Synthesis and pharmacological characterization of all sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl)glycine. Focus on (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, a novel and selective group II metabotropic glutamate receptors antagonist

Article abstract of DOI:10.1021/jm960059+

All 16 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCGs) stereoisomers 32-47 have been prepared from the corresponding racemic aldehydes 12-15 following an enantiodivergent synthetic protocol. Compounds 32-47 were evaluated by a number of binding and functional experiments as potential ligands for several classes of excitatory amino acid receptors, including metabotropic glutamate receptors (mGluR1a, mGluR2, mGluR4) and ionotropic glutamate receptors (NMDA, KA, AMPA) as well as sodium-dependent and calcium/chloride-dependent glutamate transport systems. The stereolibrary of compounds 32-47 appears to be endowed with a peculiar pharmacological profile. PCCG-2 (33) and PCCG-3 (34) displaced labeled kainate at low micromolar concentration; PCCG-9 (40) and PCCG-11 (42) weakly interacted with the NMDA site; PCCG-5 (36), PCCG-10 (41), and PCCG-12 (43) showed to be potent inhibitors of Ca²⁺/Cl^--dependent glutamate transport system. Most interestingly, PCCG-4(35) has been shown to be able to antagonize (IC₅₀ = 8 μM) the effects of glutamate on forskolin-stimulated cAMP formation in BHK cells expressing mGluR2. Uneffective at mGluR1, 35 is a weak mGluR4 agonist (EC₅₀ = 156 μM) and has no effect on either ionotropic receptors or glutamate transport systems, thus demonstrating to be a novel selective mGluR2 antagonist with a 6-fold increase in potency over previously reported antagonists.

Full text of DOI:10.1021/jm960059+

J . Med. Chem. 1996, 39, 2259-2269
2259
Syn th esis a n d P h a r m a cologica l Ch a r a cter iza tion of All Sixteen Ster eoisom er s
of 2-(2′-Ca r boxy-3′-p h en ylcyclop r op yl)glycin e. F ocu s on
(2S,1′S,2′S,3′R)-2-(2′-Ca r boxy-3′-p h en ylcyclop r op yl)glycin e, a Novel a n d Selective
Gr ou p II Meta botr op ic Glu ta m a te Recep tor s An ta gon ist
Roberto Pellicciari,*,† Maura Marinozzi,† Benedetto Natalini,† Gabriele Costantino,† Roberto Luneia,†
Gianluca Giorgi,‡ Flavio Moroni,§ and Christian Thomsen∇
Istituto di Chimica e Tecnologia del Farmaco, Universita` di Perugia, Via del Liceo 1, 06123 Perugia, Italy,
Centro Interdipartimentale di Analisi e Determinazioni Strutturali, Universita` di Siena, Via P. A. Mattioli 10,
53100 Siena, Italy, Dipartimento di Farmacologia Preclinica e Clinica, Universita` di Firenze, Viale G. B. Morgagni 65,
50134 Firenze, Italy, and Health Care Discovery, Novo Nordisk A/ S, Novo Nordisk Park, DK-2760 Ma˚løv, Denmark
Received J anuary 17, 1996X
All 16 2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCGs) stereoisomers 32-47 have been
prepared from the corresponding racemic aldehydes 12-15 following an enantiodivergent
synthetic protocol. Compounds 32-47 were evaluated by a number of binding and functional
experiments as potential ligands for several classes of excitatory amino acid receptors, including
metabotropic glutamate receptors (mGluR1a, mGluR2, mGluR4) and ionotropic glutamate
receptors (NMDA, KA, AMPA) as well as sodium-dependent and calcium/chloride-dependent
glutamate transport systems. The stereolibrary of compounds 32-47 appears to be endowed
with a peculiar pharmacological profile. PCCG-2 (33) and PCCG-3 (34) displaced labeled
kainate at low micromolar concentration; PCCG-9 (40) and PCCG-11 (42) weakly interacted
with the NMDA site; PCCG-5 (36), PCCG-10 (41), and PCCG-12 (43) showed to be potent
inhibitors of Ca2+/Cl--dependent glutamate transport system. Most interestingly, PCCG-4 (35)
has been shown to be able to antagonize (IC50 ) 8 µM) the effects of glutamate on forskolin-
stimulated cAMP formation in BHK cells expressing mGluR2. Uneffective at mGluR1, 35 is
a weak mGluR4 agonist (EC50 ) 156 µM) and has no effect on either ionotropic receptors or
glutamate transport systems, thus demonstrating to be a novel selective mGluR2 antagonist
with a 6-fold increase in potency over previously reported antagonists.
Ch a r t 1. Classification of Cloned Metabotropic
Glutamate Receptors
In tr od u ction
The continuing quest for selective ligands acting at
excitatory amino acid (EAA) receptors is fostered by the
important role that these receptors play in areas such
as development plasticity, learning, and neurodegen-
erative and acute diseases and by the constant dis-
closure of new receptor subtypes whose pharmacological
and physiological characterization is a key step on route
to the discovery of clinically useful agents.1 Synaptically
released glutamic acid (L-Glu) interacts with a variety
of proteins, including ionotropic receptors, metabotropic
receptors, transport systems, and metabolizing en-
zymes. The recent years have seen, in particular, a
growing interest in the field of metabotropic glutamate
receptor (mGluRs) family,2 due to the intriguing thera-
peutic opportunities offered by the modulation of its
members. Indeed, mGluRs have been shown to play
important roles in the induction of long term potentia-
tion (LTP) or long term depression (LTD) of synaptic
transmission,3 regulation of the baroceptive reflex,4
spatial learning,5 preprogramming of rapid movements,
motor learning, and the process of postural-kinetic
integration6 as well as in the pathogenesis of either
acute (e.g., ischemia, epilepsy, hipoxia) or chronic (e.g.,
Alzheimer’s disease, Parkinsonism, AIDS-related de-
mentia) diseases. After the independent discovery of
the first mGluR by Sladeczeck et al. in 1985,7 and by
Nicoletti et al. in 1986,8 the multiplicity of this class
has been disclosed by expression cloning studies.9 Cur-
rently, eight mGluRs (and several splice variants) have
been isolated and subdivided in three groups according
to sequence homology, signal transduction, and phar-
macology as indicated in Chart 1. Briefly, the first
group includes mGluR1 and mGluR5 which are coupled
† Universita` di Perugia.
‡ Universita` di Siena.
§ Universita` di Firenze.
∇ Novo Nordisk A/S.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00059-3 CCC: $12.00 © 1996 American Chemical Society

2260 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Pellicciari et al.
Ch a r t 2
proved useful for mapping the presence of still unex-
plored accessory areas in the recognition site of mem-
bers of the glutamate receptor family.
While previous knowledge of conformational require-
ments of ligands acting at various members of EAA17,20,21
receptors could have addressed the synthetic efforts
toward the preparation of 2-(2′-carboxy-3′-phenylcyclo-
propyl)glycines (PCCGs) with definite stereochemistry,
expected to interact with particular members of EAA
receptors, we decided to follow an enantiodivergent
synthetic protocol with the aim of producing a complete
stereolibrary of (phenylcarboxycyclopropyl)glycines. The
availability of this set of compounds will be useful for
either a better pharmacological definition of already
known or the characterization of still unknown members
of the metabotropic glutamate receptor family and,
evidently, also for further characterization of ionotropic
receptors and glutamate transport systems.
to IP3/Ca2+ signal transduction via activation of phos-
pholipase C (PLC), whereas the members of group II,
mGluR2 and mGluR3, as well as those of group III,
mGluR4, mGluR6 mGluR7, and mGluR8, are negatively
linked to the activity of adenylyl cyclase, thus resulting
in the inhibition of cAMP accumulation.
Several classes of glutamate analogs have been used
in the past as chemical probes for characterizing EAA
receptors: among them, (carboxycyclopropyl)glycines
(CCGs) have represented a valuable source of potent
and selective ligands for the various members of the
glutamate receptor family, including ionotropic recep-
tors, metabotropic receptors, and uptake carrier pro-
teins. The introduction of a cyclopropyl moiety on the
glutamate skeleton induces chirality, partially reduces
the conformational flexibility thus allowing its selective
interaction with a reduced number of recognition sites,
and, most importantly, defines foreseeable orientations
of the ω-carboxylate group with respect to the R-amino
acidic moiety, thus allowing the assessment of the
conformational requirements of L-Glu acting at its
receptor subtypes. Thus, among the eight possible CCG
diastereoisomers, we first reported that (2S,1′R,2′S)-
(carboxycyclopropyl)glycine [L-CGAC (CCG IV), 1]10 is
a potent and selective NMDA agonist, while the corre-
sponding isomers (2S,1′R,2′R)-CCG II (2) and
(2S,1′S,2′R)-CCG III (3) have been reported to be L-Glu
uptake inhibitors11 (Chart 2). In 1990, Shinozaki’s
group first described the (2S,1′S,2′S)-CCG isomer (CCG
I, 4)12 as a potent and rather selective group II mGluRs
agonist. 1,2,3-Trisubstituted CCG derivatives have also
been evaluated as ligands for glutamatergic pathways.
