Identification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity Relationship Study

Article abstract of DOI:10.1021/acs.jmedchem.6b01058

Screening of a small compound library at the three excitatory amino acid transporter subtypes 1-3 (EAAT1-3) resulted in the identification of compound (Z)-4-chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoic aci

Full text of DOI:10.1021/acs.jmedchem.6b01058

Article
pubs.acs.org/jmc
Identification of a New Class of Selective Excitatory Amino Acid
Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure−
Activity Relationship Study
Stinne W. Hansen, Mette N. Erichsen, Bingru Fu, Walden E. Bjørn-Yoshimoto, Bjarke Abrahamsen,
Jacob C. Hansen, Anders A. Jensen, and Lennart Bunch*
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, Copenhagen Ø 2100, Denmark
S
* Supporting Information
ABSTRACT: Screening of a small compound library at the
three excitatory amino acid transporter subtypes 1−3
(EAAT1−3) resulted in the identification of compound (Z)-
4-chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-
5-ylidene)methyl)furan-2-yl)benzoic acid (1a) that exhibited a
distinct preference as an inhibitor at EAAT1 (IC50 20 μM)
compared to EAAT2 and EAAT3 (IC50 > 300 μM). This
prompted us to subject 1a to an elaborate structure−activity
relationship study through the purchase and synthesis and
subsequent pharmacological characterization of a total of 36
analogues. Although this effort did not result in analogues with
substantially improved inhibitory potencies at EAAT1
compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this
scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently available
pharmacological tools in the EAAT field but also substantiates the notion that EAAT ligands not derived from α-amino acids
hold considerable potential in terms of subtype-selective modulation of the transporters.
expressed almost exclusively in cerebellum and retina,
respectively.1,2
INTRODUCTION
■
(S)-Glutamate (S)-(Glu) is the major excitatory neuro-
transmitter in the CNS being involved in a plethora of
physiological and pathophysiological processes. Dysfunctional
glutamatergic neurotransmission is known to constitute a core
component of a wide range of cognitive, psychiatric, and
neurotoxic/neurodegenerative disorders. This fact makes
glutamatergic targets highly interesting as drug targets. The
synaptic reuptake of (S)-Glu following its release from
presynaptic terminals into the synaptic cleft during neuro-
transmission is mediated by the family of excitatory amino acid
transporters (EAATs).1−6 The EAAT family consists of five
transporters, termed EAAT1−5. The EAAT1,2 subtypes are
predominantly expressed in glia cells, and the EAAT3−5
subtypes are predominantly expressed in neurons. The five
subtypes are also characterized by distinct expression patterns
in the CNS. EAAT2 is the major physiological subtype believed
to account for >90% of total Glu uptake in the brain, and
EAAT1 and EAAT3 are also widely expressed in the CNS. As
for EAAT1, it is the major EAAT subtype in the cerebellum
(where the highest density of the transporter is found in
Bergmann glia cells), circumventribular organs, inner ear, and
the retina, but the transport are also abundantly distributed in
other CNS regions.1 In contrast, EAAT4 and EAAT5 are
In contrast to the immense efforts made in terms of ligand
development in the glutamate receptor field over the last couple
of decades, the EAATs have been given considerably less
attention as putative drug targets. Because of this, the selection
of pharmacological tools for the transporters are rather limited
and only a few ligands displaying true subtype-selectivity
between the five EAATs have been identified. This has been an
obstacle to explorations of the physiological functions and
putative therapeutic potential of the respective subtypes.2 Most
of the ligand development in the EAAT field has been based on
the scaffolds of the endogenous substrates for the transporters,
(S)-Glu and (S/R)-aspartate (S/R)-(Asp) (Figure 1A). As
exemplified in Figure 1B, these efforts have yielded a couple of
ligands exhibiting distinct selectivity or preference for one
EAAT subtype over others. The substrate binding sites in the
EAATs are believed to be quite conserved, and this presents a
challenge to the development of orthosteric subtype-selective
ligands.3,5,6 In the light of this, we have previously applied the
strategy of identification of novel EAAT inhibitors by screening
of compound libraries followed by medicinal chemistry
Received: July 18, 2016
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of the endogenous EAAT substrates (S)-Glu and (S/R)-Asp and reported subtype preferring/selective EAAT
inhibitors: WAY213613,13 (2S)-3-((3-(trifluoromethyl)phenyl)sulfonamido)aspartic acid (40),14 L-β-BA,15 DHK,16,17 (2S,4R)-2-amino-4-(3-(2,2-
diphenylethylamino)-3-oxopropyl)pentanedioic acid (41),18 38,10 and 39.8
optimization. Compounds comprised in these library screenings
are often structurally very different from α-amino acids, which
leads to the assumption that hit compounds likely act through
allosteric sites in the transporter. A successful outcome of this
approach is discovery of the first class of EAAT1-selective
inhibitors, represented by the analogues 2-amino-5,6,7,8-
tetrahydro-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-
4H-chromene-3-carbonitrile (38, UCPH-101)7 and 2-amino-
5,6,7,8-tetrahydro-4-(4-methyl)-7-(naphthalen-1-yl)-5-oxo-4H-
chromene-3-carbonitrile (39, UCPH-102)8 (Figure 1C).9,10
This inhibitor class was developed based on a hit compound
identified in a screening of a commercial 3040-compound
library at EAAT1−3,9 and in subsequent studies the structure−
activity-relationship (SAR) of this scaffold was explored in great
detail.9−12 In the present study, we report the discovery of a
new class of selective EAAT1 inhibitors based on another hit
compound from this screening and present an elaborate
structure−activity relationship (SAR) study of this inhibitor
class.
batch of 1a synthesized by us, which exhibited IC50 values of 12
μM, >300 μM, and >300 μM at EAAT1, EAAT2, and EAAT3,
respectively (Figure 2, Tables 3−4). The fact that no significant
Figure 2. Concentration−inhibition curves for (S)-Glu and 1a at
EAAT1-, EAAT2-, and EAAT3-HEK293 cells in the [3H]-D-Asp
uptake assay. The figure depicts data from single representative
experiments, and data are given as mean SD values (in % of specific
uptake determined in the absence of test compound).
RESULTS AND DISCUSSION
■
From a library screening, compound 1a was found to be a
putative EAAT1-selective inhibitor.7 When tested at a
concentration of ∼100 μM, 1a almost completely inhibited
the EAAT1-mediated uptake of [3H]-D-Asp uptake in EAAT1-
HEK293 cells, whereas it exhibited no significant effects on
[3H]-D-Asp uptake measured in EAAT2- and EAAT3-HEK293
cells in concomitant screenings. This apparent selective
EAAT1-selectivity was subsequently verified by detailed
characterization of the compound in the [3H]-D-Asp uptake
assay. 1a was found to inhibit EAAT1-mediated uptake in a
concentration-dependent manner exhibiting an IC50 value of 20
μM, whereas it did not inhibit EAAT2- or EAAT3-mediated
uptake at concentrations up to 300 μM (Tables 1−2). These
pharmacological properties were subsequently confirmed for a
inhibition of EAAT2,3 was observed for 1a at 300 μM suggests
that the compound is at least 50-fold selective for EAAT1 over
EAAT2,3 (Figure 2).
The interesting selectivity profile exhibited by 1a prompted
us to perform an elaborate SAR study on this compound
scaffold, where a total of 36 analogues were either purchased
from commercial sources or synthesized and subsequently
tested on EAAT1−3 in a [3H]-D-Asp uptake assay. A structural
analysis of 1a identifies five rotatable bounds, of which the aryl-
B
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Figure 3. (A) Low-energy conformation of 1a. (B) Overview of disconnections and chemical modifications of 1a.
heteroaryl and the heteroaryl-alkenyl bonds are restrained by
strong rotational barriers. Thus, 1a is a highly conformationally
locked structure with a well-defined low-energy conformation
(Figure 3A). To explore the SAR of this compound class, we
disconnected 1a into synthetically feasible fragments: an ester
moiety (purple), an aryl moiety (blue), a heteroaromatic
moiety (red), and heterocycle (green) (Figure 3B).
Table 1. Investigation of the Importance of the 2-Chloro-5-
carboxyphenyl Functionality for EAAT1 Activity
b
Pharmacological Characterization of 2a−2e. We first
explored the influence of the chloro-carboxy substituted phenyl
ring on the EAAT1 activity through the testing of the
commercially available analogues 2a−2e (Table 1). Perhaps
not surprisingly, the dramatic structural changes introduced in
analogues 2b−2e resulted in complete loss of inhibitory activity
at EAAT1 (and EAAT2,3). The inactivity of analogue 2a, in
which the phenyl ring substituent is retained but with a
completely different substitution pattern compared to 1a, was
more informative. This suggested that the presence of a
carboxylate group in the 4′-position and/or the presence of a
substituent smaller than a bromo atom in the 2′-position was
important for EAAT1 activity. The exact importance of these
two substituents was investigated further in a subsequent part
of the SAR study.
Pharmacological Characterization of 3a−d. We next
turned to investigate the importance of the ester functionality
by characterizing the commercially available analogues 3a−3d
at EAAT1−3 (Table 2). The ester functionality was found to be
of key importance, and it was therefore decided to retain this
functionality throughout the rest of the SAR study.
Synthesis and Pharmacological Characterization of
1b−m. We then proceeded with a detailed SAR study of the
phenyl ring and its substituents. This work was commenced by
resynthesis of 1a by coupling of 3-bromo-4-chloro benzoic acid
(5) with boronic acid 6 to give 7 in good conversion (Scheme
1). Crude 7 was therefore protected as its benzyl ester under
standard conditions, and 8 was isolated in 65% over two steps.
Condensation with 9, readily obtained from 10, gave the
desired target compounds 11, and after deprotection, 1a in 58%
yield.
a
b
Compound obtained from commercial supplier. None of the
Compounds Displayed Inhibitory Activity at EAAT2,3 (IC50 > 300
μM)
boronic acid 6 gave the desired target compound 1d in 24%.
The synthesis of 1g was achieved starting from 3-bromo-4-
fluoro-benzoic acid which underwent Pd-catalyzed coupling
with 6 (Scheme 1, step b) to give aldehyde 12 in 60%.
Following the same strategy, (Scheme 1, step b) Pd-catalyzed
coupling of 3-(5-formyl-2-furyl)-4-methoxybenzoic acid with
C
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Pharmacological evaluation of 1a−1m at EAAT1−3 (Table
3) revealed a number of interesting observations: Repositioning
of the 2′-chlorine atom to the 4′-position (compound 1b) led
to complete loss of EAAT1 inhibitory activity. The same was
observed when the carboxylate group was relocated from the
5′-position to the 4′-position (compound 1c). Substitution of
the chlorine for a methoxy group (compound 1d) was well
accepted, whereas the benzyloxy group introduced in this
position in analogue 1e evidently was too bulky (IC50 > 300
μM). Analogues with a methyl group (compound 1f) or a
fluorine atom (compound 1g) in this position both exhibited
∼5-fold drop in inhibitory activity. Interestingly, a 1-naphthyl
group (compound 1h) was well accepted, which opens up for
introduction of additional substitutions. Analogues 1i−1m were
all inactive, which further underlines the necessity of a
substituent in the 2′-position and a carboxylate group in the
5′-position. Importantly, the size of the 2′-substituent is clearly
essential for the EAAT1 inhibitory activity, as evidenced by the
equipotent EAAT1 activity of the Cl- and MeO-analogues 1a
and 1d compared to the inactivity of analogues with smaller
(Me, F) or bigger (benzyloxy) substituents. It was decided to
retain the 2′-chloro functionality throughout the rest of the
SAR study.
