Synthesis of Urea-Based Inhibitors as Active Site Probes of Glutamate Carboxypeptidase II: Efficacy as Analgesic Agents

Article abstract of DOI:10.1021/jm0306226

The neuropeptidase glutamate carboxypeptidase II (GCPII) hydrolyzes N-acetyl-L-aspartyl-L-glutamate (NAAG) to liberate N-acetylaspartate and glutamate. GCPII was originally cloned as PSMA, an M_r 100 000 type II transmembrane glycoprotein highly expressed in prostate tissues. PSMA/GCPII is located on the short arm of chromosome 11 and functions as both a folate hydrolase and a neuropeptidase. Inhibition of brain GCPII may have therapeutic potential in the treatment of certain disease states arising from pathologically overactivated glutamate receptors. Recently, we reported that certain urea-based structures act as potent inhibitors of GCPII (J. Med. Chem. 2001, 44, 298). However, many of the potent GCPII inhibitors prepared to date are highly polar compounds and therefore do not readily penetrate the blood-brain barrier. Herein, we elaborate on the synthesis of a series of potent, urea-based GCPII inhibitors from the lead compound 3 and provide assay data for these ligands against human GCPII. Moreover, we provide data revealing the ability of one of these compounds, namely, 8d, to reduce the perception of inflammatory pain. Within the present series, the γ-tetrazole bearing glutamate isostere 7d is the most potent inhibitor with a K_i of 0.9 nM. The biological evaluation of these compounds revealed that the active site of GCPII likely comprises two regions, namely, the pharmacophore subpocket and the nonpharmacophore subpocket. The pharmacophore subpocket is very sensitive to structural changes, and thus, it appears important to keep one of the glutamic acid moieties intact to maintain the potency of the GCPII inhibitors. The site encompassing the nonpharmacophore subpocket that binds to glutamate's α-carboxyl group is sensitive to structural change, as shown by compounds 6b and 7b. However, the other region of the nonpharmacophore subpocket can accommodate both hydrophobic and hydrophilic groups. Thus, an aromatic ring can be introduced to the inhibitor, as in 8b and 8d, thereby increasing its hydrophobicity and thus potentially its ability to cross the blood-brain barrier. Intrathecally administered 8d significantly reduced pain perception in the formalin model of rat sensory nerve injury. A maximal dose of morphine (10 mg) applied in the same experimental paradigm provided no significant increase in analgesia in comparison to 8d during phase 1 of this pain study and modestly greater analgesia than 8d in phase 2. These urea-based inhibitors of GCPII thus offer a novel approach to pain management.

Full text of DOI:10.1021/jm0306226

J . Med. Chem. 2004, 47, 1729-1738
1729
Syn th esis of Ur ea -Ba sed In h ibitor s a s Active Site P r obes of Glu ta m a te
Ca r boxyp ep tid a se II: Effica cy a s An a lgesic Agen ts
Alan P. Kozikowski,*,† J iazhong Zhang,† Fajun Nan,† Pavel A. Petukhov,† Ewa Grajkowska,‡
J arda T. Wroblewski,‡ Tatsuo Yamamoto,§ Tomasz Bzdega,# Barbara Wroblewska,# and J oseph H. Neale#
Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, 539 College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, Department of Pharmacology,
Georgetown University Medical Center, 3970 Reservoir Road, N.W., Washington, D.C. 20057,
Departments of Anesthesiology and Neurobiology, Graduate School of Medicine, Chiba University,
Chiba 260-8670, J apan, and Department of Biology, Georgetown University, Washington, D.C. 20057
Received December 23, 2003
The neuropeptidase glutamate carboxypeptidase II (GCPII) hydrolyzes N-acetyl-L-aspartyl-L-
glutamate (NAAG) to liberate N-acetylaspartate and glutamate. GCPII was originally cloned
as PSMA, an Mr 100 000 type II transmembrane glycoprotein highly expressed in prostate
tissues. PSMA/GCPII is located on the short arm of chromosome 11 and functions as both a
folate hydrolase and a neuropeptidase. Inhibition of brain GCPII may have therapeutic potential
in the treatment of certain disease states arising from pathologically overactivated glutamate
receptors. Recently, we reported that certain urea-based structures act as potent inhibitors of
GCPII (J . Med. Chem. 2001, 44, 298). However, many of the potent GCPII inhibitors prepared
to date are highly polar compounds and therefore do not readily penetrate the blood-brain
barrier. Herein, we elaborate on the synthesis of a series of potent, urea-based GCPII inhibitors
from the lead compound 3 and provide assay data for these ligands against human GCPII.
Moreover, we provide data revealing the ability of one of these compounds, namely, 8d , to
reduce the perception of inflammatory pain. Within the present series, the γ-tetrazole bearing
glutamate isostere 7d is the most potent inhibitor with a Ki of 0.9 nM. The biological evaluation
of these compounds revealed that the active site of GCPII likely comprises two regions, namely,
the pharmacophore subpocket and the nonpharmacophore subpocket. The pharmacophore
subpocket is very sensitive to structural changes, and thus, it appears important to keep one
of the glutamic acid moieties intact to maintain the potency of the GCPII inhibitors. The site
encompassing the nonpharmacophore subpocket that binds to glutamate’s R-carboxyl group is
sensitive to structural change, as shown by compounds 6b and 7b. However, the other region
of the nonpharmacophore subpocket can accommodate both hydrophobic and hydrophilic groups.
Thus, an aromatic ring can be introduced to the inhibitor, as in 8b and 8d , thereby increasing
its hydrophobicity and thus potentially its ability to cross the blood-brain barrier. Intrathecally
administered 8d significantly reduced pain perception in the formalin model of rat sensory
nerve injury. A maximal dose of morphine (10 mg) applied in the same experimental paradigm
provided no significant increase in analgesia in comparison to 8d during phase 1 of this pain
study and modestly greater analgesia than 8d in phase 2. These urea-based inhibitors of GCPII
thus offer a novel approach to pain management.
In tr od u ction
covery of selective agonists and antagonists of glutamate
receptors that possess therapeutic potential for these
conditions. Although several drugs designed to attenu-
ate the pathological consequences of excessive glutamate
activation have been shown to reduce injury in experi-
mental models of cerebral ischemia, so far none of these
compounds has proven to be effective in the clinical
treatment of stroke.1
L-Glutamate is one of the most abundant excitatory
neurotransmitters found in the mammalian brain.
Through its actions on ionotropic and metabotropic
receptors, glutamate plays an important role in a variety
of physiological functions including learning, memory,
and developmental plasticity. Excessive glutamatergic
transmission has been implicated in the pathogenesis
of a host of neurological disorders, including excitotoxic
cell death associated with ischemia and central nervous
system trauma, inflammatory and neuropathic pain,
and chronic neurodegenerative diseases. Over the past
decade, considerable effort has been given to the dis-
N-Acetyl-L-aspartyl-L-glutamate (NAAG), a major
peptide neurotransmitter in the mammalian nervous
system,2 acts as an agonist at group II metabotropic
glutamate receptors on neurons and glia.3 The neuro-
peptidase glutamate carboxypeptidase II (GCPII) is an
extracellular enzyme that hydrolyzes NAAG to N-
acetylaspartate and glutamate following the release of
the peptide into the synaptic space.4 There appears to
be a high level of NAAG metabolism in the brain, and
inhibition of the peptidase activity has been shown to
* To whom correspondence should be addressed. Phone: 202 687
0686. Fax: 202 687 5065. E-mail: kozikowa@uic.edu.
† University of Illinois at Chicago.
‡ Georgetown University Medical Center.
§ Chiba University.
#
Georgetown University.
10.1021/jm0306226 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/02/2004

1730 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Kozikowski et al.
substantially reduce extracellular levels of glutamate
under conditions of intense synaptic release.5 Thus, the
inhibition of peptidase activity against NAAG has the
potential to provide protection in clinical conditions in
which glutamate mediates and mGluR3 activation
reduces clinical pathology.5a,6 Reduced blood flow to the
brain as occurs during stroke and cardiac arrest results
in a high level of excitatory transmitter release in the
brain, including the release of glutamate and NAAG.
The high glutamate levels are responsible for a cascade
of events that lead to a delayed nerve cell death.
