Design of remarkably simple, yet potent urea-based inhibitors of glutamate carboxypeptidase II (NAALADase) [1]

Full text of DOI:10.1021/jm000406m

298
J . Med. Chem. 2001, 44, 298
Letters
NAAG while decreasing the levels of glutamate. In fact,
recent work by Slusher et al. led to the demonstration
that the GCPII inhibitor 2-PMPA provides significant
protection against injury in rats after transient middle
cerebral artery occlusion (MCAO).7 Furthermore, in the
rat MCAO model, 2-PMPA decreased glutamate levels
while increasing NAAG levels, as would be predicted
for a compound working as a GCPII inhibitor. As a
therapeutic target, GCPII inhibition has been suggested
to have potential benefits over receptor-based strategies,
as it represents an upstream mechanism of glutamate
regulation that could reduce transmission at a number
of glutamatergic receptors rather than inhibiting a
single receptor subtype.8 Equally important, NAAG is
colocalized in neurons with small amine transmitters
including GABA and dopamine, and it has been shown
to act on presynaptic receptors to regulate transmitter
release.9
Design of Rem a r k a bly Sim p le, Yet P oten t
Ur ea -Ba sed In h ibitor s of Glu ta m a te
Ca r boxyp ep tid a se II (NAALADa se)
Alan P. Kozikowski,*,† Fajun Nan,† Paola Conti,†
J iazhong Zhang,† Epolia Ramadan,‡ Tomasz Bzdega,‡
Barbara Wroblewska,‡ J oseph H. Neale,‡
Sergey Pshenichkin,# and J arda T. Wroblewski#
Drug Discovery Program, Department of Neurology, and
Department of Pharmacology, Georgetown University
Medical Center, 3900 Reservoir Road, N.W.,
Washington, D.C. 20007, and Department of Biology,
Georgetown University, Washington, D.C. 20057
Received September 15, 2000
In tr od u ction . The amino acid glutamate is present
in high concentrations in the mammalian brain, and it
acts as the major excitatory neurotransmitter in the
CNS. Through its actions on both ionotropic and me-
tabotropic receptors, glutamate plays an important role
in a variety of physiological functions including learning,
memory, and developmental plasticity. Excessive acti-
vation of glutamate receptors or disturbances in the
cellular mechanisms that protect against the adverse
consequences of physiological glutamate receptor acti-
vation have been implicated in the pathogenesis of a
host of neurological disorders. Although several drugs
designed to attenuate the pathological consequences of
excessive glutamate activation have been shown to
reduce injury in experimental models of cerebral is-
chemia, so far none of these compounds has proven to
be effective in the clinical treatment of stroke.1
In our previous work,10 starting from NAAG, we
designed a dually acting ligand, 4,4′-phosphinicobis-
(butane-1,3-dicarboxylic acid) (1), which acts both as an
mGluR3-selective agonist (∼30 µM) and as a potent
inhibitor of GCPII (21.7 ( 2.1 nM). From this novel lead
compound, we now chose to investigate the activity of
structures comprising two amino acids joined through
their NH2 groups by a urea linkage (Figure 1). We
envisaged that the urea group would serve as a suitable
replacement for the central CH2P(O)(OH)CH2 present
in the lead structure. The impetus to pursue this
chemistry was driven largely by the ease of synthesizing
such structures, thereby facilitating further SAR analy-
sis.
N-Acetyl-L-aspartyl-L-glutamate (NAAG) is a peptide
neurotransmitter that is widely distributed in the
mammalian nervous system.2 NAAG is both an agonist
at metabotropic glutamate receptors (mGluR3)3 and a
mixed agonist/antagonist at the N-methyl-D-aspartate
(NMDA) receptor.4 NAAG is hydrolyzed by the neuro-
peptidase glutamate carboxypeptidase II (GCPII; also
known as N-acetylated R-linked acidic dipeptidase,
NAALADase, or NAAG peptidase) to liberate N-acetyl-
aspartate and glutamate both in vitro and in vivo.5 The
role of this metalloprotease GCPII is thus thought to
be twofold: (1) to terminate the neurotransmitter activ-
ity of NAAG and (2) to liberate glutamate which is then
able to act at the various glutamate receptor subtypes.
