4-Hydroxy-1,2,5-oxadiazol-3-yl moiety as bioisoster of the carboxy function. Synthesis, ionization constants, and molecular pharmacological characterization at ionotropic glutamate receptors of compounds related to glutamate and its homologues

Article abstract of DOI:10.1021/jm1001452

In order to investigate the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as a carboxylic acid bioisoster at ionotropic glutamate receptors (iGluRs), a series of acidic α-aminocarboxylic acids in which the distal carboxy group was replaced by the 4-hydroxy-1,2,5-oxadiazol-3-yl group was synthesized. Ionization constants were determined. All target compounds, except the Asp analogue 12, were resolved using chiral HPLC. Whereas 12 showed good affinity exclusively at NMDA receptors, the Glu analogue, (+)-10, was an unselective, though potent AMPA receptor preferring agonist (EC₅₀ = 10 μM at iGluR2) showing only low stereoselectivity. The two higher Glu homologues, (+)-15 and (+)-18, turned out to be weak agonists at iGluR2 as well as weak antagonists at NR1/NR2A, whereas the corresponding (-)-isomers were selective NR1/NR2A antagonists with somewhat higher potency. The results proved the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety to be a useful bioisoster at all three classes of iGluRs, capable of being integrated into agonists as well as antagonists.

Full text of DOI:10.1021/jm1001452

4110 J. Med. Chem. 2010, 53, 4110–4118
DOI: 10.1021/jm1001452
4-Hydroxy-1,2,5-oxadiazol-3-yl Moiety as Bioisoster of the Carboxy Function. Synthesis, Ionization
Constants, and Molecular Pharmacological Characterization at Ionotropic Glutamate Receptors of
Compounds Related to Glutamate and Its Homologues
Marco L. Lolli,† Cecilia Giordano,† Darryl S. Pickering,‡ Barbara Rolando,† Kasper B. Hansen,§, Antonio Foti,†
Alberto Contreras-Sanz,‡ Ahmad Amir,‡ Roberta Fruttero,† Alberto Gasco,*,† Birgitte Nielsen,§ and Tommy N. Johansen*,§
†
‡
Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy, Department of
ꢀ
Pharmacology and Pharmacotherapy, and §Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,
2 Universitetsparken, DK-2100 Copenhagen, Denmark, and Department of Molecular Biology, H. Lundbeck A/S, Copenhagen, Denmark
Received February 3, 2010
In order to investigate the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as a carboxylic acid bioisoster at
ionotropic glutamate receptors (iGluRs), a series of acidic R-aminocarboxylic acids in which the distal
carboxy group was replaced by the 4-hydroxy-1,2,5-oxadiazol-3-yl group was synthesized. Ionization
constants were determined. All target compounds, except the Asp analogue 12, were resolved using
chiral HPLC. Whereas 12 showed good affinity exclusively at NMDA receptors, the Glu analogue, (þ)-
10, was an unselective, though potent AMPA receptor preferring agonist (EC50 = 10 μM at iGluR2)
showing only low stereoselectivity. The two higher Glu homologues, (þ)-15 and (þ)-18, turned out to be
weak agonists at iGluR2 as well as weak antagonists at NR1/NR2A, whereas the corresponding (-)-
isomers were selective NR1/NR2A antagonists with somewhat higher potency. The results proved the
4-hydroxy-1,2,5-oxadiazol-3-yl moiety to be a useful bioisoster at all three classes of iGluRs, capable of
being integrated into agonists as well as antagonists.
Introduction
In the design of selective receptor ligands, isosteric replacement
of moieties in a lead structure is one of the most frequently used
approaches.3-7 This approach should be carried out taking
into account that a particular isosteric group proven to be a
bioisoster in one lead compound may not necessarily be
successful in another. In the designing of mimetics of endogen-
ous acidic amino acids, a number of bioisosteric replacements
of the carboxylic group have been described8 and especially 3-
hydroxyisoxazol-4-yl has been a well studied example at
iGluRs.9 Quite recently, another hydroxy-substituted hetero-
cycle, 4-hydroxy-1,2,5-thiadiazol-3-yl, was substituted for the
distal carboxylic group in Glu to give 2-amino-3-(4-hydroxy-
1,2,5-thiadiazol-3-yl)propionic acid (TDPA, 2, Figure 1) which
shows a preference for the AMPA subtype of iGluRs.10 Re-
cently, we proposed the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety
as a potential bioisoster of the COOH function and we explored
the possibility of obtaining biomimetics of γ-aminobutyric acid
(GABA), the major inhibitory neurotransmitter in the CNS.
This approach gave rise to compounds with partial agonist
profiles at GABAA receptors.11 In this paper we describe the
synthesis, ionization properties, resolution, and molecular
pharmacological characterization at native and recombinant
(S)-Glutamate ((S)-Glu,a Figure 1) plays a major roleinthe
transmission of excitatory signals in the central nervous
system (CNS) and is implicated in epilepsy, pain, memory,
excitotoxicity, and brain development.1 It mediates its actions
through two classes of receptors (GluRs). The first class
consists of ionotropic glutamate receptors (iGluRs) that are
classified according to their selective agonists into NMDA
(N-methyl-D-aspartate), AMPA((RS)-2-amino-3-(3-hydroxy-
5-methylisoxazol-4-yl)propionic acid, 1, Figure 1) and KA
(kainic acid) receptors. The second class consists of metabo-
tropic glutamate receptors (mGluRs) that are divided into
three groups (I, II, III) each containing at least two subtypes.
The vast majority of both iGluR and mGluR subtypes are
considered interesting therapeutic targets for the treatment of a
number of neurologic and psychiatric diseases.1,2
Specific agonists and antagonists are necessary in order to
characterize the physiological roles of the individual receptors.
*To whom correspondence should be addressed. For A.G.: phone,
þ39 0116707670; fax, þ39 0116707687; e-mail, alberto.gasco@unito.it.
For T.N.J.: phone, þ45 35336412; fax, þ45 35336040; e-mail, tnj@
farma.ku.dk.
a Abbreviations: (S)-Glu, (S)-glutamate; CNS, central nervous sys-
tem; iGluRs, ionotropic glutamate receptors; NMDA, N-methyl-D-
aspartate; AMPA, (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)-
propionic acid; KA, kainic acid; mGluRs, metabotropic glutamate
receptors; TDPA, (RS)-2-amino-3-(4-hydroxy-1,2,5-thiadiazol-3-yl)-
propionic acid; GABA, γ-aminobutyric acid; [3H]CGP 39653,
[3H](RS)-(E)-2-amino-4-phosphonomethyl-3-heptenoic acid; THF, tet-
rahydrofurane; NBS, N-bromosuccinimide; Tf2O, triflic anhydride;
TLC, thin layer chromatography; CD, circular dichroism.
Figure 1. Structures of (S)-Glu and selected heterocyclic analogues.
r
pubs.acs.org/jmc
Published on Web 04/21/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4111
Scheme 1. Synthesis of Target Compounds 10, 12, 15, and 18
Table 1. pKa Values ((SD) of Compounds 10, 12, 15, 18 as Determined
iGluRs of the target compounds 10, 12, 15, and 18. These
compounds can be formally derived from Glu, its inferior
homologue, and its two superior homologues by substitution
of the distal COOH group with the 4-hydroxy-1,2,5-oxadiazol-
3-yl moiety.
by Potentiometrya
pKa ( SD
þ
compd
pKa1 (R-COOH)
pKa2
pKa3 (NH3
)
Glu
Asp
Gly
12
2.2
2.0
4.2
3.9
9.6
10.0
Results and Discussion
2.3
9.8
1.88 ( 0.27
1.90 ( 0.19
2.12 ( 0.05
2.48 ( 0.04
3.37 ( 0.03
3.48 ( 0.02
3.68 ( 0.03
3.69 ( 0.02
7.60 ( 0.01
8.81 ( 0.01
9.10 ( 0.02
9.39 ( 0.01
Chemistry. The synthetic pathways used to prepare the
target compounds 10, 12, 15, 18 are reported in Scheme 1.
The strict analogue 10 of Glu was prepared starting from (4-
hydroxy-1,2,5-oxadiazol-3-yl)acetic acid (3) that was trans-
formed into the corresponding ethyl ester by action of
gaseous HCl in dry ethanol. The ester was purified by flash
chromatography and immediately treated with benzyl bro-
mide in acetone in the presence of K2CO3 to afford the ester
4. Basic hydrolysis of 4 afforded 4a that by KMnO4 lateral
chain oxidative demolition produced the benzyloxyl substi-
tuted 1,2,5-oxadiazolecarboxylic acid 5. Methyl ester 6 was
obtained from 5 in dry methanol solution saturated with
gaseous HCl. Reduction of the ester function of 6 carried out
with NaBH4 in tetrahydrofuran (THF) yielded the alcohol 7.