Additionally, glutamate has long been recognized as a
spinal sensory transmitter with a significant role in pain
perception and the physiological events that mediate
chronic pain perception associated with inflammation
or peripheral nerve irritation (allodynia). Activation of
group II mGluRs (mGluR2 and mGluR3) has been
shown to reduce perception of this type of pain.7 For
these reasons, decreasing extracellular glutamate and
increasing the concentration of the group II agonist
NAAG by inhibition of brain peptidase activity against
this peptide represent a significant new therapeutic
strategy.
F igu r e 1. Previously reported GCPII inhibitors.
GCPII inhibitors with enhanced lipophilicity. The syn-
thesis, enzyme inhibitory activities, and analgesic quali-
ties of our new compounds are described herein.
Additionally, in light of the identity of human GCPII
with prostate specific membrane antigen, a type II
transmembrane glycoprotein highly expressed in pros-
tate hyperplasia, inhibitors with high affinity for the
active site of GCPII may also find applications in
prostate tumor diagnosis and therapy.
2-PMPA (1) is a potent inhibitor of GCPII8 and has
been shown to provide significant protection against
injury in rats after transient middle cerebral artery
occlusion.5a In this model system, 2-PMPA substan-
tially reduced the large increase in extracellular
glutamate levels that occurs immediately after induction
of ischemia while having no detectable effect on
glutamate levels under normal conditions. 2-PMPA also
has been found to reduce the perception of chronic pain
in rodent models.9 As a therapeutic target, GCPII
inhibition has been suggested to have potential benefits
over the existing receptor-based strategies because it
represents an upstream mechanism for regulation of
synaptic glutamate that could reduce transmission at
a number of glutamatergic receptors rather than inhib-
iting a single receptor subtype. Equally important,
NAAG is colocalized in neurons with small amine
transmitters, including glutamate, GABA, acetylcholine,
and dopamine,10 and it has been shown to act on
presynaptic mGluR3 receptors to inhibit transmitter
release.11 Thus, endogenous NAAG may play a signifi-
cant role in inhibiting the release of glutamate in
pathological conditions where excess glutamate is a
central factor.
Ch em istr y
The compounds that have been prepared are shown
in Figure 2. In our previous work,12b we have demon-
strated the specificity of GCPII for ligands containing
S-configured amino acids (or R in the case of cysteine)
by comparing the activity of compound 3 with that of
its two stereoisomers. We therefore limited our present
efforts to the synthesis of analogues possessing the
S-configuration in both amino acid moieties. In general,
symmetrical ureas were obtained by treating the ap-
propriate amino acid benzyl esters (in free form or as
their tosylates), which were prepared according to a
published procedure,13 with triphosgene/Et3N. The un-
symmetrical ureas were prepared through the reaction
of isocyanate 11, generated in situ from the tosylate salt
of glutamic acid dibenzyl ester and triphosgene/Et3N,
with the second amino acid benzyl ester. The benzyl
ester intermediates were then converted to the free
acids by catalytic hydrogenation (Scheme 1).
To construct the bioisosteric tetrazole analogues, the
2-cyanoethyl group was initially chosen as the N1-
protecting group of the tetrazole. Starting from com-
mercially available R-benzyl-N-Boc-L-glutamate 12, the
tetrazole moiety was elaborated according to a published
procedure as shown in Scheme 2.14 Deprotection of 14
with CF3COOH afforded the free amine, which was used
to prepare symmetrical and unsymmetrical tetrazole-
containing urea intermediates following the procedures
described above (Schemes 2 and 3). Hydrogenolysis of
the resulting benzyl ester intermediates afforded the
cyanoethyl-protected tetrazole acids 6c and 7c, which
were then further deprotected with DBU to afford, after
purification on a cation exchange column (Dowex-
50WX8-400), the free tetrazole acids 6d and 7d . Ana-
logues 6a ,b and 7a ,b, in which one or both R-carboxyl
groups are replaced by tetrazole moieties, were prepared
in a similar fashion using γ-benzyl-N-Boc-L-glutamate
as the starting material. To avoid the cation exchange
treatment necessary after cleavage of the cyanoethyl
group, we switched to benzyl as the N1-protecting group
of the tetrazole. The final deprotection could then be
Our studies are directed toward the development of
GCPII inhibitors using NAAG as the lead compound.12
We designed and synthesized a dually acting ligand,
4,4′-phosphinicobis(butane-1,3-dicarboxylic acid) (2),
which acts both as an mGluR3 selective agonist (∼30
µM) and as a potent inhibitor of GCPII (21.7 ( 2.1 nM).
Starting from this lead compound, we then designed and
synthesized several urea-based ligands, including 3 and
4, which display potent inhibitory activity toward GCPII
and possess a strong neuroprotective action. However,
many of the potent GCPII inhibitors (shown in Figure
1) prepared to date are highly polar compounds and as
such may not readily penetrate the blood-brain barrier.
Because of the relative ease with which compounds of
this structural type can be prepared, we chose to
synthesize additional examples to elucidate their SAR,
which may, in turn, lead to the discovery of more potent

Urea-Based Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1731
F igu r e 2. New inhibitors synthesized in the present study.
Sch em e 1. Synthetic Route to Compounds 5e,fa
a
Reagents: (a) triphosgene, Et3N, CH2Cl2, -78 °C to room temperature; (b) H2, 20% Pd(OH)2/C, t-BuOH; (c) 5b or 5c, Et3N, CH2Cl2.
Sch em e 2. Synthetic Scheme for the Preparation of 6d a
a
Reagents: (a) 3-aminopropionitrile fumarate, BOP, Et3N, DMF; (b) TMSN3, DIAD, Ph3P, CH3CN; (c) (i) CF3COOH, CH2Cl2; (ii)
triphosgene, Et3N, CH2Cl2, -78 °C to room temperature; (d) H2, 20% Pd(OH)2/C, t-BuOH; (e) DBU, CH2Cl2, 2 h.
carried out in a single step by catalytic hydrogenation
(Scheme 4).
Finally, a series of urea analogues containing aro-
matic substituents were prepared as shown in Scheme
5 using commercially available amino acids. The benzyl
esters of the aromatic amino acids were reacted with
isocyanate 11 to give urea intermediates 21a -d . Cata-
lytic hydrogenation of the resulting intermediates pro-

1732 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sch em e 3. Synthetic Scheme for the Preparation of 7d a
Kozikowski et al.
a
Reagents: (a) (i) CF3COOH, CH2Cl2; (ii) isocyanate 11, Et3N, CH2Cl2; (b) H2, 20% Pd(OH)2/C, t-BuOH, 1 h; (c) DBU, CH2Cl2, 2 h.
Sch em e 4. Alternative Synthetic Scheme for the Preparation of 6d a
a
Reagents: (a) BnNH2, BOP, Et3N, DMF; (b) TMSN3, DIAD, Ph3P, CH3CN; (c) (i) CF3COOH, CH2Cl2, room temp, 1 h; (ii) triphosgene,
Et3N, CH2Cl2, -78 °C to room temperature; (d) H2, 20% Pd(OH)2/C, t-BuOH.
Sch em e 5. Route to 8a -d a
a
Reagents: (a) isocyanate 11, Et3N, CH2Cl2, overnight; (b) H2, 20% Pd(OH)2/C, t-BuOH.
Ta ble 1. Inhibitory Activity of Urea Analogues against
vided the free acids 8a -d . It should be noted here that
Expressed Human GCPII
for compound 8a , partial epimerization occurred when
a
compd
ClogP
Ki (nM)
SEM
L-phenylglycine benzyl ester was used to prepare the
urea as evidenced by the 13C NMR spectra of the
tribenzyl ester intermediate and the final free acid.
However, epimerization can be avoided by reacting the
isocyanate 11 directly with free L-phenylglycine.