Alterations in the levels of GCPII and NAAG have been
observed in disorders that are linked to abnormalities
in glutamatergic neurotransmission.6
Resu lts a n d Discu ssion . The compounds that have
been prepared are shown in Table 1. For comparison
purposes, we also provide published data6a for some
related dipeptide structures. First, we explored the
activity of Glu-C(O)-Glu, where the Glu’s are of the
S-configuration. In general, these symmetrical ureas
were prepared (Scheme 1) by reacting the appropriate
amino acid benzyl ester with triphosgene/Et3N at -78
As a consequence of these findings, it has been
hypothesized that the inhibition of GCPII might provide
an effective strategy for achieving neuroprotection in
cases of cerebral ischemia by increasing the levels of
* To whom correspondence should be addressed. Tel: 202-687-0686.
Fax: 202-687-5065. E-mail: kozikowa@giccs.georgetown.edu.
† Department of Neurology.
‡ Department of Biology.
Department of Pharmacology.
#
F igu r e 1. Rational design of urea-based GCPII inhibitors.
10.1021/jm000406m CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/03/2001

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 299
Ta ble 1. Inhibitory Activity of Dipeptides and Ureas Against
Expressed Rat GCPIIa
compound
IC50
5.1 ( 0.6 nM
PMPA
[HO2C(CH2)2CH(CO2H)CH2]2P(O)(OH) 21.7 ( 2.1 nM (ref 10)
(S)-Glu-(S)-Glu
0.75 µM (ref 6a)
47 ( 4.5 nM
67% inhib at 100 µM
25% inhib at 1 µM
9% inhib at 1 µM
42% inhib at 100 µM (ref 6a)
3.8 µM
(S)-Glu-C(O)-(S)-Glu
(R)-Glu-C(O)-(R)-Glu
(R)-Glu-C(O)-(S)-Glu
(S)-Glu-C(O)C(O)-(S)-Glu
(S)-Asp-(S)-Asp
(S)-Asp-C(O)-(S)-Asp
(S)-Asp-(S)-Glu
(S)-Asp-C(O)-(S)-Glu
t-BuNHC(O)-(S)-Glu
Gly-C(O)-(S)-Glu
2.4 µM (ref 6a)
46.1 ( 1.4 nM
F igu r e 2. Protective effects of novel ureas against neuronal
cell death induced by NMDA in primary cultures of mouse
cortical neurons. Cultures of cortical neurons were treated for
10 min with 20 µM NMDA, followed by treatment with culture
medium collected from cultures of cortical astrocytes pre-
treated for 24 h with the indicated compounds at 1 µM
concentration. Neuronal viability was assessed 24 h later by
measurements of LDH accumulation. Results are expressed
as a percent of cell death induced by NMDA alone. Bars
represent mean values ( SEM from 16-32 determinations.
10% inhib at 1 µM
46% inhib at 1 µM
inactive at 1 µM
29 ( 6 nM
(R)-Cys-C(O)-(R)-Cys
(t-Bu)Cys-C(O)-(S)-Glu
(R)-Cys-C(O)-(S)-Glu
6.9 ( 0.4 nM
a
IC50 data were obtained using a substrate concentration of 5
µM.
Sch em e 1. Synthesis of Unsymmetrical Ureas:
AA-C(O)-AA′
tive. Last, we examined the ability to replace one of the
Glu fragments by other amino acids, or even a simple
amine. The preparation of these unsymmetrical ureas
was brought about by first treating the tosylate salt of
dibenzyl glutamate with triphosgene/Et3N at -78 °C
followed by addition of the second amine component and
warming to room temperature. Deprotection was then
effected through catalytic hydrogenation as well as the
use of TFA/Hg(OAc)2/anisole followed by H2S in the case
of cleavage of the t-Bu group from Cys (Scheme 1).
