Structure-activity relationship study of selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor 2-amino-4-(4-methoxyphenyl)-7- (naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101) and absolute configurational assignment using infrared and vibrational circular dichroism spectroscopy in combination with ab initio hartree-fock calculations

Article abstract of DOI:10.1021/jm300345z

The excitatory amino acid transporters (EAATs) play essential roles in regulating the synaptic concentration of the neurotransmitter glutamate in the mammalian central nervous system. To date, five subtypes have been identified, named EAAT1-5 in humans, and GLAST, GLT-1, EAAC1, EAAT4, and EAAT5 in rodents, respectively. In this paper, we present the design, synthesis, and pharmacological evaluation of seven 7-N-substituted analogues of UCPH-101/102. Analogue 9 inhibited EAAT1 in the micromolar range (IC₅₀ value 20 μM), whereas analogues 8 and 10 were inactive (IC₅₀ values >100 μM). The diastereomeric pairs 11a/11b and 12a/12b were separated by HPLC and the absolute configuration assigned by VCD technique in combination with ab initio Hartree-Fock calculations. Analogues 11a (RS-isomer) and 12b (RR-isomer) inhibited EAAT1 (IC₅₀ values 5.5 and 3.8 μM, respectively), whereas analogues 11b (SS-isomer) and 12a (SR-isomer) failed to inhibit EAAT1 uptake (IC₅₀ values >300 μM).

Full text of DOI:10.1021/jm300345z

Article
pubs.acs.org/jmc
Structure−Activity Relationship Study of Selective Excitatory Amino
Acid Transporter Subtype 1 (EAAT1) Inhibitor 2-Amino-4-(4-
methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (UCPH-101) and Absolute Configurational
Assignment Using Infrared and Vibrational Circular Dichroism
Spectroscopy in Combination with ab Initio Hartree−Fock
Calculations
Tri H. V. Huynh,† Irene Shim,‡ Henrik Bohr,§ Bjarke Abrahamsen,† Birgitte Nielsen,† Anders A. Jensen,†
and Lennart Bunch*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
‡Department of Chemistry, Technical University of Denmark, Kemitorvet build. 206, 2800 Kgs. Lyngby, Denmark
§Quantum Protein Centre, Department of Physics, Technical University of Denmark, Fysikvej build. 309, 2800 Kgs. Lyngby,
Denmark
S
* Supporting Information
ABSTRACT: The excitatory amino acid transporters (EAATs)
play essential roles in regulating the synaptic concentration of
the neurotransmitter glutamate in the mammalian central
nervous system. To date, five subtypes have been identified,
named EAAT1−5 in humans, and GLAST, GLT-1, EAAC1,
EAAT4, and EAAT5 in rodents, respectively. In this paper, we
present the design, synthesis, and pharmacological evaluation of
seven 7-N-substituted analogues of UCPH-101/102. Analogue 9
inhibited EAAT1 in the micromolar range (IC50 value 20 μM),
whereas analogues 8 and 10 were inactive (IC50 values >100
μM). The diastereomeric pairs 11a/11b and 12a/12b were
separated by HPLC and the absolute configuration assigned by
VCD technique in combination with ab initio Hartree−Fock
calculations. Analogues 11a (RS-isomer) and 12b (RR-isomer) inhibited EAAT1 (IC50 values 5.5 and 3.8 μM, respectively),
whereas analogues 11b (SS-isomer) and 12a (SR-isomer) failed to inhibit EAAT1 uptake (IC50 values >300 μM).
astrocytes, whereas EAAT3,4 are expressed almost exclusively
in neurons.3 Finally, EAAT1−3 are high-capacity Glu trans-
porters, while EAAT4,5 are considered to be low-capacity Glu
transporters, functioning primarily as Glu-gated chloride ion
channels.3 Malfunction of the EAATs has been suggested to be
a contributing factor in neurotoxic states and neurodegenerative
diseases such as Alzheimer’s,4 Huntington’s,5 amyotrophic
lateral sclerosis (ALS),6 cerebral stroke7, and epilepsy,1,8,9 as
well as in psychiatric disorders like depression10 and
schizophrenia.11,12
INTRODUCTION
■
In the central nervous system (CNS), the excitatory amino acid
transporters (EAATs) are responsible for the uptake of the
major excitatory neurotransmitter (S)-glutamate (Glu) from
the synaptic cleft into glial cells and neurons. Thus, the EAATs
are key players in the maintenance of synaptic as well as
extrasynaptic Glu concentrations below levels of neurotoxicity.1
To date, five EAAT subtypes have been identified, termed
EAAT1−5 in humans, whereas they are termed GLAST, GLT-
1, EAAC1, EAAT4, and EAAT5, respectively, in rodents. The
five EAAT subtypes exhibit distinct expression patterns in the
CNS: while EAAT1−3 (GLAST, GLT-1, and EAAC1,
respectively) are expressed throughout the CNS, the EAAT4
subtype is expressed exclusively in Purkinje cells of the
cerebellum and EAAT5 only in the retina.2 At the cellular
level, EAAT1,2 are expressed predominantly in glia cells and
We have recently reported the first class of selective EAAT1
inhibitors and a first structure−activity-relationship (SAR)
study.13,14 The analogues UCPH-101 and UCPH-102 were the
Received: March 13, 2012
© XXXX American Chemical Society
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Figure 1. Chemical structures of selective EAAT1 inhibitors UCPH-101 and UCPH-102 together with key analogues from previously reported SAR
studies (* indicates stereogenic centers).
most potent inhibitors in the series (IC50 values 0.66 and 0.43
μM, respectively) (Figure 1).13,14 Comprising two chiral
centers, all analogues in the series were synthesized and
characterized pharmacologically as a mixture of four stereo-
isomers (Figure 2), however, the inhibitory activity resides in
complete loss of inhibitory activity at EAAT1 (compounds 4−
5, Figure 1). In regard to the stereochemical configuration at
the 4 and 7-positions, an in silico study concluded that the
stereochemical configuration at C7 (R1) has little influence on
the spatial orientation of the substituent, whereas the
substituent at 4-position (R2) adapts distinct spatial orientation
on inverting the stereochemistry (Figure 3). This finding is
Figure 3. Superimposition of low-energy conformations of 1. (a)
Stereochemistry at C4 (R1) is conserved: syn-isomer (green) and anti-
isomer (gray); (b) stereochemistry at C7 (R2) is conserved: syn-isomer
(green) and anti-isomer (gray).
Figure 2. Three component reaction of diketone A, aldehyde B, and
malononitrile for the preparation of the 2-amino-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile parental skeleton C with
various substituents R1 in the 7-positions and R2 in the 4-position.
intriguing, and together with the fact that 4,4-dimethyl
analogue 4 is inactive allowed for the conclusion that the
stereochemical configuration at C4 is essential for inhibitory
activity.13 To elucidate the stereochemical requirements for
inhibitory activity, desymmetrization of the diketone fragment
by introduction of a heteroatom seemed as an attractive
only two of these.13 In this paper, we present the elucidation of
the stereochemical configuration in correlation with inhibitory
activity for this new class of selective EAAT1 inhibitors.
approach. Starting with the enantiopure keto-lactam,15,16
a
RESULTS AND DISCUSSION
■
Design and Synthesis. The synthesis of the UCPH-101/
102 compound class (Figure 1) is in general terms carried out
by a three-component reaction (Figure 2).13 The R1 substituent
in the 7-position of the parental skeleton C originates from
diketone A, whereas the R2 substituent is derived from aldehyde
B.