The related bromo derivative 8 was obtained by treating 7
with N-bromosuccinimide (NBS) in THF in the presence of
Ph3P. A THF solution of this last intermediate was allowed
to react with diethyl [(tert-butoxycarbonyl)amino]malonate
and NaH mineral oil dispersion to give 9. Pd/C, H2 reduction
of 9 afforded 9a that was refluxed in HBr water solution to
afford, after ion exchange chromatography, the racemic
mixture 10. Synthesis of the racemic mixture of 12 was
carried out by nitrosation of the active methylene of 4 and
subsequent reduction of oxime 11 with Pd/C, H2 followed by
immediate HCl hydrolysis of the intermediate ester 11a. To
prepare the racemic mixture 15, the intermediate 4 was
transformed into the alcohol 13 by action of LiAlH4 in
THF. Alcohol 13, treated with triflic anhydride (Tf2O) in
the presence of 2,6-lutidine afforded the corresponding tri-
fluoromethanesulfonate, which was quickly purified by flash
chromatography and immediately allowed to react with
diethyl [(tert-butoxycarbonyl)amino]malonate and NaH
mineral oil dispersion to give 14. This last product, treated
under the same conditions used to prepare 10 from 9, gave
rise to the final compound 15 through the intermediate
formation of 14a. Synthesis of 17 was carried out by treating
10
15
18
a Glu, Asp, and Gly pKa values are shown for comparison.
alcohol 16 in the same conditions used to prepare 14 from 13.
Treatment of 17 with NaOH in DMSO induced nucleophilic
substitution of the phenylsulfonyl group with consequent
formation of the corresponding sulfinic acid and partial
hydrolysis of ethyl ester moieties. Treatment of this mixture
with Oxone at controlled pH, in order to oxidate phenylsul-
finic acid to the more acidic sulfonic acid, and subsequent
extraction with diethyl ether of the resulting mixture gave a
crude material that was refluxed in aqueous HBr to yield
target compound 18.
Ionization Constants. The ionization constants (pKa
values) for the final compounds 10, 12, 15, 18 were obtained
by potentiometric titration using a GLpKa apparatus. The
pKa values are collected in Table 1. The products show three
dissociation constants in the ranges pKa1 = 1.88-2.48, pKa2
=
3.37-3.69, and pKa3 = 7.60-9.39. The first two ranges are
related to the dissociation of the two acid functions, while the
third is related to pKa values of the basic -NH2 function. A
comparison of these data with the pKa values of Glu, Asp, and
Gly12 (Table 1) indicates that it is reasonable to assign the first
and the second sets to the prevalent dissociation of the COOH
function and of the hydroxyl-1,2,5-oxadiazole moiety, respec-
þ
tively. The third set is then assigned to the NH3 group. At
physiological pH, products 15 and 18 exist, similar to Glu and
ASP, predominantly in the anionic tricharged form (- - þ),
while in 12, and partly in 10, this species is in equilibrium with
the dianionic form (- -).
At AMPA receptors, groups known to behave as bioisos-
ters of the distal COOH function cover a pKa range where the
ionized form predominates in aqueous solutions.13,14 A low

4112 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Lolli et al.
degree of ionization is expected to disfavor binding by
shifting the equilibrium away from the presumably active
form carrying three charges. The high degree of acidity
exhibited by the hydroxyfurazan moiety should be favorable
in this sense.
and NMDA receptor sites were determined in receptor
binding assays at rat brain membrane preparations using
the radioligands [3H]AMPA, [3H]KA, and [3H](RS)-(E)-2-
amino-4-phosphonomethyl-3-heptenoic acid ([3H]CGP
39653). The results are reported in Table 2.
Chiral HPLC Resolution of Stereomers. The resolution of
compounds 10, 12, 15, and 18 was performed on a Crownpak
CR(þ) semipreparative column, using 15 mM aqueous
acetic acid (pH 3.0) as the mobile phase. The stereochemical
purity of each obtained enantiomer was >99%. For all the
products the first eluted enantiomers were the levorotatory
(-) ones. It is interesting to point out that the specific
Compound 12 showed affinity and preference for NMDA
receptors similar to that of NMDA itself and the correspond-
ing 5-methyl-3-hydroxyisoxazole analogue,20 whereas com-
pounds 15 and 18 displayed affinities for both NMDA and
AMPA, but not for KA, receptor sites. For compounds 15
and 18, the AMPA receptor affinity resided in the (þ)-
isomers, whereas all four 15 and 18 isomers showed affinity
for NMDA receptor sites with the (-)-enantiomers being
eutomers. For 15, the observed profile at ionotropic receptor
sites was different from the corresponding hydroxythiadia-
zole analogue, which does not show measurable affinity for
native iGluR sites.15 Compound 10, the close analogue of
Glu, not only displayed quite potent affinity for AMPA
receptors similar to that of AMPA (1) and TDPA10 (2) but
also turned out to be an unselective iGluR ligand, although
with some preference for AMPA receptors. The pharmacol-
ogy profile of 10 is quite different from those of the corre-
sponding 3-hydroxyisoxazole21 and the corresponding
hydroxythiadiazole10 (2) analogues which interact selectively
with the AMPA receptor subtype of iGluRs. At all three
classes of native iGluRs the affinity of 10 was shown to reside
mainly in the (þ)-isomer. Similar to TDPA,10 the interaction
of 10 with AMPA receptors showed low stereoselectivity, as
(þ)-10 is only approximately 6 times more potent than the
other enantiomer. Since the stereochemical purity of (-)-10
is very high (99.6% ee), the observed affinity at AMPA
receptors is not caused by a stereochemical impurity. The
lower affinity for NMDA and KA receptors prevents any
quantitative comparison of stereoselectivity at these sites.
Furthermore, (þ)- and (-)-10 were evaluated in receptor
binding at the cloned AMPA receptor iGluR2(R)o and KA
receptors iGluR5(Q)1b, iGluR6(V,C,R)A, and iGluR7A
(Table 3). At both iGluR2 and iGluR5 receptors (þ)-10
was the most potent isomer, being 6- and 10-fold more potent
compared to (-)-10, respectively. (þ)-10 was demonstrated
to be almost as potent as AMPA22 at iGluR2 and iGluR5 but
did not show any affinity for iGluR6 or iGluR7. Whereas
(þ)-10 was more than 100-fold more potent at iGluR2 than
at iGluR5 receptors, neither (þ)-15 nor (þ)-18 showed any
preference for iGluR2 receptors.
rotations [R]25 found for 10 and 15 are close to those of
D
the corresponding thio analogues.10,15 On the basis of the
observed elution order of corresponding thio analogues on
the Crownpak column10,15 as well as the general elution
order of a series of racemic R-amino acids, the first-eluting
(-)-isomers of compounds 10, 15, and 18 are believed to
possess R-configuration.16 No acceptable resolution was
achieved for compound 12 or its N-Boc protected derivative,
although a number of chiral stationary phases and eluents
were investigated. NMR experiments, performed in D2O
studying the rate of deuterium exchange of the R-H in
compound 12, showed that the R-H undergoes quick ex-
change, most likely too fast for a successful resolution of 12.
Radioligand Binding at iGluRs. The affinities of the race-
mates and the isolated enantiomers for native AMPA, KA,
Table 2. Receptor Binding Affinities at Native Rat iGluRsa
IC50 (μM)
Ki (μM)
NMDA receptors
compd
AMPA receptors
0.34
KA receptors
(S)-Glub
AMPA (1)c 0.039
0.38
>100
0.007d
>100
>100
0.20
>100
KA
NMDAb
TDPA (2)e 0.12
4.0d
>100
>100
6.2
>100 f
12
10
>100
>100
6.3 [5.20 ( 0.02]
16 [4.80 ( 0.03]
13 [4.90 ( 0.03]
>100
0.17 [6.76 ( 0.05] 49 [4.31 ( 0.05]
0.11 [6.95 ( 0.03] 18 [4.76 ( 0.03]
0.76 [6.12 ( 0.06] >100
77 [4.12 ( 0.07]
42 [4.37 ( 0.03]
>100
(þ)-10
(-)-10
15
>100
>100
>100
>100
>100
>100
4.9 [5.31 ( 0.03]
73 [4.13 ( 0.02]
3.4 [5.47 ( 0.05]
2.9 [5.54 ( 0.06]
22 [4.66 ( 0.05]
1.8 [5.75 ( 0.06]
(þ)-15
(-)-15
18
61 [4.22 ( 0.07]
42 [4.37 ( 0.01]
>100
(þ)-18
Functional Studies at Representative Recombinant iGluRs.