Table 2. Investigation of the Importance of the Ester
Functionality for EAAT1 Activity (None of the Compounds
Displayed Inhibitory Activity at EAAT2,3 (IC50 > 300 μM))
Synthesis and Pharmacological Characterization of
1n−q. The next step was a SAR study of the importance of the
furan ring and the alkene functionality for EAAT1 activity. In
these analogues, the furan ring of 1a was substituted with a
phenyl (1n and 1o) or a 2,5-thienyl (1p) ring systems and the
alkene for an alkyl bond (1q). Synthesis of the meta-substituted
phenyl analogue 1n was accomplished by coupling of
commercially available 3-bromo-4-chlorobenzoic acid with 6
to give 21, which was then condensed with 9 to give the target
compound 1n in 55% yield. Analogously, the para-substituted
analogue 1o was prepared in 49% yield, while the 2,5-thienyl
analogue 1p was obtained in 50% yield from respective
aldehydes. Synthesis of 1q (Scheme 3) was achieved by first
reducing aldehyde 8 to the alcohol 22, which was then
converted to chloride 23. N-Alkylation of heterocycle 25 under
standard conditions provided 24, which after deprotection gave
1q in 52%.
a
Compound obtained from commercial supplier.
Condensation with 9 gave target compound 1g in 37% yield.
To access benzyloxy analogue 1e, 4-(benzyloxy)-3-bromoben-
zoic acid (14) (Scheme 2) was prepared from 13 by
benzylation of both the phenol and the carboxylic acid
functionalities followed by saponification of the benzyl
ester.19 Coupling of 14 with 6 gave the desired product 4-
(benzyloxy)-3-(5-formylfuran-2-yl)benzoic acid (15) in low
yield as reported by others.20 The synthesis of 1h (Scheme 2)
commenced by regioselective bromination of 3-amino-2-
naphthoic acid (16) to give 3-amino-4-bromo-2-naphthoic
acid (17) in 89%. Subsequent deamination by treatment with
NaNO2 in H2SO4 gave 4-bromo-2-naphthoic acid (18) in 91%.
Coupling of bromine 18 with boronic acid 6 gave the desired
aldehyde 19 in 11% yield. Despite the low yield, the reaction
was not optimized further as condensation of 19 with 9 gave 1h
in quantities sufficient for spectral and pharmacological
characterization. The synthesis of 1c was carried out by
condensation of 20 with 6 to give 1c in 80% yield.
Interestingly, the meta-substituted phenyl analogue 1n
exhibited EAAT1 activity although it displayed slightly lower
inhibitory potency compared to 1a. On the other hand, the
para-substituted phenyl analogue 1o was completely inactive.
a
Scheme 1. Synthesis of 1a
a
Reagents and conditions: (a) (PPh3)2PdCl2, Na2CO3 (aq), DME:EtOH 50 °C; (b) BnBr, K2CO3, DMF, rt; (c) 9, piperidine, EtOH, reflux; (d)
FeCl3, DCM, rt; (e) ethyl 2-bromoacetate, NaH (60 w/w%), THF, reflux.
D
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a
Scheme 2. Synthesis of ortho-Benzyloxy Analogue 1e and 1-Naphthyl Analogue 1h
a
Reagents and conditions: (a) benzyl bromide, anhydrous K2CO3, acetone, reflux; (b) 4 M NaOH (aq), MeOH, rt; (c) (5-formylfuran-2-yl)boronic
acid (6), cat. Pd(PPh3)4, K2CO3, toluene:EtOH (7:3), 90 °C; (d) 9, 0.1 equiv piperidine, 0.1 equiv AcOH, EtOH, 90 °C; (e) Br2, FeBr3, CHCl3, 2 h,
rt; (g) NaNO2, H2SO4, AcOH, then Cu2O, reflux; (g) 6, cat. Pd(PPh3)4, K2CO3, toluene:EtOH (7:3), 90 °C. (h) 9, 0.1 equiv piperidine, 0.1 equiv
AcOH, EtOH, 90 °C.
The 2,5-thienyl analogue 1p was also inactive, which is
surprising given the similarity in substitution geometry
compared to 1n and 1a (Table 4). The heterocyclic analogue
1q displayed no inhibitory activity at EAAT1, which underlines
the importance of the linear geometry of the molecule for
transporter activity.
Synthesis and Pharmacological Characterization of
4b−4e. The limited success enhancing the potency of 1a led us
to explore more fundamental changes to the core skeleton.
Having learned that the carboxylate and the alkene
functionalities were important, we decided to synthesize
truncated as well as conformationally relieved analogues of 1a
wherein only the ethyl 2-(2,4-dioxothiazolidin-3-yl)acetate (9)
heterocycle was conserved. In total, 11 analogues 4b−4f and
4g−4e, respectively, were prepared by condensation of the
corresponding aldehyde (Table 5) with 9 in accordance with
previously applied conditions (Scheme 1). The pharmaco-
logical properties exhibited by these 11 analogues (Table 5)
were however disappointing, as they were all very inactive at
EAAT1−3.
Considering the distinct chemical structure of 1a compared to
the EAAT substrates, it seems plausible that the compound
analogously to 38/39 could mediate its EAAT1 inhibition
through an allosteric site in the transporter.21 However, it
should be stressed that a definite conclusion as to whether 1a
and the other inhibitors in this class are indeed allosteric
inhibitors or actually target the substrate-binding pocket in
EAAT1 will have to await further experimental data. Finally, it
is noteworthy that even though 1a clearly is not nearly as
potent and selective an EAAT1 inhibitor as 387 or 39,8 the
inhibitory potency and degree of selectivity exhibited by 1a at
EAAT1 are nevertheless roughly comparable to the properties
displayed by DHK at EAAT2.16 In view of the extensive use of
this prototypic EAAT2 inhibitor as a pharmacological tool in
studies of EAAT2 function in native tissues over the years, we
propose that 1a analogously could be a valuable tool in future
explorations of the physiological roles mediated by and the
therapeutic potential in EAAT1, be it as a supplement or
alternative to 38/39.
EXPERIMENTAL SECTION
■
CONCLUSION
■
Chemistry. All reactions involving anhydrous 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 (TLC, Merck silica gel 60 F254 aluminum sheets).
Flash chromatography was carried out using Merck silica gel 60A (35−
70 μm), and dry column chromatography was carried out using Merck
silica gel 60 (15−40 μm). 1H and 13C NMR spectra were recorded on
a Bruker Avance 400 MHz 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
We have identified a new class of EAAT1-selective inhibitors by
screening of a commercially available compound library. While
the SAR study presented herein of 1a largely failed to to
improve its EAAT1 inhibitory potency, it did disclose
substantial insight into the pharmacophore elements which
will be useful in future work within the field. More importantly,
the EAAT1-selectivity exhibited by this inhibitor class
substantiates the notion gained from the 38/39 inhibitor
class, that subtype-selective EAAT modulators can emerge from
scaffolds with no structural similarity to the α-amino acids.
E
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containing 0.1% TFA. Melting points were measured using a MPA 100
Optimelt automatic melting point system. Optical rotation was
measured using a PerkinElmer 241 polarimeter with Na lamp (589
nm). All commercial chemicals were used without further purification.
The purity of all tested compounds was determined by HPLC to be
>95%.
Table 3. Detailed SAR Study of the Phenyl Ring and Its
Substitution Pattern for EAAT1 Activity (None of the
Compounds Displayed Inhibitory Activity at EAAT2,3 (IC50
> 300 μM))
(Z)-4-Chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-
5-ylidene)methyl)furan-2-yl)benzoic Acid (1a). Under a nitrogen
atmosphere, (Z)-benzyl 4-chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-
dioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoate (11) (0.100 g,
0.190 mmol, 1 equiv) was dissolved in anhydrous CH2Cl2 (3.8 mL).
FeCl3 (0.124 g, 0.761 mmol, 4 equiv) was added during ice-cooling,
and after 10 min of stirring at that temperature the mixture was left to
come to rt for 2.5 h. The reaction was quenched by addition of H2O
(30 mL) and then extracted with CH2Cl2 (2 × 50 mL). The combined
organic phases were dried using anhydrous MgSO4, filtered, and
evaporated to give a brown solid (0.093 g). The crude product was
purified by dry column vacuum chromatography (eluent: 100:1
CH2Cl2/AcOH) to give the title compound (0.048 g, 58%) as yellow
1
solid. Rf (100:1 CH2Cl2/AcOH) 0.14. H NMR (400 MHz, DMSO-
d6) δ: 8.53 (d, 1H, J = 2.0 Hz), 7.94 (dd, 1H, J = 2.0 Hz, J = 8.3 Hz),
7.92 (s, 1H), 7.75 (d, 1H, J = 8.3 Hz), 7.52 (d, 1H, J = 3.8 Hz), 7.38
(d, 1H, J = 3.8 Hz), 4.50 (s, 2H), 4.18 (q, 2H, J = 7.3 Hz), 1.22 (t, 3H,
J = 7.3 Hz). 13C NMR (100 MHz, DMSO-d6) δ: 167.4, 166.7, 166.0,
164.6, 152.6, 149.0, 133.8, 131.6, 130.9, 130.4, 128.8, 127.1, 121.7,
119.7, 118.2, 115.0, 61.6, 42.2, 13.9. LC-MS [M + H+] calcd for
C19H15ClNO7S, 436.03; found, 436.1. Elem. Anal. Calcd for
C19H14ClNO7S: C, 52.36; H, 3.24; N, 3.21; S, 7.36. Found: C,
52.28; H, 3.31; N, 3.20; S, 7.01. Melting point: 227.0−230.5 °C (1
°C/min) (decomposes).
3-Chloro-4-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)2-furyl)benzoic Acid (1c). 3-Chloro-4-(5-formyl-2-
furyl)benzoic acid (20) (101 mg, 0.403 mmol) was suspended in
abs EtOH (15 mL), and a solution of 3-(2-ethoxy-2-oxoethanyl)2,4-
dioxothiazolidine (81 mg, 0.399 mmol) in abs EtOH (3 mL) was
added. After gentle heating and stirring for 10 min, piperidine (75 μL,
0.758 mmol) was added and the reaction mixture was heated to reflux
and stirred at that temperature for 1 h. Thereafter, the reaction mixture
was cooled to rt and quenched with satd aq NH4Cl (50 mL) and the
resulting precipitate was filtered off, washed with H2O (10 mL), and
dried giving the title compound as a light-brown solid (140 mg, 0.321
1
mmol, 80%). Rf = 0 (n-heptane/EtOAc 1:1). H NMR (400 MHz,
CDCl3) δ: 8.05−7.93 (m, 3H), 7.91 (s, 1H), 7.52 (d, J = 3.9 Hz, 1H),
7.39 (d, J = 3.9 Hz, 1H), 4.50 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t,
J = 7.1 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ: 167.5, 166.7, 166.5,
164.6, 153.3, 149.1, 131.4, 129.3, 128.5, 128.4, 127.8, 121.8, 119.6,
118.1, 115.2, 61.6, 42.2, 14.0 (one signal missing or coinciding with
others). LC-MS (Bruker): [M + H]+
436.02, [M + H]+
436.0.
calcd
found
HPLC: >98%, retention time 2.39 min, reverse phase, H2O/MeCN/
TFA gradient run, 4 mL min−1.