Among the derivatives of this class so far reported,
(2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine
(DCG IV, 5)13 has revealed to be a particularly interest-
ing compound, being a potent mGluR2 agonist endowed
with neuroprotective properties,14 also active as an
agonist at the NMDA receptor site. Attempts to in-
crease the selectivity of these molecules toward metabo-
tropic receptors have resulted in the substitution of the
3′-carboxy group of 5 with a methoxymethyl group thus
leading to (2S,1′R,2′R,3′R)-[2-carboxy-3-(methoxymeth-
yl)cyclopropyl]glycine (cis-MCG I, 6),15 also a group II
mGluRs agonist, characterized by lower activity and
increased selectivity over ionotropic receptors with
respect to 5. Interestingly, trans-MCG (7), an epimer
of 6 and 5 at C-3′, is also a group II mGluRs agonist,
with the same order of activity of 5.16 In view of the
limited exploitation of trisubstituted CCGs as potential
EAA receptor ligands and as a continuation of our
research in the field of EAA receptors and in particular
in the metabotropic one,17 we reasoned that the intro-
duction of a hydrophobic moiety, such as a phenyl ring,
in position 3′ of 2-(2′-carboxycyclopropyl)glycine can be
Ch em istr y
The problem of synthesizing all 16 (phenylcarboxy-
cyclopropyl)glycine stereoisomers 32-47 consisted of
two different tasks. The first one, the synthesis of the
four racemic aldehydes 12-15 chosen as precursors for
further enantiodivergent synthetic elaborations, was
accomplished essentially by utilizing the synthetic
protocol developed by Martin et al.,22 involving, as a key
step, the intramolecular cyclopropanation of an allylic
diazo ester precursor.23 By this route, the enantio-
selective synthesis of (+)- and (-)-13 and (+)- and (-)-
14 has recently been reported.24 As racemates, alde-
hydes (()-13 and (()-14 have been previously reported
by the same author as uncharacterized intermediates,22
while (()-12 and (()-15 are still unreported. Briefly,
the first step is the bis(N-tert-butylsalicylaldimine)-
copper(II)-catalyzed25 decomposition in refluxing toluene
of (Z)-cinnamoyl diazo ester 8a to give the racemic endo-
lactone 9a (87% yield), which was then converted26 into
the corresponding hydroxymethyl morpholine amide
10a in 90% yield (Scheme 1). Selective inversion of the
configuration at C-1 of alcohol 10a as previously de-
scribed22,24 affords the isomeric alcohol 11, which, jointly
with 10a , was oxidized (PCC, CH2Cl2, room tempera-
ture) to give aldehydes (()-13 and (()-12, respectively.
The third aldehyde, (()-14, was quantitatively obtained
by methanolic potassium carbonate-induced isomeriza-
tion at C-2 of aldehyde (()-13. Aldehyde (()-15, finally,
was synthesized starting from (E)-cinnamoyl diazo ester
8b 23 which was submitted to bis(N-tert-butylsalicyl-
aldimine)copper(II)-catalyzed25 decomposition to give
exo-lactone 9b (96% yield), which was then converted
into aldehyde (()-15 by sequential conversion into the
corresponding morpholine amide 10b (99% yield) and
oxidation (PCC, CH2Cl2, room temperature).
With all four racemic aldehydes 12-15 in hand, the
enantiodivergent transformation of each one of them
into the corresponding four possible enantiopure PCCGs
was addressed. The key step in the route (Schemes 2
and 3) was a diastereoselective Strecker synthesis
involving the nucleophilic addition of a cyanide ion to
the Schiff bases formed by condensing each racemic
aldehyde (12-15) with optically active R-phenylglyci-
nol.27 Since two new contiguous chiral centers are
present, each aldehyde was expected to yield the four
corresponding diastereoisomeric (2R)- and (2S)-N-

Stereoisomers of PCCG
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2261
Sch em e 1a
Sch em e 2a
a
(a) Cu(TBS)2, PhMe, reflux; (b) morpholine, AlMe3, CH2Cl2,
reflux; (c) Li-HMDS, THF, room temperature; (d) PCC, CH2Cl2,
room temperature; (e) K2CO3, MeOH, room temperature.
substituted R-amino nitrile derivatives in varied amounts
according to the configuration of the R-phenylglycinol
employed.27 (R)- And (S)-R-phenylglycinol preferentially
induce opposite chirality in the newly formed asym-
metric center. Accordingly, treatment of racemic alde-
hyde 12 with (S)-R-phenylglycinol in MeOH at room
temperature for 2 h, followed by reaction of the Schiff
base with TMSCN for 12 h at room temperature, yielded
a mixture of the expected four R-amino nitriles, as two
major and two minor components in ca. 8:2 ratio. The
diastereoisomeric content of each isomeric pair was ca.
1:1 (GC-MS). Medium pressure chromatography (MPC)
of the reaction mixture allowed only the separation of
the two more abundant constituents. From (S)-R-
phenylglycinol these were identified as (2R)-[(S)-(phen-
ylglycinyl)amino] nitrile 16 and (2R)-[(S)-(phenylglyci-
nyl)amino nitrile 17 in 30% and 15% yields, respectively
(for the absolute configuration assignment, see the
following section). To isolate the two remaining (2S)-
R-amino nitriles, aldehyde (()-12 was again submitted
to the diastereoselective Strecker synthesis utilizing (R)-
R-phenylglycinol for the formation of the Schiff base.
Similarly, the reaction mixture containing the four
corresponding R-amino nitriles was submitted to MPC
affording (2S)-[(R)-(phenylglycinyl)amino] nitrile 18 and
(2S)-[(R)-(phenylglycinyl)amino] nitrile 19 in 21% and
32% yields, respectively.
a
(a) i. (S)-R-Phenylglycinol, TMSCN, MeOH, room tempera-
ture, 14 h, ii. MPC; (b) i. (R)-R-phenylglycinol, TMSCN, MeOH,
room temperature, 14 h, ii. MPC; (c) i. Pb(OAc)4, MeOH-CH2Cl2,
ii. 6 N HCl, reflux, iii. Dowex 50WX2-200.
then applied to the three remaining racemic aldehydes
13-15, thus obtaining: from aldehyde (()-13,
(2R,1′R,2′S,3′S)-PCCG-5 (36), (2R,1′S,2′R,3′R)-PCCG-6
(37), (2S,1′S,2′R,3′R)-PCCG-7 (38), and (2S,1′R,2′S,3′S)-
PCCG-8 (39) in 48%, 53%, 48%, and 55% yields,
respectively; from aldehyde (()-14, (2R,1′S,2′S,3′S)-
PCCG-9
(40),
(2R,1′R,2′R,3′R)-PCCG-10
(41),
(2S,1′R,2′R,3′R)-PCCG-11 (42), and (2S,1′S,2′S,3′S)-
PCCG-12 (43) in 62%, 62%, 75%, and 63% yields,
respectively; and from aldehyde (()-15, (2R,1′S,2′R,3′S)-
PCCG-13 (44), (2R,1′R,2′S,3′R)-PCCG-14 (45),
(2S,1′R,2′S,3′R)-PCCG-15 (46), and (2S,1′S,2′R,3′S)-
PCCG-16 (47) in 49%, 75%, 50%, and 55% yields,
respectively.
The two (2R)-R-amino nitriles 16 and 17 and the two
(2S)-R-amino nitriles 18 and 19 thus obtained were then
submitted to oxidative cleavage with lead tetraacetate,28
acidic hydrolysis (6 N HCl), and ion exchange chroma-
tography on Dowex 50X2-200 to afford respectively
(2R,1′S,2′S,3′R)-PCCG-1 (32), (2R,1′R,2′R,3′S)-PCCG-2
(33), (2S,1′R,2′R,3′S)-PCCG-3 (34), and (2S,1′S,2′S,3′R)-
PCCG-4 (35) with 86%, 74%, 70%, and 73% yields.
Absolu te Con figu r a tion Assign m en t
The absolute configuration assignment to the 16
diastereoisomeric amino acids 32-47 was based upon
the single-crystal X-ray analysis performed on suitably
selected compounds chosen among the N-substituted
R-amino nitriles or the corresponding final amino acids
derived from the three racemic aldehydes (()-12, (()-
13, and (()-15, depending on the quality of their
The
synthetic
protocol
above
describeds
diastereoselective Strecker synthesis with both (S)- and
(R)-R-phenylglycinol, oxidative cleavage followed by
acidic hydrolysis of the corresponding imine, and puri-
fication by ion exchange resin chromatographyswas

2262 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Pellicciari et al.
Sch em e 3a
F igu r e 2. SHELXTL41 drawing of compound 19. The non-H
atom ellipsoids enclose 50% probability.
F igu r e 3. SHELXTL41 drawing of compound 44. The non-H
atom ellipsoids enclose 50% probability.
(S) at the R-amino nitrilic carbon and opposite
(1′R,2′R,3′S) at the three cyclopropylic carbons. More-
over, a comparison of the specific rotatory powers of 18
and 19 with those of the two N-substituted R-amino
nitriles obtained from the reaction with (S)-phenylgly-
cinol (16 and 17) revealed the corresponding enantio-
meric couples.
The full stereochemical characterization of the com-
pounds derived from aldehyde (()-13 was obtained by
submitting to X-ray analysis the hydrobromic salt of 39
(Figure 2). Also in this case, the S-chirality of the
R-amino acidic center was confirmed, and the analysis
of the specific rotatory power of the four amino acids
20-23 disclosed the enantiomeric couples.
a
(a) i. (S)-R-Phenylglycinol, TMSCN, MeOH, room tempera-
ture, 14 h, ii. MPC; (b) i. (R)-R-phenylglycinol, TMSCN, MeOH,
room temperature, 14 h, ii. MPC; (c) i. Pb(OAc)4, MeOH-CH2Cl2,
ii. 6 N HCl, reflux, iii. Dowex 50WX2-200.
