Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology

Article abstract of DOI:10.1021/jm010303j

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in

Full text of DOI:10.1021/jm010303j

J . Med. Chem. 2002, 45, 19-31
19
Novel 1-Hyd r oxya zole Bioisoster es of Glu ta m ic Acid . Syn th esis, P r otolytic
P r op er ties, a n d P h a r m a cology
Tine B. Stensbøl,† Peter Uhlmann,† Sandrine Morel,† Birgitte L. Eriksen,† J akob Felding,† Hasse Kromann,†
Mette B. Hermit,† J eremy R. Greenwood,† Hans Brau¨ner-Osborne,† Ulf Madsen,† Finn J unager,‡
Povl Krogsgaard-Larsen,† Mikael Begtrup,† and Per Vedsø*,†
NeuroScience PharmaBiotec Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
2 Universitetsparken, DK-2100 Copenhagen, Denmark, and Product Development, Novo Nordisk A/ S,
DK-2760 Ma˚løv, Denmark
Received J uly 3, 2001
A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid
(Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological
studies, including a series of Glu receptor binding assays, uptake studies on native as well as
cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds
7a ,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic
functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC50 ) 2.7
( 0.4 µM and IC50 ) 2.6 ( 0.6 µM, respectively), correlating with electrophysiological data
from the rat cortical wedge model (EC50 ) 280 ( 48 µM and EC50 ) 586 ( 41 µM, respectively).
1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a ,b, showed high affinity for [3H]-
AMPA receptor binding sites (IC50 ) 0.15 ( 0.03 µM and IC50 ) 0.13 ( 0.02 µM, respectively).
Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical
wedge model (EC50 > 1000 µM), whereas the corresponding 4-methyl analogue 8b was a potent
AMPA receptor agonist (EC50 ) 15 ( 2 µM). In accordance with this disparity, compound 8a
was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC50 ) 93 ( 25 µM), as well as
excitatory amino acid transporters (EAATs) EAAT1 (IC50 ) 100 ( 30 µM) and EAAT2 (IC50
)
300 ( 80 µM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites,
neither synaptosomal nor cloned. Compounds 9a -c and 10a ,b, possessing 1-hydroxyimidazole
as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic
properties of compounds 7a ,b, 8b, and 9b were determined by titration, and a correlation
between the pKa values and the activity at AMPA receptors was apparent. Optimized structures
of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2
construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed,
this could be rationalized on the basis of the ability of the ligand to fit the construct. The results
presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as
bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed
significant activity at metabotropic Glu receptors.
In tr od u ction
signaling pathways in Alzheimer’s patients may in part
explain loss of cognitive function characteristic of this
disease.6,7
(S)-Glutamic acid (Glu) (1) (Figure 1) is the main
excitatory neurotransmitter in the central nervous
system (CNS). Excitation is mediated by two major
classes of receptor systems: the G-protein-coupled me-
tabotropic receptors and the ionotropic receptors.1-4 The
ionotropic Glu (iGlu) receptors are divided into three
groups: N-methyl-D-aspartic acid (NMDA), (R,S)-2-
amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid
(AMPA), and kainate receptors. For each group of
receptors a number of subunits have been cloned.
Evidence suggests that receptor complexes are as-
sembled from either four or five subunits.5 The impor-
tance of iGlu receptors in fast synaptic signaling path-
ways, and thus in learning and memory processes, is
well established. Deficits within the glutamatergic
The actions of Glu in the mammalian brain are
carefully regulated, in part by high-efficiency uptake
systems that secure the termination of receptor activa-
tion such that concentrations of Glu do not become
neurotoxic. To date, five different high-affinity sodium-
dependent human excitatory amino acid transporters
(EAATs) have been cloned (EAAT1-5), of which
EAAT1-3 are thought to be responsible for most of the
uptake.8,9 The ability of these transporters to control
the concentration of Glu present in the synaptic cleft
suggests that they are potential drug targets for treating
a number of diseases related to hypo- or hyperactivation
of Glu receptors.
We and others have previously demonstrated that a
number of 3- and 5-hydroxyazoles serve as useful
bioisosteres for the distal carboxylic acid function of
Glu.10-15 The natural products (S)-quisqualic acid (2)16
* To whom correspondence should be addressed. Phone: (+45)
35306487. Fax: (+45) 35306040. E-mail: pv@dfh.dk.
† The Royal Danish School of Pharmacy.
‡ Novo Nordisk A/S.
10.1021/jm010303j CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/04/2001

20 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Stensbøl et al.
Sch em e 1a
a
Reagents and conditions: (a) m-CPBA, EtOAc, rt; (b) PhCH2Br,
NEtPri
.
2
Sch em e 2a
a
Reagents and conditions: (a) n-BuLi or LDA, THF, DMF, -78
°C to rt; (b) NaBH4, MeOH, 0 °C; (c) SOCl2, rt; (d) CH3CONHCH-
(COOC2H5)2, NaH, DMF, rt; (e) H2 (1 atm), Pd/C, MeOH, 0 °C; (f)
4 M HCl or 1 M TFA, reflux, 3h.
Sch em e 3a
F igu r e 1. Structures of Glu (1) and a number of heterocyclic
analogues including the synthesized N-hydroxyazoles 7a ,b ,
8a ,b, 9a -c, and 10a ,b.
a
Reagents and conditions: (a) PhCH2ONH2 (a 2:1 mixture of
and (S)-2-amino-3-(5-hydroxy-4-isoxazolyl)propionic acid
[TAN-950A (4)]15 bearing the acidic groups 1,2,4-oxa-
diazolidine-3,5-dione and 5-hydroxyisoxazole, respec-
tively, are both potent Glu receptor agonists. AMPA
(3b), bearing a 3-hydroxyisoxazole moiety as the distal
acidic function, has for long been the standard com-
pound for characterizing these receptors.17 Furthermore,
3-hydroxyisothiazole (e.g., 5)13 and 3-hydroxy-1,2,5-
thiadiazole (6)14 analogues of Glu have also been proven
to interact with Glu receptors. These results prompted
us to search for other azoles that could serve in place of
the distal carboxylic acid of Glu. This report describes
the synthesis of a series of analogues of AMPA bearing
1-hydroxypyrazole (7a ,b), 1-hydroxy-1,2,3-triazole (8a ,b;
in the following 1-hydroxytriazole is used as a synonym
for 1-hydroxy-1,2,3-triazole), or 1-hydroxyimidazole (9a,b
and 10a ,b) as the distal acid function and the pharma-
cological evaluation of the target compounds at Glu
receptors and uptake sites.
19 and 20 was obtained); (b) NH2NH2, Cu(OAc)2, pyridine, reflux
(2:1 mixture of 21 and 22); (c) n-BuLi, THF, DMF, -78 °C to rt;
(d) NaBH4, MeOH, 0 °C.
Resu lts
Ch em istr y. The synthetic approach to the target
structures 7a ,b, 8a ,b, 9a -c, and 10a ,b is outlined in
Schemes 1-7. The key step in the synthesis of these
compounds involves introduction of a formyl substituent
at the 5-position of the pyrazole or 1,2,3-triazole ring
and at the 2- or 5-position of the imidazole ring. This
was accomplished by applying our previously reported
method for ortho lithiation of 1-benzyloxypyrazole,18
1-benzyloxy-1,2,3-triazole,19 and 1-benzyloxyimidazole20,21
and subsequent formylation using DMF. The synthetic
sequences used for the transformation of the ortho-
formylated 1-benzyloxyazoles 14a ,b, 23a ,b, 34a -c, and
33a ,b into the target compounds 7a ,b, 8a ,b, 9a -c, and
10a ,b were as follows (Schemes 2, 4, 6, and 7): (1)
reduction of the formyl group using sodium borohydride,

Bioisosteres of Glutamic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 21
Sch em e 7a
Sch em e 4a
a
Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) SOCl2,
CH2Cl2, 0 °C; (c) CH3CONHCH(COOC2H5)2, NaH, DMF, rt; (d)
H2 (1 atm), Pd/C, MeOH, 0 °C; (e) 12 M HCl, reflux, 3h.
a
Reagents and conditions: (a) n-BuLi, THF, DMF, -78 °C to
rt; (b) NaBH4, MeOH, 0 °C; (c) SOCl2, 0 or 60 °C; (d) CH3CONHCH-
(COOC2H5)2, NaH, DMF, rt; (d) H2 (1 atm), Pd/C, MeOH, 0 °C; (e)
1 M TFA or 4 M HCl, reflux.
Sch em e 5a
target compounds that were isolated as their hydro-
chlorides (compounds 7a , 8-10b, and 9c) or as the
zwitterionic forms following ion exchange chromatog-
raphy (compounds 7b, 8-10a ). 1-Benzyloxy-4-meth-
ylpyrazole (13b) required for the synthesis of 7b was
prepared by N-oxidation of 4-methylpyrazole (11) fol-
lowed by O-benzylation in a fashion similar to the
previously reported18 preparation of 1-benzyloxypyra-
zole (Scheme 1). The 4-methyl-substituted triazole 22
(Scheme 3) required for preparation of 8b was not
readily available, since no existing methodology deals
with functionalization of the 4-position of 1-hydroxy-
triazoles. Thus, 22 was prepared as a 1:2 mixture with
the isomeric 1-benzyloxy-5-methyl-1,2,3-triazole (21) by
treatment of a 1:2 mixture of 19 and 20 with excess
hydrazine and by subsequent oxidative cyclization. The
isomeric methyl-substituted triazoles 21 and 22 were
inseparable by column chromatography. However, only
the 4-methyl-substituted triazole 22 underwent ortho
lithiation and subsequent formylation, producing a
mixture of 23b and unchanged 21, which were easily
separated after the aldehyde 23b had been reduced to
the corresponding 5-hydroxymethyl derivative 24b
(Scheme 3).
a
Reagents and conditions: (a) NH2OH‚HCl, aqueous CH2O,
MeOH, rt; (b) PhCH2Br, KOH, MeOH, reflux; (c) PCl3, CHCl3,
reflux (a 1:1 mixture of 29b and 30b was obtained); (d) n-BuLi,
THF, C2Cl6, -78 °C to rt (6:5 mixture of 31 and 32); (e) n-BuLi,
THF, DMF, -78 °C to rt.
Sch em e 6a
Also, the isomeric 4-methyl- and 5-methyl-1-benzyl-
oxyimidazoles 30b and 29b (Scheme 5) were not readily
available as separate isomers. However, a 1:1 mixture
of 30b and 29b was obtained from 4(5)-methyl-1-
hydroxyimidazole-3-oxide (28) by O-benzylation and
subsequent PCl3-induced deoxygenation. The insepa-
rable mixture of 30b and 29b was lithiated at C-2, and
subsequent chlorination using hexachloroethane pro-
duced the corresponding 2-chloro derivatives 31 and 32,
which could be separated by column chromatography.
1-Benzyloxy-2-chloro-4-methylimidazole (32) was selec-
tively ortho-lithiated at C-5. The chlorine at C-2 protects
this position from lithiation, and then treatment with
DMF produced the aldehyde 34b, which was converted
to the desired imidazole amino acid 9b using the
standard protocol (Scheme 6) with simultaneous and
advantageous C-2 dechlorination during the O-deben-
zylation step. When 1-benzyloxy-2-chloro-5-methylimi-
dazole (31) was treated with n-BuLi/DMF, the lithiation/
formylation occurred at C-2, via chlorine-lithium
exchange, and not at C-4 because lithiation at this
position is disfavored by the adjacent lone pair at N-3.22
The resulting 2-formylated imidazole was transformed
a
Reagents and conditions: (a) n-BuLi, THF, DMF, -78 °C to
rt; (b) NaBH4, MeOH, 0 °C; (c) SOCl2, 0 or 60 °C; (d) CH3CONHCH-
(COOC2H5)2, NaH, DMF, rt; (e) H2 (1 atm), Pd/C, MeOH, 0 °C; (f)
4 M HCl, reflux, 3h.
producing the corresponding hydroxymethyl-substituted
derivatives (compounds 15a,b, 24a,b, 35a-c, and 39a,b);
(2) preparation of the chloromethyl-substituted deriva-
tives by reaction with thionyl chloride (compounds
16a ,b, 25a ,b, 36a -c, a n d 40a ,b); (3) introduction of the
diethyl acetamidomalonate group (compounds 17a ,b ,
26a ,b, 37a -c, and 41a ,b); (4) O-debenzylation using
hydrogen (1 bar) and palladium on carbon (compounds
18a ,b, 27a ,b, 38a -c, and 42a ,b); and (5) acid depro-
tection in 4 or 12 M HCl or 1 M TFA. This gave the

