Synthesis and biological evaluation of thiol-based inhibitors of glutamate carboxypeptidase II: Discovery of an orally active GCP II inhibitor

Article abstract of DOI:10.1021/jm020515w

A series of 2-(thioalkyl)pentanedioic acids were synthesized and evaluated as inhibitors of glutamate carboxypeptidase II (GCP II, EC 3.4.17.21). The inhibitory potency of these thiol-based compounds against GCP II was found to be dependent on the number of methylene units between the thiol group and pentanedioic acid. A comparison of the SAR of the thiol-based inhibitors to that of the phosphonate-based inhibitors provides insight into the role of each of the two zinc-binding groups in GCP II inhibition. The most potent thiol-based inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (IC₅₀ = 90 nM), was found to be orally bioavailable in rats and exhibited efficacy in an animal model of neuropathic pain following oral administration.

Full text of DOI:10.1021/jm020515w

J . Med. Chem. 2003, 46, 1989-1996
1989
Syn th esis a n d Biologica l Eva lu a tion of Th iol-Ba sed In h ibitor s of Glu ta m a te
Ca r boxyp ep tid a se II: Discover y of a n Or a lly Active GCP II In h ibitor
Pavel Majer, Paul F. J ackson, Greg Delahanty, Brian S. Grella, Yao-Sen Ko, Weixing Li, Qun Liu,
Keith M. Maclin, J ana Pola´kova´, Kathryn A Shaffer, Doris Stoermer, Dilrukshi Vitharana, Eric Yanjun Wang,
Anthony Zakrzewski, Camilo Rojas, Barbara S. Slusher, Krystyna M. Wozniak, Eric Burak,
Tharin Limsakun, and Takashi Tsukamoto*
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, Maryland 21224
Received November 13, 2002
A series of 2-(thioalkyl)pentanedioic acids were synthesized and evaluated as inhibitors of
glutamate carboxypeptidase II (GCP II, EC 3.4.17.21). The inhibitory potency of these thiol-
based compounds against GCP II was found to be dependent on the number of methylene units
between the thiol group and pentanedioic acid. A comparison of the SAR of the thiol-based
inhibitors to that of the phosphonate-based inhibitors provides insight into the role of each of
the two zinc-binding groups in GCP II inhibition. The most potent thiol-based inhibitor, 2-(3-
mercaptopropyl)pentanedioic acid (IC50 ) 90 nM), was found to be orally bioavailable in rats
and exhibited efficacy in an animal model of neuropathic pain following oral administration.
Ch a r t 1
In tr od u ction
Glutamate plays a key role as an excitatory neu-
rotransmitter in both the central and peripheral nervous
systems.1,2 Excess glutamate, however, is implicated in
a number of neurological disorders, including stroke,
spinal cord injury, amyotrophic lateral sclerosis (ALS),
peripheral neuropathy, chronic pain, schizophrenia, and
epilepsy. Conventional therapeutic approaches for treat-
ing these diseases have been focused on blocking
postsynaptic glutamate receptors with small molecules.
To this end, a wide variety of glutamate receptors have
been evaluated as therapeutic targets for neurological
disorders associated with excess glutamate toxicity. Of
these glutamate receptors, the N-methyl-D-aspartate
(NMDA) receptor has been the most extensively studied
over the past decade and several NMDA receptor
antagonists are currently in clinical trials.3
An alternative therapeutic strategy to blocking
glutamate receptors would be the upstream reduction
of presynaptic glutamate. One of the major sources of
glutamate in the nervous system is believed to be
hydrolysis of N-acetylaspartyl glutamate catalyzed by
a metalloprotease glutamate carboxypeptidase II (GCP
II).4 Inhibition of GCP II, therefore, has gained consid-
erable attention as a strategy to suppress glutamate
excitotoxicity leading to neurological disorders.5 Thus,
there has been a substantial interest in the discovery
of potent and selective GCP II inhibitors.
Earlier studies on GCP II inhibitors by us and other
groups suggest that effective GCP II inhibitors are
capable of interacting with both an active site zinc ion
and the glutamate recognition site (P1′ site) of the
enzyme. Indeed, most of the published potent GCP II
inhibitors contain a glutarate moiety (including a
glutamate) in part of the molecules in addition to a zinc-
binding group.6-12 2-(Phosphonomethyl)pentanedioic
acid (2-PMPA, 1a ; Chart 1)13 is one of the earliest potent
inhibitors of GCP II with a Ki value of 0.2 nM.14 The
high potency of compound 1a can be attributed to the
strong chelation of the phosphonate group to an active
site zinc atom as well as the interaction of the glutarate
(pentanedioic acid) portion of the inhibitor with the
glutamate recognition site of GCP II. 2-PMPA has been
extensively utilized to study the mechanism and physi-
ological role of GCP II as well as the potential thera-
peutic effects of GCP II inhibition. For example, we have
demonstrated that 2-PMPA considerably reduces the
ischemia-induced rise in extracellular glutamate and
* To whom correspondence should be addressed. Phone: (410) 631-
6762. Fax: (410) 631-6797. E-mail: tsukamotot@guilfordpharm.com.
10.1021/jm020515w CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/02/2003

1990 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Sch em e 1. Synthesis of Compounds 4a and 4ca
Majer et al.
most extensively studied classes of metalloprotease
inhibitors. More importantly, many thiol-based metal-
loprotease inhibitors exhibit pharmacokinetic profiles
superior to phosphorus-based compounds. For example,
while captopril 2 is orally active, fosinoprilat 3a , a
phosphinate-based ACE inhibitor, suffers from poor oral
bioavailability and is provided as a prodrug, fosinopril
3b. The present study was driven by the idea that
replacements of the phosphonomethyl group of 1a with
thioalkyl groups could result in significant improvement
in oral bioavailability without considerable loss of
inhibitory potency against GCP II. One of the key
objectives of this study is to locate an optimal position
for the thiol group to achieve maximum affinity to GCP
II by inserting a different number of methylene units
between the thiol group and the R-position of glutarate
moiety. In this paper, we describe the design, synthesis,
and biological evaluation of 2-(thioalkyl)pentanedioic
acids 4a -f and their analogues, leading to the discovery
of a potent thiol-based GCP II inhibitor that is orally
active in an animal model of neuropathic pain.
a
Reagents and conditions: (a) THF, 2 M NaOH, 40%; (b) LDA,
ethyl 3-bromopropionate, THF, -78 °C, 16%; (c) THF, 2 M NaOH,
66%.
vigorously protects against injury in a neuronal culture
model of ischemia and in rats after transient middle
cerebral artery occlusion (MCAO).15
Subsequently, we carried out extensive SAR studies
using compound 1a as a template and identified a series
of potent phosphinate-based inhibitors of GCP II.6
Among these compounds, 2-(hydroxypentafluorophenyl-
methylphosphinoylmethyl)pentanedioic acid 1b exhib-
ited efficacy not only in the MCAO model of ischemia
(30 mg/kg iv bolus followed by 6 mg/kg/h iv infusion for
4 h) but also in the chronic constriction injury (CCI)
model of neuropathic pain (10 mg/kg ip daily). Addition-
ally, compound 1b prevents long-term diabetic neur-
opathy in type 1 diabetic BB/Wor rats.16 However, a
relatively poor pharmacokinetic profile of compound 1b,
due to its highly polar zinc-binding group phosphinate,17
limited its potential as a therapeutic agent, particularly
in the area of chronic neurological disorders such as
diabetic neuropathy.
