Synthesis, molecular modeling and preliminary biological evaluation of 1-amino-3-phosphono-3-cyclopentene-1-carboxylic acid and 1-amino-3-phosphono-2-cyclopentene-1-carboxylic acid, two novel agonists of metabotropic glutamate receptors of group III

Article abstract of DOI:10.1016/S0960-894X(00)00247-X

On the basis of a pharmacophore definition of mGlu₄ agonists, the two novel semi-rigid derivatives 12 and 13 were designed and synthesized. The preliminary biological evaluation demonstrated that both compounds interact with hmGlu(4a), while ineffective at group II receptor subtypes. In particular, derivative 13 is a full hmGlu(4a) agonist with an EC₅₀=17 μM. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00247-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1447±1450
Synthesis, Molecular Modeling and Preliminary Biological
Evaluation of 1-Amino-3-phosphono-3-cyclopentene-1-carboxylic
Acid and 1-Amino-3-phosphono-2-cyclopentene-1-carboxylic
Acid, Two Novel Agonists of Metabotropic Glutamate
Receptors of Group III
Laura Amori,a Gabriele Costantino,a Maura Marinozzi,a Roberto Pellicciari,a,*
Fabrizio Gasparini,b Peter J. Flor,b Rainer Kuhnb and Ivo Vranesicb
a
Á
Dipartimento di Chimica e Tecnologia del Farmaco, Universita di Perugia, Italy
bNovartis Pharma AG, Basel, Switzerland
Received 14 January 2000; accepted 26 April 2000
AbstractÐOn the basis of a pharmacophore de®nition of mGlu4 agonists, the two novel semi-rigid derivatives 12 and 13 were
designed and synthesized. The preliminary biological evaluation demonstrated that both compounds interact with hmGlu4a, while
ine€ective at group II receptor subtypes. In particular, derivative 13 is a full hmGlu4a agonist with an EC50=17 mM. # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
most of the interest has been devoted to group I and group
II mGlu receptor subtypes. In particular, the prevalently
postsynaptic group I receptor subtypes are involved in the
ampli®cation of the excitotoxic insult, whereas group II
receptor subtypes may contribute to the attenuation of
the neuronal damage, also by releasing neurotrophic
factors.
Most neurons and glia in the central nervous system
(CNS) express multiple receptor subtypes responding to
l-Glutamic acid, the major excitatory neurotransmitter
in the central nervous system (CNS) of vertebrates.
Among these, metabotropic glutamate (mGlu) receptors
constitute an heterogeneous family of G-protein coupled
receptors. So far, at least eight mGlu subtypes have been
cloned and classi®ed into three groups, according to
sequence homology, transduction mechanisms and ago-
nist pharmacology. Group I includes mGlu1 and mGlu5,
which are positively coupled to PI hydrolysis whereas
group II (mGlu2, mGlu3) and group III (mGlu4, mGlu6,
mGlu7, and mGlu8) are both negatively coupled to the
activity of adenylyl cyclase but are endowed with dif-
ferent pharmacology and neuronal localization.1
Recent evidences, however, point out an important role
for group III mGlu receptor subtypes, and mGlu4 in
particular, in a variety of CNS diseases.2 Indeed, it has
been observed that the presynaptically localized mGlu4
receptor subtypes play an important role in the homeo-
stasis of extracellular glutamic acid under ischemic/
hypoxic conditions and that genetically modi®ed mGlu4-
lacking mice are much more vulnerable to excitotoxic
insults than wild type animals.3 These observations bring
support to the idea that III mGlu receptor agonists may
be of potential therapeutic relevance in attenuating neu-
ronal damage following excitotoxic insults.
Over the past decade, the therapeutic potentialities
associated with the modulation of individual mGlu
subtypes have continuously increased as potent and
subtype-selective ligands have become available. So far,
l-1-Amino-4-phosphonobutyric acid (l-AP4, 1) is the
prototype of a group III agonist, and is characterized by
the substitution of the o-carboxylate of l-Glu by a phos-
phonate moiety. l-AP4 (1) is an agonist at all the group
III subtypes. Conformationally constrained analogues
*Corresponding author. Tel.: +39-075-5855120; fax: +39-075-
5855124; e-mail: rp@unipg.it
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00247-X

