7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor

Article abstract of DOI:10.1021/acs.jmedchem.0c01160

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site (Ki = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.

Full text of DOI:10.1021/acs.jmedchem.0c01160

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Article
7-Hydroxycoumarins are Affinity-based Fluorescent Probes for
Competitive Binding Studies of Macrophage Migration Inhibitory Factor
Zhangping Xiao, Deng Chen, Shanshan Song, Ramon van der Vlag, Petra
E. van der Wouden, Ronald van Merkerk, Robbert Cool, Anna K.H Hirsch,
Barbro Melgert, Wim J. Quax, Gerrit J. Poelarends, and Frank J. Dekker
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01160 • Publication Date (Web): 17 Sep 2020
Downloaded from pubs.acs.org on September 20, 2020
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-Hydroxycoumarins are Affinity-based Fluorescent
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Probes for Competitive Binding Studies of
Macrophage Migration Inhibitory Factor
a
a
a,b
d
Zhangping Xiao, Deng Chen, Shanshan Song, Ramon van der Vlag, Petra E. van der
Wouden, Ronald van Merkerk, Robbert H. Cool, Anna K. H. Hirsch, Barbro N. Melgert,b,c
a
a
a
d,e,f
a
a
a,*
Wim J. Quax, Gerrit J. Poelarends, Frank J. Dekker
a
Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP),
University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
b
Molecular Pharmacology, Groningen Research Institute of Pharmacy (GRIP), University of
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
c
University Medical Center Groningen, Groningen Research Institute of Asthma and COPD,
University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands
d
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen,
the Netherlands
e
Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research
Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI) Campus Building E8.1, 66123
Saarbrücken, Germany
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Department of Pharmacy, Saarland University, Campus Building E8.1, 66123 Saarbrücken,
Germany
*
Corresponding author: f.j.dekker@rug.nl
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KEYWORDS: 7-Hydroxycoumarin, fluorescence, macrophage migration inhibitory factor (MIF),
Cluster of Differentiation 74 (CD74), cell proliferation
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ABSTRACT
Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and
cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also
harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for
identification of MIF binding molecules that also interfere with its biological activity. However,
MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative
methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the
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MIF tautomerase active site (K = 18±1 nM) that binds with concomitant quenching of its
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fluorescence. This property enabled development of a novel competition-based assay format to
quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered
with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high
affinity MIF binder as a novel tool to advance MIF-oriented research.
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INTRODUCTION
The impact of cancer as a major public health problem is demonstrated by the estimated 9.6
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million cancer-related deaths worldwide in 2018. Although substantial progress has been
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achieved over the last decades, cancer treatment remains a challenge. This challenge can be
addressed by exploring novel molecular mechanisms involved in cell proliferation to identify
novel therapeutics. Apart from inflammation,3,4 the cytokine macrophage migration inhibitory
factor (MIF) has also been connected to several processes in the pathogenesis and progression of
cancer.5,6 Overexpression of MIF was found in several cancers, including genitourinary cancer,7
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melanoma, neuroblastoma and lung carcinoma . Both clinical and animal studies demonstrated
that MIF enhanced tumor growth, invasion and angiogenesis.11,12 Additionally, MIF gene
knockout or knockdown decreased proliferation and increased apoptosis of cancer cells.13,14 The
role of MIF in tumor development indicates that MIF represents a potential drug target for cancer
therapy.
On a molecular level, MIF operates via protein-protein interactions (PPIs) with membrane bound
receptors such as the cluster of differentiation 74 (CD74), CXCR4 and CXCR7 receptor, as well
as with intracellular targets such as p53 and Jab15–18. Binding of MIF to CD74 triggers activation
of the mitogen-activated protein kinase (MAPK) pathway and inhibition of p53, which both
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suppress apoptosis and enhance cell proliferation. Development of molecules that interfere with
MIF-receptor interactions is an attractive strategy to inhibit MIF-induced cellular signaling. The
utility of this approach has been demonstrated by the development of the MIF neutralizing
antibody Imalumab, which is currently in a phase II clinical trial for treatment of patients with
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metastatic colorectal cancer. Also the development of small molecule MIF binders to interfere
with MIF signaling has gained attention over the past years.20,21
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There is structural information available to facilitate development of MIF targeted therapeutics.
