Structural optimization of thiol-based inhibitors of glutamate carboxypeptidase II by modification of the P1′ side chain

Article abstract of DOI:10.1021/jm051019l

A series of thiol-based inhibitors containing a benzyl moiety at the P1′ position have been synthesized and tested for their abilities to inhibit glutamate carboxypeptidase II (GCP II). 3-(2-Carboxy-5-mercaptopentyl)- benzoic acid 6c was found to be the m

Full text of DOI:10.1021/jm051019l

2876
J. Med. Chem. 2006, 49, 2876-2885
Structural Optimization of Thiol-Based Inhibitors of Glutamate Carboxypeptidase II by
Modification of the P1′ Side Chain
Pavel Majer, Bunda Hin, Doris Stoermer, Jessica Adams, Weizheng Xu, Bridget R. Duvall, Greg Delahanty, Qun Liu,
Marigo J. Stathis, Krystyna M. Wozniak, Barbara S. Slusher, and Takashi Tsukamoto*
MGI Pharma, Inc., 6611 Tributary Street, Baltimore, Maryland 21224
ReceiVed October 11, 2005
A series of thiol-based inhibitors containing a benzyl moiety at the P1′ position have been synthesized and
tested for their abilities to inhibit glutamate carboxypeptidase II (GCP II). 3-(2-Carboxy-5-mercaptopentyl)-
benzoic acid 6c was found to be the most potent inhibitor with an IC50 value of 15 nM, 6-fold more potent
than 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a previously discovered, orally active GCP II inhibitor.
Subsequent SAR studies have revealed that the phenoxy and phenylsulfanyl analogues of 6c, 3-(1-carboxy-
4-mercaptobutoxy)benzoic acid 26a and 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid 26b, also possess
potent inhibitory activities toward GCP II. In the rat chronic constriction injury (CCI) model of neuropathic
pain, compounds 6c and 26a significantly reduced hyperalgesia following oral administration (1.0 mg/kg/
day).
Chart 1
Introduction
Inhibitors of glutamate carboxypeptidase II (GCP II, EC
3.4.17.21) have shown efficacy in a variety of animal models
of neurological diseases associated with glutamate excito-
toxicity.1,2 Recently, we found that 2-(3-mercaptopropyl)-
pentanedioic acid (2-MPPA, Chart 1), the first reported orally
available GCP II inhibitor, exhibits antinociceptive effects in a
rat chronic constrictive injury (CCI) model of neuropathic pain
following daily oral administration,3 thereby potentially extend-
ing the therapeutic utility of GCP II inhibitors to multiple chronic
disorders associated with glutamate excitotoxicity. Consequently,
we found that the treatment of G93A FALS (familial amyo-
trophic lateral sclerosis) transgenic mice with 2-MPPA by oral
administration resulted in statistically significant prolongation
in median survival.4 The discovery of 2-MPPA and its thera-
peutic utility in multiple animal models prompted us to further
conduct structure-activity relationship (SAR) studies on thiol-
based GCP II inhibitors.
alteration of the side chain greatly reduces GCP II inhibitory
potency.6
Over the past decades, tremendous efforts have been made
in identifying glutamate mimetics, particularly in the field of
glutamate receptor agonists and antagonists.7 A variety of
compounds have been found to elicit affinity equal to or better
than that of glutamate. Very recently, Miller’s group reported
the synthesis of conformationally constricted analogues contain-
ing a [1,2]oxazinane-3,6-dicarboxylic acid for the replacement
of the terminal glutamate residue.8 They found that IC50 values
of these compounds for GCP II were comparable to the Km value
for NAAG. More recently, crystal structures of GCP II in
complex with its inhibitors were determined at high resolution.9
Crystallographic analysis revealed that the S1′ pocket filled with
the glutarate moiety presents unoccupied hydrophobic space
formed by the side chains of Leu428, Phe209, Lys699, and
Tyr700. These new findings indicate that there is still an
opportunity to improve the potency by modifying the P1′ side
chain of 2-MPPA despite the presumed strict structural require-
ment at this part of the molecule for GCP II inhibition.
On the basis of synthetic feasibility, we have chosen to
examine thiol-based GCP II inhibitors containing a carboxy-
benzyl group at the P1′ position as a substitute for a carboxyethyl
group of 2-MPPA. In this paper, we describe the synthesis and
biological evaluation of these new thiol-based analogues, leading
to the discovery of a class of GCP II inhibitors superior to
2-MPPA in both in vitro and in vivo assessments.
Identifying more potent GCP II inhibitors using 2-MPPA as
a template, however, posed a challenge because the metallo-
peptidase has been known to have a high degree of specificity
for both substrates and inhibitors. Only two types of natural
peptides are known as substrates for GCP II, N-acetylaspartyl-
glutamate (NAAG) and folate poly-γ-glutamates. A common
feature of these peptides is the presence of acidic residues
including a C-terminal glutamate. Thus, most of the substrate-
based GCP II inhibitors contain a glutarate (pentanedioic acid)
moiety linked to a zinc binding group.5 This core structure
allows the compounds to interact with both the glutamate
recognition site of the enzyme and the active site zinc atom(s),
demonstrating a potent inhibitory effect. An attempt to further
improve the inhibitory potency by modifying the glutarate
moiety has not been vigorously pursued in the past because such
a modification is expected to cause significant loss in potency.
For instance, we have previously evaluated the influence of the
P1′ side chain variability on the binding of 2-(phosphonomethyl)-
pentanedioic acid (2-PMPA, Chart 1) and found that any
Chemistry
The synthesis of P1′-carboxybenzyl-containing analogues of
2-MPPA is outlined in Scheme 1. The previously reported
synthesis of 2-MPPA utilized monosubstituted Meldrum’s acid
1 as a key intermediate,3 which was coupled with methyl
* To whom correspondence should be addressed. Phone: (410) 631-
6762. Fax: (410) 631-6797. E-mail: takashi.tsukamoto@mgipharma.com.
10.1021/jm051019l CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/21/2006

Inhibitors of Glutamate Carboxypeptidase II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2877
Scheme 3. Synthesis of Compounds 13, 15, and 16a
Scheme 1. Synthesis of Compounds 6a-da
a Reagents and conditions: (a) K2CO3, benzyltriethylammonium chloride,
acetonitrile, 75 °C, 86% for 3a, 76% for 3b, 69% for 3c,79% for 3d; (b)
2.0 M NaOH-dioxane, 100 °C, 100% crude yield; (c) DMSO, 130 °C,
78% for 5a, 51% for 5b, 95% for 5c, 85% for 5d; (d) triisopropylsilane,
TFA, dichloromethane, room temp, 85% for 6a, 43% for 6b, 89% for 6c,
90% for 6d.
a Reagents and conditions: (a) iodomethane, sodium methoxide, metha-
nol, room temp, quantitative yield; (b) 10-camphorsulfonic acid, toluene,
reflux, 67%; (c) sodium methoxide, methanol, room temp, 83%; (d) 28%
ammonium hydroxide, room temp, 87%.
Scheme 2. Synthesis of Compounds 9 and 12a-ca
Scheme 4. Synthesis of Compounds 18, 20, and 22a
a Reagents and conditions: (a) 4.3 M NaOH-dioxane, room temp,
quantitative yield; (b) (i) thiolacetic acid, dichloromethane, DMF, room
temp; (ii) 4.3 M NaOH-dioxane, room temp, 86%; (c) methyl (3-bromo-
methyl)benzoate 2c, K2CO3, benzyltriethylammonium chloride, acetonitrile,
75 °C, 54% for 11a, 65% for 11b, 88% for 11c; (d) (i) 2.0 M NaOH-
dioxane, 100 °C; (ii) DMSO, 130 °C; (iii) triisopropylsilane, TFA,
dichloromethane, room temp, 80% for 12a, 77% for 12b, 86% for 12c.
a Reagents and conditions: (a) (i) sodium hydroxide, water-dioxane,
room temp; (ii) DMSO, 130 °C, 57%; (b) triisopropylsilane, TFA,
dichloromethane, room temp, 53%; (c) ammonium chloride, diisopropyl-
ethylamine, HATU, DMF, room temp, 98%; (d) sodium hydroxide, THF-
water, room temp, 87%; (e) oxalyl chloride, DMF, acetonitrile, 0 °C, 88%;
(f) 1 M NaOH-THF, room temp, 89%.
acrylate via Michael addition reaction. This type of coupling
reaction with 1 can also be conducted using other relatively
reactive electrophiles such as benzyl bromide as a substitute
for methyl acrylate. Indeed, reaction of 1 and benzyl bromide
2a gave disubstituted Meldrum’s acid 3a in 86% yield (Scheme
1). Hydrolysis of the acetonide ring of 3a afforded diacid 4a.
Subsequent decarboxylation in DMSO and the removal of the
trityl group by TFA/triisopropylsilane afforded 6a. The same
method was successfully applied to the synthesis of three
carboxybenzyl analogues 6b-d using the corresponding benzyl
bromide 2b-d, respectively.
Analogues of 6c with various alkyl chain lengths were
synthesized as illustrated in Scheme 2. The shortest analogue 9
was prepared from acrylate dieseter 7.10 Hydrolysis of the ester
groups provided the corresponding diacid 8. Addition of
thiolacetic acid and the subsequent deacetylation gave 9. Other
analogues 12a-c were synthesized using the same method as
described for 6c but starting with monosubstituted Meldrum’s
acids 10a-c, respectively.
methyl iodide under basic conditions. Compound 6c was
converted into the corresponding monomethyl ester 15 and
amide 16 through methanolysis and aminolysis of 14, respec-
tively. As illustrated in Scheme 4, the methyl benzoate analogue
18 was prepared from the disubstituted Meldrum’s acid 3c while
benzamide and benzonitrile analogues 20 and 22 were prepared
from a common intermediate 19.
Scheme 5 summarizes the synthesis of the ether and sulfide-
containing analogues 26a and 26b. Methyl 2,5-dibromovalerate
23 was treated with 24a or 24b and subsequently with potassium
thioacetate to give the penultimate precursor 25a or 25b,
respectively. Base-mediated hydrolysis of the methyl esters and
thioacetyl group afforded 26a and 26b.
In addition, we synthesized analogues of 6c in which its
sulfhydryl group is substituted with other zinc-binding groups
commonly used in metalloprotease inhibitors. As illustrated in
Scheme 6, the synthesis of the phosphonate-based compound
28 involves the 1,4-addition of diethyl phosphite to acrylate
dieseter 7 followed by hydrolysis of all the ester groups. The
acrylate dieseter 7 also served as a starting material for the
synthesis of the phosphinate-based compound 31 (Scheme 6).
We have also synthesized various 6c analogues where one
of its key moieties was slightly modified. As shown in Scheme
3, the S-methyl derivative 13 was prepared by treating 6c with

2878 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Scheme 5. Synthesis of 26a and 26ba
Majer et al.
