Dual function glutamate-related ligands: Discovery of a novel, potent inhibitor of glutamate carboxypeptidase II possessing mGluR3 agonist activity [3]

Full text of DOI:10.1021/jm9905559

772
J . Med. Chem. 2000, 43, 772-774
Du a l F u n ction Glu ta m a te-Rela ted
Liga n d s: Discover y of a Novel, P oten t
In h ibitor of Glu ta m a te Ca r boxyp ep tid a se
II P ossessin g m Glu R3 Agon ist Activity
F igu r e 1. Catabolism of NAAG by the peptidase.
Fajun Nan,† Tomasz Bzdega,§ Sergey Pshenichkin,‡
J arda T. Wroblewski,‡ Barbara Wroblewska,§
J oseph H. Neale,§ and Alan P. Kozikowski*,†
Drug Discovery Program, Georgetown Institute for
Cognitive & Computational Sciences, and Departments
of Pharmacology and Biology, Georgetown University
Medical Center, 3900 Reservoir Road, N.W.,
Washington, D.C. 20007-2197
F igu r e 2. Design strategy for NAAG-based mimics.
The starting point of our studies is N-acetyl-L-aspar-
tyl-L-glutamate (NAAG), which is a peptide neurotrans-
mitter that is widely distributed in the mammalian
nervous system.3 This peptide is inactivated by an
extracellular peptidase (NAAG peptidase or glutamate
carboxypeptidase II) producing glutamate and N-acetyl-
aspartate (Figure 1).4 The peptidase is concentrated, if
not exclusively localized, in glia. NAAG is a low-potency
agonist and may act as a partial antagonist at some
NMDA receptors.5 Additionally, in studies using cell
lines transfected with mGluR1-6, NAAG was found to
selectively activate the mGluR3 with an EC50 value in
the range of 65 ( 20 µM.6 NAAG therefore provides a
starting point for the design of new therapeutic drugs
that may selectively inhibit glutamate carboxypeptidase
II and/or act as mGluR3 agonists or antagonists.
Certain phosphonate analogues of NAAG, such as
2-(phosphonomethyl)pentanedioic acid, have been re-
ported to act as potent inhibitors of NAAG peptidase.7
Interestingly, while this compound was reported to show
no activity at glutamate receptors, we found that it does,
in fact, act as a weak antagonist at mGluR3. Upon the
basis of this unexpected result, we chose to explore the
activity of other NAAG analogues, the subject of the
present report.
At the onset of this work, we explored the activity of
NAAG-like analogues that were missing the amide bond
between the Asp and Glu residues (the standard ke-
tomethylene isosteric replacement). Furthermore, the
acetylamino group was deleted (1), as this particular
group was reported not to be an absolute requirement
for binding to NAAG peptidase.8 Among the compounds
synthesized, the symmetric compound 2, comprising an
acetone moiety flanked by the two pentanedioic acid
groups, proved to be the most interesting, as it retained
some mGluR3 activity albeit at high micromolar con-
centrations. Following this observation, we chose to
explore the activity of the compound in which the
central carbonyl group of 2 was replaced by P(O)OH (3,
4,4′-phosphinicobis(butane-1,3-dicarboxylic acid)) with
the idea that this compound might not only act as an
mGluR3-selective ligand but also function as an NAAG
peptidase inhibitor (Figure 2).
Received November 8, 1999
The metabotropic glutamate receptors (mGluRs) are
a heterogeneous family of G-protein-linked receptors
that couple to multiple second messengers. These
include the negative modulation of adenylate cyclase,
activation of phosphoinositide-specific phospholipase C,
and modulation of ion channel currents.1 Three types
of mGlu receptors have been identified: group I recep-
tors couple to phosphoinositide hydrolysis and include
mGluR1 and mGluR5; group II receptors couple to the
inhibition of cyclic adenosine 5′-monophosphate (cAMP)
formation and include mGluR2 and mGluR3; group III
receptors (mGluR4, mGluR6, mGluR7, and mGluR8) are
negatively coupled to cAMP. Each subtype is distin-
guished on the basis of its pharmacology and sequence
homology. Excessive activation of glutamate receptors
or disturbances in the cellular mechanisms that protect
against the adverse consequences of physiological
glutamate receptor activation have been implicated in
the pathogenesis of a host of neurological disorders.
These disorders include epilepsy, ischemia, central
nervous system trauma, neuropathic pain, and chronic
neurodegenerative diseases. Because of the ubiquitous
distribution of glutamatergic synapses, mGluRs have
the potential to participate in a wide variety of functions
in the CNS. In addition, because of the wide diversity
and heterogeneous distribution of the mGluR subtypes,
the opportunity exists for the development of highly
selective drugs that affect a limited number of CNS
functions. The mGluRs therefore provide novel targets
for the development of therapeutic agents that could
have a dramatic impact on the treatment of CNS
disorders.
To date, almost all of the commonly used agonists and
antagonists employed in biological studies of the mGluRs
are amino acids, often embodying a structurally rigidi-
fied glutamate-like core.2 During our efforts to identify
potent and selective ligands acting at these receptors,
we have discovered an mGluR3-selective agonist that
contains only acid groups and that acts simultaneously
as a potent inhibitor of NAAG peptidase.
The synthesis of 1 and 2 is outlined in Scheme 1.
Compound 4 was prepared using a literature method.7
Conjugate addition of nitromethane to 4 in the presence
of Triton B9 afforded a mixture of compounds 5 and 6
* To whom correspondence should be addressed. Tel: 202-687-0686.
Fax: 202-687-5065. E-mail: kozikowa@pop3.odr.georgetown.edu.
† Drug Discovery Program.
‡ Department of Pharmacology.
§ Department of Biology.
10.1021/jm9905559 CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/10/2000