MIF exists in a homotrimeric form, in which each monomer contains a peptide with 114 amino
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acids folding into two β-stands and four α-helics. MIF also harbors three tautomerase active sites,
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each located at the interface between two adjacent monomers, centering around Pro1 residues,15
that catalyze keto-enol conversion of substrates such as D-dopachrome and 4-
hydroxylphenylpyruvate (4-HPP). Importantly, the enzyme active sites are located in the vicinity
of amino acid residues that are involved in binding to the CD74 receptor. For instance, amino acid
residues Y36, K66, N109, I64 and W108 on the MIF surface (Figure 1A) were mapped as residues
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responsible for activation of CD74 by alanine-scanning mutagenesis. Residues of 79-86 on
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second α -helix were also identified to be responsible for MIF-CD74 binding. Interestingly, Y99
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of MIF was reported to regulate both catalytic activity and CD74 activation allosterically.26
Therefore, inhibitors of MIF tautomerase activity can be expected to interfere with MIF/CD74
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binding and MIF induced signaling. The initially discovered MIF tautomerase inhibitor ISO-1
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(
Figure 1B) also suppresses MIF cytokine activity. For instance, ISO-1 significantly inhibited
prostate cancer growth through neutralizing MIF-triggered MAPK pathway activation both on the
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cellular level and in animal models. Other inhibitors like 4-IPP and SCD-19 (Figure 1B) were
also effective in inhibition of MIF-mediated tumor cell growth or migration.30,31 However, the
currently available inhibitors are not in clinical development for various reasons, such as, lack of
potency, poor physico-chemical properties, chemical reactivity, etc. Therefore, novel inhibitors
with improved properties are needed to facilitate both basic research as well as drug development.
Several assays have been developed to study binding to the MIF tautomerase active site. The
most commonly used assay format depends on MIF catalyzed keto-enol tautomerization of D-
dopachrome methyl ester or 4-HPP that can be monitored by a corresponding change in the UV
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absorption spectrum (Figure 1C). Despite its utility, this tautomerization based assay format has
several drawbacks. The use of D-dopachrome methyl ester has the disadvantage that it can undergo
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spontaneous decarboxylation to form a 5,6-dihydroxyindole (DHI) and CO , which makes this
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substrate less convenient. The substrate 4-HPP proved to be a more stable substrate for MIF
catalyzed tautomerization, 33 which stimulated broad application in MIF tautomerase activity
assays. Nevertheless, the 4-HPP enol reaction product proved to be relatively unstable in an
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aqueous environment for both enthalpic and entropic reasons. This creates the need to perform
the MIF catalyzed tautomerization of 4-HPP in buffers with relatively low pH (~6.0) and high
concentration of boric acid and ammonium acetate to stabilize the enol reaction product. We also
note that the UV absorbance at 306 nm for detection of the 4-HPP enol reaction product is
relatively unspecific, which increases the chance for interference by UV active compounds. These
and other factors can result in irregularities in MIF tautomerase enzyme activity assays as
described previously.35,36 This demonstrates the need for complementary assays to study MIF
binding such as the fluorescence polarization competition assay with fluorescently labeled MIF
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ligand B as shown in Figure 1D. Here, we provide a fluorescent indicator displacement (FID)
assay as a convenient and sensitive assay for MIF binding studies.
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Figure 1. Structure of MIF, MIF tautomerase inhibitors and molecules used for MIF binding
studies. A) The MIF surface alanine-scanning mutagenesis study results and ISO-1 binding site
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(PDB code:1LJT ). The regions highlighted in red or pink show mutants that can not or only
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partially activate CD74, respectively. ISO-1 is shown in green. The ISO-1 binding site is
located in the vicinity of amino acid residues involved in CD74 receptor activation, such as Y36,
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K66 and N109. B) Structure of ISO-1 , 4-IPP and SCD-19 . C) 4-HPP tautomerization
catalyzed by MIF. 33 D) Structure of Ligand B for fluorescent polarization probe assay37.
The FID assay provides a convenient format for competitive binding studies.39,40 In this assay
format, a fluorescent indicator is allowed to bind reversibly to a receptor upon which binding of a
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competing ligand can be quantified by displacement of the fluorescent indicator. Development
of a fluorescent indicator that binds tightly to the target and changes fluorescence upon binding is
key to successful development of an FID assay. We envisioned that 7-hydroxycoumarin
derivatives are promising fluorophores for the development of a fluorescent MIF binding sensor
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because of their intensive fluorescence. Importantly, these fluorophores were reported to bind to
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the MIF tautomerase active site. However, their utility in an FID assay for MIF has not yet been
explored.
In this study, we describe the development of a 7-hydroxycoumarin inhibitor as fluorescent
indicator for an FID assay to study binding to the MIF tautomerase active site. Towards this aim,
a series of 7-hydroxycoumarins were synthesized and the structure-activity relationships (SARs)
for MIF binding and concomitant fluorescence quenching were explored. A 7-hydroxycoumarin
derivative with nanomolar potency was identified and used for the development of a convenient
and sensitive FID assay for MIF binder assessment. Furthermore, we also explored the 7-
hydroxycoumarin inhibitor for its potency to interfere with the MIF-CD74 interaction and with
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MIF induced ERK phosphorylation and proliferation of A549 cells. Altogether, we identified a
novel MIF binding fluorophore that can be used in competitive binding studies as well as in cell-
based assays.