Scheme 8. Synthesis of 39a
a Reagents and conditions: (a) (i) K2CO3, DMF; (ii) potassium thio-
acetate, DMF, 37% for 25a, 68% for 25b; (b) NaOH, dioxane-THF, room
temp, 69% for 26a, 86% for 26b.
a Reagents and conditions: (a) K2CO3, benzyltriethylammonium chloride,
ethyl bromoacetate, acetonitrile, 65 °C, 60%; (b) 2 M NaOH, 100 °C, 100%
crude yield; (c) DMSO, 130 °C, 68% from 37.
Scheme 6. Synthesis of 28 and 31a
Table 1. Inhibition of GCP II by Thiol-Based Inhibitors 6a-d
compd
R
IC50 (nM)a
6a
6b
6c
6d
H
1400 ( 600
1700 ( 100
15 ( 10
2-CO2H
3-CO2H
4-CO2H
63 ( 32
a Reagents and conditions: (a) diethyl phosphite, NaH, THF, room temp,
86%; (b) 12 M HCl, 100 °C, 79%; (c) BTSP, dichloromethane, room temp,
80%; (d) pentafluorobenzyl bromide, BSA, dichloromethane, 40 °C, 97%;
(e) 12 M HCl, 100 °C, 15%.
a Values are the mean ( SD of three or more independent experiments.
olefin into a carboxylic acid, followed by coupling with
benzyloxyamine gave the fully protected precursor 35. The
benzyl groups were removed by catalytic hydrogenolysis to yield
the desired compound 36.
Scheme 7. Synthesis of 36a
Compound 32 was also utilized in the synthesis of succinic
acid-based inhibitor 39 as outlined in Scheme 8. Treatment of
32 with ethyl bromoacetate gave the disubstituted Meldrum’s
acid 37. Subsequent hydrolysis and decarboxylation afforded
the desired compound 39.
Biological Results and Discussion
In Vitro GCP II Assay. The in vitro GCP II inhibitory
potencies were measured using N-acetyl-L-aspartyl-[3H]-L-
glutamate as a substrate and human recombinant GCP II13 as
previously reported.14 Table 1 summarizes the inhibitory effects
of 2-benzyl-5-mercaptopentanoic acid 6a and its carboxylated
derivatives 6b-d. The compound 6a is more than 10-fold less
potent than 2-MPPA in inhibiting GCP II. This was expected
because the loss of one of the two carboxylates weakens the
interaction with the glutamate recognition site of GCP II.
Introduction of a carboxyl group onto the phenyl ring of 6a
had an effect on the inhibitory potency in a position-dependent
manner. Ortho-substituted analogue 6b was equally potent as
6a, while the meta- and para-substituted analogues 6c and 6d
exhibited significantly improved inhibitory potency in the GCP
II assay with IC50 values of 15 and 63 nM, respectively. This
is noteworthy because these two compounds are more potent
GCP II inhibitors than 2-MPPA and represent the first successful
attempt to improve the potency by modifying the glutarate
moiety of the GCP II inhibitors.
a Reagents and conditions: (a) K2CO3, benzyltriethylammonium chloride,
allyl bromide, acetonitrile, 75 °C, 69%; (b) (i) 2 M NaOH-dioxane, 100
°C; (ii) DMSO, 130 °C; (iii) BnOH, EDC, DMAP, dichloromethane, room
temp, 77% from 33; (c) (i) RuO2, NaIO4, acetonitrile-water, room temp;
(ii) BnONH2‚HCl, EDC, DIEA, DMAP, dichloromethane, room temp, 70%;
(d) H2 (21 psi), Pd/C, MeOH, room temp, 97%.
The treatment of 7 with bis(trimethylsilyl)phosphonite (BTSP)
generated in situ from ammonium hypophosphite, chlorotri-
methylsilane, and triethylamine afforded monosubstituted phos-
phinic acid 29. Alkylation of 29 with pentafluorobenzyl bromide
was successfully accomplished using a procedure similar to the
one reported by Reiter’s group,11 providing disubstituted phos-
phinic acid 30. A subsequent acidic hydrolysis of the two methyl
ester groups gave the final product 31.
Synthesis of the hydroxamate-based compound 36 (Scheme
7) is similar to the method previously described.12 Alkylation
of 32 with allyl bromide gave the disubstituted Meldrum’s acid
33. The acetonide and methyl ester groups of 33 were
hydrolyzed by treatment with sodium hydroxide in dioxane/
water. Subsequent decarboxylation and condensation with benzyl
alcohol afforded the dibenzyl ester 34. Oxidation of the terminal
To better understand the structure-activity relationship of
this new class of thiol-based GCP II inhibitors, we selected the
most potent inhibitor 6c as a template for further structural
modification. Table 2 summarizes the effect of carbon chain

Inhibitors of Glutamate Carboxypeptidase II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2879
Table 4. Effect of P1′ Substitution on GCP II Inhibitory Potency
Table 2. Effect of Thioalkyl Chain Length on GCP II Inhibitory
Potency
compd
Z
P1′
IC50 (nM)a
compd
n
IC50 (nM)a
2-MPPA
6c
2-PMPA
28
40
31
41
36
42
HS(CH2)2
A
B
A
B
A
B
A
B
A
B
90 ( 26b
7a
7b
6c
7c
7d
1
2
3
4
5
440 ( 180
1100 ( 400
15 ( 5
390 ( 260
190 ( 70
15 ( 5
(HO)2OP
0.30 ( 0.05b
120 ( 30
(C6F5CH2)(HO)OP
HONOC
82 ( 14b
2400 ( 700
a Values are the mean ( SD of three or more independent experiments.
220 ( 40b
15000 ( 4000
20000 ( 2000b
15000 ( 5000
Table 3. Inhibition of GCP II by Analogues of 6c
HO2C
39
a Values are the mean ( SD of three or more independent experiments.
b Values have been previously reported and are included herein for reference
purposes.
compd
R
X
Y
Z
IC50 (nM)a
6c
13
5c
H
CO2H
CO2H
CO2H
CO2CH3
CONH2
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
H
CO2CH3
CONH2
CN
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
15 ( 5
CH3
Ph3C
H
H
H
H
H
H
H
>20000
>20000
15
16
6a
18
20
22
26a
26b
730 ( 300
640 ( 90
1400 ( 600
2700 ( 1700
2200 ( 400
1800 ( 800
14 ( 7
CO2H
CO2H
H
S
32 ( 14
a Values are the mean ( SD of three or more independent experiments.
length of the mercaptoalkyl group on inhibitory potency. GCP
II inhibitory potency of 7a-d and 6c toward GCP II was found
to be dependent on the length of the mercaptoalkyl group. The
compound 6c, containing a mercaptopropyl group, is the most
potent, and the shorter and longer mercaptoalkyl groups resulted
in a significant loss of potency. A similar trend was observed
in the previously reported SAR studies on the corresponding
analogues of 2-MPPA.3
In addition, we have assessed the effect of minor modifica-
tions to the molecular structure of 6c on the GCP II inhibitory
potency. The results are summarized in Table 3. Blocking of
the thiol group of 6c resulted in complete loss of potency as
shown by compounds 13 and 5c. This can be attributed to the
inability of these compounds to interact with the active site zinc
atom(s). We also tested analogues where one of the two
carboxylic groups in compound 6c is replaced with methyl ester
(compounds 15 and 18), carboxamide (compounds 16 and 20),
or cyano group (compound 22). Although the degree of effect
was not as significant as that of the thiol-blocking, these
compounds exhibited 40- to 180-fold decrease in GCP II
inhibitory potency. These results clearly indicate that the thiol
and the two carboxyl groups of 6c play key roles in interacting
with the active site of GCP II. Unlike these key functional
groups, replacement of the benzyl carbon in 6c with either an
ether or sulfide linker was well tolerated, providing equally
potent GCP II inhibitors 26a and 26b with IC50 values of 14
and 32 nM, respectively.
Figure 1. Antinociceptive effects of 6c (A) and 26a (B) in the rat
chronic constriction injury (CCI) model of neuropathic pain. Both of
these compounds were tested at 1.0 mg/kg/day by oral administration
and significantly attenuated the CCI-induced hyperalgesic state relative
to the vehicle-treated control (/, p < 0.05).
side chain from 2-carboxyethyl group (region A in Table 4) to
3-carboxybenzyl group (region B in Table 4). As summarized
in Table 4, none of the four newly synthesized 3-carboxybenzyl
group-containing compounds 28, 31, 36, and 39 exhibited
improvement in inhibitory potency over the parent 2-carboxy-
ethyl group-containing counterparts. Instead, in three cases (2-
PMPA to 28, 40 to 31, and 41 to 36), the replacement with the
3-carboxybenzyl group resulted in significant loss of potency.
These results demonstrate that the benefit of introducing a
3-carboxybenzyl group to the P1′ side chain is unique to the
thiol-based GCP II inhibitors and does not necessarily apply to
other classes of GCP II inhibitors.
Our SAR analysis was extended to other zinc-binding groups
to determine whether the same degree of improvement as with
compound 6c (6-fold greater potency than 2-MPPA) can be
achieved with the previously reported classes of GCP II
inhibitors such as phosphonate (2-PMPA), phosphinate 40,6
hydroxamate 41,12 and carboxylate 4212 by replacing their P1′
Antinociceptive Effects of 6c in the Rat Chronic Constric-
tion Injury (CCI) Model. We have subsequently tested the
two most potent GCP II inhibitors 6c and 26a for their

2880 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Majer et al.
2-[2,2-Dimethyl-4,6-dioxo-5-(3-tritylsulfanylpropyl)[1,3]dioxan-
5-ylmethyl]benzoic Acid Methyl Ester (3b). Compound 3b was
prepared as described for the preparation of 3c, except methyl (2-
bromomethyl)benzoate 2b was used in place of 2c: beige powder
(76% yield); 1H NMR (CDCl3) δ 0.93 (s, 3H), 1.15-1.28 (m, 2H),
1.51 (s, 3H), 1.95-2.04 (m, 2H), 2.11 (t, J ) 7.3 Hz, 2H), 3.78 (s,
2H), 3.89 (s, 3H), 7.15-7.45 (18H), 7.72-7.79 (m, 1H); 13C NMR
(CDCl3) δ 24.6, 28.7, 29.5, 31.3, 39.6, 39.8, 52.3, 56.3, 66.6, 105.5,
126.6, 127.6, 127.8, 129.4, 130.5, 131.5, 132.1, 132.4, 135.3, 144.6,
168.0, 168.4.
4-[2,2-Dimethyl-4,6-dioxo-5-(3-tritylsulfanylpropyl)[1,3]dioxan-
5-ylmethyl]benzoic Acid Methyl Ester (3d). Compound 3d was
prepared as described for the preparation of 3c, except methyl (4-
bromomethyl)benzoate 2d was used in place of 2c: white powder
(79% yield); 1H NMR (CDCl3) δ 0.66 (s, 3H), 1.16-1.32 (m, 2H),
1.50 (s, 3H), 1.96-2.08 (m, 2H), 2.16 (t, J ) 7.3 Hz, 2H), 3.27 (s,
2H), 3.88 (s, 3H), 7.12-7.44 (m, 17H), 7.93 (d, J ) 8.4 Hz, 2H);
13C NMR (CDCl3) δ 24.7, 29.2, 29.3, 31.3, 40.4, 43.3, 52.1, 56.9,
66.8, 105.8, 126.7, 127.9, 129.5, 129.6, 130.0, 130.5, 140.4, 144.7,
166.6, 168.3.