3-(5-{[3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene]-
methyl}-2-furyl)-4-methoxybenzoic Acid (1d). 3-(5-Formyl-2-furyl)-
4-methoxybenzoic acid (150 mg, 0.609 mmol) and 3-(2-ethoxy-2-
oxoethanyl)2,4-dioxothiazolidine (140 mg, 0.690 mmol) were
suspended in abs EtOH (40 mL), and the reaction mixture was
stirred for 5 min at rt under N2 before piperidine (114 μL, 1.157
mmol) was added. The reaction mixture was then heated to reflux and
stirred at that temperature for 5 h. Thereafter, the reaction mixture was
cooled to rt and quenched with satd aq NH4Cl (15 mL) and H2O (20
mL). The resulting dark-brown precipitate was filtered off, washed
with H2O (2 × 5 mL), and dried before being purified by column
chromatography (2 cm Ø, 100 mL SiO2 , eluent
CH2Cl2:EtOAc:AcOH, 100:10:1). The yellow band Rf = 0.31 was
isolated, giving the title compound as an orange solid (64 mg, 0.148
a
b
Compound obtained from commercial supplier. The concentra-
tion−inhibition curve for the compound was not complete within the
tested concentration range, thus the IC50 value was estimated from the
fitted curve.
mass spectrometer equipped with an electrospray ionization source.
Gradients of 5% aqueous acetonitrile containing 0.05% formic acid
(eluent A) and 95% aqueous acetonitrile containing 0.05% formic acid
(eluent 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
1
mmol, 24%). Rf = 0.31 (CH2Cl2:EtOAc:AcOH, 100:10:1). H NMR
(600 MHz, DMSO-d6) δ: 8.48 (d, J = 2.1 Hz, 1H), 7.95 (dd, J = 8.6,
2.1 Hz, 1H), 7.88 (s, 1H), 7.34 (d, J = 3.7 Hz, 1H), 7.23 (d, J = 3.7 Hz,
1H), 7.21 (d, J = 8.6 Hz, 1H), 4.49 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H),
4.01 (s, 3H), 1.22 (t, J = 7.1 Hz, 5H). 13C NMR (150 MHz, CDCl3)
(rotamers) δ: 167.6, 167.0, 166.7, 166.7, 166.5, 164.7, 162.6, 159.1,
F
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a
Scheme 3. Synthesis of Conformationally Relieved Analogue 1q
a
Reagents and conditions. (a) NaBH4, THF, rt; (b) SOCl2, pyridine, DCM; (c) NaH, DMF; (d) H2 (g), Pd/C; (e) NaH, THF, 50 °C.
obtained solid was recrystallized from EtOH which yielded the
product as a yellow solid (4 mg, 7%). 1H NMR (600 MHz, CDCl3) δ:
8.71 (d, J = 2.1 Hz, 1H), 8.09 (dd, J = 8.7, 2.1 Hz, 1H), 7.70 (s, 1H),
7.49−7.38 (m, 5H), 7.14 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 3.6 Hz, 1H),
6.87 (d, J = 3.7 Hz, 1H), 5.30 (s, 2H), 4.48 (s, 2H), 4.24 (q, J = 7.1
Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H). 13C NMR (600 MHz, CDCl3) δ:
168.4, 168.0, 166.5, 165.6, 159.2, 153.9, 148.5, 135.6, 132.3, 129.3,
129.1, 128.8, 128.0, 122.3, 121.0, 119.9, 118.7, 118.3, 114.5, 112.3,
Table 4. SAR Study of the Influence of the Furan Ring and
the Alkene Functionalities on EAAT1 Inhibitory Activity
(None of the Compounds Displayed Inhibitory Activity at
EAAT2,3 (IC50 > 300 μM))
71.3, 62.2, 42.2, 29.9, 14.3. LC-MS: [M + H]+
508.10, [M +
calcd
H]+
508.1.
found
4-Fluoro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)2-furyl)benzoic Acid (1g). 4-Fluoro-3-(5-formyl-2-
furyl)benzoic acid (12) (18 mg, 0.077 mmol) was suspended in abs
EtOH (6 mL), and a solution of 3-(2-ethoxy-2-oxoethanyl)2,4-
dioxothiazolidine (16 mg, 0.0787 mmol) in abs EtOH (3 mL) was
added followed by piperidine (15 μL, 0.151 mmol). The reaction
mixture was heated to reflux under N2 and stirred at that temperature
for 2 h. Thereafter, the reaction mixture was cooled to rt and quenched
with satd aq NH4Cl (20 mL). The resulting precipitate was filtered off,
washed with H2O (10 mL), and dried, giving the title compound as a
brown solid (12 mg, 0.029 mmol, 37%). Rf = 0 (n-heptane/EtOAc
1
1:1). H NMR (400 MHz, DMSO-d6) δ: 13.32 (s, 1H), 8.49 (dd, J =
7.4, 2.2 Hz, 1H), 8.03 (ddd, J = 8.6, 4.9, 2.2 Hz, 1H), 7.94 (s, 1H),
7.54 (dd, J = 11.1, 8.7 Hz, 1H), 7.39 (d, J = 3.8 Hz, 1H), 7.25 (t, J =
3.6 Hz, 1H), 4.50 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz,
3H). 13C NMR (150 MHz, CDCl3) δ: 167.4, 166.7, 165.8, 164.6,
161.6, 159.9, 150.5, 149.1, 131.8, 131.8, 128.1, 127.6, 127.6, 122.1,
119.7, 117.9, 117.2, 117.1, 117.0, 117.0, 114.4, 114.3, 61.6, 42.2, 13.9.
LC-MS: [M + H]+
420.06 [M + H]+
420.0. HPLC: >95%,
calcd
found
retention time 1.96 min, reverse phase, H2O/MeCN/TFA gradient
run, 4 mL min−1. Melting point: decomposition.
(Z)-4-(5-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)furan-2-yl)-2-naphthoic Acid (1h). Piperidine (2 μL,
0.02 mmol, 0.1 equiv) and AcOH (1 μL, 0.02 mmol, 0.1 equiv) were
added to a solution of 19 (45 mg, 0.17 mmol, 1.0 equiv) and 9 (35 mg,
0.17 mmol, 1.0 equiv) in EtOH (8 mL). The mixture was allowed to
stir at reflux for 3 days, and the solvent was removed under reduced
pressure. The crude product was purified by gradient flash
chromatography (heptane:EtOAc, 1:0−2:1, with 0.5% of AcOH) on
silica gel, which yielded the product as a yellow solid (33 mg, 43%). 1H
NMR (600 MHz, DMSO-d6) δ: 13.32 (s, 1H), 8.72 (s, 1H), 8.44 (d, J
= 8.6 Hz, 1H), 8.40 (d, J = 1.6 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 7.97
(s, 1H), 7.82 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 7.74 (ddd, J = 8.1, 7.0, 1.1
Hz, 1H), 7.47 (d, J = 3.7 Hz, 1H), 7.43 (d, J = 3.7 Hz, 1H), 4.49 (s,
2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (600
MHz, DMSO-d6) δ: 167.5, 166.8, 166.7, 164.6, 156.3, 149.3, 133.1,
153.3, 151.6, 148.7, 148.0, 131.6, 131.4, 129.6, 127.2, 124.5, 122.4,
121.2, 119.8, 117.4, 117.1, 116.8, 114.4, 114.3, 112.1, 112.0, 61.6, 56.3,
42.1, 13.9. LC-MS (Bruker): [M + H]+
432.07, [M + H]+
calcd
found
432.1. HPLC: >96%, retention time 2.45 min, reverse phase, H2O/
MeCN/TFA gradient run, 4 mL min−1.
(Z)-4-(Benzyloxy)-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazo-
lidin-5-ylidene)methyl)furan-2-yl)benzoic Acid (1e). Synthesis was
carried out as described for 1h, with aldehyde 15 (36 mg, 0.11 mmol,
1.0 equiv), 9 (67 mg, 0.33 mmol, 3.0 equiv), piperidine (1 μL, 0.01
mmol, 0.1 equiv), AcOH (1 μL, 0.01 mmol, 0.1 equiv), and EtOH (5
mL) and reflux for 2 days. The precipitate was taken into acetone and
filtered. The filtrate was concentrated under reduced pressure, and the
G
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Table 5. SAR Study of Conformationally Relieved Analogues 4a−g Wherein the Ethyl 2-(2,4-Dioxothiazolidin-3-yl)acetate (9)
a
Heterocycle Is Conserved from Lead Structure 1a
a
None of the compounds 4a−g displayed inhibitory activity at EAAT2,3 (IC50 > 300 μM).
132.1, 130.7, 130.5, 129.7, 127.8, 127.4, 126.6, 125.6, 124.9, 122.1,
120.0, 117.2, 114.0, 61.6, 42.1, 13.9. Melting point: 158.9−161.5 °C.
6-Chloro-3′-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)-[1,1′-biphenyl]-3-carboxylic Acid (1n). 6-Chloro-3′-
formyl-[1,1′-biphenyl]-3-carboxylic acid (21) (30 mg, 0.115 mmol)
was suspended in abs EtOH (3 mL) and a solution of 3-(2-ethoxy-2-
oxoethanyl)2,4-dioxothiazolidine (23 mg, 0.115 mmol) in abs EtOH
(3 mL) was added and the reaction mixture was stirred for 5 min at rt
under N2 before piperidine (21.6 μL, 0.219 mmol) was added. The
reaction mixture was then heated to reflux and stirred at that
temperature for 2 h. Thereafter, the reaction mixture was cooled to rt
and quenched with satd aq NH4Cl (20 mL). The resulting yellow
precipitate was filtered off, washed with H2O (10 mL), and dried,
H
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giving the title compound as a yellow solid (28 mg, 0.063 mmol, 55%).
Rf = 0 (n-heptane/EtOAc 1:1). H NMR (400 MHz, DMSO-d6) δ:
product was purified by dry column vacuum chromatography (eluent:
2:8:0.1 EtOAc/CH2Cl2/AcOH) and coevaporation with toluene to
give the title compound (0.145 g, 52%) as white solid. Rf (25:75:1
1
8.10 (s, 1H), 7.92−7.85 (m, 2H), 7.76−7.64 (m, 3H), 7.62−7.53 (m,
2H), 4.50 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H).
13C NMR (150 MHz, CDCl3) δ: 166.8, 166.7, 166.6, 164.8, 139.8,
137.7, 133.9, 132.8, 132.0, 131.7, 131.0, 130.1, 129.5, 129.3, 129.2,
1
EtOAc/CH2Cl2/AcOH) 0.32. H NMR (400 MHz, CDCl3) δ: 8.43
(d, 1H, J = 2.0 Hz), 7.85 (dd, 1H, J = 2.0 Hz, J = 8.3 Hz), 7.48 (d, 1H,
J = 8.3 Hz), 7.05 (d, 1H, J = 3.5 Hz), 6.41 (d, 1H, J = 3.5 Hz), 4.63 (s,
2H), 4.22 (s, 2H), 4.15 (q, 2H, J = 7.3 Hz), 3.99 (s, 2H), 1.21 (t, 3H, J
= 7.3 Hz). 13C NMR (100 MHz, CDCl3) δ: 170.2, 169.2, 167.0, 155.7,
149.7, 149.3, 135.7, 131.2, 129.7, 129.5, 129.1, 128.3, 112.7, 111.2,
62.0, 49.8, 39.9, 39.6, 14.1. LC-MS [M + H+] calcd for C19H18ClN2O7,
421.08; found, 421.1. Elem. Anal. Calcd for C19H17ClN2O7·0.05C7H8:
C, 54.63; H, 4.12; N, 6.58. Found: C, 54.72; H, 4.22; N, 6.65. Melting
point: 120.1−123.8 °C (1 °C/min).