22 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Stensbøl et al.
Ta ble 1. pKa Values of Selected Compounds
pKa valuesa
these transporters. Likewise, the 1-hydroxypyrazole
analogues 7a ,b also showed no or low affinity for
EAAT1-3. The affinity of 8a for EAAT1 (IC50 ) 100 µM)
correlated with data gathered from synaptosomal Glu
uptake systems using [3H]-D-aspartic acid as a substrate
(IC50 ) 93 µM).
In contrast with compounds 7a ,b and 8a ,b, the
1-hydroxyimidazole analogues of 3a and AMPA (3b)
compounds 9a -c and 10a ,b showed no detectable
affinities for or agonist or antagonist activity at iGlu
receptors.
compound
-COOH
bioisostere
-NH2
AMPA (3b)b
1.94 ( 0.05b
5.12 ( 0.03b
10.1 ( 0.01b
2.5c
4.8c
10.0c
7a
7b
8b
9b
1.78 ( 0.04
2.05 ( 0.04
1.48 ( 0.08
2.81 ( 0.02
5.78 ( 0.01
5.89 ( 0.01
5.07 ( 0.01
8.82 ( 0.01d
9.48 ( 0.01
9.81 ( 0.01
9.24 ( 0.01
10.2 ( 0.01d
a
Determined by potentiometric titration unless otherwise
stated. Determined by 13C NMR (ref 13). c Reference 23. As-
b
d
signment may be interchanged.
None of the target compounds showed appreciable
agonist or antagonist activity at 1000 µM on mGlu1,
mGlu2, or mGlu4 metabotropic glutamate receptor sub-
types (data not shown).
to the imidazole amino acid 10b using the standard
protocol (Scheme 7).
P r otolytic P r op er ties. The pKa values of compounds
7a ,b , 8b , and 9b were determined by potentiometric
titration. In general the pKa values of the carboxyl and
amino groups of compounds 7a ,b, 8b, and 9b lay in the
same range as those of AMPA (3b) (Table 1), although
minor differences were seen. The pKa value of the
carboxylic group of the R-amino acid moiety was about
one unit higher for compound 9b than for 7a ,b, 8b, and
AMPA (3b). The pKa value of the N-hydroxy group of
9b was found to be at least three pKa units higher than
those of the 1-hydroxypyrazole and the 1-hydroxytria-
zole groups of analogues 7a ,b and 8b. Thus, 9b exhib-
ited a much lower degree of acidity than the other
azoles.
Molecu la r Mod elin g. Molecular geometries were
calculated in solution for 7a ,b, 8a ,b, 9a -c, and 10a ,b
in conformations corresponding to that of the bound
ligand 3b, as found in the X-ray crystal structure of its
complex with the GluR2-S1S2J construct of Armstrong
and Gouaux.27 The optimized ligand structures were
fitted to the crystal structure of the AMPA-GluR2
complex, and 3b was then removed, as depicted for 7b,
8b, 9b, and 10b in Figure 2. Each ligand was analyzed
according to its ability to form hydrogen bonds and
favorable or unfavorable van der Waals contacts with
the receptor, taking receptor water molecules into
account. Critical internuclear distances are noted in
Figure 2.
In Vitr o P h a r m a cology. The affinities of all com-
pounds for AMPA, kainic acid, and NMDA receptor
binding sites were determined using [3H]AMPA,24 [3H]-
kainic acid,25 and [3H][3-(2-carboxypiperazin-4-yl)pro-
pyl]phosphonic acid ([3H]CPP),26 respectively (Table 2).
None of the compounds showed detectable affinities for
NMDA receptor binding sites (IC50 > 100 µM) or
appreciable affinities for kainic acid binding sites. The
two 1-hydroxypyrazoles 7a ,b showed the same affinities
for [3H]AMPA binding sites (IC50 2.7 ( 0.4 µM and 2.6
( 0.6 µM, respectively), being 50-fold weaker than that
of AMPA (3b). The two 1-hydoxytriazole analogues 8a ,b
showed affinities that were appreciably higher than
those of 7a ,b but were similar to each other (IC50 ) 0.15
( 0.03 µM and 0.13 ( 0.02 µM, respectively), thus being
slightly weaker than AMPA (3b) and significantly more
potent than 3a . On the rat cortical wedge preparation,
compounds 7a ,b were almost equipotent, displaying
potencies that correlated with their affinities for AMPA
receptors (EC50 ) 280 ( 48 µM and EC50 ) 586 ( 41
µM, respectively). Compound 8a was apparently devoid
of activity on the rat cortical wedge preparation (EC50
> 1000 µM and IC50 > 1000 µM), whereas the 4-methyl
analogue 8b proved to be a potent AMPA receptor
agonist (EC50 ) 15 ( 2 µM). Depolarization evoked by
concentrations equivalent to the EC50 values of com-
pounds 3a ,b, 7a ,b, and 8b was not antagonized by 5
µM CPP but fully antagonized by 5 µM NBQX, thus
indicating selective actions on AMPA receptors. To
further explore the disparity between the affinity and
potency of compound 8a , all compounds were evaluated
on both synaptosomal and cloned Glu uptake systems.
Whereas the non-methylated analogues 3a and 8a
showed affinities for both EAAT1 and EAAT2 uptake
sites, the corresponding methylated analogues 3b and
8b showed no and weaker affinity, respectively, toward
For compounds 7a ,b none of the important polar
contacts between the ligand and the receptor are inter-
rupted. According to the superposition, the proton at C-3
of the pyrazole ring lies just within the van der Waals
contact radius of one of the protons of Met-708 (Figure
2A).
Fitting 8a and 8b to the GluR2 crystal structure
indicates that these can bind in an AMPA-like mode,
making the same favorable contacts with the closed
binding domain of the receptor as AMPA, without
disrupting hydrogen bonds or entering sterically forbid-
den zones (Figure 2B).
When compared with 3b in the GluR2-S1S2 con-
struct, it is apparent that 1-hydroxyimidazoles 9a -c
and 10a ,b cannot form hydrogen bonds with the tightly
bound receptor water W2 or with the backbone NH of
Glu-705 (parts C and D of Figure 2). The proton at C-2
in 9a ,b and 10a also collides with Glu-705 according to
the superpositions (Figure 2C). Attempting to drive a
methyl group into this region on domain closure (9c,
10b) would interfere further with W2 and Glu-705.
Discu ssion
Recently, Armstrong and Gouaux have elucidated the
crystal structure of a construct of the GluR2 binding
domain in complex with AMPA, as well as with DNQX,
Glu, and kainic acid.27 Although the side chains of the
receptor in the binding site overlap closely between the
Glu- and AMPA-GluR2-complexes, as do the R-amino
acid portions of the ligands, the distal groups of the
ligands are not aligned. However, in combination with
water, the distal acidic moieties interact isosterically
with the receptor.27 The aim of the present project has
been to elucidate whether one or more N-hydroxyazole
scaffolds presented here could function in a fashion

Bioisosteres of Glutamic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 23
Ta ble 2. Receptor Binding, Electrophysiology, and Affinity for Glu Transportersa
IC50 (µM)
EC50 (µM)
IC50 (µM)
compound
[3H]AMPA
[3H]kainic acid
[3H]CPP
cortical wedge
[3H]D-aspartic acid
EAAT1
EAAT2
EAAT3
AMPA (3b)
3a
7a
7b
8a
8b
9a
9b
9c
10a
10b
0.04 ( 0.01
0.27 ( 0.06
2.7 ( 0.4
2.6 ( 0.6
0.15 ( 0.03
0.13 ( 0.02
>100
>100
>100
>100
>100
47 ( 7
90 ( 3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.5 ( 0.2
900 ( 60
280 ( 48
586 ( 41
>1000
>200
>200
>200
>200
93 ( 25
>200
>200
>200
>200
>200
>200
>1000
270 ( 40
>1000
>1000
100 ( 30
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
240 ( 20
430 ( 80
>1000
300 ( 80
490 ( 30
>1000
15 ( 2
>1000
>100
>1000
>1000
>100
>1000
>1000
>100
>1000
>1000
>100
>1000
>1000
a
Data represent mean ( SEM of at least three individual experiments.
similar to the 3-hydroxyisoxazole moiety of AMPA (3b),
mimicking the action, though not necessarily the bind-
ing mode, of the distal carboxylic acid of Glu. Further-
more, we wanted to investigate the relationship among
structure, the protolytic properties of these compounds,
and their ability to interact with Glu receptors and
uptake sites.
The structure-activity relationships described in this
paper indicate that the 1-hydroxypyrazole and 1-hy-
droxytriazole analogues (compounds 7a ,b and 8a ,b)
interact with Glu recognition sites, whereas the less
acidic 1-hydroxyimidazole analogues (compounds 9a -c
and 10a ,b) do not possess the structural and protolytical
requirements for activation of Glu receptors or Glu
uptake systems.
The two 1-hydroxypyrazoles 7a ,b bind to and activate
AMPA receptors to a similar extent. Of the 3-hydroxy-
isoxazoles, only AMPA (3b), bearing a 5-methyl sub-
stituent, appears to activate AMPA receptors in the rat
cortical wedge preparation. This failure of 3a to depo-
larize cortical neurones has been suggested to be due
to uptake by Glu transporters present in the cortical
wedge preparation.10 This hypothesis is reinforced by
the observed affinity of 3a toward cloned uptake sys-
tems (Table 2). Although 7a is an analogue of 3a , this
compound does not interact with EAAT1, and it retains
activity at AMPA receptors in accordance with the
proposed role of the uptake systems.
The reduced potency of 7a ,b vs 3b at AMPA receptors
(Figure 2A) can be explained by the molecular modeling
studies. To accommodate the extra proton at C-3 of 7a
and 7b, Met-708 and the heterocycle moiety of the
ligand must come to occupy positions more distant from
one another upon domain closure. Tightening the con-
formation of the methionine away from the heterocycle
will slightly increase the total free energy of the system,
as will shifting the ligand from the more optimal AMPA-
like position. However, the adjustment required is small
(1-2 kcal/mol), in accordance with only losing an order
of magnitude of potency.
are on par with one another, whereas only compounds
3a and 8a show affinities for EAAT 1. Taking into
account that the affinities of 3a and 8a for AMPA
receptors do not correlate with the potency found in the
rat cortical wedge model, whereas the affinity and
potency of 7a does, these findings could lead to the
conclusion that 3a and 8b are substrates for EAAT1 in
the cortical wedge preparation. However, substrates and
blockers cannot be distinguished by the present assay.
EAAT2 has been described as the major uptake mech-
anism in the forebrain, whereas EAAT 1 primarily is
localized in the cerebellum.28 This evidence contradicts
the above-mentioned hypothesis that EAAT1 is the
primary cause for the lack of potency of 3a and 8b
observed here and suggests that EAAT2 might be
involved. Hence, this issue remains to be clarified.
The lack of activity of 9a -c and 10a ,b as AMPA
receptor agonists can be rationalized in two ways. First,
the pKa value of the N-hydroxy group of the 1-hydroxy-
imidazole moiety is so high that only a modest propor-
tion of the molecules is expected to be ionized in
solution. To date, structures known to function as distal
carboxylate bioisosteres at AMPA receptors cover a pKa
range where the ionized form dominates in aqueous
solution. A low degree of ionization is expected to
disfavor binding by shifting the equilibrium away from
the presumably active form carrying three charges.
Second, the modeling studies show that the presence of
a proton or a methyl group at C-2 of the imidazole ring
(9a ,b, 10a , and 9c,10b, respectively) prohibits two
hydrogen bonds with the receptor environment com-
pared with the AMPA binding mode, each corresponding
to at least 4 kcal/mol in binding energy. The presence
of a methyl group directly interferes with domain
closure, thus rendering these imidazole analogues inac-
tive.
The structural requirements for interaction with Glu
uptake systems are far from being elucidated, and the
results found here do not imply a simple structure-
activity relationship. However, these observations lead
to the conclusion that the presence of a methyl group
neighboring the R-amino acid side chain of the N-
hydroxyazole hinders uptake by Glu transport mecha-
nisms. Furthermore, iGluRs as well as Glu uptake
mechanisms display a preference for hydroxyazoles with
pKa values in the same range as that of the 3-hydroxy-
isoxazole of AMPA.
The similarity of the binding modes and affinities of
8a ,b lies in stark contrast to their observed potency on
the rat cortical wedge preparation. Compound 8a ap-
parently does not activate or antagonize AMPA recep-
tors on the cortical wedge, whereas compound 8b is a
potent AMPA receptor agonist. As is the case for the
non-methylated 3-hydroxyisoxazol analogue 3a, 8a shows
affinity for EAAT1 and EAAT2, which again most likely
explains its lack of activity on the rat cortical wedge.
The affinities of compounds 3a , 7a , and 8a for EAAT 2
In conclusion, the results obtained indicate that
1-hydroxytriazoles and 1-hydroxypyrazoles may be
useful as novel carboxylic acids bioisosteres while