Active enantiomers were characterized functionally at se-
lected AMPA and NMDA receptor subtypes (Table 4). The
concentration-response data of the stereoisomers and the
calculated EC50 values generally supported the affinity data
reported in Table 3. At iGluR2, (þ)- and (-)-10 as well as
(þ)-15 and (þ)-18 turned out to be agonists (Figure 2) with
(-)-18
a AMPA, KA, and NMDA receptors were studied using [3H]AMPA,
[3H]KA, and [3H]CGP 39653 as radioligands, respectively. Mean pIC50
( SEM and pKi ( SEM (shown in brackets) and the corresponding IC50
and Ki values were calculated from full concentration-response curves
of three individual experiments. b Reference 17. c Reference 18. d Refer-
ence 19. e Reference 10. f [3H]CPP binding.
Table 3. Receptor Binding Affinities at Cloned Rat Subtypes Expressed in Sf9 Cellsa
Ki (μM)
compd
iGluR2(R)o
iGluR5(Q)1b
iGluR6(V,C,R)A
iGluR7A
(S)-Glu
(þ)-10
(-)-10
(þ)-15
(þ)-18
0.28 [6.55 ( 0.08]
0.043 [7.37 ( 0.02]
0.25 [6.60 ( 0.03]
40 [4.39 ( 0.07]
20 [4.69 ( 0.02]
0.14 [6.86 ( 0.01]
8.1 [5.09 ( 0.01]
77 [4.11 ( 0.01]
53 [4.27 ( 0.02]
67 [4.18 ( 0.06]
0.33 [6.48 ( 0.02]
>100
>100
0.49 [6.31 ( 0.03]
>100
>100
>1000
>1000
41 [4.38 ( 0.07]
>1000
a Mean pKi ( SEM (shown in brackets) and the corresponding Ki values were calculated from full concentration-response curves of three individual
experiments.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4113
Table 4. Functional Characterization at Selected Cloned AMPA and NMDA Receptors
iGluR2(Q)i expressed in Xenopus oocytesa,c
NR1-1a/NR2A expressed in BHK-21 cellsb,c
compd
EC50 (μM)
relative Imax
EC50 (μM)
relative Fmax
IC50 (μM)
(S)-Glu
(þ)-10
(-)-10
(þ)-15
(-)-15
(þ)-18
(-)-18
14 [4.86 ( 0.04]
9.4 [5.03 ( 0.05]
43 [4.37 ( 0.04]
489 [3.31 ( 0.09]
nd
1.00
0.92 ( 0.03
nd
nd
nd
nd
nd
1.8 [5.75 ( 0.12]
47 [4.33 ( 0.13]
>500
1.00
0.76 ( 0.02
nd
nr
nr
1024 [2.99 ( 0.01]
49 [4.31 ( 0.04]
245 [3.61 ( 0.04]
28 [4.55 ( 0.06]
491 [3.31 ( 0.06]
nd
nr
nr
a Parameters measured by TEVC recordings. Mean pEC50 ( SEM (shown in brackets) and the corresponding EC50 values were calculated from full
concentration-response curves. The mean relative maximal response (I/Imax ( SEM) is relative to maximal steady-state currents evoked by (S)-Glu. The
number of oocytes was between 5 and 13. b Parameters measured by [Ca2þ]i measurements. Mean pEC50 ( SEM (shown in brackets) and the
corresponding EC50 values as well as relative Fmax ( SEM (relative to Fmax of (S)-Glu) (maximal response to glutamate, 1.0) were calculated from full
concentration-response curves. For the agonist data the Hill coefficients were between 1.1 and 1.5, and the number of experiments was 3. Inhibition of
NMDA receptor subtype, NR1-1a/NR2A, by increasing concentrations of antagonists was performed using 6 μM (S)-Glu. The mean pIC50 ( SEM
(shown in brackets) and the corresponding IC50 values were calculated from full concentration-response curves. For all antagonist data, the number of
experiments was 3. c nd: not determined. nr: no response to 1000 μM.
Figure 2. Electrophysiological activity at iGluR2 expressed in
Xenopus oocytes.
relative potencies to some extent in agreement with the
affinities reported in Table 3. Efficacy measurements con-
ducted for (þ)-10 at iGluR2(Q)i indicated that the com-
pound has an efficacy that was statistically significantly
lower (paired t test, P = 0.037), 0.92 ( 0.03 (mean (
SEM, n = 8) relative to (S)-Glu (efficacy = 1.000).
Furthermore, enantiomers displaying affinity at native
NMDA receptor sites were studied at NR1/NR2A receptors
expressed in BHK-21 cells using intracellular calcium mea-
Figure 3. Activity at NR1/NR2A receptors expressed in BHK-21
surements.23 (þ)-10 displayed a partial agonist profile
cells. (a) Representative concentration-response data determined
(Figure 3a and Table 4). The enantiomers of 15 and 18 were
inactive when tested for agonistic activity. However, (-)-15
and (-)-18 behaved as antagonists with similar potencies
(Figure 3b). Also, the related (þ)-enantiomers showed an-
tagonistic activity but their IC50 values (Table 4) were
markedly higher complementing the affinity data of Table 3.
It is interesting that the stereoselectivity of compound 15,
with (-)-15 being the more potent isomer, is also seen with
the corresponding hydroxythiadiazole analogue of 15,15
although this compound is markedly less potent than (-)-
15 at NMDA receptors.
by intracellular calcium measurements at NR1-1a/NR2A receptors
expressed in BHK-21 cells. The maximal fluorescence response to
(S)-Glu (Fmax,(S)-Glu) is normalized to 1, and the minimal response
(Fmin) is normalized to 0. The fluorescence responses to the agonists
are normalized to Fmax,(S)-Glu. Data points are represented as the
mean ( SD of four to six wells. (b) Representative concentra-
tion-response curves for inhibition of (S)-Glu-induced responses
determined by intracellular calcium measurements at NR1-1a/
NR2A receptors expressed in BHK-21 cells. The responses were
induced by coapplication of 6 μM (S)-Glu. Data points are repre-
sented as the mean ( SD of four to six wells..
nized by iGluRs. Among the four new acidic amino acids, the
strict Glu analogue 10 turned out to have a pharmacological
profile very similar to that of Glu, being an unselective but
AMPA-receptor preferring agonist, with EC50 = 10 μM at
iGluR2 and a relative efficacy of 0.92. At iGluR2, iGluR5,
and NR1/NR2A, (þ)-10 was shown to be the eutomer. These
results indicate that biomimetics of Asp, Glu, and their
Conclusions
The results reported in the present work show that the
4-hydroxy-1,2,5-oxadiazol-3-yl moiety, when incorporated in
Asp and Glu-related compounds as well as the two higher
homologues, gives rise to products that are uniquely recog-

4114 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Lolli et al.
homologues can be obtained by substitution of the distal
carboxylic acid with the isoster 4-hydroxy-1,2,5-oxadiazol-3-
yl substructure.
(30 mL, 251.95 mmol) were added to an acetone (600 mL)
solution of the ester previously obtained. The resulting mixture
was refluxed for 3 h and then filtered. The filtrate was concen-
trated under reduced pressure, obtaining an oil that was purified
preliminarily by flash chromatography (eluent, petroleum ether
(40-60 °C)/EtOAc, 95:5 v/v) and then distilled (bp = 152 °C at
2.4 ꢀ 10-2 mbar) to give 4 as colorless oil. Yield 75%. 1H NMR
(CDCl3) δ 1.22 (t, J = 7.1 Hz, 3H, -OCH2CH3), 3.74 (s, 2H,
-CH2COO-), 4.17 (q, J = 7.1 Hz, 2H, -OCH2CH3), 5.36 (s,
2H, PhCH2O-), 7.38-7.42 (m, 5H, arom). 13C NMR (CDCl3) δ
14.0, 28.4, 61.8, 74.0, 128.4, 128. 7, 128.9, 134.6, 141.7, 164.2,
167.2. MS (CI) m/z 263 (M þ 1)þ. Anal. (C13H14N2O4) C, H, N.
[4-(Benzyloxy)-1,2,5-oxadiazol-3-yl]acetic Acid (4a). NaOH
(6 M, 13 mL) was added over 20 min to a solution of 4 (17.0 g,
64.82 mmol) in ethanol (96%, 230 mL). The resulting suspen-
sion was concentrated under reduced pressure, and the residue
was dissolved in water (120 mL). The solution was acidified
with 6 M HCl and then extracted with dichloromethane (2 ꢀ
150 mL). The organic layers were collected, dried, and concen-
trated under reduced pressure to obtain pure 4a as a white solid
(mp 101-102 °C from diisopropyl ether/hexane, 7:3 v/v). Yield
Experimental Section
€
Chemistry. Melting points were determined on a Buchi 530
apparatus or an SRS Optimelt apparatus and are uncorrected.
