e Sa lt (18). Compound 17
(10.51 g, 50.0 mmol), acetone (102 mL), and water (64 mL)
were combined and heated to 55 °C. (R)-(+)-1-phenylethyl-
amine (6.06 g, 50.0 mmol) was added quickly over several
minutes. The heating mantle was removed, and the solution
was allowed to cool slowly to room temperature. The product
crystallized almost immediately. The reaction was stirred at
room temperature for several hours and filtered. The cake
was washed with acetone (20 mL) and dried to give 18 (7.62
g, 23.0 mmol) in 46% yield: mp 211-217 °C; 1H NMR (DMSO-
d6) δ 1.25 (m, 1H) 1.27 (d, 3H), 1.72 (m, 5H), 1.93 (m, 1H),
4.05 (q, 1H), 7.26 (m, 5H), 7.60 (s, 2H, NH2), 7.93 (s, 1H, NH);
[R]D ) -27.00° (c ) 1.01, H2O). Anal. (C17H21N3O4‚1/2H2O)
C, H, N.
33.38 (CH), 67.40 (C), 177.13 (CdO), 180.96 (CdO); [R]D
)
23.18° (c ) 1.0, 1 N HCl). Anal. (C8H11NO4‚H2O) C, H, N.
(-)-2-Am in obicyclo[3.1.0]h exa n e-2,6-d ica r boxylic Acid
[(-)-9]. A mixture of (+)-17 (1.05 g, 5.0 mmol) and 48% HBr
(25 mL) was heated for 48 h at reflux. The reaction was
filtered while hot through diatomaceous earth. Upon the
filtrate being cooled to room temperature a crystalline product
formed and was collected by filtration. The solid was dissolved
in water (7 mL) and the pH adjusted to 3.0 using 5 N NaOH.
The product which precipitated was stirred for 1 h at a pH of
3.0 and then collected by filtration and dried to give (-)-9 (0.52
g, 2.6 mmol) in 51% yield: mp 277 °C; FDMS M+ (+H) ) 186;
1H NMR (trifluoroacetic acid-d) δ 1.75 (m, 1H), 2.13 (m, 2H),
2.40 (m, 3H), 2.57 (m, 1H); 13C NMR (D2O + KOD) δ 25.11
(CH), 27.60 (CH2), 29.13 (CH), 32.02 (CH2), 33.62 (CH), 67.40
(C), 177.85 (CdO), 181.56 (CdO); [R]D ) -22.08° (c ) 1.01, 1
N HCl). Anal. (C8H11NO4‚H2O) C, H, N.
Ack n ow led gm en t. The authors would like to thank
Mr. J ack Deeter, Ms. J ennifer Wilkie, and Dr. Gregory
Stephenson for the X-ray crystal structures of (+)-9 and
10, Mr. J on Paschal and Mr. Larry Spangle for NMR
data acquisition and interpretation, Mr. J oe Kennedy
for chiral HPLC analyses, and Deah L. Modlin, Kenneth
D. Firman, and Vanessa J . Benagh for their excellent
technical assistance.
(S)-(-)-1-P h en yleth yla m in e Sa lt (19). Compound 17
(10.51 g, 0.05 mol), acetone (102 mL), and water (64 mL) were
combined and heated to 55 °C. (S)-(-)-1-Phenylethylamine
(6.06 g, 50.0 mmol) was added quickly over several minutes.
The heating mantle was removed, and the solution was
Su p p or tin g In for m a tion Ava ila ble: Combustion analy-
ses for all numbered compounds and X-ray crystallographic
data pertaining to compound (+)-9 (6 pages). Ordering
information is given on any current masthead page.

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+
536 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Monn et al.
(23) Constantino, G.; Natalini, B.; Pellicciari, R. Definition of a
pharmacophore for the metabotropic glutamate receptors nega-
tively linked to adenylyl cyclase. Bioorg. Med. Chem. 1993, 1,
259-265.
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(1) Colinridge, G. L.; Lester, R. A. Excitatory amino acid receptors
in the vertebrate central nervous system. Pharmacol. Rev. 1989,
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+
+
mGluR Agonist Effects of LY354740
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