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L. Amori et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1447±1450
of l-AP4 (1) are endowed with agonist activity at group
III receptor subtypes in the low micromolar range of
potencies 4 (Chart 1). They include cyclopentyl derivatives
3, 4 and the cyclopropyl derivatives 5, 6. Of remarkable
interest is the activity of (RS)-PPG (7)5 and that of the 1-
amino-3-(phosphonomethylene)cyclobutanecarboxylic
acid derivative 8, which are not l-AP4 derivatives but
still retain a high potency and selectivity towards group III
receptor subtypes. Interestingly, also conformationally
constrained l-Glu derivatives display agonist activity at
mGlu4. Among these, the broad mGlu agonist (1S,3R)-
ACPD (11) and the tri-carboxy derivatives6 ACPT-I (9)
and (+)-ACPT-III (10) which have agonist potency
similar to that of o-phosphono derivatives with marked
selectivity toward mGlu4 subtype.
Tripos force ®eld (electrostatic term included, dielectric
constant of 80) and then submitted to a conformational
search performed by using the multisearch routine, as
implemented in sybyl, based on the Montecarlo algo-
rithm. One hundred conformers were generated for each
derivative. Those conformers with an energy higher than
6 Kcal/mol from the global minumum were discarded.
After completation of the conformational sampling, the
DISCO algorithm was run.7 The pharmacophore search
yielded 128 solutions that were reduced to 10 after elim-
ination of those solution endowed with a maximum toler-
ance and a RMS ®t higher than 1. These 10 ®nal solutions
were graphically examined and found to be very similar
each to the others only di€ering by slight di€erent
superimposition of individual ligands. The solution
endowed with the lowest mean energy of the contribut-
ing conformers was chosen as pharmacophore model.
The 3-D representation of the resulting pharmacophore
is shown in Figure 1. More in detail, all the derivatives
contribute to the pharmacophore with conformers less
than 2 Kcal/mol from the global minimum with the
exception of compound 2 and 9, whose proposed
bioactive conformers are 4.1 and 3.7 Kcal/mol above the
global minimum, respectively.
With the aim of understanding the conformational and
topological requirements common to all the above group
III agonists, we have undertaken a molecular modeling
study.
Group III agonist pharmacophore
Also in analogy with other glutamate receptor families,
a ®ve points pharmacophoric model was chosen. These
®ve points include a hydrogen bonding donor atom (the
positively charged amino acidic nitrogen), two acceptor
atoms (the two oxygen atoms of the carboxylic moiety of
the aminoacidic group) and two donor sites (ELPs)
belonging to the distal phosphonate or carboxylate moi-
ety. Thus, three ELPs were added to each of the phospho-
nate oxygens and two ELPs were added to each of the
carboxylate oxygens.
It should be mentioned that while this paper was in pre-
paration, a study by Bessis et al. has appeared describing a
pharmacophore model for mGlu4 agonists.8 A major dif-
ference between that study and our own is that we have
included in the search (RS)-PPG (7), which is the com-
monly used pharmacological tool for studying group III
receptors and has a potency comparable with l-AP4 deri-
vatives. Because of its unique structure among the other
group III agonists, (RS)-PPG (7) strongly in¯uences the
topology of the pharmacophore. The results of the
pharmacophore search point out that l-AP4 (1) interacts
Derivatives 1±11 were built from standard dictionary
within the sybyl program, energy minimized by the
Chart 1.