As shown in Table 1, the urea derived from tert-
butylamine + (S)-Glu proved inactive, as did Gly-C(O)-
(S)-Glu. In light of the ability of certain sulfur-
containing ligands to act as potent peptidase inhibitors
(e.g., captopril for angiotensin converting enzyme)
through the ability of the sulfur atom to coordinate with
a zinc atom present in the active site, we felt it would
be valuable to explore the activity of (R)-Cys-C(O)-(S)-
Glu. Remarkably, this tricarboxylic acid proved to be
6-fold more potent than (S)-Glu-C(O)-(S)-Glu. Even the
tert-butylthio-containing precursor molecule (t-Bu)Cys-
CO-(S)-Glu proved to be more active with a Ki of 29 nM.
Note, however, that the related urea (R)-Cys-C(O)-(R)-
Cys was inactive when tested at 1 µM. Thus the present
SAR reveals that a urea containing at least one
glutamate residue plus a second residue bearing a
carboxyl group in addition to another group (SR or
CO2H) represents the minimum requirement to achieve
effective GCPII inhibition.
For purposes of comparison, we note that PMPA has
been reported to be a potent inhibitor of GCPII with a
Ki of less than 1 nM8c against brain membrane pepti-
dase activity and Ki values up to 4.6 nM when tested
against cloned GCPIIs.8e PMPA was assayed in parallel
with the compounds reported herein and found to have
an IC50 of 5.1 ( 0.6 nM, reflecting an inhibitory activity
very close to that obtained for (R)-Cys-C(O)-(S)-Glu.
As shown in Figure 2, we have investigated the action
of some of these novel ureas for their ability to block
NMDA toxicity in vitro. This was done using a published
protocol in which cultures of cortical glial cells are first
treated with the test compound in order to induce the
release of protective factors into the medium.12 In
parallel, cultures of cortical neurons (without glia) are
°C, followed by warming to room temperature. After
purification by column chromatography or recrystalli-
zation, the intermediate tetraester was transformed to
its free acid by hydrogenolysis. All new compounds were
assayed for their ability to inhibit rat GCPII stably
expressed in Chinese hamster ovary (CHO) cells using
conditions identical to those reported previously. The
readily synthesized compound (S)-Glu-C(O)-(S)-Glu was
quite active, with an IC50 value of 47 nM against
expressed rat GCPII. Thus, this ligand is only 2-fold less
potent than our lead phosphinate. Glu-C(O)-Glu made
from (R)-Glu gave only 67% inhibition when tested at
100 µM, while (R)-Glu-CO-(S)-Glu gave 25% inhibition
at 1 µM, thus demonstrating the specificity of the
enzyme for S-configured amino acids. The corresponding
dipeptide (S)-Glu-(S)-Glu has been reported to have
some inhibitory activity toward GCPII, but it is 16-fold
less potent with an IC50 of 0.75 µM.6a,11 Interestingly,
when we examined (S)-Asp-C(O)-(S)-Asp, this compound
was found to be relatively inactive, with an IC50 of 3.8
µM. The corrresponding dipeptide (S)-Asp-(S)-Asp has
been reported to inhibit only 42% of enzyme activity at
100 µM.6a Next, we examined the activity of (S)-Asp-
C(O)-(S)-Glu and found this compound to be comparable
in activity (IC50 ) 46 nM) to (S)-Glu-C(O)-(S)-Glu. Thus,
the presence of a single fragment having a three-carbon
spacer between two of the carboxyl groups appears to
be essential for high inhibitory potency. The possibility
to replace the urea linker by a larger spacer group,
namely an oxalamide, was explored. This particular
analogue, (S)-Glu-C(O)C(O)-(S)-Glu, proved to be inac-

300 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Letters
Su p p or tin g In for m a tion Ava ila ble: Spectral and ana-
lytical data for all new compounds. This information is
available free of charge via the Internet at http://pubs.acs.org.
