Design, synthesis and pharmacological characterization of coumarin-based fluorescent analogs of excitatory amino acid transporter subtype 1 selective inhibitors, UCPH-101 and UCPH-102

Article abstract of DOI:10.1016/j.bmc.2012.09.049

The excitatory amino acid transporters (EAATs) play a pivotal role in regulating the synaptic concentration of glutamate in the mammalian central nervous system. To date, five different subtypes have been identified, named EAAT15 in humans (and GLAST, GLT-1, EAAC1, EAAT4, and EAAT5, respectively, in rodents). Recently, we have published and presented a structure-activity relationship (SAR) study of a novel class of selective inhibitors of EAAT1 (and GLAST), with the analogs UCPH-101 (IC₅₀ = 0.66 μM) and UCPH-102 (IC₅₀ = 0.43 μM) being the most potent inhibitors in the series. In this paper, we present the design, synthesis and pharmacological evaluation of six coumarin-based fluorescent analogs of UCPH-101/102 as subtype-selective inhibitors at EAAT1. Analogs 1114 failed to inhibit EAAT1 function (IC ₅₀ values >300 μM), whereas analogs 15 and UCPH-102F inhibited EAAT1 with IC₅₀ values in the medium micromolar range (17 μM and 14 μM, respectively). Under physiological pH no fluorescence was observed for analog 15, while a bright blue fluorescence emission was observed for analog UCPH-102F. Regrettably, under confocal laser scanning microscopy selective visualization of expression of EAAT1 over EAAT3 was not possible due to nonspecific binding of UCPH-102F.

Full text of DOI:10.1016/j.bmc.2012.09.049

Bioorganic & Medicinal Chemistry 20 (2012) 6831–6839
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and pharmacological characterization of
coumarin-based fluorescent analogs of excitatory amino acid transporter
subtype 1 selective inhibitors, UCPH-101 and UCPH-102
Tri H. V. Huynh, Bjarke Abrahamsen, Karsten K. Madsen, Alba Gonzalez-Franquesa, Anders A. Jensen,
⇑
Lennart Bunch
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2012
Revised 24 September 2012
Accepted 25 September 2012
Available online 2 October 2012
The excitatory amino acid transporters (EAATs) play a pivotal role in regulating the synaptic concentra-
tion of glutamate in the mammalian central nervous system. To date, five different subtypes have been
identified, named EAAT15 in humans (and GLAST, GLT-1, EAAC1, EAAT4, and EAAT5, respectively, in
rodents). Recently, we have published and presented a structure–activity relationship (SAR) study of a
novel class of selective inhibitors of EAAT1 (and GLAST), with the analogs UCPH-101 (IC50 = 0.66
lM)
and UCPH-102 (IC50 = 0.43 M) being the most potent inhibitors in the series. In this paper, we present
the design, synthesis and pharmacological evaluation of six coumarin-based fluorescent analogs of UCPH-
101/102 as subtype-selective inhibitors at EAAT1. Analogs 1114 failed to inhibit EAAT1 function (IC50 val-
l
Keywords:
Central nervous system
Coumarin-based fluorescent analogs
Excitatory amino acid transporters
Structure–activity relationship
ues >300
micromolar range (17
l
M), whereas analogs 15 and UCPH-102F inhibited EAAT1 with IC50 values in the medium
M and 14 M, respectively). Under physiological pH no fluorescence was
l
l
observed for analog 15, while a bright blue fluorescence emission was observed for analog UCPH-
102F. Regrettably, under confocal laser scanning microscopy selective visualization of expression of
EAAT1 over EAAT3 was not possible due to nonspecific binding of UCPH-102F.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
EAAT1–3 (GLAST, GLT-1, EAAC1) are expressed throughout
the CNS, the EAAT4,5 subtypes are expressed exclusively in Purkinje
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. Thereby, the transporters are cru-
cial players in the maintenance of synaptic as well as extra-synaptic
Glu concentrations below levels of neurotoxicity and consequently
for the overall firing tone in this neurotransmitter system.1 To date,
five EAAT subtypes have been identified. In humans these subtypes
are termed EAAT1–5, whereas they in rodents are termed GLAST,
GLT-1, EAAC1, EAAT4, and EAAT5, respectively. The five EAAT sub-
types exhibit distinct expression patterns in the CNS: While
cells of the cerebellum and in the retina, respectively.2 At the
cellular level, EAAT1,2 are expressed predominantly in glia cells
and astrocytes, whereas EAAT3,4 are expressed almost exclusively
on neurons.3 Finally, EAAT1–3 are high-capacity Glu transporters,
while EAAT4,5 are considered to be low-capacity Glu transporters,
functioning primarily as Glu-gated chloride channels.3 Malfunction
of the EAATs has been suggested to be a contributing factor in
neurotoxic states and neurodegenerative diseases such as Alzhei-
mer’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
Considering the therapeutic potential of the EAATs the available
pharmacological tool box for in vitro and in vivo studies is, how-
ever, limited with EAAT1 inhibitor UCPH-101 being the most
recent disclosure.3 Thus, as a part of our medicinal chemistry re-
search program within the EAATs, we focus on the discovery of
small-molecule fluorescent probes for the potential use in studies
of function, expression and trafficking of these transporters. We
here report the design and synthesis of a series of coumarin-based
fluorescent analogs of the EAAT1-selective inhibitors UCPH-101/
102 which may be useful as pharmacological tools.
Abbreviations: ALS, amyotrophic lateral sclerosis; CNS, central nervous system;
EAAC1, excitatory amino acid carrier subtype 1; EAAT(s), excitatory amino acid
transporter(s); GABAA, c-aminobutyric acid receptor A; GLAST, glutamate aspartate
transporter; GLT-1, glutamate transporter subtype 1; SAR, Structure–activity
relationship.
⇑
Corresponding author. Tel.: +45 35336244; fax: +45 35336041.
E-mail address: lebu@farma.ku.dk (L. Bunch).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.049

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T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
2.2. Design and synthesis
2. Results and discussions
2.1. Coumarins as fluorophores
In accordance with previous reported structure–activity rela-
tionship (SAR) study of UCPH-101 and UCPH-102,21,22 the presence
of an aromatic group in the 7-position is crucial for the inhibitory
activity and that a 1-naphthyl group gave the most potent
analogs (UCPH-101 and UCPH-102) (Fig. 2). On the other hand,
the 4-position was found to be able to accommodate small and
larger substituents, not being restricted to aromatics only (com-
pounds 7–10, Fig. 2).21,22
Given this SAR information, our strategy was to incorporate the
coumarin moiety into the 4- and 7-positions of the parental scaf-
fold. The coumarin analogs 11 and 12 (Fig. 3) were designed in
accordance with 10, taking advantage of the methyl attachment
point of the coumarin skeleton. In the 4-position, the coumarin
and 1-naphthyl substituents occupy the same space being flat bicy-
clic substituents. For 11, the phenyl group was kept in the 7-posi-
tion whereas for 12 a 1-naphthyl group was chosen as to mimic
UCPH-101/102. The 4-coumarin analogs 13 and 14 (Fig. 3) com-
prise a phenyl spacer and were designed based on the SAR obser-
vation that larger and elongated substituents are allowed (Fig. 2,
structure 8 and 9). As an alternative strategy, the coumarin moiety
was incorporated into the 7-position (analogs 15, 16 and UCPH-
102F) with the methyl group being the point of attachment as to
resemble UCPH-101/102. This attachment point is likely essential
as compound 7 which comprises a 2-naphthyl group is 10–20 fold
less active. A methyl group in the 4-position, as present in UCPH-
102, was chosen as to balance solubility properties.