Assuming that the phosphonate and phosphinate
functional groups are the primary cause of poor oral
bioavailability of 1a and 1b, respectively, our focus has
shifted to a different class of molecules in pursuit of the
discovery of orally active GCP II inhibitors. As repre-
sented by a potent inhibitor of angiotensin converting
enzyme (ACE), captopril 2,18 the thiols are one of the
Ch em istr y
The synthesis of 2-(thioalkyl)pentanedioic acids 4a -f
is outlined in Schemes 1 and 2. The shortest analogue
4a (n ) 0) was obtained by base-mediated hydrolysis of
diethyl 2-(acethylthio)glutarate 5.19 Compound 4b (n )
1) was prepared according to the previously described
procedure.20 Compound 4c (n ) 2) was synthesized by
alkylation of γ-thiobutyrolactone 6 with ethyl 3-bro-
mopropionate followed by base-mediated hydrolysis of
the alkylated thiolactone 7. In the final step of the
syntheses of these target compounds, it is essential to
remove residual oxygen from the solvent prior to the
base-mediated hydrolysis reaction in order to prevent
the formation of disulfide derivatives. An attempt to
synthesize 4d (n ) 3) by a similar manner using
δ-thiovalerolactone was unsuccessful because of the
formation of multiple unidentified byproducts. Alterna-
tively, 4d was synthesized using Meldrum’s acid as a
starting material as outlined in Scheme 2. Reductive
coupling of S-trityl-3-mercaptopropionic acid 8a to Mel-
Sch em e 2. Synthesis of Compounds 4d -fa
a
Reagents and conditions: (a) (i) DCC, DMAP, CH2Cl2, -5 °C; (ii) NaBH4, AcOH, CH2Cl2, -5 °C, 75% for 10a , 77% for 10b, 74% for
10c; (b) methyl acrylate, NaOAc, t-BuOH, 60 °C, 81% for 11a , 86% for 11b, 79% for 11c; (c) 1.75 M NaOH, 100 °C, >100% crude; (d)
DMSO, 130 °C, 96% for 13a , quantitative yields for 13b and 13c; (e) triisopropylsilane, TFA, CH2Cl2, room temp, 75% for 4d , 72% for 4e,
77% for 4f.

Thiol-Based Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1991
Ta ble 1. Inhibition of GCP II by Thiol-Based Inhibitors
Sch em e 3. Synthesis of Compounds 14-16a
compd
n
IC50 (nM)
4a
4b
4c
4d
4e
4f
0
1
2
3
4
5
2600 ( 800
6000 ( 2800
580 ( 190
90 ( 26
1100 ( 200
2600 ( 500
compounds. The shorter analogue 17 was synthesized
by hydrolysis of the fully protected precursor 19.22 The
synthesis of the longer analogue 18 involves 1,4-addition
of dimethyl methylphosphonate to 2-substituted acryl-
ate ester 2023 followed by hydrolysis of all the ester
groups.
a
Reagents and conditions: (a) triisopropylsilane, TFA, CH2Cl2,
room temp, 67%; (b) dimethyl sulfate, 2.75 M NaOH, 50 °C, 57%;
(c) oxygen, 1.75 N NaOH, room temp, 94%.
Sch em e 4. Synthesis of Compounds 17 and 18a
Resu lts a n d Discu ssion
In Vitr o GCP II In h ibition . The ability of com-
pounds 4a -f to inhibit GCP II was evaluated using
N-acetyl-L-aspartyl-[3H]-L-glutamate as a substrate.14
As summarized in Table 1, the inhibitory potency of
these compounds against GCP II was found to be
dependent on the number of methylene units between
the thiol group and pentanedioic acid. Compound 4d
was found to be the most potent inhibitor of GCP II with
an IC50 value of 90 nM. Increasing or decreasing the
number of methylene units between the thiol group and
pentanedioic acid led to a gradual loss of inhibitory
potencies of the resulting compounds 4a -c and 4e,f.
This observation was unexpected because prior SAR
studies on two other zinc metalloproteases, ACE24 and
carboxypeptidase A (CPA),25 suggest that a 3-mercap-
topropanoyl moiety is the most effective backbone for
the P1′ position in inhibiting these two enzymes. For
example, the optimal acyl chain length for mercaptoal-
kanoyl derivatives of proline for ACE inhibition is that
of 3-mercaptopropanoylproline, which led to discovery
of captopril 2.24 In the area of CPA, 2-benzyl-3-mercap-
topropanoic acid is the most potent thiol-based inhibitor
with a Ki value of 11 nM.25 In contrast to these
inhibitory profiles for ACE and CPA, the 3-mercapto-
propionic acid analogue 4b was a poor GCP II inhibitor
with an IC50 value of 6 µM, nearly 70-fold less potent
than the most potent inhibitor 4d .
a
Reagents and conditions: (a) 6 M HCl, reflux, 73%; (b)
dimethyl methylphosphonate, CuI, n-BuLi, -78 °C, 15%; (c) 6 M
HCl, reflux, 46%.
drum’s acid 9 gave 5-monosubstituted Meldrum’s acid
derivative 10a .21 Michael addition of 10a to methyl
acrylate followed by simultaneous hydrolysis of both
acetonide and methyl ester of 11a afforded triacid 12a .
Subsequent decarboxylation in DMSO and the removal
of the trityl group from 13a by TFA/triisopropylsilane
afforded 4d . The same method was successfully applied
to the synthesis of two other analogues 4e (n ) 4) and
4f (n ) 5) from S-trityl-4-mercaptobutanoic acid 8b and
S-trityl-5-mercaptopentanoic acid 8c, respectively.
The syntheses of several analogues of 4d are outlined
in Scheme 3. The truncated analogue of 4d , 5-mercap-
topentanoic acid 14, was obtained by removing a trityl
group from S-trityl-5-mercaptopentanoic acid 8c. Two
S-substituted analogues of 4d , the S-methyl derivative
15 and the disulfide derivative 16, were prepared by
treating 4d with dimethyl sulfate and oxygen, respec-
tively, under the basic conditions.
The shift of optimal chain length for the mercaptoal-
kanoyl moiety from mercaptopropanoyl (for ACE and
CPA) to mercaptopentanoyl (for GCP II) may be largely
due to a structural difference in the active site between
ACE/CPA and GCP II. Both ACE and CPA are known
to have one zinc ion at their active site. On the other
hand, the catalytic domain of GCP II is believed to
contain two zinc ions forming a cocatalytic active site
with metal ligands.26 Obviously, the structure of an
optimal pharmacophore would largely depend on which
As shown in Scheme 4, two homologues of 2-PMPA
1a , 2-phosphorylpentanedioic acid 17 and 2-(3-phospho-
noethyl)pentanedioic acid 18, were also synthesized in
order to extend our SAR studies to phosphonate-based

1992 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Majer et al.