3-(2-Carboxy-5-tritylsulfanylpentyl)benzoic Acid (5c). A sus-
pension of 3c (6.10 g, 10.0 mmol) in 2.0 M NaOH (30 mL) and
dioxane (10 mL) was stirred at 100 °C for 3 h. The resulting clear
solution was concentrated, acidified to pH 1 by adding 1.0 M
H2SO4, and extracted with EtOAc (100 mL). The organic extract
was dried over MgSO4, filtered, and concentrated to give 5.80 g of
2-(3-carboxybenzyl)-2-(3-tritylsulfanylpropyl)malonic acid 4c as a
crude material (100% crude yield). This material was dissolved in
DMSO (15 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,
filtered, and concentrated. The residual material was recrystallized
from EtOAc/hexanes to give 4.85 g of 5c as a white solid (95%
yield from 3c): mp 196-197 °C; 1H NMR (CD3OD) δ 1.23-1.60
(m, 4H), 2.08-2.20 (m, 2H), 2.43-2.55 (m, 1H), 2.65-2.77 (m,
1H), 2.80-2.93 (m, 1H), 7.15-7.45 (m, 17H), 7.80-7.92 (m, 2H);
13C NMR (CD3OD) δ 27.9, 32.8, 33.1, 39.4, 48.7, 66.1, 128.1,
129.3, 129.9, 131.2, 131.6, 132.4, 135.1, 142.6, 146.8, 170.3, 179.1.
Anal. (C32H30O4S) C, H, S.
2-Benzyl-5-tritylsulfanylpentanoic Acid (5a). Compound 5a
was prepared as described for the preparation of 5c, except 5-benzyl-
2,2-dimethyl-5-(3-tritylsulfanylpropyl)[1,3]dioxane-4,6-dione 3a was
used in place of 3c: off-white solid (78% yield from 3a); 1H NMR
(CDCl3) δ 1.28-1.65 (m, 4H), 2.13 (t, J ) 6.9 Hz, 2H), 2.45-
2.59 (m, 1H), 2.66 (dd, J ) 6.6, 13.6 Hz, 1H), 2.89 (dd, J ) 8.1,
13.6 Hz, 1H), 7.07-7.32 (m, 15H), 7.35-7.45 (m, 6H); 13C NMR
(CDCl3) δ 26.3, 31.0, 31.6, 37.9, 46.8, 66.5, 126.5, 126.6, 127.8,
128.4, 128.8, 129.6, 138.8, 144.9, 180.8.
antinociceptive effects following oral administration (1.0 mg/
kg/day) using the rat chronic constriction injury model of
neuropathic pain.15 As shown in Figure 1, both 6c and 26a
significantly reduced thermal hyperalgesia relative to the vehicle-
treated control on days 8 and 12. It should be noted that the
effective doses for 6c and 26a are lower by an order of
magnitude compared to that of 2-MPPA (10 mg/kg/day),
presumably due to their improved potency toward GCP II. The
results also suggest that both of these new thiol-based inhibitors
are orally available and suitable for the treatment of chronic
neuropathic pain.
Conclusions
Through the modifications of the P1′ side chain, we have
successfully identified a new series of thiol-based GCP II
inhibitors. Some of these compounds display in vitro potency
superior to 2-MPPA. As represented by 6c and 26a, the
improved potency was well-reflected in the subsequent assess-
ment in the animal model of neuropathic pain, where these two
compounds exhibited efficacy by daily oral dosing of 1.0 mg/
kg, 10 times lower than the effective dose of 2-MPPA.
Relatively straightforward synthetic methods, combined with
the easy availability of diverse benzyl bromides and phenols,
allow us to expand our future SAR analysis to a wide variety
of analogues within this class of GCP II inhibitors. In the
meantime, further pharmacological characterizations of 6c and
26a are currently underway in various animal models of the
neurological disorders associated with glutamate excitotoxicity.
Experimental Section
All reactions were performed under nitrogen. Unless otherwise
noted, all materials were obtained from commercial suppliers and
used without further purification. Methyl 2,5-dibromovalerate 23
was obtained from AmeriBrom, Inc., Fort Lee, NJ. Melting points
1
were obtained on a Mel-Temp apparatus and are uncorrected. H
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 analysis results were obtained from Atlantic
Microlabs, Norcross, GA.
3-[2,2-Dimethyl-4,6-dioxo-5-(3-tritylsulfanylpropyl)[1,3]dioxan-
5-ylmethyl]benzoic Acid Methyl Ester (3c). To a solution of 1
(6.91 g, 15.0 mmol) and triethylbenzylammonium chloride (3.42
g, 15.0 mmol) in acetonitrile (75 mL) was added anhydrous K2CO3
(2.07 g, 15.0 mmol), and the suspension was stirred at 75 °C for
20 min. To the mixture was added a solution of methyl (3-
bromomethyl)benzoate 2c (4.12 g, 18.0 mmol) in acetonitrile (10
mL), and the resulting mixture was stirred at the same temperature
for 5 h. The solvent was removed under reduced pressure, and the
residue was taken up in ethyl acetate (100 mL). The solution was
washed with aqueous 5% KHSO4 (100 mL), dried over MgSO4,
filtered, and concentrated. The resulting solid residue was recrystal-
lized from EtOAc/hexanes to provide 6.30 g of 3c as a white solid
(69% yield): 1H NMR (CDCl3) δ 0.66 (s, 3H), 1.12-1.35 (m, 2H),
1.50 (s, 3H), 1.98-2.07 (m, 2H), 2.17 (t, J ) 7.2 Hz, 2H), 3.27 (s,
2H), 3.89 (s, 3H), 7.15-7.50 (m, 17H), 7.81 (bs, 1H), 7.88-7.98
(m, 1H); 13C NMR (CDCl3) δ 24.7, 29.1, 29.4, 31.3, 40.3, 43.3,
52.2, 57.0, 66.8, 105.8, 126.7, 127.9, 128.9, 129.1, 129.5, 130.8,
131.2, 134.8, 135.7, 144.7, 166.5, 168.3.
2-(2-Carboxy-5-tritylsulfanylpentyl)benzoic Acid (5b). Com-
pound 5b was prepared as described for the preparation of 5c, except
2-[2,2-dimethyl-4,6-dioxo-5-(3-tritylsulfanylpropyl)[1,3]dioxan-5-
ylmethyl]benzoic acid methyl ester 3b was used in place of 3c:
yellow solid (51% from 3b); 1H NMR (CD3OD) δ 1.20-1.58 (m,
4H), 2.09 (t, J ) 7.0 Hz, 2H), 2.51-2.64 (m, 1H), 3.03 (dd, J )
9.0, 13.1 Hz, 1H), 3.20 (dd, J ) 6.0, 13.1 Hz, 1H), 7.12-7.29 (m,
11H), 7.30-7.40 (m, 7H), 7.91 (d, J ) 7.5 Hz, 1H); 13C NMR
(CD3OD) δ 27.5, 32.8, 33.0, 37.9, 40.4, 67.6, 127.6, 127.7, 128.8,
130.7, 131.3, 132.2, 132.8, 132.9, 142.5, 146.3, 170.7, 179.3.
4-(2-Carboxy-5-tritylsulfanylpentyl)benzoic Acid (5d). Com-
pound 5d was prepared as described for the preparation of 5c, except
4-[2,2-dimethyl-4,6-dioxo-5-(3-tritylsulfanylpropyl)[1,3]dioxan-5-
ylmethyl]benzoic acid methyl ester 3d was used in place of 3c:
5-Benzyl-2,2-dimethyl-5-(3-tritylsulfanylpropyl)[1,3]dioxane-
4,6-dione (3a). Compound 3a was prepared as described for the
preparation of 3c, except benzyl bromide 2a was used in place of
1
white solid (85% yield from 3d); H NMR (CDCl3) δ 1.35-1.75
(m, 4H), 2.08-2.28 (m, 2H), 2.52-2.69 (m, 1H), 2.76 (dd, J )
5.3, 13.9 Hz, 1H), 2.91 (dd, J ) 9.3, 13.9 Hz, 1H), 7.10-7.51 (m,
17H), 7.96 (d, J ) 7.8 Hz, 2H); 13C NMR (CDCl3) δ 26.3, 31.3,
31.6, 37.8, 46.5, 66.6, 126.6, 127.6, 127.8, 129.0, 129.6, 130.4,
144.9, 145.3, 171.9, 180.9.
1
2c: white powder (86% yield); H NMR (CDCl3) δ 0.60 (s, 3H),
1.15-1.32 (m, 2H), 1.49 (s, 3H), 1.96-2.07 (m, 2H), 2.15 (t, J )
7.2 Hz, 2H), 3.23 (s, 2H), 7.10-7.32 (m, 14H), 7.35-7.45 (m,
6H); 13C NMR (CDCl3) δ 24.7, 28.8, 29.4, 31.3, 40.3, 43.7, 57.3,
66.7, 105.8, 126.7, 127.8, 127.9, 128.8, 129.5, 130.3, 135.5, 144.7,
168.6.
3-(2-Carboxy-5-mercaptopentyl)benzoic Acid (6c). To a solu-
tion of 5c (4.85 g, 9.50 mmol) in dichloromethane (10 mL) were

Inhibitors of Glutamate Carboxypeptidase II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2881
added trifluoroacetic acid (10 mL) and triisopropylsilane (1.90 g,
12.0 mmol). The dark solution gradually turned light-yellow. After
the mixture was stirred for 30 min, the solvent was removed under
reduced pressure and the residual material was partitioned between
hexanes (60 mL) and 1.0 M NaOH (40 mL containing 200 mg of
tris(2-carboxyethyl)phosphine hydrochloride). The aqueous layer
was washed with hexanes (20 mL), acidified to pH 1 with 1 M
H2SO4, and extracted with EtOAc (100 mL). The organic extract
was dried over MgSO4, filtered, and concentrated. The residual
material was recrystallized from EtOAc/hexanes to give 2.27 g of
6c as a white solid (89% yield): mp 123-124 °C; 1H NMR
(CD3OD) δ 1.54-1.77 (m, 4H), 2.43-2.53 (m, 2H), 2.58-2.72
(m, 1H), 2.83 (dd, J ) 6.1, 13.5 Hz, 1H), 2.97 (dd, J ) 8.8, 13.5
Hz, 1H), 7.32-7.48 (m, 2H), 7.81-7.92 (m, 2H); 13C NMR
(CD3OD) δ 24.7, 31.9, 32.9, 39.2, 48.4, 128.8, 129.5, 131.2, 132.0,
134.7, 141.3, 169.9, 178.8. Anal. (C13H16O4S) C, H, S.