Coumarins comprise the parental skeleton 2H-chromen-2-one
(1) (Table 1) are found in a broad class of natural products, phar-
maceutical drugs and fluorophores.13 While the parental coumarin
skeleton 1 is characterized by low fluorogenic properties, addition
of an electron rich substituent in the 7-position on the phenyl ring
gives rise to electron resonance throughout the fused ring system
and thus strong fluorescent characteristics. At physiological pH,
7-hydroxycoumarin (2) (Table 1) is equally fluorescent to 1. How-
ever, when placed at pH of 10 or higher, the 7-hydroxy group is
fully deprotonated and the phenolate anion participates in reso-
nance, thereby giving rise to fluorescent properties. In contrast,
the N,N-7-diethylamino-coumarin derivative (3) (Table 1) is highly
fluorescent under physiological pH, which makes this fluorophore
attractive for use in biological studies.14
Several other coumarin derivatives have been incorporated into
compounds and used to study various biologically important tar-
gets. For example, the 7-aminocoumarin derivative 4 (Fig. 1) has
been used for preparing blue fluorescent conjugates of proteins
and nucleic acids,18 while the 7-diethylaminocoumarin derivative
5 has been incorporated into ligands used for studies of c-amino-
butyric acid type A (GABAA) receptors.19 The 7-hydroxycoumarin
derivative 6, which comprise three substituents on the benzene
ring has been applied as fluorophore for the labeling of recombi-
nant proteins inside living cells and on the cell surface by Esche-
richia coli lipoic acid ligase (LplA) mutants.20
The synthesis of the compound class profiled by UCPH-101/102
is in general terms carried out by a three-component reaction21–24
as depicted in Figure 4. 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. Thus, to synthesize analogs
with a coumarin moeity in the 7-position as in compounds 15, 16
and UCPH-102F, the coumarin is to be part of the diketone frag-
ment. On the other hand, upon its introduction in the 4-position
as in compounds 11–14, the coumarin moiety is to be employed
as an aldehyde.
Table 1
Chemical structures and absorption and emission maxima for coumarin (1) and
selected derivatives (2–3)
O
O
HO
O
O
N
O
O
1
2
3
2.3. Synthesis of analogs with a coumarin moiety in the 4-
position
Solventa
kabs (nm)
kem (nm)
The synthesis of analog 11 commenced with the preparation of
aldehyde 18 by oxidation of coumarin 17, using SeO2 in p-xylene.25,26
The aldehyde was then used in the three-component reaction with
5-phenylcyclohexane-1,3-dione, malononitrile, piperidine in abs
EtOH to give the desired product 7-methoxy-coumarin analog 11 in
77% yield (Scheme 1).21 The synthesis of the N,N-diethylamino-cou-
marin analog 12 followed the same pathway starting by oxidation
of the diethylamino coumarin 3 with SeO2 in p-xylene but afforded
the corresponding aldehyde 19 in only 32% yield (Scheme 1).25,26
With aldehyde 19 in hand, it was reacted with 5-(naphthalen-
1-yl)cyclohexane-1,3-dione, malononitrile, piperidine in abs EtOH
to afford coumarin analog 12 in 57% yield.21
115
Acetonitrile
0.1 M Phosphate buffer (pH = 10)
Abs ethanol
320
360
373
—
450
451
216
314,17
a
The experiments were performed at rt.
H2N
O
O
O
OH
4
The synthesis of analogs 13 and 14 with a coumarin moiety in the
4-position were carried out following the same strategy. The reac-
tion commenced with monobromination of coumarins 17 and 3
using N-bromosuccinimide in PEG-40027 or NH4OAc in acetoni-
trile28 to give 3-monobrominated products 20 and 21 in 41% and
51% yield, respectively (Scheme 2). Subsequently, 20 and 21 under-
went palladium cross coupling reaction with 4-formylphenylboron-
ic acid, Pd(dppf)Cl2, Na2CO3 in a mixture of DMF/H2O (4:1) to give
the aldehydes 22 and 23 in high yields (86% and 82%, respec-
tively).28 Finally, the aldehydes were employed in the three compo-
nent reaction together with 5-phenylcyclohexane-1,3-dione,
malononitrile, and piperidine base in abs EtOH to afford the
F
N
O
O
O
O
HO
F
O
O
O
O
H
N
H
N
OH
NH2
3
O
O
O
6
5
Figure 1. Coumarin derivatives 4–6 which have found use as fluorophores in
biological imaging assays.

T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
6833
O
O
O
O
CN
O
O
CN
CN
NH2
4
7
O
NH2
O
NH2
7
UCPH-101
IC50 = 0.66 µM
UCPH-102
IC50 = 0.43
IC50 = 3.2 µM
µM
Cl
F
O
O
O
O
O
CN
CN
NH2
CN
O
NH2
O
O
NH2
9
10
IC50 = 7.4 µM
8
IC50 = 4.3 µM
IC50 = 2.1 µM
Figure 2. Chemical structures of reported subtype selective inhibitors of EAAT1.21,22
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
N
O
O
CN
CN
CN
CN
NH2
NH2
O
NH2
O
NH2
11
13
14
12
O
O
O
N
CN
NH2
HO
CN
NH2
O
CN
O
O
O
NH2
O
O
O
UCPH-102F
16
15
O
O
O
Figure 3. Chemical structures of coumarin-based fluorescent analogs.
4-substituted coumarin analogs 13 and 14 in 61% and 82% yield,
respectively.
nate, NaH in THF to afford the conjugated ketones 24 and 25 in
91% and 89% yield, respectively. The ketones were subsequently
reacted with diethylmalonate, 21% w/w NaOEt in abs EtOH fol-
lowed by hydrolysis of the ester in 2 M NaOH, H2O and decar-
boxylation in 2 M H2SO4 (pHꢀ3) to provide diketones 26 and
27 in 38% and 20% yield, respectively. In the closing three-com-
ponent reaction, diketones 26 and 27 were reacted with malon-
onitrile, acetaldehyde, N-methylmorpholine in abs EtOH to
afford the coumarin analogs 15 and UCPH-102F in 68% and
77% yield, respectively.