diastereomeric pairs of the final products 6 or 7 would be
obtained. Following separation by chromatography, the
absolute configuration could be determined by X-ray
crystallography. Unfortunately, lactam 6 and lactone 7 were
both without inhibitory activity at EAAT1.13
To continue the objective, the strategy was modified as to
introduce a nitrogen atom in the 7-position (Figure 4). By this
tactic, the four stereoisomers is reduced to two, although it is
unclear what the consequence of the presence of a basic
nitrogen is for EAAT1 inhibitory activity (Figure 4). The 7-N-
analogue 8 was designed in accordance with 1, comprising a
phenyl group in the 4- and 7-positions. The basicity of the
aniline nitrogen functionality is thus limited but also the free
rotation of the 7-N-phenyl ring. For analogue 9, the more
flexible N-benzyl group was incorporated based on the SAR
The previously reported SAR study of UCPH-101 and
UCPH-102 concluded that the presence of an aromatic ring in
the 7-position (R1) is essential for inhibitory activity and that a
1-naphthyl group (UCPH-101 and UCPH-102) is superior
(Figure 1 and Figure 2).3,13 On the other hand, the 4-position
(R2) was found to be able to accommodate substituents of
various sizes, not being restricted to aromatic moieties
(compounds 1−3, Figure 1). Furthermore, two methyl groups
or no substituent in the 4-position was observed to result in
B
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Figure 4. Chemical structures of newly designed 7-N-analogues.
observation that a 7-benzyl group is allowed (Figure 2,
compound 2). To broaden the SAR study further, analogue
10 was designed comprising an amide functionality in the 7-
position. The two pairs of diastereomers, 11a/11b and 12a/
12b, comprising an enantiomerically pure substituent in the 7-
position (R1), were designed to resemble benzyl analogue 2
(Figure 2). The diastereomeric mixtures 11a/b and 12a/b were
planned to be separated by HPLC and subsequently the
absolute stereochemistry assigned by X-ray crystallography or
IR/VCD investigations.
Scheme 1. Synthetic Pathway towards 7-N-Phenyl Analogue
8
a
The synthesis of the 7-N-analogues was to be carried out by
the three component reaction already described (Figure 1).
Thus the synthesis of aminodiketone D comprising the
appropriate N-substituent was to be pursued first and
subsequently react it with 2-benzylidenemalononitrile (13) to
afford the final product, compounds 8−12b (Figure 5).3,13
a
Reagents and conditions: (a) DIEA, dry acetonitrile, 60 °C, 3 h, 46%;
(b) chloroacetone, NaI, K2CO3, dry THF, 60 °C, 3 days, 31%, (c) t-
BuOK, dry THF, 19 h; (d) 2-benzylidinemalononitrile, piperidine, abs
EtOH/H2O (3:1), 19 h, 51%.
Figure 5. Various diketones D react with 13 to afford 8−12b.
Scheme 2. Reactions and Conditions for the Synthesis of 7-
N-Benzyl Analogue 9
a
The synthesis of target structure 7-N-phenyl 8 commenced
with the synthesis of secondary amine 14 by N-alkylation of
commercially available aniline and ethyl 2-bromoacetate in 46%
yield (Scheme 1).17 Subsequently, N-alkylation of 14 with
chloroacetone, NaI, and K2CO3 in THF afforded tertiary amine
15 in 31% yield.
With tertiary amine 15 in hand, reaction with t-BuOK in
THF afforded diketone 16. Because 16 was found to be
unstable on contact with silica,18 crude 16 was immediately
converted to the target compound 7-N-phenyl 8 upon reaction
with 1313 (Scheme 1).
The synthesis of 7-N-benzyl 9 (Scheme 2) followed the same
strategy as for 8. Diketone 19, also decomposed on silica gel,18
thus crude 19 was converted directly into 7-N-benzyl analogue
9 in 57% yield (Scheme 2).
The synthesis of 7-N-benzoyl analogue 10 was first explored
by N-debenzylation of 9. However, all attempts failed (BzCl,
Pd/C and H2 (g), Pd/C, H2 (g) and TFA, Pd/C, H2 (g) and
concd HCl) at room temperature and atmospheric pressure
were tried.19,20 Either full N-debenzylation was not achieved or
a complex reaction mixture was obtained, including reduction
of the two double bonds (observed by NMR).
Alternatively, N-debenzylation of tertiary amine 18 by
hydrogenolysis gave the hydrochloride salt of secondary
amine 20 in 97% yield (Scheme 3).20 Subsequently, amine
20 was reacted with benzoyl chloride and Et3N as base, in THF,
a
Reagents and conditions: (a) dry THF, rt, 3.5 h, 90%; (b)
chloroacetone, NaHCO3, abs EtOH, 60 °C, 18 h, 68%; (c) t-BuOK,
dry THF, 17.5 h; (d) 2-benzylidinemalononitrile, piperidine, abs
EtOH/H2O (10:1), 24 h, 57%.
C
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material. A diastereomeric mixture of 12a/12b was obtained in
a 1:1 ratio in 58% yield, and separation by HPLC gave
enantiopure 12a and 12b. To assign the stereochemical
configuration at C4 of 11a/11b and 12a/12b, an X-ray
crystallography study seemed attractive but it proved
impossible to obtain crystals of 11a/11b or 12a/12b of
sufficient quality.
Scheme 3. Reagents and Conditions for the Synthesis of 7-N-
Benzoyl Analogue 10
a
Vibrational Circular Dichroism (VCD) and Fast Fourier
Transform Infrared (FTIR) Spectroscopy. In combination
with ab initio Hartree−Fock (HF) calculations, VCD is a
valuable experimental method for the unambiguous assignment
of absolute stereochemical configuration of chiral mole-
cules.22−28 For reasons of compound quantities, it was decided
to undertake 12a and 12b for IR/VCD study. The geometries
of (S)-2-amino-5-oxo-4-phenyl-7-((R)-1-phenylethyl)-5,6,7,8-
tetrahydro-4H-pyrano[2,3-c]pyridine-3-carbonitrile (S1, Figure
6) and (R)-2-amino-5-oxo-4-phenyl-7-((R)-1-phenylethyl)-
a
Reagents and conditions: (a) Pd/C, concd HCl, H2(g), abs EtOH, 2
h, 97%; (b) Et3N, benzoyl chloride, dry THF, 14 h, 88%; (c) t-BuOK,
dry THF, 20 h; (d) 13, piperidine, abs EtOH/H2O (3:1), 26 h, 55%.
to give amide 21 in 88% yield. Cyclization of amide 21 using t-
BuOK in THF afforded the unstable diketone 22, which was
immediately converted to the 7-N-benzoyl analogue 10 by
condensation with 1313 (Scheme 3).
The synthesis of the diastereomeric pairs 11a/11b
commenced with the preparation of amine 23 from alkylation
of (S)-1-phenylethylamine with ethyl 2-bromoacetate.21
A
second alkylation with chloroacetone afforded tertiary amine
24. Basic cyclization of tertiary amine 24 afforded crude
diketone 25, which was condensed with 1313 to give a
diastereomeric mixture of 11a/11b in a 1:1 ratio in overall 41%
yield (Scheme 4). Separation of the diastereomeric mixture by
chiral HPLC afforded enantiopure 11a and 11b. The synthesis
of the diastereomeric pair 12a/12b followed the same strategy
as for 11a/11b but with (R)-1-phenylethylamine as starting
Figure 6. Chemical structures of S1 and S2 as well as optimized
geometries.
Scheme 4. Synthetic Pathway Towards the Diastereomeric
Pairs, 11a and 11b
a
5,6,7,8-tetrahydro-4H-pyrano[2,3-c]pyridine-3-carbonitrile (S2,
Figure 6) were optimized (low energy conformation) and used
for calculation of IR (see Supporting Information) and VCD
spectra (Figure 7).
The calculated IR spectra of S1 and S2 are for all importance
similar (see Supporting Information). The absorption in the
2850−3000 cm−1 range is due to sp3 C−H stretching, whereas
absorption over 3000 cm−1 is from sp3 N−H, sp2 C−H, and sp
C−H stretching. The absorption peak at 2550 cm−1 originates
from the triple bond of CN group, whereas the absorption in
the 1450−2000 cm−1 is stretching, bending, and scissoring of
alkanes, alkenes, aromatic rings, and ketones. The complexity of
absorption in the 500−1450 cm−1 region (fingerprint region)
makes it difficult to assign all of the absorption bands for S1
and S2 (see Supporting Information). Knowing the IR
frequencies, the VCD spectra for S1 and S2 could be calculated
(Figure 7).