Ta ble 3. Inhibition of GCP II by Phosphonate-Based Inhibitors
compd
n
IC50 (nM)
17
1
18
0
1
2
1400 ( 500
0.30 ( 0.05
1200 ( 600
or inability to bind to an active site zinc ion. Significant
loss of potency was observed even with the S-methyl
analogue 15. Kozikowski’s group recently discovered a
thiol-containing urea-based GCP II inhibitor, (R)-Cys-
C(O)-(S)-Glu.10 Interestingly, neither S-methyl28 nor
S-tert-butyl10 analogues exhibited significant loss of
inhibitory potency, indicating a distinct role for the free
sulfhydryl group of 4d as a zinc-binding group in the
interaction with GCP II. One of the potential metabo-
lites, the disulfide analogue 16, also showed significantly
lower potency than 4d against GCP II.
F igu r e 1. Double-reciprocal plot of the hydrolysis of NAAG
by GCP II in the presence of compound 4d . The units of
substrate concentration are µM, while the units of velocity are
pM/s. Concentrations of 4d were 0 (O), 50 (4), 200 (2), and
400 nM (9). Inset: Replot of the double-reciprocal plot Kmapp
Vmax vs [I].
/
Comparison of the GCP II inhibitory profile of the
thiol-based inhibitors to that of the phophonate-based
inhibitors revealed fundamental differences between the
two classes of compounds. Table 3 summarizes the effect
of the phosphonoalkyl chain length of phosphonate-
based compounds on GCP II inhibition. In striking con-
trast to thiol-based GCP II inhibitors whose potency
changes gradually upon deletion or insertion of one
methylene unit from/to 4d , both truncation (compound
17) and extension (compound 18) of 2-PMPA by one
methylene group increased IC50 value by over 3 orders
of magnitude. This difference in inhibitory profiles can
be explained by a distinct mode of interaction with the
catalytic zinc by each of the two zinc-binding groups.
Maximum interaction of the phosphonate group with a
catalytic zinc ion can be attained when the phosphonate
group bivalently coordinates with the two active site
zinc ions. Using molecular modeling studies, Kozikows-
ki’s group demonstrated that the phosphonate group of
compound 1a is finely positioned to achieve this interac-
tion while its glutarate group optimally interacts with
the glutamate recognition site of the enzyme, resulting
in extremely potent GCP II inhibition.29 The consider-
able loss of potency by its homologues 17 and 18 is
presumably due to their inability to take advantage of
both of the two key interactions simultaneously. None
of the thiol-based GCP II inhibitors are nearly as potent
as 2-PMPA because of the inability of monodentate thi-
olate to coordinate with the zinc ions. The effect of chain
length on potency is relatively modest for this class of
compounds because contribution of the thiol-zinc in-
teraction to the overall affinity is less significant
compared to contribution of the phosphonate-zinc
interaction of 1a .
Ta ble 2. Inhibition of GCP II by S-Substituted Analogues
of 4d
compd
R1
R2
IC50 (nM)
4d
14
13a
15
16
H
H
Ph3C
CH3
(CH2)2CO2H 90 ( 26
H
1400 ( 500
(CH2)2CO2H >10000
(CH2)2CO2H 6500 ( 1500
4,6-dicarboxyhexylsulfanyl (CH2)2CO2H 3100 ( 1500
zinc ion the sulfhydryl group of GCP II inhibitor
interacts with. Further discussion on this issue, how-
ever, would best await an X-ray crystal structure of a
GCP II inhibitor complex.
We conducted further kinetic studies on 4d to deter-
mine its mode of GCP II inhibition. The double-recipro-
cal plot of velocity (v) vs substrate concentration ([S])
for inhibitor 4d shows a pattern characteristic of
competitive inhibition (Ki ) 30 nM), suggesting that the
compound 4d occupies the substrate-binding pocket of
GCP II and takes advantage of bindings to both the
active site zinc and the glutamate recognition site
(Figure 1). The results demonstrate that attaching a
thiol group to the R-position of glutarate using a
methylene linker is a valid strategy to identify potent
thiol-based GCP II inhibitors.
Inhibition of GCP II by several analogues of 4d is
summarized in Table 2. A truncated analogue of 4d ,
5-mercaptopantanoic acid 14, was nearly 16-fold less
potent than 4d in inhibiting GCP II. The notable decline
in potency upon removal of a propionate portion from
4d suggests critical contribution of the P1′ side chain
of 4d to its strong affinity to GCP II. A similar trend
was observed during our SAR studies on phosphonate-
based GCP II inhibitors. A significant loss of inhibitory
potency was observed with 3-phosphonopropionic acid,
a truncated analogue that lacks the propionate P1′ side
chain of 1a .27 Lack of inhibitory potency observed with
S-trityl analogue 13a , a synthetic precursor for 4d , can
be attributed to the increased size of the molecule and/
Selectivity of Com p ou n d 4d . Since compound 4d
contains a zinc-binding group as well as a portion that
mimics glutamate, it is important to assess the selectiv-
ity of this compound for GCP II over other potentially
relevant proteins such as metalloproteases and glutamate
receptors. Thus, compound 4d was evaluated in a panel
of assays listed in Table 4 at 10 µM, over 100-fold higher

Thiol-Based Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1993
Ta ble 4. Selectivity of Compound 4d a
glutamate receptor
AMPA
kainate
NMDA (agonist)
NMDA (glycine)
zinc metalloprotease
angiotensin converting enzyme (ACE)
matrix metalloproteinase 1 (MMP-1)
matrix metalloproteinase 2 (MMP-2)
matrix metalloproteinase 3 (MMP-3)
NMDA (phencyclidine) matrix metalloproteinase 7 (MMP-7)
NMDA (polyamine)
matrix metalloproteinase 9 (MMP-9)
neutral endopeptidase (NEP)
a
Compound 4d showed significant responses (g50% stimulation
or inhibition) in none of the assays listed in this table.
than its IC50 value for GCP II (90 nM). At this
concentration, compound 4d exhibited significant activ-
ity (g50% stimulation or inhibition) against neither
glutamate receptors nor various metalloproteases. The
results confirm that compound 4d is a selective inhibitor
of GCP II and useful in evaluating pharmacological
benefit of GCP II inhibition.
F igu r e 2. Chronic constriction injury (CCI) model of neuro-
pathic pain. Compound 4d (10 mg kg-1day-1, po) significantly
attenuated CCI-induced hyperalgesic state relative to the
vehicle-treated control.
P h a r m a cok in et ic St u d ies. The phamacokinetic
data for 4d in fasted male Sprague-Dawley rats are
summarized in Table 5. Following intravenous or oral
administration (10 mg/kg) of 4d to rats, blood samples
were analyzed for plasma concentration of both 4d and
16. Compound 4d is rapidly absorbed after oral admin-
istration as indicated by a short Tmax (less than 30 min)
with a mean peak plasma concentration value of 2.23
µg/mL. As expected from pharmacokinetic studies on
captopril,30 the disulfide derivative 16 was also detected
in plasma samples after both intravenous (AUC ) 0.19
µg‚h/mL) and oral (AUC ) 0.053 µg‚h/mL) administra-
tions of 4d . These AUC values, however, are signifi-
cantly lower than those of 4d , 2.55 and 1.82 µg‚h/mL
for intravenous and oral administrations, respectively.