121.2, 61.7, 42.3, 14.0. LC-MS (Bruker): [M + H]+
446.05 [M +
calcd
H]+
446.0. HPLC: >95%, retention time 2.67 min, reverse phase,
found
H2O/MeCN/TFA gradient run, 4 mL min−1.
6-Chloro-4′-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)-[1,1′-biphenyl]-3-carboxylic Acid (1o). 6-Chloro-4′-
formyl-[1,1′-biphenyl]-3-carboxylic acid (30 mg, 0.115 mmol) was
suspended in abs EtOH (3 mL) and a solution of 3-(2-ethoxy-2-
oxoethanyl)2,4-dioxothiazolidine (23 mg, 0.115 mmol) in abs EtOH
(3 mL) was added and the reaction mixture was stirred for 5 min at rt
under N2 before piperidine (21.6 μL, 0.219 mmol) was added. The
reaction mixture was then heated to reflux and stirred at that
temperature for 4 h. Thereafter, the reaction mixture was cooled to rt
and quenched with satd aq NH4Cl (20 mL). The resulting yellow
precipitate was filtered off, washed with H2O (10 mL), and dried,
giving the title compound as a yellow solid (28 mg, 0.063 mmol, 55%).
Rf = 0 (n-heptane/EtOAc 1:1), 0.3 (CH2Cl2:EtOAc:AcOH, 100:10:1).
1H NMR (600 MHz, DMSO-d6) δ: 8.08 (s, 1H), 7.98−7.90 (m, 2H),
(Z)-4-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)-
methyl)benzoic Acid (4a). Synthesis was carried out as described for
1h, with commercially available 4-formylbenzoic acid (100 mg, 0.67
mmol, 1 equiv), 9 (136 mg, 0.67 mmol, 1 equiv), piperidine (7 μL,
0.07 mmol, 0.1 equiv), AcOH (4 μL, 0.07 mmol, 0.1 equiv), and
EtOH (5 mL) and reflux for 4 days. The precipitate was washed with
EtOH and dried, which yielded the product as an off-white solid (98
1
mg, 44%). H NMR (400 MHz, DMSO-d6) δ: 13.24 (s, 1H), 8.06 (s,
1H), 8.10−7.75 (m, 4H), 4.51 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22
(t, J = 7.1 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ: 167.1, 167.0,
166.9, 165.2, 137.1, 133.3, 132.7, 130.7, 130.5, 123.3, 62.2, 42.8, 14.4;
Melting point: 200.9−205.1 °C.
7.82−7.76 (m, 2H), 7.72−7.64 (m, 3H), 4.52 (s, 2H), 4.19 (q, J = 7.1
Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H), signal from COOH is missing. 13C
NMR (150 MHz, CDCl3) δ: 166.8, 166.7, 166.3, 164.9, 140.4, 138.6,
133.5, 132.4, 131.9, 130.3, 130.2, 121.1, 61.7, 42.3, 14.0, six Ar-C
signals are missing or coinciding with others. LC-MS (Bruker): [M +
H]+calcd 446.05 [M + H]+found 446.0. HPLC: >96%, retention time 2.70
min, reverse phase, H2O/MeCN/TFA gradient run, 4 mL min−1.
4-Chloro-3-(5-{[3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene]methyl}thiophen-2-yl)benzoic Acid (1p). 4-Chloro-3-(5-
formylthiophen-2-yl)benzoic acid (17.6 mg, 0.065 mmol) was
suspended in abs EtOH (2 mL) and a solution of 3-(2-ethoxy-2-
oxoethanyl)2,4-dioxothiazolidine (15 mg, 0.072 mmol) in abs EtOH
(1 mL) was added and the reaction mixture was stirred for 5 min at rt
under N2 before piperidine (12 μL, 0.125 mmol) was added. The
reaction mixture was then heated to reflux and stirred at that
temperature for 2.5 h. Thereafter, the reaction mixture was cooled to rt
and quenched with satd aq NH4Cl (20 mL). The resulting orange
precipitate was filtered off, washed with H2O (10 mL), and dried. The
solid was subsequently redissolved in abs EtOH (2 mL), a solution of
3-(2-ethoxy-2-oxoethanyl)2,4-dioxothiazolidine (9 mg, 0.044 mmol)
in abs EtOH (1 mL) was added, and the reaction mixture was stirred
for 5 min at rt under N2 before piperidine (20 μL, 0.202 mmol) was
added. The reaction mixture was then heated to reflux and stirred at
that temperature for 5 h. Thereafter, the reaction mixture was cooled
to rt and quenched with satd aq NH4Cl (5 mL) and H2O (5 mL). The
resulting orange precipitate was filtered off, washed with H2O (5 mL),
and dried, giving the title compound as an orange solid (15 mg, 0.033
(Z)-4-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)-
methyl)-3-hydroxybenzoic Acid (4b). Synthesis was carried out as
described for 1h, with commercially available 4-formyl-3-hydrox-
ybenzoic acid (200 mg, 1.20 mmol, 1.0 equiv), 9 (244 mg, 1.20 mmol,
1.0 equiv), piperidine (12 μL, 0.12 mmol, 0.1 equiv), AcOH (7 μL,
0.12 mmol, 0.1 equiv), and EtOH (7 mL) and reflux for 4 days.
Purified by gradient flash chromatography (heptane:EtOAc, 1:1−1:1.5,
with 0.5% of AcOH) on silica gel, which yielded the product as a
yellow solid (130 mg, 31%). 1H NMR (600 MHz, DMSO-d6) δ: 10.99
(s, 1H), 8.14 (s, 1H), 7.55−7.50 (m, 3H), 4.49 (s, 2H), 4.18 (q, J = 7.1
Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ:
166.9, 166.7, 166.5, 165.0, 157.1, 134.0, 129.0, 128.3, 123.6, 121.5,
120.4, 116.5, 61.7, 42.2, 13.9. Melting point: 225.3−229.1 °C.
(Z)-3-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)-
methyl)benzoic Acid (4c). Synthesis was carried out as described for
1h, with commercially available 3-formylbenzoic acid (28) (330 mg,
2.20 mmol, 1.0 equiv), 9 (447 mg, 2.20 mmol, 1.0 equiv), piperidine
(22 μL, 0.22 mmol, 0.1 equiv), AcOH (13 μL, 0.22 mmol, 0.1 equiv),
and EtOH (8 mL) and reflux for 16 h. The reaction mixture was
allowed to cool to rt, and the precipitate was filtered out and
recrystallized from EtOH, which yielded the product as a white solid
1
(334 mg, 45%). H NMR (400 MHz, DMSO-d6) δ: 13.31 (s, 1H),
8.20 (t, J = 1.2 Hz, 1H), 8.10 (s, 1H), 8.05 (dt, J = 7.8, 1.2 Hz, 1H),
7.91 (dt, J = 7.9, 1.2 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 4.51 (s, 2H),
4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (400
MHz, DMSO-d6) δ: 166.6, 166.5, 166.4, 164.7, 134.3, 133.2, 133.1,
131.8, 131.2, 130.4, 129.8, 121.7, 61.7, 42.3, 13.9. Melting point:
198.3−202.1 °C.
(Z)-5-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)-
methyl)-2-hydroxybenzoic Acid (4d). Synthesis was carried out as
described for 1h, with commercially available 5-formyl-2-hydrox-
ybenzoic acid (100 mg, 0.60 mmol, 1.0 equiv), 1 (122 mg, 0.60 mmol,
1.0 equiv), piperidine (6 μL, 0.06 mmol, 0.1 equiv), AcOH (4 μL, 0.06
mmol, 0.1 equiv), and EtOH (5 mL) and reflux for 11 days. Purified
by flash chromatography (heptane:EtOAc, 1:5, with 0.5% of AcOH)
on silica gel, which yielded the product as a yellow solid (75 mg, 36%).
1H NMR (600 MHz, DMSO-d6) δ: 8.08 (d, J = 2.4 Hz, 1H), 7.97 (s,
1H), 7.77 (dd, J = 8.7, 2.4 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 4.48 (s,
2H), 4.17 (q, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). 13C NMR (600
MHz, DMSO-d6) δ: 170.9, 166.7, 166.6, 165.0, 163.4, 137.0, 133.6,
132.9, 123.5, 118.5, 117.5, 114.9, 61.6, 42.2, 13.9. Melting point:
199.1−202.8 °C.
(Z)-3-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)-
methyl)-4-hydroxybenzoic Acid (4e). Synthesis was carried out as
described for 1h, with commercially available 3-formyl-4-hydrox-
1
mmol, 50%). Rf = 0.31 (CH2Cl2:EtOAc:AcOH, 100:10:1). H NMR
(600 MHz, DMSO-d6) δ: 13.44 (s, 1H), 8.32 (s, 1H), 8.17 (d, J = 2.1
Hz, 1H), 7.96 (dd, J = 8.3, 2.1 Hz, 1H), 7.83 (d, J = 4.0 Hz, 1H), 7.78
(d, J = 8.4 Hz, 1H), 7.73 (d, J = 3.9 Hz, 1H), 4.50 (s, 2H), 4.18 (q, J =
7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ:
167.1, 166.5, 166.4, 165.0, 145.4, 138.5, 136.1, 135.9, 132.2, 131.9,
131.7, 131.2, 130.9, 130.1, 127.4, 119.0, 62.1, 42.9, 14.4. LC-MS: [M +
H]+calcd 452.00 [M + H]+found 451.9. HPLC: >96%, retention time 2.08
min, reverse phase, H2O/MeCN/TFA gradient run, 4 mL min−1.
4-Chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxoimidazolidin-1-
yl)methyl)furan-2-yl)benzoic Acid (1q). Under a nitrogen atmos-
phere, 10% Pd/C (0.136 g) was added to anhydrous THF (20 mL).
H2 was bobbled through the mixture, and it was allowed to stir under a
H2 atmosphere at rt for 20 min. Then a solution of benzyl 4-chloro-3-
(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxoimidazolidin-1-yl)methyl)-
furan-2-yl)benzoate (0.340 g, 0.665 mmol) in anhydrous THF (20
mL) was added, and the mixture was stirred at rt for 2 h. Then the H2
atmosphere was exchanged with nitrogen, and the mixture was filtered
through a plug of Celite, which was then washed with THF. The
filtrate was evaporated to give slightly yellow oil (0.327 g). The crude
I
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ybenzoic acid (250 mg, 1.50 mmol, 1.0 equiv), 9 (305 mg, 1.50 mmol,
1.0 equiv), piperidine (15 μL, 0.15 mmol, 0.1 equiv), AcOH (9 μL,
0.15 mmol, 0.1 equiv), and EtOH (8 mL) and reflux for 2 days.
