Synthesis and pharmacological characterization of aminocyclopentanetricarboxylic acids: New tools to discriminate between metabotropic glutamate receptor subtypes

Article abstract of DOI:10.1021/jm970207b

The four stereoisomers of 1-aminocyclopentane-1,3,4-tricarboxylic acid {ACPT-I (18) and -II (19), (3R,4R)-III [(-)-20], and (3S,4S)-III [(+)-20]} have been synthesized and evaluated for their effects at glutamate receptors subtypes. ACPTs are ACPD analogues in which a third carboxylic group has been added at position 4 in the cyclopentane ring. None of the ACPT isomers showed a significant effect on ionotropic NMDA, KA, and AMPA receptors. On the other hand, ACPT-III (19) was found to be a general competitive antagonist for metabotropic receptors (mGluRs) and exhibited a similar affinity for mGluR1a (K(B) = 115 ± 2 μM), mGluR2 (K(B) = 88 ± 21 μM), and mGluR4a (K(B) = 77 ± 9 μM), the representative members of group I, II and III mGluRs, respectively. Two other isomers, ACPT-I (18) and (+)-(3S,4S)-ACPT-III [(+)- 20], were potent agonist at the group III receptors mGluR4a (EC₅₀ = 7.2 ± 2.3 and 8.8 ± 3.2 μM) and competitive antagonists with low affinity for mGluR1a and mGluR2 (K(B) > 300 μM). Finally, (-)-(3R,4R)-ACPT-III [(-)-20] was a competitive antagonist with poor but significant affinity for mGluR4a (K(B) = 220 μM). These results demonstrate that the addition of a third carboxylic group to ACPD can change its activity (from agonist to antagonist) and either increase or decrease its selectivity and/or affinity for the various mGluR subtypes.

Full text of DOI:10.1021/jm970207b

J . Med. Chem. 1997, 40, 3119-3129
3119
Syn th esis a n d P h a r m a cologica l Ch a r a cter iza tion of
Am in ocyclop en ta n etr ica r boxylic Acid s: New Tools to Discr im in a te betw een
Meta botr op ic Glu ta m a te Recep tor Su btyp es
Francine C. Acher,† Fre´de´rique J . Tellier,†,‡ Robert Azerad,*,† Isabelle N. Brabet,§ Laurent Fagni,§ and
J ean-Philippe R. Pin§
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Universite´ Rene´ Descartes-Paris V,
45 rue des Saints-Pe`res, 75270 Paris Cedex 06, France, and Centre INSERM-CNRS de Pharmacologie-Endocrinologie,
UPR 9023, rue de la Cardonille, 34094 Montpellier Cedex 5, France
Received March 27, 1997X
The four stereoisomers of 1-aminocyclopentane-1,3,4-tricarboxylic acid {ACPT-I (18) and -II
(19), (3R,4R)-III [(-)-20], and (3S,4S)-III [(+)-20]} have been synthesized and evaluated for
their effects at glutamate receptors subtypes. ACPTs are ACPD analogues in which a third
carboxylic group has been added at position 4 in the cyclopentane ring. None of the ACPT
isomers showed a significant effect on ionotropic NMDA, KA, and AMPA receptors. On the
other hand, ACPT-II (19) was found to be a general competitive antagonist for metabotropic
receptors (mGluRs) and exhibited a similar affinity for mGluR1a (KB ) 115 ( 2 µM), mGluR2
(KB ) 88 ( 21 µM), and mGluR4a (KB ) 77 ( 9 µM), the representative members of group I,
II and III mGluRs, respectively. Two other isomers, ACPT-I (18) and (+)-(3S,4S)-ACPT-III
[(+)-20], were potent agonists at the group III receptor mGluR4a (EC50 ) 7.2 ( 2.3 and 8.8 (
3.2 µM) and competitive antagonists with low affinity for mGluR1a and mGluR2 (KB > 300
µM). Finally, (-)-(3R,4R)-ACPT-III [(-)-20] was a competitive antagonist with poor but
significant affinity for mGluR4a (KB ) 220 µM). These results demonstrate that the addition
of a third carboxylic group to ACPD can change its activity (from agonist to antagonist) and
either increase or decrease its selectivity and/or affinity for the various mGluR subtypes.
In tr od u ction
proteins.9-11 Although generally not directly involved
in the fast synaptic transmission, these receptors modu-
late the efficiency of these synapses either by modulat-
ing postsynaptic ionic channels and receptors or by
regulating the release of Glu.9-11 mGluRs constitute
therefore an excellent target for drugs designed to
modulate the action of Glu in the brain.12-14
Most of the fast excitatory synapses in the brain use
the common amino acid glutamate (Glu) as a transmit-
ter. Accordingly, Glu is involved in many brain func-
tions such as motor control, vision, or central control of
heart for example. Glu synapses are also extremely
plastic, i.e. the synaptic efficiency can be modified in a
long term range, a property that is thought to be
essential for brain development, learning, and
memory.1-4 Glu is also the main endogenous neuro-
toxin, being responsible for the neuronal death observed
after ischemia, hypoxia, epileptic seizures, or brain
trauma.5,6 It is therefore not surprising that Glu is also
supposed to be involved in several brain disorders such
as epilepsy, Parkinson’s or Alzeimer’s disease, and
Huntington’s chorea.6
Numerous potential therapeutic applications are there-
fore expected from drugs modulating the different
effects of Glu. Two main types of Glu receptors have
been characterized: the ionotropic and metabotropic
receptors.7,8 The ionotropic receptors (iGluRs) are Glu-
gated cationic channels directly responsible for the fast
depolarization of postsynaptic cells. They are consti-
tuted of different subunits and classified into three
groups based on their pharmacology and functional
properties: the N-methyl-D-aspartate (NMDA), R-amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA),
and kainate receptors. The metabotropic Glu receptors
(mGluRs) regulate the activity of ionic channels or
enzymes producing second messengers via GTP-binding
Eight cloned mGluRs have been characterized and
classified into three groups based on sequence homology,
pharmacology, and transduction mechanism.9-11 Group
I includes mGluR1 and mGluR5 and their splice vari-
ants (1a, 1b, 1c, 1d, 5a, and 5b) that all activate
phospholipase C (PLC). Group II contains mGluR2 and
mGluR3 that both inhibit adenylyl cyclase. Group III
includes mGluR4, mGluR6, mGluR7, and mGluR8 and
their splice variants (4a, 4b, 7a, 7b, 8a, and 8b), all being
also negatively coupled to adenylyl cyclase when ex-
pressed in heterologous expression systems. Among the
compounds acting specifically on mGluRs, some ACPD
stereoisomers have been the first selective mGluR
agonists characterized:15,16 (1S,3R)-ACPD [(-)-1] be-
haves as an agonist at both group I and group II
mGluRs, and as a low-affinity agonist at group III;
(1S,3S)-ACPD [(+)-2] is more potent on group II mGluRs
than on the members of the other groups.10,17,18 Later
several more specific ligands were described.11,12,19 It
was shown that group I mGluRs are selectively acti-
vated by (3,5-dihydroxyphenyl)glycine (3,5-DHPG, 4),
while (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate
(2R,4R-APDC, 7), (2S,1′S,2′S)-2-(carboxycyclopropyl)-
glycine (L-CCG-I, 8), (2S,2′R,3′R)-2-(2,3-dicarboxycyclo-
propyl)glycine (DCG-IV, 9), and 2-aminobicyclo[3.1.0]-
hexane-2,6-dicarboxylic acid (LY354740, 13)20 are
selective agonists for group II mGluRs and 2-amino-4-
† URA 400 CNRS, Paris.