2.4. Synthesis of analogs with a coumarin moiety in the 7-
position
The synthesis of three coumarin analogs 15, 16 and UCPH-
102F (Scheme 3and 4) commenced with the building of dike-
tones 26 and 27. The previously prepared aldehydes 18 and
19 (Scheme 1) were reacted with diethyl-2-oxopropyl-phospho-

6834
T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
O
A
R
O
O
O
R
O
O
O
a
b, c
Base or Acid
+
+
NC
CN
R2
H
R1
O
O
B
O
R2
O
24 R= OMe
25 R= NEt2
18 R= OMe
19 R= NEt2
CN
NC
H
CN
R2
4
+
7
R1
O
NH2
R1
O
O
R
O
O
R
CN
NH2
C
d
Figure 4. 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-car-
bonitrle parental skeleton C with various substituents R1 in the 7-positions and R2
in the 4-position.
O
O
O
O
O
26 R= OMe
27 R= NEt2
15
R= OMe
O
O
O
R
UCPH-102F R= NEt2
R
O
O
R
O
O
a
b
Scheme 3. Reagents and conditions for the synthesis of coumarin analogs 15 and
UCPH-102F reagents and conditions: (a) towards 24: diethyl-2-oxopropyl-phos-
phonate, NaH, THF, 5 h, 91%; towards 25: diethyl-2-oxopropyl-phosphonate, NaH,
THF, 20 h, 89%; (b) towards 26 and 27: diethylmalonate, 21% w/w NaOEt, abs EtOH,
reflux, 3 h; (c) towards 26 and 27: 2 M NaOH, H2O, 100 °C, 5 h, 2 M H2SO4 (pHꢀ3),
100 °C, 1 h, 38% and 20%. (d) towards both 15 and UCPH-102F: malononitrile,
acetaldehyde, N-methylmorpholine, abs EtOH, rt, 4–5 h, 68% and 77%.
CN
O
R1
NH2
O
17 R= OMe
3 R= NEt2
11 R= OMe, R1 = Ph
12 R= NEt2, R1 = Naphthalene
18 R= OMe
19 R= NEt2
romethane,31–33 the desired product 16 was identified in low yield
but isolation from the product mixture and starting coumarin 15
was unsuccessful and thus this analog was given up.
Scheme 1. Synthetic pathway towards the coumarin analogs 11 and 12 reagents
and conditions: (a) for 18: SeO2, p-xylene, 145 °C, 22 h, 84%; for 19: SeO2, p-xylene,
145 °C, 24 h, 32%; (b) for 11: 5-phenylcyclohexane-1,3-dione, malononitrile,
piperidine, abs EtOH, rt, 21 h, 77%; for 12: 5-(naphthalen-1-yl)cyclohexane-1,3-
dione, malononitrile, piperidine, abs EtOH, rt, 20 h, 57%.
3. Characterization of fluorescent and pharmacological
properties
R
O
O
R
O
O
a
b
The absorption and emission wavelengths of the coumarin-
based fluorescent analogs 12, 14 and UCPH-102F were measured
using the Jasco FP-6200 Spectrofluorometer, in abs EtOH at rt
and reported in Table 2 (fluorescence spectra are available in the
Supplementary data). The observed absorption and emission
Br
R
20 R= OMe
21 R= NEt2
17 R= OMe
3 R= NEt2
wavelengths are close in range (kabs: 386 nm, 383 nm and
383 nm, kem: 465 nm, 468 nm and 461 nm) giving a characteristic
bright blue fluorescence.
O
O
The six coumarin-based analogs 11–15 and UCPH-102F were
characterized pharmacologically at EAAT1,2,3 stably expressed in
HEK293 cells in a [3H]- -aspartate uptake assay (Table 3).34 None
D
R
O
O
O
of the coumarin analogs 11–14, comprising the coumarin moiety
in the 4-position, displayed significant inhibitory activity at EAAT1
c
CN
at concentrations up to 300 lM. In contrast, analogs 15 and UCPH-
102F which comprise a coumarin moieties in the 7-position both
O
O
NH2
displayed inhibitory potencies at EAAT1 in medium- micromolar
range (IC50 values 17
six analogs displayed significant inhibitory activity at subtypes
EAAT2,3 at the highest possible concentrations (IC50 > 300 lM).
lM and 14 lM, respectively). None of the
22 R= OMe
23 R= NEt2
13 R= OMe
14 R= NEt2
Scheme 2. Reagents and conditions for the synthesis of 4-substituted coumarin
analogs 13 and 14 reagents and conditions: (a) for 20: N-bromosuccinimide,
PEG-400, rt, 2 h, 46%; for 21: N-bromosuccinimide, NH4OAc, acetonitrile, rt, 68 h,
51%; (b) for 22: 4-formylphenylboronic acid, Pd(dppf)Cl2, Na2CO3, DMF/H2O (4:1),
95 °C, 5 h, 86%; for 23: 4-formylphenylboronic acid, Pd(dppf)Cl2, Na2CO3, DMF/H2O
(4:1), 95 °C, 5 h, 82%; (c) for 13: 5-phenylcyclohexane-1,3-dione, malononitrile,
piperidine, abs EtOH, rt, 18 h, 61%; for 14: 5-phenylcyclohexane-1,3-dione, malon-
onitrile, piperidine, abs EtOH, rt, 19 h, 82%.
Table 2
Absorption and emission wavelengths for N,N-diethylamino-coumarin analogs 12, 14
and UCPH-102F
Coumarin analog
Solventa
Absorption kabs (nm)
Emission kem (nm)
12
14
Abs EtOH
Abs EtOH
Abs EtOH
386b
383b
383b
465c
468c
461c
UCPH-102F
However, demethylation of the 7-methoxy group of coumarin
analog 15 proved to be troublesome. Using 48% HBr29 in water,
only a trace amount of 16 was observed while pyridine–HCl in
dioxane30 gave a complex reactions mixtures. With BBr3 in dichlo-
a
b
c
The experiments were performed at rt.
Measured with a Ultrospec 4300 Pro UV/visible spectrophotometer.
Using a Jasco FP-6200 spectrofluorometer to measure. Emission spectra are
available in the Supplementary data.

T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
6835
Table 3
logically at EAATs. Analogs 11–14 which comprise a coumarin
substituents in the 4-position showed no EAAT1 inhibitory. How-
ever, coumarin analogs 15 and UCPH-102F holding the coumarin
moiety in the 7-position inhibit EAAT1, displaying IC50 values in
micromolar range. As expected, analog 15 did not display fluores-
cence properties under physiological pH, whereas a characteristic
blue fluorescence emission was observed for analog UCPH-102F.