1
3
-1.38
-1.54
-0.70
+ 2.71
-0.70
-0.63
+ 0.59
-1.12
-4.50
-4.62
-3.66
-3.78
-3.02
-3.08
-2.60
-2.66
-0.07
+ 0.43
-0.55
-0.05
1.4
8.0
1285
392
1109
939
95.3
14.4
>100000
4388
4434
14.9
2711
335
(0.09
(0.6
(67
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
(56
(62
(37
(7.7
(0.7
Resu lts a n d Discu ssion
All new compounds were tested for their ability to
inhibit human GCPII using conditions described in the
Experiment Section. For comparison purposes, 2-PMPA
1 and urea 3 were assayed in parallel with the com-
pounds reported herein and found to have Ki values of
1.4 and 8.0 nM, respectively. The inhibitory activities
of the new compounds and their calculated log P values
are listed in Table 1 with full dose-response curves
shown in Figure 4. All four symmetrical alkylated urea
analogues 5a -d were found to be almost inactive, with
Ki values of greater than 300 nM against expressed
human GCPII. In comparison, the unsymmetrical
monoalkylated ureas 5e and 5f showed some inhibitory
activity with Ki values of 95.3 and 14.4 nM, respectively.
Thus, incorporation of alkyl substituents into the urea
structure decreases enzyme inhibitory activity.
One possible explanation is that the alkyl substituent
cannot be sterically accommodated by the binding
pocket or alter the inhibitor’s conformation and conse-
quently the location of the carboxylic acid group. Our
modeling study15 of the active site of GCPII complexed
with the phosphonate/phosphinate inhibitors 1 and 2
indicates that there are two binding regions in the
enzyme active site, one for each pentanedioic acid
moiety. The two carboxyl groups of one of these moieties
interact with the residues of the enzyme through
hydrogen bonds in one region, namely, the pharmoco-
(211
(165
(0.8
(211
(35
(0.3
(0.05
(0.1
(0.9
(0.3
(0.2
5.3
0.9
2.1
12.0
5.9
3.0
a
Calculated using the KowWin program (http://esc.syrres.com/
interkow/kowdemo.htm).
phore subpocket. Within the other region, referred to
as the nonpharmocophore subpocket, another pentane-
dioic acid moiety can interact with the enzyme active
site through hydrogen bonding or hydrophobic interac-
tions. Each region has two binding sites as shown in
Figure 3. On the basis of the inhibitory data obtained
in the present work, we can hypothesize that the
pharmacophore subpocket has low tolerance to struc-
tural changes of the inhibitors while the nonpharma-
cophore subpocket has a higher tolerance to structural
changes. For good enzyme inhibitory action, both of the
subpockets should be engaged by the inhibitor. There-
fore, it appears necessary to keep one glutamic acid unit
intact.

Urea-Based Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1733
nM, while the other three were found to be almost
inactive. The low activity of the 2-cyanoethyl protected
tetrazoles 6a and 6c is readily explained by steric
interference in the low-tolerance pharmacophore sub-
pocket. The result that the R-tetrazole 6b was almost
inactive while the γ-tetrazole 6d was fairly active may
indicate that one part (S1 or S1′) of the enzyme active
site can accommodate a limited amount of steric bulk
while the other part (S2 or S2′) is intolerant even of
minor steric effects. To obtain further insight into the
active site of the enzyme, the unsymmetrical tetrazole-
containing ureas 7a -d were prepared. Compound 7d
exhibits high inhibitory activity with a Ki value of 0.9
nM. Interestingly, the 2-cyanoethyl-protected γ-tetrazole
7c is also fairly potent (Ki ) 5.3 nM). This result
supports our hypothesis that the nonpharmacophore
subpocket (site S1′) is relatively large. The free and
2-cyanoethyl-protected R-tetrazoles 7a and 7b exhibit
low activity, suggesting that the sites S2 and S2′ are
intolerant to structural changes.
F igu r e 3. Proposed binding domains of GCPII inhibitors in
the active site of GCPII.
The above results indicate that only the sites S1 and
S1′ in the enzyme active site permit structural modifi-
cations that increase steric bulk. In particular, the site
S1′ in the nonpharmacophore subpocket is tolerant of
relatively large hydrophobic groups, as demonstrated
by the relatively high activity of compound 7c, whereas
only small changes are tolerated at the site S1 in the
pharmacophore subpocket. In response to this finding,
we investigated the introduction of an aromatic ring into
one of the amino acid moieties, which would increase
the lipophilicity of the inhibitors and possibly facilitate
their penetration of the blood-brain barrier. The un-
symmetrical phenyl-substituted ureas 8a and 8b were
prepared from commercially available amino acids. To
our pleasant surprise, both compounds are fairly potent
inhibitors of expressed GCPII, with Ki values of 2.1 and
12.0 nM, respectively. Compound 8b is particularly
interesting in light of its positive ClogP value (+0.43).
Since the S1′ subpocket readily accommodates certain
hydrophilic groups, as demonstrated by compounds 2,
3, 6d , and 7d , we next prepared the p-hydroxylated
ureas 8c and 8d from the corresponding amino acids.
Both exhibit high GCPII inhibitory activity, with Ki
values of 5.9 and 3.0 nM, respectively.
Because a decision was made to explore the effects of
ligand 8d in an animal model of pain, we chose to better
assess the druglikeness of 8d . Both its Caco2 and
blood-brain barrier (BBB) permeability were calculated
and compared to those of certain known drugs. The
results of these calculations employing commercial
software available from Tripos are shown in Figure 5.
According to the calculations, compound 8d has Caco2
and BBB permeability comparable to that of cefotaxime,
chlortetracycline, and dimethylchlortetracycline. At least
one of these drugs, cefotaxime, a well-known antibiotic
for the treatment of meningitis, penetrates the BBB.
F igu r e 4. Inhibition of human GPCII by urea analogues.
Dose-response curves of the indicated compounds were gener-
ated in the presence of 3 µM NAAG, as described in the
methods section. In each figure, 2-PMPA (1) and lead com-
pound 3 were used as the reference compounds. Points
represent the mean ( SEM from 8 to 12 experiments.
Because the introduction of even a small alkyl sub-
stituent (analogue 5f) reduced activity, we turned our
attention to the preparation of bioisosteres of the lead
urea 3. The tetrazole moiety is an often-used surrogate
for the carboxyl group. First, we examined the activity
of the symmetrical N-protected and unprotected tetra-
zole-containing urea analogues 6a -d . Among those
compounds, only compound 6d exhibited relatively high
binding affinity to the enzyme, with a Ki value of 14.9
In Vivo Assa y. F or m a lin Test. In the saline-treated
rats, a subcutaneous injection of formalin in the footpad
resulted in a highly reliable biphasic display of flinching
of the injected paw (Figure 6), and this behavior was
comparable to that previously reported.9a Intrathecal
administration of 8d 10 min prior to formalin injection
decreased both the phase 1 and phase 2 flinching
response in a dose-dependent manner (phase 1, p <

1734 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Kozikowski et al.
F igu r e 5. (A) Projection of BBB PLS scores of 8d (red circle) on BBB PLS scores of compounds with known BBB properties (red,
high permeability; blue, low permeability). (B) Projection of Caco2 PLS scores of 8d (red circle) on Caco2 PLS scores of compounds
with known Caco2 properties (red, high permeability; blue, low permeability).
spinal cord rather than the periphery. To confirm this,
1000 µg of 8d was injected peripherally without effect
in this pain model. Further, the data in Figure 6 as well
as the dose-response data indicate that 8d was com-
parably effective in the two distinct phases of the
inflammatory pain response.
Con clu sion s
A series of urea-based compounds have been synthe-
sized and screened for their inhibitory activity in vitro
against expressed human GCPII. Several conclusions
can be drawn from these studies: (1) the active site of
GCPII comprises two regions, namely, the pharmaco-
phore subpocket and the nonpharmacophore subpocket;
(2) the pharmacophore subpocket is very sensitive to
structural changes, and it is important to keep one of
the glutamate moieties intact to maintain the potency
F igu r e 6. Biphasic pain responses following footpad injection
of formalin in the rat. Phase 1 (0-10 min) and phase II (10-
of the GCPII inhibitors; (3) the active site within the
nonpharmacophore subpocket that binds to glutamate’s
R-carboxyl group is sensitive to structural change, as
shown by compound 7b; (4) the other active site of the
nonpharmacophore subpocket can accommodate both
hydrophobic and hydrophilic groups. Importantly, an
aromatic ring can be introduced into the inhibitor,
thereby increasing its hydrophobicity and thus poten-
tially its ability to cross the blood-brain barrier. Among
the compounds tested, compounds 7d , 8a , and 8d show
an inhibitory activity in vitro that is comparable or even
slightly superior to that of the reported inhibitor,
2-PMPA (1). In vivo, intrathecal administration of 8d
also mimics the effects of 2-PMPA and morphine in
reducing the response to inflammatory pain evoked by
footpad injection of formalin,9a thus further supporting
the development of GCPII inhibitors for therapeutic
purposes. In light of our findings that these compounds
significantly interact with GCPII/PSMA, the exploration
of the present class of ligands for use in prostate tumor
therapy and diagnosis is also underway.17
60 min) responses were assayed for 1 min at 5 min intervals.