‡ Current address: Unite´ de Phytopharmacie et des Me´diateurs
Chimiques, INRA, route de Saint-Cyr, 78026 Versailles, France.
§ UPR 9023 CNRS, Montpellier.
X Abstract published in Advance ACS Abstracts, August 15, 1997.
S0022-2623(97)00207-0 CCC: $14.00 © 1997 American Chemical Society

3120 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Acher et al.
generates (2R,4R)-APDC (7).26 Other tricarboxylic cy-
cloamino acids, derived from iGluR agonists, have
occasionally been described.27-30 Their affinities, com-
pared to the corresponding dicarboxylic analogues, were
generally decreased; however no activity on metabotro-
pic receptors has been reported.
As a continuation of our efforts to obtain new confor-
mationally constrained cyclic analogues of glutamic
acid,31 in order to provide new and useful data for the
elaboration of the pharmacophores of metabotropic
receptors, we have prepared the four stereoisomers of
1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT) (18-
20) and determined their in vitro affinities for glutamate
receptors (iGluR and mGluR).32 We expected that the
introduction of a third carboxylic group on ACPD may
possibly increase its affinity and/or selectivity for some
mGluRs, as observed for DCG-IV (9) compared to
L-CCG-I (8) (see above). In addition, we were fascinated
by the fact that, depending on the relative stereochem-
istry of the 3,4-dicarboxy groups of ACPTs, we might
be dealing in each case with an ambivalent molecule,
related simultaneously either to a 1:1 mixture of D and
L cis or trans ACPD units, for the meso cis ACPT-I (18)
or the meso trans ACPT-II (19), respectively, or to a 1:1
mixture of D or L cis and trans ACPD units, for each
enantiomer of the trans ACPT-III (20). Such features
made their biological study particularly exciting.
phosphonobutyrate (L-AP4, 15) and L-serine O-phos-
phate (L-SOP, 16) for group III mGluRs. Antago-
nists11,12,19 include (4-carboxy-3-hydroxyphenyl)glycine
(4C3HPG, 5), R-methyl(4-carboxyphenyl)glycine (MCPG,
6) for group I mGluRs, (2S,1′S,2′S,3′R)-2-(2′-carboxy-
3′-phenylcyclopropyl)glycine (PCCG4, 10), (2S,1′S,2′S)-
2-(9′-methylxanthene)-2-(carboxycyclopropyl)glycine
(LY341495, 12),21 and (2S,4S)-2-amino-4-(4,4′-diphenyl-
but-1-yl)pentane-1,5-dioic acid (LY307452, 14) for group
II mGluRs and 2-amino-2-methyl-4-phosphonobutyrate
(MAP4, 17) for group III mGluRs. With the discovery
of LY341495 (12) and LY354740 (13), a large increase
in affinity was gained. However, there are still needs
for more selective and potent drugs acting on mGluRs
that could help define the specific physiological roles of
the different mGluR subtypes. One possible approach
is to introduce additional functional groups, possibly
forming new ionic or hydrogen bonds with the receptor,
on already characterized effectors. For example the
addition of a second hydroxyl group on (3-hydroxyphe-
nyl)glycine (3-HPG, 3) generates 3,5-DHPG (4) and
results in an increased affinity for group I mGluRs.22-24
Similarly, selectivity for group II mGluRs was increased
when adding a third carboxylic group on L-CCG-I (8),
which generates DCG-IV (9),25 or when replacing C-4
of (1S,3R)-ACPD [(-)-1] by a nitrogen atom, which
Ch em istr y
ACPT-I (18), ACPT-II (19), (()-ACPT-III (20), (3R,4R)-
ACPT-III [(-)-20], and (3S,4S)-ACPT-III [(+)-20] have
been synthesized via Bucherer-Bergs or Strecker reac-
tions starting from the known keto diesters 21 or 22,
followed by hydrolysis of the resulting spirohydantoins
and separation of isomeric amino acids by ion exchange
chromatography.
Keto diesters 21 and (()-22 were prepared following
literature procedures33-36 as depicted in Scheme 1 and
their diastereomeric purities (dr > 99.8:0.2) checked by
GC. Pig liver esterase (PLE) resolution of (()-22
according to Rosenquist et al.36 afforded (-)-(3R,4R)-
22 and (+)-(3S,4S)-22 (Scheme 2). When the reaction
was stopped at 48% hydrolysis, these authors rose the
ee of the enriched keto diester by crystallizing the
racemate and recovering the mother liquors. We ob-
tained variable ee’s by this method, and as a moderate
enantiomeric ratio (E ) 40)37 was measured, we chose
to carry out hydrolysis up to 55% and then recycle the
reesterified enriched monoacid (see the Experimental

Characterization of Aminocyclopentanetricarboxylic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3121
Sch em e 1a
a
(a) H2SO4/MeOH; (b) KMNO4; (c) NaOAc/Ac2O, 140 °C; (d) sealed tube, 100 °C.
Sch em e 2a
Sch em e 3a
a
(a) PLE, pH 7.0; (b) CH2N2/CH2Cl2.
Section). Thus, both enantiomers (-)-22 and (+)-22 had
ee > 98%, as deduced from the enantiomeric excesses
of the corresponding monoacids 23, measured by GC
after derivatization to their (R)-R-methylbenzylamides.38
At this step, Pybrop was preferred to isobutyl chloro-
formate38 as a coupling reagent to ensure a complete
reaction and an adequate measurement of enantiomeric
purity.
Hydantoins 24 and 2539 were prepared from keto
diesters 21 or 22 by the Bucherer-Bergs reaction
(Scheme 3, Table 1). Assuming that the first step of
the mechanism was an addition of cyanide on the imine
derived from 22,40,41 attack on either face of the ring
must lead to a single isomer 25c and/or 25d . However,
the same addition on the imine derived from 21 af-
forded, as expected, two meso isomers 25a and 25b.
Attack on the less hindered face was favored and gave
a major spirohydantoin 25b with a carbonyl group at
C-1 in a trans position to the preexisting carboxylates,
as previously described for cis-3,4-dimethylcyclopen-
tanone.41 Conversely, in the Strecker reaction, the
formation of the opposite meso isomer 25a was fa-
vored,41 when avoiding an alkaline equilibration.42 In
addition we observed that compounds exhibiting a cis
relation between C-3 and C-4 substituents (21, 24a , 24b,
18, and 19) were easily epimerized to the corresponding
isomers with a trans relation (22, 24c, 24d , and 20).
Thus besides hydantoins 24a and 24b, racemate (24c
+ 24d ) was also formed starting from 21 (Table 1).
Purification was achieved at the amino acid stage, so
hydantoin proportions were estimated from those of
amino acids obtained under mild hydrolytic conditions,
a
(a) (NH4)2CO3, KCN, 55 °C; (b) HCl/MeOH 60 °C; (c) NH4Cl,
KCN, rt; (d) (NH4)2CO3, rt.
assuming that a minimal epimerization had occured at
this step (see below).
Hydantoins 24 and 25 could be hydrolyzed in strong
acidic (6 N HCl, 110 °C, 5 d) or alkaline (BaO, 120 °C,
2 d) conditions (Scheme 4, Table 1). The resulting
ACPTs were purified and completely separated by ion
exchange chromatography to afford 18 (ACPT-I), 19
(ACPT-II), and (()-20 (ACPT-III) in about the same
equilibrated proportions (5:15:80) in all cases (see the
Experimental Section). Epimerization of 18 and/or 19
gave the most stable epimer (()-20. When 18 and/or
19 were the desired compounds, a milder hydrolysis of
hydantoins was required. Thus hydrolysis of intermedi-
ate diBoc-hydantoins 26 by LiOH at room temperature
(Scheme 4), as previously described,43,44 significantly

3122 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Acher et al.