24 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Stensbøl et al.
F igu r e 2. Glu analogues docked into the binding site of GluR2-S1S2J :27 (A) 7b; (B) 8b; (C) 9b; (D) 10b. Some residues lining
the binding site (Tyr-723 in front and Leu-650 behind) have been removed for clarity. The binding site is highly conserved across
AMPA-receptor subunits GluR1-4.42 Selected hydrogen bonds are shown in black, and unfavorable van der Waals interactions
are shown in dark red. W1-3 are binding site water molecules.
2. P r ep a r a tion of P yr a zole Am in o Acid 7a . 2.1. 1-Ben -
zyloxy-5-h ydr oxym eth ylpyr azole (15a). A solution of 1-ben-
zyloxy-5-formylpyrazole (14a )18 (1.88 g, 9.31 mmol) in MeOH
(30 mL) was added with sodium borohydride (0.45 g, 11.9
mmol) and stirred at 0 °C for 30 min. The MeOH was
evaporated, and the residue was taken up in CH2Cl2 and
washed with brine. The organic phase was dried (MgSO4) and
concentrated. FC (EtOAc/heptane 1:6 f 1:1) gave 1.86 g (98%)
of 15a as a viscous oil; 1H NMR (CDCl3) δ 7.40-7.26 (m, 5H),
7.22 (d, J ) 2.4 Hz, 1H), 6.07 (d, J ) 2.4 Hz, 1H), 5.29 (s, 2H),
4.24 (d, J ) 4.8 Hz, 2H), 2.35 (br s, 1 H); 13C NMR (CDCl3) δ
135.6 (s), 133.5 (s), 132.7 (d), 130.0 (d), 129.4 (d), 128.7 (d),
102.7 (d), 80.2 (t), 53.8 (t). Anal. (C11H12N2O2) C, H, N.
N-hydroxyazoles of high pKa, or lacking a hydrogen bond
acceptor R to the acidic hydroxy group, are likely to be
poor carboxylic acid bioisosters at iGlu receptors.
Exp er im en ta l Section
1. Ch em istr y. All reactions involving air-sensitive reagents
were performed under N2 using syringe-septum cap tech-
niques. All glassware was flame-dried prior to use. Flash
column chromatography (FC) was performed using silica gel
(Merck, 40-63 mesh). TLC was performed using Merck silica
gel 60 F254 aluminum sheets. The plates were visualized under
UV light (254 nm) and by spraying with ammonium cerium
molybdate. Compounds containing amino groups were visual-
ized using a ninhydrin spraying reagent. Melting points are
uncorrected. All new compounds were colorless, unless other-
wise stated. If not otherwise stated, NMR spectra were
2.2. 1-Ben zyloxy-5-ch lor om eth ylp yr a zole (16a ). Com-
pound 15a (1.68 g, 8.2 mmol) was treated with thionyl chloride
(3.7 mL, 50 mmol) for 1 h at room temperature. The mixture
was concentrated, coevaporated three times with toluene, and
dried (room temp (rt), 0.1 mmHg) to give 1.75 g of crude 16a .
FC (EtOAc/heptane 1:10 f 1:2) gave 1.60 g (87%) of 16a as
an oil: 1H NMR (CDCl3) δ 7.42-7.34 (m, 5H), 7.26 (d, J ) 2.4
Hz, 1H), 6.16 (d, J ) 2.4 Hz, 1H), 5.35 (s, 2H), 4.26 (s, 2H);
13C NMR (CDCl3) δ 133.3 (s), 132.8 (d), 131.8 (s), 129.9 (d),
recorded on
a 200 MHz Bruker or a 300 MHz Varian
spectrometer in CDCl3 (tetramethylsilane as internal stan-
dard) or D2O with dioxane as the internal standard at 3.70
ppm (1H spectra) and 67.4 ppm (13C spectra). The multiplicity
of 13C NMR signals was assigned from APT (attached proton
test) spectra. Microanalyses were within (0.4% of the theo-
retical values if not otherwise stated.
129.4 (d), 128.7 (d), 103.8 (d), 80.4 (t), 32.9 (t). Anal. (C11H11
ClN2O) C, H, N.
-
2.3. Eth yl 2-Aceta m id o-3-(1-ben zyloxy-5-p yr a zolyl)-2-
eth oxyca r bon ylp r op ion a te (17a ). Diethyl acetamidomalo-
nate (2.61 g, 12.0 mmol) was slowly added to a suspension of
sodium hydride (485 mg, 60% suspension, 12.1 mmol) in dry
DMF (30 mL) at 0 °C. The mixture was allowed to warm to
All solvents and reagents were obtained from Fluka or
Aldrich and used without further purification except for THF,
which was distilled from Na/benzophenone under nitrogen, and
DMF, which was sequentially dried with and stored over 3 Å
molecular sieves.29