8d and morphine were administered 10 min before formalin
injection. Each point represents the mean ( SEM for five (8d )
or six (morphine) rats. 8d reduced phase 1 and phase 2
flinching behavior at p < 0.05 by one-way ANOVA. Saline
versus 8d or morphine (phase 1, p < 0.01; phase 2, p < 0.001;
by one-way ANOVA). An amount of 10 µg of morphine is
significantly different in phase II from the amount of 8d (p <
0.05 by one-way ANOVA). There is no difference in the phase
I response for these two compounds.
0.01; phase 2, p < 0.001; by one-way ANOVA). There
was no difference between the phase 1 dose-response
curve and the phase 2 dose-response curve in 8d
treated rats (p > 0.6, by two-way ANOVA). A maximal
dose of morphine (10 µg) applied in the same experi-
mental paradigm provided no significant increase in
analgesia in comparison to 8d during phase 1 of this
pain study and modestly greater analgesia than 8d
during phase II (p < 0.05 by one-way ANOVA). 8d
reduced phase 1 and phase 2 flinching behavior in a
dose-dependent manner at doses between 10 and 1000
µg (p < 0.05 for both phases between 10 and 1000 mg).
While the inflammatory pain model can be affected by
both central and peripheral drug actions, the efficacy
of 8d by intrathecal injection clearly supports the
conclusion that the drug was acting directly in the
Exp er im en ta l Section
VolSu r f Ca lcu la tion s. VolSurf Caco2 and BBB properties
of 8d were calculated using precalculated Caco2 and BBB
models in VolSurf.16 Compound 8d was projected on the plot

Urea-Based Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1735
with the compounds with known Caco2 and BBB properties
for comparison. The following parameters were used for the
VolSurf setup: Caco2 model contained 396 compounds, BBB
model contained 297 compounds, prediction type was set to
PLS, and two first principal components (PC 1 and 2) were
used to create plots.
2.47 (m, 2H), 2.28-2.18 (m, 2H); 13C NMR δ 172.33, 157.64,
154.92, 135.20, 128.64, 128.45, 128.04, 116.61, 67.32, 52.44,
42.25, 29.27, 19.43, 18.33.
Rep r esen ta tive P r oced u r e for Ben zyl Ester Hyd r o-
gen olysis (Meth od D): (S,S)-4,4′-Bis[1-(2-cya n oeth yl)-1H-
tetr a zol-5-yl]-2,2′-u r eylen ed ibu tyr ic Acid (6c). To a solu-
tion of 15 (11 mg, 0.17 mmol) in tert-butyl alcohol (5 mL) was
added 11 mg of 20% Pd(OH)2/C (Aldrich, e50% H2O), and the
mixture was stirred under 1 atm of H2 for 1 h. The catalyst
was removed by filtration through Celite, and the filtrate was
concentrated. The residue was dissolved in 5 mL of water and
lyophilized to afford product 6c (8 mg). [R]D +5.5° (c 0.22,
Gen er a l Meth od s. CH2Cl2 was distilled from calcium
1
hydride. H and 13C NMR spectra were acquired at a proton
frequency of 300 MHz, using CDCl3 as the solvent unless noted
otherwise. 1H chemical shifts (parts per million, ppm) were
obtained using CHCl3 (δ ) 7.26, for CDCl3 as the solvent) or
HDO (δ ) 4.80, for D2O as the solvent) as the internal
standard. 13C chemical shifts (ppm) were determined with
CHCl3 (central peak δ ) 77.00, for CDCl3 as the solvent) or
MeOH (δ ) 49.15, for D2O as the solvent) as the internal
standard. TLC was performed on Merck silica gel 60F254 glass
plates. Optical rotations were measured at room temperature.
1
MeOH); H NMR (D2O) δ 4.72 (t, 4H, J ) 6.3 Hz), 4.29 (dd,
2H, J ) 4.8, 9.6 Hz), 3.19 (t, 4H, J ) 6.3 Hz), 3.10 (t, 4H, J )
7.2 Hz), 2.56-2.41 (m, 2H), 2.28-2.16 (m, 2H); 13C NMR (D2O)
δ 176.23, 159.40, 156.08, 118.42, 52.75, 42.82, 28.44, 19.49,
18.21.
R ep r esen t a t ive P r oced u r e for t h e R em ova l of t h e
2-Cya n oeth yl Gr ou p (Meth od E): (S,S)-4,4′-Di-(1H-tet-
r a zol-5-yl)-2,2′-u r eylen ed ibu tyr ic Acid (6d ). To a solution
of 6c (8 mg) in CH2Cl2 (3 mL) was added DBU (0.1 mL). The
reaction mixture was stirred at room temperature for 2 h and
then concentrated in vacuo. Filtration over a Dowex-50WX8-
400 column (H+ form; water as the eluent) afforded the product
6d (4 mg) after lyophilization. [R]D +12.0° (c 0.60, MeOH); 1H
NMR (D2O) δ 4.24-4.19 (m, 2H), 3.10 (t, 4H, J ) 7.2 Hz),
2.24-2.32 (m, 2H), 2.23-2.10 (m, 2H); 13C NMR (D2O) δ
176.48, 159.34, 156.33, 52.97, 28.96, 19.71.
Rep r esen ta tive P r oced u r e for th e Syn th esis of Un -
sym m etr ica l Ur ea s (Meth od F ): (S)-2-[3-[(S)-1-(Ben zyl-
oxyca r b on yl)-3-[1-(2-cya n oet h yl)-1H -t et r a zol-5-yl]p r o-
p yl]u r eid o]p en ta n ed ioic Acid Diben zyl Ester (16). To a
stirred solution tosylate of dibenzyl glutamate (250 mg, 0.50
mmol) and triphosgene (50 mg, 0.17 mmol) in CH2Cl2 (5 mL)
at -78 °C were added Et3N (0.23 mL, 1.64 mmol) in CH2Cl2
(1.0 mL). The reaction mixture was allowed to warm to room
temperature to afford isocyanate 11. Then the free amine,
obtained from compound 14 (127 mg, 0.30 mmol) as before,
was added to the isocyanate solution, and the mixture was
stirred overnight. The reaction mixture was diluted with
CH2Cl2, washed with H2O, and dried over Na2SO4. Filtration,
concentration, and column chromatography (SiO2, 1:20 MeOH/
CHCl3) afforded 16 (85 mg, 43%). 1H NMR (CDCl3) δ 7.35-
7.24 (m, 15H), 5.83-5.79 (m, 2H), 5.13 (s, 2H), 5.09 (s, 2H),
5.05 (s, 2H), 4.59-4.27 (m, 4H), 2.91-2.83 (m, 4H), 2.80-2.36
(m, 3H), 2.15 (m, 1H), 2.04-1.88 (m, 2H). 13C NMR (CDCl3) δ
172.99, 172.74, 172.11, 157.26, 154.81, 135.61, 135.29, 135.03,
128.59, 128.51, 128.47, 128.36, 128.24, 128.11, 127.86, 116.38,
67.40, 67.14, 66.47, 52.68, 52.24, 42.14, 30.32, 30.04, 27.16,
19.28, 18.35.