The oral bioavailability (F) of 4d in rats was 71%. The
pharmacokinetic evaluation in rats clearly demonstrates
that compound 4d is capable of achieving high blood
levels following oral administration. Compound 4d
represents, to the best of our knowledge, the first orally
bioavailable inhibitor of GCP II.
Con clu sion
A series of 2-(thioalkyl)glutaric acids were synthesized
and evaluated as inhibitors of GCP II. The inhibitory
potency of these compounds against GCP II was found
to be dependent on the number of methylene units
between the thiol group and pentanedioic acid. Our SAR
analysis clearly indicates that both a 5-mercaptopen-
tanoic acid backbone and a propionic acid P1′ side chain
are essential for the potent inhibition of GCP II.
Although none of the thiol-based compounds are as
nearly potent as 2-PMPA, 1a , we have shown that 4d
is orally bioavailable and efficacious in an animal model
of peripheral neuropathy by oral administration, offer-
ing ideal approaches to the management of neuropathic
pain. Further pharmacological evaluations of 4d are
currently underway in various animal models of the
neurological disorders associated with glutamate exci-
totoxicity.
Ch r on ic Con str iction In ju r y Mod el of Neu r o-
p a th ic P a in . We have subsequently tested 4d for its
antinociceptive effects using the rat chronic constriction
injury (CCI) model31 of neuropathic pain by oral admin-
istration. As shown in Figure 2, compound 4d (10 mg
kg-1 day-1) significantly reduced thermal hyperalgesia
relative to the vehicle-treated control. The antinocice-
ptive effect of the compound was significant starting
from 8 days of drug treatment and persisted to the end
of the study. Although a similar level of potency was
achieved by intraperitoneal administration of the phos-
phinate-based GCP II inhibitor 1b,6 compound 4d offers
a major advantage over 1b with regard to its route of
administration, since an orally active drug is much
preferred in the treatment of chronic pain.
Exp er im en ta l Section
Gen er a l. All reactions were performed under nitrogen.
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification.
Melting points were obtained on a Mel-Temp apparatus and
are uncorrected. 1H NMR spectra were recorded at 300 or 400
MHz. 13C NMR spectra were recorded at 75 or 100 MHz. 31P
NMR spectra were recorded at 162 MHz. Elemental analyses
were obtained from Atlantic Microlabs, Norcross, GA. S-Trityl-
3-mercaptopropionic acid 8a was purchased from Bachem,
King of Prussia, PA. Preparation of compound 10a from 8a
was previously described.21 Bioassays listed in Table 4 were
performed by MDS Pharma Services, Bothell, WA.
2-Mer ca p top en ta n ed ioic Acid (4a ). To a solution of
diethyl 2-acetysulfanylpentanedioic acid 519 (0.20 g, 0.76 mmol)
in THF (4 mL) was added 2 M NaOH (1.5 mL) at 0 °C, and
the resulting mixture was stirred under nitrogen at room
Ta ble 5. Pharmacokinetic Profile of 4d in Rats
compd
Cmax (µg/mL)
Tmax (h)
AUCinf (µg‚h/mL)
CL/F ((L/h)/kg)
T1/2 (h)
F (%)
Intravenous Administration of 4d (10 mg/kg, n ) 8)
4d
16
2.55 ( 0.51
0.19 ( 0.36
1.02 ( 0.23
1.05 ( 0.38
0.37 ( 0.06
Oral Administration of 4d (10 mg/kg, n ) 10)
4d
16
2.23 ( 1.06
0.044 ( 0.054
0.16 ( 0.19
0.90 ( 0.22
1.82 ( 0.22
1.39 ( 0.18
1.18 ( 0.30
1.94 ( 0.77
71
0.053 ( 0.034

1994 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Majer et al.
temperature overnight. After addition of water (8 mL), the
reaction mixture was washed with EtOAc (5 mL). The aqueous
layer was acidified to pH 1 with 6 M HCl and extracted with
EtOAc (20 mL × 3). The combined extracts were washed with
brine, dried over MgSO4, and concentrated. The residual solid
was recrystallized from EtOAc/hexanes to give 0.050 g of 4a
as a white solid (40% yield): mp 88.6-89.6 °C. 1H NMR (THF-
d8) δ 2.08-1.95 (m, 2H), 2.29-2.17 (m, 1H), 2.50-2.46 (m, 2H),
3.48-3.40 (m, 1H); 13C NMR (THF-d8) δ 30.2, 30.3, 39.3, 172.4,
172.9. Anal. (C5H8O4S) C, H, S.
15H); 13C NMR (CDCl3) δ 26.0, 26.1, 26.8, 28.4, 28.5, 31.6, 46.0,
66.5, 104.8, 126.5, 127.8, 129.6, 144.9, 165.4.
2,2-Dim et h yl-5-(5-t r it ylsu lfa n ylp en t yl)[1,3]d ioxa n e-
4,6-d ion e (10c). Compound 10b was prepared as previously
described for the preparation of 10a ,21 except 8c was used in
place of 8a : white solid (74% yield); 1H NMR (CDCl3) δ 1.25-
1.45 (m, 6H), 1.74 (s, 3H), 1.76 (s, 3H), 1.98-2.05 (m, 2H),
2.12-2.18 (m, 2H), 3.44 (t, J ) 5.5 Hz, 2H), 7.17-7.42 (m,
15H); 13C NMR (CDCl3) δ 26.1, 26.3, 26.9, 28.2, 28.4, 28.7, 31.8,
46.0, 66.4, 104.8, 126.5, 127.8, 129.6, 145.0, 165.5.
3-[2,2-Dim et h yl-4,6-d ioxo-5-(3-t r it ylsu lfa n ylp r op yl)-
[1,3]d ioxa n -5-yl]p r op ion ic Acid Meth yl Ester (11a ). A
solution of 10a (4.6 g, 10 mmol), methyl acrylate (4.5 mL, 50
mmol), and sodium acetate (1.64 g, 20 mmol) in tert-butyl
alcohol (15 mL) was stirred at 60 °C overnight. The solvent
was removed under reduced pressure, and the residue was
taken up with EtOAc (50 mL). The solution was washed with
aqueous 5% KHSO4 (50 mL × 3) and brine (50 mL), dried over
MgSO4, and concentrated. The solid residue was recrystallized
from EtOAc/hexanes to provide 4.42 g of 11a as a white solid
(81% yield): 1H NMR (CDCl3) δ 1.23-1.33 (m, 2H), 1.67 (s,
3H), 1.74 (s, 3H), 1.84-1.92 (m, 2H), 2.10-2.16 (m, 2H), 2.21-
2.28 (m, 2H), 2.34-2.40 (m, 2H), 3.65 (s, 3H), 7.17-7.43 (m,
15H); 13C NMR (CDCl3) δ 24.5, 29.4, 29.5, 29.6, 31.4, 32.6, 37.8,
51.9, 52.8, 66.7, 105.6, 126.7, 127.9, 129.5, 144.7, 168.4, 172.0.