2-Benzyl-5-mercaptopentanoic Acid (6a) Compound 6a was
prepared as described for the preparation of 6c, except 2-benzyl-
5-tritylsulfanylpentanoic acid 5a was used in place of 5c. The crude
material was purified by silica gel chromatography (EtOAc/hexanes,
1:1) to give 6a as a colorless oil (85% yield): 1H NMR (CDCl3)
δ 1.31 (t, J ) 7.9 Hz, 1H), 1.50-1.80 (m, 4H), 2.42-2.58 (m,
2H), 2.61-2.74 (m, 1H), 2.75 (dd, J ) 7.1, 13.4 Hz, 1H), 3.00
(dd, J ) 7.3, 13.4 Hz, 1H), 7.10-7.35 (m, 5H), 10.5-11.5 (br,
1H); 13C NMR (CDCl3) δ 24.3, 30.2, 31.5, 38.0, 46.8, 126.5, 128.5,
128.8, 138.7, 181.6. Anal. (C12H16O2S) C, H, S.
2-(2-Carboxy-5-mercaptopentyl)benzoic Acid (6b). Compound
6b was prepared as described for the preparation of 6c, except 2-(2-
carboxy-5-tritylsulfanylpentyl)benzoic acid 5b was used in place
of 5c. The crude material was purified by silica gel chromatography
(EtOAc/hexanes, 1:4 containing 1% AcOH) to give 6b as a colorless
oil (43% yield): 1H NMR (CD3OD) δ 1.55-1.81 (m, 4H), 2.41-
2.57 (m, 2H), 2.67-2.80 (m, 1H), 3.09 (dd, J ) 9.5, 12.6 Hz, 1H),
3.37 (dd, J ) 5.5, 13.1 Hz, 1H), 7.24-7.37 (m, 2H), 7.44, (t, J )
7.3 Hz, 1H), 7.95 (d, J ) 7.5 Hz, 1H); 13C NMR (CD3OD) δ 24.7,
32.4, 32.8, 38.1, 48.3, 127.6, 131.3, 132.2, 132.8, 132.9, 142.5,
170.8, 179.4. Anal. (C13H16O4S) C, H, S.
4-(2-Carboxy-5-mercaptopentyl)benzoic Acid (6d). Compound
6d was prepared as described for the preparation of 6c, except 4-(2-
carboxy-5-tritylsulfanylpentyl)benzoic acid 5d was used in place
of 5c: white solid (90% yield); mp 152-154 °C; 1H NMR (CDCl3)
δ 1.34 (t, J ) 7.9 Hz, 1H), 1.57-1.88 (m, 4H), 2.53 (q, J ) 6.7
Hz, 2H), 2.67-2.81 (m, 1H), 2.87 (dd, J ) 6.2, 13.6 Hz, 1H),
3.05 (dd, J ) 8.6, 13.7 Hz, 1H), 7.29 (d, J ) 8.2 Hz, 2H), 8.01 (d,
J ) 8.2 Hz, 2H); 13C NMR (CDCl3) δ 24.2, 30.6, 31.4, 38.1, 46.6,
127.6, 129.0, 130.5, 145.2, 172.1, 181.2. Anal. (C13H16O4S) C, H,
S.
3-(2-Carboxyallyl)benzoic Acid (8). A solution of 3-(2-meth-
oxycarbonylallyl)benzoic acid methyl ester 710 (10.1 g, 43.1 mmol)
in dioxane (50 mL) and 4.3 M NaOH (50 mL) was stirred at room
temperature overnight. The solvents were removed under reduced
pressure, and the residue was partitioned between aqueous 10%
KHSO4 (50 mL) and EtOAc (100 mL). The organic layer was dried
over Na2SO4, filtered, and concentrated to give 8.90 g of 8 as a
white solid (100% crude yield). This material was used in the next
step without further purification: 1H NMR (CD3OD) δ 3.66 (s,
2H), 5.58 (s, 1H), 6.24 (s, 1H), 7.32-7.44 (m, 2H), 7.81-7.90
(m, 2H); 13C NMR (CD3OD) δ 38.7, 127.2, 128.7, 129.5, 131.1,
132.0, 134.7, 141.1, 141.8, 170.0.
3-(2-Carboxy-3-mercaptopropyl)benzoic Acid (9). To a solu-
tion of 8 (8.90 g, 43.1 mmol) in dichloromethane (50 mL) and
DMF (15 mL) was added thiolacetic acid (6.2 mL, 86.2 mmol) at
room temperature, and the mixture was stirred at room temperature
for 2 days. The solvents were removed under reduced pressure,
and the residue was partitioned between aqueous 10% KHSO4 (60
mL) and EtOAc (75 mL). The organic layer was dried over Na2SO4,
filtered, and concentrated to give a colorless oil. This material was
dissolved in dioxane (50 mL) and 4.3 M NaOH (50 mL), and the
mixture was stirred at room temperature for 4 h. The solvents were
removed under reduced pressure, and the residue was acidified to
pH 2 with 1 M H2SO4 and taken up in EtOAc (100 mL). The
organic layer was dried over Na2SO4, filtered, and concentrated.
The crude material was purified by silica gel chromatography
(EtOAc/hexanes, 1:2 containing 2% AcOH) to give 8.90 g of 9 as
a white solid (86% from 7): mp 104-106 °C; 1H NMR (CD3OD)
δ 2.67 (d, J ) 6.3 Hz, 2H), 2.78-2.90 (m, 1H), 2.94-3.05 (m,
2H), 7.34-7.48 (m, 2H), 7.82-7.90 (m, 2H); 13C NMR (CD3OD)
δ 26.1, 37.8, 52.1, 129.0, 129.6, 131.3, 132.1, 134.7, 140.6, 169.8,
176.9. Anal. (C11H12O4S) C, H, S.
2,2-Dimethyl-5-(2-tritylsulfanylethyl)[1,3]dioxane-4,6-dione
(10a). Compound 10a was prepared as previously described for
the preparation of 1,3 except S-tritylmercaptoacetic acid was used
in place of S-trityl-3-mercaptopropionic acid: white solid (93%
yield); mp 104-106 °C; 1H NMR (CDCl3) δ 1.70 (s, 3H), 1.75 (s,
3H), 2.04 (t, J ) 6.5 Hz, 2H), 2.58 (t, J ) 6.8 Hz, 2H), 3.60 (t, J
) 6.0 Hz, 1H), 7.18-7.32 (m, 9H), 7.37-7.45 (m, 6H); 13C NMR
(CDCl3) δ 25.1, 26.5, 28.5, 29.3, 44.5, 66.9, 104.9, 126.8, 128.0,
129.5, 144.6, 164.9.
3-[2,2-Dimethyl-4,6-dioxo-5-(2-tritylsulfanyl-ethyl)[1,3]dioxan-
5-ylmethyl]benzoic Acid Methyl Ester (11a). Compound 11a was
prepared as described for the preparation of 3c, except 2,2-dimethyl-
5-(2-tritylsulfanyl-ethyl)[1,3]dioxane-4,6-dione 10a was used in
1
place of 1: white solid (54% yield); mp 154-156 °C (dec); H
NMR (CDCl3) δ 0.62 (s, 3H), 1.25 (s, 3H), 2.03-2.11 (m, 2H),
2.17-2.25 (m, 2H), 3.23 (s, 2H), 3.88 (s, 3H), 7.17-7.39 (m, 17H),
7.75-7.80 (brs, 1H), 7.87-7.94 (m, 1H); 13C NMR (CDCl3) δ 27.6,
28.9, 29.0, 39.2, 43.0, 52.2, 56.9, 67.2, 105.8, 126.8, 128.0, 128.9,
129.1, 129.4, 130.7, 131.2, 134.8, 135.3, 144.4, 166.5, 167.7.
3-[2,2-Dimethyl-4,6-dioxo-5-(5-tritylsulfanylbutyl)[1,3]dioxan-
5-ylmethyl]benzoic Acid Methyl Ester (11b). Compound 11b was
prepared as described for the preparation of 3c, except 2,2-dimethyl-
5-(5-tritylsulfanylbutyl)[1,3]dioxane-4,6-dione 10b was used in
1
place of 1 (65% yield): white solid; mp 130-132 °C; H NMR
(CDCl3) δ 0.51 (s, 3H), 1.00-1.26 (m, 4H), 1.38 (s, 3H), 1.77-
1.88 (m, 2H), 1.96 (t, J ) 7.1 Hz, 2H), 3.14 (s, 2H), 3.73 (s, 3H),
6.98-7.28 (m, 17H), 7.66 (brs, 1H), 7.77 (dt, J ) 6.3, 2.0 Hz,
1H); 13C NMR (CDCl3) δ 24.8, 28.4, 29.1, 29.4, 31.3, 40.7, 43.3,
52.2, 57.2, 66.6, 105.8, 126.6, 127.9, 128.9, 129.1, 129.6, 130.8,
131.3, 134.8, 135.8, 144.9, 166.6, 168.6.
3-[2,2-Dimethyl-4,6-dioxo-5-(5-tritylsulfanylpentyl)[1,3]dioxan-
5-ylmethyl]benzoic Acid Methyl Ester (11c). Compound 11c was
prepared as described for the preparation of 3c, except 2,2-dimethyl-
5-(5-tritylsulfanylpentyl)[1,3]dioxane-4,6-dione 10c was used in
1
place of 1: white foam (88% yield); H NMR (CDCl3) δ 0.68 (s,
3H), 1.10-1.42 (m, 6H), 1.52 (s, 3H), 2.02-2.15 (m, 4H), 3.33
(s, 2H), 3.90 (s, 3H), 7.16-7.43 (m, 17H), 7.82-7.85 (m, 1H),
7.90-7.96 (m, 1H); 13C NMR (CDCl3) δ 25.1, 28.1, 28.6, 29.1,
29.3, 31.7, 41.0, 43.3, 52.2, 57.3, 66.5, 105.7, 126.5, 127.8, 128.9,
129.1, 129.6, 130.8, 131.2, 134.8, 135.8, 145.0, 166.5, 168.6.
3-(2-Carboxy-4-mercaptobutyl)benzoic Acid (12a). Compound
12a was prepared in three steps from 11a as described for the
preparation of 6c from 3c: white solid (80% yield from 11a); mp
1
124-126 °C; H NMR (CD3OD) δ 1.65-1.80 (m, 1H), 1.83-
2.01 (m, 1H), 2.40-2.62 (m, 2H), 2.75-2.90 (m, 2H), 2.90-3.04
(m, 1H), 7.28-7.49 (m, 2H), 7.77-7.92 (m, 2H); 13C NMR
(CD3OD) δ 22.8, 37.5, 38.9, 47.5, 128.9, 129.5, 131.2, 132.0, 134.7,
141.0, 169.8, 178.3. Anal. (C12H14O4S) C, H, S.