Analogously, the absolute stereochemistry of the
compounds derived from aldehyde (()-15 was obtained
by submitting to X-ray analysis the hydrobromic salt
of 44 (Figure 3). In this case, the R-chirality of the
R-amino acidic center was confirmed, and the analysis
of the specific rotatory power of the four corresponding
amino acids 44-47 revealed the enantiomeric couples.
In the impossibility to obtain suitable crystals for
X-ray analysis with compounds derived from aldehyde
(()-14, we decided to synthesize the chiral aldehyde (+)-
14 [(1R,2R,3R)-1-(4-morpholinylcarbonyl)-2-formyl-3-
phenylcyclopropane] by following Martin’s procedure24
in order to obtain, from the subsequent diastereoselec-
tive Strecker synthesis, only two R-amino nitriles.
According to the above considerations on the inductive
F igu r e 1. SHELXTL41 drawing of compound 39. The non-H
atom ellipsoids enclose 50% probability.
crystals, and on the independent, enantioselective syn-
thesis of R-amino nitrile 26 which disclosed the absolute
stereochemistry of the four R-amino nitriles derived
from aldehyde (()-14. Thus, the N-substituted R-amino
nitrile 19 was selected among the compounds derived
from aldehyde (()-12 and submitted to X-ray analysis
which confirmed the S-chirality of the R-amino nitrilic
center and allowed to fix the absolute configuration at
the three cyclopropylic carbon atoms [(1′S,2′S,3′R),
Figure 1]. Conversely, the N-substituted R-amino ni-
trile 18, proceeding from the same racemic aldehyde 12,
resulted endowed with the same absolute configuration
effect of (R)-R-phenylglycinol,
a more abundant
(2S,1′R,2′R,3′R)-N-[(R)-R-phenylglycinyl]-2-[2′-(4-mor-
pholinylcarbonyl)-3′-phenylcyclopropyl]glycinonitrile was
obtained. A comparison of the spectroscopic data as well
as the specific rotatory power values of this compound

Stereoisomers of PCCG
Ta ble 1. Glutamate Receptor Selectivity of the Stereoisomers of 2-(2′-Carboxy-3′-phenylcyclopropyl)glycine (32-47)a
glutamate receptor binding Ki (µM) glutamate uptake IC50 (µM)
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2263
AMPA
NMDA
KA
mGluR1
mGluR4a
Na+-dep
Ca2+/Cl--dep
PCCG-1 (32)
PCCG-2 (33)
PCCG-3 (34)
PCCG-4 (35)
PCCG-5 (36)
PCCG-6 (37)
PCCG-7 (38)
PCCG-8 (39)
PCCG-9 (40)
PCCG-10 (41)
PCCG-11 (42)
PCCG-12 (43)
PCCG-13 (44)
PCCG-14 (45)
PCCG-15 (46)
PCCG-16 (47)
212 ( 32
125 ( 15
124 ( 25
>220
>220
251 ( 24
>220
>220
>220
>300
>220
>220
>220
193 ( 38
129 ( 23
107 ( 15
>190
>270
>280
>280
>280
50 ( 6
>280
>280
>280
>280
>280
>280
>280
>280
>280
>280
>280
>280
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
15 ( 3
25 ( 3
135 ( 21
60 ( 15
>270
>300
41 ( 7
>300
>200
>190
>200
>200
>200
>200
29 ( 5
>200
25 ( 5
192 ( 21
>200
123 ( 24
>200
>200
117 ( 14
122 ( 10
155 ( 25
>270
44 ( 3
>270
32 ( 6
143 ( 23
270 ( 38
233 ( 29
138 ( 19
23 ( 2
85 ( 11
7 ( 2
>190
260 ( 31
>270
>190
>190
>190
>270
205 ( 29
>270
126 ( 18
>190
155 ( 22
144 ( 13
221 ( 29
>270
>300
160 ( 30
141 ( 24
285 ( 41
>270
64 ( 5
213 ( 45
185 ( 35
>270
>270
a
The values (mean ( SEM) are the inhibitory constants (Ki) for displacement of radioligand binding or potencies (IC50) for inhibiting
uptake from rat cortical homogenates. IC50 values were calculated from at least three data points which were fitted to a sigmoidal curve
by a nonlinear regression analysis using the GraphPad Prism program (ISI, Philadelphia). Competition binding experiments were
performed as described in the Experimental Section, and Ki values were calculated from at least two individual experiments performed
in triplicate using the equation Ki ) IC50/(1 + [L])/Kd, where [L] is the concentration of the radioligand.31,46 In experiments (n ) 2 in
triplicate) measuring inhibition of [3H]glutamate uptake (sodium-dependent glutamate uptake) or [3H]-L-AP4 (calcium/chloride-dependent
glutamate uptake) into rat cortical synaptosomes, nonspecific uptake was determined by the uptake in the presence of 1 mM glutamate.
Ch a r t 3
with those derived from R-amino nitriles 26 and 27
enabled us to assign to nitrile 26 the same absolute
configuration of the above enantioselectively synthe-
sized R-amino nitrile.
Biologica l Resu lts
The pharmacological profiles of the 16 stereoisomers
of 2-(2′-carboxy-3′-phenylcyclopropyl)glycine (32-47)
were examined by evaluating (1) the inhibition of the
binding of selective ligands for AMPA, NMDA, and
kainate receptors to rat brain membranes,29 (2) the
inhibition of the binding of labeled glutamate to mem-
branes prepared from baby hamster kidney (BHK) cells
expressing mGluR1a30 or of the binding of labeled L-AP4
to membranes obtained from cells expressing mGluR4,31
(3) the inhibition of the sodium-dependent glutamate
transport into synaptosomes32 and the inhibition of the
calcium/chloride-dependent glutamate uptake,31 and (4)
the antagonism of glutamate effects on PI hydrolysis
or forskolin-stimulated cAMP formation33 in BHK cells
expressing mGluR1a, mGluR2, or mGluR4.
antagonists such as 1-aminoindan-1,5-dicarboxylic acid
(UPF 523, 48)18 or (S)-(4-carboxyphenyl)glycine (S-
4CPG, 49) (Chart 3).30,34
One of the stereoisomers we considered interesting
for the goals of the present study, was PCCG-4 (35)
which was not able to interact with the ionotropic
glutamate receptors or the uptake sites (see Table 1)
but which was able to antagonize, with an IC50 of 8 µM
(see Table 2), the effects of glutamate on forskolin-
induced cAMP formation in BHK cells expressing
mGluR2. 35 was also a weak agonist of mGluR4a (EC50
) 156 µM) but was ineffective at mGluR1a (Tables 1
and 2) and mGluR5a.35 This compound seems therefore
quite selective for mGluRs negatively coupled to aden-
ylate cyclase and in particular for mGluR2. Currently,
the most potent mGluR2 antagonist is (+)-R-methyl-(4-
carboxyphenyl)glycine [(+)-M4CPG, 50] (IC50 ) 50 µM),
which is not selective because it has a similar affinity
for mGluR1a.34 Thus, 35 is about 6-fold more potent
than 50 and much more selective since it does not
interact with mGluR1a or mGluR5.
As shown in Table 1, some of the 16 stereoisomers
displaced the selective ligands from the ionotropic
glutamate receptors. Thus, low micromolar concentra-
tions of 33 and 34 displaced labeled kainate, but while
33 was a relatively selective displacer, 34 also interacted
with mGluR1 and the Ca2+/Cl--dependent glutamate
uptake sites. Similarly, 40 and 42 interacted with the
NMDA recognition sites and the Ca2+/Cl--dependent
uptake sites. Concentrations of up to 300 µM of the 16
stereoisomers did not significantly reduce the sodium-
dependent glutamate uptake. On the contrary, the
Ca2+/Cl--dependent glutamate uptake was significantly
reduced by low micromolar concentrations of 43, 41, and
36. (2S,1′S,2′S,3′S)-PCCG-12 (43) in particular, with
an IC50 of 7 µM, is the most potent and selective
inhibitor of this class so far reported. As far as the
mGluRs are concerned, Tables 1 and 2 show that 37,
with an IC50 of 140 µM, had an acceptable affinity for
mGluR1a, and functional measurements revealed that
37 was an antagonist at this receptor subtype. How-
ever, 37 was less potent and selective than other mGluR
Table 2 shows also that larger concentrations of 35
(EC50 ) 156 µM) activate mGluR4, thus indicating that
the affinity of the compound toward this receptor is ca.
1 order of magnitude lower than that toward mGluR2.
We have no information on whether or not 35 interacts
with mGluR3 receptors. However cloned mGluR2 and

2264 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Pellicciari et al.