Bioisosteres of Glutamic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 25
room temperature, and stirring was continued until a clear
yellow solution was obtained and no more H2 evolved (ca. 2
h). Then a solution of 16a (1.36 g, 6.13 mmol) in dry DMF (12
mL) was added, and the mixture was stirred at room temper-
ature. After 18 h, the reaction was quenched with saturated
NH4Cl (100 mL) and the product was extracted with AcOEt
(3 × 100 mL). The combined organic phases were washed twice
with an ice-cold 5% solution of NaOH, dried (MgSO4), and
concentrated. FC (EtOAc/heptane 1:4 f 1:1) gave 2.22 g (90%)
of 17a as a solid. A small sample was recrystallized from
EtOAc/heptane: mp 85-86 °C; 1H NMR (CDCl3) δ 7.39-7.34
(m, 5H), 7.21 (d, J ) 2.4 Hz, 1H), 6.57 (br s, NH), 5.78 (d, J )
2.4 Hz, 1H), 5.20 (s, 2H), 4.23 (dq, J ) 10.8, 7.2 Hz, 2H), 4.15
(dq, J ) 10.8, 7.2 Hz, 2H), 3.46 (s, 2H), 1.86 (s, 3H), 1.21 (t, J
) 7.2 Hz, 6H); 13C NMR (CDCl3) δ 169.3 (s), 166.7 (s), 132.8
(s), 132.5 (s), 129.9 (d), 129.8 (d), 129.1 (d), 128.3 (d), 103.3
(d), 80.1 (t), 65.4 (s), 62.3 (t), 26.6 (t), 22.2 (q), 13.4 (q). Anal.
(C20H25N3O6) C, H, N.
2.4. Eth yl 2-Acetam ido-2-eth oxycar bon yl-3-(1-h ydr oxy-
5-p yr a zolyl)p r op ion a te (18a ). A mixture of 17a (504 mg,
1.25 mmol), 10% Pd/C (44 mg), and MeOH (25 mL) was stirred
under H2 (1 atm) at 0 °C for 30 min. Filtration through Celite
and removal of MeOH gave 377 mg (96%) of 18a as a solid:
mp >210 °C; 1H NMR (CDCl3) δ 7.08 (d, J ) 2.4 Hz, 1H), 6.69
(s, 1H), 5.86 (d, J ) 2.4 Hz, 1H), 4.31 (dq, J ) 10.8, 7.2 Hz,
2H), 4.27 (dq, J ) 11.1, 7.1 Hz, 2H), 3.79 (s, 2H), 2.01 (s, 3H),
1.29 (t, J ) 7.1 Hz, 6H). The crude product was used in the
next step without further purification.
2.5. (R,S)-2-Am in o-3-(1-h yd r oxy-5-p yr a zolyl)p r op ion ic
Acid Hyd r och lor id e (7a ‚HCl). A solution of 18a (373 mg,
1.19 mmol) in 4 M HCl was heated to reflux for 3 h. The
mixture was concentrated and evaporated twice with water
and twice with toluene to give 267 mg of crude 7a , which was
washed with glacial acetic acid to give 205 mg (83%) of 7a as
the hydrochloride: mp >210 °C; 1H NMR (D2O) δ 7.16 (d, J )
2.4 Hz, 1H), 6.15 (d, J ) 2.4 Hz, 1H), 4.28 (dd, J ) 5.4, 7.2 Hz,
1H), 3.34 (dd, J ) 5.4, 15.6 Hz, 1H), 3.24 (dd, J ) 7.2, 15.6
Hz, 1H); 13C NMR (D2O) δ 172.1 (s), 133.1 (d), 130.6 (s), 105.4
(d), 53.1 (d), 25.5 (t). Anal. (C6H9N3O3‚HCl, 15 mol % H2O) C,
H, N.
(95%) of 13b as a yellow oil: 1H NMR (CDCl3) δ 7.35-7.28
(m, 5H), 7.05 (br s, 1H), 6.80 (t, J ) 0.9 Hz, 1H), 5.21 (s, 2H),
1.93 (t, J ) 0.6 Hz, 3H); 13C NMR (CDCl3) δ 133.7, 132.8, 129.2,
128.7, 128.2, 120.9, 113.2, 79.9, 8.7. Anal. (C11H12N2O) C, H,
N.
3.3. 1-Ben zyloxy-5-for m yl-4-m eth ylp yr a zole (14b). A
freshly prepared solution of LDA (12 mmol) in THF (10 mL)
was added at -78 °C over 2 min to a solution of 13b (1.40 g,
5.2 mmol) in THF (50 mL). After the mixture was stirred at
-78 °C for 5 min, DMF (3.1 mL) was added. Stirring was
continued for 1 h, and the solution was allowed to warm to
room temperature over 10 min and stirred for another 1 h. To
the mixture was added 2 M HCl (100 mL), and the mixture
was stirred for another 1 h. The organic phase was separated
and the aqueous phase extracted with CH2Cl2. Drying of the
combined organic phases and evaporation followed by FC
(EtOAc/heptane 1:4 f 1:1) gave 14b (1.22 g, 75%) as colorless
crystals: mp 48-50 °C. A small sample was recrystallized from
1
heptane: mp 52-53 °C; H NMR (CDCl3) δ 9.59 (d, J ) 0.6
Hz, 1H), 7.40-7.25 (m, 5H), 7.16 (br s, 1H), 5.37 (s, 2H), 2.21
(d, J ) 0.6 Hz, 3H); 13C NMR (CDCl3) δ 179.4 (d), 133.1(d),
132.7 (s), 130.5 (s), 129.9 (d), 129.8 (d), 128.7 (d), 119.7 (s),
81.1 (t), 9.4 (q). Anal. (C12H12N2O2) C, H, N.
3.4. 1-Ben zyloxy-5-h yd r oxym eth yl-4-m eth ylp yr a zole
(15b). Compound 15b was prepared from 14b (820 mg, 3.8
mmol) by the method described for compound 15a . FC (CH2-
Cl2/Et2O/heptane 1:1:1) gave 740 mg (89%) of 15b: mp 81-82
°C; 1H NMR (CDCl3) δ 7.40-7.30 (m, 5H), 7.08 (br s, 1H), 5.31
(s, 2H), 4.26 (s, 2H), 2.00 (d, J ) 0,5 Hz, 3H), 1.35 (br s, 1H);
13C NMR (CDCl3) δ 133.7 S), 132.5 (d), 132.0 (s), 130.0 (d),
129.4 (d), 128.7 (d), 112.6 (s), 80.0 (t), 52.3 (t), 8.5 (q). Anal.
(C12H14N2O2) C, H, N.
3.5.
1-Ben zyloxy-5-ch lor om et h yl-4-m et h ylp yr a zole
(16b). Compound 16b was prepared from 15b (943 mg, 4.32
mmol) by the method described for compound 16a . FC (EtOAc)
gave 979 mg (99%) of 16b as yellow crystals: mp 30-33 °C. A
small sample was recrystallized from pentane: mp 36-37 °C;
1H NMR (CDCl3) δ 7.39 (br s, 5H), 7.10 br s, 1H), 5.34 (s,2H),
4.33 (s, 2H), 2.04 (d, J ) 0.5 Hz, 3H); 13C NMR (CDCl3) δ 133.2
(s), 132.3 (d), 129.8 (d), 129.3 (d), 129.0 (s), 128.6 (d), 113.5
Ss), 80.6 (t), 31.8 (t), 8.5 (q). Anal. (C12H13ClN2O): C, H, N.
3. P r ep a r a tion of P yr a zole Am in o Acid 7b. 3.1. 1-Hy-
d r oxy-4-m eth ylp yr a zole (12). A mixture of 4-methylpyrazole
(11) (40 g, 0.49 mol) and m-chloroperbenzoic acid (70%, 140 g,
0.57 mol) in EtOAc (3.4 L) was stirred for 5 days. Evaporation
of the EtOAc produced a solid that was extracted with water
(ca. 200 mL and 4 × 100 mL) on a sintered glass filter. The
combined water extracts were extracted with CH2Cl2 (4 × 100
mL). The volume of the CH2Cl2 phase was reduced to ca. 50
mL and extracted with concentrated HCl (50 mL). The HCl
phase was washed with dichloromethane (2 × 50 mL) and
combined with the aqueous phase from above. Na3PO4‚12H2O
(30 g) was added, and the pH was adjusted to 10.5 by the
addition of 33% aqueous sodium hydroxide. Continuous ex-
traction with Et2O for 24 h using a Kutscher-Steudel ap-
paratus gave 16 g (40%) of unchanged starting material (11)
after removal of the solvents. The pH of the aqueous solution
was adjusted to 2 by addition of concentrated HCl. Continuous
extraction with Et2O for 12 h produced 20.2 g of crude
1-hydroxy-4-methylpyrazole (12) as brown crystals after re-
moval of the solvents: mp 62-69 °C. Recrystallization from
EtOAc/heptane (1:5) gave 12 as colorless crystals (15.5 g,
32%): mp 75-78 °C (lit. 81-82 °C);30 1H NMR (CDCl3) δ 11.8
(br s, 1H), 7.13 (q, J ) 0.6 Hz, 1H), 6.90 (br s, 1H), 2.02 (d, J
) 0.5 Hz, 3H); 13C NMR (CDCl3) δ 130.9 (d), 121.5 (d), 113.6
(s), 9.0 (q). Anal. (C3H4N2O) C, H, N.
3.6. Eth yl 2-Acetam ido-3-(1-ben zyloxy-4-m eth yl-5-pyr a-
zolyl)-2-eth oxyca r bon ylp r op ion a te (17b). Diethyl aceta-
midomalonate (1.01 g, 4.6 mmol) was slowly added to a
suspension of sodium hydride (214 mg, 55% suspension, 4.6
mmol) in dry DMF (1 mL) at room temperature. After the
mixture was stirred for 15 min, a solution of 16b (0.94 g, 4.0
mmol) in dry DMF (0.5 mL) was added, and the mixture was
then stirred for 24 h more. The reaction mixture was worked
up as described for compound 17a . FC (EtOAc/heptane 1:1)
gave 0.96 g (50%) of 17b as a solid: mp 84-86 °C. A small
sample was recrystallized from EtOAc/heptane: mp 91.5-92
°C; 1H NMR (CDCl3) δ 7.37 (br s, 5H), 7.07 (s, 1H), 6.51 (br s,
1H), 5.13 (s, 2H), 4.24 (dq, J ) 10.8, 7.1 Hz, 2H), 4.06 (dq, J
) 10.8, 7.1 Hz, 2H), 3.41 (s, 2H), 1.86 (s, 3H), 1.81 (s, 3H),
1.19 (t, J ) 7.1 Hz, 6H); 13C NMR (CDCl3) δ 169.3 (s), 167.0
(s), 133.0 (s), 132.6 (d), 130.2 (d), 129.3 (d), 128.4 (d), 127.1
(s), 113.3 (s), 80.4 (t), 65.3 (s), 62.5 (t), 26.0 (t), 22.6 (q), 13.6
(q), 8.6 (q). The compound was used in the next step without
further purification.
3.7. Eth yl 2-Aceta m id o-3-(1-h yd r oxy-4-m eth yl-5-p yr a -
zolyl)-2-eth oxyca r bon ylp r op ion a te (18b). Compound 18b
was prepared from 17b (409 mg, 0.98 mmol) by the method
described for compound 18a . This gave 337 mg (100%) of 18b
as gray crystals: mp 132-134 °C. A small sample was
recrystallized from EtOAc/heptane: mp 133-134 °C; 1H NMR
(acetone-d6) δ 7.41 (br s, 1H), 6.87 (s, 1H), 4.24 (dq, J ) 10.8,
7.2 Hz, 2H), 4.16 (dq, J ) 10.8, 7.2 Hz, 2H), 3.62 (s, 2H), 1.98
(s, 3H), 1.86 (s, 3H), 1.22 (t, J ) 7.1 Hz, 6H); 13C NMR (acetone-
d6) δ 168.6 (s), 166.3 (s), 129.8 (d), 125.7 (s), 112.5 (s), 64.6 (s),
61.0 (t), 25.4 (t), 20.8 (q), 12.4 (q), 7.2 (q). Anal. (C14H21N3O6)
C, H, N.
3.2. 1-Ben zyloxy-4-m eth ylp yr a zole (13b). Benzyl bro-
mide (10.8 mL, 90 mmol) was added to a solution of 12 (8.5 g,
86 mmol) and N-ethyldiisopropylamine (15.4 mL, 90 mmol)
in 80 mL of dry CH2Cl2 at 0 °C. Stirring was continued at room
temperature for 16 h. To the mixture was added NaOH (2 M,
100 mL), and the aqueous phase was extracted with CH2Cl2
(3 × 50 mL). The combined organic phases were washed with
2 M NaOH and water, dried (MgSO4), and evaporated to
dryness. FC (heptane f EtOAc/heptane 1:4) provided 15.3 g
3.8. (R,S)-2-Am in o-3-(1-h yd r oxy-4-m eth yl-5-p yr a zolyl)-
p r op ion ic Acid (7b). A solution of 18b (200 mg, 0.61 mmol)