The spectra show high similarity in the 3000−4000 cm−1
region (see Supporting Information), but a clear divergence
was observed in the 1450−2000 cm−1 region (Figure 7). For
S1, positive VCD bands at 1455 cm−1 (C−H and N−H bend)
and 1814 cm−1 (CO stretch) were observed, whereas
negative VCD bands were observed for S2. Furthermore, at
1552 cm−1 (CC, C−H, and N−H bend) a negative VCD
band for S1 and a positive VCD band for S2 were observed.
a
Reagents and conditions: (a) dry THF, rt, 2.5 h, 92%; (b)
chloroacetone, NaHCO3, abs EtOH, 60 °C, 72 h, 21%; (c) t-BuOK,
dry THF, 2 h; (d) 13, piperidine, abs EtOH/H2O (10:3), 18 h, 41%.
D
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Figure 7. Calculated low-energy conformational VCD of S1 and S2 using Gaussian 09. HF method and 6-31G* basis set in gas phase were used.
VCD range from 800 to 2000 cm−1, and arrows indicate the three major differences in the VCD bands.
Figure 8. Experimentally determined IR spectra of 12a and 12b in DMSO using a FTIR apparatus. (A) IR spectra at rt of 12a (green) and 12b (red)
at 8 cm−1 resolution (20 min) in DMSO (blue) solution (150 mg/mL) using a CaF2 cell and 6 μm spacer. (B) IR spectra of 12a at 30 °C (blue), 40
°C (green) and 50 °C (red).
Experimental IR spectra for 12a/12b were necessary to
determine the possibility of measuring the VCD spectra in the
desired interval (1500−2000 cm−1) and to find the optimum IR
intensity. The measured IR spectra for 12a/12b, using a fast
transform infrared (FTIR) apparatus are depicted in figure 8.
To determine the optimal IR frequency, several solvents were
tried out, of which DMSO gave the best result (Figure 8).
Optimal conditions for the 1500−2000 cm−1 region were
shown to be a sample concentration of 150 mg/mL in DMSO
at 8 cm−1 resolution using a calcium fluoride cell and a 6 μm
spacer (Figure 8A). Next, an IR temperature-dependent
experiment was conducted for 12a at 30−50 °C, which
confirmed that frequencies and absorbance were not critically
influenced (Figure 8B). With these conditions in hand, VCD
spectra for 12a/12b were measured using a FTIR appara-
tus.25,29
frequencies. Frequencies 1299 and 1385 cm−1, in the
fingerprint region, were not detected because the experimental
VCD spectral range was 1500−2000 cm−1. Thus only
frequency 1619 cm−1 was useful in determining the absolute
configurations of 12a and 12b.
First, VCD spectrum of DMSO was measured and subtracted
from the VCD spectra of 12a/12b. However, no differences in
the VCD spectra of 12a and 12b were observed, which was
understandable because no IR signal was detected at 1500−
2000 cm−1 for DMSO. Fortunately, a positive VCD band was
observed for 12a and a negative VCD band was detected for
12b at 1614 cm−1 for two different concentrations (Figure
9A,B). A minor shift to lower frequency (1604 cm−1) was
observed at higher temperatures (Figure 9C,D). This shift in
frequency was also detected in the IR spectra (Figure 8).
By comparison of the calculated VCD spectra with the
experimentally determined, the stereochemistry of 12a and 12b
could now be assigned. S1 and 12a both displayed a positive
VCD band at 1614 and 1619 cm−1, respectively, which
unambiguously assigned the S,R-configuration to 12a. Analogue
12b displayed a negative VCD band at that frequency which
Three major differences were observed from the calculated
VCD spectra for S1 and S2 (Figure 7B). These three
frequencies (1455, 1552, and 1814 cm−1) have to multiply
with 0.8929 to give the experimental frequencies (1299, 1385,
and 1619 cm−1), which thus can compare with the measured
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Figure 9. Experimentally determined VCD spectra for 12a and 12b at different concentrations and temperatures. (A) VCD spectra at rt. for 12a
(blue) and 12b (red) in DMSO (150 mg/mL). (B) VCD spectra of 12a (blue) and 12b (red) in DMSO (300 mg/mL) at rt. (C) VCD spectra of
12a (blue) and 12b (red) in DMSO (150 mg/mL) at 40 °C. (D) VCD spectra of 12a (blue) and 12b (red) in DMSO (150 mg/mL) at 50 °C.
Arrows indicate the major differences in the VCD bands for 12a and 12b.
was in line with S2 and is thus assigned the RR-configuration
(Table 1). The absolute configurations of 11a and 11b were
assigned by comparison of melting points. Given the fact that
enantiomers have the same melting point analogues 11a and
11b were assigned the RS- and SS-configuration, respectively
(Table 1). Furthermore, comparison of HPLC retention times
and NMR data confirmed this assignment.
Table 1. Melting Point and the Absolute Configuration of
Chiral Analogues 11a−12b
Pharmacological Characterization. The seven 7-N-
substituted analogues 8−12b were characterized pharmacolog-
ically at EAAT1−3 in a [3H]-D-Aspartate uptake assay (Table
2).30 7-N-Phenyl analogue 8 displayed no significant inhibitory
activity at EAAT1 at a concentration up to 100 μM. In
comparison with 1, it can be concluded that nitrogen atom
lonepair delocalization induces a disfavored spatial orientation
of the phenyl group.
In comparison with 7-benzyl analogue 2, the 7-N-benzyl
analogue 9 retained inhibitory activity at EAAT1 in the medium
micromolar range (IC50 = 20 μM). This is explainable because
free rotation of the benzyl groups is conserved. Interestingly,
the N-benzoyl analogue 10 failed to inhibit EAAT1 mediated
uptake (Table 2). The enantiopure analogues 11a and 12b
displayed inhibitory activity at EAAT1 (IC50 values 5.5 and 3.8
μM, respectively), while 11b and 12a failed to inhibit EAAT1
uptake (Table 2). This confirmed the hypothesis that only one
configuration at the 4-position was allowed for (Table 2). None
of the synthesized analogues displayed any inhibitory activity at
a
entry
melting point [°C]
R/S isomer
11a
11b
12a
12b
121−123
102−104
121−123
102−104
RS
SS
SR
RR
a
Melting points were measured using a MPA 100 Optimelt automatic
melting point system.
F
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(TLC, Merck silica gel 60 F254 aluminum sheets). Flash chromato-
graphy was carried out using Merck silica gel 60A (35−70 μm). H
Table 2. Pharmacological Characterization of 7-N-
1
Substituted Compounds 8−12b as Inhibitors at EAAT1 in a
and 13C NMR spectra were recorded on a 300 MHz Varian Mercury
300BB in CDCl3 using CHCl3 as internal standard unless otherwise
noted. MS spectra were recorded using LC-MS performed using an
Agilent 1200 solvent delivery system equipped with an autoinjector
coupled to an Agilent 6400 triple quadrupole mass spectrometer
equipped with an electrospray ionization source. Gradients of 5%
aqueous acetonitrile ≤0.05% formic acid (buffer A) and 95% aqueous
acetonitrile +0.043% formic acid (buffer B) were employed. Analytical
HPLC was performed using a Dionex UltiMate 3000 pump and
photodiode array detector (200 and 210 nm, respectively) installed
with an XTerra MS C18 3.5 μm, 4.6 mm × 150 mm column, using a
5→95% MeCN gradient in H2O containing 0.1% TFA. Melting points
were measured using a MPA 100 Optimelt automatic melting point
system. Optical rotation was measured using a Jasco DIP-370 digital
polarimeter, with Na lamp (589 nm). All commercial chemicals were
used without further purification. The purity of all tested compounds
was determined by elementary analysis and HPLC to be >95%.