Ta ble 2. Summary of the Potencies for Subtypes mGluR1a,
mGluR2, and mGluR4 of the Stereoisomers of
2-(2′-Carboxy-3′-phenylcyclopropyl)glycine (32-47)a
mGluR1a
mGluR2
EC50 IC50 EC50
(µM) (µM) (µM)
>300 >300 >300
mGluR4
IC50
(µM)
IC50
(µM)
EC50
(µM)
PCCG-1 (32) >300
>300 >300
122 ( 8 >300 >300
PCCG-2 (33) >300
PCCG-3 (34) >300
PCCG-4 (35) >300
PCCG-5 (36) >300
PCCG-6 (37) 140 ( 11
PCCG-7 (38) >300
PCCG-8 (39) >300
PCCG-9 (40) >300
PCCG-10 (41) >300
PCCG-11 (42) >300
PCCG-12 (43) >300
PCCG-13 (44) >300
PCCG-14 (45) >300
PCCG-15 (46) >300
PCCG-16 (47) >300
>300
>300 >300 >300
>300 8 ( 2
>300 >300
156 ( 25
>300 >300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
>300 >300 >300
F igu r e 4. Solution, NMR-derived, and molecular mechanics
preferred conformation of PCCG-4 (35).
a
The concentrations for half-maximal inhibition (IC50) or
stimulation (EC50) of PI hydrolysis (mGluR1a) or cAMP formation
(mGluR2 and mGluR4) were determined from two to four experi-
ments performed in triplicate. Compounds were tested for agonist
and antagonist activity as described previously.31
mGluR3 are considered to possess a similar pharmacol-
ogy,36 and it is certainly possible that 35 does not
discriminate between these group II mGluR subtypes.
It is also not certain whether 35 is able to interact with
the other group III mGluR subtypes (mGluR6, mGluR7,
and mGluR8).
Figu r e 5. Proposed pharmacophoric requirements for mGluR2
recognition site.
mGluR2 antagonist, can be used to gain some light on
the pharmacophore requirements for antagonist at
mGluR2 receptor subtype. First of all, the equivalence
of spatial disposition of R-amino acidic moiety and
ω-carboxylate group between 35 and 4 (or 5) implies
that there exists a unique recognition site on mGluR2
where both agonists and antagonists bind. The antago-
nist profile is then given by the phenyl group of 35 that
is likely to occupy an accessory site on the binding
pocket. When superimposed, both the phenyl ring of
35 and the methoxymethyl group of 6 showed to occupy
the same region of space. The observation that 6 retains
the agonist profile of 4 (with lower potency) may be
explained by assuming that the methoxymethyl group
simply enters the pocket without making additional
bonds and without occupying accessory areas respon-
sible for antagonist activity. On the other hand, the 3′
epimerization of 6 yields 7 which displays a higher
potency as an agonist.16 The corresponding epimer on
our PCCGs series is completely inactive, thus suggesting
that in the case of 7, the methoxymethyl moiety
interacts (probably via hydrogen bond) with the binding
site but larger substituents cannot be accommodated.
This allows for a preliminary mapping of steric
requirements of mGluR2 binding site (Figure 5), being
assumed the full extended conformation as the bioactive
one. The extended disposition of R-amino acidic moiety
and ω-carboxylate group defines the spatial require-
ments for the mGluR2 binding site. A bulky group trans
to the ω-carboxylate can occupy an accessory region on
the receptor site and confers the antagonist profile. A
hydrogen-bonding acceptor group, such as methoxy-
methyl, cis to the ω-carboxylate increases the agonist
potency and confers improved selectivity toward other
mGluR subtypes. Nevertheless, it should be mentioned
that other structures than (carboxycyclopropyl)glycines
Discu ssion
The results of the testing of the 16 diastereoisomeric
2-(2′-carboxy-3′-phenylcyclopropyl)glycines 32-47 for
binding affinity and functional activity at ionotropic and
metabotropic EAA receptors and for glutamate uptake
inhibition have indicated that some of them possess an
interesting pharmacological profile. Among the active
compounds, we have chosen (2S,1′S,2′S,3′R)-PCCG-4
(35), the most potent and selective group II mGluRs
antagonist so far reported, to carry out a research study
aimed at evaluating the structure and conformational
features underlying its pharmacological properties. As
a general consideration, the introduction of the phenyl
ring into the (carboxycyclopropyl)glycine core reduces
the already limited conformational freedom of the
molecule. In fact, a Monte Carlo random search analy-
sis performed on 35 gave only four stable conformers
in comparison with the eight obtained for the parent
compound L-CCG I (4). The most stable conformer is
characterized by a 178° torsional angle around the
H-C(2)-C(1′)-H rotatable bond. This is consistent
with the X-ray structure of the parent R-amino nitrile
derivative 19, and it is also worth noticing that this
conformation is the preferred one in solution, as indi-
cated by the H-C(2)-C(1′)-H proton vicinal coupling
constant of 11.2 Hz which, within the limitation of the
modified Karplus-Altona equation,37 corresponds to a
torsional angle of 172° (Figure 4). This preferred
conformation matches the full extended disposition of
R-amino acidic moiety and ω-carboxylate group that has
been proposed by us17 and others21 as the bioactive
conformation of L-Glu when acting at group II mGluRs.38
The structural features displayed by 35, together with
its interesting profile of the first relatively selective

Stereoisomers of PCCG
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2265
J ) 0.6, 9.8 Hz, 4-CHa), 4.35 (1H, dt, J ) 2.7, 9.8 Hz, 4-CHb),
7.20-7.35 (5H, m, aromatics).
have been reported to have both agonist or antagonist
profile at mGluR2, although with lower potency, namely,
(carboxyphenyl)glycines 49-52.34 For example, (S)-(4-
carboxy-3-hydroxyphenyl)glycine [(S)-4C3HPG, 51] is
1-(4-Mo r p h o lin y lc a r b o n y l)-2-(h y d r o x y m e t h y l)-3-
p h en ylcyclop r op a n e (10a ). A 2.0 M solution of trimethyl-
aluminum in hexane (22.35 mL) was added dropwise in 20
min to a solution of morpholine (3.9 g, 44.8 mmol) in dry
CH2Cl2 (108 mL) magnetically stirred under argon at room
temperature. Stirring was continued for 20 min after which
a solution of 9a (2.59 g, 14.88 mmol) in dry CH2Cl2 (67 mL)
was added dropwise in 20 min, and the resulting mixture was
then heated at 40 °C for 20 h. The reaction mixture was
carefully acidified with 1 N HCl, the organic phase was
separated, and the aqueous layer was extracted with CH2Cl2
(2 × 60 mL). The combined organic phases were dried over
anhydrous Na2SO4, and after evaporation of the solvent, the
residue (3.7 g) was submitted to flash chromatography; elution
with CH2Cl2-MeOH (95:5) gave 10a (3.50 g, 90%): mp 102-3
reported to be an agonist at mGluR2 with an EC50
)
30 µM, while 50 is an antagonist with an IC50 ) 50 µM.
Since there is apparently no way to superimpose (car-
boxyphenyl)glycines to (carboxycyclopropyl)glycines ei-
ther in their proposed bioactive conformations or in
otherwise energetically accessible conformations, one
should argue that there exist at least two binding sites
on mGluR2 receptors.
Con clu sion
1
This study reports the synthesis and preliminary
pharmacological characterization of a stereolibrary of
conformationally constrained glutamate analogs, useful
probes for studies on the structural requirements of
glutamate binding to carriers and ionotropic or metabo-
tropic receptors. Low micromolar concentrations of
several of the above-mentioned compounds were able
to interact with the NMDA (40 and 42) or kainate (33
and 34) receptors or with the Ca2+/Cl--dependent
glutamate uptake sites (43). While studies on the
structural profile and the precise pharmacological char-
acterization of these compounds are in progress, we
report here that PCCG-4 (35) is a potent and relatively
selective group II mGluRs antagonist having an IC50 of
8 µM. Although larger concentrations of 35 interact also
with mGluR4, where the compound behaves as an
agonist (EC50 ) 156 µM), its lack of activity on the
uptake sites, ionotropic receptors, and mGluRs linked
to the IP3/Ca2+ transduction pathways should facilitate
the studies on the role that group II mGluRs have in
physiology or pathology.
°C; H-NMR (CDCl3) δ 1.95 (1H, m, 2-CH), 2.15 (1H, t, J )
9.4 Hz, 1-CH), 2.60 (1H, t, J ) 9.4 Hz, 3-CH), 3.15 (1H, m,
CHaOH), 3.40-4.00 (9H, m, morpholine ring, CHbOH), 4.20
(1H, br, OH), 7.15-7.40 (5H, m, aromatics).
Ep im er iza tion of th e Am id ic F u n ction of 10a (11). A
solution of 10a (3.40 g, 13.03 mmol) in dry THF (200 mL) was
added dropwise in 30 min to a magnetically stirred solution
of lithium hexamethyl disilazide [from addition of 1.5 M
butyllithium in hexane (26 mL) to a solution of dry 1,1,1,3,3,3-
hexamethyldisilazane (7.0 g, 43.5 mmol) in THF (200 mL)]
kept under argon at room temperature. Stirring was contin-
ued for 1 h after which the reaction mixture was diluted with
saturated NH4Cl (500 mL) and extracted with CH2Cl2 (3 ×
200 mL). The combined organic extracts were then dried
(Na2SO4) and evaporated to give a residue (3.4 g) which was
submitted to flash chromatography; elution with CH2Cl2-
MeOH (95:5) yielded 11 (3.00 g, 88%): 1H-NMR (CDCl3) δ 2.05
(2H, m, 2-CH, OH), 2.25 (1H, t, J ) 5.0 Hz, 1-CH), 2.85 (1H,
dd, J ) 5.0, 12.0 Hz, 3-CH), 3.35 (1H, dd, J ) 6.7, 12 Hz,
CHaOH) 3.50-3.90 (9H, m, morpholine ring, CHbOH), 7.15-
7.40 (5H, m, aromatics).