3-[2,2-Dim eth yl-4,6-d ioxo-5-(4-tr itylsu lfa n ylbu tyl)[1,3]-
d ioxa n -5-yl]p r op ion ic Acid Meth yl Ester (11b). Compound
11b was prepared as described for the preparation of 11a ,
except 10b was used in place of 10a : white solid (86% yield);
1H NMR (CDCl3) δ 1.16-1.40 (m, 4H), 1.71 (s, 3H), 1.75 (s,
3H), 1.81-1.87 (m, 2H), 2.07-2.15 (m, 2H), 2.26-2.34 (m, 2H),
2.36-2.42 (m, 2H), 3.66 (s, 3H), 7.16-7.44 (m, 15H); 13C NMR
(CDCl3) δ 24.7, 28.4, 29.4, 29.5, 29.6, 31.3, 32.6, 38.3, 51.9,
53.0, 66.5, 105.7, 126.6, 127.8, 129.5, 144.8, 168.6, 172.1.
3-[2,2-Dim et h yl-4,6-d ioxo-5-(5-t r it ylsu lfa n ylp en t yl)-
[1,3]d ioxa n -5-yl]p r op ion ic Acid Meth yl Ester (11c). Com-
pound 11c was prepared as described for the preparation of
11a , except 10c was used in place of 10a : white solid (79%
yield); 1H NMR (CDCl3) δ 1.11-1.38 (m, 6H), 1.69 (s, 3H), 1.76
(s, 3H), 1.88-1.94 (m, 2H), 2.08-2.13 (m, 2H), 2.28-2.34 (m,
3-(2-Oxotetr a h yd r oth iop h en -3-yl)p r op ion ic Acid Eth yl
Ester (7). To a solution of lithium diisopropylamide (2.0 M
solution, 100 mL, 200 mmol) in THF (100 mL) was added
dropwise a solution of γ-thiobutyrolactone 6 (20.0 g, 196 mmol)
in THF (20 mL) at -75 °C, and the resulting mixture was
stirred at -75 °C for 30 min. To the mixture was added
dropwise a solution of ethyl 3-bromopropionate (39.0 g, 216
mmol) in THF (20 mL) at -75 °C. The reaction mixture was
allowed to gradually warm to room temperature overnight. The
mixture was then poured into H2O (200 mL) and extracted
with EtOAc (100 mL × 3). The combined organic layers were
dried over NaSO4 and concentrated under reduced pressure.
The residue was purified by column chromatography (10%
EtOAc/hexanes) to give 6.50 g of 7 as a colorless oil (16%
yield): 1H NMR (CDCl3) δ 1.26 (t, J ) 7.3 Hz, 3H), 1.68-1.74
(m, 1H), 1.85-1.98 (m, 1H), 2.07-2.18 (m, 1H), 2.45 (t, J )
7.8 Hz, 2H), 2.43-2.50 (m, 2H), 3.26-3.32 (m, 2H), 4.13 (q, J
) 7.0 Hz, 2H); 13C NMR (CDCl3) δ 14.6, 25.4, 30.6, 32.2, 32.2,
51.1, 60.9, 173.2, 210.1.
2-(2-Mer ca p toeth yl)p en ta n ed ioic Acid (4c). To a solu-
tion of 7 (2.10 g, 10.4 mmol) in THF (15 mL) was added 2 M
NaOH (35 mL), and the resulting mixture was stirred under
an inert atmosphere at room temperature overnight. The
reaction mixture was washed with Et2O (20 mL × 2), acidified
with 2 M HCl to pH 2, and extracted with EtOAc (20 mL ×
3). The combined organic layers were dried over MgSO4 and
concentrated under reduced pressure to give 1.33 g of 4c as a
white solid (66% yield): 1H NMR (CDCl3) δ 1.43 (t, J ) 8.3
Hz, 1H), 1.65-1.75 (m, 1H), 1.85-2.15 (m, 3H), 2.48 (t, J )
7.3 Hz, 2H), 2.40-2.80 (m, 3H), 11.0-11.7 (br, 2H); 13C NMR
2H), 2.38-2.44 (m, 2H), 3.66 (s, 3H), 7.17-7.42 (m, 15H); 13
C
(CDCl3) δ 22.6, 26.8, 32.0, 36.4, 43.6, 179.9, 181.8. Anal. (C7H12
SO4) C, H, S.
-
NMR (CDCl3) δ 25.0, 28.1, 28.7, 29.4, 29.5, 29.7, 31.7, 32.6,
38.6, 51.9, 53.1, 66.4, 105.6, 126.5, 127.8, 129.5, 144.9, 168.7,
172.1.
4-Tr itylsu lfa n ylbu tyr ic Acid (8b). To a solution of tri-
tylmercaptan (15.18 g, 55 mmol) in toluene (50 mL) was added
sodium methoxide (4.37 M solution in methanol, 27.5 mL, 120
mmol). To the mixture was slowly added a solution of 4-bro-
mobutyric acid (10.02 g, 60 mmol) in methanol (22.5 mL) at
5-10 °C. The mixture was allowed to warm to 50 °C and was
stirred for 2 h. The solvent was removed under reduced
pressure, and the residue was taken up with water (200 mL).
The aqueous solution was acidified with 1 M H2SO4 and
extracted with EtOAc (200 mL × 3). The combined extracts
were dried over MgSO4 and concentrated under reduced
pressure. The crude material was recrystallized from EtOAc/
hexanes to afford 15.20 g of 8b as a white solid (76% yield):
1H NMR (CDCl3) δ 1.67 (quint, J ) 7.1 Hz, 2H), 2.22 (t, J )
2-(3-Tr itylsu lfa n ylp r op yl)p en ta n ed ioic Acid (13a ). A
suspension of 11a (5.47 g, 10 mmol) in 1.75 N NaOH (40 mL)
was stirred at 100 °C for 3 h. The resulting homogeneous
solution was cooled to room temperature, acidified to pH 1 by
1 M H2SO4, and extracted with EtOAc. The extract was
washed with brine (100 mL × 3), dried over MgSO4, and
concentrated to give 12a as a crude material (>100% crude
yield). This material was dissolved in DMSO (30 mL), and the
solution was stirred at 130 °C for 3 h. The solvent was removed
under reduced pressure, and the residue was taken up in
EtOAc (50 mL). The organic solution was washed with water
(50 mL × 3) and brine (50 mL), dried over MgSO4, and
concentrated. The residual material was recrystallized from
EtOAc/hexanes to give 4.30 g of 13a as a white solid (96% yield
for two steps): 1H NMR (CD3OD) δ 1.26-1.43 (m, 3H), 1.44-
1.54 (m, 1H), 1.61-1.80 (m, 2H), 2.08-2.32 (m, 5H), 7.16-
7.41 (m, 15H); 13C NMR (CD3OD) δ 27.5, 28.2, 32.5, 32.6, 32.7,
45.5, 67.7, 127.7, 128.8, 130.7, 146.3, 176.7, 178.9. Anal.
(C27H28O4S) C, H, S.
7.1 Hz, 2H), 2.31 (t, J ) 7.1 Hz, 2H), 7.18-7.44 (m, 15H); 13
C
NMR (CDCl3) δ 24.0, 31.5, 33.2, 67.1, 127.0, 128.3, 130.0, 145.2,
178.9.