2-Amino-5-oxo-4,7-diphenyl-5,6,7,8-tetrahydro-4H-pyrano[2,3-
c]pyridine-3-carbonitrile (8). A solution of ethyl 2-((2-oxopropyl)-
(phenyl)amino)acetate (15) (269 mg, 1.14 mmol) in dry THF (4 mL)
was added dropwise over 10 min to a suspension of potassium tert-
butoxide (192 mg, 1.71 mmol) in dry THF (5 mL) at 0 °C under a N2
atmosphere. The reaction mixture was stirred at rt for 15 h and
quenched with satd NaHCO3 (2 mL). After concentration in vacuo,
the crude product was dissolved in EtOH/H2O (8 mL, 3:1) and 13
(176 mg, 1.14 mmol) and piperidine (45 μL, 456 μmol) were added.
The reaction mixture was stirred at rt for 19 h. The reaction mixture
was concentrated with silica gel in vacuo and purified by column
chromatography on silica gel to afford the title compound as a pale-
yellow solid (200 mg, 581 μmol, 51% yield); Rf 0.31 (EtOAc/heptane
1:2). 1H NMR (300 MHz, DMSO-d6) δ: 7.26−7.08 (m, 9H), 6.98 (d,
J = 9.0 Hz, 2H), 6.85 (t, J = 7.6 Hz, 1H), 4.46 (d, J = 17.1 Hz, 1H),
4.27 (s, 1H), 4.15 (d, J = 17.1 Hz, 1H), 4.03 (d, J = 17.4 Hz, 1H), 3.76
(dd, J = 17.1, 1.2 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ: 193.1,
162.7, 159.0, 149.3, 144.7, 130.0, 129.2, 128.1, 127.6, 120.9, 120.3,
116.6, 113.4, 59.1, 56.6, 48.6, 35.6; mp 203−205 °C (decomposed).
LC-MS (m/z) calcd for C21H17N3O2 [M + H+], 344.1; found, 344.1.
2-Amino-7-benzyl-5-oxo-4-phenyl-5,6,7,8-tetrahydro-4H-
pyrano[2,3-c]pyridine-3-carbonitrile (9). A solution of ethyl 2-
(benzyl(2-oxopropyl)amino)acetate (18) (381 mg, 1.5 mmol) in dry
THF (4 mL) was added dropwise over 15 min to a solution of
potassium tert-butoxide (188 mg, 1.68 mmol) in dry THF (5 mL) at 0
°C under a N2 atmosphere. The reaction mixture was stirred at rt for
18 h and quenched with satd NaHCO3 (2 mL). After concentration in
vacuo, the crude product was dissolved in abs EtOH (10 mL) and
H2O (1 mL) and 13 (150 mg, 1.1 mmol) and piperidine (20 μL, 216
μmol) were added. The reaction mixture was stirred at rt for 22 h and
then concentrated with silica gel in vacuo and purified by column
chromatography on silica gel. This afforded the title compound as a
pale-yellow solid (222 mg, 619 μmol, 57% yield); Rf 0.20 (EtOAc/
a
[3H]-D-Aspartate Uptake Assay
a
Data are given as IC50 values in μM with pIC50 SEM in brackets.
None of the synthesized analogues displayed inhibitory activity at
EAAT2 or EAAT3 when applied at the highest possible concentrations
(8, 10: IC50 > 100 μM. 9, 11a, 11b, 12a, 12b: IC50 > 300 μM).
the EAAT2,3 subtypes (8, 10: IC50 > 100 μM. 9, 11a, 11b, 12a,
12b: IC50 > 300 μM).
CONCLUSION
■
In conclusion, seven 7-N-substituted analogues 8−12b of
EAAT1-selective inhibitors UCPH-101/102 were designed and
synthesized. The absolute configuration of enantiopure 11a,
11b, 12a, and 12b was assigned by use of VCD technique in
combination with ab initio HF calculations. In an EAAT1
uptake assay, N-benzyl analogue 9 displayed inhibitory activity
in the midmicromolar range (IC50 = 20 μM), whereas the N-
phenyl analogue 8 and N-benzoyl analogue 10 displayed no
EAAT1 inhibitory activity. Enantiopure 11a and 12b inhibited
EAAT1 uptake in the low micromolar range (IC50 values 5.5
and 3.8 μM, respectively), whereas their respective diaster-
eomer 11b and 12a was inactive. These results allow for the
conclusion that the R-configuration in the 4-position is essential
for EAAT1 inhibitory activity. This insight may advance future
design and synthesis of selective EAAT1 inhibitors.
1
heptane 3:5). H NMR (300 MHz, CDCl3) δ: 7.34−7.18 (m, 10H),
4.53 (s, 2H), 4.42 (s, 1H), 3.65 (d, J = 3.0 Hz 2H), 3.48 (dd, J = 16.8,
0.9 Hz, 1H), 3.32−3.24 (m, 2H), 3.07 (dd, J = 16.2, 2.4 Hz, 1H). 13C
NMR (75 MHz, CDCl3) δ: 192.8, 161.2, 157.1, 142.3, 135.8, 129.1,
128.6, 128.5, 127.8, 127.6, 127.3, 118.3, 113.5, 63.6, 61.1, 60.5, 51.3,
34.9; mp 180−182 °C (decomposed). LC-MS (m/z) calcd for
C22H19N3O2 [M + H+], 358.1; found, 358.1.
2-Amino-7-benzoyl-5-oxo-4-phenyl-5,6,7,8-tetrahydro-4H-
pyrano[2,3-c]pyridine-3-carbonitrile (10). A solution of ethyl 2-(N-
(2-oxopropyl)benzamido)acetate (21) (350 mg, 1.32 mmol) in dry
THF (10 mL) was added dropwise to a suspension of potassium tert-
butoxide (224 mg, 2 mmol) in dry THF (8 mL) at 0 °C under a N2
atmosphere. The reaction mixture was stirred at rt for 20 h and
quenched with satd NH4Cl (3 mL). After concentration in vacuo, the
crude product was dissolved in EtOH/H2O (12 mL, 3:1) and 13 (113
mg, 0.73 mmol) and piperidine (18 μL, 184 μmol) were added. The
reaction mixture was stirred at rt for 26 h, concentrated with silica gel
in vacuo, and purified by column chromatography on silica gel. The
EXPERIMENTAL SECTION
■
Chemistry. All reactions involving dry solvents or sensitive agents
were performed under a nitrogen atmosphere and glassware was dried
prior to use. Solvents were dried according to standard procedures,
and reactions were monitored by analytical thin-layer chromatography
G
dx.doi.org/10.1021/jm300345z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
title compound was obtained as a pale-yellow solid (148 mg, 399
μmol, 55% yield); Rf 0.32 (EtOAc/heptane 5:1). 1H NMR (300 MHz,
CDCl3) δ: 7.43−7.21 (m, 10H), 4.97 (s, 3H), 4.18 (s, 1H), 4.25 (d, J
= 18.3 Hz, 2H), 3.94 (d, J = 17.7 Hz, 1H). 13C NMR (75 MHz,
CDCl3) δ: 189.6, 160.7, 157.3, 141.9, 133.4, 130.9, 130.8, 128.9, 128.8,
128.7, 127.7, 127.6, 127.3, 127.2, 118.3, 114.1, 62.5, 54.7, 41.5, 35.0;
mp 179−181 °C (decomposed). LC-MS (m/z) calcd for C22H17N3O3
[M + H+], 372.1; found, 372.1.
2.1, 16.5 Hz, 1H), 3.20 (dd, J = 1.5, 16.2 Hz, 1H), 3.01 (dd, J = 2.1,
16.5 Hz, 1H), 1.40 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ: 193.0, 161.8, 157.2, 142.3, 141.2, 128.7, 128.6, 127.7, 127.6, 127.4,
127.3, 118.4, 113.3, 63.8, 63.6, 58.0, 49.7, 34.9, 19.3; mp 102−104 °C
(decomposed). [α]28 −34.5° (c 0.23, abs EtOH). LC-MS (m/z)
D
calcd for C23H21N3O2 [M + H+], 372.2; found, 372.2.