26 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Stensbøl et al.
in 1 M TFA (30 mL) was heated to 120 °C for 12 h. The mixture
was concentrated and dried. The brown oil was taken up in
water, neutralized to pH ) 7 with 2 M NH3, and applied to an
ion exchange column (Amberlite IR-120 (H+), 5 g). The column
was eluted with water to remove TFA and then with 0.5 M
NH3 to liberate the amino acid as its ammonium salt (after
evaporation). The ammonium salt of 7b was taken up in water
and applied to an ion exchange column (Amberlite IR-420
(OH-), 5 g). The column was eluted with water and 0.5 M
AcOH, and the fractions containing the product were concen-
trated and dried to give 103 mg (91%) of the free amino acid
7b as a solid. A small sample was recrystallized from H2O:
mp >240 °C; 1H NMR (D2O) δ 6.94 (1H, s), 3.83 (t, J ) 6.3 Hz,
1H), 3.13 (dd, J ) 15.4, 5.2 Hz, 1H), 3.02 (dd, J ) 15.4, 6.6
Hz, 1H), 2.84 (s, 3H); 13C NMR (D2O) δ 174.05 (s), 131.47 (d),
129.13 (s), 114.68 (s), 54.49 (d), 24.90 (t), 8.57 (q). Anal.
(C7H11N3O3) C, H, N.
4. P r ep a r a t ion of 1,2,3-Tr ia zole Am in o Acid 8a . 4.1.
1-Ben zyloxy-5-h yd r oxym eth yl-1,2,3-tr ia zole (24a ). Com-
pound 24a was prepared from 1-benzyloxy-5-formyl-1,2,3-
triazole (23a )19 (1.37 g, 6.75 mmol) by the method described
for compound 15a . FC (EtOAc/heptane 1:2) gave 1.29 g (93%)
of 24a as a solid. A small sample was recrystallized from
EtOAc/heptane: mp 71-72 °C; 1H NMR (CDCl3) δ 7.48 (s, 1H),
7.42-7.32 (m, 5H), 5.47 (s, 2H), 4.34 (d, J ) 4.8 Hz, 2H), 3.08
(br t, J ≈ 5 Hz, OH); 13C NMR (CDCl3) δ 132.5 (s), 131.6 (s),
131.2 (d), 130.1 (d), 130.0 (d), 128.9 (d), 82.6 (t), 52.1 (t). Anal.
(C10H11N3O2) C, H, N.
benzylhydroxylamine,31 was dissolved in MeOH (250 mL),
cooled to 0 °C, and treated with hydrazine hydrate (40 mL,
0.84 mol) for 1 h at 0 °C. The mixture was concentrated to
give a colorless oil, which was redissolved in pyridine (200 mL)
and heated to reflux. Then Cu(OAc)2 hydrate (33 g, 0.17 mol)
in pyridine (300 mL) was added, and the reaction mixture was
refluxed for 1 h, cooled to room temperature, and evaporated
in vacuo. The black slurry was extracted with Et2O (6 × 150
mL), and the combined organic phases were washed with
precooled sulfuric acid (100 mL, 4 M) and then water (200 mL).
The Et2O phase was dried (MgSO4) and evaporated in vacuo.
The brown oil (11.5 g) was purified by FC using EtOAc/heptane
(1:3 f 1:1), which gave 5.62 g (36%) of an inseparable 2:1
mixture of 1-benzyloxy-5-methyl-1,2,3-triazole (21)19 and 1-ben-
zyloxy-4-methyl-1,2,3-triazole (22). A total of 3.04 g (5.78
mmol) of this mixture was dissolved in THF (80 mL), cooled
to -78 °C, and treated with n-BuLi (1.6 M in hexanes, 4.5
mL, 7.2 mmol). After 2 min, dry DMF (0.67 mL, 9.17 mmol)
was added. The reaction mixture was stirred for 30 min at
-78 °C and then allowed to warm to room temperature over
30 min. The reaction was quenched with saturated NH4Cl (50
mL) and extracted with EtOAc (3 × 25 mL). The combined
organic phases were dried (MgSO4) and evaporated in vacuo
to give crude 23b and unchanged 21. Data for 23b: 1H NMR
(CDCl3) δ 9.56 (s, 1H), 7.42-7.20 (m, 5H), 5.56 (s, 2H), 2.47
(s, 3H); 13C NMR (CDCl3) δ 177.8 (s), 146.4 (s), 131.7 (s), 130.4
(d), 130.1 (d), 129.0 (d), 125.8 (s), 83.1 (t), 11.7 (q). The solid
was redissolved in MeOH (20 mL), cooled to 0 °C, and treated
with NaBH4 (0.33 g, 8.7 mmol) for 30 min at room tempera-
ture, then evaporated in vacuo. The crude product was
redissolved in CH2Cl2 (20 mL) and washed with brine and
water. The organic phase was dried (MgSO4) and evaporated
in vacuo. FC (EtOAc/heptane 1:3 f 1:2) gave 0.96 g (75%) of
24b and 1.71 g of unchanged 21. Data for 24b: mp 127-129
4.2. 1-Ben zyloxy-5-ch lor om eth yl-1,2,3-tr ia zole (25a ). A
solution of 24a (0.45 g, 2.2 mmol) in CH2Cl2 (8 mL) was treated
with thionyl chloride (1 mL, 13.7 mmol) and stirred for 1 h at
0 °C. The mixture was concentrated, coevaporated three times
with toluene, and dried (rt, 0.1 mmHg) to give 473 mg (97%)
of 25a as a solid, which was used in the next step without
further purification: 1H NMR (CDCl3) δ 7.61 (s, 1H), 7.45-
7.35 (m, 5H), 5.52 (s, 2H), 4.24 (s, 2H); 13C NMR (CDCl3) δ
132.2 (s), 131.9 (d), 130.1 (d), 130.0 (d), 128.9 (d), 128.4 (s),
82.7 (t), 30.6 (t).
1
°C; H NMR (CDCl3) δ 7.45-7.30 (m, 5H), 5.47 (s, 2H), 4.34
(s, 2H), 2.33 (br s, 1H), 2.48 (s, 3H); 13C NMR (CDCl3) δ 140.4
(s), 132.8 (s), 130.1 (d), 129.9 (d), 128.9 (d), 127.4 (s), 82.3 (t),
51.2 (t), 10.5 (q). Anal. (C11H13N3O2) C, H, N.
5.2. Eth yl 2-Aceta m id o-3-(1-ben zyloxy-4-m eth yl-1,2,3-
t r ia zol-5-yl)-2-et h oxyca r b on ylp r op ion a t e (26b ). Com-
pound 26b was prepared from 24b (0.93 g, 4.25 mmol), using
the method described for the preparation of 25a followed by
the method described for the preparation of 17a . FC (EtOAc/
heptane 1:3 f 1:0) gave 1.47 g (83%) of 26b as a solid: mp
4.3. Eth yl 2-Aceta m id o-3-(1-ben zyloxy-1,2,3-tr ia zol-5-
yl)-2-eth oxyca r bon ylp r op ion a te (26a ). Compound 26a was
prepared from 25a (144 mg, 0.64 mmol) by the method
described for compound 17a . FC (EtOAc/heptane 1:2) gave 246
mg (94%) of 26a as a solid. A small sample was recrystallized
from Et2O: mp 130-131 °C; 1H NMR (CDCl3) δ 7.42-7.37 (m,
5H), 7.25 (s, 1H), 6.63 (br s, 1H), 5.38 (s, 2H), 4.29-411 (m,
4H), 3.46 (s, 2H), 1.91 (s, 3H), 1.22 (t, J ) 7.1 Hz, 6H); 13C
NMR (CDCl3) δ 169.6 (s), 166.3 (s), 131.9 (s), 131.6 (s), 131.6
(d), 130.1 (d), 129.9 (d), 128.6 (d), 126.3 (s), 82.5 (t), 65.3 (s),
62.8 (t), 25.0 (t), 22.5 (q), 13.7 (q). Anal. (C19H24N4O6) C, H, N.
4.4. Eth yl 2-Acetam ido-2-eth oxycar bon yl-3-(1-h ydr oxy-
1,2,3-tr ia zol-5-yl)p r op ion a te (27a ). Compound 27a was
prepared from 26a (457 mg, 1.13 mmol) by the method
described for compound 18a . This gave 331 mg (93%) of 27a
as a solid. Recrystallization (acetone/Et2O) gave mp 144-145
°C: 1H NMR (D2O) δ 7.83 (s, 1H), 4.24 (q, J ) 7.1 Hz, 4H),
3.64 (s, 2H), 1.99 (s, 3H), 1.19 (t, J ) 7.1 Hz, 6H); 13C NMR
(D2O) δ 174.4 (s), 168.7 (s), 127.4 (d), 126.6 (s), 66.3 (s), 65.1
(t), 26.7 (t), 22.3 (q), 13.9 (q). Anal. (C12H18N4O6) C, H, N.
4.5. (R,S)-2-Am in o-3-(1-h yd r oxy-1,2,3-tr ia zole-5-yl)p r o-
p ion ic Acid (8a ). A solution of 27a (157 mg, 0.50 mmol) in
12 M HCl was heated to reflux for 3 h. The mixture was
concentrated and dried (rt, 0.1 mmHg) to give 126 mg of 8a
as the hydrochloride. The free amino acid was obtained by ion
exchange chromatography as described for 7b to give 84 mg
(96%) of 8a as a solid: mp >210 °C; 1H NMR (D2O) δ 7.94 (s,
1H), 4.13 (dd, J ) 5.1, 6,6 Hz, 1H), 3.35 (dd, J ) 5.0, 16.0 Hz,
1H), 3.25 (dd, J ) 6.6, 15.9 Hz, 1H); 13C NMR (D2O) δ 173.3
(s), 127.8 (s), 127.0 (d), 54.0 (d), 24.4 (t). Anal. (C5H8N4O3, 130
mol % H2O) C, H, N.
5. P r ep a r a t ion of 1,2,3-Tr ia zole Am in o Acid 8b . 5.1.
1-Be n zyloxy-5-h yd r oxym e t h yl-4-m e t h yl-1,2,3-t r ia zole
(24b). A 2:1 mixture of 2-O-benzyloximepropane-1,2-dione
(19) and 1-O-benzyloximepropane-1,2-dione (20) (14.9 g, 83.7
mmol), obtained by condensation of methyl glyoxal and O-
1
133-135 °C; H NMR (CDCl3) δ 7.42-7.32 (m, 5H), 6.50 (br
s, 1H), 5.34 (s, 2H), 4.25 (dq, J ) 10.8, 7.2 Hz, 2H), 4.07 (dq,
J ) 10.8, 7.2 Hz, 2H), 3.42 (s, 2H), 2.14 (s, 3H), 1.85 (s, 3H),
1.21 (t, J ) 7.2 Hz, 3H); 13C NMR (CDCl3) δ 169.7 (s), 166.8
(s), 141.0 (s), 132.0 (s), 130.4 (d), 130.0 (d), 128.8 (d), 122.7 (s),
82.5 (t), 64.9 (s), 62.8 (t), 24.8 (t), 22.5 (q), 13.6 (q), 10.5 (q).
Anal. (C20H26N4O6) C, H, N.
5.3. E t h yl 2-Acet a m id o-3-(1-h yd r oxy-4-m et h yl-1,2,3-
t r ia zol-5-yl)-2-et h oxyca r b on ylp r op ion a t e (27b ). Com-
pound 27b was prepared from 26b (292 mg, 0.70 mmol) by
the method described for compound 18a . This gave 227 mg
1
(99%) of 27b as a solid: mp 144-146 °C; H NMR (acetone-
d6) δ 7.62 (s, 1H), 4.20-4.10 (m, 4H), 3.61 (s, 2H), 2.17 (s, 3H),
1.99 (s, 3H), 1.22 (t, J ) 7.2 Hz, 3H); 13C NMR (CDCl3) δ 168.3
(s), 166.0 (s), 136.8 (s), 121.5 (s), 63.9 (s), 60.8 (t), 24.4 (t), 20.4
(q), 12.0 (q), 7.9 (q). Anal. (C13H20N4O6) C, H, N.
5.4. (R,S)-2-Am in o-3-(1-h yd r oxy-4-m eth yl-1,2,3-tr ia zol-
5-yl)p r op ion ic Acid Hyd r och lor id e (8b‚HCl). Compound
27b (572 mg, 1.74 mmol) was dissolved in 12 M HCl (5 mL)
and heated to reflux for 3 h. The reaction mixture was
evaporated in vacuo, and the resulting crystals were recrystal-
lized from 2-propanol, which gave 201 mg (62%) of 8b‚HCl:
1
mp >210 °C; H NMR (D2O) δ 4.37 (t, J ) 6 Hz, 1H), 3.31 (d,
J ) 6 Hz, 2H), 2.29 (s, 3H); 13C NMR (D2O) δ 171.8 (s), 137.2
(s), 124.8 (s), 52.3 (d), 23.3 (t), 8.7 (q). Anal. (C6H10N4O3, 115
mol % HCl, 5 mol % PriOH) C, H, N.
6. P r ep a r a tion of Im id a zole Am in o Acid 9a . 6.1. Eth yl
2-Acet a m id o-3-(1-b en zyloxy-5-im id a zolyl)-2-et h oxyca r -
bon ylp r op ion a te (37a ). A solution of 34a (814 mg, 2.02
mmol)20 dissolved in MeOH (20 mL) at 0 °C was treated with