5-Tr itylsu lfa n ylp en ta n oic Acid (8c). By the same meth-
ods described above and by use of 5-bromovaleric acid,
compound 8c was produced as a white solid (71% yield): mp
124-125 °C; 1H NMR (CDCl3) δ 1.41 (quint, J ) 7.3 Hz, 2H),
1.58 (quint, J ) 7.4 Hz, 2H), 2.16 (t, J ) 7.3 Hz, 2H), 2.20 (t,
J ) 7.3 Hz, 2H), 7.1-7.3 (m, 9H), 7.3-7.5 (m, 6H); 13C NMR
(CDCl3) δ 23.9, 27.9, 31.4, 33.4, 66.52, 126.6, 127.8, 129.6,
144.9, 179.3.
2-(4-Tr itylsu lfa n ylbu tyl)p en ta n ed ioic a cid (13b). Com-
pound 13b was prepared as described for the preparation of
13a , except 11b was used in place of 11a : white solid
1
(quantitative yield); H NMR (CD3OD) δ 1.18-1.48 (m, 6H),
1.66-1.83 (m, 2H), 2.08-2.18 (m, 2H), 2.21-2.32 (m, 3H),
7.15-7.42 (m, 15H); 13C NMR (CD3OD) δ 27.6, 28.4, 29.5, 32.6,
32.7, 32.8, 45.8, 67.7, 127.7, 128.8, 130.8, 146.5, 176.8, 179.3.
2-(5-Tr itylsu lfan ylpen tyl)pen tan edioic Acid (13c). Com-
pound 13c was prepared as described for the preparation of
13a , except 11c was used in place of 11a : white solid
2,2-Dim eth yl-5-(4-tr itylsu lfa n ylbu tyl)[1,3]d ioxa n e-4,6-
d ion e (10b). Compound 10b was prepared as previously
described for the preparation of 10a ,21 except 8b was used in
place of 8a : white solid (77% yield); 1H NMR (CDCl3) δ 1.34-
1.45 (m, 4H), 1.74 (s, 3H), 1.75 (s, 3H), 1.92-2.02 (m, 2H),
2.10-2.19 (m, 2H), 3.41 (t, J ) 5.2 Hz, 1H), 7.15-7.45 (m,
1
(quantitative yield); H NMR (CD3OD) δ 1.08-1.56 (m, 8H),

Thiol-Based Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1995
1.70-1.83 (m, 2H), 2.07-2.15 (m, 2H), 2.22-2.34 (m, 3H),
7.15-7.42 (m, 15H); 13C NMR (CD3OD) δ 27.8, 28.4, 29.6, 29.8,
32.7, 32.8, 33.1, 45.9, 67.7, 127.7, 128.8, 130.8, 146.5, 176.8,
179.4.
by TLC (hexane/EtOAc ) 2:1, containing 2% AcOH). After
completion of the reaction, the mixture was acidified by 1 M
H2SO4 and extracted with EtOAc. The extract was dried over
MgSO4 and concentrated to give 1.95 g of 16 as a colorless oil
(94% yield): 1H NMR (CD3OD) δ 1.55-1.94 (m, 12H), 2.25-
2.47 (m, 6H), 2.62-2.78 (m, 4H); 13C NMR (CD3OD) δ 27.8,
28.4, 32.0, 32.6, 39.3, 45.6, 176.8, 179.1. Anal. (C16H26O8S2‚
0.2H2O) C, H, S.
2-P h osp h on op en ta n ed ioic Acid (17). A solution of 2-(di-
ethoxyphosphoryl)pentanedioic acid 5-tert-butyl ester 1-ethyl
ester 1922 (0.10 g, 0.28 mmol) in 6 M HCl (20 mL) was refluxed
overnight. The solvent was removed under reduced pressure
and dried by concentrating from toluene (50 mL × 3). The
residual material was lyophilized to give 0.05 g (73% yield) of
17 as a semisolid (76% yield): 1H NMR (D2O) δ 1.8-1.7 (m,
2H), 2.1-2.0 (m, 2H), 2.6-2.5 (m, 1H); 31P NMR (D2O) δ 19.4.
Anal. (C5H9O7P‚1.7H2O) C, H.
2-[2-(Dim eth oxyp h osp h or yl)eth yl]p en ta n ed ioic Acid
Di-ter t-bu tyl Ester (21). To a solution of dimethyl meth-
ylphosphonate (2.50 g, 20.0 mmol) in THF (100 mL) were
added CuI (0.380 g, 2.0 mmol) and n-BuLi (1.6 M solution,
12.5 mL, 20.0 mmol) at -78 °C, and the mixture was stirred
at that temperature for 10 min. To the mixture was slowly
added 2-methylenepentanedioic acid di-tert-butyl ester 2023
(5.13 g, 20.0 mmol) over 20 min, and the reaction mixture was
stirred at -78 °C for an additional 3 h. The mixture was
poured into ice-cold 1 M HCl (100 mL) and extracted with
EtOAc (100 mL × 3). The combined extracts were washed with
water, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(EtOAc/hexanes, 1:1) to give 1.10 g of 21 as a colorless oil (15%
yield): 1H NMR (CDCl3) δ 1.50 (m, 18H), 1.70 (m, 6H), 2.33
(m, 3H), 3.79 (m, 6H); 31P NMR (CDCl3) δ 32.6.
2-(3-Mer ca p top r op yl)p en ta n ed ioic Acid (4d ). To a solu-
tion of 13a (4.49 g, 10 mmol) in dichloromethane (15 mL) was
added trifluoroacetic acid (5 mL). The reaction mixture turned
yellowish brown. To this mixture was slowly added triisopro-
pylsilane (1.74 g, 11.0 mmol). The brown color gradually turned
light-yellow, and the temperature of the reaction spontane-
ously rose because of the exothermic reaction. After the
mixture was stirred for 30 min, the solvent was removed under
reduced pressure and the residual material was partitioned
into water and hexanes (1:1 by volume, 100 mL). The aqueous
layer was washed with hexanes (50 mL × 2), basified to pH
13 with 40% aqueous NaOH, and allowed to stand for 3 h
under an atmosphere of nitrogen to convert the cyclized
byproduct (thiolactone) back to 4d . The reaction mixture was
then acidified to pH 1 with 1 M H2SO4, saturated with sodium
chloride, and extracted with EtOAc (100 mL × 3). The
combined extracts were dried over MgSO4, concentrated, and
purified by silica gel chromatography (EtOAc/hexanes) to give
1
1.55 g of 4d as a white solid (75% yield): mp 51-53 °C; H
NMR (CD3OD) δ 1.55-1.73 (m, 4H), 1.74-1.88 (m, 2H), 2.25-
2.42 (m, 3H), 2.44-2.54 (m, 2H); 13C NMR (CD3OD) δ 24.7,
28.3, 31.9, 32.6, 32.8, 45.5, 176.8, 179.2. Anal. (C8H14O4S) C,
H, S.
Although compound 4d is stable at 25 °C in the solid state,
its aqueous solution stability is largely dependent on pH and
oxygen level of the media. To avoid formation of disulfide 16,
it is essential to degas the media by bubbling argon prior to
the preparation of an aqueous solution of 4d for biological
evaluation.