exposed for 10 min to the excitotoxic action of NMDA
(20 µM). The NMDA is removed by washing, and the
neurons are treated with the medium collected from the
treated glial cells. Toxicity is then assessed 24 h after
NMDA treatment by the measurement of lactate dehy-
drogenase (LDH) activity, which serves as a marker for
dying cells. In this test system 1 µM PMPA afforded
only about 25% neuroprotection. Consistent with data
published on PMPA in a similar NMDA-based neuro-
toxicity system, 1 µM PMPA afforded about 25% neu-
roprotection in our experiments.13
Refer en ces
(1) Wahlgren, N. G. Neuroprotective agents and cerebral ischemia.
In International Review of Neurobiology; Green, A. R., Cross, A.
J ., Eds.; Academic Press: San Diego, CA, 1997; pp 337-363.
(2) Neale J . H.; Bzdega, T.; Wroblewska, B. N-Acetylaspartyl-
glutamate: The most abundant peptide neurotransmitter in the
mammalian central nervous system. J . Neurochem. 2000, 75,
443-452.
(3) (a) Wroblewska, B.; Wroblewski, J . T.; Pshenichkin, S.; Surin,
A.; Sullivan, S. E.; Neale, J . H. N-Acetylaspartylglutamate
selectively activates mGluR3 receptors in transfected cells. J .
Neurochem. 1997, 69, 174-182. (b) Wroblewska, B.; Santi, M.
R.; Neale, J . H. N-Acetylaspartylglutamate activates cyclic-AMP
coupled metabotropic glutamate receptors in cerebellar astro-
cytes. Glia 1998, 24, 172-180. (c) Wroblewska, B.; Wroblewski,
J . T.; Saab, O.; Neale, J . H. N-Acetylaspartylglutamate inhibits
Among the potent GCPII inhibitors tested herein,
three compounds, [HO2C(CH2)2CH(CO2H)CH2]2P(O)-
(OH), (S)-Glu-C(O)-(S)Glu, and (S)-Asp-C(O)-(S)Glu,
produced at 1 µM a similarly modest neuroprotection
ranging from 21% to 31%. In contrast, the two Cys-
containing compounds, (t-Bu)Cys-C(O)-(S)-Glu and (R)-
Cys-C(O)-(S)-Glu, produced 69% and 50% neuroprotec-
tion, respectively. It should be noted that PMPA is
substantially more effective in producing neuroprotec-
tion in anoxia-based models of neurotoxicity in which
the compounds described here have not been tested.7
Given that PMPA and the compounds described herein
each act as peptidase inhibitors, we speculate that their
modest to more substantial neuroprotective actions are
related to blockade of NAAG hydrolysis. It may be
however, that these compounds, including PMPA, have
an action in this NMDA-induced toxicity model that is
independent of or additive to the inhibition of NAAG
hydrolysis. Both the neurons and glia express the
peptide and may release it during the medium changes
that are associated with this cell culture system. Ad-
ditionally, the peptidase inhibitors are presented first
to the glia and then transferred to the neurons together
with whatever factors might be released from the glial
cells. Other potential mechanisms include activation of
mGluR3 receptors. We have found, for example, that
â-NAAG functions as both a peptidase inhibitor and a
highly selective mGluR3 antagonist at micromolar
concentrations.14 The precise mechanism of this neuro-
protective effect in the widely used NMDA toxicity
model therefore remains to be rigorously defined. These
data argue in favor of further study of these compounds
in other models of neurotoxicity, both in vitro and in
vivo, as well as assessment of their potential interac-
tions with metabotropic glutamate receptors.
forskolin-stimulated cyclic AMP levels via
a metabotropic
glutamate receptor in cultured cerebellar granule cells. J .
Neurochem. 1993, 61, 943-948.
(4) Westbrook, G.; Mayer, M. L.; Namboodiri, M. A. A.; Neale, J .