Ta ble 1. ACPT (18:19:20) Ratios Obtained under Different
level of activation of each receptor subtype was then
determined by measuring total inositol phosphate ac-
cumulation. Although mGluR2 and mGluR4a do not
normally couple to this transduction cascade, this was
made possible by co-expressing these receptors with the
chimeric G-proteins Gqo5 and Gqi9.18 We previously
reported that the pharmacological profiles of these
receptors was not altered using this assay.18 None of
the ACPT molecules activated mGluR1a or mGluR2
(Figure 1a). However, both ACPT-I (18) and ACPT-III
(20) activated mGluR4a in a dose-dependent manner
(Figure 1b) with EC50 values of 7.2 ( 2.3 and 40 ( 8
µM (n ) 3), respectively. Among the enantiomers of
ACPT-III (20), only (+)-ACPT-III [(+)-20] was an ago-
nist at mGluR4a with an EC50 of 8.8 ( 3.2 µM (n ) 2)
(Figure 1c).
ACPT-I (18), -II (19), and -III (20) inhibited Glu-
induced IP formation in cells expressing mGluR1a
(Figure 2), and all induced a shift to the right of the
Glu dose-response curve as expected for competitive
antagonists (Figure 3 and data not shown). ACPT-II
(19) and -III (20), but not -I (18) also inhibited Glu-
induced responses in mGluR2 expressing cells, ACPT-
II (19) being more potent than ACPT-III (20). ACPT-
II (19) also antagonized the Glu effect on mGluR4a
(Figure 2), and appeared therefore to be a general
mGluR antagonist. A Shild plot analysis indicated that
ACPT-II (19) has a similar potency on mGluR1a,
mGluR2, and mGluR4a, and that in each case the slope
is close to unity as expected for a competitive antagonist
(Figure 3). The respective KB values of ACPT-II (19)
were 115 ( 2, 88 ( 21, 77 ( 9 µM (n ) 3) for mGluR1a,
mGluR2, and mGluR4a, respectively.
Since ACPT-III (20) that consists of two enantiomers
antagonized the effect of Glu on mGluR1a and mGluR2,
the effect of both (+)- and (-)-ACPT-III (20) were
examined. Both (+)- and (-)-ACPT-III (20) inhibited
with a low potency Glu responses in cells expressing
mGluR1a (Figure 4a) or mGluR2 (Figure 4b). We also
examined the possible antagonist effect of (-)-ACPT-
III [(-)-20] on mGluR4a because, as described above
(Figure 1c), only the (+) enantiomer was found to be
an agonist on this receptor. (-)-ACPT-III [(-)-20]
antagonized the effect of Glu on mGluR4a (Figure 4c)
at high concentration, and the Shild plot analysis
indicated a competitive inhibition (slope ) 0.87) with a
KB value of 220 µM (data not shown).
Chemical Conditionsa
hydantoin 24
keto
18:19:20
ratio (%)
diester synthesisb
obtained
hydrolysis
HCl
21
B
24a + 24b +
(24c + 24d )
6:19:75
B
B
S
B
B
B
BaO
mild
mild
HCl
mild
mild
5:8:87
19:65:16
32:50:18
(()-22
(-)-22
(+)-22
(24c + 24d )
24c
24d
8:11:81
0.6:1.9:97.5c,d
0.3:1.8:97.9e,d
a
b
See the Experimental Section. B stands for Bucher-Bergs
synthesis and S for Strecker synthesis. c (-)-20 isomer. Ratio
d
measured by GC after derivatization. e (+)-20 isomer.
reduced epimerization at C-3 and C-4. When 21 was
treated by the Bucherer-Bergs or Strecker procedure,
followed by mild hydrolysis then separation, the isomer
ratios 18:19:20 were respectively 19:65:16 or 32:50:18,
with 36% or 39% total overall yields (Table 1). As
mentioned above, Bucherer-Bergs conditions favored the
meso isomer 19 (65%), whereas Strecker conditions
increased the meso isomer 18 proportion from 19 to 32%,
next to some unavoidable (()-20 (16-18%). Although
double epimerization at C-3 and C-4 was less probable,
mild hydrolytic conditions were also applied to 25c and
25d to ensure chiral integrity (ee > 98%) and higher
yields of (-)- and (+)-20 (50% and 49% from 22,
respectively).
Absolu te Con figu r a tion Assign m en ts
The relative stereochemistry of functional groups in
18 and 19 was determined by NOE analysis on their
N-t-Boc trimethyl esters derivatives 27 and 28 (Scheme
5). Significant NOEs were observed between the N-H
proton and H-3 and H-4 of 27, when no such effect was
noted with 28. Absolute configurations of (+)- and (-)-
20 were deduced from known configurations of (+)- and
(-)-22.34,36
P h a r m a cologica l Resu lts
We first examined whether the ACPTs could activate
or antagonize ionotropic receptors. For that reason, the
different ACPT molecules were applied alone (at 1 mM)
or in combination with the ionotropic glutamate receptor
agonists, NMDA or kainate (both at 100 µM), on
cerebellar granule neurons maintained in culture for
7-9 days;45 at this concentration, kainate activates both
AMPA and kainate receptors. Currents evoked by these
molecules were recorded using the whole cell configu-
ration of the patch-clamp technique, at a membrane
potential of -60 mV, as previously described.15 None
of the ACPT isomers induced currents (data not shown).
On the same patches both NMDA and kainate induced
inward currents as expected from activation of the
NMDA and non-NMDA receptors. The antagonistic
effects of ACPT-I (18), -II (19) and -III (20) were also
examined on NMDA- or kainate-induced responses. No
significant inhibition was observed with ACPT-II (19),
and ACPT-III (20) (data not shown). A small inhibition
(10%) of both NMDA and kainate responses was how-
ever observed with ACPT-I (18) (data not shown).
The effect of the different ACPTs was then analyzed
on representative members of group I, II, and III
mGluRs, mGluR1a, mGluR2, and mGluR4a, respec-
tively, transiently expressed in HEK 293 cells.18 The
Discu ssion
In the present study we described the synthesis of the
four isomers of ACPT and their activity at Glu receptor
subtypes, both ionotropic and metabotropic (Table 2).
These compounds are analogues of ACPDs, the first
described selective agonists for mGluRs, in which an
additional carboxylic group has been introduced at
position 4 in the cyclopentane ring.
None of these compounds showed potent activity at
the NMDA and non-NMDA receptors. Among these,
only ACPT-I (18) slightly decreased kainate responses
at high concentration. However, the effect was too small
to be analyzed in details so that nonspecific effect cannot
be excluded. Among the four isomers of ACPD, only the
(1R,3R) is an agonist at the NMDA receptor subtype,46,47
and ACPT-I (18) and (+)-ACPT-III [(+)-20] correspond
to (1R,3R)-ACPD [(-)-2] with an additional carboxyl
group at position 4 (Table 3). Therefore, this additional

Characterization of Aminocyclopentanetricarboxylic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3123
Sch em e 4a
a
(a) 6 N HCl, 110 °C or BaO, 120 °C; (b) ion-exchange chromatography; (c) HCl/MeOH 60 °C; (d) Boc2O, DMAP, CH3CN; (e) 1 N
LiOH/CH3CN; (f) 2 N HCl/AcOH.
Sch em e 5
determine whether a mGluR rather than an ionotropic
glutamate receptor is involved in a specific physiological
function, avoiding the use in a first set of experiments
of a battery of selective antagonists.