Ethyl 2-(Phenylamino)acetate (14). Ethyl bromoacetate (2.43 mL,
22 mmol) was added dropwise over 2 h to a stirred solution of aniline
(2 mL, 22 mmol) and N,N-diisopropylethylamine (8 mL, 46 mmol) in
acetonitrile (20 mL) at 60 °C. The reaction mixture was stirred for an
additional 3 h at 60 °C and then concentrated to dryness. Addition of
H2O (5 mL) to the residue and the solid was filtered and washed with
H2O several times. Recrystallization from toluene afforded the title
2-Amino-5-oxo-4-phenyl-7-((S)-1-phenylethyl)-5,6,7,8-tetrahy-
dro-4H-pyrano[2,3-c]pyridine-3-carbonitrile (11a/11b). A suspen-
sion of potassium tert-butoxide (78 mg, 0.69 mmol) in dry THF (5
mL) was added dropwise to a solution of (S)-ethyl 2-((2-oxopropyl)-
(1-phenylethyl)amino)acetate (24) (122 mg, 0.46 mmol) in dry THF
(8 mL) at 0 °C under a N2 atmosphere. The reaction mixture was
stirred at rt for 2 h and quenched with satd NH4Cl (2 mL). After
concentration in vacuo, the crude product was dissolved in EtOH/
H2O (6.5 mL, 10:3) and 13 (71 mg, 0.49 mmol) were added. The
reaction mixture was stirred at rt for 18 h. After concentration with
silica gel in vacuo, the crude product was purified by column
chromatography on silica gel. This afforded the title compound as a
pale-yellow solid (69 mg, 189 μmol, 41% yield); Rf 0.24 (EtOAc/
heptane 1:1). The diastereoisomic mixture was separated by HPLC
using a Chiralpak AD column (n-heptane/2-PrOH 80:20) to give 11a
and 11b (1:1) in 88% yield. Analytical data for 11a (99.7% de): tR =
14.6 min (n-heptane/2-PrOH 80:20). 1H NMR (300 MHz, CDCl3) δ:
7.36−7.19 (m, 10H), 4.50 (s, 2H), 4.41 (s, 1H), 3.54 (q, J = 6.6 Hz,
1H), 3.44 (d, J = 16.5 Hz, 1H), 3.38 (d, J = 15.9 Hz, 1H), 3.18 (d, J =
16.5 Hz, 1H), 2.98 (d, J = 15.9 Hz, 1H), 1.40 (d, J = 6.6 Hz, 3H). 13C
NMR (75 MHz, CDCl3) δ: 193.0, 161.8, 157.2, 142.2, 141.5, 128.7,
128.6, 127.7, 127.6, 127.4, 127.3, 118.4, 113.3, 63.8, 63.5, 58.3, 49.7,
34.8, 19.6; mp 121−123 °C (decomposed). [α]28D +10.3° (c 0.12, abs
EtOH). LC-MS (m/z) calcd for C23H21N3O2 [M + H+], 372.2; found,
372.2. Analytical data for 11b (99.7% de): tR = 12.3 min (n-heptane/2-
1
compound as a beige solid (1.83 g, 10.1 mmol, 46% yield). H NMR
(300 MHz, CDCl3) δ: 7.23−7.13 (m, 2H), 6.74 (t, J = 7.2, 1H), 6.59
(dd, J = 7.8, 0.9 Hz, 2H), 4.23 (q, J = 7.2 Hz, 2H), 3.88 (s, 2H), 1.29
(t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ: 171.1, 147.0,
129.2, 118.1, 112.9, 61.3, 45.8, 14.2; mp 55−57 °C (decomposed).
LC-MS (m/z) calcd for C10H13NO2 [M + H+], 180.1; found, 180.1.
Ethyl 2-((2-Oxopropyl)(phenyl)amino)acetate (15). Ethyl 2-
(phenylamino)acetate (14) (1 g, 5.6 mmol) and K2CO3 (2.31 g,
16.7 mmol) were stirred in dry THF (30 mL) at rt under a N2
atmosphere. A solution of chloroacetone (489 μL, 6.14 mmol) in dry
THF (4 mL) was added dropwise to the reaction mixture and stirred
for 30 min. Sodium iodide (920 mg, 6.14 mmol) was added, and the
reaction mixture was stirred at 60 °C for 3 days. The crude reaction
was quenched with H2O (10 mL) and extracted with EtOAc (3 × 30
mL), and the combined organic phases were washed with H2O (1 ×
30 mL) and brine (1 × 20 mL). The organic phase was dried over
MgSO4. After concentration in vacuo, the crude product was purified
by column chromatography on silica gel. This afforded the title
compound as a pale-yellow oil (448 mg, 1.73 mmol, 31% yield); Rf
1
0.25 (EtOAc/heptane 1:2). H NMR (300 MHz, CDCl3) δ: 7.24−
1
PrOH 80:20). H NMR (300 MHz, CDCl3) δ: 7.35−7.17 (m, 10H),
7.16 (m, 2H), 6.76 (dt, J = 7.2, 0.6 Hz, 1H), 6.53 (dd, J = 7.8, 0.9 Hz,
2H), 4.19 (q, J = 7.2 Hz, 2H), 4.12 (s, 2H), 4.09 (s, 2H), 2.19 (s, 3H),
1.27 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ: 207.7, 170.8,
147.7, 129.3, 118.3, 112.4, 62.3, 61.1, 53.8, 27.0, 14.2. LC-MS (m/z)
calcd for C13H17NO3 [M + H+], 236.1; found, 236.1.
4.51 (s, 2H), 4.39 (s, 1H), 3.58 (q, J = 6.6 Hz, 1H), 3.55 (dd, J = 1.5,
16.2 Hz, 1H), 3.28 (dt, J = 2.1, 16.5 Hz, 1H), 3.20 (dd, J = 1.5, 16.2
Hz, 1H), 3.02 (dd, J = 2.1, 16.5 Hz, 1H), 1.41 (d, J = 6.6 Hz, 3H). 13C
NMR (75 MHz, CDCl3) δ: 192.9, 161.7, 157.2, 142.3, 141.2, 128.7,
128.6, 127.7, 127.6, 127.5, 127.3, 118.3, 113.3, 63.7, 63.6, 58.0, 49.7,
34.9, 19.2; mp 102−104 °C (decomposed). [α]28D +31.5° (c 0.13, abs
EtOH). LC-MS (m/z) calcd for C23H21N3O2 [M + H+], 372.2; found,
372.2.
Ethyl 2-(Benzylamino)acetate (17). A solution of ethyl bromoa-
cetate (924 μL, 8 mmol) in dry THF (4 mL) was added dropwise over
10 min to a cooled solution of benzylamine (2 mL, 18 mmol) in dry
THF (20 mL) at 0 °C under a N2 atmosphere. The reaction mixture
was stirred for 3.5 h at rt, where after it was concentrated and
resuspended in diethyl ether. The white solid was filtered off. The
crude was concentrated and purified by column chromatography on
silica gel. This afforded the title compound as a pale-yellow oil (1.45 g,
2-Amino-5-oxo-4-phenyl-7-((R)-1-phenylethyl)-5,6,7,8-tetrahy-
dro-4H-pyrano[2,3-c]pyridine-3-carbonitrile (12a/12b). A suspen-
sion of potassium tert-butoxide (93 mg, 0.82 mmol) in dry THF (5
mL) was added dropwise to a solution of (R)-ethyl 2-((2-
oxopropyl)(1-phenylethyl)amino)acetate (145 mg, 0.55 mmol) in
THF (5 mL) at 0 °C under a N2 atmosphere. The reaction mixture
was stirred at rt for 3 h and quenched with satd NH4Cl (1 mL). After
concentration in vacuo, the crude product was dissolved in EtOH/
H2O (6 mL, 5:1) and 13 (84 mg, 0.55 mmol) and piperidine (11 μL,
0.20 mmol) were added. The reaction mixture was stirred at rt for 22
h. After concentration with silica gel in vacuo, the crude product was
purified by column chromatography on silica gel. This afforded the
title compound as a pale-yellow solid (117 mg, 0.32 mmol, 58% yield);
Rf 0.33 (EtOAc/heptane 1:1). The diastereoisomic mixture was
separated by HPLC using a Chiralpak AD column (n-heptane/2-
PrOH 80:20) to give 12a and 12b (1:1) in 82% yield. Analytical data
1
7.50 mmol, 90% yield); Rf 0.26 (EtOAc/heptane 1:1). H NMR (300
MHz, CDCl3) δ: 7.34−7.20 (m, 5H), 4.18 (q, J = 7.2 Hz, 2H), 3.80 (s,
2H), 3.40 (s, 2H), 1.89 (s, 1H), 1.27 (t, J = 7.2 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ: 172.3, 139.5, 128.4, 128.2, 127.1, 60.7, 53.2, 50.1,
14.2. LC-MS (m/z) calcd for C11H15NO2 [M + H+], 194.1; found,
194.1.