1-(4-Mor p h olin ylca r bon yl)-2-for m yl-3-p h en ylcyclop r o-
p a n e (12). PCC (4.20 g, 19.48 mmol) was added to a solution
of 11 (3.00 g, 11.49 mmol) in dry CH2Cl2 (130 mL), and the
resulting mixture was stirred at room temperature in an argon
atmosphere for 16 h. The reaction mixture was then diluted
with Et2O and filtered and the solvent evaporated off. Flash
chromatography of the residue (2.5 g) and elution with AcOEt-
light petroleum (8:2) afforded 12 (1.90 g, 64%): mp 89-91 °C;
1H-NMR (CDCl3) δ 2.85 (1H, m, 2-CH), 3.12 (1H, t, J ) 6.4
Hz, 1-CH), 3.35 (1H, dd, J ) 6.4, 9.4 Hz, 3-CH), 3.50-3.90
(8H, m, morpholine ring), 7.18-7.40 (5H, m, aromatics), 9.20
(1H, d, J ) 5.1 Hz, CHO).
1-(4-Mor p h olin ylca r bon yl)-2-for m yl-3-p h en ylcyclop r o-
p a n e (13). PCC (4.76 g, 22.08 mmol) was added to a solution
of 10a (3.40 g, 13.03 mmol) in dry CH2Cl2 (130 mL), and the
resulting mixture was stirred at room temperature in an argon
atmosphere for 48 h. The reaction mixture was then diluted
with Et2O and filtered and the solvent evaporated off. Flash
chromatography of the residue (3 g) and elution with AcOEt-
light petroleum (6:4) afforded 13 (2.20 g, 65%): mp 155-7 °C;
1H-NMR (CDCl3) δ 2.30 (1H, m, 2-CH), 2.72 (1H, t, J ) 9.0
Hz, 1-CH), 3.05 (1H, t, J ) 9.0 Hz, 3-CH), 3.45-3.90 (8H, m,
morpholine ring), 7.20-7.40 (5H, m, aromatics), 9.60 (1H, d,
J ) 8.1 Hz, CHO).
1-(4-Mor p h olin ylca r bon yl)-2-for m yl-3-p h en ylcyclop r o-
p a n e (14). K2CO3 (1.51 g, 10.96 mmol) was aded to a solution
of 13 (0.710 g, 2.74 mmol) in MeOH (30 mL, degassed with
nitrogen for 30 min), and the resulting suspension was stirred
under argon at room temperature for 24 h. The reaction
mixture was then diluted with saturated NH4Cl (20 mL),
extracted with Et2O (4 × 30 mL), and dried (Na2SO4).
Evaporation of the solvent yielded the aldehyde 14 (0.690 g,
97%): 1H-NMR (CDCl3) δ 2.70-2.90 (2H, m, 1-CH, 2-CH), 2.99
(1H, dd, J ) 5.6, 9.1 Hz, 3-CH), 3.10-3.80 (8H, m, morpholine
ring), 7.10-7.40 (5H, m, aromatics), 9.90 (1H, d, J ) 2.7 Hz,
CHO).
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined by the
capillary method on a Bu¨chi 535 electrothermal apparatus and
are uncorrected. 1H- and 13C-NMR spectra were taken on a
Bruker AC 200 spectrometer as solutions in CDCl3 unless
otherwise indicated. Carbon magnetic resonance of the final
amino acids was performed on the corresponding hydrochloric
salts. Proton chemical shifts are reported in ppm downfield
from tetramethylsilane, except with D2O which was also used
as an internal standard. Carbon chemical shifts are reported
in ppm using MeOH as an internal standard (δ 49.0). The
abbreviations used are as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; dd, double doublet; td, triple
doublet; br s, broad signal. GC-MS was performed on a
Hewlett-Packard HP 5890 gas chromatograph (column and
conditions: HP-1, 12 m, 0.20 mm i.d., 0.33 µm ft, 150(1′)/280
°C, 10 °C/min) equipped with a mass detector (HP 5971).
Flash chromatography was performed on Merck silica gel
(0.040-0.063 mm). Medium pressure chromatography (MPC)
was performed on Merck LiChroprep Si 60 and LiChroprep
RP-8 (reversed phase) lobar columns. Specific rotations were
recorded on a J asco Dip-360 digital polarimeter.
en d o-6-P h en yl-3-oxa bicyclo[3.1.0]h exa n -2-on e (9a ). A
solution of cis-3-phenyl-2-propen-1-yl diazoacetate22 (8a ; 1.00
g, 4.95 mmol) in dry toluene (165 mL) was added via syringe
pump to a refluxing solution of bis(N-tert-butylsalicylald-
iminato)copper(II) (0.104 g, 0.25 mmol) in dry toluene (165 mL)
magnetically stirred under argon over a period of 12 h. After
cooling, the reaction mixture was evaporated to dryness and
the residue submitted to flash chromatography; elution with
light petroleum containing 10-40% AcOEt afforded the lactone
9a (0.75 g, 87%): mp 112-3 °C; 1H-NMR (CDCl3) δ 2.60 (2H,
m, 1-CH, 5-CH), 2.78 (1H, t, J ) 8.8 Hz, 6-CH), 4.05 (1H, dd,
tr a n s-3-P h en yl-2-p r op en -1-yl Dia zoa ceta te (8b). p-
Tolylsulfonylhydrazone of glyoxylic acid chloride (22.8 g, 87.7

2266 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Pellicciari et al.
mmol) was added to a cold (0 °C) solution of (E)-cinnamic
alcohol (10.0 g, 74.6 mmol) in anhydrous CH2Cl2 (380 mL).
N,N-Dimethylaniline (10.17 g, 84.1 mmol) was then added, and
the resulting mixture was stirred at 0 °C for 15 min, after
which Et3N (39.2 g, 388 mmol) was added dropwise in 40 min.
After addition was complete, stirring was continued for 15 min
at 0 °C and for 3.5 h at room temperature. The reaction
mixture was then diluted with water (300 mL), the organic
phase was separated, and the aqueous layer was extracted
with CH2Cl2 (2 × 60 mL). The combined organic phases were
dried (Na2SO4), and after evaporation of the solvent, the
residue was submitted to flash chromatography; elution with
light petroleum-AcOEt (95:5) afforded 8b (14.5 g, 96%): IR
(2S,1′S,2′S,3′R)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(19): 32% yield as a white solid; mp 145-8 °C; [R]20 -158°
D
(c 0.80, CH2Cl2).
(2R,1′R,2′S,3′S)-N-[(S)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′p h en ylcyclop r op yl]glycin on it r ile
(20): AcOEt-light petroleum (8:2); 21% yield as an oil; 1H-
NMR (CDCl3) δ 1.75 (1H, q, J ) 10 Hz, 1′-CH), 2.12 (1H, br s,
OH), 2.35 (1H, t, J ) 10 Hz, 2′-CH), 2.58 (1H, t, J ) 10 Hz,
3′-CH), 3.25-3.80 (11H, m, morpholine, CH2OH, CHCH2OH),
4.30 (1H, d, J ) 10.5 Hz, 2-CH), 7.20-7.40 (10H, m, aromatics);
13C-NMR (CDCl3) δ 22.95, 26.93, 27.66, 41.98, 45.77, 46.10,
62.78, 65.64, 66.54, 120.60, 127.21, 127.35, 127.75, 128.46,
(film) 2110, 1700 cm-1 1H-NMR (CDCl3) δ 4.80 (3H, d, J )
;
128.72, 134.23, 140.42, 167.11; [R]20 +105° (c 0.82, CH2Cl2).
D
6.4 Hz, CHN2, CH2), 6.25 (1H, dt, J ) 16.0, 6.5 Hz, 2-CH),
6.65 (1H, d, J ) 16.0 Hz, 3-CH), 7.35 (5H, m, aromatics); 13C-
NMR (CDCl3) δ 45.73, 64.82, 122.84, 126.22, 127.67, 128.19,
133.76, 135.79, 166.06.
exo-6-P h en yl-3-oxa bicyclo[3.1.0]h exa n -2-on e (9b): pre-
pared as for 9a starting from 8b with 81% yield; mp 93-4 °C;
1H-NMR (CDCl3) δ 2.30 (2H, m, 1-CH, 5-CH), 2.50 (1H, m,
6-CH), 4.45 (2H, m, 4-CH2), 7.00-7.40 (5H, m, aromatics).
1-(4-Mo r p h o lin y lc a r b o n y l)-2-(h y d r o x y m e t h y l)-3-
p h en ylcyclop r op a n e (10b ): prepared as for 10a starting
from 9b with 99% yield; 1H-NMR (CDCl3) δ 2.00 (2H, m, 1-CH,
2-CH), 2.50 (1H, t, J ) 6.3 Hz, 3-CH), 3.40-3.75 (8H, m,
morpholine ring), 4.00-4.15 (2H, m, CH2OH), 7.00-7.35 (5H,
m, aromatics).
1-(4-Mor p h olin ylca r bon yl)-2-for m yl-3-p h en ylcyclop r o-
p a n e (15). PCC (2.00 g, 9.20 mmol) was added to a solution
of 10b (1.60 g, 6.10 mmol) in dry CH2Cl2 (62 mL), and the
resulting mixture was stirred at room temperature in an argon
atmosphere for 7 h. Et2O (180 mL) was then added, the
reaction mixture was filtered, and the solvent was evaporated
off. Flash chromatography of the residue (1.2 g) and elution
with AcOEt-light petroleum (6:4) afforded 15 (1.00 g, 62%):
1H-NMR (CDCl3) δ 2.40 (1H, ddd, J ) 9.0, 6.2, 6.0 Hz, 2-CH),
2.70 (1H, dd, J ) 9.0, 6.2 Hz, 1-CH), 3.40 (1H, t, J ) 6.0 Hz,
3-CH), 3.50-3.75 (8H, m, morpholine ring), 7.10-7.35 (5H,
m, aromatics), 9.35 (1H, d, J ) 6.2 Hz, CHO).