The compounds were routinely checked by mass spectrometry
1
(Finnigan-MatTSQ-700 spectrometer, 70 eV, direct inlet). H
NMR spectra and proton decoupled 13C NMR spectra were
recorded on a Bruker AC-300 spectrometer. The following
abbreviations are used to indicate peak multiplicity: s = singlet;
d = doublet; dd = doublet of doublet; t = triplet; q = quartet;
m = multiplet. Flash column chromatography was performed
on silica gel (Merck Kieselgel 60, 230-400 mesh ASTM) using
the indicated eluents. Thin layer chromatography (TLC) was
carried out on 5 cm ꢀ 20 cm plates with a layer thickness of
0.25 mm. When necessary, they were developed using UV light,
iodine, KMnO4, and ninhydrin. Anhydrous magnesium sulfate
was used as drying agent of the organic extracts. Elemental
analyses of new compounds were within (0.4% of the theore-
tical values unless otherwise stated. The moisture sensitive
reactions were performed under dry N2 or Ar. Tetrahydrofuran
(THF) was distilled immediately before use from sodium and
benzophenone under nitrogen. Compounds 324 and 1611 and
diethyl [(tert-butoxycarbonyl)amino]malonate25 were synthe-
sized according to the literature methods. The chemicals NaH
(60% w/w), EtONO (15% v/v in ethanol), and Oxone were
purchased from Aldrich. The ionization constants of com-
pounds were determined by potentiometric titration with the
GlpKa apparatus (Sirius Analytical Instruments Ltd., Forest
Row, East Sussex, U.K.). At least three separate 20 mL aqueous
solutions of the compounds (about 1 mM) were initially acid-
ified to pH 1.8 with HCl (0.5 N). The solutions were then titrated
with standardized 0.5 N KOH to pH 12.2. The titrations were
performed under nitrogen at 25.0 ( 0.1 °C. Optical rotations of
the isolated enantiomers were measured on a Perkin-Elmer 241
polamimeter in themostated cuvettes. Circular dichroism (CD)
spectra were recorded at 20 °C on an OLIS instrument in 0.1 M
HCl in 1.0 cm cuvettes.
1
88%. H NMR (CDCl3) δ 3.81 (s, 2H, -CH2COOH), 5.36 (s,
2H, PhCH2O-), 7.36-7.43 (m, 5H, arom), 11.34 (broad signal,
1H, -COOH). 13C NMR (CDCl3) δ 28.0, 74.2, 128.4, 128.7,
129.0, 134.4, 140.9, 164.1, 175.5. MS (CI) m/z 235 (M þ 1)þ,
Anal. (C11H10N2O4) C, H, N.
4-(Benzyloxy)-1,2,5-oxadiazole-3-carboxylic Acid (5). KMnO4
(12.28 g, 77.72 mmol) was added portionwise over 15 min to an ice
cooled solution of 4a (9.1 g, 38.96 mmol) in acetone (90 mL). The
reaction mixture was stirred at room temperature for 40 min and
then poured into 2 M HCl (90 mL). The resulting mixture was
filtered, and the solid residue was extensively washed with dichlor-
omethane. The aqueous layer was extracted with dichloromethane
(2 ꢀ 80 mL), and the organic layers were collected, dried, and
concentrated under reduced pressure to give 5 as a white solid (mp
106-107 °C from hexane/diisopropyl ether). Yield 71%. 1H NMR
(CDCl3) δ 5.45 (s, 2H, PhCH2O-), 7.3-7.5 (m, 5H, arom), 10.91
(broad signal, 1H, -COOH). 13C NMR (CDCl3) δ 74.8, 128.7,
128.8, 129.1, 134.0, 139.1, 161.6, 163.9. MS (CI) m/z 233 (M þ 1)þ.
Anal. (C10H8N2O4) C, H, N.
Methyl 4-(Benzyloxy)-1,2,5-oxadiazole-3-carboxylate (6). A
solution of 5 (7.10 g, 32.25 mmol) in methanol saturated with
HCl (160 mL) was stirred at room temperature over 20 h and then
concentrated under reduced pressure. The residue was treated
with a saturated solution of NaHCO3 (60 mL), and the resulting
mixture was extracted with diethyl ether (4 ꢀ 50 mL). The organic
layers were collected, dried, and concentrated under reduced
pressure to give 6 as a white solid (mp 45-46 °C from hexane).
Yield 73%. 1H NMR (CDCl3) 3.91 (s, 3H, -COOCH3), 5.35 (s,
2H, PhCH2O-), 7.29-7.43 (m, 5H, arom). 13C NMR (CDCl3) δ
53.1, 74.4, 128.3, 128.6, 128.9, 134.0, 139.2, 157.4, 163.7. MS (CI)
m/z 235 (M þ 1)þ. Anal. (C11H10N2O4) C, H, N.
Liquid Chromatography. Semipreparative chiral HPLC of 10,
15, and 18 was performed on a Crownpak CR(þ) column (150
mm ꢀ 10 mm, 5 μm) equipped with a Crownpak CR guard
column (10 mm ꢀ 4.0 mm, 5 μm) (Daicel Chemical Industries,
Ltd., Japan) connected to a Jasco 880 pump, a Rheodyne 7125
injector, a 5 mL loop, a TSP UV100 detector (230 nm), and a
Hitachi D-2000 Chromato-Integrator (Merck). The flow rate
was 0.7 mL/min for 10 and 1.5 mL/min for 15 and 18. The
column was kept at 0 °C using an ice bath for compound 10, and
elution was at room temperature for 15 and 18. The mobile
phase was aqueous AcOH (15 mM).
Analytical chiral HPLC, used for the determination of en-
antiomeric excess (ee) of the enantiomers of 10, 15, and 18,
was performed using a Crownpak CR(-) column (150 mm ꢀ 4.0
mm, 5 μm) (Daicel Chemical Industries, Ltd., Japan). The
column was eluted with aqueous AcOH (15 mM), and the
flow-rate was 0.4 mL/min. The column was thermostated at
1 °C for (þ)- and (-)-10 and at 10 °C for (þ)- and (-)-15 and
(þ)- and (-)-18 using a Hetofrig thermostat. A TSP HPLC
system consisting of a P2000 pump, an AC 3000 autosampler,
and an SM 5000 PDA detector was connected to the column.
Ethyl [4-(Benzyloxy)-1,2,5-oxadiazol-3-yl]acetate (4). A solu-
tion of 3 (59.4 g, 412.3 mmol) in ethanol saturated with gaseous
HCl (180 mL) was stirred at room temperature overnight. The
mixture was concentrated under reduced pressure and the
resulting crude material was purified by flash chromatography
(eluent, CH2Cl2/EtOH (96% v/v), 9:1 v/v) to give the ethyl ester
of 3 that was directly used in the next step without any further
purification. K2CO3 (70 g, 507.0 mmol) and benzyl bromide
[4-(Benzyloxy)-1,2,5-oxadiazol-3-yl]methanol (7). NaBH4
(2.59 g, 68.32 mmol) was added portionwise over 15 min to a
solution of 6 (4.00 g, 17.08 mmol) in dry ethanol (100 mL), and the
mixture was stirred under inert atmosphere at room temperature.
After 1 h, the reaction mixture was poured into iced water
(300 mL). The resulting mixture was extracted with diethyl ether
(2 ꢀ 100 mL). The organic layers were collected, dried, and
concentrated under reduced pressure. The residue was purified
by flash chromatography (eluent, dichloromethane) to afford 7 as
a colorless oil. Yield 73%. 1H NMR (CDCl3) δ 2.19 (broad t, J =
6.5 Hz, 1H, -CH2OH), 4.77 (d, J = 6.4 Hz, 2H, -CH2OH), 5.37
(s, 2H, PhCH2O-), 7.32-7.70 (m, 5H, arom). 13C NMR (CDCl3)
δ 53.7, 74.2, 128.7, 128.8, 129.1, 134.4, 146.4, 163.6. MS (CI) m/z
207 (M þ 1)þ. Anal. (C10H10N2O3) C, H, N.