3-(2-Carboxy-6-mercaptohexyl)benzoic Acid (12b). Compound
12b was prepared in three steps from 11b as described for the
preparation of 6c from 3c (77% yield from 11b): white solid; mp
1
81-83 °C; H NMR (CDCl3) δ 1.32 (t, J ) 7.8 Hz, 1H), 1.40-
1.79 (m, 6H), 2.51 (q, J ) 7.5 Hz, 2H), 2.63-2.74 (m, 1H), 2.88
(dd, J ) 6.0, 13.6 Hz, 1H), 2.99 (dd, J ) 9.0, 13.6 Hz, 1H), 7.33-
7.46 (m, 2H), 7.85-7.95 (m, 2H); 13C NMR (CDCl3) δ 24.3, 26.0,
31.3, 33.7, 38.1, 47.7, 128.5, 128.7, 129.4, 130.5, 134.4, 139.3,
172.2, 181.6. Anal. (C14H18SO4) C, H, S.
3-(2-Carboxy-7-mercaptoheptyl)benzoic Acid (12c). Com-
pound 12c was prepared in three steps from 11c as described for
the preparation of 6c from 3c: white solid (86% yield from 11c);
1
mp 80-82 °C; H NMR (CD3OD) δ 1.26-1.47 (m, 4H), 1.47-
1.72 (m, 4H), 2.45 (t, J ) 7.0 Hz, 2H), 2.56-2.72 (m, 1H), 2.82
(dd, J ) 5.9, 13.6 Hz, 1H), 2.95 (dd, J ) 8.8, 13.5 Hz, 1H), 7.32-

2882 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Majer et al.
7.48 (m, 2H), 7.81-7.93 (m, 2H); 13C NMR (CD3OD) δ 24.8, 27.8,
29.1, 33.1, 34.9, 39.2, 48.8, 128.8, 129.5, 131.2, 132.0, 134.7, 141.4,
169.9, 179.1. Anal. (C15H20O4S) C, H, S.
10% aqueous KHSO4 (10 mL) and EtOAc (25 mL). The organic
layer was washed with brine (15 mL), dried over Na2SO4, filtered,
and concentrated. The crude material was purified by silica gel
chromatography (hexanes/EtOAc, 2:1 containing 1% AcOH) to give
0.210 g of 17 as a colorless oil (57% yield): 1H NMR (CDCl3) δ
1.15-1.60 (m, 4H), 2.00-2.20 (m, 2H), 2.50-2.62 (m, 1H), 2.72
(dd, J ) 6.5, 13.8 Hz, 1H), 2.91 (dd, J ) 8.6, 13.8 Hz, 1H), 3.90
(s, 3H), 7.16-7.44 (m, 17H), 7.79-7.83 (m, 1H), 7.85-7.96 (m,
1H).
3-(2-Carboxy-5-mercaptopentyl)benzoic Acid Methyl Ester
(18). To a solution of 17 (0.21 g, 0.40 mmol) in dichloromethane
(2 mL) were added triisopropylsilane (0.11 mL, 0.49 mmol) and
trifluoroacetic acid (0.5 mL). The dark solution gradually turned
light-yellow. After the mixture was stirred for 1.5 h, the solvent
was removed under reduced pressure and the residual material was
suspended in hexanes/EtOAc (95:5, 10 mL). The product was
extracted with saturated aqueous NaHCO3 (20 mL containing 10
mg of tris(2-carboxyethyl)phosphine hydrochloride). The aqueous
layer was washed with hexanes (10 mL), acidified to pH 5 with
10% aqueous KHSO4, and extracted with EtOAc (20 mL). The
organic extract was washed with brine (15 mL), dried over Na2SO4,
filtered, and concentrated to give 0.06 g of 18 as a colorless oil
(53% yield): 1H NMR (CD3OD) δ 1.55-1.80 (m, 4H), 2.40-2.55
(m, 2H), 2.58-2.72 (m, 1H), 2.83 (dd, J ) 6.1, 13.6 Hz, 1H), 2.96
(dd, J ) 9.0, 13.6 Hz, 1H), 3.88 (s, 3H), 7.33-7.50 (m, 2H), 7.80-
7.91 (m, 2H); 13C NMR (CD3OD) δ 24.7, 31.9, 32.5, 39.2, 48.4,
52.6, 128.6, 129.6, 131.0, 131.4, 134.9, 141.5, 168.6, 178.8. Anal.
(C14H18O4S) C, H, S.
3-(2-Oxotetrahydrothiopyran-3-ylmethyl)benzamide (19). To
a solution of 14 (1.61 g, 6.43 mmol) and ammonium chloride (0.35
g, 6.54 mmol) in DMF (12 mL) were added diisopropylethylamine
(2.26 mL, 13.0 mmol) and HATU (2.75 g, 7.23 mmol) at 0 °C.
The mixture was stirred at room temperature for 3 days. The solvent
was removed under reduced pressure, and the residue was taken
up in EtOAc (30 mL). The organic solution was washed with
saturated aqueous NaHCO3 (25 mL), 10% KHSO4 (20 mL), and
brine (20 mL). The extract was dried over Na2SO4, filtered, and
concentrated. The crude material was recrystallized from EtOAc/
hexanes, and the recrystallized material was washed with water to
remove any residual NaCl to give 1.57 g of 19 as a white solid
(98% yield): 1H NMR (CDCl3) δ 1.52-1.70 (m, 1H), 1.86-2.15
(m, 3H), 2.71 (dd, J ) 9.0, 13.2 Hz, 1H), 2.72-2.86 (m, 1H), 3.13
(t, J ) 5.9 Hz, 2H), 3.37 (dd, J ) 3.6, 13.2 Hz, 1H), 5.55-5.85
(brs, 1H), 5.95-6.25 (brs, 1H), 7.32-7.43 (m, 2H), 7.59-7.69 (m,
2H).
2-(3-Carbamoylbenzyl)-5-mercaptopentanoic Acid (20). A
solution of 19 (0.16 g, 0.64 mmol) and sodium hydroxide (0.052
g, 1.3 mmol) in THF/water (1:1, 4 mL containing 30 mg of tris-
(2-carboxyethyl)phosphine hydrochloride) was stirred at room
temperature for 2 h. Additional sodium hydroxide (0.026 g, 0.65
mmol) was added, and the mixture was stirred for another 30 min.
The reaction mixture was partitioned between aqueous 10% KHSO4
(10 mL) and EtOAc (10 mL). The organic layer was washed with
brine (10 mL), dried over Na2SO4, filtered, and concentrated. The
crude material was recrystallized from EtOAc/hexanes to give 0.15
g of 20 as a white powder (87% yield): mp 137-139 °C; 1H NMR
(CD3OD) δ 1.51-1.79 (m, 4H), 2.37-2.55 (m, 2H), 2.59-2.75
(m, 1H), 2.83 (dd, J ) 6.3, 13.5 Hz, 1H), 2.97 (dd, J ) 8.8, 13.5
Hz, 1H), 7.30-7.44 (m, 2H), 7.63-7.73 (m, 2H); 13C NMR
(CD3OD) δ 24.7, 31.9, 32.9, 39.3, 48.4, 126.7, 129.3, 129.6, 133.6,
135.0, 141.3, 172.4, 178.9. Anal. (C13H17NO3S) C, H, N, S.
3-(2-Oxotetrahydrothiopyran-3-ylmethyl)benzonitrile (21).
Oxalyl chloride (0.42 mL, 4.8 mmol) was slowly added to a solution
of DMF (0.45 mL, 5.8 mmol) in acetonitrile (10 mL) at 0 °C. The
resulting white suspension was stirred at 0 °C for 40 min. A solution
of 19 (1.00 g, 4.01 mmol) in DMF (7 mL) was added to the mixture
at 0 °C. After the mixture was stirred for 55 min, triethylamine
(1.23 mL, 8.8 mmol) was added and the reaction mixture was
concentrated under reduced pressure. The residual material was
dissolved in EtOAc (30 mL), washed with water (25 mL) and brine
(25 mL), dried over Na2SO4, filtered, and concentrated. The crude
3-(2-Carboxy-5-methylsulfanylpentyl)benzoic Acid (13). To
a solution of 6c (2.00 g, 7.45 mmol) in methanol (20 mL) were
added sodium methoxide (25 wt % solution in methanol, 6.5 mL)
and iodomethane (0.51 mL, 8.20 mmol). The reaction mixture was
stirred at room temperature for 2 h. The solvent was removed under
reduced pressure, and the residue was partitioned between aqueous
10% KHSO4 (30 mL) and EtOAc (50 mL). The organic layer was
washed with saturated aqueous sodium thiosulfate (50 mL) and
brine (50 mL), dried over Na2SO4, filtered, and concentrated to
give 2.10 g of 13 as a white solid (quantitative yield): mp 72-74
°C; 1H NMR (CD3OD) δ 1.54-1.81 (m, 4H), 2.03 (s, 3H), 2.38-
2.55 (m, 2H), 2.60-2.74 (m, 1H), 2.83 (dd, J ) 5.9, 13.7 Hz, 1H),
2.97 (dd, J ) 9.0, 13.7 Hz, 1H), 7.33-7.48 (m, 2H), 7.82-7.93
(m, 2H); 13C NMR (CD3OD) δ 15.2, 27.8, 32.2, 34.7, 39.2, 48.5,
128.8, 129.5, 131.2, 132.0, 134.7, 141.3, 169.9, 178.8. Anal.
(C14H18O4S) C, H, S.
3-(2-Oxotetrahydrothiopyran-3-ylmethyl)benzoic Acid (14).
A solution of 6c (5.12 g, 19.1 mmol) and 10-camphorsulfonic acid
(0.50 g, 2.2 mmol) in toluene (30 mL) was refluxed for 6 h in a
flask equipped with a Dean-Stark trap. The solvent was removed
under reduced pressure, and the residual material was purified by
chromatography (EtOAc/hexanes, 1:4). The resulting white solid
was recrystallized from EtOAc/hexanes to give 3.20 g of 14 as a
white solid (67% yield): 1H NMR (CDCl3) δ 1.55-1.69 (m, 1H),
1.89-2.01 (m, 2H), 2.03-2.14 (m, 1H), 2.72 (dd, J ) 9.0, 13.6
Hz, 1H), 2.77-2.86 (m, 1H), 3.07-3.19 (m, 2H), 3.41 (dd, J )
4.0, 13.6 Hz, 1H), 7.38-7.48 (m, 2H), 7.93 (s, 1H), 7.98 (dd, J )
1.5, 7.5 Hz, 1H); 13C NMR (CDCl3) δ 22.2, 27.4, 30.6, 36.3, 51.5,
128.3, 128.7, 129.4, 130.8, 134.8, 139.7, 172.2, 203.2.
3-(5-Mercapto-2-methoxycarbonylpentyl)benzoic Acid (15).
To a solution of 14 (0.325 g, 1.30 mmol) in MeOH (3 mL) was
added sodium methoxide (25 wt % solution in methanol, 0.3 mL).