(2R,1′S,2′R,3′R)-N-[(S)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′p h en ylcyclop r op yl]glycin on it r ile
1
(21): AcOEt-light petroleum (8:2), 33% yield as an oil; H-
NMR (CDCl3) δ 1.93 (1H, q, J ) 10 Hz, 1′-CH), 2.20 (1H, br s,
OH), 2.30 (1H, t, J ) 10 Hz, 2′-CH), 2.68 (1H, t, J ) 10 Hz,
3′-CH), 3.15-3.78 (10H, m, morpholine, CH2OH), 3.85 (1H, d,
J ) 10.5 Hz, 2-CH), 4.08 (1H, dd, J ) 4.5, 8.8 Hz, CHCH2-
OH), 6.80-7.30 (10H, 2 × m, aromatics); 13C-NMR (CDCl3) δ
23.04, 26.56, 27.61, 41.88, 44.93, 46.02, 62.89, 66.47, 67.01,
120.44, 126.79, 127.32, 127.54, 128.42, 133.88, 137.98, 167.00;
[R]20 -59° (c 1.35, CH2Cl2).
D
(2S,1′S,2′R,3′R)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(22): 23% yield as an oil; [R]20 -97° (c 0.90, CH2Cl2).
D
(2S,1′R,2′S,3′S)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(23): 36% yield as an oil; [R]20 +56° (c 1.35, CH2Cl2).
D
(2R,1′S,2′S,3′S)-N-[(S)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(24): AcOEt-light petroleum (8:2); 31% yield as a white solid;
1
mp 161-2 °C; H-NMR (CDCl3) δ 2.35 (1H, dd, J ) 5.6, 10.4
Hz, 2′-CH), 2.50-2.80 (3H, m, 1′-CH, 3′-CH, 2-CH), 3.00-3.80
(11H, 2 × m, morpholine, CH2OH, OH), 4.10 (1H, dd, J ) 4.5,
10.4 Hz, CHCH2OH), 7.00-7.40 (10H, 2 × m, aromatics); 13C-
NMR (CDCl3) δ 25.01, 28.11, 42.28, 45.70, 49.87, 63.28, 66.48,
67.18, 117.77, 127.09, 127.30, 127.71, 128.23, 128.39, 128.83,
135.33, 138.41, 165.90; [R]20 + 56° (c 0.30, CH2Cl2).
D
Gen er a l P r oced u r e for th e Ster eoselective Str eck er
Syn th esis To Give Su bstitu ted r-Am in o Nitr iles. (S)- Or
(R)-R-phenylglycinol (3.86 mmol) was added to a solution of
the aldehyde (3.86 mmol) in MeOH (38 mL), and the resulting
solution was magnetically stirred at room temperature for 2
h. After cooling to 0 °C, TMSCN (7.72 mmol) was added, and
the resulting mixture was stirred for 12 h at room tempera-
ture. Evaporation of the solvent gave a residue which was
submitted to medium pressure chromatography using AcOEt-
light petroleum mixtures as eluent to give the corresponding
N-substituted R-amino nitriles.
(2R,1′R,2′R,3′R)-N-[(S)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(25): AcOEt-light petroleum (8:2); 29% yield as a white solid;
1
mp 149-51 °C; H-NMR (CDCl3) δ 2.20 (1H, dd, J ) 4.8, 9.7
Hz, 2′-CH), 2.65 (3H, m, 1′-CH, 3′-CH, 2-CH), 3.00-3.80 (11H,
2 × m, morpholine, CH2OH, OH), 4.10 (1H, dd, J ) 3.2, 8.1
Hz, CHCH2OH), 7.10-7.40 (10H, 2 × m, aromatics); 13C-NMR
(CDCl3) δ 24.96, 28.11, 28.44, 42.10, 45.47, 50.12, 62.99, 66.35,
66.95, 117.77, 126.97, 127.31, 127.61, 128.26, 128.73, 135.19,
138.13, 165.47; [R]20 +40° (c 0.10, CH2Cl2).
D
(2S,1′R,2′R,3′R)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(2R,1′S,2′S,3′R)-N-[(S)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(16): AcOEt-light petroleum (8:2); 30% yield as a white solid;
mp 120-1 °C; 1H-NMR (CDCl3) δ 2.20 (2H, m, 1′-CH, 2′-CH),
2.75 (1H, d, J ) 9.8 Hz, 2-CH), 3.10 (2H, m, 3′-CH, OH), 3.40-
3.90 (10H, m, morpholine, CH2OH), 3.95 (1H, dd, J ) 2.9, 8.8
Hz, CHCH2OH), 6.70-7.40 (10H, 2 × m, aromatics); 13C-NMR
(CDCl3) δ 20.97, 30.37, 42.55, 45.96, 46.50, 62.83, 66.37, 66.85,
119.09, 127.12, 127.26, 127.65, 128.53, 133.71, 137.32, 168.83;
(26): 30% yield as a white solid: mp 161-2 °C; [R]20 -59° (c
D
0.45, CH2Cl2).
(2S,1′S,2′S,3′S)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(27): 31% yield as a white solid: mp 146-8 °C; [R]20 -42° (c
D
0.10, CH2Cl2).
(2R,1′S,2′R,3′S)-N-[(R)-rP h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(28): AcOEt-light petroleum (55:45); 23% yield as an oil; 1H-
NMR (CDCl3) δ 2.01 (1H, td, J ) 4.7, 9.4, 15.6 Hz, 1′-CH),
2.18 (1H, dd, J ) 4.7, 9.4 Hz, 2′-CH), 2.55 (1H, br s, OH), 2.68
(1H, t, J ) 4.7 Hz, 3′-CH), 3.30-4.30 (11H, m, morpholine,
CH2OH, 2-CH), 4.05 (1H, dd, J ) 4.7, 9.4 Hz, CHCH2OH),
7.05-7.40 (10H, m, aromatics); 13C-NMR (CDCl3) δ 26.79,
29.79, 31.06, 42.43, 46.04, 46.94, 62.95, 66.34, 67.04, 119.52,
126.24, 126.73, 127.20, 127.93, 128.50, 128.55, 138.31, 138.65,
[R]20 -30° (c 0.90, CH2Cl2).
D
(2R,1′R,2′R,3′S)-N-[(S)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(17): AcOEt-light petroleum (8:2); 15% yield as a white solid;
mp 148-9 °C; 1H-NMR (CDCl3) δ 2.20 (2H, m, 1′-CH, 2′-CH),
2.61 (1H, d, J ) 8 Hz, 2-CH), 2.90 (2H, m, 3′-CH, OH), 3.50-
4.05 (11H, m, morpholine, CH2OH, CHCH2OH), 6.90-7.40
(10H, 2 × m, aromatics); 13C-NMR (CDCl3) δ 22.94, 29.63,
29.89, 42.77, 46.15, 63.05, 66.67, 118.52, 127.24, 127.62,
167.03; [R]20 +105° (c 1.69, CH2Cl2).
D
128.17, 128.34, 128.56, 128.73, 133.89, 137.99, 169.19; [R]20
+155° (c 0.80, CH2Cl2).
D
(2R,1′R,2′S,3′R)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(29): AcOEt-light petroleum (55:45); 24% yield as an oil; 1H-
NMR (CDCl3) δ 2.00 (1H, m, 1′-CH), 2.18 (1H, dd, J ) 5.5, 9.2
Hz, 2′-CH), 2.84 (1H, t, J ) 5.5 Hz, 3′-CH), 3.07 (1H, br s,
OH), 3.35-3.80 (11H, m, morpholine, CH2OH, 2-CH), 4.05 (1H,
(2S,1′R,2′R,3′S)-N-[(R)-rP h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(18): 21% yield as a white solid; mp 124-5 °C; [R]20 +25° (c
D
0.70, CH2Cl2).

Stereoisomers of PCCG
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2267
dd, J ) 3.0, 7.7 Hz, CHCH2OH), 7.05-7.35 (10H, m, aromat-
ics); 13C-NMR (CDCl3) δ 26.85, 27.58, 31.23, 42.41, 46.02, 46.36,
62.86, 66.38, 67.07, 119.15, 126.51, 126.70, 127.28, 127.46,
(2H, m, 1′-CH, 2′-CH), 2.85 (1H, t, J ) 8.6 Hz, 3′-CH), 3.30
(1H, d, J ) 8.6 Hz, 2-CH), 7.25 (5H, m, aromatics); 13C-NMR
(D2O + DCl) δ 25.05, 26.70, 31.45, 55.35, 127.57, 128.57,
128.95, 134.60, 170.57, 173.05; [R]20D + 38° (c 0.40, 2.5 N HCl).
(2S,1′R,2′R,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (42, P CCG-11): 75% yield; mp 222-3 °C; [R]20D +85°
(c 0.22, 2.5 N HCl).
(2S,1′S,2′S,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (43, P CCG-12): 63% yield; mp 237-8 °C; [R]20D -33°
(c 0.30, 2.5 N HCl).
128.01, 128.41, 137.68, 138.29, 167.17; [R]20 +24° (c 1.00,
D
CH2Cl2).