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RESULTS
Design and synthesis. In order to identify a suitable fluorophore for fluorescence quenching
binding studies, we set out to investigate a focused compound collection around the 7-
hydroxycoumarin scaffold with carbonyl or phenyl substitution in the 3-position. 2,4-
dihydroxybenzaldehyde 1 was employed as starting material to provide the desired product using
the Knoevenagel condensation as a key step as outlined in Scheme 1. Condensation of 1 with
diethylmalonate, using piperidine as base, provided 2 in 76% yield. Condensation of 1 with
malonic acid afforded intermediate 3 that was converted to compounds 4a and 4b by an amidation
reaction in yields of 78% and 85%, respectively. Tert-butyldimethylsilyl (TBDMS) protection of
1 provided 5 as a starting material for condensation with the corresponding 2-phenylacetic acids
to obtain compounds 6a–l. Condensation proceeded with cyanuric chloride and N-
methylmorpholine (NMM) followed by TBDMS deprotection using TBAF. The yields for these
subsequent two reaction steps were 20–90%. All final compounds were purified with
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chromatography and characterized by H and C NMR spectroscopy and LC-HRMS (Supporting
information).
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Scheme 1. Synthesis of 7-hydroxycoumarins as MIF inhibitors from 2,4-dihydroxybenzaldehyde
1 as a key precursor. Reagents and conditions: i. Diethyl malonate, piperidine, rt; ii. malonic acid,
pyridine, aniline, rt; iii. EDCI, HOBt, DMF, rt; iv. TBDMSCl, imidazole, CH Cl , rt; v. 1)
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Cyanuric chloride, NMM, DMF, reflux; 2) TBAF, THF, rt.
SARs. The focused compound collection described above was tested for inhibition of MIF
tautomerase activity employing 4-HPP as a substrate as described previously.35 In brief, the
compound stock solution in DMSO was subsequently mixed with aqueous EDTA solution and
MIF solution in assay buffer. After 10 min pre-incubation, the assay was started by mixing the
inhibitor-enzyme mixture with 4-HPP solution. The final reaction mixture contained 225 nM MIF,
0
.5 mM 4-HPP, 2.5% (v/v) DMSO and a variable inhibitor concentration. Enzyme activity was
monitored by the change in UV absorbance at 306 nm over time. The residual enzyme activity was
determined with reference to the positive control for which a corresponding amount of DMSO was
used, which was set to 100%. A control reaction in the absence of the enzyme was used as negative
control to correct for the spontaneous conversion of the substrate, which was set to 0%. The linear
regression parameters were determined to calculate the IC using GraphPad Prism. The IC
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values were transformed to K values using the Cheng-Prusoff equation: K =IC /(1+[S]/K ), in
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which [S] is the substrate concentration (0.5 mM) and K is the Michaelis-Menten constant (1.0
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mM) .
The SARs for inhibition of the MIF tautomerase activity by the 7-hydroxycoumarins are shown
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in Table 1. For compound 2 a K of 12.4±1.3 μM was observed, which is in line with previous
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reports (7.4±2.0 μM).43 Changing the ester to a substituted amide in compounds 4a and 4b
decreased the inhibitory potency. In contrast, phenyl substitution at the 7-hydroxycoumarin 3
position in compound 6a provided tenfold enhanced potency (K of 1.17±0.10 μM) compared to
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inhibitor 2. Subsequently, compound 6a was used as a starting point to evaluate changes in potency
upon substitution on the phenyl ring (6b–l). Substitution of the phenyl para-position with a chloro-
(6b), methyl- (6c), methoxyl- (6d), fluoro- (6j), bromo- (6k) or iodo- (6l) functionality provided
two- to three-fold improvements in inhibitory potency. In contrast, replacement of the phenyl with
a naphthalene (6e) or substitution at the meta- or ortho-position (6f–i) did not improve the potency
compared to 6a. Among the para-halogen substituted analogues, bromo-substitution in 6k
provided, with a K value of 0.31±0.02 μM, the highest inhibitory potency.
i
Table 1. Inhibition of the MIF tautomerase enzyme activity by 3-substituted 7-hydroxycoumarin
derivatives as determined by the conversion of 4-HPP as a substrate. (n = 3, standard deviation of
the non-linear curve fitting are reported).