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 773
Sch em e 1a
Ta ble 1. EC50 Values for 3 and Its Stereoisomers at NAAG
Peptidase and mGluR3
NAAG peptidase
inhib (EC50, nM)
mGluR3 agonism
compound
(EC50, µM)
1
2
35% inhib at 100 µM NAb
NAb
>300
3 (mixture of isomers) 21.7 ( 2.1
39 ( 8
33 ( 9
45 ( 7
51 ( 10
(R,R)/(S,S)-3a
(S,S)/(R,R)-3a
meso-3
84.0 ( 20.8
6.9 ( 0.7
22.3 ( 5.8
a
b
Absolute stereochemistry not determined. NA ) no activity.
a
Reagents and conditions: (a) CH3NO2, Triton B, rt, 24 h; (b)
benzyl acrylate, Triton B, CH2Cl2, rt, 24 h; (c) cetyltrimethylam-
monium permanganate, Et3N, CH2Cl2, rt, 4 h; (d) H2 (1 atm), 20%
Pd(OH)2/C, rt, 12 h.
Sch em e 2a
F igu r e 3. Effect of 3 (1000 µM) at mGluRs. Results are means
( SEM from 4-8 experiments.
a
Reagents and conditions: (a) NaH2PO2, TMSCl, Et3N, then 4,
rt, 24 h; (b) PivCl, BnOH, CH2Cl2/py (10:1), rt, 2 h; (c) NaH, THF,
0 °C, then 4, rt, 2 h; (d) 70 psi H2, 20% Pd(OH)2/C, rt, 24 h; (e)
PivCl, (R)-(+)-1-phenyl-1-butanol, CH2Cl2/py (10:1), rt, 2 h.
as previously described.12 In brief, group I receptors
(mGluR1a and mGluR5a) are coupled to phospholipase
C, and their activity was determined by accumulation
of inositol phosphates. Group II receptors included the
cAMP-coupled mGluR2, as well as the chimeric receptor
mGluR3/1a which, as shown previously,6 combines the
pharmacological properties of mGluR3 with the activa-
tion of phospholipase C. Group III receptors were
represented by mGluR4a and mGluR6, both coupled to
the inhibition of cAMP formation. As is apparent from
Table 1 and Figure 3, compound 3 exhibits good potency
and selectivity as an agonist for mGluR3 in comparison
to the other mGluR subtypes. Upon the basis of its EC50
value, it is roughly 2-fold more potent than NAAG.
In summary, we describe the discovery of a dually
acting ligand that offers a novel approach for the
generation of possible neuroprotective agents for the
treatment of neurodegenerative disorders. As the tet-
raacid 3 acts both as an mGluR3-selective agonist and
as a potent inhibitor of the enzyme involved in the
cleavage of NAAG to glutamate, it may simultaneously
be able to control levels of glutamate and to activate an
mGluR subtype known to confer neuroprotection.13
Although the pharmacological profiles of the mGluR2
and mGluR3 subtypes are very similar, compound 3
proved to be more potent for mGluR3 than for mGluR2,
indicating that it is possible to develop selective drugs
that are able to discriminate between these two recep-
tors. The mGluR selectivity of 3 may make it a useful
pharmacological agent for defining mGluR3 function at
identified synapses. Further efforts aimed at improving
compound potency as well as selectivity are in progress.
in a ratio of 2:1.5. After separation, 5 underwent a
second conjugate addition with benzyl acrylate to afford
compound 7. Intermediates 6 and 7 were converted to
carbonyl compounds 8 and 9,10 respectively, which were
finally transformed to 1 and 2 by hydrogenolysis.
The synthesis of 3 is outlined in Scheme 2. Conjugate
addition of sodium hypophosphite to dibenzyl R-meth-
yleneglutarate (4) afforded the phosphonate 10, which
was converted to the benzyl-protected compound 11a
with benzyl alcohol in the presence of trimethylacetyl
chloride.11 Deprotonation of 11a with NaH followed by
conjugate addition to 4 provided the completely pro-
tected pentaester 12a , which was finally transformed
to 3 by hydrogenolysis (Scheme 2).
As the above synthesis proceeds in a stereorandom
fashion, we also prepared the three optically pure
isomers of 3 through the use of the chiral alcohol (R)-
(+)-1-phenyl-1-butanol (see Supporting Information).
These compounds were assayed for their ability to
inhibit rat NAAG peptidase stably expressed in Chinese
hamster ovary (CHO) cells using conditions identical to
those reported previously.4d Of the compounds tested,
compound 3 proved to be the most potent and inhibited
NAAG peptidase with an EC50 of 21.7 ( 2.1 nM. The
ketone 2 showed no ability to inhibit this enzyme, while
1 was only weakly active, inhibiting about 35% of
enzyme activity at 100 µM. The individual stereoisomers
of 3 show some differences for the inhibition of NAAG
peptidase compared to the isomeric mixture. However,
the isomeric mixture is only 3-fold less potent than the
best of the optically pure isomers.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (NS 35449) and Depart-
ment of Defense (DAMD17-93-V-3018).
The mGluR activity of compound 3 was tested in cell
lines expressing the individual mGluRs using conditions

774 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
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