The mixture was stirred at room temperature for 30 min. The solvent
was removed under reduced pressure, and the residue was parti-
tioned between 10% KHSO4 (30 mL) and EtOAc (50 mL). The
organic layer was dried over Na2SO4, filtered, and concentrated.
The residual material was purified by chromatography (EtOAc/
hexanes, 7:3 containing 1% AcOH) to give 0.308 g of 15 as a white
1
solid (83% yield): mp 51-53 °C; H NMR (CD3OD) δ 1.51-
1.80 (m, 4H), 2.47 (t, J ) 6.4 Hz, 2H), 2.64-2.77 (m, 1H), 2.85
(dd, J ) 5.9, 13.5 Hz, 1H), 2.94 (dd, J ) 9.0, 13.5 Hz, 1H), 3.57
(s, 3H), 7.33-7.44 (m, 2H), 7.79-7.89 (m, 2H); 13C NMR
(CD3OD) δ 24.7, 31.9, 32.8, 39.2, 48.5, 52.0, 128.9, 129.6, 131.1,
132.1, 134.6, 141.0, 169.8, 177.2. Anal. (C14H18O4S‚0.2H2O) C,
H, S.
3-(2-Carbamoyl-5-mercaptopentyl)benzoic Acid (16). A solu-
tion of 14 (0.42 g, 1.7 mmol) in 28% ammonium hydroxide (5
mL) was stirred at room temperature for 40 min. The reaction
mixture was purged with nitrogen to remove excess ammonia and
acidified to pH 4 with aqueous 10% KHSO4. The product was
extracted with EtOAc (25 mL), washed with brine (20 mL), dried
over Na2SO4, filtered, and concentrated. The crude material was
recrystallized from EtOAc/hexanes to give 0.39 g of 16 as a white
powder (87% yield): mp 163-165 °C; 1H NMR (CD3OD) δ 1.49-
1.80 (m, 4H), 2.37-2.69 (m, 3H), 2.77 (dd, J ) 5.7, 13.4 Hz, 1H),
2.91 (dd, J ) 9.3, 13.4 Hz, 1H), 7.31-7.47 (m, 2H), 7.79-7.92
(m, 2H); 13C NMR (CD3OD) δ 24.8, 32.3, 33.0, 39.8, 49.2, 128.7,
129.4, 131.3, 132.0, 134.8, 141.5, 169.9, 180.2. Anal. (C13H17NO3S)
C, H, N, S.
3-(2-Carboxy-5-tritylsulfanylpentyl)benzoic Acid Methyl Es-
ter (17). To a solution of 3c (0.43 g, 0.71 mmol) in water (3 mL)
and dioxane (3 mL) was added sodium hydroxide (0.068 g, 1.70
mmol) at 0 °C, and the mixture was stirred at room temperature
for 3.5 h. The reaction mixture was acidified with 10% aqueous
KHSO4 (20 mL), extracted with EtOAc (25 mL), washed with brine
(15 mL), dried over Na2SO4, filtered, and concentrated. The residual
material was dissolved in DMSO (1 mL) and heated at 130 °C for
1.5 h. After cooling, the reaction mixture was partitioned between

Inhibitors of Glutamate Carboxypeptidase II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2883
material was purified by chromatography (hexanes/EtOAc, 1:1) to
give 0.82 g of 21 as a colorless oil (88% yield): 1H NMR (CDCl3)
δ 1.52-1.70 (m, 1H), 1.88-2.18 (m, 3H), 2.65-2.85 (m, 2H), 3.11
(dd, J ) 5.0, 12.6 Hz, 1H), 3.18 (dd, J ) 5.5, 12.6 Hz, 1H), 3.25-
3.38 (m, 1H), 7.37-7.50 (m, 3H), 7.53 (dt, J ) 6.8, 1.8 Hz, 1H).
2-(3-Cyanobenzyl)-5-mercaptopentanoic Acid (22). A solution
of 21 (0.77 g, 3.3 mmol) in 1 M NaOH/THF (1:1, 20 mL containing
30 mg of tris(2-carboxyethyl)phosphine hydrochloride) was stirred
at room temperature for 30 min. The reaction mixture was
partitioned between aqueous 10% KHSO4 (20 mL) and EtOAc (30
mL). The organic layer was washed with brine (20 mL), dried over
Na2SO4, filtered, and concentrated. The crude material was purified
by chromatography (hexanes/EtOAc, 1:1 containing 1% AcOH)
to give 0.74 g of 22 as a colorless oil (89% yield): 1H NMR
(CD3OD) δ 1.54-1.80 (m, 4H), 2.40-2.57 (m, 2H), 2.58-2.72
(m, 1H), 2.85 (dd, J ) 5.8, 13.5 Hz, 1H), 2.94 (dd, J ) 9.3, 13.5
Hz, 1H), 7.40-7.60 (m, 4H); 13C NMR (CD3OD) δ 24.7, 32.0,
32.7, 38.8, 48.2, 113.3, 119.8, 130.5, 131.2, 133.6, 135.0, 142.7,
178.4. Anal. (C13H15NO2S‚0.3H2O) C, H, N, S.
3-(4-Acetylsulfanyl-1-methoxycarbonylbutoxy)benzoic Acid
Methyl Ester (25a). To a solution of methyl 2,5-dibromovalerate
23 (22.00 g, 80.3 mmol) and methyl 3-hydroxybenzoate 24a (10.18
g, 66.9 mmol) in DMF (80 mL) at room temperature was added
K2CO3 (12.94 g, 93.7 mmol). The mixture was stirred at room
temperature for 12 h and then at 70 °C for 1 h. To this reaction
mixture was added potassium thioacetate (22.93 g, 200.8 mmol),
and the mixture was stirred at 70 °C for another 1 h. The reaction
mixture was allowed to cool to room temperature, diluted with
EtOAc (1000 mL), washed with water (300 mL × 3) and brine
(300 mL × 2), dried over MgSO4, filtered, and concentrated. The
crude product was purified by chromatography (EtOAc/hexanes,
1:9 to 1:4) to give 8.40 g of 25a as a yellow oil (37% yield): 1H
NMR (CDCl3) δ 1.73-1.88 (m, 2H), 2.01-2.08 (m, 2H), 2.32 (s,
3H), 2.93 (t, J ) 7.2 Hz, 2H), 3.75 (s, 3H), 3.89 (s, 3H), 4.69 (t,
J ) 6.2 Hz, 1H), 7.07 (dd, J ) 2.1, 8.0 Hz, 1H), 7.32 (t, J ) 7.9
Hz, 1H), 7.49 (s, 1H), 7.65 (d, J ) 7.9 Hz, 1H); 13C NMR (CDCl3)
δ 25.4, 28.5, 30.6, 31.4, 52.2, 52.3, 76.2, 115.9, 120.0, 123.0, 129.6,
131.7, 157.7, 166.6, 171.5,195.5.
(t, J ) 7.9 Hz, 1H), 1.99-1.83 (m, 2H), 2.22-2.11 (m, 2H), 2.63
(m, 2H), 4.76 (dd, J ) 7.3, 5.0 Hz, 1H), 7.23 (m, 1H), 7.38 (t, J )
7.8 Hz, 1H), 7.49 (m, 1H), 7.72 (d, J ) 7.7 Hz, 1H); 13C NMR
(CDCl3) δ 24.1, 29.6, 31.2, 75.5, 114.8, 122.1, 123.9, 129.9, 130.5,
157.6, 171.8, 177.1. Anal. (C12H14O5S) C, H, S.
3-[(1-Carboxy-4-mercaptobutyl)thio]benzoic Acid (26b). A
solution of 25b (0.94 g, 2.64 mmol) and tris(2-carboxyethyl)-
phosphine hydrochloride (0.76 g, 2.65 mmol) in THF (30 mL) was
purged of oxygen by bubbling nitrogen gas through the solution
for 1 h. To the solution was added 2 M NaOH (13 mL), and the
mixture was stirred at room temperature for 24 h. The reaction
mixture was acidified to pH 2 with 2 M HCl and extracted with
ether (80 mL × 3). The combined extracts were washed with water
(250 mL) and brine (250 mL), dried over MgSO4, filtered, and
concentrated to give 0.65 g of 26b as a white solid (86% yield):
1
mp 116-118 °C; H NMR (CDCl3) δ 1.39 (t, J ) 8.0 Hz, 1H),
1.72-1.96 (m, 4H), 2.52-1.65 (m, 2H), 3.53 (t, J ) 7.8 Hz, 1H),
7.48 (t, J ) 7.6 Hz, 1H), 7.80 (dt, J ) 7.6, 1.2 Hz, 1H), 8.07-8.17
(m, 2H); 13C NMR (CDCl3) δ 24.1, 29.0, 31.4, 50.6, 129.3, 130.2,
131.4, 131.6, 137.8, 141.1, 171.4, 177.5. Anal. (C12H14O4S2) C, H,
S.
3-[3-(Diethoxy-phosphoryl)-2-methoxycarbonylpropyl]ben-
zoic Acid Methyl Ester (27). To a solution of 7 (0.19 g, 0.81 mmol)
and diethyl phosphite (0.11 mL, 0.85 mmol) in THF (10 mL) was
added sodium hydride (0.008 g, 60% dispersion in mineral oil) at
0 °C, and the mixture was stirred at room temperature for 2 h. The
reaction mixture was diluted with 1 M HCl (30 mL) and extracted
with EtOAc (30 mL × 2). The combined extracts were washed
with water (30 mL) and brine (30 mL), dried over MgSO4, filtered,
and concentrated. The crude product was purified by chromatog-
raphy (EtOAc/hexanes, 9:1) to give 0.26 g of 27 as a colorless oil
(86% yield): 1H NMR (CDCl3) δ 1.25 (t, J ) 7.1 Hz, 3H), 1.27 (t,
J ) 7.1 Hz, 3H), 1.83 (m, 1H), 2.19 (m, 1H), 2.92-3.10 (m, 3H),
3.59 (s, 3H), 3.88 (s, 3H), 3.95-4.09 (m, 4H), 7.32-7.35 (m, 2H),
7.82 (s, 1H), 7.88 (m, 1H); 13C NMR (CDCl3) δ 16.2, 16.3, 27.2
(d, J ) 142.0 Hz), 39.0 (d, J ) 12.3 Hz), 41.8 (d, J ) 3.5 Hz),
51.8, 52.0, 62.1 (d, J ) 6.5 Hz), 62.2 (d, J ) 6.5 Hz), 128.5, 128.6,
130.6, 130.4, 134.0, 138.3, 166.9, 174.2.
3-(4-Acetylsulfanyl-1-methoxycarbonylbutylsulfanyl)-
benzoic Acid Methyl Ester (25b). To a solution of 3-mercapto-
benzoic acid methyl ester 24b (0.71 g, 4.22 mmol) and K2CO3 (0.82
g, 5.91 mmol) in DMF (10 mL) was added methyl 2,5-dibromo-
valerate 23 (1.50 g, 5.48 mmol), and the mixture was stirred at
room temperature for 12 h. The reaction mixture was poured into
water (50 mL) and extracted with EtOAc (40 mL × 3). The
combined extracts were washed with water (80 mL × 3) and brine
(80 mL). The organic layer was dried over MgSO4, filtered, and
concentrated. The residual material was dissolved in DMF (10 mL).