a
Compound
R
IC (µM)
K (µM)
i
5
0
2
(Orita-1)
4a
-OCH CH
18.6±1.9
11% b
12.4±1.3 c
2
3
O
O
O
OH
R
-NH-C H
--
--
6
5
4b
-NH-CH -C H
5
12% b
2
6
6
a
H
1.76±0.14
1.17±0.10
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O
O
OH
6
b
4’-Cl
0.92±0.07
0.69±0.06
0.56±0.05
0.61±0.05
0.46±0.04
0.37±0.03
6
c
4’-CH3
R
6
d
4’-OCH3
1
1
1
1
1
1
1
1
1
1
2
2
2
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3
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6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
e
2.19±0.30
1.46±0.20
6f
3’-OCH3
2’-OCH3
3’-Cl
1.85±0.14
2.78±0.41
1.93±0.10
19.1±3.4
1.23±0.09
1.85±0.27
1.29±0.07
12.7±2.3
6
g
6
h
6
i
2’-Cl
6j
4’-F
2.54±0.27
0.47±0.03
0.72±0.06
1.69±0.18
0.31±0.02
0.48±0.04
6k
4’-Br
6
l
4’-I
a
b
c
43
K =IC /(1+0.5/K ); % inhibition at 25 μM; 7.4±2.0 μM (Orita-1) in literature.
i
50
m
Fluorescence quenching and indicator-displacement assay. From the focused compound
collection, inhibitor 6d was chosen for the initial exploration of an FID assay for MIF binding.
Inhibitor 6d has a UV absorption maximum at 340 nm in PBS (pH 7.4, Sigma) and a fluorescence
emission maximum at 460 nm, resulting in a Stokes shift of 120 nm (Figure 2A). The fluorescence
quantum yield was determined to be 0.25 (Figure S1), which is sufficiently high for the
4
2
development of a fluorescent sensor for binding studies. Importantly, the fluorescence intensity
of 6d is linearly correlated to its concentration at concentrations below 200 nM (Figure 2B).
Fluorescence quenching of 6d (200 nM) was observed upon addition of MIF (1 µM) (Figure 2C)
and the fluorescence quenching is concentration-dependent (Figure 2D). Quantification of the
change in fluorescence enabled determination of a dissociation constant of 0.39±0.04 µM for 6d
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6
to MIF (Figure 2E), which is in line with the K value calculated from the MIF tautomerase enzyme
i
activity assay. Taken together, this demonstrates that 7-hydroxycoumarin 6d shows affinity-
dependent fluorescence quenching upon binding to the MIF tautomerase active site, which enables
quantification of binding.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. The fluorescence of compound 6d is quenched upon binding to MIF. A) UV absorbance
(50 µM) and fluorescence emission spectra (200 nM) of 6d; B) Concentration-fluorescence
intensity correlation of 6d, n=3; C) Fluorescence and fluorescence quenching of 6d (200 nM) in
absence or presence of MIF (1 µM); D) Decrease in fluorescence intensity for 6d (100 nM) with
increasing concentration of MIF (10 nM to 4 µM); E) Concentration-dependent decrease of the
fluorescence of 6d (100 nM) in response to increased concentration of MIF, n=3. All experiments
were conducted in pH 7.4 PBS buffer.
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The affinity-dependent fluorescence quenching of 6d upon binding to MIF creates chances for
development of a FID assay to quantify binding to the MIF tautomerase active site. The quenched
fluorescence of 6d (100 nM) by MIF (1.0 µM) can be recovered by using the non-fluorescent MIF
inhibitor 8 in a concentration-dependent manner (Figure 3A). Plotting the fluorescence intensity
against the concentration of 8 provided a sigmoidal curve with an effective concentration for half
1
1
1
1
1
1
1
1
1
1
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5
5
5
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6
0
1
2
3
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5
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7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
recovery of the fluorescence (EC ) of 0.386±0.044 µM as derived by non-linear curve fitting.
50
Figure 3. Fluorescence recovery of 6d in presence of MIF by 8. A) Fluorescence spectra of 6d
(100 nM) together with MIF (1.0 µM) increased with the addition of 8; B) Nonlinear regression
for log concentration of 8 vs response, n=3.