ACPT-II (19) corresponds to (1R,3S)- or (1S,3R)-
ACPD [(+)-1 or (-)-1] with an additional carboxylic
group at position 4 in a cis relationship with the
3-carboxylate (Table 3). Comparing the effect of ACPT-
II (19) on mGluR1 and mGluR2 to its parent ACPD’s
effect, we note that agonists are turned into antagonists
and affinity maintained or decreased. On mGluR4 the
same comparison shows that affinity is markedly in-
creased. We suspect that the additional group lies in a
sterically unfavorable location at mGluR binding site.
Accordingly, this steric hindrance would be associated
with a decrease in affinity of the ligand. A steric
hindrance has already been speculated for the change
of agonist to antagonist properties of L-CCG-I (8) and
L-AP4 (15) resulting from the addition of a methyl group
on the alpha carbon [MCCG-I (11) and MAP4 (17),
respectively]. This was also associated with a decrease
in the apparent affinity of the compounds.18 It can be
also noticed that since ACPT-II (19) and (1R,3S)-ACPD
[(+)-1] (D configuration) exhibit a similar affinity at
mGluR1 and mGluR2 (KB 77-115 µM vs EC50 110-127
µM, Table 2) it cannot be excluded that ACPT-II (19)
binds to the receptor as (1R,3S)-ACPD [(+)-1] does. In
the case of mGluR4, the sterical reason for ACPT-II (19)
to be an antagonist has to be modulated by an additional
factor since the added carboxylic group increases the
affinity of the ligand compared to that of its parent
ACPD molecules. As discussed below, this strongly
suggests that this additional group promotes additional
interactions (likely hydrogen bonds) with the receptor.
group dramatically affects the activity and affinity of
this compound on ionotropic Glu receptors, as already
described for a similar modification of well-characterized
iGluR agonists.27-30
All ACPT isomers display interesting properties on
mGluRs (Table 2). ACPT-II (19) is a nonselective
mGluR antagonist, acting with a similar affinity on the
representative members of each group of mGluRs.
Since ACPT-II (19) is devoid of activity at ionotropic Glu
receptors, this compound will be useful to examine the
properties of the glutamatergic transmission in the total
absence of mGluR activation. It will also be useful to

3124 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Acher et al.
F igu r e 1. Agonist activity of the different ACPT isomers. (a)
IP formation was determined in cells expressing mGluR1a,
mGluR2 (plus Gqi9), or mGluR4a (plus Gqo5) under control
condition (Basal) or after 30 min stimulation with Glu (1 mM)
or ACPT (3 mM), as indicated on the figure. (b) Agonist dose-
response curves of ACPT-I (18) (closed triangles) and ACPT-
III (20) (open circles) on mGluR4a-expressing cells. IP forma-
tion was determined after a 30 min stimulation of cells
expressing mGluR4a and Gqo5 with the indicated concentra-
tions of ACPT-I (18) and -III (20). (c) Agonist dose-response
curves of (+)-ACPT-III [(+)-(20)] (open circles) and (-)-ACPT-
III [(-)-(20)] (closed circles) on mGluR4a-expressing cells. Data
are expressed as the IP formation over radioactivity remaining
in the membranes (a), or as the percentage of the maximal
ACPT-induced IP formation (b and c). Data are means ( SEM
of three or four independent experiments performed in trip-
licates (a and b), or means ( SEM of triplicate determination
from a typical experiment (c).
F igu r e 3. Antagonist activity of ACPT-II (18) on mGluR1a,
mGluR2, and mGluR4a. Graphs on the left represent the IP
formation in cells expressing mGluR1a (top), mGluR2 and Gqi9
(middle), or mGluR4a and Gqo5 (bottom) induced by different
concentrations of Glu applied alone (closed circles) or in
combination with ACPT-II (18) at a concentration of 200
(mGluR1a) or 100 (mGluR2 or mGluR4a) µM (open squares);
400 (mGluR1a) or 300 (mGluR2 or mGluR4a) µM (closed
triangles); 1000 µM (open circles). The EC50 value for Glu
applied alone (A) or in combination with a fixed concentration
(B) of ACPT-II (18) (A′) were determined as described in the
text. The Shild plots obtained from these values are presented
on the right. Data are means of triplicate determinations from
typical experiments.
prevent the ligand from being adequately positioned but
prevents further events leading to the activation of the
receptor.
ACPT-I (18) and (+)-ACPT-III [(+)-20] represent a
new series of specific agonists for mGluR4 (and possibly
other group III mGluRs), in addition to the phosphono
and phosphate derivatives L-AP4 (15) and L-SOP (16)
that are inactive on group I and group II mGluRs. They
display weak antagonist properties (except ACPT-I (18)
which is devoid of activity on mGluR2 in the range of
concentration examined) on mGluR1a and mGluR2.
Compounds with agonist properties at one mGluR
subtype and with antagonist properties on the members
of another group of mGluRs have already been de-
scribed. One of these, (S)-4-carboxy-3-hydroxyphenylg-
lycine (4C3HPG) (5) is a potent competitive antagonist
at mGluR1 and a potent agonist at mGluR2. Such a
property has been proposed to be of special interest13,14
since group I mGluRs like mGluR1a often potentiate
fast excitatory transmission, whereas group II mGluRs
often inhibit glutamatergic transmission.10 Accordingly,
4C3HPG (5) can decrease an excess of Glu excitation
by both inhibiting mGluR1 and activating mGluR2.
Such a property makes therefore 4C3HPG (5) a good
candidate for protection against neurotoxicity and epi-
leptic seizure, two properties that have been verified
experimentally.48,49 Since L-AP4-sensitive group III
F igu r e 2. Antagonist activity of the different ACPT isomers.
IP formation was determined in cells expressing mGluR1a,
mGluR2 (plus Gqi9), or mGluR4a (plus Gqo5) under control
condition (Basal) or after 30 min stimulation with Glu alone
or in combination with 1 mM ACPT, as indicated on the figure.
The glutamate concentration used was 3 µM for mGluR1a-
expressing cells, and 30 µM for mGluR2- and mGluR4a-
expressing cells. Data are means ( SEM of three or four
independent experiments performed in triplicates.
Similarly, the high affinity of PCCG4 (10), LY341495
(12), and LY307452 (14) mGluR2 antagonists could be
explained by a stabilization of the ligand-receptor
complex resulting from a positive interaction between
the added aromatic groups and protein residues. In all
cases the steric bulk of an additional group does not

Characterization of Aminocyclopentanetricarboxylic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3125
DCG-IV (9) results in an increased selectivity of the
compound at group II mGluRs (but no increase in
affinity), and the addition of a second hydroxyl group
on 3-HPG (3) to obtain 3,5-DHPG (4) increases its
affinity on group I mGluRs. However, the addition of
a carboxylic group on 3-HPG (3) to generate 4C3HPG
(5) does not modify the affinity of the compound on
mGluR1, but changes its property from an agonist to
an antagonist. On mGluR2, this additional acidic group
dramatically increases the affinity of the compound
since 3-HPG (3) has no effect on this receptor subtype,
but 4C3HPG (5) is a potent agonist of this receptor.
On mGluR1a and mGluR2, this additional carboxylic
group changes in all cases the agonist properties of
ACPD into antagonist properties and results in most
cases in a decrease in affinity which could be due to
steric or ionic effects, as discussed above for ACPT-II
(19). In contrast, on mGluR4a, this additional carboxy-
lic group results in an increased affinity in most cases,
with a conserved agonist property in some cases {ACPT-
I (18) and (+)-ACPT-III [(+)-20]} and a switch from
agonist to antagonist in others {ACPT-II (19) and (-)-
ACPT-III [(-)-20]}. Moreover, it has been shown that
replacement of the carboxylic group at position 3 on
trans-ACPD by a phosphono group increases the affinity
of this cyclopentane derivatives.52 These observations
suggest that stabilizing interaction (probably hydrogen
bonds) between oxygen atoms of the additional carboxy-
late or of the phosphonate function and protein residues
could be involved. This hypothesis is reinforced by the
large decrease of affinity of 2-amino-4-methylphosphi-
nobutanoic acid compared to L-AP4 (15).52
F igu r e 4. Antagonist activity of (+)-ACPT-III [(+)-(20)] and
(-)-ACPT-III [(-)-(20)] on mGluR1a, mGluR2, and mGluR4a.