Bioisosteres of Glutamic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 27
NaBH4 (0.27 g, 3.5 mmol). The mixture was stirred at 0 °C
for 3 h and worked up as described for compound 15a to give
crude 35a . This was stirred with thionyl chloride (10 mL, 140
mmol) at 60 °C for 1.5 h. The mixture was concentrated and
evaporated twice with toluene to give 1.1 g of crude 36a as
crystals that were used directly in the following step. The
crystals were dissolved in dry DMF (10 mL) and added to a
solution of the sodium salt of diethyl acetamidomalonate (12
mmol in 30 mL of DMF), prepared as described for 17a . The
solution was stirred for 16 h at room temperature and worked
up as described for 17a to give 1.1 g of crude 37a . FC (EtOAc/
heptane 1:1 f 1:0, then EtOAc/MeOH 1:0 f 1:5) gave 451 mg
(28%) of 37a as a semicrystalline compound that became an
oil when exposed to air: 1H NMR (CDCl3) δ 7.43-7.29 (m, 5H),
6.63 (br s, 1H), 6.68 (br d, J ) 0.9 Hz, 1H), 5.00 (s, 2H), 4.25
(dq, J ) 10.8, 7.2 Hz, 2H), 4.18 (dq, J ) 10.8, 7.2 Hz, 2H),
3.65 (s, 2H), 1.90 (s, 3H), 1,24 (t, J ) 7.1 Hz, 6 H); 13C NMR
(CDCl3) δ 169.7, 167.2, 132.9, 131.9, 130.1, 129.9, 128.9, 124.8,
122.5, 82.9, 66.1, 62.8, 26.0, 22.7, 13.8. Anal. (C21H26ClN3O3)
C, N, H: calcd, 6.46; found, 5.95.
30b. Attempts to separate the two isomers failed. A total of
2.00 g (10.6 mmol) of this mixture was dissolved in THF (60
mL), cooled to -78 °C, and treated with n-BuLi (1.6 M in
hexanes, 8.0 mL, 12.8 mmol). After 2 min, hexachloroethane
(4.98 g, 21.2 mmol) was added and the reaction mixture was
stirred for 1 h at -78 °C and then allowed to warm to room
temperature over 1 h. Stirring was continued for 30 min more.
The reaction was quenched with saturated NaHCO3 (50 mL)
and extracted with CH2Cl2. The combined organic phases were
dried (MgSO4) and evaoporated in vacuo. FC (heptane/Et2O)
gave 0.59 g (25%) of 32 as an oil: 1H NMR (CDCl3) δ 7.38-
7.30 (m, 5H), 6.59 (s, 1H), 5.04 (s, 2H), 2.08 (s, 3H); 13C NMR
(CDCl3) δ 132.9 (s), 132.6 (s), 129.4 (d), 129.3 (d), 128.4 (d),
125.5 (s), 113.2 (d), 81.4 (t), 13.9 (q). The crude product was
used in the next step without further purification. Further
elution gave 0.71 g (30%) of 31 as an oil: 1H NMR (CDCl3) δ
7.40-7.32 (m, 5H), 6.54 (s, 1H), 5.11 (s, 2H), 1.98 (s, 3H); 13
C
NMR (CDCl3) δ 132.8 (s), 130.02(d), 130.0 (s), 129.7 (d), 128.8
(d), 126.9 (s), 121.2 (d), 81.1 (t), 8.6 (q). The crude product was
used in the next step without further purification.
6.2. (R,S)-2-Am in o-3-(1-h yd r oxy-5-im id a zolyl)p r op ion -
ic Acid (9a ). A mixture of 37a (422 mg, 1.05 mmol), 10% Pd/C
(39 mg), and MeOH (15 mL) was stirred under H2 (1 atm) at
0 °C for 30 min and at 20 °C for 30 min. Filtration through
Celite and removal of MeOH gave 292 mg of crude 38a as a
solid, which was dissolved in 4 M HCl (20 mL) and heated to
reflux for 16 h. The mixture was concentrated and evaporated
twice with water and twice with toluene to give 272 mg of
crude 9a . The crude product was taken up in water and applied
to ion exchange chromatography as described for 7b to give
crude 9a as a solid, which was dissolved in warm 2-propanol/
water and left for 2 days at 5 °C for crystallization. The crystals
were filtered off and gave 125 mg (63%) of 9a : mp >220 °C
7.3. 1-Ben zyloxy-2-ch lor o-5-h yd r oxym et h yl-4-m et h -
ylim id a zole (35b). At -78 °C a solution of n-BuLi (1.6 M in
hexanes, 7.2 mL, 11.5 mmol) was added dropwise over 2 min
to 32 (2.14 g, 9.6 mmol) dissolved in THF (60 mL). The mixture
was stirred for 2 min before DMF (3.7 mL, 48 mmol) was
added. Stirring was continued for 1 h at -78 °C, and the
mixture was allowed to warm to room temperature over 1 h.
Stirring was continued for 30 min more before the reaction
was quenched with 2 M HCl (25 mL) and neutralized (pH )
8) by the addition of saturated NaHCO3. The organic layer was
separated, and the aqueous layer was extracted with CH2Cl2.
The combined organic phases were dried (MgSO4) and evaopo-
rated in vacuo. FC (heptane/Et2O) gave 2.20 g (92%) of 34b
as an oil, which was used without further purification: 1H
NMR (CDCl3) δ 9.68 (s, 1H), 7.45-7.38 (m, 5H), 5.27 (s, 2H),
2.5 (s, 3H). Compound 34b (2.20 g, 8.78 mmol) was dissolved
in MeOH (50 mL), cooled to 0 °C, and treated with NaBH4
(360 mg, 9.5 mmol). The mixture was stirred at 0 °C for 3 h
and worked up as described for compound 15a . FC (EtOAc)
gave 1.96 g (89%) of 35b as a solid. A small sample was
recrystallized from EtOAc/heptane: mp 110-111 °C; 1H NMR
δ (CDCl3) 7.42 (br s, 5H), 6.60 (br s, 1H), 5.25 (s, 2H), 4.61 (s,
2H), 2.06 (s, 3H); 13C NMR (CDCl3) δ 133.1 (s), 131.9 (s), 130.2
(d), 130.0 (d), 129.0(d), 126.7 (s), 125.5 (s), 81.9 (t), 52.4 (t),
13.0 (q). Anal. (C12H13ClN2O2) C, H, N.
1
(dec); H NMR (D2O) δ 8.11 (d, J ) 1.8 Hz, 1H), 7.00 (d, J )
1.8 Hz, 1H), 3.99 (dd, J ) 3.9, 6.3 Hz, 1H), 3.23 (dd, J ) 3.9,
16 Hz, 1H), 3.15 (dd, J ) 6.6, 16 Hz, 1H). Anal. (C6H9N3O3‚
H2O) C, H, N.
7. P r ep a r a tion of Im id a zole Am in o Acid 9b. 7.1. 4(5)-
Meth yl-1-h yd r oxyim id a zole-3-oxid e (28). A mixture of 40%
aqueous methyl glyoxal (42 mL, 0.50 mol), 37% aqueous
formaldehyde (27.3 mL, 0.6 mol), and MeOH (15 mL) was
cooled to 0 °C, and a solution of hydroxylammonium hydro-
chloride (70.1 g, 0.5 mol) in water (85 mL) was added over 10
min. Then concentrated HCl (10 mL, 37%) was added over 1
min, and the mixture was stirred for 24 h at room temperature.
The reaction mixture was cooled to 0 °C, and the pH was
adjusted to 4 by cautious addition of 33% aqueous NaOH. After
evaporation of MeOH, the flask was left overnight at 5 °C.
The precipitate formed was filtered, washed with water,
MeOH, and Et2O, and dried under vacuum. Recrystallization
from water gave 15.7 g (28%) of 28 as a solid: 1H NMR (D2O)
δ 8.24 (d, J ) 2.0 Hz, 1H), 6.91 (br s, 1H), 2.13 (s, 3H). The
compound was used in the following step without further
purification.
7.2. 1-Ben zyloxy-2-ch lor o-5-m eth ylim id a zole (31) a n d
1-Ben zyloxy-2-ch lor o-4-m eth ylim id a zole (32). Benzyl bro-
mide (13.5 mL, 0.114 mol) was added to a solution of 28 (13 g,
0.116 mol) and potassium hydroxide (4.78 g, 0.085 mol) in
MeOH (40 mL), and the mixture was refluxed for 1 h. After
filtration and removal of the solvent, the yellow oil was
suspended in CHCl3 (40 mL) and cooled to 0 °C. Then PCl3
(50 mL) was carefully added at such a rate that the internal
temperature was kept under 25 °C. The mixture was allowed
to warm to reflux and refluxed for 1.5 h. Caution: At ca. 40
°C a vigorous exothermic reaction takes place. The mixture was
cooled to room temperature. Solvents and excess PCl3 were
removed in vacuo. The residue was added to toluene (100 mL),
and the mixture was evaporated, with repeated addition of
toluene and evaporation. Then water (250 mL) and Et2O (200
mL) were added and the pH was adjusted to 10 by careful
addition of 33% aqueous NaOH. Separation of the organic
layer, followed by extraction with Et2O (3 × 50 mL), drying of
the combined organic phases (MgSO4), filtration, evaporation,
and FC (EtOAc) gave 7.0 g (32%) of a 1:1 mixture of 29b and
7.4. Eth yl 2-Acetam ido-3-(1-ben zyloxy-2-ch lor o-4-m eth -
yl-5-im idazolyl)-2-eth oxycar bon ylpr opion ate (37b). A mix-
ture of 35b (250 mg, 1.00 mmol) and thionyl chloride (1.0 mL,
14 mmol) in toluene (6 mL) was stirred at 0 °C for 1 h. The
mixture was concentrated to give 0.28 g of crude 36b, which
was used directly in the following step. The crude 36b was
dissolved in dry DMF (3 mL) and added to a solution of the
sodium salt of diethyl acetamidomalonate (2 mmol in 2 mL of
DMF) prepared as described for 17a . The solution was stirred
for 40 h at room temperature and worked up as described for
17a . FC (EtOAc/heptane 1:1 f 1:0) gave 222 mg (49%) of 37b
as a solid. A small sample was recrystallized from EtOAc/
1
heptane: mp 149-151 °C; H NMR (CDCl3) δ 7.48-7.40 (m,
5H), 6.58 (br s, 1H), 4.98 (s, 2H), 4.18-4.06 (m, 4H), 3.57 (s,
2H), 2.01 (s, 3H), 1.74 (s, 3H), 1.18 (t, J ) 7.1 Hz, 6 H); 13C
NMR (CDCl3) δ 169.62 (s), 167.05 (s), 132.41 (s), 132.14 (s),
130.42 (d), 129.76 (d), 128.60 (d), 125.51 (s), 119.7 (s), 81.69
(t), 62.47 (t), 62.47 (s), 26.37 (t), 22.37 (q), 13.49 (q), 12.83 (q).
Anal. (C21H26ClN3O3) C, H, N.
7.5. Eth yl 2-Aceta m id o-3-(1-h yd r oxy-4-m eth yl-5-im i-
dazolyl)-2-eth oxycar bon ylpr opion ate Hydr och lor ide (38b‚
HCl). Compound 38b‚HCl was prepared from 37b (222 mg,
0.48 mmol) by the method described for compound 18a , using
2 h at 0 °C for the hydrogenolysis. This gave 112 mg (70%) of
38b‚HCl as a solid. Recrystallization from MeOH/Et2O af-
1
forded crystalline 38b‚HCl: mp 209-211 °C; H NMR (D2O)
δ 8.61 (s, 1H), 4.34-4.17 (m, 4H), 3.62 (s, 2H), 2.13 (s, 3H),
1.98 (s, 3H), 1.21 (t, J ) 7.2 Hz, 6 H); 13C NMR (D2O) δ 175.1,