Regrettably nonspecific binding prevents its use to visualize EAAT1
expression selectively in a HEK cell setup. Despite this discourag-
ing result, the design and synthesis of fluorescent analogs of
UCPH-101/102 remains to be an attractive strategy for the visual-
ization and quantification of EAAT1 expression.
Pharmacological characterization of coumarin analogs of UCPH-101/102 as inhibitors
at EAAT1 in a [3H]-D-aspartate uptake asssaya
O
5
R2
CN
4
2
7
R1
O
NH2
Entry
R1
R2
EAAT1 IC50
M)
(
l
5. Experimental section
5.1. Chemistry
O
O
O
O
N
11
>300 [<3.5]
>300 [<3.5]
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 chromatog-
raphy (TLC, Merck silica gel 60 F254 aluminum sheets). Flash chro-
O
12
O
matography was carried out using Merck silica gel 60A (35–70 l.
1H NMR spectra were recorded on a 300 MHz Varian Mercury
300BB or a 400 MHz Avance Bruker and 13C NMR spectra on a
75 MHz Varian Gemini 2000BB or a 100 MHz Avance Bruker. MS
spectra were recorded using LC–MS performed using an Agilent
1200 series solvent delivery system equipped with an autoinjector
coupled to an Agilent 6400 series triple quadrupole mass spec-
trometer equipped with an electrospray ionization source. Gradi-
ents of 5% aqueous acetonitrile 60.05% formic acid (solvent A)
and 95% aqueous acetonitrile +0.043% formic acid (solvent B) were
employed. Melting points were measured using a MPA 100 opti-
melt automatic melting point system. All commercial chemicals
were used without further purification.
O
O
13
>300 [<3.5]
N
O
14
15
>300 [<3.5]
O
5.1.1. 20-Amino-7-methoxy-2,50-dioxo-70-phenyl-50,60,70,
80-tetrahydro-2H,40H-4,40-bichromene-30-carbonitrile (11)
Piperidine (10 lL, 0.10 mmol) was added to a suspension
of 7-methoxy-2-oxo-2H-chromene-4-carbaldehyde (18) (100 mg,
0.49 mmol), 5-phenylcyclohexane-1,3-dione (92 mg, 0.49 mmol)
and malononitrile (32 mg, 0.49 mmol) in abs EtOH (7 mL) at rt.
The reaction mixture was stirred for 21 h. The white precipitate
was filtered off and washed three times with cold abs EtOH. This
afforded the titled compound as an off-white solid (166 mg,
0.38 mmol, 77% yield): mp 281–283 °C; 1H NMR (300 MHz,
DMSO-d6) (ꢁ3:4 ratio of diastereomers) d 2.42–2.51 (m, 1H),
2.67–3.24 (m, 3H), 3.46–3.57 (m, 1H), 3.87 (s, 1.5H), 3.88 (s,
1.5H), 4.85 (s, 1H), 5.95 (s, 0.5), 6.12 (s, 0.5H), 7.02–6.95 (m, 2H),
7.38–7.22 (m, 7H), 8.02 (d, J = 9.0 Hz, 0.5H), 8.07 (d, J = 9.0 Hz,
0.5H); 13C NMR (100 MHz, DMSO-d6) d 33.6, 33.8, 37.2, 37.6,
42.9, 43.0, 55.6, 55.8, 55.9, 100.9, 109.5, 109.7, 111.7, 111.8,
112.0, 118.9, 126.5, 126.6, 126.8, 126.9, 126.9, 128.5, 142.5,
142.6, 155.1, 155.2, 159.0, 159.1, 160.7, 160.8, 162.5, 162.6,
164.9, 165.4, 195.0, 195.1; LC–MS [M+H]+ calcd for C26H20N2O5:
441.2, found: 441.2; Anal. calcd for C26H20N2O5: C, 70.90; H,
4.58; N, 6.36, found: C, 70.61, H, 4.64; N, 6.22.
O
O
O
O
O
N
17
–CH3
–CH3
[4.79 0.12]
UCPH-
14
102F
[4.85 0.05]
a
Data are given as IC50 values in
analogs displayed significant inhibitory activity at EAAT2 or EAAT3 when tested at
the highest possible concentrations (300 M).
lM with pIC50 SEM in brackets. None of the
l
Finally, UCPH-102F was applied to HEK cells expressing EAAT1
over EAAT3 to investigate if selectivity could be detected under
confocal laser scanning microscopy. Regrettably, discrimination
of EAAT1 expression over EAAT3 could not be shown in this exper-
imental setup due to nonspecific binding of UCPH-102F. All at-
tempts to block this were unsuccessful (see Supplementary data).
5.1.2. 20-Amino-7-(diethylamino)-70-(naphthalen-1-yl)-2,50-
dioxo-50,60,70,80-tetrahydro-2H,40H-4,40-bichromene-30-
carbonitrile (12)
4. Conclusion
Piperidine (4 lL, 34 lmol) was added to a solution of 7-
(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (19) (42 mg,
0.17 mmol) and malononitrile (11.3 mg, 0.17 mmol) in abs EtOH
(4 mL) at rt and stirred for 2 min. 5-(Naphthalen-1-yl)cyclohex-
ane-1,3-dione (30 mg, 0.13 mmol) was added and the reaction
In conclusion, seven coumarin analogs of EAAT1-selective
inhibitors UCPH-101/102 were designed on the basis of previously
reported SAR for this class of compounds. Of these, six were suc-
cessfully synthesized and subsequently characterized pharmaco-

6836
T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
mixture was stirred at rt for 20 h. The precipitated solid was fil-
tered off and washed four times with ice-cold abs EtOH to afford
(26) (50 mg, 175
ononitrile (23 mg, 350
l
mol), acetaldehyde (20
lL, 350 lmol) and mal-
l
mol) in abs EtOH (4 mL) at rt and stirred
the titled compound as a pale-yellow solid (38 mg, 72
l
mol, 57%
for 5 h. After concentration in vacuo the crude product was puri-
yield); mp 264–266 °C; 1H NMR (300 MHz, CDCl3) (ꢁ1:3 ratio of
diastereomers) d 1.21 (t, J = 7.2 Hz, 6H), 2.72–3.10 (m, 4H), 3.41
(q, J = 7.2 Hz, 4H), 4.24–4.36 (m, 1H), 4.86 (s, 1H), 4.69 (s, 2H),
5.90 (s, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.66 (dd, J = 9.0, 2.4 Hz, 1H),
7.29–7.60 (m, 4H), 7.75–8.07 (m, 4H); 13C NMR (75 MHz, CDCl3)
d 12.0, 12.5, 29.4, 33.1, 34.2, 41.2, 43.2, 44.7, 61.3, 96.3, 97.7,
106.9, 107.3, 108.9, 113.6, 122.5, 122.6, 125.3, 125.7, 126.0,
126.8, 128.1, 129.2, 130.8, 134.0, 137.1, 151.0, 156.5, 158.4,
158.6, 163.2, 164.3, 195.0; LC–MS [M+H]+ calcd for C33H29N3O4:
532.2, found: 532.2; Anal. calcd for C33H29N3O4: C, 74.56; H,
5.50; N, 7.90, found: C, 74.12; H, 5.28; N, 7.84.