The EC for fluorescence recovery was used to calculate the corresponding K value for binding
5
0
d
of 8 to MIF by application of equation 1 according to literature procedures.39,45 In this equation,
K is the disassociation binding constant between MIF and the nonfluorescent inhibitor; H is total
d
t
host (MIF) concentration; I is the total concentration of the fluorescent indicator (6d); EC is the
t
50
concentration of the competitor that provides half-maximal fluorescence recovery; K is the
S
dissociation binding constant of the interaction between MIF and fluorescent indicator (6b); F is
b
fraction of bound indicator (6b). Using this equation, a K value of 0.156±0.018 µM was calculated
d
for the binding of 8 to MIF. We note that this value corresponds well with the K value 0.100±0.010
i
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6
µM as determined by the MIF tautomerase activity assay using 4-HPP as a substrate. Binding
properties of 8 are also in line with the SARs of a group of structurally similar MIF inhibitors.37
At this point, we conclude that the FID assay provides a viable alternative for the MIF tautomerase
enzyme activity assay used for analysis of MIF binding molecules.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
2
3
4
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7
8
9
0
2
[
( ― 퐻 ∗ 퐹 ) + (퐾 + 퐼 ― 퐸퐶 + 퐻 ) ∗ 퐹 + 퐸퐶 ― 퐼 ] ∗ 퐹 ∗ 퐾
푡
푏
푆
푡
50
푡
푏
50
푡
푏
푆
퐾 =
푑
Equation ퟏ
2
(
1 ― 퐹 ) ∗ [(퐼 ∗ 퐹 ) ― ((퐾 + 퐼 + 퐻 ) ∗ 퐹 ) + 퐻 ]
푏 푡 푏 푆 푡 푡 푏 푡
Assay optimization. In the next step, we set out to improve the FID assay by development of a
7
-hydroxycoumarin with a higher affinity for MIF to gain sensitivity and enable reduction of the
concentrations of both the fluorophore and MIF. Improved affinity was achieved by expansion of
the substituent on the para-position of the 3-phenyl-7-hydroxycoumarin scaffold with a 4’-
carboxyphenyl functionality. This functionality was installed using a Suzuki cross-coupling
reaction on the para-iodophenyl derivative 6l to provide 7-hydroxycoumarin 7 (Scheme 2).
Interestingly, 7 exhibited a 10-fold increased potency compared to its precursor 6l in the MIF
tautomerase activity assay to provide a IC value of 71±3 nM, which indicates tight binding
5
0
properties. Because of the tight-binding properties the Morrison equation was used to calculate the
binding constant of 7, which proved to be 16±1 nM (see Figure S2).46
Scheme 2. Synthesis of 7. Reagents and conditions: vi. 4-boronobenzoic acid, Pd(AcO)2,
Na CO , EtOH, rt.
2
3
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7
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To rationalize binding of 7 to MIF, a docking study was performed for compound 7 using the
4
3
crystal structure of MIF bound to inhibitor 2 (Orita-1) (PDB code:1GCZ). Inhibitor 2 (Orita-1)
was removed from the binding site and redocked to validate the docking protocol. The 7-
hydroxylcoumarin part of 2 (Orita-1) occupies the active site that harbors the MIF tautomerase
activity. There is a second hydrophobic region at the rim of the tautomerase active site, to which
1
1
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1
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1
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2
2
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2
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6
0
1
2
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6
7
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9
0
1
2
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7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
(Orita-1) does not bind. The highest-scoring docking poses for compound 7 can occupy the same
position as observed for 2 (Orita-1) to provide similar interactions of the coumarin core (Figure
A and S8). The major difference between 2 (Orita-1) and 7 resides in the substitution at the
4
coumarin 3-position. 2 (Orita-1) formed a hydrophobic interaction between the ethyl group of the
ester and Lys32. In contrast, for compound 7 both phenyl rings at the coumarin 3-position formed
π–π stacking interactions with Tyr 36 and other interactions with Pro1 and Lys32 (Figure 4B) at
the rim of the tautomerase active site. Thus, compound 7 provides additional interactions with the
hydrophobic rim of the MIF tautomerase active site, which explains the higher MIF binding
potency observed for inhibitor 7.
Figure 4. Molecular modeling of compound 7 in the MIF tautomerase active site (PDB
4
3
code:1GCZ) . A) Overlay for binding of 2 (Orita-1) (green) and 7 (yellow) to the MIF tautomerase
active site. The protein is shown in ribbon diagram. Inhibitors are displayed as sticks; B) Binding
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of 7 to the MIF tautomerase active site shown in surface representation. The interactions between
phenyl rings of 7 and rim residues Tyr36, Lys32 are highlighted.
Meanwhile, 7 also demonstrated favorable fluorescence properties with a quantum yield of 0.32,
a Stokes shift of 100 nm (Figure 5A) and concentration dependent fluorescence quenching upon
binding to MIF nm (Figure 5B). Using this fluorescence quenching experiment, the binding
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0
constant K of 7 was determined to be 16±3 nM (Figure 5C), which is again in line with the
s
inhibition of the MIF tautomerase activity (K = 18±1 nM) . The high affinity of 7 for MIF enabled
i
the reduction of both the fluorophore (50 nM) and MIF (100 nM) concentrations in the FID assay.