Cells expressing mGluR1a (a), mGluR2 (plus Gqi9) (b), or
mGluR4a (plus Gqo5) (c) were stimulated with Glu (3 µM in a
and b, 20 µM in c) and various concentrations of (+)-ACPT-
III [(+)-(20)] (closed circles) or (-)-ACPT-III [(-)-(20)] (open
circles). Data are means of triplicate determinations from
typical experiments.
An examination of the concept that ACPTs may
represent ambivalent di-ACPD molecules leads to a
similar conclusion to those evoked above, considering a
simple addition of a third carboxylic group on ACPD.
None of the results observed with ACPTs reflects any
effect originating from the superposition of the constitu-
tive ACPD isomers. Further studies on other ACPD
analogues as well as molecular modeling are under
current investigations.
mGluRs, especially mGluR4, mGluR7, and mGluR8 are
also mostly presynaptic, inhibiting Glu release at many
synapses,10 the properties of ACPT-I (18) and (+)-ACPT-
III [(+)-20] on mGluRs make them good candidates for
neuroprotective and anticonvulsant agents.
Whereas the racemic mixture of ACPT-III (20) has
agonist properties at mGluR4a, the (+) enantiomer is
an agonist and the (-) enantiomer is a competitive
antagonist. Similarly, the (S) enantiomer of 2-amino-
3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid (APPA)
is a full AMPA receptor agonist, whereas the (R)
enantiomer is a competitive antagonist with a similar
affinity,50 and this explained why the racemic mixture
(RS)-APPA behaved as a partial agonist. Since then it
has been demonstrated that the mixture of a full agonist
and a competitive antagonist behaves as a partial
agonist, the maximal response obtained being depend-
ent on the affinity of the two molecules and on the molar
ratio used.51 Accordingly, it is not surprising that on
mGluR4a, (()-ACPT-III (20) behaves as a full agonist
since the agonist enantiomer is about 20 times more
potent than the enantiomer with antagonist property.
It is interesting to compare the activities of the
different ACPD isomers with those of ACPT, since these
later correspond to the formers in which a new carboxy-
lic group has been introduced at position 4 on the
cyclopentane ring (Table 3). When a polar group has
been introduced in other conformationally constrained
mGluR agonists, variable effects have resulted. The
addition of a carboxyl group on L-CCG-I (8) to obtain
Su m m a r y
The four epimers of 1-aminocyclopentane-1,3,4-tri-
carboxylic acid have been prepared and evaluated as
agonist or antagonist of glutamate receptor subtypes.
We have shown that all ACPTs are inactive on ionotro-
pic glutamate receptors, that both ACPT-II (19) and
(3R,4R)-ACPT-III [(-)-20] are general antagonists of
mGluR1, mGluR2, and mGluR4, the representative
members of group I, group II, and group III, respec-
tively, and that ACPT-I (18) and (3S,4S)-ACPT-III [(+)-
20] are potent agonists at mGluR4 and antagonists at
the representative members of group I and II mGluRs.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
obtained using a Bu¨chi capillary melting point apparatus and
are uncorrected. 1H (250.13 MHz) and 13C (62.9 MHz) NMR
spectra were recorded on an ARX 250 Bruker spectrometer.
Chemical shifts (δ) are reported in ppm downfield from
tetramethylsilane. NOE experiments were performed on an
ARX 500 Bruker spectrometer. Optical rotations were mea-
sured at the sodium D line (589 nm), at room temperature
(rt), with a Perkin-Elmer 241 polarimeter using a 1-dm
pathlength cell. Gas chromatography (GC) was performed on
Varian 3700 or 3400 chromatographs equipped with a flame

3126 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Ta ble 2. Activities of the Different Isomers of ACPT at mGluR1a, mGluR2, and mGluR4a, and Comparison with ACPD Isomersa
mGluR1a mGluR2 mGluR4a
Acher et al.
EC50
KB
EC50
KB
neb
EC50
KB
ACPT-I (18)
ACPT-II (19)
(()-ACPT-III [(()-20]
(+)-ACPT-III [(+)-20]
(-)-ACPT-III [(-)-20]
neb
>1000
115 ( 2
>300
ndc
neb
7.2 ( 2.3
neb
40 ( 8
8.8 ( 3.2
neb
neb
neb
neb
neb
neb
neb
neb
neb
88 ( 21
>300
ndc
77 ( 9
ndc
ndc
220
(1S,3R-)-ACPD [(-)-1]
(1R,3S)-ACPD [(+)-1]
(1S,3S)-ACPD [(+)-2]
(1R,3R)-ACPD [(-)-2]
9.3 ( 2.0d
127 ( 15d
>300d
18 ( 1e
>1000e
>1000e
48 ( 3e
neb,e
110 ( 10e
13 ( 3e
>1000d
g1000e
ndc
a
Means (µM) ( SEM of the EC50 (for the agonist activity) or KB (for the antagonist activity) values determined as described in the text.
ne: no effect at 1 mM. c nd: not determined. Values from ref 18. e Values from ref 22.
b
d
Ta ble 3. Stereochemical Relationship between ACPT and ACPD Isomers from Which the Formers can be Derived by Stereospecific
Addition of a Third Carboxylic Group at C-4
ACPT
(1S,3R,4S)-I (18)
(1R,3R,4S)-II (19)
(3R,4R)-III [(-)-20]
(3S,4S)-III [(+)-20]
ACPD
(1R,3R) (-)-2
(1S,3S) (+)-2
(1R,3S) (+)-1
(1S,3R) (-)-1
(1R,3S) (+)-1
(1S,3S) (+)-2
(1R,3R) (-)-2
(1S,3R) (-)-1
ionisation detector, using helium (1 bar) as carrier gas and
fitted with a Flexibond OV-1701 capillary column (15 m × 0.25
mm, Pierce Chemical Co.) or a Chirasil-Val capillary column
(50 m × 0.32 mm, Alltech). GC of amino acids was performed
after derivatization to N-(trifluoroacetyl)-O-isopropyl esters.53
Mass spectra were recorded on a Hewlett-Packard 5890-II/
5972 instrument equipped with a GC-mass coupling (30 m ×
0.2 mm capillary Ultra-2 column): injection temperature 250
°C; detection temperature 280 °C. Method A: column tem-
perature 90 °C (2 min), 90-250 °C (8 °C/min), 250 °C (5 min).
Method B: column temperature 120 °C (2 min), 120-300 °C
(10 °C/min), 300 °C (5 min). TLC was performed on Merck
60F254 precoated silica gel plates (0.2 mm thick, 8 cm migra-
tion) with the indicated solvent systems. Products were
visualized by UV light (254 nm), alcaline potassium perman-
ganate solution [KMnO4 (1 g), K2CO3 (5 g), KOH (0.5 g) in 100
mL of H2O], 2% (w/v) ninhydrin in ethanol and TDM reagent.54
Merck 60H silica gel (230-400 mesh) was used for flash
chromatography. Pig liver esterase (PLE, EC 3.1.1.1, 260
units mg-1) was purchased from Sigma.
cis-Dim eth yl Cyclop en ta n on e-3,4-d ica r boxyla te (21).