Purified by gradient flash chromatography (heptanes:EtOAc, 2:1−1:1,
with 0.5% of AcOH) on silica gel, which yielded the product as a
yellow solid (180 mg, 34%), 1H NMR (600 MHz, DMSO-d6) δ: 12.85
(s, 1H), 11.52 (s, 1H), 8.14 (s, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.91 (dd,
J = 8.6, 2.1 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 4.50 (s, 2H), 4.18 (q, J =
7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H), 13C NMR (600 MHz, DMSO-
d6) δ: 166.7, 166.6, 166.4, 165.0, 161.1, 133.8, 130.3, 128.4, 122.1,
120.3, 119.6, 116.2, 61.6, 42.2, 13.9;, Melting point: 200.9−204.0 °C.
(Z)-Ethyl 2-(5-(3-(1H-Tetrazol-5-yl)benzylidene)-2,4-dioxothiazo-
lidin-3-yl)acetate (4f). Piperidine (10 μL, 0.10 mmol, 0.1 equiv) was
added to a solution of 3-(1H-tetrazol-5-yl)benzaldehyde (31) (170
mg, 0.98 mmol, 1.0 equiv) and 9 (199 mg, 0.98 mmol, 1.0 equiv) in
EtOH (4 mL). The mixture was allowed to stir at reflux for 18 h then
cooled to 0 °C. Then 1 M HCl (5 mL) was added and the resulting
precipitate was filtered off, washed with water (8 mL) and hepetanes
(8 mL), and dried, which yielded the product as a white solid (162 mg,
1.0 equiv), 9 (103 mg, 0.51 mmol, 1.0 equiv), piperidine (5 μL, 0.05
mmol, 0.1 equiv), AcOH (3 μL, 0.05 mmol, 0.1 equiv), and EtOH (5
mL) and reflux for 2 days. Purified by flash chromatography
(heptane:EtOAc, 1:1, with 0.5% of AcOH) on silica gel and
recrystallized from a mixture (1:1:0.01) of MeCN, H2O and DMSO,
1
which yielded the product as a yellow solid (84 mg, 43%). H NMR
(600 MHz, DMSO-d6) δ: 12.14 (s, 1H), 10.47 (s, 1H), 8.13 (s, 1H),
7.23−6.89 (m, 3H), 4.48 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 2.78 (t, J =
7.4 Hz, 2H), 2.52 (t, J = 7.3 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C
NMR (600 MHz, DMSO-d6) δ: 173.7, 167.3, 166.7, 165.1, 155.8,
133.0, 132.1, 129.5, 128.1, 119.4, 118.9, 116.2, 61.6, 42.1, 35.3, 29.3,
13.9. Melting point: 185.1−188.3 °C.
(E)-3-(3-((Z)-(3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)phenyl)acrylic Acid (4k). Synthesis was carried out as
described for 1h, with 36 (100 mg, 0.57 mmol, 1.0 equiv), 9 (115 mg,
0.57 mmol, 1.0 equiv), piperidine (6 μL, 0.06 mmol, 0.1 equiv), AcOH
(4 μL, 0.06 mmol, 0.1 equiv), and EtOH (5 mL) and reflux for 3 days.
Purified by flash chromatography (heptane:EtOAc, 1:1.25, with 0.5%
of AcOH) on silica gel, which yielded the product as a white solid (101
1
1
mg, 49%). H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 8.03 (s,
45%). H NMR (400 MHz, DMSO-d6) δ: 8.28 (s, 1H), 8.14 (d, J =
1H), 7.96 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H),
7.64 (d, J = 15.6 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 6.63 (d, J = 16.1
Hz, 1H), 4.51 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz,
3H). 13C NMR (600 MHz, DMSO-d6) δ: 167.4, 166.8, 166.6, 164.8,
142.5, 135.4, 133.5, 133.4, 130.7, 130.4, 130.1, 130.0, 121.4, 121.0,
61.7, 42.3, 13.9. Melting point: 172.6−175.1 °C.
7.7 Hz, 1H), 8.08 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.8 Hz,
1H), 4.52 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H).
13C NMR (400 MHz, DMSO-d6) δ: 166.6, 166.5, 164.7, 155.4, 133.8,
132.9, 132.4, 130.5, 128.9, 128.1, 125.6, 122.2, 61.7, 42.3, 13.9. Melting
point: 220.5−223.8 °C.
(Z)-2-(3-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)phenyl)acetic Acid (4g). Synthesis was carried out as
described for 1h, with 32 (175 mg, 1.07 mmol, 1.0 equiv), 9 (217 mg,
1.07 mmol, 1.0 equiv), piperidine (10 μL, 0.11 mmol, 0.1 equiv),
AcOH (6 μL, 0.11 mmol, 0.1 equiv), and EtOH (5 mL) and reflux for
5 days. Purified by flash chromatography (heptane:EtOAc, 1:1, with
0.5% of AcOH) on silica gel and recrystallized from MeOH, which
4-Chloro-3-(5-formylfuran-2-yl)benzoic Acid (7). A flask was
charged with (5-formylfuran-2-yl)boronic acid (5.95 g, 42.5 mmol, 2
equiv), 3-bromo-4-chlorobenzoic acid (4.96 g, 21.2 mmol, 1 equiv),
and (PPh3)2PdCl2 (0.75 g, 1.06 mmol, 0.05 equiv) and then evacuated
and refilled with N2 three times. DME (70 mL, degassed), EtOH (70
mL, degassed), and 2 M Na2CO3 (64 mL, 128 mmol, 6 equiv,
degassed) were added, and the mixture was heated to 50 °C and
stirred at that temperature for 20 h. The mixture was allowed to cool
to rt and was then evaporated in vacuo. The residue was suspended in
H2O (700 mL) and filtered through a plug of Celite, which was then
washed with H2O (700 mL). The combined aqueous filtrates were
acidified (pH 1−2) using 1 M HCl. The solids were filtered off,
washed with H2O, and freeze-dried to give off-white solid (5.4 g). The
crude product was used in the next step without further purification.
Benzyl 4-Choro-3-(5-formylfuran-2-yl)benzoate (8). Crude 4-
chloro-3-(5-formylfuran-2-yl)benzoic acid (7) (5.44 g, 21.2 mmol, 1
equiv) was dissolved in DMF (125 mL). K2CO3 (4.41 g, 31.8 mmol,
1.5 equiv) was added, followed by benzyl bromide (2.8 mL, 23.4
mmol, 1.1 equiv). The mixture was stirred at rt for 2.5 h, and then the
DMF was reduced in vacuo. The residue was suspended in Et2O (750
mL) and H2O (750 mL). The phases were separated, and the aqueous
phase was washed with H2O (750 mL). Precipitation occurred in the
organic phase, therefore additional Et2O (250 mL) was added followed
by EtOAc (750 mL). The organic phase was washed with H2O (250
mL). The combined aqueous phases were then extracted with EtOAc
(3 × 250 mL). The combined organic phases were dried using
anhydrous MgSO4, filtered, and evaporated to give orange solid (7.29
g). The crude product was purified by dry column vacuum
chromatography (EtOAc/heptane) to give the title compound (4.70
g, 65%) as orange solid. Rf (10:48 EtOAc/petroleum ether (40−65
°C)) 0.54. 1H NMR (300 MHz, CDCl3) δ: 9.73 (s, 1H), 8.64 (d, 1H, J
= 2.0 Hz), 7.98 (dd, 1H, J = 2.2 Hz, J = 8.5 Hz), 7.55 (d, 1H, J = 8.5
Hz), 7.49−7.36 (m, 5H), 7.36 (d, 1H, J = 3.9 Hz), 7.31 (d, 1H, J = 3.6
Hz), 5.41 (s, 2H). 13C NMR (75 MHz, CDCl3) δ: 177.9, 165.1, 154.3,
152.1, 136.3, 135.7, 131.3, 130.9, 130.5, 129.5, 128.8, 128.6, 128.5,
128.1, 122.1, 113.9, 67.5. LC-MS [M + H+] calcd for C19H14ClO4,
341.06; found, 341.1. Melting point: 120.0−121.8 °C (1 °C/min).
Ethyl 2-(2,4-Dioxothiazolidin-3-yl)acetate (9). In an ice bath, NaH
(60 w/w%, 2.22 g, 55.5 mmol, 1.3 equiv) was slowly added to a stirred
solution of thiazolidine-2,4-dione (5.00 g, 42.7 mmol, 1.0 equiv) in
anhydrous THF (25 mL). The stirring was continued at rt until the
evolution of gas ceased. In an ice bath, ethyl 2-bromoacetate (7.00 mL,
64.0 mmol, 1.5 equiv) was added to the obtained solution. The
reaction mixture was heated to reflux and stirred at that temperature
1
yielded the product as a white solid (60 mg, 16%). H NMR (400
MHz, DMSO-d6) δ: 12.44 (s, 1H), 7.98 (s, 1H), 7.57−7.40 (m, 4H),
4.50 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 3.68 (s, 2H), 1.22 (t, J = 7.1 Hz,
3H). 13C NMR (400 MHz, DMSO-d6) δ: 172.8, 167.4, 167.1, 165.4,
136.9, 134.4, 133.2, 132.6, 131.5, 129.8, 129.1, 121.1, 62.1, 42.7, 40.7,
14.4. Melting point: 154.9−158.5 °C.
(Z)-2-(3-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)-4-hydroxyphenyl)acetic Acid (4h). Synthesis was
carried out as described for 1h, with 33 (320 mg, 1.78 mmol, 1.0
equiv), 9 (362 mg, 1.78 mmol, 1.0 equiv), piperidine (18 μL, 0.18
mmol, 0.1 equiv), AcOH (10 μL, 0.18 mmol, 0.1 equiv), and EtOH (8
mL) and reflux for 5 days. Purified by flash chromatography
(MeOH:EtOAc, 1:5, with 0.5% of AcOH) on silica gel and
recrystallized from MeOH, which yielded the product as a yellow
1
solid (149 mg, 23%). H NMR (600 MHz, DMSO-d6) δ: 12.32 (s,
1H), 10.58 (s, 1H), 8.14 (s, 1H), 7.29 (d, J = 2.0 Hz, 1H), 7.23 (dd, J
= 8.4, 2.1 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 4.48 (s, 2H), 4.17 (q, J =
7.1 Hz, 2H), 3.54 (s, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (600
MHz, DMSO-d6) δ: 173.2, 167.7, 167.2, 165.6, 156.7, 134.5, 129.8,
129.7, 126.8, 119.8, 119.5, 116.6, 62.1, 42.6, 40.5, 14.4. Melting point:
204.3−208.6 °C.
(Z)-4-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)-
methyl)-3-hydroxybenzoic Acid (4i). Synthesis was carried out as
described for 1h, with aldehyde 34 (140 mg, 0.79 mmol, 1.0 equiv), 9
(161 mg, 0.79 mmol, 1.0 equiv), piperidine (8 μL, 0.08 mmol, 0.1
equiv), AcOH (5 μL, 0.08 mmol, 0.1 equiv), and EtOH (5 mL) and
reflux for 3 days. Purified by gradient flash chromatography
(heptane:EtOAc, 4:1−2.5:1, with 0.5% of AcOH) on silica gel,
1
which yielded the product as an off-white solid (115 mg, 40%). H
NMR (600 MHz, DMSO-d6) δ: 12.16 (s, 1H), 7.96 (s, 1H), 7.54−
7.36 (m, 4H), 4.50 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 2.89 (t, J = 7.5
Hz, 2H), 2.59 (t, J = 7.5 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR
(600 MHz, DMSO-d6) δ: 173.6, 166.9, 166.7, 164.9, 142.2, 134.2,
132.8, 131.1, 130.1, 129.4, 128.0, 120.5, 61.7, 42.2, 34.8, 30.0, 13.9.