H. High concentrations of N-acetylaspartylglutamate (NAAG)
selectively activate NMDA receptors on mouse spinal cord
neurons in cell culture. J . Neurosci. 1986, 6, 3385-3392.
(5) (a) Robinson, M. B.; Blakely, R. D.; Couto, R.; Coyle, J . T.
Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-Lglutamate.
J . Biol. Chem. 1987, 262, 14498-14506. (b) Serval, V.; Barbeito,
L.; Pittaluga, A.; Cheramy, A.; Lavielle, S.; Glowinski, J .
Competitive inhibition of N-acetylated-R-linked acidic dipepti-
dase activity by N-acetyl-L-aspartyl-R-linked L-glutamate. J .
Neurochem. 1990, 55, 39-46. (c) Carter, R. E.; Feldman, A. R.;
Coyle, J . T. Prostate-specific membrane antigen is a hydrolase
with substrate and pharmacologic characteristics of a neuropep-
tidase. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 749-753. (d)
Bzdega, T.; Turi, T.; Wroblewska, B.; She, D.; Chung, H. S.; Kim,
H.; Neale, J . H. Molecular cloning of a peptidase against
N-acetylaspartylglutamate (NAAG) from
a rat hippocampal
cDNA library. J . Neurochem. 1997, 69, 2270-2278.
(6) (a) Tsai, G.; Coyle, J . T.; Kleinman, J . E.; Baer, L.; Carter, R.;
Slusher, B. S.; Passani, L. A. Abnormal excitatory neurotrans-
mitter metabolism in schizophrenic brains. Arch. Gen. Psychiatry
1996, 52, 829-836. (b) Tsai, G. C.; Stauch-Slusher, B.; Sim, L.;
Hedreen, J . C.; Rothstein, J . D.; Kunc, R.; Coyle, J . T. Reductions
in acidic amino acids and N-acetylaspartylglutamate in amyo-
tropic lateral sclerosis CNS. Brain Res. 1991, 556, 151-156. (c)
Passani, L. A.; Vonsattel, J . P.; Coyle, J . T. Distribution of
N-acetylaspartylglutamate immunoreactivity in human brain
and its alteration in neurodegenerative disease. Brain Res. 1997,
772, 9-22. (d) Meyerhoff, J . L.; Koller, K. J .; Walczak, D. D.;
Coyle, J . T. Regional brain levels of N-acetyl-aspartyl-
glutamate: the effect of kindled seizures. Brain Res. 1985, 46,
392-396.
(7) (a) Slusher, B. S.; Vornov, J . J .; Thomas, A. G.; Hurn, P. D.;
Harukuni, I.; Bhardwaj, A.; Traystman, R. J .; Robinson, M. B.;
Britton, P.; Lu, X.-C. M.; Tortella, F. C.; Wozniak, K. M.; Yudkoff,
M.; Potter, B. M.; J ackson, P. F. Selective inhibition of NAALA-
Dase, which converts NAAG to glutamate, reduces ischemic
brain injury. Nat. Med. 1999, 5, 1396-1402. (b) Thomas, A. G.;
Vornov, J . J .; Olkowski, J . L.; Merion, V. T.; Slusher, B. S.
N-Acetylated alpha-linked acidic dipeptidase converts N-acetyl-
aspartylglutamate from a neuroprotectant to a neurotoxin. J .
Pharmacol. Exp. Ther. 2000, 295, 16-22. (c) J ackson, P. F.; Cole,
D. C.; Slusher, B. S.; Stetz, S. L.; Ross, L. E.; Donzati, B. A.;
Trainor, D. A. Design, synthesis, and biological activity of a
potent inhibitor of neuropeptidase N-acetylated-alpha-linked
acidic dipeptidase. J . Med. Chem. 1996, 39, 619-622. (d) Tiffany,
C. W.; Merion, A.; Calvin, D. C.; Lapidus, R. G.; Slusher, B. S.
Characterization of NAALADase activity in the prostate. Soc.
Neurosci. Abstr. 1998, 24, 582.