2-(4-Mer ca p tobu tyl)p en ta n ed ioic Acid (4e). Compound
4e was prepared as described for the preparation of 4d , except
13b was used in place of 13a : white solid (72% yield); 1H NMR
(CD3OD) δ 1.38-1.55 (m, 3H), 1.56-1.66 (m, 3H), 1.74-1.88
(m, 2H), 2.24-2.40 (m, 3H), 2.46-2.53 (m, 2H); 13C NMR (CD3-
OD) δ 24.7, 27.0, 28.4, 32.6, 32.8, 35.0, 45.9, 176.8, 179.3. Anal.
(C9H16O4S‚0.1H2O) C, H, S.
2-(5-Mer ca p top en tyl)p en ta n ed ioic Acid (4f). Compound
4f was prepared as described for the preparation of 4d , except
13c was used in place of 13a : white solid (77% yield); 1H NMR
(CD3OD) δ 1.26-1.68 (m, 8H), 1.72-1.88(m, 2H), 2.24-2.41
(m, 3H), 2.43-2.52 (m, 2H); 13C NMR (CD3OD) δ 24.8, 27.8,
28.4, 29.2, 32.7, 33.2, 35.0, 45.9, 176.8, 179.4. Anal. (C10H18O4S)
C, H, S.
5-Mer ca p top en ta n oic Acid (14). To a solution of 8c (0.753
g, 2.0 mmol) in dichloromethane (5 mL) were added trifluo-
roacetic acid (5 mL) and trisopropylsilane (0.348 g, 2.2 mmol)
at 0 °C, and the mixture was stirred at room temperature for
2 h. The solvent was removed under reduced pressure, and
the resulting light-yellow solid was dissolved in dichlo-
romethane and passed through a column of silica gel (dichlo-
romethane) to give 0.179 g of a colorless oil (67% yield): 1H
NMR (CD3OD) δ 1.37 (t, J ) 7.8 Hz, 1H), 1.6-1.8 (m, 4H),
2.39 (t, J ) 7.1 Hz, 2H), 2.56 (q, J ) 7.2 Hz, 2H), 7.0-8.0 (br,
1H); 13C NMR (CD3OD) δ 23.3, 24.2, 33.2, 33.3, 179.2. Anal.
(C5H10O2S) C, H. S: calcd, 23.89; found, 23.20.
2-(3-Meth ylsu lfa n ylp r op yl)p en ta n ed ioic Acid (15). To
a solution of 4d (1.00 g, 4.8 mmol) in 2.75 M NaOH (8 mL)
was added dimethyl sulfate (0.61 g, 4.8 mmol) at 0 °C, and
the mixture was stirred at 50 °C for 1 h. The reaction mixture
was acidified to pH 1 with 1 M HCl and extracted with EtOAc
(50 mL × 2). The combined extracts were dried over sodium
sulfate and concentrated to give 0.60 g of 15 as a white solid:
57% yield; mp 48-49 °C; 1H NMR (CDCl3) δ 1.57-2.04 (m,
6H), 2.09(s, 3H), 2.42-2.52 (m, 5H); 13C NMR (CDCl3) δ 15.5,
26.6, 26.9, 30.8, 32.0, 33.9, 44.5, 179.7, 182.0. Anal. (C9H16O4S)
C, H, S.
2-(2-P h osp h on oeth yl)p en ta n ed ioic Acid (18). A solution
of 21 (0.60 g, 1.6 mmol) in 6 M HCl (40 mL) was refluxed
overnight. The solvent was removed under reduced pressure
and dried by concentrating from toluene (50 mL × 3). The
residue was then purified by ion-exchange resin to give 0.20 g
of 18 as a colorless oil (46% yield): 1H NMR (D2O) δ 1.51-
1.63 (m, 6H), 2.14-2.27 (m, 3H); 31P NMR (D2O) δ 31.6. Anal.
(C7H13O7P‚1.7H2O) C, H.
GCP II Assa y. The GCP II assay was carried out as
outlined previously.14 Briefly, radiolabeled NAA[3H]G was
incubated with purified recombinant GCP II32 (20 pM) in Tris
buffer (pH 7.6, 50 mM) containing CoCl2 (1 mM) in a total
volume of 50 µL for 15 min at 37 °C. The reaction was
terminated with phosphate buffer (0.1 M, pH 7.4, 50 µL), and
the material was applied to a strong anion exchange minicol-
umn (AG 1-X8 anion exchange resin, 200-400 mesh, formate
form). The [3H]glutamate was eluted with 180 µL of formate
(1.0 M) while unreacted NAA[3H]G remained bound to the
column. The eluate was transferred to a lumaplate and dried.
The radioactivity was measured with
a top scintillation
counter. IC50 values were determined at the NAAG concentra-
tion of 30 nM. The values are the mean ( SD of two or more
independent experiments. The Ki value of compound 4d was
determined from the initial velocity of the hydrolysis at NAAG
concentrations ranging from 60 nM to 1 µM. An apparent value
of Km (Km app) was obtained from the reciprocal plot (1/v vs 1/[S])
for each set of data. The Ki value was obtained directly from
a replot of the slopes of the reciprocal plots (Kmapp/Vmax) vs
inhibitor concentration.33
CCI Stu d ies. The chronic constriction injury model of
neuropathic pain was performed as previously reported.31 In
brief, sciatic nerve ligation, consisting of four ligatures being
tied loosely around the sciatic nerve at 1 mm intervals
proximal to the nerve trifurcation, was performed on rats.
Following this treatment, rats exhibited a unilateral thermal
hyperalgesia and allodynia. Animals received compound 4d
(10 mg/kg po daily) or vehicle, starting 10 days after surgery.
Animals were habituated to the Hargreaves apparatus. The
infrared heat source was directed onto the dorsal surface of
the hindpaw, and the time taken for the animal to withdraw
its paw was noted. The difference score (between the latency
2-[3-(4,6-Dica r b oxyh exyld isu lfa n yl)p r op yl]p en t a n e-
d ioic Acid (16). A solution of 4d (2.06 g, 10.0 mmol) in 1.75
M NaOH (20 mL) was stirred vigorously in an oxygen
atmosphere for 1-2 days. Progress of the reaction was followed

1996 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Majer et al.
Dase, which converts NAAG to glutamate, reduces ischemic
brain injury. Nat. Med. 1999, 5, 1396-1402.
of the response for the paw on the operated side versus the
control side) was determined. Treatment with compound 4d
normalized the difference scores between the two paws com-
pared to the continued hyperalgesic vehicle-treated con-
trols.
(16) Zhang, W.; Slusher, B.; Murakawa, Y.; Wozniak, K. M.; Tsuka-
moto, T.; J ackson, P. F.; Sima, A. A. GCPII (NAALADase)
inhibition prevents long-term diabetic neuropathy in type 1
diabetic BB/Wor rats. J . Neurol. Sci. 2002, 194, 21-28.
(17) Krise, J . P.; Stella, V. J . Prodrugs of phosphates, phosphonates,
and phosphinates. Adv. Drug Delivery Rev. 1996, 19, 287-
310.
Refer en ces
(1) Watkins, J . C. L-Glutamate as
a central neurotransmitter:
(18) Cushman, D. W.; Ondetti, M. A. Design of angiotensin converting
enzyme inhibitors. Nat. Med. 1999, 5, 1110-1113.