Melting point: 148.2−152.1 °C.
(Z)-3-(3-((3-(2-Ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-
ylidene)methyl)-4-hydroxyphenyl)propanoic Acid (4j). Synthesis was
carried out as described for 1h, with aldehyde 35 (100 mg, 0.51 mmol,
J
DOI: 10.1021/acs.jmedchem.6b01058
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
for 5 h before the solvent was removed under reduced pressure. The
residue was diluted with cold water (50 mL) and extracted with EtOAc
(3 × 50 mL). The combined organic phases were dried over
anhydrous MgSO4, filtered, and concentrated under reduced pressure.
The crude was purified by flash chromatography (DCM) on silica gel,
mg, 0.05 mmol, 0.05 equiv), 2 M aqueous K2CO3 solution (1.4 mL,
2.73 mmol, 3 equiv), and a mixture (7:3) of toluene and EtOH (10
mL) and reflux for 5 days. Purified by gradient flash chromatography
(heptane:EtOAc, 1:0−2.5:1, with 0.5% of AcOH) on silica gel, which
1
yielded the product as a yellow solid (45 mg, 15%). H NMR (600
1
which yielded the product as a colorless oil (6.75 g, 84%). H NMR
MHz, CD3OD) δ: 9.63 (s, 1H), 8.70 (d, J = 2.2 Hz, 1H), 8.09 (dd, J =
8.7, 2.2 Hz, 1H), 7.58−7.40 (m, 5H), 7.47 (d, J = 3.8 Hz, 1H), 7.35 (d,
J = 8.8 Hz, 1H), 7.14 (d, J = 3.7 Hz, 1H), 5.38 (s, 2H). Spectral data in
agreement with earlier reported.20
(400 MHz, CDCl3) δ: 4.34 (s, 2H), 4.23 (q, J = 7.1 Hz, 2H), 4.03 (s,
2H), 1.29 (t, J = 7.1 Hz, 3H). 13C NMR (400 MHz, CDCl3) δ: 171.2,
170.8, 166.3, 62.3, 42.3, 34.0, 14.2.
(Z)-Benzyl 4-Chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothia-
zolidin-5-ylidene)methyl)furan-2-yl)benzoate (11). 2,4-Dioxothiazo-
lidine 9 (0.244 g, 1.2 mmol, 1 equiv) was dissolved in absolute EtOH
(15 mL). Benzyl 4-chloro-3-(5-formylfuran-2-yl)benzoate (8) (0.41 g,
1.2 mmol, 1 equiv) and piperidine (119 μL, 1.2 mmol, 1 equiv) were
added. The mixture was heated to reflux and stirred that temperature
for 1.5 h and then allowed to cool to rt. The solids was filtered off and
washed with cold EtOH. Drying in vacuum oven gave the title
compound (0.55 g, 88%) as a yellow solid. Rf (1:5 EtOAc:heptanes)
3-Amino-4-bromo-2-naphthoic Acid (17). In a flask wrapped up
with foil, FeBr3 (1.36 g, 4.60 mmol, 0.1 equiv) was added to a well-
stirred solution of 3-amino-2-naphthoic acid (16) (8.57 g, 45.8 mmol,
1.0 equiv) in CHCl3 (220 mL). A solution of bromine (2.58 mL, 50.4
mmol, 1.1 equiv) in CHCl3 (120 mL) was added dropwise. The
reaction mixture was allowed to stir for 16 h at rt. The precipitate was
collected and washed with CHCl3 (200 mL). The crude was dissolved
in 1 M aqueous NaOH (250 mL) and then acidified with AcOH. The
precipitate was filtered out and dried, which yielded the product as a
1
1
0.30. H NMR (300 MHz, CDCl3) δ: 8.64 (d, 1H, J = 2.2 Hz), 7.96
yellow solid (10.85 g, 89%). H NMR (400 MHz, DMSO-d6) δ: 8.57
(dd, 1H, J = 2.2 Hz, J = 8.5 Hz), 7.70 (s, 1H), 7.55 (d, 1H, J = 8.5 Hz),
7.53−7.47 (m, 2H), 7.43−7.35 (m, 3H), 7.34 (d, 1H, J = 3.9 Hz), 6.95
(d, 1H, J = 3.9 Hz), 5.42 (s, 2H), 4.49 (s, 2H), 4.26 (q, 2H, J = 7.2
Hz), 1.32 (t, 3H, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3) δ: 168.0,
166.3, 165.4, 165.1, 153.4, 149.2, 135.7, 135.5, 131.4, 130.3, 129.63,
129.60, 128.9, 128.8, 128.7, 128.0, 120.3, 119.6, 114.9, 67.6, 62.4, 42.4,
14.5. LC-MS [M + H+] calcd for C26H21ClNO7S, 526.07; found,
526.1. Melting point: 177.8−178.4 °C (1 °C/min).
(s, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.61 (t, J =
7.9 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H). 13C NMR (400 MHz, DMSO-
d6) δ: 169.2, 144.5, 135.2, 133.8, 130.9, 130.5, 125.9, 124.2, 123.0,
116.1, 103.7.
4-Bromo-2-naphthoic Acid (18). 3-Amino-4-bromo-2-naphthoic
acid (17) (4.20 g, 15.8 mmol, 1.0 equiv) was added to AcOH (100
mL). The mixture was heated to boil then rapidly cooled to 15 °C. In
an ice bath, the obtained suspension and NaNO2 (1.33 g, 18.9 mmol,
1.2 equiv) was added to a mixture (1:1) of concentrated H2SO4 and
AcOH (20 mL). The resulting mixture was allowed to stir for 2.5 h
before it was poured into a well-stirred suspension of Cu2O (6.33 g,
44.2 mmol, 2.8 equiv) in MeOH (90 mL) and allowed to stir at 60 °C
for 1 h before it was cooled to rt. The obtained mixture was filtered,
and the filtrate was concentrated under reduced pressure. The residue
was diluted with Et2O (100 mL) and extracted with saturated aqueous
NaHCO3 (3 × 250 mL). The combined aqueous phases were acidified
with 4 M HCl. The precipitate was filtered off and dried, which yielded
4-Fluoro-3-(5-formyl-2-furyl)benzoic Acid (12). 3-Bromo-4-fluo-
robenzoic acid (55 mg, 0.251 mmol), (5-formyl-2-furyl)boronic acid
(71 mg, 0.508 mmol), and Pd(PPh3)2Cl2 (9 mg, 0.013 mmol) were
mixed in a vial, which was subsequently evacuated and backfilled with
N2 several times. DME (1 mL, degassed), aq Na2CO2 (2M, 1 mL,
degassed), and EtOH (1 mL, degassed) were added and the two-phase
reaction mixture was heated to 50 °C and stirred at that temperature
for 17 h, after which it was cooled to rt and the solvent was removed in
vacuo. The resulting black solid was resuspended in H2O (50 mL) and
filtered through a plug of Celite (3 cm Ø, 1.5 cm), and the Celite was
washed through with additional H2O (2 × 10 mL). The combined aq
solution was extracted with EtOAc (3 × 20 mL) and CH2Cl2 (20 mL)
before it was acidified with aq HCl (2M, app 10 mL) until pH ≈ 1.
The resulting orange precipitate was filtered off, washed with H2O (3
× 5 mL), and dried, giving the title compound as a yellow solid (35
1
the product as a brown solid (3.62 g, 91%). H NMR (400 MHz,
DMSO-d6) δ: 13.37 (s, 1H), 8.67 (s, 1H), 8.25 (d, J = 1.5 Hz, 1H),
8.23 (d, J = 8.1 Hz, 1H), 8.20 (t, J = 8.4 Hz, 1H), 7.85 (ddd, J = 8.4,
6.9, 1.3 Hz, 1H), 7.73 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H).
4-(5-Formylfuran-2-yl)-2-naphthoic Acid (19). 4-Bromo-2-naph-
thoic acid (18) (100 mg, 0.40 mmol, 1.0 equiv) was dissolved in a
mixture (7:3) of toluene and EtOH (10 mL) and degassed with argon.
Pd(PPh3)4 (23 mg, 0.02 mmol, 0.1 equiv) and (5-formylfuran-2-
yl)boronic acid (6) (62 mg, 0.44 mmol, 1.1 equiv) were added to the
obtained solution and degassed with argon for. Degassed 2 M aqueous
K2CO3 solution (0.60 mL, 1.20 mmol, 3 equiv) was then added, and
the mixture was allowed to stir at reflux for 4 days. The reaction
mixture was allowed to cool to rt, then filtered through Celite, and the
filtrate was concentrated under reduced pressure. The residue was
acidified with 4 M HCl, then extracted with EtOAc (3 × 10 mL) and
washed with water (2 × 20 mL) and brine (20 mL). The organic phase
was dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by gradient flash
chromatography (heptanes:EtOAc, 1:0−2:1, with 0.5% of AcOH) on
1
mg, 0.149 mmol, 60%). H NMR (400 MHz, DMSO-d6) δ: 13.37 (s,
1H), 9.70 (s, 1H), 8.45 (dd, J = 7.3, 2.3 Hz, 1H), 8.05 (ddd, J = 8.6,
5.0, 2.3 Hz, 1H), 7.70 (d, J = 3.8 Hz, 1H), 7.55 (dd, J = 11.0, 8.6 Hz,
1H), 7.21 (t, J = 3.8 Hz, 1H). 13C NMR (100 MHz, MeOD) δ: 179.5,
168.0, 164.8, 162.2, 153.6, 153.6, 153.5, 133.8, 133.7, 130.0, 130.0,
129.3, 125.1, 118.8, 118.7, 117.8, 117.6, 114.2, 114.1. LC-MS: [M +
H]+calcd 235.03 [M + H]+found 235.0. HPLC: >98%, retention time 1.62
min, reverse phase, H2O/MeCN/TFA gradient run, 4 mL min−1.
4-(Benzyloxy)-3-bromobenzoic Acid (14). Benzyl bromide (157
mg, 0.92 mmol, 2.0 equiv) and anhydrous K2CO3 (159 mg, 1.15
mmol, 2.5 equiv) were added to a solution of 3-bromo-4-
hydroxybenzoic acid (100 mg, 0.46 mmol, 1.0 equiv) in anhydrous
acetone (10 mL). The mixture was allowed to stir at reflux for 16 h
and filtered hot. The filtrate was concentrated under reduced pressure.
Then 4 M aq NaOH (345 μL, 1.38 mmol, 3.0 equiv) was added to a
solution of the obtained crude product in MeOH (8 mL), and the
mixture was allowed to stir at rt for 16 h. The solvent was removed
under reduced pressure, and the residue was acidified with 4 M HCl
and extracted with EtOAc (3 × 15 mL). The combined organic phases
were dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure, which yielded the product as a white solid (95 mg,
1
silica gel, yielding the product as a yellow solid (12 mg, 11%). H
NMR (400 MHz, DMSO-d6) δ: 9.79 (s, 1H), 8.76 (s, 1H), 8.49 (d, J =
8.6 Hz, 1H), 8.46 (d, J = 1.6 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.75
(ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.66 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H),
7.46 (d, J = 3.6 Hz, 1H), 7.02 (d, J = 3.6 Hz, 1H).