Heating cis-1,2,3,6-tetrahydrophthalic anhydride (45.6 g, 0.3
mol) in H2SO4/MeOH afforded its dimethyl ester [53.4 g, 0.27
mmol, 90.0%, GC (OV-1701, 150 °C) tR 4.51 min] which was
oxidized by KMnO4 (59.1 g, 0.225 mol, 83.7%, mp 141-142
°C, lit.35 mp 147 °C), then cyclized to 21 (22.9 g, 0.114 mol,
50.7%, 21:22 dr > 99.8:0.2) as previously described:35 mp 55-
57 °C (lit.35 mp 58 °C); TLC (CH2Cl2/EtOAc, 3:2) Rf 0.58; GC
(OV-1701, 120-240 °C at 6 °C/min) tR 9.5 min.
tr a n s-Dim eth yl Cyclopen tan on e-3,4-dicar boxylate [(()-
22]. trans-1,2,3,6-tetrahydrophthalic dimethyl ester [7 g, 0.035
mol, 71%, GC (OV-1701, 150 °C) tR 4.72 min] was prepared
from 3-sulfolene (6 g, 0.05 mol) and dimethyl fumarate (7.21
g, 0.05 mol) as previously described.33 Oxidation with KMnO4
[6.6 g, 0.025 mol, 72%, mp 164 °C (lit.36 mp 168-169 °C)] and
then cyclization as described35 for 21 afforded (()-22 (2.6 g,
0.013 mol, 52%, 22:21 dr > 99.8:0.2): mp 63-64 °C (lit.36 mp
64-65 °C); TLC (CH2Cl2/EtOAc, 3:2) Rf 0.66; GC (OV-1701,
120-240 °C at 6 °C/min) tR 8.4 min.
tr a n s-Dim eth yl Cyclopen tan on e-3,4-dicar boxylate [(-)-
22] a n d [(+)-22]. To a solution of (()-22 (0.4 g, 2 mmol) in a
0.01 M pH 7.0 phosphate buffer (200 mL) was added PLE (15
µL, 11 mg‚mL-1) at rt.36 A solution of 0.1 N NaOH was added
with a pH-stat to maintain the pH at 7.0 until 0.55 equiv of
base (11 mL) had been consumed. Hydrolysis was stopped by
cooling to 0 °C, acidification to pH 2 with 1 N HCl, and
saturation with NaCl. The mixture was extracted with EtOAc
(3 × 60 mL). The combined organic layers were dried and
evaporated, and the residue was purified by flash chromatog-
raphy. Elution with increasing amounts of EtOAc (0-10%)
in CH2Cl2 afforded (3R,4R)-22 (0.172 g, 0.859 mmol, ee > 98%),
and further elution with CH2Cl2/EtOAc/AcOH (60:40:0.3)
afforded (3S,4S)-23 (0.195 g, 1.05 mmol, 79% ee). Monoacid
(3S,4S)-23 was esterified with CH2N2. Crystallization in ether/
pentane of the resulting diester (3S,4S)-22 gave variable
amounts of (()-22 crystals, raising the ee of the mother liquor
from 79% to 83-98%.36 When (3S,4S)-22 (0.284 g, 1.42 mmol,
88.6% ee) was reprocessed with PLE as described above and
hydrolysis stopped at 65% (9.1 mL 0.1 N NaOH added),
(3R,4R)-22 (0.089 g, 0.445 mmol) and (3S,4S)-23 (0.160 g, 0.86
mmol, ee > 99.8%) were obtained, TLC (CH2Cl2/EtOAc/AcOH,
70:30:1) Rf (22) 0.68, (23) 0.25. (3R,4R)-22 (ee > 98%): [R]D
-135.7° (c 0.56, CHCl3) (lit.34 [R]D -133.3°). (3S,4S)-22 (ee >
99.8%): [R]D + 130.1° (c 0.69, CHCl3) (lit. [R]D +134.4°,34
+
136° 55).
Deter m in a tion of Op tica l P u r ity of 22 a n d 23.
A
solution of diester 22 (3 mg, 0.015 mmol) in 0.02 N NaOH (0.75
mL, 1 equiv) was stirred for 1.5 h, acidified to pH 4 with 1 N
HCl (7.5 µL), evaporated, and dried under vacuum to yield
23.
To a solution of 3 mg (0.016 mmol) of 23 in THF (0.5 mL)
was added Et3N (5 µL, 2.2 equiv), (R)-(+)-R-methylbenzylamine
(4 µL, 2 equiv, ee > 99.6%), a few crystals of DMAP and
PyBrop (9 mg, 1.2 equiv). After being stirred overnight at rt,
the solvent was removed and the residue partitioned between
CH2Cl2 (2 mL) and saturated aqueous NaCl (1.5 mL) to which
were added crystals of KHSO4 (pH 3). The emulsion was
stirred for 15 min, and then the organic phase was separated,
washed with saturated aqueous NaCl (2 × 1 mL), and dried
on Na2SO4. GC (OV-1701, 120-240 °C at 6 °C/min) tR (3R,4R)
26.5 min and (3S,4S) 27.5 min; (Ultra-2, method A) tR 23.4
and 23.8 min; GC MS m/ z (%) 289 [M]+ (7), 274 [M - CH3]+
(3), 258 [M - OCH3]+ (0.9), 214 (7), 120 (100), 105 (86.4).
Gen er a l P r oced u r e for th e P r ep a r a tion of Hyd a n toin s
by th e Bu ch er er -Ber gs Syn th esis. KCN (0.154 g, 1.2 equiv)
was added to a solution of 21 or 22 (0.4 g, 2 mmol) and (NH4)2-
CO3 (0.96 g, 5 equiv) in MeOH (4 mL) and water (3 mL). The
resulting mixture was heated with a reflux condenser at 55-
60 °C for 2.5 h with 21 or 5 h with 22. The solution was cooled,
acidified to pH 3-4 with dilute HCl, stirred for 15 min, and
concentrated or evaporated and dried under vacuum for
reesterification. When reaction lasted over 2.5 h the partially
hydrolyzed mixture was reesterified with a solution of dry HCl
in MeOH in a screw-cap bottle at 60 °C for 3 h and evaporated.
In both cases, the crude mixture was extracted with EtOAc
(100 mL), the organic phase washed with saturated NaCl
solution (20 mL) to which solid NaHCO3 was added, if needed,
to maintain neutral pH, washed again with brine (20 mL),
dried over Na2SO4, and evaporated to afford 25 (76-82% yield),
TLC (CH2Cl2/EtOAc, 3:2) Rf (25a or 25b) 0.20, (25c) 0.15.
Gen er a l P r oced u r e for th e P r ep a r a tion of Hyd a n toin s
by th e Str eck er Syn th esis. NH4Cl (1.08 g, 10 equiv) and

Characterization of Aminocyclopentanetricarboxylic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3127
KCN (0.13 g, 1 equiv) were added to a solution of 21 (0.4 g, 2
mmol) dissolved in water (5 mL). After 3 days of stirring at
rt, (NH4)2CO3 (0.211 g, 1.1 equiv) was added, and the mixture
was stirred for an additional 30 h and then acidified to pH 4
with 1 N HCl, evaporated, and dried under vacuum. The
residue was esterified with HCl/MeOH and purified as de-
scribed in the Bucherer-Bergs synthesis to afford 25 (70% yield
from 21).
described above: TLC (n-BuOH/AcOH/H2O, 4:1:1) Rf 0.15; 1H
NMR (D2O, NH4+ salt) δ 3.20 (m, 2H, H-3, H-4), 2.63 (m, 2H,
+
H-2, H-5), 2.24 (m, 2H, H-2, H-5); 13C NMR (D2O, NH4 salt)
δ 183.8 and 179.0 (CO), 70.0 (C-1), 50.8 (C-3, C-4), 42.1 (C-2,
C-5); GC (OV-1701, 190 °C) tR 19.2 min, (Ultra 2, method B)
tR 13.9 min, (Chirasil-Val, 190 °C) tR 22.4 min; GC MS m/ z
352 [M - CO2iPr]+ (4), 296 (30), 250 (26), 222 (100). Anal.