(S)-2-[(ter t-Bu toxycar bon yl)am in o]-4-[1-(2-cyan oeth yl)-
1H-tetr azol-5-yl]bu tyr ic Acid Ben zyl Ester (14). (a) Am ide
F or m a t ion (Met h od A). To a stirred solution of 3-amino-
propionitrile fumarate (1.10 g, 8.59 mmol) in DMF (30 mL)
was added N-Boc-glutamic acid R-benzyl ester (12) (2.31 g, 6.85
mmol) followed by BOP reagent (benzotriazol-1-yloxytris-
(dimethylamino)phosphonium hexafluorophosphate; 3.50 g,
7.94 mmol). The reaction mixture was cooled to 0 °C with an
ice bath, and then Et3N (2.20 mL, 15.8 mmol) was added. After
being stirred overnight at ambient temperature, the reaction
mixture was poured into ice-cold water and extracted with
EtOAc. The combined organic layers were washed successively
with 1 N HCl, H2O, saturated NaHCO3, and brine, dried over
Na2SO4, filtered, and concentrated. The residue was purified
by column chromatography (SiO2, 1:4 EtOAc/hexane) to afford
the carboxamide intermediate 13 (1.60 g, 60%).
(b) Tetr a zole F or m a tion (Meth od B). To a stirred
suspension of 13 (1.60 g, 4.11 mmol) and Ph3P (2.82 g, 10.8
mmol) in ice-cold anhydrous CH3CN (45 mL) were added
diisopropyl azodicarboxylate (DIAD) (2.20 mL, 11.2 mmol) and,
2 min later, trimethylsilyl azide (1.60 mL, 11.8 mmol) over 5
min. The heterogeneous reaction mixture was allowed to warm
to ambient temperature and then stirred overnight. To the
mixture cooled to 0 °C was added a solution of NaNO2 (0.31 g,
4.5 mmol) in H2O (1.5 mL). After 30 min, a solution of ceric
ammonium nitrate (2.50 g, 4.50 mmol) in H2O (7.0 mL) was
added, and the mixture was stirred for 20 min. The mixture
was poured into cold water and extracted with CH2Cl2. The
combined organic layers were washed with H2O, dried over
Na2SO4, and concentrated in vacuo. The residue was purified
by column chromatography (SiO2, 1:10 MeOH/CHCl3) to afford
1
14 (1.05 g, 62%). H NMR δ 7.39 (s, 5H), 5.33 (d, 1H, J ) 6.0
Hz), 5.21, 5.15 (ABq, 2H, J ) 12.3 Hz), 4.47 (t, 2H, J ) 6.9
Hz), 4.37 (m, 1H), 3.05 (t, 2H, J ) 6.9 Hz), 3.00-2.93 (m, 2H),
2.57 (m, 1H), 2.27 (m, 1H), 1.44 (s, 9H); 13C NMR δ 171.49,
155.56, 154.43, 134.99, 128.61, 128.54, 128.40, 115.90, 80.28,
67.42, 52.72, 42.23, 29.84, 28.17, 19.45, 18.46.
(2S,2′S,4R,4′R)-N,N′-Ca r bon yl-4,4′-d im eth yld iglu ta m -
ic Acid Tetr a ben zyl Ester (10a ). 10a was synthesized from
9a according to method C. 1H NMR δ 7.34-7.27 (m, 20H),
5.15-5.03 (m, 10H), 4.60-4.53 (m, 2H), 2.63-2.52 (m, 2H),
2.28-2.17 (m, 2H), 1.76-1.66 (m, 2H), 1.15 (d, 6H, J ) 7.2
Hz); 13C NMR δ 175.61, 172.74, 156.36, 135.96, 135.29, 128.53,
128.50, 128.31, 128.13, 128.08, 128.03, 67.05, 66.33, 51.61,
36.14, 17.47.
Rep r esen ta tive P r oced u r e for th e Syn th esis of Sym -
m et r ica l Ur ea s (Met h od C): (S,S)-4,4′-Bis[1-(2-cya n o-
eth yl)-1H-tetr a zol-5-yl]-2,2′-u r eylen ed ibu tyr ic Acid Di-
ben zyl Ester (15). To a stirred solution of 14 (205 mg, 0.50
mmol) in dry CH2Cl2 (3.0 mL) was added CF3COOH (1.5 mL).
After being stirred at ambient temperature for 3 h, the reaction
mixture was poured into CH2Cl2 (20 mL) and washed succes-
sively with saturated NaHCO3 and H2O. The organic layer was
dried over Na2SO4 and concentrated to afford the crude free
amine (145 mg, 92%).
To a stirred solution of the above free amine (145 mg, 0.46
mmol) in CH2Cl2 (5 mL) at -78 °C were added Et3N (64 µL,
0.46 mmol) and triphosgene (22.9 mg, 0.23 mmol) in CH2Cl2
(0.50 mL). The reaction mixture was then allowed to warm to
room temperature, stirred for an additional 2 h, diluted with
CH2Cl2, washed with H2O, and dried over Na2SO4. Filtration,
concentration, and column chromatography (SiO2, 1:10 MeOH/
CHCl3) afforded 15 (70 mg, 47%). 1H NMR δ 7.34-7.29 (m,
10H), 6.23 (d, 2H, J ) 8.1 Hz), 5.12 (t, 4H, J ) 12.9 Hz), 4.59-
4.48 (m, 2H), 4.47-4.36 (m, 4H), 2.95-2.89 (m, 8H), 2.56-
(2S,2′S,4R,4′R)-N,N′-Ca r bon yl-4,4′-d im eth yld iglu ta m -
ic Acid (5a ). 5a was synthesized from 10a according to
1
method D. [R]D +2.3° (c 0.17, MeOH); H NMR (D2O) δ 4.37
(dd, 2H, J ) 4.5, 9.9 Hz), 2.73-2.59 (m, 2H), 2.24-2.15 (m,
2H), 1.86-1.76 (m, 2H), 1.20 (d, 6H, J ) 7.2 Hz); 13C NMR
(D2O) δ 180.74, 176.84, 159.22, 51.99, 36.47, 34.88, 17.20.
(2S,2′S,4R,4′R)-4,4′-Dib en zyl-N,N′-ca r b on yld iglu t a m -
ic Acid Tetr a ben zyl Ester (10b). 10b was synthesized from
9b according to method C. 1H NMR δ 7.30-6.94 (m, 30H),
5.06-4.93 (m, 10H), 4.59-4.52 (m, 2H), 2.91-2.64 (m, 6H),
2.25-2.16 (m, 2H), 1.75-1.66 (m, 2H); 13C NMR δ 174.37,
172.53, 156.26, 138.23, 135.74, 135.21, 128.92, 128.49, 128.34,
128.24, 128.10, 128.08, 127.96, 126.42, 67.00, 66.33, 51.56,
43.58, 38.33, 34.13.
(2S,2′S,4R,4′R)-4,4′-Dib en zyl-N,N′-ca r b on yld iglu t a m -
ic Acid (5b). 5b was synthesized from 10b according to

1736 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Kozikowski et al.
method D. [R]D +11.7° (c 0.33, MeOH); 1H NMR (D2O) δ 7.26-
7.15 (m, 10H), 4.32-4.28 (m, 2H), 2.98-2.75 (m, 6H), 2.20-
2.11 (m, 2H), 1.72-1.61 (m, 2H); 13C NMR (D2O) δ 178.49,
176.32, 160.30, 140.39, 130.30, 129.57, 127.60, 53.00, 45.41,
39.78, 35.75.
(2S,2′S,4S,4′S)-N,N′-Ca r b on yl-4,4′-d im et h yld iglu t a m -
ic Acid Tetr a ben zyl Ester (10c). 10c was synthesized from
9c according to method C. 1H NMR δ 7.36-7.26 (m, 20H),
5.14-4.98 (m, 10H), 4.55 (dt, 2H, J ) 4.8, 13.2 Hz), 2.65-
2.54 (m, 2H), 2.10-2.00 (m, 2H), 1.93-1.85 (m, 2H), 1.19 (d,
6H, J ) 6.9 Hz); 13C NMR δ 176.06, 172.75, 156.70, 135.89,
135.22, 128.56, 128.54, 128.37, 128.20, 128.11, 67.19, 66.42,
51.54, 36.32, 35.92, 17.11.
(s, 2H), 4.34 (m, 1H), 2.84-2.66 (m, 2H), 2.30 (m, 1H), 2.05
(m, 1H), 1.41 (s, 9H); 13C NMR δ 171.48, 154.00, 134.96, 133.10,
129.20, 128.92, 128.61, 128.56, 128.37, 127.48, 79.95, 67.40,
52.83, 50.67, 29.77, 28.20, 19.81.