3-Chloro-4-(5-formyl-2-furyl)benzoic Acid (20). 4-Bromo-3-chlor-
obenzoic acid (600 mg, 2.55 mmol), (5-formyl-2-furyl)boronic acid
(713 mg, 5.10 mmol), and Pd(PPh3)2Cl2 (90 mg, 0.128 mmol) were
mixed in a round-bottom flask which was subsequently evacuated and
backfilled with N2 four times. DME (11 mL, degassed), aq Na2CO2
(2M, 11 mL, degassed), and EtOH (11 mL, degassed) were added,
and the reaction mixture was heated to 50 °C and stirred at that
temperature for 19 h, after which it was cooled to rt and the solvent
was removed in vacuo. The resulting black solid was resuspended in
H2O (100 mL) and filtered through a plug of Celite (4 cm Ø, 2 cm),
1
67% for two steps). H NMR (400 MHz, CDCl3) δ: 8.08 (d, J = 2.1
Hz, 1H), 7.92 (dd, J = 8.6, 2.1 Hz, 1H), 7.51−7.33 (m, 5H), 7.31 (d, J
= 8.7 Hz, 1H), 5.30 (s, 2H). Spectral data in agreement with reported
by Kulkarni et al.19
4-(Benzyloxy)-3-(5-formylfuran-2-yl)benzoic Acid (15). Synthesis
was carried out as described for 19, with bromine 14 (280 mg, 0.91
mmol, 1.0 equiv), 6 (140 mg, 1.00 mmol, 1.1 equiv), Pd(PPh3)4 (58
K
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Journal of Medicinal Chemistry
Article
and the Celite was washed through with H2O (2 × 20 mL). The
combined aq solution was extracted with EtOAc (3 × 50 mL) before it
was acidified with aq HCl (1M, app 20 mL) until pH ≈ 1. The
voluminous yellow precipitate was filtered off, washed with H2O (3 ×
10 mL), and dried, giving the title compound as a yellow solid (497
150.3, 150.0, 135.9, 135.0, 131.1, 129.5, 129.2, 129.1, 128.8, 128.5,
128.4, 112.9, 112.1, 67.3, 37.7. LC-MS [M + H+] calcd for
C19H14ClO3, 325.06; found, 325.1. Melting point: 87.5−89.2 °C (1
°C/min).
Benzyl 4 Chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazoli-
din-1-yl)methyl)furan (24). Under a nitrogen atmosphere, NaH (60%
dispersion in mineral oil) (0.062 g, 1.523 mmol, 1.1 equiv) was
suspended in anhydrous DMF (5 mL) and stirred at rt for 30 min. A
solution of ethyl 2-(2,5-dioxoimidazolidin-1-yl)acetate (25) (0.260 g,
1.384 mmol, 1 equiv) in anhydrous DMF (5 mL) was added, and the
mixture was stirred at rt for 30 min. A solution of benzyl 4-chloro-3-(5-
(chloromethyl)furan-2-yl)benzoate (23) (0.500 g, 1.384 mmol, 1
equiv) in anhydrous DMF (5 mL) was added, and the mixture was
stirred at rt for 19 h. The mixture was quenched by addition of H2O
(100 mL). The phases were separated, and the aqueous phase was
extracted with EtOAc (3 × 150 mL), diluted with H2O (50 mL), and
extracted with EtOAc (3 × 150 mL). The combined organic phases
were dried using anhydrous MgSO4, filtered, and evaporated to give
yellow oil (0.734 g). The crude product was purified by dry column
vacuum chromatography (EtOAc/heptane (0−30%)) to give the title
compound (0.503 g, 71%) as yellow oil. Rf (1:1 EtOAc/heptane) 0.37.
1H NMR (400 MHz, CDCl3) δ: 8.48 (d, 1H, J = 2.0 Hz), 7.91 (dd,
1
mg, 1.983 mmol, 78%). H NMR (400 MHz, DMSO-d6) δ: 13.49 (s,
1H), 9.70 (s, 1H), 8.09−7.97 (m, 3H), 7.70 (d, J = 3.8 Hz, 1H), 7.51
(d, J = 3.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 178.6, 165.5,
153.1, 151.9, 132.5, 131.4, 130.5, 130.4, 129.2, 128.4, 124.1, 114.8. LC-
MS: [M + H]+
251.01 [M + H]+
251.0. HPLC: >96%,
calcd
found
retention time 1.66 min, reverse phase, H2O/MeCN/TFA gradient
run, 4 mL min−1.
6-Chloro-3′-formyl-[1,1′-biphenyl]-3-carboxylic Acid (21). 3-
Bromo-4-chlorobenzoic acid (99 mg, 0.420 mmol, (3-formylphenyl)-
boronic acid (126 mg, 0.840 mmol), and Pd(PPh3)2Cl2 (15 mg, 0.021
mmol) were mixed in a vial which was subsequently evacuated and
backfilled with argon several times. DME (1.2 mL, degassed), aq
Na2CO2 (2M, 1.3 mL, degassed), and abs EtOH (1.2 mL, degassed)
was added, and the two-phase reaction mixture was heated to 50 °C
and stirred at that temperature for 17 h. Thereafter, it was cooled to rt
and the solvent was removed in vacuo. The resulting black solid was
resuspended in H2O (50 mL) and filtered through a plug of Celite (3
cm Ø, 2 cm), and the Celite was washed through with additional H2O
(2 × 10 mL). The combined aq solution was acidified with aq HCl
(2M, app 10 mL) until pH ≈ 1. The resulting white precipitate was
filtered off, washed with H2O (3 × 10 mL), and dried, giving the title
1H, J = 2.0 Hz, J = 8.3 Hz), 7.53 (d, 1H, J = 8.3 Hz), 7.51−7.35 (m,
5H), 7.11 (d, 1H, J = 3.5 Hz), 6.49 (d, 1H, 3.5 Hz), 5.42 (s, 2H), 4.70
(s, 2H), 4.28 (s, 2H), 4.24 (q, 2H, J = 7.3 Hz), 4.04 (s, 2H), 1.30 (t,
3H, J = 7.3 Hz). 13C NMR (100 MHz, CDCl3) δ: 169.1, 167.0, 165.4,
155.6, 149.8, 149.2, 135.8, 135.0, 131.1, 129.3, 129.1, 129.0, 128.7,
128.4, 128.3, 112.5, 111.1, 67.1, 62.0, 49.8, 39.8, 39.6, 14.1. LC-MS [M
+ H+] calcd for C26H24ClN2O7, 511.13; found, 511.2.
1
compound as a yellow solid (100 mg, 0.384 mmol, 91%). H NMR
(600 MHz, DMSO-d6) δ: 13.32 (s, 1H), 10.09 (s, 1H), 8.02−7.94 (m,
4H), 7.86−7.81 (m, 1H), 7.78−7.71 (m, 2H). 13C NMR (150 MHz,
DMSO-d6) δ: 193.0, 192.9, 166.2, 138.8, 138.7, 136.3, 136.3, 135.9,
135.2, 132.0, 130.4, 130.4, 130.3, 130.2, 129.3, 128.9. Rotamers of this
compound gives double signals for some of the protons. LC-MS: [M +
H]+calcd 261.03 [M + H]+found 261.0. HPLC: >94%, retention time 2.17
min, reverse phase, H2O/MeCN/TFA gradient run, 4 mL min−1.
Benzyl 4-Chloro-3-(5-(hydroxymethyl)furan-2-yl)benzoate (22).
Under a nitrogen atmosphere, benzyl 4-chloro-3-(5-formylfuran-2-
yl)benzoate (8) (0.638 g, 1.87 mmol, 1 equiv) was suspended in
anhydrous MeOH (40 mL). NaBH4 (0.215 g, 5.62 mmol, 3 equiv) was
added, and the mixture was stirred at rt for 45 min. The reaction was
quenched by addition of saturated NaHCO3 (80 mL). MeOH was
removed by evaporation in vacuo. The residue was partitioned
between H2O (200 mL) and EtOAc (200 mL), and the aqueous phase
was extracted with EtOAc (2 × 200 mL). The combined organic
phases were dried using anhydrous MgSO4, filtered, and evaporated to
give yellow oil (0.58 g). The crude product was purified by dry column
vacuum chromatography (EtOAc/heptane) to give the title compound
(0.46 g, 71%) as slightly yellow solid. Rf (3:7 EtOAc/heptane) 0.18.
1H NMR (300 MHz, CDCl3) δ: 8.54 (d, 1H, J = 2.2 Hz), 7.85 (dd,
1H, J = 2.2 Hz, J = 8.3 Hz), 7.48 (d, 1H, J = 8.5 Hz), 7.48−7.32 (m,
5H), 7.11 (d, 1H, J = 3.6 Hz), 6.45 (d, 1H, J = 3.4 Hz), 5.40 (s, 2H),
4.70 (d, 2H, J = 6.3 Hz), 1.90 (t, 1H, J = 6.3 Hz). 13C NMR (75 MHz,
DMSO-d6) δ: 165.2, 156.9, 147.8, 136.5, 134.1, 132.1, 129.5, 129.4,
129.3, 129.2, 128.9, 128.7, 128.4, 113.5, 110.1, 67.3, 56.3. LC-MS [M +
(−OH)] calcd for C19H14ClO3, 325.06; found, 325.1. Melting point:
72.2−74.2 °C (1 °C/min).
Ethyl 2-(2,5-Dioxoimidazolidin-1-yl)acetate (25). Under a nitro-
gen atmosphere, NaH (60% in mineral oil) (0.882 g, 22.0 mmol, 1.1
equiv) was suspended in anhydrous THF (90 mL). Hydantoin (2.00 g,
20.0 mmol, 1 equiv) was added, and the mixture was stirred at rt for 1
h. Ethyl 2-bromoacetate (2.25 mL, 20.0 mmol, 1 equiv) dissolved in
anhydrous THF (8 mL) was added, and the mixture was heated to 50
°C and stirred at that temperature for 17.5 h. The mixture was allowed
to cool to rt and was then quenched by addition of aqueous Na2CO3
(10%, 100 mL). The two phases were separated, and the aqueous
phase was neutralized (pH 7) using concentrated aqueous HCl and
then extracted with EtOAc (100 mL). The combined organic phases
were dried using anhydrous MgSO4, filtered, and evaporated to give
off-white solid (2.45 g). The crude product was purified by dry column
vacuum chromatography (EtOAc/heptane) to give the title compound
1
(0.68 g, 18%) as white solid. Rf (1:1 EtOAc/heptane) 0.09. H NMR
(300 MHz, CDCl3) δ: 5.67 (br s, 1H), 4.26 (s, 2H), 4.23 (q, 2H, J =
7.2 Hz), 4.08 (d, 2H, 1.1 Hz), 1.30 (t, 3H, J = 7.2 Hz). 13C NMR (75
MHz, CDCl3) δ: 170.8, 167.1, 157.6, 62.2, 46.9, 39.6, 14.4. LC-MS [M
+ H+] calcd for C7H11N2O4, 187.07; found, 187.0. Melting point:
118.6−119.8 °C (1 °C/min).
3-(1H-Tetrazol-5-yl)benzaldehyde (31). Commercially available 3-
formylbenzonitrile (50 mg, 0.38 mmol, 1.0 equiv) and TBAF·3H2O
(180 mg, 0.57 mmol, 1.5 equiv) were added to TMSN3 (227 μL, 1.71
mmol, 4.5 equiv). The mixture was allowed to stir at 90 °C for 15 h.
The reaction mixture was diluted with EtOAc (10 mL) and washed
with 1 M HCl (3 × 5 mL). The organic phase was dried over
anhydrous MgSO4, filtered, and concentrated under reduced pressure,
Benzyl 4-Chloro-3-(5-(chloromethyl)furan-2-yl)benzoate (23).