fied by column chromatography on silica gel. This afforded the
titled compound as a yellow solid (53 mg, 140 lmol, 80% yield):
Rf = 0.12 (EtOAc/heptane 1:1); mp 193–195 °C (decomposed); 1H
NMR (300 MHz, CDCl3) (ꢁ1:1 ratio of diastereomers) d 1.25 (d,
J = 6.9 Hz, 1.46H), 1.30 (d, J = 6.9 Hz, 1.54H), 2.48–2.89 (m, 4H),
3.39 (q, J = 6.6 Hz, 1H), 3.68–3.84 (m, 1H), 3.89 (s, 3H), 4.54 (s,
2H), 6.13 (s, 0.43H), 6.16 (s, 0.57H), 6.84–6.91 (m, 2H), 7.48 (d,
J = 8.7 Hz, 0.45H), 7.53 (d, J = 8.7 Hz, 0.55H); 13C NMR (100 MHz,
CDCl3) d 22.3, 22.9, 24.8, 24.9, 31.5, 31.6, 32.3, 32.8, 41.3, 55.9,
56.0, 57.5, 57.8, 101.1, 101.2, 109.3, 111.1, 111.2, 112.3, 114.7,
114.8, 119.9, 126.0, 126.1, 155.1, 156.5, 156.7, 158.6, 158.7,
160.2, 160.3, 162.4, 162.5, 162.8, 194.7, 194.9; LC–MS [M+H]+ calcd
for C21H18N2O5: 379.1, found: 379.1. Anal. calcd C, 66.66; H, 4.79; N
7.40, for C21H18N2O5: found: C, 66.23; H, 4.90; N, 7.03.
5.1.3. 2-Amino-4-(4-(7-methoxy-4-methyl-2-oxo-2H-chromen-
3-yl)phenyl)-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene-
3-carbonitrile (13)
4-(7-Methoxy-4-methyl-2-oxo-2H-chromen-3-yl)benzalde-
hyde (22) (55 mg, 0.19 mmol), 5-phenylcyclohexane-1,3-dione
(35 mg, 0.19 mmol) and malononitrile (12 mg, 0.19 mmol) were
5.1.6. 7-Methoxy-2-oxo-2H-chromene-4-carbaldehyde (18)
7-Methoxy-4-methyl-2H-chromen-2-one (17) (1 g, 5.26 mmol)
and selenium dioxide (875 mg, 7.89 mmol) were stirred in p-xylene
(50 mL) at 145 °C for 18 h. The reaction mixture was cooled to rt and
the white precipitate was filtered and purified by column chroma-
tography on silica gel. This afforded the titled compound as a yellow
solid (902 mg, 4.42 mmol, 84% yield): Rf = 0.59 (100% EtOAc); mp
200–202 °C (decomposed); 1H NMR (300 MHz, CDCl3) d 3.89 (s,
3H), 6.69 (s, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.90 (dd, J = 9.0, 2.4 Hz,
1H), 8.47 (d, J = 9.0 Hz, 1H), 10.05 (s, 1H); 13C NMR (75 MHz, CDCl3)
d 55.8, 101.1, 108.1, 113.3, 122.2, 127.3, 143.7, 156.5, 160.7, 163.3,
191.7; LC–MS [M+H]+ calcd for C11H8O4: 205.1, found: 205.1.
stirred in abs EtOH (5 mL) at rt. Piperidine (4 lL, 37 lmol) was
added and the reaction mixture was stirred for 16 h at rt. The white
precipitate was filtered off and washed three times with cold abs
EtOH. This afforded the titled compound as a pale-yellow solid
(60 mg, 0.11 mmol, 61% yield): mp 241–243 °C (decomposed); 1H
NMR (300 MHz, CDCl3) (ꢁ1:1 ratio of diastereomers) d 2.27 (s,
1.5H), 2.29 (s, 1.5H), 2.53–2.95 (m, 4H), 3.35–3.55 (m, 1H), 3.87
(s, 3H), 4.53 (s, 0.5H), 4.55 (s, 0.5H), 4.63 (s, 1H), 4.65 (s, 1H),
6.83–6.89 (m, 2H), 7.15–7.38 (m, 9H), 7.56 (d, J = 8.4 Hz, 1H); 13C
NMR (100 MHz, CDCl3) d 16.8, 16.9, 34.5, 34.6, 35.0, 35.2, 37.9,
38.8, 43.7, 43.9, 55.8, 62.9, 63.1, 100.6, 112.3, 112.4, 114.2, 115.2,
115.3, 118.7, 118.7, 123.9, 126.2, 126.6, 126.7, 127.4, 127.6,
128.9, 128.9, 130.5, 130.6, 133.4, 133.5, 141.7, 141.8, 142.5,
142.7, 148.1, 148.2, 154.3, 157.9, 158.1, 161.3, 161.4, 161.9,
162.3, 162.4, 162.8, 194.9, 195.2; LC–MS [M+H]+ calcd for
5.1.7. 7-(Diethylamino)-2-oxo-2H-chromene-4-carbaldehyde
(19)
Selenium dioxide (720 mg, 6.48 mmol) was added to a solution
of 7-(diethylamino)-4-methylcoumarine (3) (1 g, 4.32 mmol) in
p-xylene (50 mL) at rt under a N2 atmosphere. The reaction mix-
ture was stirred at 145 °C for 21 h and cooled to rt. The dark sus-
pension was filtered through celite and concentrated in vacuo.
The crude product was purified by column chromatography on
silica gel to afford the titled compound as a dark red solid
(320 mg, 1.38 mmol, 32% yield): Rf = 0.20 (100% dichloromethane);
1H NMR (300 MHz, CDCl3) d 1.22 (t, J = 7.2 Hz, 6H), 3.42 (q,
J = 7.2 Hz, 4H), 6.42 (s, 1H), 6.49 (d, J = 2.7 Hz, 1H), 6.60 (dd,
J = 9.0, 2.7 Hz, 1H), 8.26 (d, J = 9.0 Hz, 1H), 9.99 (s, 1H); 13C NMR
(75 MHz, CDCl3) d 12.4, 44.8, 97.5, 103.7, 109.5, 117.2, 126.9,
143.8, 150.9, 157.3, 161.8, 192.5; LC–MS [M+H]+ calcd for
C33H26N2O5: 531.2, found: 531.3; Anal. calcd for C33H26N2O5: C,
74.7; H, 4.94; N, 5.28, found: C, 74.29; H, 4.62; N, 5.57
5.1.4. 5,6,7,8-Tetrahydro-4H-chromene-3-carbonitrile (14)
4-(7-(Diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)benz-
aldehyde (23) (100 mg, 0.30 mmol), 5-phenylcyclohexane-1,3-
dione (57 mg, 0.30 mmol) and malononitrile (20 mg, 0.30 mmol)
were stirred in abs EtOH (7 mL) at rt. Piperidine (6 lL, 60 lmol)
was added and the reaction mixture was stirred for 19 h at rt.