This provides an assay format that is sufficiently sensitive to quantify the binding affinity of MIF
ligands with nanomolar potencies.
The FID assay was established with fluorophore 7. Firstly, a control experiment for non-MIF
dependent fluorescence quenching was performed with a corresponding amount of bovine serum
albumin (BSA). In clear contrast to MIF, no fluorescence quenching was observed upon the
addition of 800 nM BSA to a 50 nM solution of fluorophore 7 (Figure 5C). Enzyme kinetics
experiments demonstrated that 7 binds in competition with 4-HPP (Figure 5D). As a next step, we
aimed to distinguish static versus dynamic fluorescence quenching by determination of the
fluorescence lifetime of 7 (100 nM) in the absence (τ ) or in the presence (τ) of MIF (500 nM),
0
which proved to be 4.2 ns and 4.0 ns, respectively (Figure 5E). The constant value for the
fluorescence lifetime demonstrates that fluorescence quenching is static, which implies that
quenching occurs by specific binding of the fluorophore to a cavity in the protein rather than by
random collision-induced energy transfer. In addition, the influence of the solvent on the
fluorescence intensity of 7 (200 nM) was investigated. The fluorescence proved to be the strongest
in PBS buffer (Figure 5F), which indicates that an aqueous environment is most suitable for this
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fluorophore. The steep decrease in fluorescence in hydrophobic solvents, such as THF and toluene,
indicates that hydrophobicity of the binding site of MIF could play a role in fluorescence
quenching upon binding.
1
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1
1
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2
2
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7
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9
0
We previously observed that the MIF tautomerase activity assay using 4-HPP as a substrate was
2
+ 35
2+
sensitive to the presence of heavy metal ions such as Cu . Therefore, the influence of Cu ions
on this FID assay for MIF binding using 7 as a fluorophore was investigated. Importantly, no
2
+
influence of Cu ion was observed up to a concentration of 200 µM (Figure S5), which indicates
that this assay format is not sensitive to interference by this heavy metal ion.
Figure 5. Fluorescence properties of 7 and its fluorescence quenching upon MIF binding. A)
UV absorbance and fluorescence emission spectra of 7; B) Fluorescence quenching of 50 nM 7 by
MIF (12.5-200 nM); C) Quantification of fluorescence quenching of 50 nM 7 by MIF or BSA; D)
Lineweaver-Burk plot of inhibition of 7 against MIF tautomerase; E) Fluorescence lifetime study
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6
in 1 mL pH 7.4 PBS; half life of 100 nM fluorephore is 4.2 ns and together with 500 nM MIF is
4
.0 ns; F) Fluorescence intensity of 200 nM 7 in different solvents, Ex/Em=355/455 nm.
Thus, the FID assay using fluorescent indicator 7 was established as a method for competition
0
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7
8
9
0
based binding studies to the MIF tautomerase active site. This assay was used to determine the
affinity of a series of structurally diverse MIF tautomerase inhibitors (Figure 6)35,47 for validation.
This assay was performed in a 96-well format in which each well contained 100 µL MIF (200 nM,
in pH 7.4 PBS buffer) and 50 µL inhibitor in various concentrations in PBS (pH 7.4 with 10%
(v/v) DMSO). The mixture was incubated for ten minutes at room temperature and subsequently
5
0 µL indicator 7 (200 nM) in PBS buffer (pH 7.4) was added and incubated for ten minutes before
the fluorescence intensity was recorded at Ex/Em=355/455 nm. The window coefficient (Z’-factor)
of this assay was evaluated for an inhibition curve of 8 and proved to be 0.75 in this setup (SI 6),
which indicates the quality of this assay is sufficient for medium- to high-throughput applications
(0.5–1).48 Using the FID assay, the EC50 values for the structurally diverse series of MIF
tautomerase inhibitors were determined and used to calculate the K values using equation 1.
d
Comparison of the K values as determined by the FID assay to the K values as calculated from
d
i
the MIF tautomerase enzyme activity assay indicated that both methods provide comparable
affinity values (Table 2), thus indicating that the FID assay is a reliable and accurate alternative
for the MIF tautomerase enzyme activity assay.