Potassium thioacetate (0.79 g, 6.92 mmol) was added to the solution,
and the mixture was stirred at 70 °C for 2 h. The reaction mixture
was allowed to cool to room temperature and diluted with EtOAc
(60 mL). The mixture was washed with water (50 mL × 3) and
brine (60 mL), dried over MgSO4, filtered, and concentrated. The
crude product was purified by chromatography (EtOAc/hexanes,
3-(2-Carboxy-3-phosphonopropyl)benzoic Acid (28). A solu-
tion of 27 (0.24 g, 0.64 mmol) in 12 M HCl (8 mL) was stirred at
100 °C for 12 h. The reaction mixture was concentrated to dryness,
and the residue was washed with ether and dried under vacuum to
give 0.15 g of 28 as a white solid (79% yield): mp 205-207 °C;
1H NMR (D2O) δ 1.83-1.96 (m, 1H), 2.07-1.18 (m, 1H), 2.90-
3.05 (m, 3H), 7.42 (t, J ) 7.6 Hz, 1H), 7.48 (d, J ) 7.8 Hz, 1H),
7.75 (s, 1H), 7.78 (d, J ) 7.6 Hz, 1H); 13C NMR (D2O) 27.9 (d, J
) 136.5 Hz), 38.0 (d, J ) 14.6 Hz), 41.8 (d, J ) 3.8 Hz), 127.3,
128.1, 128.9, 129.2, 133.5, 137.8, 169.6, 177.5; 31P NMR (D2O) δ
27.4. Anal. (C11H13O7P) C, H.
3-(3-Hydroxyphosphinoyl-2-methoxycarbonylpropyl)benzo-
ic Acid Methyl Ester (29). To a suspension of ammonium
hypophosphate (1.77 g, 21.4 mmol) in dichloromethane (30 mL)
were added chlorotrimethylsilane (7.00 mL, 55.5 mmol) and
triethylamine (7.14 mL, 51.2 mmol) while maintaining the tem-
perature below 10 °C. A solution of 7 (1.00 g, 4.27 mmol) in
dichloromethane (10 mL) was added to the mixture at a rate such
that the temperature remained below 10 °C. The reaction mixture
was allowed to warm to room temperature and was stirred for 18
h. The reaction mixture was then quenched by the careful addition
of 3 M HCl (50 mL) and diluted with dichloromethane (100 mL).
The organic layer was washed with 3 M HCl (50 mL × 4) and
H2O (50 mL × 2) and concentrated to give 1.01 g of 29 as a light-
yellow viscous oil (80% yield). This material was used in the next
step without further purification: 1H NMR (CDCl3) δ 1.73-1.90
(m, 1H), 2.02-2.17 (m, 1H), 2.87-3.00 (m, 1H), 3.02-3.18 (m,
2H), 3.64 (s, 3H), 3.89 (s, 3H), 5.0-5.5 (br, 1H), 7.10 (d, J )
564.8 Hz, 1H), 7.30-7.38 (m, 2H), 7.81 (s, 1H), 7.87-7.92 (m,
1H); 13C NMR (CDCl3) δ 28.6 (d, J ) 94.3 Hz), 36.9 (d, J ) 12.3
Hz), 38.6, 50.2 (2C), 126.3, 126.8, 128.2, 128.6, 131.7, 136.0, 164.9,
171.9 (d, J ) 5.4 Hz); 31P NMR (CDCl3) δ 35.2 (ddt, J ) 15.9,
562.8, 13.9 Hz).
1
1:9 to 1:3) to give 1.03 g of 25b as a yellow oil (68%). H NMR
(CDCl3) δ 1.70-1.94 (m, 4H), 2.33 (s, 3H), 2.88 (t, J ) 6.5 Hz,
2H), 3.68 (m, 4H), 3.93 (s, 3H), 7.40 (t, J ) 8.0 Hz, 1H), 7.62 (d,
J ) 7.9 Hz, 1H), 7.95 (d, J ) 8.0 Hz, 1H), 8.11 (s, 1H); 13C NMR
(CDCl3) δ 27.1, 28.4, 30.4, 30.6, 50.1, 52.3, 52.3, 129.0, 129.0,
131.0, 133.8, 133.8, 137.1, 166.3, 172.1, 195.5.
3-(1-Carboxy-4-mercaptobutoxy)benzoic Acid (26a). A solu-
tion of 25a (8.00 g, 23.5 mmol) in THF (60 mL) was purged of
oxygen by bubbling nitrogen gas through the solution for 1 h. To
the solution was added 3 M NaOH (47 mL), and the mixture was
stirred at room temperature for 24 h. The reaction mixture was
acidified to pH 2 with 1 M HCl and extracted with EtOAc (300
mL × 3). The combined extracts were washed with water (300
mL) and brine (300 mL), dried over MgSO4, filtered, and
concentrated. The residual oil was dissolved in ether, and the
solution was slowly concentrated to precipitate 4.40 g of 26a as a
white solid (69% yield): mp 90-92 °C; 1H NMR (CDCl3) δ 1.40

2884 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Majer et al.
3-[3-(Hydroxylpentafluorophenylmethylphosphinoyl)-2-meth-
oxycarbonylpropyl]benzoic Acid Methyl Ester (30). A solution
of pentafluorobenzyl bromide (0.478 g, 1.83 mmol) and 29 (0.499
g, 1.66 mmol) in dichloromethane (10 mL) was purged with
nitrogen for 30 min by bubbling a stream of nitrogen through the
solution. To the solution was added N,O-bis(trimethylsilyl)acet-
amide (1.23 mL, 4.98 mmol), and the reaction mixture was stirred
at 40 °C for 3 h. The reaction mixture was diluted with dichloro-
methane (50 mL) and washed with 0.5 M HCl (100 mL). The
organic layer was dried over Na2SO4, filtered, and concentrated.
The viscous oil was then dissolved in methanol and concentrated
to give 0.774 g of 30 as a colorless oil (97% yield). This material
was used in the next step without further purification: 1H NMR
(CDCl3) δ 1.74-1.88 (m, 1H), 2.07-2.24 (m, 1H), 2.86-3.20 (m,
3H), 3.15 (d, J ) 16.2 Hz, 2H), 3.59 (s, 3H), 3.89 (s, 3H), 4.57 (s,
1H), 7.28-7.38 (m, 2H), 7.80 (s, 1H), 7.84-7.93 (m, 1H); 13C
NMR (CDCl3) δ 25.1 (d, J ) 89.0 Hz), 29.9 (d, J ) 95.1 Hz),
39.2 (d, J ) 11.5 Hz), 40.9 (d, J ) 3.8 Hz), 50.7, 52.1 (d, J ) 6.1
Hz), 128.2, 128.6, 130.1, 130.4, 133.6, 137.9, 135-152 (multiple
fluoroaromatic peaks), 166.9, 174.1 (d, J ) 6.1 Hz); 31P NMR
(CDCl3) δ 49.3 (m).
filtered, and concentrated. The residual material was purified by
chromatography (EtOAc/hexanes, 1:5) to give 0.670 g of 34 as a
colorless oil (77% yield from 33): 1H NMR (CDCl3) δ 2.22-2.35
(m, 1H), 2.35-2.48 (m, 1H), 2.77-2.91 (m, 2H), 2.99 (dd, J )
10.5, 15.1 Hz, 1H), 5.01 (s, 2H), 5.01-5.12 (m, 2H), 5.35 (s, 2H),
5.74 (ddt, J ) 10.1, 17.0, 7.1 Hz, 1H), 7.13-7.22 (m, 2H), 7.26-
7.48 (m, 10H), 7.85-7.95 (m, 2H).
3-(3-Benzyloxycarbamoyl-2-benzyloxycarbonylpropyl)ben-
zoic Acid Benzyl Ester (35). To a solution of 34 (0.660 g, 1.59
mmol) in acetonitrile (30 mL) and water (30 mL) were added
sodium periodate (2.74 g, 12.8 mmol) and a catalytic amount of
ruthenium oxide. The mixture was stirred at room temperature for
20 h. The reaction mixture was filtered, and the filtrate was
concentrated. The resulting aqueous solution was extracted with
EtOAc (40 mL × 2), and the combined extracts were washed with
brine (40 mL), dried over Na2SO4, filtered, and concentrated. The
residual material was dissolved in dichloromethane (30 mL). To
the solution were added EDC (0.268 g, 1.4 mmol), O-benzyl-
hydroxylamine hydrochloride (0.208 g, 1.3 mmol), diisopropyl-
ethylamine (0.168 g, 1.3 mmol), and DMAP (0.012 g, 0.1 mmol).
The mixture was stirred at room temperature for 3 h. The solvent
was removed under reduced pressure, and the residue was taken
up in EtOAc (80 mL). The organic solution was washed with 1 M
HCl (40 mL) and brine (40 mL), dried over Na2SO4, filtered, and
concentrated. The residual material was purified by silica gel
chromatography to give 0.600 g of 35 as a colorless oil (70% yield
from 34): 1H NMR (CDCl3) δ 2.30 (dd, J ) 4.6, 18.2 Hz, 1H),
2.60 (dd, J ) 9.0, 18.2 Hz, 1H), 2.76 (dd, J ) 9.0, 13.9 Hz, 1H),
3.04 (tt, J ) 4.4, 9.0 Hz, 1H), 3.20 (dd, J ) 4.6, 13.9 Hz, 1H),
4.67-4.73 (m, 2H), 5.03 (d, J ) 10.1 Hz, 1H), 5.09 (d, J ) 10.1
Hz, 1H), 5.35 (s, 2H), 7.22-7.48 (m, 18H), 7.85 (brs, 1H), 7.97
(dt, J ) 7.3, 1.5 Hz, 1H).
3-(2-Carboxy-3-hydroxycarbamoylpropyl)benzoic Acid (36).
To a solution of 35 (0.581 g, 1.08 mmol) in MeOH (30 mL) was
added 100 mg of palladium on carbon (10%), and the mixture was
shaken under hydrogen (21 psi) for 3 h. The catalyst was removed
by filtration, and the filtrate was concentrated. The residual material
was dissolved in water and lyophilized to give 0.280 g of 36 as a
glassy solid (97% yield): mp 163-165 °C (dec); 1H NMR
(CD3OD) δ 2.40 (dd, J ) 4.0, 17.9 Hz, 1H), 2.70 (dd, J ) 8.2,
17.9 Hz, 1H), 2.96 (dd, J ) 9.9, 14.9 Hz, 1H), 3.16-3.28 (m, 2H),
7.35-7.47 (m, 2H), 7.84-7.91 (m, 2H); 13C NMR (CD3OD) δ 31.5,
36.7, 39.5, 129.2, 129.8, 131.3, 134.1, 134.2, 139.0, 171.0, 173.8,
176.6; MS ES- m/z 266 (M - H)-; MS ES+ m/z 268 (M + 1)+.