3-(Benzyloxy)-4-(bromomethyl)-1,2,5-oxadiazole (8). NBS (1.03 g,
5.82 mmol) was added portionwise over 35 min under inert atmo-
sphere to a cooled (-10 °C) solution of 7 (1.00 g, 4.85 mmol) and
triphenylphosphine (1.52 g, 5.82 mmol) in dry dichloromethane

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4115
(20 mL). The reaction mixture was directly loaded onto a flash
chromatography column (eluent, dichloromethane) to give 8 as a
white solid (mp 37-38 °C triturated from diisopropyl ether). Yield
(C5H7N3O4 0.2 H2O) C, H, N. The collected fractions of the
3
secondeluting peakwerepooled, evaporated, re-evaporatedthree
times from water, and dried in vacuo to give (þ)-10 (mp 176-
177 °C dec). Yield (117 mg, 95%). 1H NMR spectrum of (þ)-10
was identical to that of (-)-10. ee = 98.9%. [R]2D5 þ18° (c 0.43, 0.1
1
88%. H NMR (CDCl3) δ 4.41 (s, 2H, -CH2Br), 5.40 (s, 2H,
PhCH2O-), 7.40-7.49 (m, 5H, arom). 13C NMR (CDCl3) δ 15.1,
74.3, 128.5, 128.7, 129.1, 134.4, 144.4, 163.3. MS (CI) m/z 269/271
(M þ 1)þ. Anal. (C10H9BrN2O2) C, H, N.
M HCl). Δε (210 nm) = þ0.36 m2/mol. Anal. (C5H7N3O4
3
0.2H2O) C, H. N: calcd, 23.78; found, 23.19.
Diethyl [(tert-Butoxycarbonyl)amino][(4-(benzyloxy)-1,2,5-
oxadiazol-3-yl)methyl]malonate (9). NaH (60% w/w, 535 mg,
13.38 mmol) was added portionwise to an ice cooled solution of
diethyl [(tert-butoxycarbonyl)amino]malonate (3.68 g, 13.38
mmol) in dry THF (45 mL) kept under inert atmosphere. The
reaction mixture was stirred at room temperature for 1 h, and
then a solution of 8 (3.00 g, 11.15 mmol) in dry THF (13 mL) was
added over 15 min. The resulting mixture was stirred at room
temperature for 18 h and then poured into a saturated solution
of NH4Cl (150 mL). The resulting mixture was extracted with
diethyl ether (100 þ 50 mL). The organic layers were collected,
dried, and concentrated under reduced pressure to afford a solid
white material. The crude material was triturated with diisopro-
pyl ether to afford pure 9 as a white solid (mp 111-112 °C).
Yield 94%. 1H NMR (CDCl3) δ 1.20 (t, J = 7.1 Hz, 6H,
-COOCH2CH3), 1.38 (s, 9H, -NHCOOCH(CH3)3), 3.76 (s,
2H, furazanCH2-), 4.06-4.30 (m, 4H, -COOCH2CH3), 5.31
(s, 2H, PhCH2O-), 5.88 (s, 1H, -NHCOOC(CH3)3), 7.36-7.41
(m, 4H, arom). 13C NMR (CDCl3) δ 13.8, 26.3, 28.1, 63.3, 65.2,
74.0, 80.7, 128.4, 128.7, 129.1, 134.6, 142.9, 154.0, 164.6, 166.9.
MS (CI) m/z 464 (M þ 1)þ. Anal. (C22H29N3O8) C, H, N.
Diethyl [(tert-Butoxycarbonyl)amino][(4-hydroxy-1,2,5-oxa-
diazol-3-yl)methyl]malonate (9a). Pd/C (10% w/w, 750 mg)
was added to a solution of 9 (4.63 g, 9.99 mmol) in dry THF
(90 mL). The resulting mixture was vigorously stirred at room
temperature under hydrogen for 1 h. The resulting mixture was
filtered on Celite, and the filtrate was concentrated under
reduced pressure. The crude material was triturated with diiso-
propyl ether to afford pure 9a as a white solid (mp 145-146 °C
from diisopropyl ether). Yield 59%. 1H NMR (DMSO) δ 1.33
(t, J = 7.8 Hz, 6H, -COOCH2CH3), 1.50 (broad s, 9H,
-NHCOOCH(CH3)3), 3.66 (s, 2H, furazanCH2-), 4.2-4.5
(m, 4H, -COOCH2CH3), 6.79 (s, 1H, -NHCOOC(CH3)3),
12.68 (s, 1H, furazanOH). 13C NMR (CDCl3) δ 13.9, 26.74,
28.0, 63.3, 66.5, 83.9, 142.8, 158.1, 163.6, 166.4. MS (CI) m/z 374
(M þ 1)þ. Anal. (C15H23N3O8) C, H, N.
Ethyl 2-[4-(Benzyloxy)-1,2,5-oxadiazol-3-yl](hydroxyimino)-
acetate (11). A solution of 4 (5.00 g, 19.03 mmol) in dry ethanol
(60 mL) was added over 5 min to a cooled (0 °C) solution of NaH
(60% w/w, 913 mg, 22.8 mmol) in dry ethanol (75 mL), kept
under inert atmosphere. The mixture was stirred at 0 °C for
30 min, and then an ethanolic solution of EtONO (15% w/w,
24 mL, 38.2 mmol) was added dropwise. The reaction mixture
was allowed to reach room temperature and then stirred for 20 h.
After addition of glacial acetic acid (1.3 mL), the mixture was
poured into water (250 mL). The resulting mixture was extracted
with diethyl ether (3 ꢀ 100 mL). The organic layers were
collected, dried, and concentrated under reduced pressure. The
resulting crude material was purified by flash chromatography
(eluent, petroleum ether (40-60 °C)/EtOAc, 8:2 v/v) to afford 11
as a white solid as a pure mixture of the two geometrical isomers
in variable ratio. Yield 85%. 1H NMR (most abundant isomer,
CDCl3) δ 1.28 (t, J = 7.1 Hz, 3H, -OCH2CH3), 4.36 (q, J = 7.2
Hz, 2H, -OCH2CH3), 5.39 (s, 2H, PhCH2O-), 7.37-7.41 (m,
5H, arom), 10.24 (s, 1H, dNOH). 1H NMR (less abundant
isomer, CDCl3) δ 1.36 (t, J = 7.1 Hz, 3H, -OCH2CH3), 4.44 (q,
J = 7.1 Hz, 2H, -OCH2CH3), 5.36 (s, 2H, PhCH2O-),
7.27-7.31 (m, 5H, arom), 10.90 (broad signal, 1H, dNOH).
MS (CI) m/z 292 (M þ 1)þ. Anal. (C13H13N3O5) C, H, N.
Ethyl (RS)-2-Amino-(4-hydroxy-1,2,5-oxadiazol-3-yl)acetate
Hydrochloride (11a). Dry ethanol saturated with gaseous HCl
(15 mL) and Pd/C (10% w/w, 400 mg) were added to a solution
of 11 (1.00 g, 3.43 mmol) in dry ethanol (35 mL). The resulting
mixture was kept in a sealed Paar reactor under 20 atm of
hydrogen for 4 h. The mixture was filtered on Celite and the
filtrate was concentrated under reduced pressure, obtaining a
solid crude material. The crude was triturated with diisopropyl
ether to afford pure 11a as a white solid (mp 162 °C dec). Yield
90%. 1H NMR (DMSO) δ 1.19 (t, J = 7.1 Hz 3H, -OCH2CH3),
4.25 (q, J = 7.1 Hz 2H, -CH2CH3), 5.76 (s, 1H, -CHCOO),
10.34 (broad signal). 13C NMR (DMSO) δ 13.6, 45.1, 62.9,
142.4, 162.3, 164.9. MS (CI) m/z 188 (M þ 1)þ. Anal.
(C6H9N3O4 HCl 0.1H2O) C, H, N.
(RS)-2-Amino-3-(4-hydroxy-1,2,5-oxadiazol-3-yl)propionic
Acid (10). A suspension of 9a (2.00 g, 5.36 mmol) in hydro-
bromic acid (48% w/w, 50 mL) was refluxed for 24 h and then
concentrated under reduced pressure. The crude material was
triturated with dry EtOAc and dissolved in water (26 mL, 5% w/w).
The solution was percolated on IRA400 (conditioned with
1 M AcOH) using water as eluent until neutrality and then with
AcOH (from 1 to 3 M). The ninhydrin positive fractions were
collected and concentrated under reduced pressure. The resulting
crude material was finally purified by trituration with water to
afford pure 10 as a white solid (mp 205-206 °C dec from water).
Yield 53%. 1H NMR (D2O) δ 3.14-3.28 (m, 2H, -CH2CH-),
4.10 (dd, J = 5.7 Hz, J = 6.8 Hz, 1H, -CH2CH-). 13C NMR
(D2O) δ 24.1, 52.4, 145.5, 165.1, 172.6. MS (CI) m/z 174 (M þ 1)þ.
3
3
(RS)-2-Amino-(4-hydroxy-1,2,5-oxadiazol-3-yl)acetic Acid
(12). A solution of 11a (760 mg, 2.01 mmol) in 6 M HCl (30
mL) was stirred at 70 °C for 4 h. The resulting mixture was
concentrated under reduced pressure. The crude material
was purificated by trituration with water to afford pure 12 as
a white solid (mp 156-158 °C dec from water). Yield 86%. 1H
NMR (D2O) δ 5.11 (s, 1H, -CHCOOH). 13C NMR (D2O/
NaOD) δ 50.5, 151.8, 168.6, 177.7. Anal. (C4H5N3O4 H2O)
3
C, H, N.