The white precipitate was filtered off and washed three times with
cold abs EtOH. This afforded the titled compound as a yellow solid
(139 mg, 0.25 mmol, 82% yield): mp 241–243 °C (decomposed); 1H
NMR (300 MHz, CDCl3) (ꢁ1:1 ratio of diastereomers) d 1.21 (t,
J = 7.2 Hz, 6H), 2.21 (s, 1.5H), 2.23 (s, 1.5H), 2.52–2.92 (m, 4H),
3.32–3.48 (m, 5H), 4.51 (s, 0.5H), 4.53 (s, 0.5H), 4.68 (s, 1H), 4.69
(s, 1H), 6.51 (t, J = 2.1 Hz, 1H), 6.60 (dd, J = 9.0, 2.1 Hz, 1H), 7.35–
7.13 (m, 9H), 7.42 (dd, J = 6.9, 2.1 Hz, 1H); 13C NMR (75 MHz,
CDCl3) d 12.9, 17.0, 35.0, 35.4, 35.7, 38.3, 39.2, 44.2, 45.2, 63.0,
63.2, 97.8, 109.0, 109.9, 115.5, 115.7, 119.4, 121.0, 126.6, 127.0,
127.1, 127.7, 127.9, 129.2, 129.3, 131.2, 131.3, 134.3, 134.4,
142.1, 142.2, 142.5, 142.7, 149.0, 149.1, 150.6, 155.4, 158.3,
158.5, 162.3, 162.6, 163.2, 195.4, 195.6; LC–MS [M+H]+ calcd for
C14H15NO3: 246.1, found: 246.1.
5.1.8. 3-Bromo-7-methoxy-4-methyl-2H-chromen-2-one (20)
N-Bromosuccinimide (224 mg, 1.26 mmol) was added slowly to
a
suspension of 7-methoxy-4-methyl-2H-chromen-2-one (17)
(200 mg, 1.05 mmol) in PEG-400 (6 mL). The reaction mixture
was stirred at rt for 2 h. The reaction mixture was extracted with
H2O (25 mL) and EtOAc (3 ꢂ 25 mL) and the combined organic
phases were washed with brine (30 mL). The organic phase was
dried over Na2SO4. After concentration in vacuo the crude product
was purified by column chromatography on silica gel. This afforded
the titled compound as a white solid (130 mg, 0.48 mmol, 46%
yield); Rf = 0.35 (EtOAc/heptane 1:2); mp 144–146 °C (decom-
posed); 1H NMR (300 MHz, CDCl3) d 2.60 (s, 3H), 3.88 (s, 3H),
6.81 (d, J = 2.7 Hz, 1H), 6.88 (dd, J = 9.0, 2.7 Hz, 1H), 7.54 (d,
J = 9.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 19.5, 55.8, 100.7,
109.7, 112.9, 113.4, 126.0, 151.1, 153.6, 157.3, 162.7; LC–MS
[M+H]+ calcd for C11H9BrO3: 269.0, found: 269.0; Anal. calcd for
C36H33N3O4: 572.2, found: 572.2; Anal. calcd for C36H33N3O4: C,
75.64; H, 5.82; N, 7.35, found: C, 75.44, H, 5.59; N, 7.12.
5.1.5. 20-Amino-7-methoxy-40-methyl-2,50-dioxo-50,60,70,80-
tetrahydro-2H,40H-4,70-bichromene-30-carbonitrile (15)
N-Methylmorpholine (10
lL, 88 lmol) was added to a solution
of 5-(7-methoxy-2-oxo-2H-chromen-4-yl)cyclohexane-1,3-dione
C11H9BrO3: C, 49.10, H, 3.37, found: C, 48.76; H, 3.13.

T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
6837
5.1.9. 3-Bromo-7-(diethylamino)-4-methyl-2H-chromen-2-one
(21)
7-(Diethylamino)-4-methyl-2H-chromen-2-one
5.1.12. (E)-7-Methoxy-4-(3-oxobut-1-enyl)-2H-chromen-2-one
(24)
(3)
(1.5 g,
A
solution of diethyl-2-oxopropylphosphonate (518 lL,
6.48 mmol), ammonium acetate (500 mg, 6.48 mmol) and N-bro-
mosuccinimide (1.16 g, 6.48 mg) were stirred in dry acetonitrile
(25 mL) at rt under a N2 atmosphere. The reaction mixture was
stirred at rt for 68 h. The crude reaction was concentrated and
H2O (50 mL) was added. The water phase was extracted with
EtOAc (3 ꢂ 50 mL) and the combined organic phases were washed
with brine (30 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 titled compound as
a yellow solid (1.02 g, 3.31 mmol, 51% yield): Rf = 0.07 (EtOAc/hep-
tane 1:10); 1H NMR (300 MHz, CDCl3) d 1.21 (t, J = 7.2 Hz, 6H), 2.51
(s, 3H), 3.40 (q, J = 7.2 Hz, 4H), 6.45 (d, J = 2.7 Hz, 1H), 6.58 (dd,
J = 9.0, 2.7 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H); 13C NMR (75 MHz,
CDCl3) d 12.9, 19.5, 45.2, 97.6, 105.9, 109.3, 109.4, 126.5, 151.0,
151.9, 154.8, 158.5; LC–MS [M+H]+ calcd for C14H16BrNO2: 310.1,
found: 310.1; Anal. calcd for C14H16NO2: C, 54.21; H, 5.20; N,
4.52, found: C, 53.80; H, 4.90; N, 4.90.