Anal. (C12H13NO6) H. C: calcd, 53.93; found, 55.09. N: calcd,
5.24; found, 6.35.
3-(5-Ethoxycarbonylmethyl-2,2-dimethyl-4,6-dioxo[1,3]dioxan-
5-ylmethyl)benzoic Acid Methyl Ester (37). To a solution of 32
(0.877 g, 3.0 mmol) and benzyltriethylammonium chloride (0.683
g, 3.0 mmol) in acetonitrile (15 mL) was added anhydrous K2CO3
(0.415 g, 3.0 mmol), and the suspension was stirred at 65 °C for
20 min. To the mixture was added ethyl bromoacetate (0.551 g,
3.3 mmol), and the resulting mixture was stirred at the same
temperature for 20 h. The solvent was removed under reduced
pressure, and the residue was partitioned between aqueous 10%
KHSO4 (10 mL) and EtOAc (10 mL). The organic layer was
separated, and the aqueous layer was extracted with EtOAc (10
mL × 2). The combined extracts were dried over Na2SO4, filtered,
and concentrated. The crude material was recrystallized from
EtOAc/hexanes to give 0.680 g of 37 as a white solid (60% yield):
1H NMR (CDCl3) δ 0.74 (s, 3H), 1.23 (t, J ) 7.1 Hz, 3H), 1.76 (s,
3H), 3.21 (s, 2H), 3.29 (s, 2H), 3.89 (s, 3H), 4.12 (q, J ) 7.1 Hz,
2H), 7.33 (d, J ) 7.8 Hz, 1H), 7.40 (t, J ) 7.8 Hz, 1H), 7.79 (s,
1H), 7.97 (d, J ) 7.8 Hz, 1H); 13C NMR (CDCl3) δ 14.0, 28.1,
28.6, 41.6, 44.9, 52.3, 52.4, 61.7, 107.5, 129.0, 129.6, 130.9, 131.0,
133.8, 134.6, 166.4, 167.7, 170.7.
3-[2-Carboxy-3-(hydroxypentafluorophenylmethylphosphin-
oyl)propyl]benzoic Acid (31). A solution of 30 (0.470 g, 0.98
mmol) in 12 M HCl (30 mL) was stirred at 100 °C for 12 h. The
reaction solution was cooled to room temperature and concentrated
in vacuo to dryness. The residual material was recrystallized from
H2O to give 0.058 g of 31 as a crystalline white solid (13% yield):
1
mp 178-179 °C; H NMR (DMSO-d6) δ 1.80 (dt, J ) 5.4, 14.9
Hz, 1H), 2.07 (ddd, J ) 6.8, 12.6, 14.9 Hz, 1H), 2.86-3.08 (m,
3H), 3.20 (d, J ) 15.2 Hz, 2H), 7.38-7.46 (m, 2H), 7.75-7.84
(m, 2H); 13C NMR (DMSO-d6) δ 25.4 (d, J ) 84.4 Hz), 30.4 (d,
J ) 93.6 Hz), 38.3 (d, J ) 10.0 Hz), 40.8 (d, J ) 3.1 Hz), 127.5,
128.5, 129.9, 130.8, 133-136 (multiple fluoroaromatic peaks),
133.6, 139.2, 167.3, 174.9 (d, J ) 8.4 Hz); 31P NMR (DMSO-d6)
δ 41.3 (m). Anal. (C18H14F5O6P‚H2O) C, H.
3-(5-Allyl-2,2-dimethyl-4,6-dioxo[1,3]dioxan-5-ylmethyl)ben-
zoic Acid Methyl Ester (33). To a solution of 32 (1.17 g, 4.0 mmol)
and benzyltriethylammonium chloride (0.911 g, 4.0 mmol) in
acetonitrile (30 mL) was added anhydrous K2CO3 (0.553 g, 4.0
mmol), and the suspension was stirred at 75 °C for 20 min. To the
mixture was added allyl bromide (0.42 mL, 4.8 mmol), and the
resulting mixture was stirred at the same temperature for 5 h. The
solvent was removed under reduced pressure, and the residue was
taken up in EtOAc (50 mL). The solution was washed with 1 M
HCl (50 mL) and brine (50 mL), dried over MgSO4, filtered, and
concentrated. The residual material was purified by silica gel
chromatography (EtOAc/hexanes, 1:10) to give 0.918 g of 33 as a
white solid (69% yield): mp 57-59 °C; Rf ) 0.32 (EtOAc/hexanes,
1
1:4); H NMR (CDCl3) δ 0.73 (s, 3H), 1.52 (s, 3H), 2.87 (d, J )
7.5 Hz, 2H), 3.36 (s, 2H), 3.88 (s, 3H), 5.21 (d, J ) 9.9 Hz, 1H),
5.25 (d, J ) 17.1 Hz, 1H), 5.69 (ddt, J ) 9.9, 17.1, 7.5 Hz, 1H),
7.30-7.43 (m, 2H), 7.83 (s, 1H), 7.87-7.96 (m, 1H); 13C NMR
(CDCl3) δ 29.0, 29.6, 43.5, 44.2, 52.2, 57.6, 105.9, 121.6, 129.0,
129.1, 130.4, 130.8, 131.1, 134.7, 135.5, 166.6, 168.1.
3-(2-Benzyloxycarbonylpent-4-enyl)benzoic Acid Benzyl Ester
(34). A suspension of 33 (0.698 g, 2.10 mmol) in 2 M NaOH (10
mL) and dioxane (5 mL) was stirred at 100 °C for 3 h. The resulting
clear solution was concentrated, acidified to pH 1 by adding 1 M
HCl, and extracted with EtOAc (25 mL). The extract was dried
over Na2SO4, filtered, and concentrated. The resulting white solid
was dissolved in DMSO (10 mL), and the solution was stirred at
130 °C for 3 h. The reaction mixture was taken up in EtOAc (80
mL), washed with water (40 mL × 2), dried over Na2SO4, filtered,
and concentrated to give 0.62 g of glassy liquid. This material was
dissolved in dichloromethane (50 mL). To the solution of this
material in dichloromethane (50 mL) were added EDC (0.959 g,
5.0 mmol), benzyl alcohol (0.541 g, 5.0 mmol), and DMAP (0.06
g, 0.5 mmol). The mixture was stirred at room temperature for 20
h. Solvent was removed under reduced pressure, and the residue
was dissolved in EtOAc (80 mL). The organic solution was washed
with 1 M HCl (40 mL) and brine (40 mL), dried over Na2SO4,
2-Carboxy-2-(3-carboxybenzyl)succinic Acid (38). A suspen-
sion of 37 (0.570 g, 1.51 mmol) in 2 M NaOH (8 mL) was stirred
at 100 °C for 3 h. The resulting clear reaction mixture was acidified
to pH 1 by 1 M H2SO4 and extracted with EtOAc (25 mL). The
organic extract was dried over MgSO4, filtered, and concentrated

Inhibitors of Glutamate Carboxypeptidase II
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2885
(4) Ghadge, G. D.; Slusher, B. S.; Bodner, A.; Canto, M. D.; Wozniak,
K.; Thomas, A. G.; Rojas, C.; Tsukamoto, T.; Majer, P.; Miller, R.
J.; Monti, A. L.; Roos, R. P. Glutamate carboxypeptidase II inhibition
protects motor neurons from death in familial amyotrophic lateral
sclerosis models. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9554-
9559.
(5) Zhou, J.; Neale, J. H.; Pomper, M. G.; Kozikowski, A. P. NAAG
peptidase inhibitors and their potential for diagnosis and therapy.
Nat. ReV. Drug DiscoVery 2005, 4, 1015-1026.
(6) Jackson, 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) Stefanic, P.; Dolenc, M. S. Aspartate and glutamate mimetic structures
in biologically active compounds. Curr. Med. Chem. 2004, 11, 945-
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O. Syntheses of conformationally constricted molecules as potential
NAALADase/PSMA inhibitors. Org. Lett. 2004, 6, 1805-1808.
(9) Mesters, J. R.; Barinka, C.; Li, W.; Tsukamoto, T.; Majer, P.; Slusher,
B. S.; Konvalinka, J.; Hilgenfeld, R. Structure of glutamate carboxy-
peptidase II, a drug target in neuronal damage and prostate cancer.
EMBO J. 2006, 25, 1375-1384.
(10) Hin, B.; Majer, P.; Tsukamoto, T. Facile synthesis of R-substituted
acrylate esters. J. Org. Chem. 2002, 67, 7365-7368.
(11) Reiter, L. A.; Jones, B. P. Amide-assisted hydrolysis of â-carbox-
amido-substituted phosphinic acid esters. J. Org. Chem. 1997, 62,
2808-2812.
(12) Stoermer, D.; Liu, Q.; Hall, M. R.; Flanary, J. M.; Thomas, A. G.;
Rojas, C.; Slusher, B. S.; Tsukamoto, T. Synthesis and biological
evaluation of hydroxamate-based inhibitors of glutamate carboxy-
peptidase II. Bioorg. Med. Chem. Lett. 2003, 13, 2097-2100.
(13) Barinka, C.; Rinnova, M.; Sacha, P.; Rojas, C.; Majer, P.; Slusher,
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logical analysis of recombinant human glutamate carboxypeptidase
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(15) 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.
to give 0.450 g of 38 (100% crude yield) as a colorless oil. This
material was used in the next step without further purification: 1H
NMR (CD3OD) δ 2.78 (s, 2H), 3.46 (s, 2H), 7.35-7.42 (m, 2H),
7.80-7.93 (m, 2H).
2-(3-Carboxybenzyl)succinic Acid (39). A solution of 38 (0.450
g, 1.51 mmol) in DMSO (6 mL) was stirred at 130 °C for 3 h. The
solvent was removed under reduced pressure, and the residual oil
was dissolved in EtOAc (25 mL). The organic solution was washed
with water (25 mL × 3), dried over MgSO4, filtered, and
concentrated. The crude material was recrystallized from EtOAc/
hexanes to give 0.260 g of 39 as a white solid (68% yield from
1
37): mp 188-190 °C; H NMR (CD3OD) δ 2.40 (dd, J ) 4.9,
16.8 Hz, 1H), 2.60 (dd, J ) 8.6, 16.9 Hz, 1H), 2.85-2.97 (m, 1H),
2.99-3.13 (m, 2H), 7.33-7.49 (m, 2H), 7.82-7.92 (m, 2H); 13C
NMR (CD3OD) δ 36.1, 38.4, 44.4, 129.1, 129.6, 131.4, 132.1,
134.8, 140.5, 169.8, 175.4, 177.6. Anal. (C12H12O6) C, H.
Biological Studies. The GCP II assay was carried out as outlined
previously.14 The chronic constrictive injury models were performed
following the procedure reported by Bennett’s group.15
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
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Synthesis and biological evaluation of thiol-based inhibitors of
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