(S,S)-4,4′-Bis(1-ben zyl-1H-tetr a zol-5-yl)-2,2′-u r eylen e-
d ibu tyr ic Acid Diben zyl Ester (19). 19 was synthesized
from 18 according to method C. 1H NMR δ 7.30-7.12 (m, 20H),
6.36 (d, 2H, J ) 8.4 Hz), 5.39, 5.33 (AB, 4H, J ) 15.6 Hz),
5.04 (s, 4H), 4.58-4.51 (m, 2H), 2.89-2.72 (m, 4H), 2.35-2.24
(m, 2H), 2.17-2.05 (m, 2H); 13C NMR δ 172.11, 157.45, 154.38,
135.09, 133.14, 129.05, 128.74, 128.47, 128.30, 128.02, 127.64,
67.06, 52.47, 50.55, 29.39, 19.79.
(S)-4-[(ter t-Bu toxycar bon yl)am in o]-4-[1-(2-cyan oeth yl)-
1H-tetr a zol-5-yl]bu tyr ic Acid Ben zyl Ester . This com-
pound was prepared from N-Boc-glutamic acid R-benzyl ester
and 3-aminopropionitrile fumarate according to methods A and
(2S,2′S,4S,4′S)-N,N′-Ca r b on yl-4,4′-d im et h yld iglu t a m -
ic Acid (5c). 5c was synthesized from 10c according to method
1
D. [R]D +8.0° (c 0.21, MeOH); H NMR (D2O) δ 4.27 (dd, 2H,
1
J ) 4.8, 9.6 Hz), 2.60 (dt, 2H, J ) 6.6, 21.0 Hz), 2.11-1.90 (m,
4H), 1.18 (d, 6H, J ) 6.6 Hz); 13C NMR (D2O) δ 181.17, 176.98,
159.39, 51.93, 36.69, 34.73, 16.40.
B. H NMR δ 7.35 (s, 5H), 5.71 (d, 1H, J ) 8.1 Hz), 5.19, 5.14
(AB, 1H, J ) 7.5 Hz), 5.11 (s, 2H), 4.80-4.64 (m, 2H), 3.06 (t,
2H, J ) 6.9 Hz), 2.70-2.43 (m, 2H), 2.37-2.30 (m, 2H); 13C
NMR δ 172.28, 155.61, 155.29, 135.37, 128.42, 128.18, 128.10,
115.91, 80.68, 66.47, 43.27, 42.45, 29.77, 28.31, 27.98, 18.21.
(S,S)-4,4′-Bis[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-4,4′-ureyl-
en ed ibu tyr ic Acid Diben zyl Ester . This compound was
prepared from the preceding intermediate according to method
C. 1H NMR δ 7.40-7.25 (m, 10H), 6.52 (d, 2H, J ) 8.1 Hz),
5.29-5.21 (m, 2H), 5.08, 5.03 (AB, 4H, J ) 12.3 Hz), 4.76-
(S,S)-N,N′-Car bon yl-2,2′-dim eth yldiglu tam ic Acid Tetr a-
ben zyl Ester (10d ). 10d was synthesized from 9d according
to method C. 1H NMR δ 7.32-7.30 (m, 20H), 5.33 (s, 2H),
5.13-5.06 (m, 8H), 2.46-2.16 (m, 8H), 1.53 (s, 6H); 13C NMR
δ 174.28, 172.96, 155.24, 135.75, 135.50, 128.48, 128.45,
128.20, 128.16, 128.12, 128.08, 67.24, 66.30, 59.00, 32.11,
29.21, 23.53.
(S,S)-N,N′-Ca r bon yl-2,2′-d im eth yld iglu ta m ic Acid (5d ).
5d was synthesized from 10d according to method D. [R]D
+5.2° (c 0.57, MeOH); 1H NMR (D2O) δ 2.43-2.20 (m, 6H),
2.12-2.02 (m, 2H), 1.41 (s, 6H); 13C NMR (D2O) δ 178.57,
178.03, 157.56, 58.06, 31.46, 28.81, 22.70.
4.60 (m, 4H), 3.02 (t, 4H, J ) 6.9 Hz), 2.25-2.20 (m, 8H); 13
C
NMR δ 172.48, 156.70, 156.17, 135.51, 128.68, 128.49, 128.36,
116.01, 66.75, 42.90, 42.73, 30.01, 28.76, 18.41.
(S,S)-4,4′-Bis[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-4,4′-ureyl-
en ed ibu tyr ic Acid (6a ). 6a was prepared from the preceding
intermediate according to method D. [R]D -31.2° (c 0.67,
(2S,2′S,4R)-4-Ben zyl-N,N′-ca r b on yld iglu t a m ic Acid
Tetr a ben zyl Ester (10e). 10e was synthesized from 5b and
1
MeOH); H NMR (D2O) δ 5.22 (t, 2H, J ) 7.2 Hz), 4.82-4.77
1
(m, 4H), 3.25 (t, 4H, J ) 6.6 Hz), 2.49 (t, 4H, J ) 6.9 Hz),
2.31-2.24 (m, 4H); 13C NMR (D2O) δ 177.19, 158.04, 156.75,
118.10, 43.63, 43.16, 30.02, 27.71, 18.00.
isocyanate 11. H NMR δ 7.36-6.99 (m, 25H), 5.16-4.98 (m,
10H), 4.63-4.49 (m, 2H), 2.93 (m, 1H), 2.84-2.67 (m, 2H),
2.47-2.10 (m, 4H), 1.93 (m, 1H), 1.77 (m, 1H); 13C NMR δ
174.43, 172.71, 172.59, 172.49, 156.42, 138.22, 135.73, 135.19,
128.95, 128.55, 128.53, 128.49, 128.42, 128.40, 128.36, 128.30,
128.16, 128.04, 126.47, 67.16, 67.09, 66.40, 66.38, 52.40, 51.70,
43.62, 38.33, 33.98, 30.16, 27.97.
(S,S)-4,4′-Di-(1H -t et r a zol-5-yl)-4,4′-u r eylen ed ib u t yr ic
Acid (6b). 6b was synthesized from 6a according to method
E. [R]D -26.0° (c 0.10, MeOH); 1H NMR (D2O) δ 5.16 (dd, 2H,
J ) 6.0, 9.0 Hz), 2.52 (t, 4H, J ) 6.9 Hz), 2.38-2.29 (m, 4H);
13C NMR (D2O) δ 177.10, 158.58, 158.37, 45.22, 29.94, 27.97.
(S)-2-[3-[(S)-3-(Ben zyloxycar bon yl)-1-[1-(2-cyan oeth yl)-
1H-tetr azol-5-yl]pr opyl]u r eido]pen tan edioic Acid Diben -
zyl Ester . This compound was prepared from (S)-4-[(tert-
butoxycarbonyl)amino]-4-[1-(2-cyanoethyl)-1H-tetrazol-5-
yl]butyric acid benzyl ester and isocyanate 11. 1H NMR δ
7.37-7.26 (m, 15H), 6.35 (d, 1H, J ) 9.0 Hz), 5.98 (d, 1H, J )
7.8 Hz), 5.36, 5.31 (AB, 1H, J ) 8.1 Hz), 5.18-5.08 (m, 4H),
5.00 (s, 2H), 4.80-4.58 (m, 2H), 4.45 (m, 1H), 2.95 (t, 2H, J )
6.6 Hz), 2.61-1.90 (m, 8H); 13C NMR δ 172.61, 172.53, 172.29,
156.79, 156.38, 135.69, 135.52, 135.20, 128.57, 128.55, 128.53,
128.39, 128.33, 128.28, 128.24, 128.13, 115.97, 67.18, 66.71,
66.36, 52.69, 42.70, 42.55, 30.13, 29.99, 28.92, 27.31, 18.34.