(19) Garbiras, B. J .; Stephen, M. Preparation of Carboxythioalactones
and Their Active Derivatives. Synthesis 1999, 270-274.
(20) Kalman, T. I. 2-Mercaptomethylglutaric acid derivatives. U.S.
Patent 4,927,966.
(21) Hin, B.; Majer, P.; Tsukamoto, T. Facile Synthesis of R-Substi-
tuted Acrylate Esters. J . Org. Chem. 2002, 67, 7365-7368.
(22) Petter, R. C.; Banerjee, S.; Englard, S. Inhibition of γ-Butyro-
betaine Hydroxylase by Cyclopropyl-Substituted γ-Butyrobe-
taines. J . Org. Chem. 1990, 55, 3088-3097.
(23) Amri, H.; Rambaud, M.; Villieras, J . A short large scale synthesis
of (() sarkomycin esters. Tetrahedron Lett. 1989, 30, 7381-
7382.
(24) Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A.
Design of potent competitive inhibitors of angiotensin-converting
enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino acids.
Biochemistry 1977, 16, 5484-5491.
(25) Ondetti, M. A.; Condon, M. E.; Reid, J .; Sabo, E. F.; Cheung, H.
S.; Cushman, D. W. Design of potent and specific inhibitors of
carboxypeptidases A and B. Biochemistry 1979, 18, 1427-
1430.
(26) Rawlings, N. D.; Barrett, A. J . Structure of membrane glutamate
carboxypeptidase. Biochim. Biophys. Acta 1997, 1339, 247-
252.
(27) J ackson, P. F.; Slusher, B. S. Design of NAALADase inhibitors:
A novel neuroprotective strategy. Curr. Med. Chem. 2001, 8,
949-957.
Looking back. Biochem. Soc. Trans. 2000, 28, 297-309.
(2) Carpenter, K. J .; Dickenson, A. H. Amino acids are still as
exciting as ever. Curr. Opin. Pharmacol. 2001, 1, 57-61.
(3) Excitatory Amino Acids: Clinical Results with Antagonists;
Herrling, P., Ed.; Academic Press: San Diego, CA, 1997.
(4) Carter, R. E.; Coyle, J . T. Glutamate Carboxypeptidase II.
Handbook of Proteolytic Enzymes; Academic: New York, 1998;
pp 1434-1437.
(5) Whelan, J. NAALADase inhibitors: A novel approach to glutamate
regulation. Drug Discovery Today 2000, 5, 171-172.
(6) J ackson, P. F.; Tays, K. L.; Maclin, K. M.; Ko, Y. S.; Li, W.;
Vitharana, D.; Tsukamoto, T.; Stoermer, D.; Lu, X. C.; Wozniak,
K.; Slusher, B. S. Design and pharmacological activity of
phosphinic acid based NAALADase inhibitors. J . Med. Chem.
2001, 44, 4170-4175.
(7) Serval, V.; Barbeito, L.; Pittaluga, A.; Cheramy, A.; Lavielle, S.;
Glowinski, J . Competitive inhibition of N-acetylated-alpha-
linked acidic dipeptidase activity by N-acetyl-L-aspartyl-beta-
linked L-glutamate. J . Neurochem. 1990, 55, 39-46.
(8) Subasinghe, N.; Schulte, M.; Chan, M. Y.; Roon, R. J .; Koerner,
J . F.; J ohnson, R. L. Synthesis of acyclic and dehydroaspartic
acid analogues of Ac-Asp-Glu-OH and their inhibition of rat
brain N-acetylated R-linked acidic dipeptidase (NAALA dipep-
tidase). J . Med. Chem. 1990, 33, 2734-2744.
(9) 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.
(10) Kozikowski, A. P.; Nan, F.; Conti, P.; Zhang, J .; Ramadan, E.;
Bzdega, T.; Wroblewska, B.; Neale, J . H.; Pshenichkin, S.;
Wroblewski, J . T. Design of remarkably simple, yet potent urea-
based inhibitors of glutamate carboxypeptidase II (NAALADase).
J . Med. Chem. 2001, 44, 298-301.
(11) Rodriguez, C. E.; Lu, H.; Martinez, A. R.; Hu, Y.; Brunelle, A.;
Berkman, C. E. Inhibition of glutamate carboxypeptidase II by
phosphonamidothionate derivatives of glutamic acid. J . Enzyme
Inhib. 2001, 16, 359-365.
(12) Tsukamoto, T.; Flanary, J . M.; Rojas, C.; Slusher, B. S.; Valiaeva,
N.; Coward, J . K. Phosphonate and phosphinate analogues of
N-Acylated gamma-glutamylglutamate. Potent inhibitors of
glutamate carboxypeptidase II. Bioorg. Med. Chem. Lett. 2002,
12, 2189-2192.
(13) J ackson, P. F.; Cole, D. C.; Slusher, B. S.; Stetz, S. L.; Ross, L.
E.; Donzanti, B. A.; Trainor, D. A. Design, synthesis, and
biological activity of a potent inhibitor of the neuropeptidase
N-acetylated R-linked acidic dipeptidase. J . Med. Chem. 1996,
39, 619-622.
(14) Rojas, C.; Frazier, S. T.; Flanary, J .; Slusher, B. S. Kinetics and
inhibition of glutamate carboxypeptidase II using a microplate
assay. Anal. Biochem. 2002, 310, 50-54.
(15) 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.; Tortella, F. C.; Wozniak, K. M.; Yudkoff,
M.; Potter, B. M.; J ackson, P. F. Selective inhibition of NAALA-
(28) Pomper, M. G.; Musachio, J . L.; Zhou, Y.; Scheffel, U.; Hilton,
J .; Maini, A.; Dannals, R. F.; Wong, D. F.; Zhang, J .; Kozikowski,
A. P. 11C-MCG: Synthesis, Uptake Selectivity, and Primate PET
of a Probe for Glutamate Carboxypeptidase II (NAALADase).
Mol. Imaging 2002, 1, 96-101.
(29) Rong, S. B.; Zhang, J .; Neale, J . H.; Wroblewski, J . T.; Wang,
S.; Kozikowski, A. P. Molecular modeling of the interactions of
glutamate carboxypeptidase II with its potent NAAG-based
inhibitors. J . Med. Chem. 2002, 45, 4140-4152.
(30) Migdalof, B. H.; Antonaccio, M. J .; McKinstry, D. N.; Singhvi,
S. M.; Lan, S. J .; Egli, P.; Kripalani, K. J . Captopril: pharmacol-
ogy, metabolism and disposition. Drug Metab. Rev. 1984, 15,
841-869.
(31) Bennett, G. J .; Xie, Y. K. A peripheral mononeuropathy in rat
that produces disorders of pain sensation like those seen in man.
Pain 1988, 33, 87-107.
(32) Barinka, C.; Rinnova, M.; Sacha, P.; Rojas, C.; Majer, P.; Slusher,
B. S.; Konvalinka, J . Substrate specificity, inhibition and enzy-
mological analysis of recombinant human glutamate carboxy-
peptidase II. J . Neurochem. 2002, 80, 477-487.
(33) Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady-state Enzyme Systems, 1st ed.; J ohn
Wiley & Sons: New York, 1975.
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