(C8H11NO6‚1.8H2O) C, H, N.
Gen er a l P r oced u r e for th e Hyd r olysis of Hyd a n toin s
w ith HCl or Ba O. A mixture of hydantoins 24 or 25 was
obtained by reaction of 21 or 22 (0.2 g, 1 mmol) with KCN/
(NH4)2CO3 as described previously. Partial purification was
achieved by further heating the mixture 1 h at 80 °C without
a reflux condenser, cooling, acidifying to pH 3, evaporating to
dryness, and extracting the resulting residue with MeOH.
After removal of the solvent, the crude mixture of hydantoins
was dissolved in 6 N HCl (20 mL) or in a suspension of BaO
(1.5 g) in water (20 mL), transferred to a screw-cap bottle and
heated 5 days at 110 °C or 2 days at 120 °C, respectively. HCl
was removed by evaporation, and BaO by neutralization with
dilute H2SO4 aqueous solution and filtration through a Celite
pad. Purification of the ACPT mixture was carried out as
described below. Using HCl hydrolysis, 18 (0.01 g), 19 (0.03
g), (()-20 (0.12 g) in 64.1% overall yield were obtained from
21, and 18 (0.015 g), 19 (0.02 g), (()-20 (0.15 g) in 74.2% overall
yield from (()-22. Using BaO hydrolysis, 18 (0.009 g), 19
(0.016 g), (()-20 (0.162 g) in 75.1% overall yield were obtained
from 21.
Gen er a l P r oced u r e for Mild Hyd r olysis of Hyd a n to-
in s. To the mixture of hydantoin esters 25 prepared from 21
or 22 (2 mmol) following the Bucherer-Bergs or Strecker
procedure and dissolved in CH3CN (20 mL) were added di-
tert-butyl dicarbonate (1.15 mL, 3 equiv) and DMAP (0.015 g,
0.08 equiv).43,44 Solvent was removed after 1.5 h. The residue
was then purified on a short silica gel column (2 × 13 cm) by
rapid elution with CH2Cl2 (60 mL) and CH2Cl2/EtOAc (4:1, 150
mL). Fractions of di-Boc diester 26 were detected by TLC {-
(CH2Cl2/EtOAc, 9:1) Rf (meso-26) 0.59 and [(RS,RS)-26] 0.51}
with TDM reagent and evaporated to give an oily residue (76-
81% yield) which was dissolved in CH3CN (3 mL) and 1 N
LiOH (12 mL). The mixture was stirred for 5 h at rt, cooled,
neutralized with 1 N HCl (12 mL), evaporated, and dried under
vacuum. The residue was treated with a solution of dry 2 N
HCl in AcOH for 0.5 h and evaporated to dryness. Purification
of the resulting ACPTs mixture was carried out as described
below.
Starting from 21 and using the Bucherer-Bergs procedure,
18 (0.035 g), 19 (0.116 g), and (()-20 (0.029 g) in 36.1% overall
yield were obtained. Starting from 21 and using the Strecker
procedure, 18 (0.062 g), 19 (0.096 g), and (()-20 (0.035 g) in
38.8% overall yield were obtained. Starting from (-) or (+)-
22 and using the Bucherer-Bergs procedure, (-)- and (+)-20
in 50% and 49% overall yield were respectively obtained (see
below).
Gen er a l P r oced u r e for P u r ifica tion of ACP T-I (18),
ACP T-II (19), a n d ACP T-III (20). The mixture of ACPTs
obtained from 21 or 22 (2 mmol) by the Bucherer-Bergs or
Strecker procedure and by acidic, alkaline, or mild hydrolysis
of resulting hydantoins was dissolved in water (up to 1.2 L in
the case of mild hydrolysis). The solution was adjusted at pH
4 and deposited on a Dowex 50 × 4 column (H+, 20-50 mesh,
3 × 17 cm). The resin was rinsed with water (0.3 L) and the
mixture of ACPTs eluted with 0.5 M aqueous NH4OH. Nin-
hydrin positive fractions were pooled and evaporated. The
residue was dissolved in boiled water (0.4 L), pH adjusted at
9, and the solution deposited on a AG1×4 (AcO-, 200-400
mesh, 3 × 19 cm). The resin was rinsed with boiled water,
and ACPT-I (18), ACPT-II (19), and ACPT-III (20) were
respectively eluted with 0.5, 0.8, and 1 M AcOH.
(1R,3R,4S)-1-Am in ocyclop en t a n e-1,3,4-t r ica r b oxylic
Acid (19). Compound 19 was prepared by the Bucherer-Bergs
procedure followed by HCl hydrolysis (12% yield from 21 and
8% yield from 22) or BaO hydrolysis (6.4% yield from 21) or
mild hydrolysis (23.2% yield from 21). When it was prepared
by the Strecker procedure followed by mild hydrolysis, a 19.2%
yield was obtained from 21. Purification was carried out as
described above. TLC (n-BuOH/AcOH/H2O, 4:1:1) Rf 0.15; 1H
NMR (D2O, NH4+ salt) δ 3.33 (m, 2H, H-3, H-4), 2.52 (m, 2H,
+
H-2, H-5), 2.32 (m, 2H, H-2, H-5); 13C NMR (D2O, NH4 salt)
δ 183.6 and 180.4 (CO), 69.2 (C-1), 53.7 (C-3, C-4), 42.3 (C-2,
C-5); GC (OV-1701, 190 °C) tR 16.4 min, (Ultra 2, method B)
tR 14.2 min, (Chirasil-Val, 190 °C) tR 18.3 min; GC MS m/ z
380 [M - OiPr]+ (1), 352 [M - CO2iPr]+ (12), 296 (21), 250
(30), 222 (100). Anal. (C8H11NO6‚1.8H2O) C, H, N.
(3RS,4RS)-1-Am in ocyclop en t a n e-1,3,4-t r ica r b oxylic
Acid [(()-20]. Compound (()-20 was prepared by the
Bucherer-Bergs procedure followed by HCl hydrolysis (48.1%
yield from 21 and 60.1% yield from 22), BaO hydrolysis (65%
yield from 21), or mild hydrolysis (5.8% yield from 21). When
it was prepared by the Strecker procedure followed by mild
hydrolysis, a 7% yield was obtained from 21. Purification was
carried out as described above. TLC (n-BuOH/AcOH/H2O, 4:1:
1) Rf 0.15; 1H NMR (D2O, NH4+ salt) δ 3.19 (m, 2H, H-3, H-4),
+
2.59 (m, 1H), 2.42 (m, 2H), 2.10 (m, 1H); 13C NMR (D2O, NH4
salt) δ 185.9, 183.9 and 179.2 (CO), 69.9 (C-1), 53.2 (C-3, C-4),
43.3 and 42.8 (C-2, C-5); GC (OV-1701, 190 °C) tR 13.5 min,
(Ultra 2, method B) tR 13.7 min, (Chirasil-Val, 190 °C) tR (3S,-
4S) 15.0 min and (3R,4R) 15.1 min; GC MS m/ z 440 [M +
H]+ (0.01), 380 [M - OiPr]+ (2), 352 [M - CO2iPr]+ (15), 296
(12), 250 (20), 222 (100). Anal. (C8H11NO6‚1.8H2O) C, H, N.