2-[4-(Benzyloxy)-1,2,5-oxadiazol-3-yl]ethanol (13). LiAlH4
(2.60 g, 68.49 mmol) was added over 150 min to a cooled (-50 °C)
solution of 4 (11.0 g, 41.94 mmol) in dry THF (65 mL) kept under
inert atmosphere. The reaction mixture was stirred at -50 °C for
14 h, then allowed to reach room temperature and poured into ice
cooled 2 M HCl (160 mL). The resulting mixture was extracted
with diethyl ether (2 ꢀ 100 mL). The organic layers were
collected, dried, and concentrated under reduced pressure to
afford a crude solid material which was purified by flash chro-
matography (eluent, petroleum ether (40-60 °C)/EtOAc, 8:2 v/
v), obtaining 13 as a white solid (mp 47-48 °C triturated from
diisopropyl ether). Yield 53%. 1H NMR (CDCl3) δ 2.17 (s, 1H,
-CH2CH2OH), 2.90 (t, J = 6.1 Hz 2H, -CH2CH2OH), 3.97 (t,
J = 6.1 Hz, 2H, -CH2CH2OH), 5.32 (s, 2H, PhCH2O-),
7.35-7.46 (m, 5H, arom). 13C NMR (CDCl3) δ 26.1, 59.3,
74.0, 128.5, 128.7, 129.0, 134.6, 145.3, 164.2. MS (CI) m/z 221
Anal. (C5H7N3O4 0.1H2O) C, H, N.
3
(-)-2-Amino-3-(4-hydroxy-1,2,5-oxadiazol-3-yl)propionic Acid
[(-)-10] and (þ)-2-Amino-3-(4-hydroxy-1,2,5-oxadiazol-3-yl)pro-
pionic Acid [(þ)-10]. Compound 10 (250 mg, 1.43 mmol) was
dissolved in aqueous AcOH (15 mM) (100 mL), filtered through
an MV filter (0.45 μm, Waters), and resolved in 5 mg injections.
The collected fractions of the first eluting peak were pooled,
evaporated, re-evaporated three times from water, and dried in
vacuo to give (-)-10 (mp 175-176 °C dec). Yield (119 mg, 95%).
1H NMR (D2O) δ 3.30 (dd, J = 6.9 Hz, J = 5.4 Hz, 1H,
-CHH-), δ 3.31 (dd, J = 6.9 Hz, J = 5.4 Hz, 1H, -CHH-), δ
4.18 (dd, J = 7.2 Hz, J = 6.9 Hz, 1H, -CH-). ee = 99.6%. [R]D25
-16° (c 0.42, 0.1 M HCl). Δε (210 nm) = -0.36 m2/mol. Anal.
(M þ 1)þ. Anal. (C11H12N2O3 0.1H2O) C, H, N.
3

4116 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Lolli et al.
Diethyl [(tert-Butoxycarbonyl)amino][2-(4-(benzyloxy)-1,2,5-
oxadiazol-3-yl)ethyl]malonate (14). Triflic anhydride (1.95 mL,
11.8 mmol, 1.3 equiv) was added over 1 h to a solution of 13
(2,00 g, 9.08 mmol) and 2,6-lutidine (1.27 mL, 10.90 mmol) in
dry dichloromethane (50 mL), and the mixture was stirred under
inert atmosphere at -45 °C. The reaction mixture was allowed
to reach room temperature and then stirred for 1 h. The reaction
mixture was directly loaded onto a flash chromatography
column (eluent, dichloromethane) to give the moisture sensitive
trifluoromethylsulfonyl derivative of 13 as a colorless oil in
acceptable purity to be used immediately in the next step. NaH
(60% w/w, 400 mg, 9.99 mmol) was added portionwise to an ice
cooled solution of diethyl [(tert-butoxycarbonyl)amino]-
malonate (2.75 g, 9.99 mmol) in dry THF (40 mL) kept under
inert atmosphere. The mixture was stirred at room temperature
for 1 h, and then a solution in dry dichloromethane (50 mL) of
the triflate, obtained in the previous step, was slowly added. The
mixture was stirred for 15 h and then poured into a saturated
solution of NH4Cl (100 mL). The aqueous layer was extracted
with dichloromethane (100 mL). The organic layers were col-
lected, dried, and concentrated under reduced pressure. The
crude material was purified by flash chromatography (eluent,
petroleum ether (40-60 °C)/EtOAc, 9:1 v/v) to afford 14 as a
white solid (mp 62 °C from hexane/diisopropyl ether, 5:1 v/v).
Yield 73%. 1H NMR (CDCl3) δ 1.21 (t, J = 7.1 Hz, 6H,
-(COOCH2CH3)2), 1.40 (s, 9H, -NHCOOCH(CH3)3), 2.58-
2.60/2.70-2.73 (m, 4H, furazanCH2CH2-/m, 4H, furazan-
CH2CH2-), 4.13-4.24 (m, 4H, -COOCH2CH3), 5.33 (s, 2H,
PhCH2O-), 5.96 (broad signal, 1H, -NHCOOC(CH3)3),
7.37-7.47 (m, 5H, arom). 13C NMR (CDCl3) δ 13.9, 17.2,
28.1, 29.9, 62.7, 65.9, 73.9, 80.5, 128.5, 128.7, 128.9, 134.7,
146.3, 153.9, 164.0, 167.7. MS (CI) m/z 478 (M þ 1)þ. Anal.
(C23H31N3O8) C, H, N.
Diethyl [(tert-Butoxycarbonyl)amino][2-(4-hydroxy-1,2,5-ox-
adiazol-3-yl)ethyl]malonate (14a). Pd/C (10% w/w, 300 mg)
was added to a solution of 14 (2.12 g, 4.44 mmol) in dry ethanol
(40 mL). The resulting suspension was vigorously stirred at
room temperature under hydrogen for 1 h. The resulting
mixture was filtered on Celite, and the filtrate was concentrated
under reduced pressure. The crude material was purified by flash
chromatography (eluent, petroleum ether (40-60 °C)/EtOAc
from 8:2 v/v to 1:1 v/v) to give 14 as a white solid (mp 125 °C,
from diisopropyl ether). Yield 63%, 1H NMR (CDCl3) δ 1.25 (t,
J = 7.2 Hz, 6H, -COOCH2CH3), 1.44 (s, 9H, -NHCOOCH-
(CH3)3), 2.69-2.77 (m, 2H, furazanCH2CH2-), 4.15-4.34 (m,
4H, -COOCH2CH3), 6.04 (s, 1H, -NHCOOC(CH3)3), 10.3
(broad signal, 1H, furazanOH). 13C NMR (CDCl3) δ 13.9, 17.1,
28.1, 30.0, 62.9, 65.9, 80.9, 147.0, 154.2, 162.4, 167.7. MS (CI)
m/z 388 (M þ 1)þ. Anal. (C16H25N3O8) C, H, N.
evaporated, re-evaporated three times from water, and dried in
vacuo to give (-)-15 (mp 176-177 °C dec). Yield (122 mg, 98%).
1H NMR (D2O) δ 2.25-2.34 (m, 2H, -CH2CH-), 2.81-2.88
(m, 2H, furazan-CH2CH2-), 3.86 (t, J = 6.3 Hz, 1H, -CH-).
ee = 99.9%. [R]2D5 -32° (c 0.43, 0.1 M HCl). Δε (210 nm) =
-0.46 m2/mol. Anal. (C6H9N3O4 0.85H2O) C, H, N. The col-
3
lected fractions of the second eluting peak were pooled, evapo-
rated, re-evaporated three times fromwater, anddriedin vacuo to
give(þ)-15(mp 175-176 °C dec). Yield (121 mg, 97%). 1H NMR
spectrum of (þ)-15 was virtually identical with that of (-)-15.
ee = 99.5%. [R]2D5 þ35° (c 0.46, 0.1 M HCl). Δε (210 nm) =
þ0.42 m2/mol. Anal. (C6H9N3O4 0.85H2O) C, H, N.