Ethyl 2-(Benzyl(2-oxopropyl)amino)acetate (18). Ethyl 2-
(benzylamino)acetate (17) (165 mg, 853 μmol) and NaHCO3 (72
mg, 853 μmol) were stirred in abs EtOH (3 mL) at 60 °C under a N2
atmosphere. A solution of chloroacetone (68 μL, 853 μmol) in abs
EtOH (1 mL) was added dropwise to the reaction mixture and stirred
for 18 h. The crude reaction was quenched with H2O (5 mL) and
extracted with EtOAc (3 × 10 mL), and the combined organic phases
were washed with H2O (1 × 10 mL) and brine (1 × 10 mL). The
organic phase was dried over MgSO4. After concentration in vacuo, the
crude product was purified by column chromatography on silica gel to
afford the title compound as a pale-yellow oil (137 mg, 580 μmol, 68%
yield); Rf 0.51 (EtOAc/heptane 1:1). 1H NMR (300 MHz, CDCl3) δ:
7.34−7.20 (m, 5H), 4.15 (q, J = 6.9 Hz, 2H), 3.83 (s, 2H), 3.52 (s,
2H), 3.45 (s, 2H), 1.26 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ: 207.8, 171.0, 138.1, 128.9, 128.4, 127.4, 63.1, 60.4, 58.4,
54.3, 27.5, 14.2. LC-MS (m/z) calcd for C14H19NO3 [M + H+], 250.1;
found, 250.1.
1
for 12a (99.8% de): tR = 11.0 min (n-heptane/2-PrOH 80:20). H
NMR (300 MHz, CDCl3) δ: 7.36−7.17 (m, 10H), 4.58 (s, 2H), 4.40
(s, 1H), 3.53 (q, J = 6.6 Hz, 1H), 3.43 (d, J = 16.5 Hz, 1H), 3.37 (d, J
= 15.9 Hz, 1H), 3.17 (dt, J = 1.5, 16.5 Hz, 1H), 2.98 (dd, J = 2.4, 16.2
Hz, 1H), 1.39 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ:
193.0, 161.9, 157.3, 142.3, 141.5, 128.7, 128.6, 127.6, 127.4, 127.3,
118.5, 113.2, 63.8, 63.1, 58.3, 49.7, 34.8, 19.6; mp 121−123 °C
(decomposed). [α]28D +15.9° (c 0.23, abs EtOH). LC-MS (m/z) calcd
for C23H21N3O2 [M + H+], 372.2; found, 372.2. Analytical data for
1
12b (99.2% de): tR = 15.5 min (n-heptane/2-PrOH 80:20). H NMR
(300 MHz, CDCl3) δ: 7.35−7.17 (m, 10H), 4.55 (s, 2H), 4.38 (s, 1H),
3.57 (q, J = 6.6 Hz, 1H), 3.54 (dd, J = 1.5, 16.2 Hz, 1H), 3.27 (dt, J =
H
dx.doi.org/10.1021/jm300345z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Ethyl 2-(2-Oxopropylamino)acetate (20). Ethyl 2-(benzyl(2-
oxopropyl)amino)acetate (18) (1.19 g, 4.76 mmol) was stirred in
abs EtOH (20 mL) at rt. under and a N2 atmosphere. Concd HCl (1
mL) and Pd/C (118 mg, 476 μmol) were added, and the reaction
mixture was purged with H2 (g) for 2 h. The black solid material was
filtered through Celite, and the residue was washed several times with
abs EtOH. The filtrate was concentrated to afford the title compound
as a beige solid (900 mg, 4.62 mmol, 97% yield). 1H NMR (300 MHz,
MeOH-d4) δ: 4.30 (q, J = 6.9 Hz, 2H), 4.19 (s, 2H), 3.96 (s, 2H), 2.24
(s, 3H), 1.32 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, MeOH-d4) δ:
200.2, 166.6, 62.7, 54.8, 46.9, 26.2, 13.4; mp 132−134 °C
(decomposed). LC-MS (m/z) calcd for C7H13NO3 [M + H+],
160.1; found, 160.1.
HPLC. Analytical HPLC for determination of the diastereomeric
excess (de) was performed using a Chiralpak AD column (4.6 mm ×
250 mm) equipped with a Chiralpak AD guard column (4 mm × 10
mm) (Daicel) and eluted at 1.0 mL/min with n-heptane/2-PrOH
(80:20). The column was connected to a Dionex Ultimate 3000 pump,
a TSP AS-3000 autosample and a Dionex Ultimate 3000 photodiode
array detector. For HPLC control, data collection and data handling,
Chromeleon software v. 6.80 was used.
Preparative HPLC. Separation of the diastereomeric mixtures was
achieved using a Chiralpak AD column (20 mm × 250 mm) and flow
rate 6 mL/min with n-heptane/2-PrOH (80:20). Sample concen-
tration was 6 mg/mL and injection volume was 2 mL.
IR and VCD. The IR was measured using a fast transform infrared
(FTIR) apparatus. VCD spectra were measured using a FTIR
apparatus with interferometers and with an IR source operating at
Phi-M 400 Hz. A Nicolet Nexus 870 FT-IR spectrometer with the
PEM module from Thermo Electron Corporation that has a wide
spectral range from 200 to 7000 cm−1 was used. A sample
concentration of 150 mg/mL in DMSO at 8 cm−1 resolution using
a calcium fluorite (CaF2) cell and a 6 μm spacer gave an absorbance at
0.7. Calculated VCD frequencies using HF and 6-31G* basis set come
out uniformly higher than experimental frequencies. Thus, a scaling
factor of 0.8929 for harmonic vibrational frequencies is proposed as
being appropriate for predictive proposes.31−35
In Silico Study. The modeling study was performed using the
software package MOE (Molecular Operating Environment, Chemical
Computing Group, 2010) using the built-in mmff94x forcefield and
the GB/SA continuum solvent model. General procedure: The
compound of interest was submitted to a stochastic conformational
search (standard setup) to determine its low-energy conformation.
Superimposition of selected low-energy conformations was done using
the built-in function by fitting the three atoms amino groups and the
C1′-carbons of the phenyl rings.