28 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Stensbøl et al.
169.3, 129.9, 128.2, 122.7, 66.8, 65.4, 26.2, 22.4, 14.0, 9.8. Anal.
(C14H21N3O6‚HCl) C, H, N.
to reflux for 16 h. The mixture was concentrated and coevapo-
rated twice with toluene (25 mL). Recrystallization (twice) from
EtOH/Et2O gave 77 mg (54%) of 9c‚2HCl as a solid. According
to 1H NMR, the crystals contained 60 mol % ethanol: mp 142-
144 °C (dec); 1H NMR (D2O) δ 7.19 (s,1H), 4.18 (t, J ) 6.4 Hz,
1H), 3.41-3.22 (m, 2H), 2.53 (s, 3H); 13C NMR (D2O) δ 172.1
(s), 143.7 (s), 126.8 (s), 116.7 (d), 53.4 (d), 25.1 (t), 10.9 (q).
Anal. (C7H11N3O3‚2HCl‚H2O, 60 mol % EtOH) C, H, N.
7.6. (R,S)-2-Am in o-3-(1-h yd r oxy-4-m eth yl-5-im id a zol-
yl)p r op ion ic Acid Dih yd r och lor id e (9b‚2HCl). A solution
of 38b‚HCl (100 mg, 0.27 mmol) in 4 M HCl (4 mL) was heated
to reflux for 16 h. The mixture was concentrated and coevapo-
rated twice with toluene (25 mL) to give 62 mg (79%) of 9b‚
1
2HCl as a hygroscopic solid: mp 170-172 °C (dec); H NMR
(D2O) δ 8.74 (s,1H), 4.36 (t, J ) 6.4 Hz, 1H), 3.46-3.31 (m,
2H), 2.25 (s, 3H); 13C NMR (D2O) δ 171.2 (s), 130.2 (d), 127.8
(s), 122.1 (s), 52.0 (d), 22.9 (t), 9.5 (q). Anal. (C7H11N3O3‚2HCl,
150 mol % H2O) C, H, N.
9. P r ep a r a t ion of Im id a zole Am in o Acid 10a . 9.1.
1-Ben zyloxy-2-h yd r oxym eth ylim id a zole (39a ). At -78 °C
a solution of n-BuLi (1.6 M in hexanes, 10.8 mL, 17.2 mmol)
was added dropwise over 2 min to 1-benzyloxyimidazole (29a )21
(2.48 g, 14.3 mmol) dissolved in THF (80 mL). The mixture
was stirred for 5 min before DMF (5.52 mL, 71.4 mmol) was
added. The solution was stirred for 1 h at -78 °C and allowed
to warm to room temperature over 30 min. The reaction
mixture was quenched with 2 M HCl (25 mL) and neutralized
(pH 8) by addition of saturated NaHCO3. The organic layer
was separated, and the aqueous layer was extracted with CH2-
Cl2 (5 × 40 mL). Drying of the combined organic phases and
evaporation gave the crude aldehyde 33a 21 as a yellow oil. This
was dissolved in MeOH (75 mL), cooled to 0 °C, and treated
with NaBH4 (1.06 g, 28.6 mmol). The mixture was stirred at
0 °C for 2 h and worked up as described for compound 15a .
FC (EtOAc/heptane 1:1) gave 2.91 g (89%) of 39a as a solid. A
small sample was recrystallized from EtOAc/heptane: mp 80-
8. P r ep a r a tion of Im id a zole Am in o Acid 9c. 8.1. 1-Ben -
zyloxy-5-for m yl-2-m eth ylim id a zole (34c). At -78 °C a
solution of n-BuLi (1.57 M in hexanes, 3.5 mL, 5.5 mmol) was
added dropwise over 2 min to 1-benzyloxy-2-methylimidazole
(30b)21 (1.00 g, 5.50 mmol) dissolved in THF (30 mL). The
mixture was stirred for 5 min before DMF (1.95 mL, 25 mmol)
was added. Stirring was continued for 1 h at -78 °C, and the
mixture was allowed to warm to room temperature over 30
min. The reaction mixture was quenched with 2 M HCl (25
mL) and neutralized (pH 8) by addition of saturated NaHCO3.
The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (2 × 40 mL). Drying of the combined
organic phases and removal of solvents in vacuo, followed by
FC (EtOAc/heptane 1:2 f 1:0) gave 567 mg (50%) of 34c as
an oil: 1H NMR δ (CDCl3) 9.67 (s, 1H), 7.64 (s, 1H), 7.50-
7.30 (m, 5H), 5.21 (s, 2H), 2.09 (s, 3H); 13C NMR (CDCl3) δ
176.5 (s), 147.6 (s), 138.4 (s), 132.7 (s), 130.2 (d), 129.8 (d),
128.8 (d), 128.0 (d), 81.5 (t), 11.5 (q). The compound was used
in the next step without further purification.
1
81 °C; H NMR δ (CDCl3) 7.40 (s, 5H), 6.79 (s, 2 H), 6.12 (s,
1H), 5.25 (s, 2H), 4,59 (s, 2H); 13C NMR (CDCl3) δ 143.5 (s),
133.4 (s), 129.7 (d), 129.4 (d), 128.7 (d), 123.0 (d), 116.0 (d),
82.5 (t), 53.6 (t). Anal. (C11H12N2O2) C, H, N.
9.2. 1-Ben zyloxy-2-ch lor om eth ylim id a zole Hyd r och lo-
r id e (40a ‚HCl). A mixture of compound 39a (1.49 g, 7.3
mmol), thionyl chloride (2.13 mL, 29.2 mmol), and N-ethyldi-
isopropylamine (2.54 mL, 14.6 mmol) in CHCl3 (35 mL) was
refluxed for 2 h. The mixture was concentrated. FC (EtOAc/
heptane 1:4) provided pure fractions of unstable 40a , which
were immediately added to 4 M HCl (40 mL). The mixture
was coevaporated three times with toluene to give 1.39 g (74%)
of 40a ‚HCl as a brown crystalline compound. A small sample
was recrystallized from EtOAc/MeOH to give light-brown
8.2. 1-Ben zyloxy-5-h yd r oxym eth yl-2-m eth ylim id a zole
(35c). A solution of 34c (520 mg, 2.40 mmol) dissolved in
MeOH (25 mL) at 0 °C was treated with NaBH4 (0.32 g, 4.2
mmol). The mixture was stirred at 0 °C for 3 h and worked up
as described for compound 15a . FC (EtOAc/MeOH 9:1) gave
400 mg (76%) of 35c as a solid: mp 91-92 °C; 1H NMR δ
(CDCl3) 7.40-7.30 (m, 5H), 6.68 (s, 1H), 5.25 (s, 2H), 4.75 (br
s, 1H), 4.56 (s, 2H), 2.11 (s, 3H); 13C NMR (CDCl3) δ 141.0 (s),
133.5(s), 130.0 (d), 129.6 (d), 128.7 (d), 128.4(s), 122.2 (d), 81.4
(t), 52.6 (t), 11.5 (q). The compound was used in the next step
without further purification.
1
crystals: mp 114-115 °C; H NMR (D2O) δ 7.67 (d, J ) 1.5
Hz, 1 H), 7.43-7.40 (m, 6 H), 5.42 (s, 2 H), 4.53 (s, 2 H); 13C
NMR (D2O) δ 134.0 (s), 132.8 (d), 132.5 (d), 131.4 (d), 121.3
(d), 112.0 (d), 85.7 (t), 32.0 (t). Anal. (C11H11ClN2O‚HCl) C, H,
N.
8.3. Eth yl 2-Aceta m id o-3-(1-ben zyloxy-2-m eth yl-5-im i-
d a zolyl)-2-eth oxyca r bon ylp r op ion a te (37c). A mixture of
35c (369 mg, 1.68 mmol) and thionyl chloride (1.5 mL, 21
mmol) in toluene (9 mL) was stirred at 60 °C for 1 h. The
mixture was concentrated to give 386 mg of crude 36c‚HCl as
a powder, which was used without further purification: 1H
NMR δ (DMSO) 7.71 (s, 1H), 7.58-7.66 (m, 5H), 5.47 (s, 2H),
4.92 (s, 2H), 2.49 (s, 3H); 13C NMR (DMSO) δ 143.0 (s), 132.4
(s), 130.5 (d), 130.2 (d), 129.0 (d), 126.9 (s), 116.4 (d), 82.4 (t),
32.4 (t), 9.8 (q). Crude 36c‚HCl was dissolved in dry DMF (5
mL), and the mixture was added to a solution of the sodium
salt of diethyl acetamidomalonate (3.4 mmol in 4 mL of DMF),
prepared as described for 17a . The solution was stirred for 40
h at room temperature and worked up as described for 17a .
FC (EtOAc/heptane 1:1 f 1:0) gave 263 mg (38%) of 37c as a
solid: mp 162-163 °C; 1H NMR (CDCl3) δ 7.41 (br s, 5H), 6.65
(br s, 1H), 6.50 (s, 1H), 4.93 (s, 2H), 4.30-4.03 (m, 4H), 3.69
9.3. E t h yl 2-Acet a m id o-3-(1-b en zyloxy-2-im id a zolyl)-
2-eth oxyca r bon ylp r op ion a te (41a ). A solution of diethyl
acetamidomalonate (1.41 g, 6.48 mmol) in dry DMF (5 mL)
was added to a stirred solution of sodium hydride (55%, 389
mg, 16.2 mmol) in dry DMF (5 mL). After 2 h, compound 40a
(1.39 g, 5.4 mmol) in dry DMF (10 mL) was added, and the
mixture was stirred for 24 h more at room temperature. The
reaction mixture was quenched with saturated NaHCO3 (25
mL) and worked up as described for compound 17a . FC
(EtOAc/MeOH 9:1) provided pure fractions of 41a , which after
evaporation and drying at 0.1 mmHg for several days afforded
2.04 g of a thick oil as a mixture of 41a and EtOAc (ratio 1:0.57
1
according to H NMR) corresponding to 1.81 g (83%) of 41a :
1H NMR (CDCl3) δ 7.42-7.35 (m, 5H), 7.0 (s, 1 H), 6.85 (d, J
) 1.5 Hz, 1 H), 6.78 (d, J ) 1.5 Hz, 1 H), 5.02 (s, 2 H), 4.28
(dq, J ) 10.7 and 7.1 Hz, 2 H), 4.22 (dq, J ) 10.7 and 7.2 Hz,
2 H), 3.64 (s, 2 H), 1.92 (s, 3 H), 1.25 (t, J ) 7.1 Hz, 6 H); 13C
NMR (CDCl3) δ 169.4, 167.0, 138.1, 133, 129.8, 129.6, 128.7,
123.7, 115.5, 82.0, 65.2, 62.7, 28.7, 22.7, 13.8. The compound
was used in the next step without further purification.
(s, 2H), 2.24 (s, 3H), 2.05 (s, 3H), 1.24 (t, J ) 7.1 Hz, 6 H); 13
C
NMR (CDCl3) δ 169.5 (s), 167.0 (s), 140.1 (s), 132.6 (s), 130.2
(d), 129.7 (d), 128.9 (d), 123.0 (d), 121.8 (s), 81.5 (t), 66.25 (s),
62.8 (t), 26.7 (t), 22.8 (q), 13.9 (q), 12.1 (q). The compound was
used in the next step without further purification.
8.4. (R,S)-2-Am in o-3-(1-h yd r oxy-2-m eth yl-5-im id a zol-
yl)p r op ion ic Acid Hyd r och lor id e (9c‚2HCl). Compound
38c was prepared from 37c (195 mg, 0.48 mmol) by the method
described for compound 18a . After recrystallization from
acetone, this provided 124 mg of 38c as a solid: mp 179 °C;
1H NMR (D2O) δ 6.81 (s, 1H), 4.20 (q, J ) 7.2 Hz, 4H), 3.50 (s,
2H), 2.32 (s, 3H), 1.99 (s, 3H), 1.19 (t, J ) 7.1 Hz, 6 H); 13C
NMR (D2O) δ 170.2 (s), 167.6 (s), 137.2 (s), 125.5 (d), 115.8
(s), 66.1 (q), 62.6 (t), 27.0 (t), 22.8 (q), 13.8 (q), 9.5 (q).
Compound 38c was dissolved in 4 M HCl (4 mL) and heated
9.4. E t h yl 2-Acet a m id o-3-(1-h yd r oxy-2-im id a zolyl)-2-
eth oxyca r bon ylp r op ion a te (42a ). Compound 42a was pre-
pared from 41a (1.01 g, 2.50 mmol) by the method described
for compound 18a . This gave 773 mg (99%) of 42a as a white
foam. Recrystallization from acetone afforded crystals: mp
1
141-142 °C; H NMR (D2O) δ 7.04 (br s, 2 H), 4.28 (dq, J )
10.7 and 7.1 Hz, 2 H), 4.24 (dq, J ) 10.7 and 7.1 Hz, 2 H),
3.73 (s, 2H), 1.98 (s, 3 H), 1.18 (t, J ) 7.14 Hz, 6 H); 13C NMR