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7
8
9
0
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6
7
8
9
0
Figure 6. Structurally diverse MIF inhibitors that were used to compare the MIF tautomerase
3
7
enzyme activity assay to the FID assay. Compound 8 is an analogue of NVS-2; 9 and 10 are two
4
9
50
typical biaryltrizoles synthesized by the Jorgensen lab; 11 is a phenolic hydrazone analogue;
35
1
2 and 13 were reported by our lab. 14 (Benzyl isothiocyanate, BITC) is a covalent inhibitor of
MIF.51,52
Table 2. Results for MIF tautomerase activity inhibition (K ) from the 4-HPP conversion assay
i
and for MIF binding (K ) from the fluorescent indicator displacement assay using compound 7 as
d
a probe. Data are shown as mean±SD of three independent experiments.
Compound
K (µM)
i
K (µM)
d
ISO-1
44±4.9
0.10±0.01
5.0±0.6
47±6.3
0.11±0.01
4.0±0.5
8
9
10
0.44±0.03
8.0±0.6
0.47±0.08
5.6±0.5
1
1
1
2
3.3±0.6
4.4±0.3*
1.4±0.1
13
4#
0.96±0.2
4.3±0.3
1
2.9±0.4
*K value measured by microscale thermophoresis (MST) was 3.6 µM.35 #Measured after 10
d
minutes incubation.
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6
Inhibition of the MIF-CD74 PPI. The high affinity of 7 for the MIF tautomerase active site
raised the question if this compound also interferes with the MIF-CD74 PPI. To address this
question, a previously published ELISA assay to monitor the interaction between MIF and sCD74
0
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was used. In this assay, human recombinant MIF was coated on a 96-well ELISA plate by
incubation at 4 °C overnight. After removal of the unbound MIF and blocked with 2% (w/v) BSA,
the plate was incubated with a mixture of MBP-sCD74 (500 nM) and inhibitor for half an hour.
Subsequently, bound MBP-sCD74 was detected using an anti-CD74 primary antibody and a signal
amplifying secondary antibody. The signal for MBP-sCD74 in the absence of inhibitor was set to
1
00% and the signal in which MBP-sCD74 was replaced by blank PBS buffer was used as a
negative control and set to 0%. Compound 7 provided a dose-dependent inhibition of the sCD74
binding to MIF with an IC of 36±3 µM (Figure 7). The MIF tautomerase inhibitors ISO-1 and
5
0
compound 8 were subjected to the same assay but did not demonstrate inhibition at concentrations
up to 50 µM (Figure S8). Thus, compound 7 interferes with the MIF-sCD74 interaction in this
assay format.
Figure 7. Compound 7 inhibits the MIF-sCD74 binding as determined by an ELISA assay.
Binding of MBP-sCD74 to MIF coated ELISA plates was detected using rabbit anti-CD74
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8
9
antibody as primary and goat anti-rabbit horseradish peroxidase conjugate as secondary antibody.
Data are displayed as mean±SD (n=3).
Cell-based study. MIF-induced signaling proceeds through binding to the CD74 receptor,
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activation of the MAPK signaling pathway and can result in cell proliferation. The ability of
5
4
inhibitor 7 to interfere with MIF-induced signaling was investigated in A549 cells. As a first
step, an MTS assay was performed, which demonstrated that compound 7 did not inhibit cell
viability at concentrations below 20 µM upon 24 hours of treatment (Figure S9). Next, the growth
inhibitory potency of compound 7 in A549 was studied by a colony-forming assay. Compounds
3
5
ISO-1 and 13 were used as references to prior literature and they inhibited colony formation at
100 µM and 10 µM, respectively (Figure 8). Treatment with inhibitor 7 in doses of 2.5 µM or 10
µM resulted in significantly lower numbers of colonies. These data demonstrate that 7 is a more
potent inhibitor of A549 cell proliferation than ISO-1 and 13.
Figure 8. Inhibition of cell proliferation by MIF inhibitors. A) Representative pictures of
clonogenic assays. A549 cells (100 cells per well) were treated with appropriate inhibitors and
stained with crystal violet; B) Colony quantification provided a bar graph showing inhibition of
colony formation upon treatment with MIF inhibitors ISO-1, 13 or 7. Colonies were counted by
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ImageG and confirmed by manual counting. One colony was estimated as an aggregate of >50
cells. Data were shown as mean±SD of three independent experiments. *p<0.05, **p<0.01 and
***p<0.001 vs control group.
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As a next step the influence of 7 on MIF-induced ERK phosphorylation was investigated in
A549 cells. Towards this aim, A549 cells were stimulated with MIF or compound 7 pre-incubated
MIF and subsequently ERK phosphorylation was detected by Western blot. We found that
compound 7 attenuated MIF-induced ERK phosphorylation in A549 cells (Figure 9) at a
concentration of 10 µM.