Ethyl 2-(N-(2-Oxopropyl)benzamido)acetate (21). Ethyl 2-(2-
oxopropylamino)acetate (20) (300 mg, 1.53 mmol) was stirred in
dry THF (20 mL) at 0 °C under a N2 atmosphere. A solution of Et3N
(446 μL, 3.22 mmol) in dry THF (2 mL) was added dropwise and
stirred for an additional 5 min. A solution of benzoyl chloride (196 μL,
1.69 mmol) in dry THF (1 mL) was added dropwise, and the reaction
mixture was stirred at rt for 14 h. The crude reaction was quenched
with H2O (10 mL) and extracted with EtOAc (3 × 30 mL), and the
combined organic phases were washed with H2O (1 × 20 mL) and
brine (1 × 20 mL). The organic phase was dried over MgSO4 and
concentrated in vacuo. The crude product was purified by column
chromatography on silica gel to afford the title compound as a pale-
yellow oil (354 mg, 1.35 mmol, 88% yield); Rf 0.29 (EtOAc/heptane
1
2:1). H NMR (300 MHz, DMSO-d6) δ: 7.43−7.18 (m, 5H, minor
and m, 5H, major), 4.30 (s, 2H, minor), 4.22 (s, 2H, major), 4.13−
4.04 (m, 2H, minor and m, 2H, major), 4.09 (s, 2H, minor), 3.99 (s,
2H, major), 2.12 (s, 3H, minor), 1.94 (s, 3H, major), 1.21 (t, J = 6.0
Hz, 3H, minor), 1.13 (t, J = 6.0 Hz, 3H, major). 13C NMR (75 MHz,
CDCl3, M = major conformer, m = minor conformer) δ: 202.9 (M and
m), 172.2 (M and m), 169.3 (M and m), 134.9 (m), 134.7 (M), 130.2
(M and m), 128.6 (M and m), 126.8 (M), 126.6 (m), 61.6 (M), 61.3
(m), 59.6 (m), 55.6 (M), 51.8 (M), 47.4 (m), 27.4 (M), 27.0 (m),
14.1 (M and m). LC-MS (m/z) calcd for C14H17NO4 [M + H+],
264.1; found, 264.1.
Pharmacology. Cell culture of the EAAT1,2,3-HEK293 cell lines
and the [3H]-D-Aspartate uptake assay were performed essentially as
previously described.30 The experimental procedures are described in
detail in Supporting Information.
(S)-Ethyl 2-(1-Phenylethylamino)acetate (23). A solution of ethyl
bromoacetate (1 mL, 9 mmol) in dry THF (4 mL) was added
dropwise over 10 min to a cooled solution of (S)-1-phenylethanamine
(2.41 mL, 19 mmol) in dry THF (25 mL) at 0 °C under a N2
atmosphere. The reaction mixture was stirred for 2.5 h at rt. After
concentration in vacuo, the crude product was purified by column
chromatography on silica gel. This afforded the title compound as a
colorless oil (1.71 g, 8.3 mmol, 92% yield); Rf 0.49 (EtOAc/heptane
2:1). 1H NMR (300 MHz, CDCl3) δ: 7.30−7.19 (m, 5H), 4.14 (q, J =
ASSOCIATED CONTENT
* Supporting Information
IR and VCD values in table format for S1 and S2, as well as
pharmacology experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +45 35336244. Fax: +45 35336041. E-mail: lebu@
farma.ku.dk.
■
2
6.9 Hz, 2H), 3.78 (q, J = 6.9 Hz, 1H), 3.24 (AB system, J = 3.3 Hz,
2H), 1.92 (s, 1H), 1.38 (d, J = 6.9 Hz, 3H), 1.23 (t, J = 6.9 Hz, 3H).
13C NMR (75 MHz, CDCl3) δ: 172.9, 145.0, 128.9, 127.5, 127.1, 61.1,
58.1, 49.3, 24.7, 14.6. LC-MS (m/z) calcd for C12H17NO2 [M + H+],
208.1; found, 208.1.
Notes
The authors declare no competing financial interest.
(S)-Ethyl 2-((2-Oxopropyl)(1-phenylethyl)amino)acetate (24).
(S)-Ethyl-2-(1-phenylethylamino)-acetate (23) (1.59 g, 7.67 mmol)
and NaHCO3 (773 mg, 9.21 mmol) were stirred in abs EtOH (25
mL) at 60 °C under a N2 atmosphere. A solution of chloroacetone
(733 μL, 9.21 mmol) in abs EtOH (4 mL) was added dropwise to the
reaction mixture and stirred for 3 days. The crude reaction was
quenched with H2O (10 mL) and extracted with EtOAc (3 × 50 mL),
and the combined organic phases were washed with H2O (1 × 50 mL)
and brine (1 × 40 mL). The organic phase was dried over MgSO4.
After concentration in vacuo, the crude product was purified by
column chromatography on silica gel. This afforded the title
compound as a pale-yellow oil (432 mg, 1.61 mmol, 21% yield); Rf
0.13 (100% dichloromethane). 1H NMR (300 MHz, CDCl3) δ: 7.40−
7.22 (m, 5H), 4.17−4.08 (m, 3H), 3.57 (d, J = 5.4 Hz, 1H), 3.50 (d, J
= 5.4 Hz, 1H), 3.43 (d, J = 4.2 Hz, 1H), 3.38 (d, J = 4.2 Hz, 1H), 2.11
(s, 3H), 1.34 (d, J = 6.9 Hz, 3H), 1.25 (t, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 208.8, 171.6, 143.9, 128.4, 127.4, 127.3, 61.6,
60.9, 60.4, 52.4, 27.5, 19.8 14.2. LC-MS (m/z) calcd for C15H21NO3
[M + H+], 264.1; found, 264.1.
ACKNOWLEDGMENTS
■
We thank Postdoc Karin Stibius Jensen, Quantum Protein
Centre, Department of Physics, Technical University of
Denmark, for the technical assistance with the VCD
spectrometer. We express our gratitude to the Lundbeck
Foundation, the Novo Nordisk Foundation, the Augustinus
Foundation, the Danish Medical Council, and the Carlsberg
Foundation for financial support.
ABBREVIATIONS USED
■
ALS, amyotrophic lateral sclerosis; CNS, central nervous
system; EAAC1, excitatory amino acid carrier subtype 1;
EAAT(s), excitatory amino acid transporter(s); FTIR, Fast
transform infrared; GLAST, glutamate aspartate transporter;
GLT-1, glutamate transporter subtype 1; HF, Hartree−Fock;
I
dx.doi.org/10.1021/jm300345z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(19) Muller, D.; Zeltser, I.; Bitan, G.; Gilon, C. Building Units for N-
Backbone Cyclic Peptides. 3. Synthesis of Protected N(alpha)-(omega-
Aminoalkyl)amino Acids and N(alpha)-(omega-Carboxyalkyl)amino
Acids. J. Org. Chem. 1997, 62, 411−416.
(20) Clinch, K.; Evans, G. B.; Frohlich, R. F.; Furneaux, R. H.; Kelly,
P. M.; Legentil, L.; Murkin, A. S.; Li, L.; Schramm, V. L.; Tyler, P. C.;
Woolhouse, A. D. Third-generation immucillins: syntheses and
bioactivities of acyclic immucillin inhibitors of human purine
nucleoside phosphorylase. J. Med. Chem. 2009, 52, 1126−1143.
(21) Porzi, G.; Sandri, S. Enantioselective Synthesis of (R)- and (S)-
α-Amino Acids Using (6S)- and (6R)-6-Methyl-morpholine-2,5-dione
Derivatives. Tetrahedron: Asymmetry 1996, 7, 189−196.
(22) Monde, K.; Taniguchi, T.; Miura, N.; Vairappan, C. S.; Suzuki,
M. Absolute configurations of endoperoxides determined by vibra-
tional circular dichroism (VCD). Tetrahedron Lett. 2006, 47, 4389−
4392.
(23) Devlin, F. J.; Stephens, P. J.; Osterle, C.; Wiberg, K. B.;
Cheeseman, J. R.; Frisch, M. J. Configurational and conformational
analysis of chiral molecules using IR and VCD spectroscopies:
spiropentylcarboxylic acid methyl ester and spiropentyl acetate. J. Org.
Chem. 2002, 67, 8090−8096.
(24) Bringmann, G.; Busemann, S. Quantumchemical Calculation of
CD Spectra: The Absolute Configuration of Palmarumycins CP3 and
C2. Tetrahedron 1997, 53, 1655−1664.
(25) Ma, S.; Freedman, T. B.; Dukor, R. K.; Nafie, L. A. Near-infrared
and mid-infrared Fourier transform vibrational circular dichroism of
proteins in aqueous solution. Appl. Spectrosc. 2010, 64, 615−626.