2.45 mmol) in dry THF (4 mL) was added dropwise over 40 min
to a solution of NaH (60% dispersion in mineral oil, 108 mg,
2.70 mmol) in dry THF (20 mL) at rt under a N2 atmosphere. The
lightly yellow solution was stirred for an additional 30 min at rt,
after which a solution of 7-methoxy-2-oxo-2H-chromene-4-carb-
aldehyde (18) (500 mg, 2.45 mmol) in dry THF (30 mL) was added
dropwise over 40 min at 0 °C. The reaction mixture was stirred at rt
for 3 h. The reaction mixture was quenched with H2O (10 mL) and
extracted with EtOAc (3 ꢂ 30 mL) and the combined organic
phases were washed with brine (30 mL). The organic phase was
dried over Na2SO4. After concentration in vacuo the crude product
was purified by column chromatography on silica gel. This afforded
the titled compound as a yellow solid (542 mg, 2.23 mmol, 91%
yield): Rf = 0.25 (EtOAc/heptane 1:1); mp 144–146 °C; 1H NMR
(300 MHz, CDCl3) d 2.44 (s, 3H), 3.89 (s, 3H), 6.38 (s, 1H), 7.80 (d,
J = 16.2 Hz, 1H), 6.83–6.89 (m, 2H), 7.56 (d, J = 8.4 Hz, 1H), 7.71
(d, J = 16.2 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 28.8, 55.9, 101.3,
110.0, 111.3, 112.7, 125.4, 133.7, 134.8, 148.2, 155.6, 160.7,
163.1, 196.7; LC–MS [M+H]+ calcd for C14H12O4: 245.1, found:
245.1; Anal. calcd for C14H12O4: C, 68.85, H, 4.94, found: C, 68.51;
H, 4.80.
5.1.10. 4-(7-Methoxy-4-methyl-2-oxo-2H-chromen-3-
yl)benzaldehyde (22)
3-Bromo-7-methoxy-4-methyl-2H-chromen-2-one
(120 mg, 0.45 mmol), 4-formyl-phenylboronic acid (67 mg,
0.45 mmol), PdCl2(dppf) (6.5 mg, 9 mol) and Na2CO3 (123 mg,
(20)
l
5.1.13. (E)-7-(Diethylamino)-4-(3-oxobut-1-enyl)-2H-chromen-
1.16 mmol) were stirred in a mixture of DMF/H2O (5 mL, 4:1) at
90 °C under a N2 atmosphere for 5 h. The reaction mixture was
cooled to rt and H2O (30 mL) was added. The reaction mixture
was extracted with dichloromethane (3 ꢂ 30 mL) and the combined
organic phases were washed with H2O (3 ꢂ 30 mL) and brine
(30 mL). The organic phase was dried over Na2SO4. After concentra-
tion in vacuo the crude product was purified by column chromatog-
raphy on silica gel. This afforded the titled compound as an off-
white solid (113 mg, 0.39 mmol, 86% yield): Rf = 0.36 (EtOAc/hep-
tane 1:1); mp 188–190 °C (decomposed); 1H NMR (300 MHz, CDCl3)
d 3.89 (s, 3H), 2.30 (s, 3H), 6.85 (d, J = 2.7 Hz, 1H), 6.90 (dd, J = 9.0,
2.7 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 9.0 Hz, 1H), 7.95 (d,
J = 8.1 Hz, 2H), 10.04 (s, 1H); 13C NMR (75 MHz, CDCl3) d 16.7,
55.8, 100.7, 112.6, 113.7, 123.0, 126.2, 129.6, 129.7, 131.1, 131.2,
135.7, 141.1, 148.6, 154.5, 160.8, 162.7, 191.8; LC–MS [M+H]+ calcd
for C18H14O4: 295.1, found: 295.2; Anal. calcd for C18H14O4: C, 73.46;
H, 4.79, found: C, 73.64, H, 4.61.
2-one (25)
A
solution of diethyl-2-oxopropylphosphonate (259 lL,
1.34 mmol) in dry THF (4 mL) was added dropwise over 1 h to a
solution of NaH (60% dispersion in mineral oil, 54 mg, 1.34 mmol)
in dry THF (10 mL) at rt under a N2 atmosphere. The light yellow
solution was stirred for an additional 45 min at rt, after which a
solution of 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde
(19) (300 mg, 1.22 mmol) in dry THF (5 mL) was added dropwise
over 30 min at 0 °C. The reaction mixture was stirred at rt for
2 days. The crude reaction was quenched with H2O (5 mL) and ex-
tracted with dichloromethane (3 ꢂ 25 mL) and the combined or-
ganic phases were washed with brine (25 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 titled compound as a red solid (312 mg, 1.08 mmol,
89% yield): Rf = 0.38 (dichloromethane/EtOAc 10:1); mp 143–
144 °C (decomposed); 1H NMR (300 MHz, CDCl3)
d 1.22 (t,
J = 6.9 Hz, 6H), 2.43 (s, 3H), 3.42 (q, J = 6.9 Hz, 4H), 6.16 (s, 1H),
6.50 (d, J = 2.1 Hz, 1H), 6.58 (dd, J = 9.0, 2.1 Hz, 1H), 6.77 (d,
J = 15.6 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 15.6 Hz, 1H);
13C NMR (75 MHz, CDCl3) d 12.4, 28.7, 44.8, 97.9, 106.0, 106.8,
108.7, 125.3, 133.1, 135.6, 148.1, 150.9, 156.4, 161.8, 197.2; LC–
MS [M+H]+ calcd for C17H19NO3: 286.1, found: 286.1; Anal. calcd
for C17H19NO3: C, 71.56; H, 6.71; N, 4.91, found: C, 71.40; H,
6.48; N, 4.85.
5.1.11. 4-(7-(Diethylamino)-4-methyl-2-oxo-2H-chromen-3-
yl)benzaldehyde (23)
3-Bromo-7-(diethylamino)-4-methyl-2H-chromen-2-one (21)
(400 mg, 1.29 mmol), 4-formyl-phenylboronic acid (194 mg,
1.29 mmol), PdCl2(dppf) (19 mg, 26 lmol) and Na2CO3 (355 mg,
3.35 mmol) were stirred in a mixture of DMF/H2O (10 mL, 4:1) at
90 °C under a N2 atmosphere for 5 h. The reaction mixture was
cooled to rt and H2O (30 mL) was added. The reaction mixture
was extracted with dichloromethane (3 ꢂ 30 mL). The combined
organic phases were washed with H2O (3 ꢂ 30 mL) and brine
(30 mL). The organic phase was dried over Na2SO4. After concen-
tration in vacuo the crude product was purified by column chro-
matography on silica gel. This afforded the titled compound as a
yellow solid (354 mg, 1.06 mmol, 82% yield): Rf = 0.30 (EtOAc/hep-
tane 8:10); mp 140–142 °C; 1H NMR (300 MHz, CDCl3) d 1.23 (t,
J = 6.9 Hz, 6H), 2.25 (s, 3H), 3.44 (q, J = 6.9 Hz, 4H), 6.55 (d,
J = 2.4 Hz, 1H), 6.64 (dd, J = 9.0, 2.4 Hz, 1H), 7.46–7.52 (m, 3H),
7.92–7.96 (m, 2H), 10.10 (s, 1H); 13C NMR (75 MHz, CDCl3) d
12.9, 16.8, 45.2, 97.8, 109.2, 109.5, 120.1, 126.7, 130.0, 131.9,
135.8, 142.4, 149.4, 151.0, 155.7, 161.9, 192.3; LC–MS [M+H]+ calcd
for C21H21NO3: 336.2, found: 336.2; Anal. calcd for C21H21NO3: C,
75.20; H, 6.31; N, 4.52, found: C, 74.92; H, 5.92; 4.24.