3
Diethyl [(tert-Butoxycarbonyl)amino]{3-[4-(phenylsulfonyl)-
1,2,5-oxadiazol-3-yl)]propyl}malonate (17). Triflic anhydride
(2.22 mL, 13.42 mmol) was added to a solution of 16 (3.00 g,
11.18 mmol) and 2,6-lutidine (1.56 mL, 13.42 mmol) in dry
dichloromethane (60 mL) cooled (-45 °C) under inert atmo-
sphere. The reaction mixture was allowed to reach room tem-
perature and then stirred for 1 h. The reaction mixture was then
loaded onto a flash chromatography column (dichloro-
methane), obtaining the moisture sensitive trifluoromethansul-
fonate of 16 as colorless oil in acceptable purity to be used
immediately in the next step. NaH (60% w/w, 492 mg) was
added portionwise to a solution of diethyl [(tert-butoxycarbo-
nyl)amino]malonate (3.39 g, 12.30 mmol) in dry THF (24 mL),
kept under inert atmosphere. The mixture was stirred at room
temperature for 1 h. Then a solution of triflate, obtained in the
previous step, in dry dichloromethane (60 mL) was added over
1.5 h. The reaction mixture was stirred at room temperature and
then poured into a saturated solution of NH4Cl (100 mL). The
resulting mixture was extracted with dichloromethane (100 þ 20
mL), and the collected organic layers were dried and concen-
trated under reduced pressure. The residue was purified by flash
chromatography (eluent, petroleum ether (40-60 °C)/diisopro-
pyl ether, 1:1 v/v) to give a crude that by trituration with EtOH
(96% w/w) afforded pure 17 as a white solid (mp 78 °C from
hexane/diisopropyl ether, 5:1 v/v). Yield 43%. 1H NMR (CDCl3)
δ 1.26 (t, J = 7.1 Hz 6H, -CH2CH3), 1.44 (s, 9H, -C(CH3)3),
1.68-1.79 (m, 2H, furazanCH2CH2CH2), 2.42-2.47 (m, 2H,
furazanCH2CH2CH2), 3.02 (t, J = 7.5 Hz, 2H, furazanCH2CH2-
CH2), 4.17-4.34 (m, 4H, -CH2CH3), 5.98 (s, 1H, -NHCOOC-
(CH3)3), 7.63-8.08 (m, 5H, arom). 13C NMR (CDCl3) δ 14.0,
21.6, 23.3, 28.2, 32.1, 62.6, 66.2, 80.4, 128.8, 129.8, 135.4, 138.1,
152.5, 153.9, 156.6, 168.0. MS (CI) m/z 526 (M þ 1)þ. Anal.
(C23H31N3O9S) C, H, N.
(RS)-2-Amino-5-(4-hydroxy-1,2,5-oxadiazol-3-yl)pentanoic Acid
(18). NaOH (5 M, 8 mL) was added to a solution of 17 (1.046 g,
1.99 mmol) in DMSO (8 mL). The reaction mixture was heated at
70 °C for 2 h, diluted with water (10 mL), and neutralized with 6 M
HCl until pH 7.5 was obtained. Oxone (2.43 g, 3.95 mmol) was
added to the mixture, maintaining the pH near 1.7 by adding 2 M
NaOH. The resulting mixture was filtered, and the filtrate was
extracted with diethyl ether (20 mL ꢀ 13). The organic layers were
collected, dried, and concentrated under reduced pressure. The
crude material was dissolved in hydrobromic acid (48% w/w, 15
mL), and the resulting solution was refluxed for 72 h. The mixture
was concentrated under reduced pressure, and the resulting residue
was dissolved in water. After repeating this step three times, the
resulting crude material was triturated with dry EtOAc. The
obtained crude solid was dissolved in water (7 mL) and the
resulting solution was percolated on IRA400 (conditioned with 1
M AcOH) using water as eluent until neutrality was obtained and
then aqueous AcOH (from 1 to 3 M). Ninhydrin positive fractions
were collected and concentrated under reduced pressure. Tritura-
tion with water of the resulting crude material afforded pure 18 as a
(RS)-2-Amino-4-(4-hydroxy-1,2,5-oxadiazol-3-yl)butanoic Acid
(15). A suspension of 14a (1.28, 3.29 mmol) in hydrobromic acid
(48% w/w, 30 mL) was refluxed for 24 h and then concentrated
under reduced pressure. The resulting crude solid material was
triturated with dry EtOAc and then dissolved in water (26 mL, 5%
w/w). The solution was percolated on IRA400 (conditioned
with 1 M AcOH) using water as eluent until neutrality and then
aqueous AcOH (from 1 to 3 M). Ninhydrin positive fractions were
collected and concentrated under pressure. The resulting crude
material was triturated with water to afford 15 as a white solid (mp
202 °C dec from water). Yield 70%. 1H NMR (D2O) δ 1.98-2.30
(m, 2H, -CH2CH-), 2.52-2.82 (m, 2H, -CH2CH2-), 3.78 (t,
J = 6.3 Hz, 1H, -CH2CH-). 13C NMR (D2O) δ 18.2, 27.3, 53.5,
147.8, 163.5, 173.4. MS (CI) m/z 188 (M þ 1)þ. Anal. (C6H9N3O4)
C, H, N.
(-)-2-Amino-4-(4-hydroxy-1,2,5-oxadiazol-3-yl)butanoic Acid
[(-)-15] and (þ)-2-Amino-4-(4-hydroxy-1,2,5-oxadiazol-3-yl)but-
anoic Acid [(þ)-15]. A solution of 15 (250 mg, 1.34 mmol) in
aqueous AcOH (15 mM) (100 mL) was filtered through an MV
filter (0.45 μm, Waters) and resolved in 15 mg injections.
The collected fractions of the first eluting peak were pooled,
1
white solid (mp 200-202 °C dec, from water). Yield 47%. H
NMR (D2O) δ 1.63-1.90 (m, 4H, -CH2CH2CH-), 2.65 (t, J =
6.0 Hz, 2H, furazanCH2-), 3.71 (t, J = 6.0 Hz, 1H, -CH2CH-).
13C NMR (D2O) δ 22.3, 23.0, 34.6, 56.1, 151.9, 169.3, 183.8, MS
(CI) m/z 188 (M þ 1)þ. Anal. (C7H11N3O4) C, H, N.

Article
(-)-2-Amino-5-(4-hydroxy-1,2,5-oxadiazol-3-yl)pentanoic Acid
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4117
prepared using column purification (Qiagen, Chatsworth, CA).
cRNA was synthesized from these cDNAs using the mMessage
mMachine T7 mRNA-capping transcription kit (Ambion Inc.,
Austin, TX) and purified using RNeasy spin columns (Qiagen).
Restriction and other molecular biological enzymes were
obtained from New England BioLabs (Beverly, MA).
[(-)-18] and (þ)-2-Amino-5-(4-hydroxy-1,2,5-oxadiazol-3-yl)pen-
tanoic Acid [(þ)-18]. Compound 18 (250 mg, 1.24 mmol) was
dissolved in aqueous AcOH (15 mM) (100 mL), filtered through an
MV filter (0.45 μm, Waters), and resolved in 9 mg injections. The
collected fractions of the first eluting peak were pooled, evapo-
rated, re-evaporated three times from water, and dried in vacuo to
give (-)-18 (mp 182-184 °C dec). Yield (117 mg, 94%). 1H NMR
(D2O): δ 1.75-2.00 (m, 4H, -CH2CH2CH-), δ 2.74 (t, J =
7.0 Hz, 2H, furazanCH2-), δ 3.80 (t, J = 5.8 Hz, 1H, -CH--.
ee = 99.9%. [R]2D5 -18° (c 0.45, 0.1 M HCl). Δε (210 nm) =
-0.42 m2/mol. Anal. (C7H11N3O4) C, H, N. The collected frac-
tions of the second eluting peak were pooled, evaporated, re-
evaporated three times from water, and dried in vacuo to give
Cloned iGluR TEVC Electrophysiology. Surgical procedures
were conducted under the approval of the Danish Ministry of
Justice Animal Experiments Inspectorate (2004/561-876). Ma-
ture female Xenopus laevis (Nasco, Fort Atkinson, WI) were
anesthetized using 0.1% ethyl 3-aminobenzoate methanesulfo-
nate (tricaine), and ovaries were surgically removed. The ovar-
ian tissue was dissected and treated with 2 mg/mL collagenase
(type I; Worthington Biochemical Corp., Lakewood, NJ) in
nominally Ca2þ-free Barth’s medium (in mM: 88 NaCl, 1 KCl,
0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 2.4 NaHCO3, 10
HEPES, pH 7.4) for 2 h at room temperature. Oocytes were
kept overnight in Barth’s medium containing 0.10 mg/mL
gentamicin (Sigma) and 1% penicillin-streptomycin (Life
Technologies, Paisley, U.K.) at 16 °C. On the following day,
oocytes were injected with 25-50 nL of cRNA (∼1 mg/mL).
Oocytes were typically used for recordings from 3 to 10 days
postinjection and were voltage-clamped with the use of a two-
electrode voltage clamp (GeneClamp 500B, Axon Instruments,
Union City, CA) with both microelectrodes filled with 3 M KCl.