(26) Minick, D. J.; Copley, R. C.; Szewczyk, J. R.; Rutkowske, R. D.;
Miller, L. A. An investigation of the absolute configuration of the
potent histamine H3 receptor antagonist GT-2331 using vibrational
circular dichroism. Chirality 2007, 19, 731−740.
(27) Shin-ya, K.; Sugeta, H.; Shin, S.; Hamada, Y.; Katsumoto, Y.;
Ohno, K. Absolute configuration and conformation analysis of 1-
phenylethanol by matrix-isolation infrared and vibrational circular
dichroism spectroscopy combined with density functional theory
calculation. J. Phys. Chem. A 2007, 111, 8598−8605.
(28) Munoz, M. A.; Munoz, O.; Joseph-Nathan, P. Absolute
configuration of natural diastereoisomers of 6beta-hydroxyhyoscy-
amine by vibrational circular dichroism. J. Nat. Prod. 2006, 69, 1335−
1340.
(29) Guo, C.; Shah, R. D.; Mills, J.; Dukor, R. K.; Cao, X.; Freedman,
T. B.; Nafie, L. A. Fourier transform near-infrared vibrational circular
dichroism used for on-line monitoring the epimerization of 2,2-
dimethyl-1,3-dioxolane-4-methanol: a pseudo racemization reaction.
Chirality 2006, 18, 775−782.
(30) Jensen, A. A.; Brauner-Osborne, H. Pharmacological character-
ization of human excitatory amino acid transporters EAAT1, EAAT2
and EAAT3 in a fluorescence-based membrane potential assay.
Biochem. Pharmacol. 2004, 67, 2115−2127.
(31) Pople, J. A.; Schlegel, H. B.; Krishnan, R.; DeFrees, D. J.;
Binkley, J. S.; Frisch, M. J.; Whiteside, R. A.; Hout, R. F.; Hehre, W. J.
Molecular Orbital Studies of Vibrational Frequencies. Int. J. Quantum
Chem., Quantum Chem. Symp. 1981, 15, 269−278.
(32) Hout, R. F.; Levi, B. A.; Hehre, W. J. Effect of Electron
Correlation on Theoretical Vibrational Frequencies. J. Comput. Chem.
1982, 3, 234−250.
(33) Pople, J. A.; Head-Gordon, M.; Fox, D. J.; Raghavachari, K.;
Curtiss, L. A. Gaussian-1 Theory: A General Procedure for Prediction
of Molecular Energies. J. Chem. Phys. 1989, 90, 5622−5629.
(34) Curtiss, L. A.; Raghavachari, K.; Trucks, G. W.; Pople, J. A.
Gaussian-2 Theory for Molecular Energies of First- and Second-Row
Compounds. J. Chem. Phys. 1991, 94, 7221−7230.
(35) Curtiss, L. A.; Carpenter, J. E.; Raghavachari, K.; Pople, J. A.
Validity of addivity approximations used in Gaussian-2 theory. J. Chem.
Phys. 1992, 96, 9030−9034.
HPLC, high-performance liquid chromatography; SAR, struc-
ture−activity relationship; VCD, vibrational circular dichroism
REFERENCES
■
(1) Beart, P. M.; O’Shea, R. D. Transporters for L-glutamate: an
update on their molecular pharmacology and pathological involve-
ment. Br. J. Pharmacol. 2007, 150, 5−17.
(2) Foster, A. C.; Kemp, J. A. Glutamate- and GABA-based CNS
therapeutics. Curr. Opin. Pharmacol. 2006, 6, 7−17.
(3) Bunch, L.; Erichsen, M. N.; Jensen, A. A. Excitatory amino acid
transporters as potential drug targets. Expert Opin. Ther. Targets 2009,
13, 719−731.
(4) Lee, S. G.; Su, Z. Z.; Emdad, L.; Gupta, P.; Sarkar, D.; Borjabad,
A.; Volsky, D. J.; Fisher, P. B. Mechanism of ceftriaxone induction of
excitatory amino acid transporter-2 expression and glutamate uptake in
primary human astrocytes. J. Biol. Chem. 2008, 283, 13116−13123.
(5) Estrada Sanchez, A. M.; Mejia-Toiber, J.; Massieu, L. Excitotoxic
neuronal death and the pathogenesis of Huntington’s disease. Arch.
Med. Res. 2008, 39, 265−276.
(6) Corona, J. C.; Tovar-y-Romo, L. B.; Tapia, R. Glutamate
excitotoxicity and therapeutic targets for amyotrophic lateral sclerosis.
Expert Opin. Ther. Targets 2007, 11, 1415−1428.
(7) Muir, K. W. Glutamate-based therapeutic approaches: clinical
trials with NMDA antagonists. Curr. Opin. Pharmacol. 2006, 6, 53−60.
(8) Sheldon, A. L.; Robinson, M. B. The role of glutamate
transporters in neurodegenerative diseases and potential opportunities
for intervention. Neurochem. Int. 2007, 51, 333−355.
(9) Alexander, G. M.; Godwin, D. W. Metabotropic glutamate
receptors as a strategic target for the treatment of epilepsy. Epilepsy
Res. 2006, 71, 1−22.
(10) Sanacora, G.; Treccani, G.; Popoli, M. Towards a glutamate
hypothesis of depression: an emerging frontier of neuropsychophar-
macology for mood disorders. Neuropharmacology 2012, 62, 63−77.
(11) Karlsson, R. M.; Tanaka, K.; Heilig, M.; Holmes, A. Loss of glial
glutamate and aspartate transporter (excitatory amino acid transporter
1) causes locomotor hyperactivity and exaggerated responses to
psychotomimetics: rescue by haloperidol and metabotropic glutamate
2/3 agonist. Biol. Psychiatry 2008, 64, 810−814.
(12) Karlsson, R. M.; Tanaka, K.; Saksida, L. M.; Bussey, T. J.; Heilig,
M.; Holmes, A. Assessment of glutamate transporter GLAST
(EAAT1)-deficient mice for phenotypes relevant to the negative and
executive/cognitive symptoms of schizophrenia. Neuropsychopharma-
cology 2009, 34, 1578−89.
(13) Erichsen, M. N.; Huynh, T. H.; Abrahamsen, B.; Bastlund, J. F.;
Bundgaard, C.; Monrad, O.; Bekker-Jensen, A.; Nielsen, C. W.;
Frydenvang, K.; Jensen, A. A.; Bunch, L. Structure−activity relation-
ship study of first selective inhibitor of excitatory amino acid
transporter subtype 1: 2-Amino-4-(4-methoxyphenyl)-7-(naphthalen-
1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-
101). J. Med. Chem. 2010, 53, 7180−7191.
(14) Jensen, A. A.; Erichsen, M. N.; Nielsen, C. W.; Stensbol, T. B.;
Kehler, J.; Bunch, L. Discovery of the first selective inhibitor of
excitatory amino acid transporter subtype 1. J. Med. Chem. 2009, 52,
912−915.
(15) Kawecki, R. Addition of dienolates to sulfinimines. Stereo-
selective synthesis of dihydropyridones. Tetrahedron 2001, 57, 8385−
8390.
(16) Davis, F. A.; Fang, T. N.; Chao, B.; Burns, D. M. Asymmetric
synthesis of the four stereoisomers of 4-hydroxypipecolic acid.
Synthesis 2000, 2106−2112.
(17) Kotake, Y.; Okauchi, T.; Iijima, A.; Yoshimatsu, K.; Nomura, H.
Novel 6−5 fused ring heterocycle antifolates with potent antitumor
activity: bridge modifications and heterocyclic benzoyl isosters of 2,4-
diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidine antifolate. Chem.
Pharm. Bull. 1995, 43, 829−841.
(18) Olofsson, B.; Bogar, K.; Fransson, A. B.; Backvall, J. E. Divergent
asymmetric synthesis of 3,5-disubstituted piperidines. J. Org. Chem.
2006, 71, 8256−8260.
J
dx.doi.org/10.1021/jm300345z | J. Med. Chem. XXXX, XXX, XXX−XXX