L. Amori et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1447±1450
1449
III agonist activity.9 The synthesis and the preliminary
biological evaluation of two new conformationally con-
strained analogues of l-AP4, namely 1-amino-3-phos-
phono-2-cyclopentene- and 1-amino-3-phosphono-3-
cyclopentene-1-carboxylic acids 12 and 13, respectively,
are reported herein.
Chemistry
The synthesis of the two phosphonocyclopentene amino
acids (12 and 13) is depicted in Scheme 1.
The diester 15, readily obtained from dimethyl malonate
and Z-1,4-dichloro-2-butene,10 was submitted to mono-
hydrolysis to give the corresponding monoester 16 which
was then transformed into the protected amino acid 18 via
the Curtius rearrangement of the corresponding acyl
azide. Hydroboration±oxidation reaction of 18 gave the
alcohol 1911 which was in turn oxidized (20) and reacted
with N-phenyl bis(tri¯uoromethanesulfonimide)12 thus
a€ording an inseparable mixture of the two corresponding
enol tri¯ates 21 and 22. Pd(0)-catalyzed coupling of the
mixture of 21 and 22 with diethyl phosphite13 yielded the
two protected phosphonocyclopentene amino acids (23
and 24) which were then separated (¯ash chromatography)
Figure 1. (a) Depiction of group III pharmacophore model. PPG (8)
(S-con®guration at the a-carbon atom) is included for clarity; (b).
Superimposition of l-AP4 (1) to PPG (8).
with group III receptor in a g g conformation (N-C2-
C3-C4= 56.9ꢀ; C2-C3-C4-C5= 60ꢀ). The syn ELPs
of the phosphonate group are superimposed to the cor-
responding anti ELPs of the phosphonate of PPG (7). On
the basis of the pharmacophore model, we reasoned that
the two cyclopentene derivatives 12 and 13, which are
endowed with the required relative disposition between
pharmacophoric groups can potentially display group
Scheme 1.

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L. Amori et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1447±1450
Figure 2. (a) Agonist activity of 12 and 13 determined by stimulation of [35S]-GTPgS binding to membranes prepared from CHO-hmGlu4a cells.
Data points were normalized to the stimulation obtained by 1 mM of l-Glu, and represent the mean Æ SEM of three determinations from one
experiment; (b) and (c) Inhibition of forskolin-stimulated cAMP accumulation in CHO cells expressing hmGlu4a. Forskolin (10 mM) stimulated
cAMP formation by about 40-fold (taken as control). All values are given as fraction of control. The e€ect of forskolin is inhibited by 1mM l-AP4;
this submaximal agonist concentration represents approximately EC80. To test for agonist activity, 12 and 13 were applied to forskolin-stimulated
CHO cells Antagonist activity of the compounds was assessed by co-application with the sub-maximal concentration of l-AP4. Bars represent mean
Æ SEM of two independent experiments, nꢁ5. Asterisks indicate statistically signi®cant agonist activity (2P<0.01; Dunnett's t-test).
Table 1. Activity of 12 and 13 on two mGlu subtypes expressed in recombinant mammalian cells
Assay
Receptor subtype
Human mGlu2
12
13
EC50 (mM)
IC50 (mM)
EC50 (mM)
IC50 (mM)
Group II mGlu
>200
Group III mGlu
GTPgS binding
>300
>300
>300
GTPgS binding
Human mGlu4a
Human mGlu4a
>30 (Partial agonist)
>30
>100
>100
17 Æ 8
>100
>100
# Forskolin-stimulated cAMP
ꢂ30 No full inhibition
References
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Preliminary biological evaluation
The novel derivatives 12 and 13 were evaluated for their
ability to interact group II or group III mGlu in CHO cell
lines expressing hmGlu2 and hmGlu4a. cAMP Accumula-
tion14,15 and [35S]-GTPgS16,17 assays were employed as
previously described.
While ine€ective at group II receptor subtypes, both 12
and 13 signi®cantly interact with human mGlu4a, either
in the GTPgS binding assays or in the cAMP assays
(Fig. 2 and Table 1). In particular, in the GTPgS assay
12 is a partial agonist, with an EC50>3 0 mM, while 13 is
a full hmGlu4a agonist with an EC50=17 mM.
In conclusion, we have reported that the cyclopentene
derivatives 12 and 13 are endowed with activity at the
mGlu4a receptor subtype. Compound 12 behaves as a
partial agonist, while 13 is a moderately potent mGlu4a
agonist, thus con®rming our hypothesis on the group III
pharmacophore requirements. It should be noted that
both 12 and 13 have been tested as racemic mixture and
that their anity and functional pro®le (in the case of
the partial agonism of 12) may change when the pure
enantiomers will be synthesized and tested.