(3R,4R)-1-Am in ocyclop en ta n e-1,3,4-tr ica r boxylic Acid
[(-)-20]. Compound (-)-20 was prepared from (-)-22 (0.134
g, 0.67 mmol, ee > 98%) by the Bucherer-Bergs procedure
followed by mild hydrolysis (26, 0.209 g, 0.445 mmol) and
purification as described above. After cation exchange chro-
matography a mixture (0.091g, 0.362 mmol) containing mostly
(-)-20 (97.5%) and small amounts of 18 (0.6%) and 19 (1.9%)
as diammonium salts was recovered. Pure (-)-20 (0.083 g,
0.333 mmol, 49.8% overall yield, ee 99.2%) was obtained after
anion exchange chromatography: GC (Chirasil-Val, 150 °C)
tR 86.1 min; [R]D -32.3° (c 0.73, H2O).
(3S,4S)-1-Am in ocyclop en ta n e-1,3,4-tr ica r boxylic Acid
[(+)-20]. Compound (+)-20 was prepared from (+)-22 (0.172
g, 0.858 mmol, ee > 99.8%) by the Bucherer-Bergs procedure
followed by mild hydrolysis (26, 0.235 g, 0.5 mmol) and
purification as described above. After cation exchange chro-
matography a mixture (0.109 g, 0.434 mmol) containing mostly
(+)-20 (98.2%) and small amounts of 18 (< 0.4%) and 19 (1.8%)
as diammonium salts were recovered. Pure (+)-20 (0.105 g,
0.421 mmol, 49.1% overall yield, ee 98.3%) was obtained after
anion exchange chromatography: GC (Chirasil-Val, 150 °C)
tR 84.7 min; [R]D +26.4° (c 0.64, H2O).
(1S,3R,4S)-Tr im et h yl N-t-Boc-1-a m in ocyclop en t a n e-
1,3,4-tr ica r boxylic Ester (27). ACPT-I (18, 10 mg, 0.046
mmol) was suspended in dry 3 M HCl in MeOH (5 mL), heated
5 h at 70 °C in a screw-cap bottle, evaporated, and kept
overnight under vacuum on KOH. To the resulting triester
in CH2Cl2 (2 mL) was added Et3N (10 µL, 2 equiv), DMAP (7
mg, 1.2 equiv), and Boc-on (23 mg, 2 equiv), and the solution
was stirred 2 months at rt. Purification by flash chromatog-
raphy (19 × 1 cm, EtOAc/cyclohexane, 3:7) afforded 27 (8.2
mg, 0.023 mmol) in 50% overall yield: TLC (CH2Cl2/MeOH,
(1S,3R,4S)-1-Am in ocyclop en t a n e-1,3,4-t r ica r b oxylic
Acid (18). Compound 18 was prepared by the Bucherer-Bergs
procedure followed by HCl hydrolysis (4% yield from 21 and
6% yield from 22) or BaO hydrolysis (3.6% yield from 21) or
mild hydrolysis (7% yield from 21). When it was prepared by
the Strecker procedure followed by mild hydrolysis, a 12.4%
yield was obtained from 21. Purification was carried out as
1
95:5) Rf 0.31; H NMR (CDCl3) δ 4.90 (br s, 1H, NH), 3.71 (s,
3H, OCH3), 3.66 (s, 6H, 2 × OCH3), 3.36 (m, 2H, H-3, H-4),
2.67 and 2.66 (2 dd, 2H, J ) 6.4, 14.0 Hz, H-2, H-5), 2.34 (br
dd, 2H, J ) 6.4, 14.0 Hz, H-2, H-5), 1.41 [s, 9H, C(CH3)3]; 13
C
NMR (CDCl3) δ 173.1 (CO), 155.1 (NCO), 80.5 [C(CH3)3], 64.9

3128 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Acher et al.
(9) Nakanishi, S. Metabotropic glutamate receptors: synaptic trans-
mission, modulation, and plasticity. Neuron 1994, 13, 1031-
1037.
(C-1), 52.7 and 52.0 (C-3, C-4), 45.2 (3 × OCH3), 39.6 (C-2,
C-5), 28.2 [C(CH3)3].
(1R,3R,4S)-Tr im eth yl N-t-Boc-1-a m in ocyclop en ta n e-
1,3,4-tr ica r boxylic Ester (28). ACPT-II (19), 12.3 mg, 0.061
mmol) was esterified, N-protected (3 weeks), and purified as
described above to afford 28 (11.0 mg, 0.031 mmol) in 54%
overall yield: TLC (CH2Cl2/MeOH, 95:5) Rf 0.27; 1H NMR
(CDCl3) δ 5.21 (br s, 1H, NH), 3.71 (s, 3H, OCH3), 3.66 (s, 6H,
2 × OCH3), 3.26 (m, 2H, H-3, H-4), 2.65 and 2.64 (2 dd, 2H, J
) 6.0, 14.0 Hz, H-2, H-5), 2.29 (br dd, 2H, J ) 6.0, 14.0 Hz,
H-2, H-5), 1.39 [s, 9H, C(CH3)3]; 13C NMR (CDCl3) δ 174.3 and
173.4 (CO), 155.0 (NCO), 80.2 [C(CH3)3], 64.3 (C-1), 52.7 and
52.0 (C-3, C-4), 45.5 (3 × OCH3), 39.7 (C-2, C-5), 28.2 [C(CH3)3].
Biologica l Assa ys. Cu ltu r e a n d Tr a n sfection of HEK
293 Cells. HEK 293 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco BRL) supplemented with 10%
fetal calf serum and transfected by electroporation as previ-
ously described.18 Electroporation was carried out in a total
volume of 300 µL with 10 µg of carrier DNA, plasmid DNA
containing mGluR1a (0.3 µg), mGluR2 (2 µg), or mGluR4a (5
µg), and 10 million cells. To allow mGluR2 and mGluR4a to
activate PLC, an effect easier to measure than the inhibition
of cAMP production, these receptors were co-expressed with
the chimeric G-proteins Gqo5 and Gqi9 as previously de-
scribed.18 We previously reported that the pharmacological
profiles of these two receptors was identical to that character-
ized by measuring the inhibition of cAMP formation.
Deter m in a tion of In ositol P h osp h a tes (IP ) Accu m u la -
tion . Determination of IP accumulation in transfected cells
was performed as previously described after labeling the cells
overnight with [3H]myoinositol (23.4 Ci/mol, NEN, France).18
The stimulation was conducted for 30 min in a medium
containing 10 mM LiCl and the agonist at the indicated
concentration. The basal IP formation was determined after
30 min incubation in the presence of 10 mM LiCl and the Glu-
degrading enzyme glutamate pyruvate transaminase (1 unit/
mL) and 2 mM pyruvate to avoid the possible action of Glu
released from the cells. Results are expressed as the amount
of IP produced over the radioactivity present in the mem-
branes. The dose-response curves were fitted using the
equation y ) (ymax - ymin)/[1 + (x/EC50)n] + ymin and the
kaleidagraph program.
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Cu ltu r e a n d Recor d in gs of Cer ebella r Gr a n u le Neu -
r on s. Cerebellar granule cells were cultured from 7 days old
mice as previously described.45 Neurons were recorded using
the patch-clamp technique in the whole cell configuration, and
drugs were applied using a fast application technique as
previously described.15
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Synthesis of the four Isomers of 4-Aminopyrrolidine-
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Ack n ow led gm en t. We are indebted to Odile Con-
vert (Universite´ Pierre et Marie Curie, Paris) for NOE
experiments and their interpretation. This work was
supported by grants from the French Ministry of
Education, Research and Professional Insertion (ACC-
SV5, no. 9505077), the European community Biomed2
(BMH4-CT96-0228) and Biotech2 (BIO4-CT96-0049)
programs, and the Fondation pour la Recherche Me´di-
cale.
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