5.1.14. 5-(7-Methoxy-2-oxo-2H-chromen-4-yl)cyclohexane-1,3-
dione (26)
A solution of NaOEt (688
added dropwise over 40 min to a stirred solution of diethylmalo-
nate (290 L, 1.84 mmol) in abs EtOH (7 mL) at rt under a N2 atmo-
lL, 1.84 mmol) in abs EtOH (3 mL) was
l
sphere. This yellow solution was stirred at rt for an additional 1 h,
after which it was added dropwise over 1 h to a yellow suspension
of (E)-7-methoxy-4-(3-oxobut-1-enyl)-2H-chromen-2-one (24)
(300 mg, 1.22 mmol) in abs EtOH (15 mL). The dark red solution
was left to stir at rt for 1 h and refluxed for 3 h at 90 °C. The reac-
tion mixture was concentrated and the brown sticky residue was
dried in vacuo. To this residue H2O (3 mL) and 2 M NaOH (3 mL,
6.1 mmol) were added and the reaction mixture was stirred at
100 °C for 5 h, where after it was cooled to rt. The pH was adjusted

6838
T. H. V. Huynh et al. / Bioorg. Med. Chem. 20 (2012) 6831–6839
to 2 by adding 2 M H2SO4 and stirred at 100 °C for 1 h. The crude
reaction was extracted with EtOAc (3 ꢂ 30 mL) and the combined
organic phases were washed with H2O (20 ml) and brine (30 mL).
The organic phase was dried over Na2SO4. After concentration in
vacuo the crude product was purified by column chromatography
on silica gel. This afforded the titled compound as a pale-yellow so-
lid (132 mg, 0.46 mmol, 38% yield): Rf = 0.28 (EtOAc/heptane
(10:1) ? EtOAc/MeOH (10:1)); mp 200–202 °C (decomposed); 1H
NMR (300 MHz, DMSO-d6) d 2.24–2.82 (m, 4H), 3.76–3.92 (m,
1H), 3.85 (s, 3H), 5.30 (s, 1H), 6.26 (s, 1H), 6.94 (dd, J =9.0,
2.7 Hz, 1H), 7.00 (d, J = 2.7 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 11.30
(s, 1H); (100 MHz, DMSO-d6) d 33.3, 55.9, 56.0, 101.2, 103.4,
109.1, 111.3, 112.3, 125.9, 155.1, 157.3, 160.4, 162.3; LC–MS
[M+H]+ calcd for C16H14O5: 287.1, found: 287.1; Anal. calcd C,
67.13; H, 4.93, for C16H14O5: C, 67.40; H, 4.97.
156.5, 157.6, 157.7, 161.4, 161.9, 162.0, 162.1, 194.4, 194.6; LC–MS
[M+H]+ calcd for C24H25N3O4: 420.2, found: 420.2; Anal. calcd for
C24H25N3O4: C, 68.72; H, 6.01; N, 10.02, found: C, 68.35; H, 6.10;
N, 9.67.
5.2. Pharmacology
Cell culture of the EAAT1,2,3-HEK293 cell lines and the
[3H]-
D-Aspartate uptake assay were performed essentially as previ-
ously described.34 The experimental procedures are described in
detail in Supplementary data.
Acknowledgements
We would like to thank Camilla Jensen, Department of Pharma-
ceutics and Analytical Chemistry, Faculty of Pharmaceutical Sci-
ences, University of Copenhagen, for the technical assistance
with the Ultrospec 4300 Pro UV/visible Spectrophotometer. We
would like to express our gratitude to the Lundbeck Foundation,
the Novo Nordisk Foundation, the Augustinus Foundation, the
Carlsberg Foundation, Drug Research Academy—University of
Copenhagen, and the Danish Medical Research Council for financial
support.
5.1.15. 5-(7-(Diethylamino)-2-oxo-2H-chromen-4-
yl)cyclohexane-1,3-dione (27)
A solution of NaOEt (491
added dropwise over 40 min to a stirred solution of diethylmalo-
nate (206 L, 1.31 mmol) in abs EtOH (7 mL) at rt under a N2 atmo-
lL, 1.31 mmol) in abs EtOH (3 mL) was
l
sphere. This yellow solution was stirred at rt for an additional 1 h,
after which it was added dropwise over 50 min to a red solution of
(E)-7-(diethylamino)-4-(3-oxobut-1-enyl)-2H-chromen-2-one
(25) (250 mg, 0.88 mmol) in a mixture of abs EtOH and THF (20 mL,
3:1). The dark red solution was left to stir at rt for 1 h and refluxed
for 3 h at 90 °C. The reaction mixture was concentrated and the
brown sticky residue was dried in vacuo. To this residue H2O
(3 mL) and 2 M NaOH (2.2 mL, 4.38 mmol) were added and the
reaction mixture was stirred at 100 °C for 6 h, where after it was
cooled to rt. The pH was adjusted to 2 by adding 2 M H2SO4 and
stirred at 100 °C for 1 h. The crude reaction was quenched with
sat NH4Cl (10 mL) and extracted with EtOAc (3 ꢂ 30 mL) and the
combined organic phases were washed with H2O (20 ml) and brine
(30 mL). The organic phase was dried over Na2SO4. After concen-
tration in vacuo the crude product was purified by column chro-
matography on silica gel. This afforded the titled compound as a
yellow solid (56 mg, 0.18 mmol, 20% yield): Rf = 0.10 (EtOAc/hep-
tane (10:1) ? EtOAc/MeOH (10:1)); mp 132–134 °C; 1H NMR
(300 MHz, CDCl3) d 1.24 (t, J = 6.9 Hz, 6H), 2.48–2.73 (m, 4H),
3.44 (q, J = 6.9 Hz, 4H), 3.65–3.76 (m, 1H), 5.51(s, 1H), 6.00 (s,
1H), 6.56 (d, J = 2.4 Hz, 1H), 6.62 (dd, J = 9.3, 2.4 Hz, 1H), 7.41 (d,
J = 9.3 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 12.3, 24.9, 29.5,
33.1, 41.9, 43.9, 97.1, 103.4, 105.2, 106.4, 108.8, 125.5, 150.2,
155.1, 157.6, 161.0, 162.9; LC–MS [M+H]+ calcd for C19H21NO4:
328.1, found: 328.1.
Supplementary data
Supplementary data associated (the emission spectra for the
coumarin analogs 12, 14 and UCPH-102F are available) with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.09.049.
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