(S)-2-[3-[(S)-3-Ca r boxy-1-[1-(2-cya n oeth yl)-1H-tetr a zol-
5-yl]p r op yl]u r eid o]p en ta n ed ioic Acid (7a ). 7a was pre-
pared from the preceding intermediate according to method
D. [R]D +10.4° (c 0.25, MeOH); 1H NMR (D2O) δ 5.25 (dd, 1H,
J ) 6.6, 8.1 Hz), 4.82-4.77 (m, 2H), 4.16 (dd, 1H, J ) 5.1, 9.3
Hz), 3.24 (t, 2H, J ) 6.6 Hz), 2.62-2.49 (m, 6H), 2.14 (m, 1H),
1.92 (m, 1H); 13C NMR (D2O) δ 177.94, 177.52, 177.50, 158.82,
156.97, 118.23, 53.77, 43.72, 43.29, 30.64, 30.25, 27.95, 26.92,
18.14.
(S )-2-[3-[(S )-3-Ca r b oxy-1-(1H -t e t r a zol-5-yl)p r op yl]-
u r eid o]p en ta n ed ioic Acid (7b). 7b was synthesized from
7a according to method E. [R]D -18.8° (c 0.46, MeOH); 1H NMR
(D2O) δ 5.18 (dd, 1H, J ) 5.4, 9.0 Hz), 4.22 (dd, 1H, J ) 5.1,
9.0 Hz), 2.57-2.46 (m, 4H), 2.39-2.11 (m, 3H), 1.98 (m, 1H);
13C NMR (D2O) δ 177.69, 177.29, 176.66, 159.15, 158.65, 53.06,
45.33, 30.43, 30.09, 28.19, 26.46.
(S)-2-[3-[(S)-1-Ca r boxy-3-[1-(2-cya n oeth yl)-1H-tetr a zol-
5-yl]p r op yl]u r eid o]p en ta n ed ioic Acid (7c). 7c was syn-
thesized from 16 according to method D. [R]D +3.7° (c 0.21,
MeOH); 1H NMR (D2O) δ 4.72 (t, 2H, J ) 6.3 Hz), 4.31 (m,
1H), 4.24 (m, 1H), 3.19 (t, 2H, J ) 6.3 Hz), 3.10 (t, 2H, J ) 7.5
(2S,2′S,4R)-4-Ben zyl-N,N′-car bon yldiglu tam ic Acid (5e).
5e was synthesized from 10e according to method D. [R]D -3.2°
(c 0.22, MeOH); 1H NMR (D2O) δ 7.39-7.26 (m, 5H), 4.25-
4.18 (m, 2H), 3.00-2.87 (m, 3H), 2.49 (t, 2H, J ) 7.2 Hz), 2.27-
2.10 (m, 2H), 2.01-1.83 (m, 2H); 13C NMR (D2O) δ 179.26,
177.64, 176.80, 176.67, 159.22, 138.74, 129.20, 128.83, 126.96,
52.96, 52.09, 44.52, 38.25, 33.07, 30.29, 26.64.
(2S,2′S,4S)-N,N′-Ca r b on yl-4-m et h yld iglu t a m ic Acid
Tetr a ben zyl Ester (10f). 10f was synthesized from 5c and
1
isocyanate 11. H NMR δ 7.33-7.31 (m, 20H), 5.18-4.96 (m,
10H), 4.59-4.50 (m, 2H), 2.60 (m, 1H), 2.49-2.31 (m, 2H),
2.24-1.85 (m, 4H), 1.20 (d, 3H, J ) 6.9 Hz); 13C NMR δ 176.08,
172.79, 172.73, 172.39, 156.59, 135.87, 135.75, 135.20, 135.17,
128.57, 128.54, 128.42, 128.39, 128.21, 128.09, 67.24, 67.22,
66.44, 66.43, 52.53, 51.62, 36.38, 35.92, 30.20, 27.88, 17.14.
(2S,2′S,4S)-N,N′-Car bon yl-4-m eth yldiglu tam ic Acid (5f).
5f was synthesized from 10f according to method D. [R]D
1
+18.0° (c 0.16, MeOH); H NMR (D2O) δ 4.17-4.09 (m, 2H),
2.48 (m, 1H), 2.37 (t, 2H, J ) 7.5 Hz), 2.10-1.78 (m, 4H), 1.06
(d, 3H, J ) 7.2 Hz); 13C NMR (D2O) δ 181.13, 177.57, 176.91,
176.67, 159.34, 52.90, 51.89, 36.71, 34.64, 30.26, 26.50, 16.42.
(S )-4-(Be n zylca r b a m oyl)-2-[(t er t -b u t oxyca r b on yl)-
a m in o]bu tyr ic Acid Ben zyl Ester (17). 17 was synthesized
1
from 12 and benzylamine according to method A. H NMR δ
7.29-7.17 (m, 10H), 6.97 (br, 1H), 5.68 (d, 1H, J ) 8.1 Hz),
5.11, 5.04 (ABq, 2H, J ) 12.3 Hz), 4.30 (d, 2H, J ) 5.4 Hz),
4.25 (m, 1H), 2.32 (t, 2H, J ) 7.2 Hz), 2.11 (m, 1H), 1.94 (m,
1H), 1.39 (s, 9H); 13C NMR δ 172.09, 171.92, 155.60, 137.90,
135.04, 128.27, 128.10, 127.94, 127.33, 127.27, 127.02, 79.64,
66.74, 53.02, 43.19, 31.94, 27.98.
(S)-4-(1-Ben zyl-1H-tetr a zol-5-yl)-2-[(ter t-bu toxyca r bon -
yl)a m in o]bu tyr ic Acid Ben zyl Ester (18). 18 was synthe-
sized from 17 according to method B. 1H NMR δ 7.37-7.12
(m, 10H), 5.44, 5.38 (ABq, 2H, J ) 15.6 Hz), 5.21 (m, 1H), 5.11

Urea-Based Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1737
Hz), 2.52-2.42 (m, 3H), 2.28-2.11 (m, 2H), 1.97 (m, 1H); 13C
NMR (D2O) δ 177.63, 176.68, 176.19, 159.48, 156.07, 118.41,
52.97, 52.68, 42.81, 30.40, 28.48, 26.51, 19.46, 18.20..
(S )-2-[3-[(S )-1-Ca r b oxy-3-(1H -t e t r a zol-5-yl)p r op yl]-
u r eid o]p en ta n ed ioic Acid (7d ). 7d was synthesized from
7c according to method E. [R]D -18.8° (c 0.46, MeOH); 1H NMR
(D2O) δ 5.18 (dd, 1H, J ) 5.4, 9.0 Hz), 4.22 (dd, 1H, J ) 5.1,
9.0 Hz), 2.57-2.46 (m, 4H), 2.39-2.11 (m, 3H), 1.98 (m, 1H);
13C NMR (D2O) δ 177.69, 177.29, 176.66, 159.15, 158.65, 53.06,
45.33, 30.43, 30.09, 28.19, 26.46.
(S)-2-[3-((S)-r-Car boxyben zyl)u r eido]pen tan edioic Acid
(8a ). 8a was prepared from isocyanate 11 and L-phenylglycine
followed by hydrogenation. [R]D +62.9° (c 0.41, MeOH); 1H
NMR (D2O) δ 7.43 (m, 5H), 5.24 (s, 1H), 4.25 (dd, 1H, J ) 5.1,
9.0 Hz), 2.47 (t, 2H, J ) 7.2 Hz), 2.17 (m, 1H), 1.95 (m, 1H);
13C NMR (D2O) δ 177.59, 176.67, 175.5, 158.96, 137.10, 129.30,
128.80, 127.41, 58.41, 52.88, 30.25, 26.55.
(S )-2-[3-[(S )-1-(Be n zyloxyca r b on yl)-2-p h e n yle t h yl]-
u r eid o]p en ta n ed ioic Acid Diben zyl Ester (21b). 21b was
synthesized from 20b and isocyanate 11. 1H NMR δ 7.36-
6.94 (m, 20H), 5.35-4.98 (m, 8H), 4.82 (m, 1H), 4.57 (m, 1H),
3.10-2.98 (m, 2H), 2.47-2.29 (m, 2H), 2.16 (m, 1H), 1.90 (m,
1H); 13C NMR δ 172.85, 172.70, 172.16, 156.31, 135.79, 135.74,
135.19, 129.45, 128.54, 128.51, 128.41, 128.20, 128.18, 126.91,
67.28, 67.11, 66.42, 53.91, 52.42, 38.49, 30.24, 27.91.