(2S,1′R,2′S,3′R)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(30): 23% yield as an oil; [R]20 -120° (c 1.40, CH2Cl2).
D
(2S,1′S,2′R,3′S)-N-[(R)-r-P h en ylglycin yl]-2-[2′-(4-m or -
p h olin ylca r b on yl)-3′-p h en ylcyclop r op yl]glycin on it r ile
(31): 20% yield as an oil; [R]20 -19° (c 0.79, CH2Cl2).
(2R,1′S,2′R,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (44, P CCG-13): Dowex 50WX2-200 (10% pyridine)
followed by Dowex 1X8 (1 N AcOH); 49% yield; mp 199-200
°C; 1H-NMR (D2O) δ 2.05 (1H, td, J ) 5.6, 9.1, 10.4 Hz, 1′-
CH), 2.30 (1H, dd, J ) 5.4, 9.1 Hz, 2′-CH), 2.70 (1H, t, J ) 5.6
Hz, 3′-CH), 4.00 (1H, d, J ) 10.4 Hz, 2-CH), 7.10-7.30 (5H,
m, aromatics); 13C-NMR (D2O + DCl) δ 28.30, 30.80, 51.02,
D
Gen er a l P r oced u r e for th e Hyd r olysis of th e N-Su b-
st it u t ed r-Am in o Nit r iles. Lead(IV) acetate (1.63 mmol)
was added to a cold (0 °C), magnetically stirred solution of
the nitrile (1.48 mmol) in anhydrous MeOH-CH2Cl2 (0.12 M,
1:1); after 10 min, water (10 mL) was added and the resulting
mixture was filtered with the aid of Celite. After evaporation
of the solvent, the residue was refluxed in 6 N HCl (30 mL)
for 6-12 h. The reaction mixture was washed with CH2Cl2 (2
× 10 mL) and evaporated to dryness. The residue was
submitted to ion exchange resin chromatography.
126.57, 127.47, 128.93, 137.45, 170.53, 174.32; [R]20 +48.5°
D
(c 0.90, 2.5 N HCl).
(2R,1′R,2′S,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (45, P CCG-14): Dowex 50WX2-200 (10% pyridine)
followed by Dowex 1X8 (1 N AcOH); 75% yield; mp 184-5 °C;
1H-NMR (D2O) δ 2.05 (1H, td, J ) 6.1, 7.8, 10.7 Hz, 1′-CH),
2.25 (1H, dd, J ) 6.1, 7.8 Hz, 2′-CH), 2.85 (1H, t, J ) 6.1 Hz,
3′-CH), 4.45 (1H, d, J ) 10.7 Hz, 2-CH), 7.10-7.30 (5H, m,
aromatics); 13C-NMR (D2O + DCl) δ 25.99, 28.61, 32.02, 51.12,
(2R,1′S,2′S,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (32, P CCG-1): Dowex 50WX2-200 (10% pyridine);
1
86% yield; mp 240-1 °C; H-NMR (D2O) δ 1.95 (1H, td, J )
5.4, 8.9, 10.8 Hz, 1′-CH), 2.55 (1H, t, J ) 5.4 Hz, 2′-CH), 2.85
(1H, dd, J ) 5.4, 8.9 Hz, 3′-CH), 3.00 (1H, d, J ) 10.8 Hz,
2-CH), 7.20-7.35 (5H, m, aromatics); 13C-NMR (D2O + DCl)
δ 23.90, 27.78, 29.97, 50.40, 128.03, 128.43, 129.17, 132.76,
126.70, 127.50, 128.86, 137.39, 170.98, 174.65; [R]20 -59.4°
D
(c 0.90, 2.5 N HCl).
170.81, 175.24; [R]20 -74° (c 0.30, 2.5 N HCl).
(2S,1′R,2′S,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
D
glycin e (46, P CCG-15): 50% yield; mp 202-4 °C; [R]20
-47.4° (c 0.55, 2.5 N HCl).
(2R,1′R,2′R,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (33, P CCG-2): Dowex 50WX2-200 (10% pyridine);
D
1
74% yield; mp 221-3 °C; H-NMR (D2O) δ 2.10 (1H, dt, J )
(2S,1′S,2′R,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (47, P CCG-16): 55% yield; mp 184-5 °C; [R]20D +65°
(c 0.80, 2.5 N HCl).
5.0, 10.5 Hz, 1′-CH), 2.40 (1H, t, J ) 5.0 Hz, 2′-CH), 3.00 (2H,
m, 3′-CH, 2-CH), 7.30 (5H, m, aromatics); 13C-NMR (D2O +
DCl) δ 22.91, 27.83, 31.39, 51.49, 127.69, 128.59, 128.96,
Str eck er Syn th esis fr om Ald eh yd e (+)-14. (R)-R-Phen-
ylglycinol (0.068 g, 0.5 mmol) was added to a solution of
(1R,2R,3R)-1-(4-morpholinylcarbonyl)-2-formyl-3-phenylcyclo-
propane [(+)-14; 0.130 g, 0.5 mmol] in MeOH (5 mL), and the
resulting solution was magnetically stirred at room temper-
ature for 2 h. After cooling to 0 °C, TMSCN (0.13 mL, 1.0
mmol) was added and the resulting mixture was stirred for
12 h. Evaporation of the solvent gave a residue which was
submitted to MPC using AcOEt-light petroleum (6:4) as
eluent to give (2S,1′R,2′R,3′R)-N-substituted amino nitrile 26
(0.040 g, 20%): mp 168-70 °C.
X-r a y An a lysis. Single crystals of 19, 39, and 44 were
obtained by dissolution in AcOEt (19) or H2O-48% HBr (10:
1) (39 and 44) and slow evaporation at room temperature.
Colorless prisms of 39, 19, and 44 of approximate dimensions
0.10 × 0.25 × 0.15, 0.20 × 0.30 × 0.25, and 0.10 × 0.25 ×
0.15 mm, respectively, were used for data collection. Lattice
parameters were determined by least-squares refinement on
36, 25, and 30 (for 39, 19, and 44, respectively) randomly
selected and automatically centered reflections. Data were
collected on a Siemens P4 four-circle diffractometer with
graphite monochromated Mo KR radiation, in the ω scan mode
for 5 e 2Θ e 50° (39), 4 e 2Θ e 50° (19), and 2 e 2Θ e 45°
(44) scan ranges. Scan widths equal to 0.80°, 0.84°, and 1.3°
and constant scan speeds equal to 2.5, 2.4, and 2.9 deg min-1
were used for the data collections of compounds 39, 19, and
44, respectively. A total of 2240 (39), 3964 (19), and 1962 (44)
independent reflections were collected at 22 °C. Three stand-
ard reflections measured in each data collection every 60 min
showed no variations. Absorption correction based on Ψ-scan
were applied for compounds 39 and 44. The structures were
solved by direct methods of the SHELXTL PC package.41
Refinements were carried out by full-matrix anisotropic least-
squares on F2 for all reflections for non-H atoms by using the
133.28, 169.70, 175.25; [R]20 +100° (c 0.20, 2.5 N HCl).
D
(2S,1′R,2′R,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (34, P CCG-3): 70% yield; mp 237-8 °C; [R]20 +72°
D
(c 0.30, 2.5 N HCl).
(2S,1′S,2′S,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (35, P CCG-4): 73% yield; 217-8 °C; [R]20 -108° (c
D
0.15, 2.5 N HCl).
(2R,1′R,2′S,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (36, P CCG-5): Dowex 50WX2-200 (10% pyridine);
48% yield; mp 219-220 °C; 1H-NMR (D2O) δ 1.80 (1H, dt, J )
9.4, 11.9 Hz, 1′-CH), 2.49 (1H, t, J ) 9.4 Hz, 2′-CH), 2.75 (1H,
t, J ) 9.4 Hz, 3′-CH), 4.10 (1H, d, J ) 11.9 Hz, 2-CH), 7.10-
7.30 (5H, m, aromatics); 13C-NMR (D2O + DCl) δ 23.99, 24.54,
28.51, 49.00, 127.86, 129.20, 129.71, 133.33, 171.27, 175.24;
[R]20 +20° (c 0.50, 2.5 N HCl).
D
(2R,1′S,2′R,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (37, P CCG-6): Dowex 50WX2-200 (10% pyridine);
1
53% yield; mp 231-3 °C; H-NMR (D2O) δ 1.90 (1H, dt, J )
8.8, 11.5 Hz, 1′-CH), 2.55 (1H, t, J ) 8.8 Hz, 2′-CH), 2.80 (1H,
t, J ) 8.8 Hz, 3′-CH), 4.15 (1H, d, J ) 11.5 Hz, 2-CH), 7.10-
7.30 (5H, m, aromatics); 13C-NMR (D2O + DCl) δ 23.67, 24.29,
28.40, 49.00, 127.65, 128.59, 129.00, 133.07, 171.05, 173.98;
[R]20 -17° (c 0.60, 2.5 N HCl).
D
(2S,1′S,2′R,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (38, P CCG-7): 48% yield; mp 218-9 °C; [R]20 -21°
D
(c 0.50, 2.5 N HCl).
(2S,1′R,2′S,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (39, P CCG-8): 55% yield; mp 235-6 °C; [R]20 +18°
D
(c 0.40, 2.5 N HCl).