Bioisosteres of Glutamic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 29
(D2O) δ 174.7, 169.0, 133.2, 121.1, 116.3, 66.4, 65.5, 29.0, 22.7,
14.2. Anal. (C13H19N3O6, 10 mol % H2O) C, H, N.
130.5 (d), 114.1 (s), 51.2 (d), 25.3 (t), 7.7 (q). Anal. (C7H11N3O3‚
2HCl‚H2O) C, H, N.
11. P oten tiom etr ic Titr a tion s. A titration method was
applied for the determination of the pKa values of the four
compounds using a GLpKa (Sirius Analytical Instruments,
Ltd.) apparatus. This instrument is specifically designed to
determine ionization constants (pKa values) by titration using
potentiometry.
9.5. (R,S)-2-Am in o-3-(1-h yd r oxy-2-im id a zolyl)p r op ion -
ic Acid (10a ). A solution of 41a (252 mg, 0.80 mmol) in 1 M
TFA (20 mL) was heated to reflux for 40 h. The mixture was
concentrated and coevaporated twice with 2 M HCl (25 mL)
and twice with toluene (25 mL). The crude product was taken
up in water and applied to ion exchange chromatography as
described for 7b to give 137 mg (100%) of 10a as a solid.
Since the water solubility of the compounds is high, the
titrations were performed without use of a cosolvent. Ap-
proximately 5 mg of the particular compound was weighed out,
15 mL of 0.15 M KCl was added, and three titrations were
performed at 25.1 ( 0.1 °C on the sample using 0.500 N HCl
and 0.500 N KOH. The first titration was in the direction of
low pH (pH 2) to high pH (pH 12), the second from high pH to
low, and the last and third from low to high pH. On the basis
of the titration curves and the corresponding blank curve, the
three pKa values were determined for each of the difference
curves by numerical refinement using the GLpKa software. A
difference curve is defined as the number of protons bound
per molecule as a function of pH. The three curves were
combined in a multiset, and the refinement was repeated. The
final results regarding the pKa values were from the multiset
analysis.
12. Recep tor Bin d in g Assa ys. Affinities for NMDA,
AMPA, and kainic acid receptors were determined using [3H]-
CPP,26 [3H]AMPA,24 and [3H]kainic acid25 with the following
modifications. The membrane preparation used in all the
receptor binding experiments were prepared according to the
method described by Ransom and Stec.32 All binding experi-
ments were carried out at 0-4 °C. On the day of experiments,
frozen homogenates were quickly thawed and resuspended in
50 volumes of buffer (pH 7.4) (50 mM Tris-HCl + 2.5 mM
CaCl2, 30 mM Tris-HCl + 2.5 mM CaCl2, or 50 mM Tris-HCl
for [3H]CPP, [3H]AMPA, or [3H]Kainic acid binding, respec-
tively) and centrifuged (48 000g, 10 min). This step was
repeated four times. In [3H]AMPA experiments 100 mM KSCN
was added to the buffer during the final wash and during
incubation. The final pellet was resuspended in ice-cold buffer,
corresponding to approximately 50, 25, or 25 mg/mL original
tissue for [3H]CPP, [3H]AMPA, or [3H]kainic acid binding,
respectively. Nonspecific binding was determined using 1 mM
Glu. For [3H]CPP binding, aliquots consisted of 50 µL of test,
50 µL of [3H]CPP, and 400 µL of membrane suspension.
Following incubation for 30 min, filtration (GF/B) was carried
out through a 48-well Brandell cell harvester, followed by
washing with 3 × 0.5 mL buffer. To the filters was added a
total of 3 mL of OptiFluor, and the amount of bound radioac-
tivity was determined using a TRI-CARB liquid scintillation
analyzer. [3H]AMPA and [3H]kainic acid binding were carried
out in aliqouts consisting of 25 µL of [3H]ligand, 25 µL of test,
and 200 µL of membrane suspension. Binding was terminated
by filtration through GF/B filters using a 96-well Packard
FilterMate cell harvester and washing with 3 × 250 µL of
buffer. Filters were dried, and 25 µL of Microscint 0 was added.
The amount of bound radioactivity was determined using a
Packard TOPCOUNT microplate scintillation counter. The
data were analyzed using Grafit 3.0, Leatherbarrow software.
Data were fitted to the equation
1
Recrystallization from MeOH gave 10a : mp 160-161 °C; H
NMR (D2O) δ 7.27 (d, J ) 2.1 Hz, 1 H), 7.16 (d, J ) 2.1 Hz, 1
H), 4.10 (t, J ) 6.4 Hz, 1 H), 3.46 (d, J ) 6.3 Hz, 2 H); 13C
NMR (D2O) δ 172.6, 136.2, 120.7, 116.5, 53.0, 26.0. An
analytical sample was obtained by evaporation of 10a with 2
M HCl. Anal. (C6H9N3O3‚HCl, 20 mol % H2O) C, H, N.
10. P r ep a r a t ion of Im id a zole Am in o Acid 10b. 10.1.
1-Ben zyloxy-2-h yd r oxym eth yl-5-m eth ylim id a zole (39b).
At -78 °C a solution of n-BuLi (1.6 M in hexanes, 6.7 mL,
10.7 mmol) was added dropwise over 2 min to 1-benzyloxy-2-
chloro-5-methylimidazole (31) (1.97 g, 8.95 mmol) dissolved in
THF (50 mL). The mixture was stirred for 5 min before DMF
(3.5 mL, 45 mmol) was added. The solution was stirred for 1
h at -78 °C and was allowed to warm to room temperature
over 30 min. The reaction mixture was quenched with 2 M
HCl (25 mL) and neutralized (pH 8) by addition of saturated
NaHCO3. The organic layer was separated, and the aqueous
layer was extracted with CH2Cl2 (5 × 30 mL). Drying of the
combined organic phases and evaporation gave 1.62 g (85%)
of the crude aldehyde 33b as a yellow oil: 1H NMR δ (CDCl3)
9.72 (s, 1H), 7.40 (s, 5H), 6.92 (s, 1H), 5.22 (s, 2H), 1.99 (s,
3H); 13C NMR (CDCl3) δ 178.4 (s), 138.4 (s), 132.5 (s), 131.7
(s), 129.9 (d), 129.4 (d), 128.4 (d), 126.0 (d), 81.5 (t), 7.6 (q).
Compound 33b was dissolved in MeOH (60 mL), cooled to 0
°C, and treated with NaBH4 (0.32 g, 4.2 mmol). The mixture
was stirred at 0 °C for 3 h and was worked up as described
for compound 15a . FC (EtOAc) gave 1.40 g (85%) of 39b as a
solid. A small sample was recrystallized from EtOAc/hep-
tane: mp 106-107 °C; 1H NMR δ (CDCl3) 7.46-7.36 (m, 5H),
6.58 (s, 1H), 5.26 (s, 2H), 4.62 (s, 2H), 2.04 (s, 3H); 13C NMR
(CDCl3) δ 142.9 (s), 133.5 (s), 130.1 (d), 129.7 (d), 128.9 (d),
125.2 (d), 120.6 (s), 82.1 (t), 54.3 (t), 8.1 (q). Anal. (C12H14N2O2)
C, H, N.
10.2. E t h yl 2-Acet a m id o-3-(1-b en zyloxy-5-m et h yl-2-
im id a zolyl)-2-et h oxyca r b on ylp r op ion a t e (41b ). Com-
pound 41b was prepared from 39b (600 mg, 2.75 mmol) and
thionyl chloride (2.0 mL, 28 mmol) in toluene (12 mL) by the
method described for 37b. FC (EtOAc/heptane 1:1 f 1:0) gave
568 mg (50%) of 41b as an oil: 1H NMR (CDCl3) δ 7.42 (br s,
5H), 6.95 (br s, 1H), 6.55 (s, 1H), 4.97 (s, 2H), 4.29 (dq, J )
10.5, 6.9 Hz, 2H), 4.23 (dq, J ) 10.5, 7.2 Hz, 2H), 3.71 (s, 2H),
2.11 (s, 3H), 1.89 (s, 3H), 1.25 (t, J ) 7 Hz, 6H); 13C NMR
(CDCl3) δ 169.4 (s), 167.1 (s), 137.0 (s), 132.7 (s), 129.9 (d),
129.4 (d), 128.6 (d), 124.1 (d), 121.4 (s), 80.9 (t), 65.2 (s), 62.3
(t), 29.1 (t), 22.4 (q), 13.5 (q), 8.0 (q).
10.3. Eth yl 2-Aceta m id o-3-(1-h yd r oxy-5-m eth yl-2-im i-
d a zolyl)-2-eth oxyca r bon ylp r op ion a te (42b). A mixture of
41b (515 mg, 1.24 mmol), 10% Pd/C (50 mg), and MeOH (5
mL) was stirred under H2 (1 atm) at 0 °C for 1 h. Filtration
through Celite and removal of MeOH gave 367 mg (91%) of
42b as a solid. A small sample was recrystallized from
EtOAc: mp 66-67 °C; 1H NMR (D2O) δ 6.81 (s, 1H), 4.32-
4.18 (m, 4H), 3.71 (s, 2H), 2.07 (s, 3H), 1.97 (s, 3H), 1.18 (t, J
) 7 Hz, 6H); 13C NMR (CDCl3) δ 174.6(s), 168.9 (s), 132.1 (s),
129.5 (d), 112.4 (s), 66.1 (s), 65.1 (t), 29.0 (t), 22.2 (q), 13.7 (q),
8.2 (q). Anal. (C12H21N3O6‚H2O) C, H, N.
10.4. (R,S)-2-Am in o-3-(1-h ydr oxy-5-m eth yl-2-im idazolyl)-
p r op ion ic Acid Dih yd r och lor id e (10b‚2HCl). A solution
of 42b (180 mg, 0.55 mmol) in 4 M HCl was heated to reflux
for 16 h. The mixture was concentrated and evaporated twice
with water and twice with toluene to give 144 mg (92%) of
10b‚2HCl as a hygroscopic solid: mp 150-152 °C (dec); 1H
NMR (D2O) δ 7.09 (s, 1H), 4.42 (t, J ) 7.1 Hz, 1H), 3.68-3.53
(m, 2H), 2.24 (s, 3H); 13C NMR (D2O) δ 171.0 (s), 137.2 (s),
100 × [inhibitor]n
B ) 100 -
IC50n + [inhibitor]n
where B is the binding as a percentage of total specific binding
and n is the Hill coefficient.
13. Syn a p tosom a l Glu Up ta k e. The experiments were
carried out essentially as previously described.33 Rat cortical
synaptosomes were prepared from male Sprague-Dawley rats
(200-249 g). The final synaptosomal pellet was resuspended
in assay buffer containing 128 mM NaCl, 10 mM glucose, 5
mm KCl, 1.5 mM NaH2PO4, 1.77 mM CaCl2, 1 mM MgSO4,
and 10 mM Tris (pH 7.4) in a final concentration of 2 mg/mL.
The experiment was carried out at room temperature following
a 5 min preincubation of the synaptosomes with a solution

30 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Stensbøl et al.
containing [3H]-(R)-aspartic acid (30 nM) and (R)-aspartic acid
(3 µM). The assay was initiated by adding 800 µL of membrane
solution to a mixture of test substance (100 µL) and a solution
(100 µL) containing [3H]-(R)-aspartic acid (30 nM) and (R)-
aspartic acid (3 µM). Follwing a 10 min incubation, the reaction
was terminated by filtering though a 48-well Brandell cell
harvester using GF/B filters. Filters were washed with 3 × 3
mL of 50 mM Tris buffer. Total binding was determined in
the presence of buffer, and nonspecific binding was determined
in the presence of 1 mM (R)-aspartic acid.
14. Testin g a t m eta botr op ic Glu r ecep tor s. Three
metabotropic Glu receptor subtypes, mGlu1R, mGlu2, and
mGlu4a, were stably expressed in Chinese hamster ovary cells
and used as representatives for groups I, II, and III, respec-
tively. Cell maintenance and pharmacological assays were
performed as previously described.34
15. In Vitr o Electr op h ysiology. The rat cortical prepara-
tion35 in a modified version36 was used for the determination
of the depolarizing effects of the analogues under study.
Compounds that did not show agonist properties were tested
as antagonists toward 5 µM AMPA, 5 µM kainic acid, and 10
µM NMDA. Agonists were applied for 90 s. Receptor selectivity
was determined by antagonizing agonist responses, evoked
with a concentration approximately corresponding to the EC50
values of the agonists in question, with 5 µM CPP and 5 µM
NBQX for NMDA and AMPA receptors, respectively. Antago-
nists were applied for 90 s, followed by coapplication of agonist
and antagonist for 90 s. The data were fitted to the equation
intermediate character between, high-level gas-phase ab initio
structures and X-ray crystal structures.38 The appropriate ring-
methyls of the optimized heterocycles were resubstituted with
the glycine moiety in zwitterionic form, and the three dihedral
angles defining the R-amino acid conformation were adjusted
to match those recorded by Armstrong and Gouaux27 for AMPA
bound to GluR2-S1S2J . The structures were reoptimized in
solution, loosely constraining these dihedrals and freezing the
ring atoms plus those atoms directly bound to the ring. This
optimization was performed using the Cramer-Truhlar AM1-
SM2 semiempirical method implemented in Spartan 5.1.40 The
result was aqueous 3D geometries for the negatively charged
tri-ionized forms of 7a ,b, 8a ,b, 9a -c, and 10a ,b in solution,
corresponding to the bound conformation of 3b and close to
the aqueous local energy minima.
The nine structures were docked into the binding site of the
centrally located protein of the three almost identical subunit
constructs found in the crystal structure of the GluR2-S1S2J -
AMPA complex of Armstrong and Gouaux.27 This procedure
involved superimposing each structure in turn on 3b, using
least-squares fitting. Five atoms of each structure were used:
both of the oxygens and the nitrogen of the R-amino acids, the
distal phenolate-like oxygen, and the ring atom attached to
it. Protons were added to the proximal residues using the
builder in InsightII.41 Crucial water molecules were proto-
nated, and the waters were rotated to maximize hydrogen
bonding. The dihedral angles of rotatable side chain hydroxyls
were also adjusted to complete the hydrogen bond network.
Ack n ow led gm en t. This work was supported by the
Lundbeck Foundation and the Danish Medical Council.
The mGlu-expressing cell lines were a generous gift
from Prof. Shigetada Nakanishi (Kyoto University,
J apan). We also thank Ms. Heidi Petersen and Ms.
Lisbeth Eriksen for technical assistance.
E
max[agonist]n
n
% response )
EC50 + [agonist]n
where Emax is the relative maximal response and n is the Hill
coefficient.
16. EAAT Up ta k e P r oced u r e. Cell maintenance and
EAAT uptake assay was performed as previously described.37
Cos-7 cells were maintained in Dulbecco’s modified eagle’s
medium (DMEM) containing 10% fetal bovine serum (both
GIBCO, Paisley, Scotland) in a humidified incubator contain-
ing 5% CO2 and 95% atmosphere. A total of 1 million cells were
split into a 10 cm tissue culture dish, and the following day,
cells were transfected with 5 µg of EAAT DNA inserted in
pcDNA3 (Invitrogen, La J olla, CA) using SuperFect (Qiagen,
Hilden, Germany) as a DNA carrier. The day after transfec-
tion, cells from one 10 cm dish were transferred to 48-well
tissue culture plates, and 2 days after transfection, uptake
assays were performed.
Su p p or tin g In for m a tion Ava ila ble: Detailed description
of the molecular modeling. This material is available free of
charge via the Internet at http://pubs.acs.org.
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