(S )-2-[3-((S)-1-Ca r b oxy-2-p h e n yle t h yl)u r e id o]p e n -
ta n ed ioic Acid (8b). 8b was synthesized from 21b according
to method D. [R]D +40.2° (c 0.18, MeOH); 1H NMR (D2O) δ
7.42-7.24 (m, 5H), 4.47 (m, 1H), 4.15 (m, 1H), 3.19 (m, 1H),
3.02 (m, 1H), 2.48-2.40 (m, 2H), 2.11 (m, 1H), 1.90 (m, 1H);
13C NMR (D2O) δ 177.69, 176.88, 159.07, 137.02, 129.50,
128.82, 127.19, 55.18, 53.03, 37.52, 30.33, 26.64.
were washed three times by centrifugation, and the final
aliquots of membranes were adjusted to a protein concentra-
tion of 20 mg/mL and stored at -80 °C. The GPCPII activity
was assayed in a two-step process. In the first step, aliquots
of membranes (4 µg) were incubated for 2 h at 37 °C in 50
mM Tris-HCl, pH 7.4, in the presence of 4 µM NAAG and the
given inhibitor, in a volume of 100 µL, to allow for the
accumulation of glutamate produced in the GPCII reaction.
In the second step, the amount of accumulated glutamate was
measured using the Amplex Red glutamic acid assay kit
(Molecular Probes), and the fluorescence was read with a
Dynex fluorescent plate reader using excitation at 530 nm and
emission at 590 nm. For all inhibitors, dose-response curves
were obtained and were used to calculate IC50 values by
nonlinear regression using the Sigma-Plot software. In each
series of experiments a concentration curve of NAAG was used
to determine the Km. The final Ki values for each inhibitor were
calculated from the respective IC50 and Km values using the
Cheng and Prusoff equation.
P a in Assa y Meth od s. The following investigations were
performed according to a protocol approved by the Institutional
Animal Care Committee of Chiba University, Chiba, J apan,
consistent with international standards for care and use of
experimental animals. Male Sprague-Dawley rats weighing
250-300 g were fitted with long-term intrathecal catheters
and examined for the effects of ZJ 17 on the formalin test.
In tr a th eca l Ca th eter s. Chronic intrathecal catheters were
inserted, during halothane anesthesia, by passing a PE-10
catheter through an incision in the atlanta-occipital membrane
to a position 8 cm caudal to the cisterna at the level of lumbar
enlargement.9a,b The catheter was externalized on the top of
the skull and sealed with a steel wire, and the wound was
closed with 3-0 silk sutures. The animals were allowed to
recover for 1 week before being used experimentally. All
animals displayed normal feeding and drinking behavior
postoperatively. Rats showing neurological deficits were not
studied.
(S)-2-[3-[(S)-r-(Ben zyloxyca r bon yl)-4-h yd r oxyben zyl]-
u r eid o]p en ta n ed ioic Acid Diben zyl Ester (21c). 21c was
1
synthesized from 20c and isocyanate 11. H NMR (CDCl3) δ
7.37-7.16 (m, 15H), 7.09 (d, 2H. J ) 8.7 Hz), 6.58 (d, 2H, J )
8.7 Hz), 6.21 (br, 1H), 5.79 (br, 1H), 5.42-5.38 (m, 2H), 5.16-
5.05 (m, 6H), 4.54 (m, 1H), 2.48-2.30 (m, 2H), 2.16 (m, 1H),
1.96 (m, 1H).
F or m a lin Test. To carry out the formalin test, the animal
was briefly anesthetized with halothane (4%). When there was
momentary loss of spontaneous movement with preservation
of the deep spontaneous respiration, blink and pinnae reflexes,
50 µL of 5% formalin was injected subcutaneously into the
dorsal surface of the right hind paw with a 27-gauge needle.
Immediately after the formalin injection, the animal was
placed in an open Plexiglas box (10 cm × 20 cm × 24 cm),
which permitted observation. Within 1 min after the formalin
injection, the rat held its injected pay just off the floor, a
behavior typical of this model. During this period, spontaneous
flinching of the injected paw could also be observed. Flinching
is readily discriminated and is characterized as a rapid and
brief withdrawal or flexion of the injected paw. This pain-
related behavior was quantified by counting the number of
flinches for 1 min periods at 1-2 and 5-6 min and then for 1
min periods at 5 min intervals during the period from 10 to
60 min after the injection. Two phases of spontaneous flinching
behavior were observed. An initial acute phase (phase 1,
during the first 6 min after the formalin injection) was followed
by a relatively short quiescent period and then by a prolonged
tonic response (phase 2, beginning about 10 min after the
formalin injection). After the observation period, the animals
were immediately killed with an overdose of barbiturate.
(S)-2-[3-((S)-r-Ca r b oxy-4-h yd r oxyb en zyl)u r eid o]p en -
ta n ed ioic Acid (8c). 8c was synthesized from 21c according
to method D. [R]D +85.7° (c 0.64, MeOH); 1H NMR (D2O) δ
7.29 (d, 2H, J ) 8.1 Hz), 6.89 (d, 2H, J ) 8.1 Hz), 5.18 (s, 1H),
4.25 (dd, 1H, J ) 5.1, 8.7 Hz), 2.46 (t, 2H, J ) 6.9 Hz), 2.13
(m, 1H), 1.94 (m, 1H); 13C NMR (D2O) δ 177.57, 176.52, 175.46,
159.09, 156.20, 129.26, 128.22, 116.18, 57.52, 52.83, 30.29,
26.57.
(S )-2-[3-[(S )-1-(B e n zy lo x y c a r b o n y l)-2-(4-h y d r o x y -
p h en yl)eth yl]u r eid o]p en ta n ed ioic Acid Diben zyl Ester
1
(21d ). 21d was synthesized from 20d and isocyanate 11. H
NMR δ 7.55 (m, 1H), 7.32-7.22 (m, 15H), 6.67 (d, 2H, J ) 8.1
Hz), 6.58 (d, 2H, J ) 8.1 Hz), 5.88 (m, 1H), 5.65 (m, 1H), 5.14-
4.90 (m, 6H), 4.82 (br, 1H), 4.61 (m, 1H), 2.98-2.86 (m, 2H),
2.44-2.25 (m, 2H), 2.11 (m, 1H), 1.81 (m, 1H); 13C NMR δ
173.34, 172.79, 172.31, 156.80, 155.31, 135.58, 135.03, 134.98,
130.54, 128.66, 128.51, 128.48, 128.44, 128.40, 128.36, 128.16,
126.71, 115.42, 67.51, 67.14, 66.46, 53.74, 52.10, 37.52, 30.18,
28.10.
(S )-2-[3-[(S )-1-Ca r b oxy-2-(4-h yd r oxyp h e n yl)e t h yl]-
u r eid o]p en ta n ed ioic Acid (8d ). 8d was synthesized from
1
21d according to method D. [R]D +34.9° (c 0.48, MeOH); H
NMR (D2O) δ 7.14 (d, 2H, J ) 7.2 Hz), 6.83 (d, J ) 7.2 Hz),
4.43 (m, 1H), 4.17 (m, 1H), 3.07 (m, 1H), 2.93 (m, 1H), 2.43 (t,
2H, J ) 6.9 Hz), 2.11 (m, 1H), 1.90 (m, 1H); 13C NMR (D2O) δ
177.49, 176.54, 176.48, 159.10, 154.52, 130.81, 128.62, 115.55,
54.99, 52.74, 36.50, 30.17, 26.43.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (Grants NS 35449, NS
38080, and NS 42672). We also thank Drs. Werner
Tueckmantel and S.-B. Rong for their help in the
preparation of this manuscript.
Biologica l Assa y Meth od s. The inhibitory activity of all
compounds was determined using a fluorescent assay of
human GCPII activity. The enzyme used in the assay was
obtained from
a Chinese hamster ovary (CHO) cell line
Refer en ces
transfected with the cloned human cDNA for GCPII using the
calcium phosphate method, as described previously.5d Cells
grown to confluency, were harvested in 50 mM Tris-HCl, pH
7.4, and subjected to freezing and thawing. Cell membranes
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