In conclusion, the present investigation reveals the
ability of some remarkably simple compounds to act as
potent inhibitors of GCPII, thus offering a new avenue
in the rational design of GCPII inhibitors that may lead
to effective neuroprotective agents. It is likely that the
appendage of other functionality, particularly hydro-
phobic groups, may lead to further improvements in
potency through interaction with accessory hydrophobic
pockets. Moreover, because of the possibility to employ
GCPII inhibitors in stroke therapy, it will be essential
to explore prodrugs or analogues containing carboxylic
acid isosteres so as to facilitate blood-brain barrier
penetration.
(8) (a) Rothman, S. M.; Olney, J . W. Glutamate and the pathophysi-
ology of hypoxic-ischemic brain death. Ann. Neurol. 1986, 19,
105-111. (b) Choi, D. W.; Rothman, S. M. The role of glutamate
neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev.
Neurosci. 1990, 13, 171-182. (c) Meldrum, B. S. Protection
against ischaemic neuronal damage by drugs acting on excita-
tory neurotransmission. Cerebrovasc. Brain Metab. Rev. 1990,
2, 27-57. (d) Bruno, V.; Battaglia, G.; Copani, A.; Giffard, R.
G.; Raciti, G.: Raffaele, R.; Shinozaki, H.; Nicoletti, F. Activation
of class II or III metabotropic glutamate receptors protects
cultured cortical neurons against excitotoxic degeneration. Eur.
J . Pharmacol. 1995, 7, 1906-1913.
(9) Zhao, J .; Ramadan, E.; Cappiello, M.; Wroblewska, B.; Bzdega,
T.; Neale, J . H.; NAAG inhibits KCl-induced [3H]-GABA release
via mGluR3, cAMP, PKA, and the L-type calcium channel. Eur.
J . Neurosci. 2001, in press.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (NS 35449 and NS 39080)
and Department of Defense (DAMD17-93-V-3018).

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 301
(10) Nan, F.; Bzdega, T.; Pshenichkin, S.; Wroblewski, J . T.; Wrob-
lewska, B.; Neale, J . H.; Kozikowski, A. P. Dual function
glutamate-related ligands: Discovery of a novel, potent inhibitor
of glutamate carboxypeptidase II possessing mGluR3 agonist
activity. J . Med. Chem. 2000, 43, 772-774.
(11) Subasinghe, N.; Schulte, M.; Chan, M. Y.-M.; Roon, R. J .;
Koerner, J . F.; J ohnson, R. L. Synthesis of acyclic and dehy-
droaspartic acid analogues of Ac-Asp-Glu-OH and their inhibi-
tion of rat brain N-acetylated R-linked acidic dipeptidase (NAA-
LA Dipeptidase). J . Med. Chem. 1990, 33, 2734-2744.
neuroprotective properties. Bioorg. Med. Chem. Lett. 1999, 9,
1721-1726. (b) Bruno, V.; Wroblewska, B.; Wroblewski, J . T.;
Fiore, L.; Nicoletti, F. Neuroprotective activity of N-acetylas-
partylglutamate in cultured cortical cells. Neuroscience 1998,
85, 751-757.
(13) Tortella, F. C.; Lin, Y.; Ved, H.; Slusher, B. B.; Dave, J . R.
Neuroprotection produced by the NAALADase inhibitor 2-PMPA
in rat cerebellar neurons. Eur. J . Pharmacol. 2000, 402, 31-
37.
(14) Lea, P. M.; Wroblewska, B.; Sarvey, J . M.; Neale, J . H. Beta-
NAAG rescues LTP from blockade by NAAG in rat dentate gyrus
via the type 3 metabotropic glutamate receptor. J . Neurophys.
2001, in press.
(12) (a) Kozikowski, A. P.; Araldi, G. L., Tu¨ckmantel, W.; Pshenich-
kin, S.; Serina, E.; Wroblewski, J . T. 1-Amino-APDC, a partial
agonist of group II metabotropic glutamate receptors with
J M000406M