Recordings were made while the oocytes were continuously
superfused with nominally Ca2þ-free frog Ringer’s solution (in
mM: 115 NaCl, 2 KCl, 1.8 BaCl2, 5 HEPES, pH 7.0). Drugs
were dissolved in Ca2þ-free frog Ringer’s solution and added by
bath application. Recordings were made at room temperature.
Efficacy measurements of (þ)-10 were made at iGluR2(Q)i
at holding potentials from -15 to -20 mV in the presence of
100 μM cyclothiazide in order to block receptor desensitization
(cyclothiazide EC50: iGluR2(Q)i = 5.2 μM). As a control for
current rundown, oocytes were stimulated with 1 mM (S)-Glu
plus 100 μM cyclothiazide immediately prior to application of
300 μM (þ)-10 plus 100 μM cyclothiazide, with a washout
period of 5-10 min between applications. The maximum re-
sponse of (þ)-10 was then expressed as a fraction of the (S)-Glu
stimulation. (S)-Glu is defined as having an efficacy of unity.
Concentration-response curves for agonists were analyzed to
determine the EC50 and Hill value (nH), using the logistic
1
(þ)-18 (mp 179-181 °C dec). Yield (117 mg, 94%). H NMR
spectrum of (þ)-18 was identical with that of (-)-18. ee = 99.1%.
[R]2D5 þ19° (c 0.42, 0.1 M HCl). Δε (210 nm) = þ0.46 m2/mol.
Anal. (C7H11N3O4) C, H, N.
Native iGluR Binding Assays. AMPA, KA, and NMDA
receptor binding assays were performed using rat brain synaptic
membranes from male Sprague-Dawley rats, and tissue pre-
paration was prepared as earlier described.26 Affinities for native
AMPA, KA, and NMDA receptors were determined using 5 nM
[3H]AMPA (45.5 Ci/mmol),27 5 nM[3H]KA (58 Ci/mmol),28 and
2 nM [3H]CGP39653 (50 Ci/mmol, Kd = 6 nM),29 respectively,
with minor modifications as previously described.17 Nonspecific
binding was determined using1 mM (S)-Glu. Data were analyzed
using GraphPadPrism, version4.0 (GraphPadSoftwareInc., San
Diego, CA) and fit to the equation Y = Ymax - (Ymax[inhibitor]n)/
(IC50 þ [inhibitor]n), where Y is the binding as a percentage of total
n
specific binding and n is the Hill coefficient.
Recombinant iGluR Binding Assays. Rat iGluR2(R)o,
iGluR5(Q)1b, iGluR6(V,C,R)A, and iGluR7A were inserted into
recombinant baculoviruses, receptors expressed by infection of
Sf9 insect cells and infected Sf9 cell membranes utilized for
radioligand binding assays. Cells were maintained in Baculo-
Gold Max-XP serum-free medium (BD Biosciences-Pharmin-
gen, San Diego, CA) according to standard protocols in “Guide
to Baculovirus Expression Vector Systems and Insect Cell
Culture Techniques” (Life Technologies, Paisley, U.K.) and
“Baculovirus Expression Vector System: Procedures and Meth-
ods Manual”, second edition (Pharmingen). Drug affinities
were determined by radioligand binding competition assays.
Sf9 insect cell membranes expressing iGluR2(R)o were incu-
bated with 2-5 nM (RS)-[3H]AMPA (43.5-55.5 Ci/mmol;
Perkin-Elmer, Boston, MA) in the presence of 1 nM to
0.1 mM drug for 1-2 h at 4 °C in assay buffer (50 mM Tris-
HCl, 100 mM KSCN, 2.5 mM CaCl2, pH 7.2 at 4 °C). Samples
were filtered through Whatman (Kent, U.K.) GF/B filters and
washed twice with 4 °C assay buffer. Sf9 membranes expressing
iGluR5(Q)1b, iGluR6(V,C,R)A, and iGluR7A were incubated
with [3H]SYM2081 ([3H](2S,4R)-4-methylglutamic acid) (47.9
Ci/mmol; ARC Inc., St. Louis, MO) and 1-1000 μM drug in
assay buffer (50 mM Tris-HCl, pH 7.1 at 4 °C) for 1-2 h at 4 °C.
Samples were filtered through GF/B filters (UniFilter-96,
PerkinElmer, Waltham MA) on a PerkinElmer FilterMate
manifold using two washes with 4 °C assay buffer. Nonspecific
binding was determined in the presence of 1 mM (S)-Glu.
Radioactivity was determined as DPM by liquid scintillation
counting. Competition data were analyzed using Grafit, version
3.00 (Erithacus Software Ltd., Horley, U.K.) and fit to equa-
tions as previously described for the determination of Ki.30
Molecular Biology. The rat AMPA-R clone iGluR2(Q)i in the
vector pGEMHE31 was used for preparation of high-expression
cRNA transcripts for functional expression in oocytes. The
iGluR2(Q)i variant was selected for efficacy measurements,
since the Q-isoform of GluR2 allows for functional homomeric
channels while the flip isoforms have greatest sensitivity to the
desensitization-blocking drug cyclothiazide. cDNAs were
grown in XL1 Blue bacteria (Stratagene, La Jolla, CA) and
(log[agonist]) nH
equation I = Imax/(1 þ 10(log[EC ])/10
) , where I is
the measured current and Imax is the maximal steady-state
current.
50
NMDA Receptor Pharmacology. Concentration-response
data at the NMDA receptor subtype NR1/NR2A were gener-
ated essentially as previously described.23 Briefly, rat NR1
(GenBank accession no. U11418) and rat NR2A (D13211) were
stably expressed in BHK-21 cells. These cells were then used for
intracellular calcium measurements to detect changes in the
concentration of intracellular calcium upon activation of the
NMDA receptors. The measurements were performed using a
fluometric imaging plate reader (FLIPR) (FLIPR384; Molecu-
lar Devices, Sunnyvale, CA) and the fluorescent calcium indi-
cator Fluo-4 (Molecular Probes Invitrogen, Carlsbad, CA) as
previously described.23 For the concentration-response data,
each data point was averaged from four to six wells. These data
points were fitted to the Hill equation: F = Fmin þ ((Fmax
-
F
min)/(1 þ 10(log EC - log[A])nH)). Fmax is the maximal response
50
to the agonist, nH denotes the Hill coefficient, [A] is the agonist
concentration, and EC50 is the agonist concentration that
produces half-maximal response. The log EC50 and nH from
the individual experiments were used to calculate the mean (
standard error of mean (SEM). For graphical presentation, a
representative data set ( standard deviation (SD) from an
individual experiment was selected. The maximal response
evoked by (S)-Glu (Fmax,(S)-Glu) in the same experiment was
normalized to 1 and the minimal response (Fmin) was normalized
to 0. The normalized data points were then fitted to the Hill

4118 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Lolli et al.
equation and plotted together with the resulting curve. Antago-
nist concentration-response data were calculated using a simi-
lar protocol, where instead of calculating the EC50 value, the
concentration required to inhibit a response induced by 6 μM
(S)-Glu by 50% (IC50) was calculated.
agonists at group II metabotropic glutamate receptors: synthesis,
stereochemistry, and molecular pharmacology of (S)- and (R)-2-
amino-4-(4-hydroxy[1,2,5]thiadiazol-3-yl)butyric acid. J. Med.
Chem. 2002, 45, 4240–4245.
(16) Shinbo, T.; Yamagushi, T.; Nishimura, K.; Sugiura, M. Chroma-
tographic separation of racemic amino acids by use of chiral crown
ether-coated reversed-phase packings. J. Chromatogr. 1987, 405,
145–153.
Acknowledgment. This work was supported by MIUR
(Grant COFIN 2003), the Lundbeck Foundation, and the
Villum Kann Rasmussen Foundation
(17) Clausen, R. P.; Hansen, K. B.; Cali, P.; Nielsen, B.; Greenwood,
€
J. R.; Begtrup, M.; Egebjerg, J.; Brauner-Osborne, H. The respec-
tive N-hydroxypyrazole analogues of the classical glutamate
receptor ligands ibotenic acid and (RS)-2-amino-2-(3-hydroxy-
5-methyl-4-isoxazolyl)acetic acid. Eur. J. Pharmacol. 2004, 499,
35–44.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
(18) Vogensen, S. B.; Frydenvang, K.; Greenwood, J. R.; Postorino, G.;
Nielsen, B.; Pickering, D. S.; Ebert, B.; Bolcho, U.; Egebjerg, J.;
Gajhede, M.; Kastrup, J. S.; Johansen, T. N.; Clausen, R. P.;
Krogsgaard-Larsen, P. A tetrazolyl-substituted subtype-selective
AMPA receptor agonist. J. Med. Chem. 2007, 50, 2408–2414.
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