(2R,1′S,2′S,3′S)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (40, P CCG-9): Dowex 50WX2-200 (10% pyridine)
followed by reversed phase (RP-8) MPC and elution with H2O-
MeOH (75:25); 62% yield; mp 222-4 °C; 1H-NMR (D2O) δ 2.30
(2H, m, 1′-CH, 2′-CH), 2.80 (1H, dd, J ) 6.8, 9.8 Hz, 3′-CH),
3.45 (1H, d, J ) 8.7 Hz, 2-CH), 7.25 (5H, m, aromatics); 13C-
NMR (D2O + DCl) δ 24.40, 27.61, 31.10, 54.98, 127.61, 128.59,
128.95, 134.45, 170.44, 172.86; [R]20D -93° (c 0.25, 2.5 N HCl).
(2R,1′R,2′R,3′R)-2-(2′-Ca r b oxy-3′-p h en ylcyclop r op yl)-
glycin e (41, P CCG-10): Dowex 50WX2-200 (10% pyridine)
followed by reversed phase (RP-8) MPC and elution with H2O-
MeOH (75:25); 62% yield; mp 240-2 °C; 1H-NMR (D2O) δ 2.20
2
2 2
SHELXL-93 program.42 Minimized function ∑w(F0 - Fc
)
with weighting scheme w ) 1/[σ2(Fo)2 + aP2 + bP], where P )
(Fo2 + 2Fc2)/3, a ) 0.0546 (39), 0.0610 (19), or 0.0766 (44), and
b ) 0.0000 (39), 0.2282 (19), or 0.0000 (44). Since 44
crystallizes in a polar space group, polar axis restraints were
applied by the method of Flack and Schwarzenbach.43 Atomic
scattering factors including f′ and f′′ were taken from ref 44.
The absolute configurations of the four chiral centers were

2268 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Pellicciari et al.
assigned by Flack parameter45 for compounds 39 and 44, while
for 19 it was derived by the presence of the known absolute
configuration at the phenylglycinyl moiety. Most of the
hydrogen atoms were located on the final difference Fourier
synthesis, while the others were placed in calculated positions.
The C-H, N-H, and O-H distances were constrained to 0.96,
0.90, and 0.82 Å, respectively. Conventional R factor values
are 0.061, 0.048, and 0.070, while Rw values, based on F2, are
(3) (a) Bortolotto, Z. A.; Bashir, Z. I.; Davies, C. H.; Collingridge,
G. L. A molecular switch activated by metabotropic glutamate
receptors regulates induction of long term potentiation. Nature
1994, 368, 740-743. (b) Linden, D. J . Long term synaptic
depression in the rat mammalian brain. Nature 1994, 12, 457-
472.
(4) Pawloski-Dahm, C.; Gordon, F. Evidence for
a kynurenate-
insensitive glutamate receptor in the nucleus tractus solitarius.
J . Am. Physiol. 1992, 363, 1611-1615.
(5) Riedel, G.; Wetzel, W.; Reymann, K. G. (R,S)-alpha-methyl-4-
carboxyphenylglycine (MCPG) blocks spatial learning in rats and
long-term potentiation in the dentate gyrus in vivo. Neurosci.
Lett. 1994, 167, 141-144.
0.108, 0.109, and 0.143 for 39, 19, and 44, respectively.
R
factors based on F2 (Rw) are statistically about 2 times as large
as those (R) based on F.42
(6) Sacaan, A. I.; Bymaster, F. P.; Schoepp, D. D. Metabotropic
glutamate receptor activation produces extrapyramidal motor
system activation that is mediated by striatal dopamine. J .
Neurochem. 1992, 59, 245-251.
(7) Sladeczeck, F.; Pin, J .-P.; Re´casens, M.; Bockaert, J .; Weiss, S.
Glutamate stimulates inositol phosphate formation in striatal
neurons. Nature 1985, 317, 717-719.
(8) Nicoletti, F.; Meek, J . L.; Iadarola, M. J .; Chuang, D. M.; Roth,
B. L.; Costa, E. Coupling of inositol phospholipid metabolism
with excitatory aminoacid recognition site in rat hippocampus.
J . Neurochem. 1986, 46, 40-46.
(9) Nakanishi, S. Molecular diversity of glutamate receptors and
implications for brain function. Science 1992, 258, 597-603.
(10) (a) Pellicciari, R.; Curini, M.; Natalini, B.; Ceccherelli, P. Abstract
of the IX International Symposium on Medicinal Chemistry,
Berlin, Germany, 1986; p 118. (b) Pellicciari, R.; Natalini. B.;
Monahan, J . B.; Lanthorn, T. H.; Pilipauskas, D.; Snyder, J . P.
6th Camerino-Noordwijkerout Symposium on Recent Advances
in Receptor Chemistry, Camerino, Italy, 1987; pp 73-74. (c)
Pellicciari, R.; Natalini, B.; Marinozzi, M.; Selvi, L.; Chiorri, C.;
Monahan, J . B.; Lanthorn, T. H.; Snyder, J . P. In Frontiers in
Excitatory Amino Acid Research; Cavalheiro, E. A., Lehman, J .;
Turksi, L., Eds.; Alan R. Liss, Inc.: New York, 1988; p 67. (d)
Shinozaki, H.; Ishida, M.; Shimamoto, K.; Ohfune, Y. Potent
NMDA-like actions and potentiation of glutamate responses by
conformational variants of a glutamate analogue in the rat
spinal cord. Br. J . Pharmacol. 1989, 98, 1213-1224.
(11) Kwai, M.; Horikawa, Y.; Ishihara, T.; Shimamoto, K.; Ohfune,
Y. 2-(Carboxycyclopropyl)glycines: binding, neurotoxicity, and
free Ca2+ increase. Eur. J . Pharmacol. 1992, 211, 195-202.
(12) Ishida, M.; Akagi, H.; Shimamoto, K.; Ohfune, Y.; Shinozaki,
H. A potent metabotropic glutamate receptor agonist: electro-
physiological actions of a conformationally restricted glutamate
analogue in the rat spinal cord and Xenopus oocytes. Brain Res.
1990, 537, 311-314.
Biologica l Assa ys. Receptor binding experiments to rat
cerebral cortical membranes using [3H]-R-amino-3-hydroxy-5-
methylisoxazole-4-propionate (AMPA) (47 Ci/mmol), [3H]kain-
ate (47 Ci/mmol), and [3H]-3-(2-carboxypiperazin-4-yl)propane-
1-phosphonic acid (CPP) (58 Ci/mmol) all from New England
Nuclear) were performed as previously described.29 Receptor
binding experiments using [3H]glutamate (specific activity 50
Ci/mmol; Amersham) to membranes prepared from baby
hamster kidney cells (BHK) expressing mGluR1a were con-
ducted as previously reported.30 Sodium-dependent [3H]glu-
tamate uptake into rat cortical synaptosomes was determined
according to the method by Fletcher and J ohnston.32 Calcium/
chloride-dependent glutamate uptake was measured as de-
scribed46 using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-
AP4) binding to rat cortical membranes in the presence of 2.5
mM CaCl2 at 37 °C (specific activity of [3H]-L-AP4 50 Ci/mmol;
Tocris Cookson, U.K.). [3H]-L-AP4 binding was also used to
label mGluR4a when expressed in BHK cells.31 In brief,
membranes from mGluR4a-transfected cells were incubated
with 30 nM [3H]-L-AP4 and test compounds in a buffer (30
mM HEPES-Na, pH 8.0, 110 mM NaCl, 1.2 mM MgCl2, 5 mM
KCl, 2.5 mM CaCl2, and 0.1 mM phenylmethanesulfonyl
fluoride) for 30 min at 0 °C. Bound and free radioactivity was
separated by centrifugation (40000g, 3 min, 0 °C). The pellets
were quickly rinsed with ice cold buffer (2 × 2 mL), solubilized
in 2 M NaOH (0.5 mL), and transferred to scintillation vials.
Nonspecific binding was defined as the binding in the presence
of 0.1 mM L-serine O-phosphate.
BHK cells stably expressing mGluR1a, mGluR2, or mGluR4
were used for measurements of phosphoinositide (PI) hydroly-
sis or cAMP formation.33 For measurements of PI hydrolysis,
cells expressing mGluR1a were cultured for 2 days in 24-well
plates (Costar) and then incubated with 4 µCi/mL [3H]myo-
inositol (specific activity 17 Ci/mmol; Amersham). After 24 h
of prelabeling with [3H]myoinositol, the cells were washed
twice with a Krebs-Henseleit buffer (pH 7.4) supplemented
with 2.5 mM CaCl2 and 10 mM LiCl and subsequently
incubated with test compounds for 30 min in a similar buffer.
The incubation was stopped by quickly washing each well with
ice cold buffer (1 mL) and adding ice cold 10% perchloric acid
(1 mL). Fractions of inositol monophosphates were separated
from the neutralized extracts on ion exchange minicolumns
(Amersham, RPN 1908). Measurements of cAMP formation
were performed as described previously.33 The protein con-
tents were determined using a BioRad assay with γ-globulin
as a standard.
(13) (a) Ohfune, Y.; Shimamoto, K.; Ishida, M.; Shinozaki, H.
Synthesis of L-2-(2,3-dicarboxycyclopropyl)glycines, novel con-
formationally restricted glutamate analogues. Bioorg. Med.
Chem. Lett. 1993, 3, 15-18. (b) Ishida, M.; Saitoh, T.; Shima-
moto, K.; Ohfune, Y.; Shinozaki, H. A novel metabotropic
glutamate receptor agonist: marked depression of monosynaptic
excitation in the newborn rat isolated spinal cord. Br. J .
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