Under a nitrogen atmosphere, benzyl 4-chloro-3-(5-(hydroxymethyl)-
furan-2-yl)benzoate (22) (1.00 g, 2.92 mmol, 1 equiv) was dissolved in
anhydrous CH2Cl2 (20 mL). The solution was cooled to
approximately 0 °C. Pyridine (0.26 mL, 3.21 mmol, 1.1 equiv) was
added, followed by thionyl chloride (1.3 mL, 17.2 mmol, 5.9 equiv).
The mixture was stirred at rt for 2 h and was then diluted with CH2Cl2
(80 mL) and quenched by addition of H2O (80 mL). The phases were
separated, and the organic phase was washed with H2O (3 × 80 mL),
dried using anhydrous MgSO4, filtered, and evaporated to give the title
compound (1.05 g, quant) as slightly red solid. Rf (3:7 EtOAc/
1
which yielded the product as a white solid (55 mg, 83%). H NMR
(400 MHz, DMSO-d6) δ: 10.13 (s, 1H), 8.58 (t, J = 1.5 Hz, 1H), 8.37
(dt, J = 7.7, 1.3 Hz, 1H), 8.13 (dt, J = 7.7, 1.3 Hz, 1H), 7.86 (t, J = 7.7
Hz, 1H).
2-(3-Formylphenyl)acetic Acid (32). Benzoyl peroxide (7 mg, 0.03
mmol, 0.01 equiv) was added to a solution of commercially available 2-
(3-methylphenyl)acetic acid (0.50 g, 3.33 mmol, 1.0 equiv) and N-
bromosuccinimide (0.89 g, 5.00 mmol, 1.5 equiv) in CHCl3 (20 mL).
The mixture was heated to reflux and stirred at that temperature for 24
h before the solvent was removed under reduced pressure. The residue
was dissolved in a mixture (1:1) of EtOH and H2O (20 mL) and
hexamethylenetetramine (1.26 g, 8.99 mmol, 2.7 equiv) was added,
and the mixture was refluxed for for 4 h. Concentrated HCl (1.6 mL,
20.0 mmol, 6 equiv) was slowly added to the mixture at reflux. The
1
heptane) 0.60. H NMR (300 MHz, CDCl3) δ: 8.56 (d, 1H, J = 2.2
Hz), 7.88 (dd, 1H, J = 2.2 Hz, J = 8.5 Hz), 7.49 (d, 1H, J = 8.3 Hz),
7.49−7.32 (m, 5H), 7.12 (d, 1H, J = 3.4 Hz), 6.52 (d, 1H, J = 3.4 Hz),
5.40 (s, 2H), 4.47 (s, 2H). 13C NMR (75 MHz, CDCl3) δ: 165.5,
L
DOI: 10.1021/acs.jmedchem.6b01058
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mixture was allowed to stir at reflux for 30 min and then cooled to rt.
DCM (10 mL) and H2O (10 mL) were added, and the phases were
separated. The organic phase was dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue was
taken into saturated aqueous NaHCO3 (10 mL) and washed with
DCM (2 × 10 mL). The aqueous phase was acidified with 4 M HCl (8
mL) and extracted with DCM (3 × 20 mL). The organic phases were
dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure, which yielded the product as an off-white solid
(275 mg, 51%). 1H NMR (400 MHz, CDCl3) δ: 10.02 (s, 1H), 7.84−
7.50 (m, 4H), 3.76 (s, 2H).
2-(3-Formyl-4-hydroxyphenyl)acetic Acid (33). First, 1 M aqueous
LiOH (30 mL, 30 mmol, 9.0 equiv) was added to a solution of 37
(660 mg, 3.40 mmol, 1 equiv) in THF (6 mL). The mixture was then
allowed to stir at reflux for 15 h. The reaction mixture was acidified
with 4 M HCl and extracted with EtOAc (3 × 40 mL). The combined
organic phases were dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash chromatography (heptane:EtOAc, 1:1), which yielded the
product as a white solid (254 mg, 42%). 1H NMR (400 MHz,
DMSO-d6) δ: 10.24 (s, 1H), 7.53 (d, J = 2.3 Hz, 1H), 7.40 (dd, J = 8.5,
2.4 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 3.53 (s, 2H). 13C NMR (400
MHz, DMSO-d6) δ: 191.4, 172.7, 159.5, 137.6, 129.5, 126.1, 121.9,
117.2, 39.5.
3-(3-Formylphenyl)propanoic Acid (34). NaBH4 (168 mg, 4.44
mmol, 3.0 equiv) was added portionwise over 5 min to a well-stirred
solution of 36 (260 mg, 1.48 mmol, 1.0 equiv) and NiCl2·6H2O (352
mg, 1.48 mmol, 1.0 equiv) in MeOH (20 mL) at rt. The reaction
mixture was allowed to stir at rt for 1 h and quenched with saturated
aqueous NH3·HCl (10 mL). The mixture was filtered through a plug
of Celite, and the filtrate was extracted with EtOAc (3 × 20 mL). The
combined organic phases were dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue was purified by
flash chromatography (heptane:EtOAc, 1:1, with 0.5% of AcOH) on
silica gel, which yielded the product as a white solid (140 mg, 53%).
1H NMR (400 MHz, DMSO-d6) δ: 9.98 (s, 1H), 7.77 (t, J = 1.5 Hz,
1H), 7.73 (dt, J = 7.5, 1.5 Hz, 1H), 7.59 (dt, J = 7.5, 1.5 Hz, 1H), 7.51
(t, J = 7.5 Hz, 1H), 2.80 (t, J = 7.6 Hz, 2H), 2.51 (t, J = 7.6 Hz, 2H).
3-(3-Formyl-4-hydroxyphenyl)propanoic Acid (35). First 4 M
NaOH (7.5 mL, 30 mmol, 10 equiv) was added to a solution of 3-(4-
hydroxyphenyl)propanoic acid (500 mg, 3.01 mmol, 1.0 equiv) in
CHCl3 (10 mL). The mixture was then heated to reflux and stirred at
that temperature for 6 h before it was acidified with 4 M HCl. The
solvent was removed under reduced pressure, and the residue was
extracted with EtOAc (3 × 20 mL). The combined organic phases
were dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The crude was purified by flash chromatography
(heptane:EtOAc, 1:1, with 0.5% of AcOH), which yielded a mixture
(1:2) of the product and starting material (363 mg). The mixture was
used in the next step without further purification. 1H NMR (400 MHz,
CDCl3) δ: 10.22 (s, 1H), 9.12 (s, 1H), 7.49 (d, J = 2.3 Hz, 1H), 7.39
(dd, J = 8.5, 2.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 2.77 (t, J = 7.5 Hz,
2H), 2.51 (t, J = 7.5 Hz, 2H).
heated to reflux and stirred at that temperature for 2 h. The reaction
mixture was acidified with 6 M HCl and concentrated under reduced
pressure. The residue was extracted with diethyl ether (3 × 30 mL),
and the combined organic phases were dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The crude product
was purified by flash chromatography (heptanes:EtOAc, 6:1), which
1
yielded the product as a white solid (1.07 g, 61%). H NMR (400
MHz, CDCl3) δ: 10.94 (s, 1H), 9.88 (s, 1H), 7.48 (d, J = 2.2 Hz, 1H),
7.44 (dd, J = 8.5, 2.3 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 3.71 (s, 3H),
3.62 (s, 2H). 13C NMR (400 MHz, CDCl3) δ: 196.5, 171.9, 160.9,
138.2, 134.2, 125.6, 120.6, 118.1, 52.3, 40.0.
[3H]-D-Asp Uptake Assay. The screening of a commercial
compound library consisting of 3040 compounds (Chembridge
Corporation, San Diego, CA) at stable HEK293 cell lines expressing
human EAAT1, EAAT2, and EAAT3 in a conventional [3H]-D-Asp
uptake assay has been described previously.16 The pharmacological
properties of the identified hit 1a, and its analogues were subsequently
characterized at the three cell lines in the assay essentially as described
previously.16 Briefly, the cell lines were maintained in culture medium
[Dulbecco’s Modified Eagle Medium (DMEM) supplemented with
penicillin (100 U/mL), streptomycin (100 μg/mL), and 5% dialyzed
fetal bovine serum] supplemented with 1 mg/mL G-418 at 37 °C in a
humidified 5% CO2 incubator. The day before the assay, cells were
split into poly-D-lysine-coated white 96-well plates (PerkinElmer,
Boston, MA). Then 16−24 h later, the culture medium was aspirated
and cells were washed twice with 100 μL of assay buffer (Hank’s
Buffered Saline Solution supplemented with 20 mM HEPES, 1 mM
CaCl2 and 1 mM MgCl2, pH 7.4). Then 50 μL of assay buffer
supplemented with 30 nM [3H]-D-aspartate (PerkinElmer, Boston,
MA) and various concentrations of test compounds were added to the
wells, and the plate was incubated at 37 °C for 5 min. Nonspecific
[3H]-D-Asp uptake in the cells was determined in the presence of 3
mM (S)-Glu. The assay mixtures was quickly removed from the wells,
which were then washed with 3 × 75 μL of ice-cold assay buffer, and
150 μL of Microscint20 scintillation fluid (PerkinElmer, Boston, MA)
was added to each well. The plate was shaken for at least 1 h and
counted in a TopCounter (PerkinElmer, Boston, MA). The
experiments were performed in duplicate 3−4 times for each
compound.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01058.
■
S
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +45 35 33 62 44. E-mail: lebu@sund.ku.dk.
■
Notes
(E)-3-(3-Formylphenyl)acrylic Acid (36). Commercially available
isophthalaldehyde (1.0 g, 7.46 mmol, 1.0 equiv) and malonic acid
(0.80 g, 7.46 mmol, 1.0 equiv) were added to a mixture of pyridine and
EtOH (1:1, 20 mL). The mixture was heated to reflux and stirred at
that temperature for 16 h. The reaction mixture was allowed to cool to
rt and acidified with 6 M HCl (20 mL). The precipitate was filtered off
and purified by flash chromatography (heptane:EtOAc, 1:1.25, with
0.5% of AcOH) on silica gel, which yielded the product as a white solid
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Lundbeck Foundation, the Novo Nordisk Foundation and
the Danish Medical Research Council are thanked for financial
support.
1
ABBREVIATIONS USED
(0.75 g, 58%). H NMR (400 MHz, DMSO-d6) δ: 12.52 (s, 1H),
■
10.04 (s, 1H), 8.22 (s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 7.6
Hz, 1H), 7.68 (d, J = 15.9 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 6.66 (d, J
= 16.1 Hz, 1H).
Methyl 2-(3-Formyl-4-hydroxyphenyl)acetate (37). Paraformalde-
hyde (1.90 g, 63.2 mmol, 7 equiv), MgCl2 (1.72 g, 18.1 mmol, 2.0
equiv), and triethylamine (2.74 g, 27.1 mmol, 3.0 equiv) were added to
a solution of methyl 2-(4-hydroxyphenyl)acetate (1.50 g, 9.03 mmol,
1.0 equiv) in MeCN (25 mL). The obtained yellow mixture was
DHK, dihydrokainic acid; CNS, central nercous system; EAAT,
excitatory amino acid transporter; SAR, structure−activity-
relationship; TBOA, threo-benzyloxy aspartate
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