Figure 9. Effect of MIF inhibitor 7 on MIF induced ERK phosphorylation in A549 cells. A) A
representative result of western blot experiment. 150 ng/mL MIF was incubated with or without
appropriate concentrations of MIF inhibitors for 10 min, followed by the stimulation at 37°C for
1
0 min; B) Quantification of the pERK level using pERK:GAPDH ratio. In the control group,
vehicle was applied without MIF. In the vehicle group, MIF was incubated with appropriate
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amount of DMSO. Data are shown as mean±SD of four independent experiments. *p<0.05,
*
*p<0.01 and ***p<0.001 vs vehicle group.
DISCUSSION AND CONCLUSION
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In this study, we set out to develop an FID assay for competitive binding studies on the MIF
tautomerase active site. Towards this aim, we employed 7-hydroxycoumarins as fluorophores that
quench fluorescence upon binding to the tautomerase active site.42,43 In order to identify 7-
hydroxycoumarins with high potency, a focused collection of 3-substituted 7-hydroxycoumarins
was synthesized and inhibition of MIF tautomerase enzyme activity was investigated. We observed
that compounds with a phenyl substituent in the 7-hydroxycoumarin 3-position were more potent
than derivatives with an ester or amide functionality in this position. This result is in line with
literature in which the hydrophobic surface at the rim of active site of MIF was described to be
4
3
involved in the enhanced potency of 3-phenyl substituted 7-hydroxycoumarins. Further analysis
of the SARs indicated that para- phenyl substitutions in the 3-position of 7-hydroxycoumarins that
add bulk and are weakly electron-withdrawing are favorable for the potency of inhibitors. This
inspired the design of an inhibitor with a para-benzoic acid functionality. Inhibitor 7 proved to be
the most potent inhibitor of this series with a K of 47±2 nM. This confirms that MIF tautomerase
i
inhibitors that bind both in the active site to residues such as Asn97 and Tyr95 as well as on the
4
3
hydrophobic edge of the active site to residue Tyr36 provide low nanomolar potency. Taken
together, we obtained one of the most potent MIF tautomerase inhibitors by exploiting the
interactions with both the MIF tautomerase active site as well as the rim of the active site.
7
-hydroxy- and 7-aminocoumarins are widely used fluorescent sensors in biological
4
2
applications. Therefore, the fluorescence properties of the 7-hydroxycoumarin MIF inhibitors
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were exploited for the development of an FID assay for binding to the MIF tautomerase active site.
The fluorogenic properties of MIF inhibitors 6d and 7 are favorable with a Stokes shift exceeding
100 nm and a quantum yield of 0.25 and 0.32, respectively. Importantly, the fluorescence of both
0
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d and 7 quenched upon MIF addition in a concentration-dependent manner. Binding of the 7-
hydroxycoumarin fluorophores to the MIF tautomerase active site proved to be reversible, which
enabled development of competitive binding assay to recover the 7-hydroxycoumarin
fluorescence. Using fluorescence lifetime analysis, we found fluorescence quenching of 7 upon
binding to MIF to be static rather than dynamic. This excludes fluorescence quenching by random
collision, which also qualifies 7 as an appropriate fluorophore for FID assay development.
Therefore, 7 was used as the most potent MIF binder with suitable fluorogenic properties for the
development of an FID assay to quantify binding to the MIF tautomerase active site. This assay
employs the same format as applied for other targets such as carbonic anhydrase and retinoid X
receptor.40,55 Most fluorescent indicators contain three parts: a specific high-affinity target binding
moiety, a linker and a fluorophore. In contrast, 7-hydroxycoumarin 7 is a fluorophore and a high-
affinity binder at the same time. Furthermore, 7 is a small molecule that can be synthesized in only
four steps including TBDMS protection and deprotection, which makes this molecule and assay
format easily accessible.
The FID assay using 7 as fluorophore was validated through measuring binding affinity of a
group of structurally diverse MIF tautomerase inhibitors. The consistency between K values
d
assessed by the FID assay and the K values measured by the enzymatic tautomerase assay (Table
i
2
) demonstrates that the FID assay is a reliable substitution for the MIF tautomerase assay.
Notably, the FID assay has several advantages. For example, in contrast to the 4-HPP tautomerase
assay, the FID assay can be performed in PBS buffer and does not require reduced pH and borate
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2+
buffer (Figure S10). The presence of metal ions such as Cu did not interfere with the FID assay
in contrast to the tautomerase activity assay, which reduces the chance for irregularities in the
3
5
assay. In addition, the FID assay employs 100 nM MIF, which is more sensitive compared to the
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47
MIF tautomerase assay that typically requires 200-400 nM MIF. We note that the FID assay
could provide false positive results by fluorescence quenching through the competing ligand, for
which proper controls need to be included. We also note that K values of reference compounds
i
measured by 4-HPP tautomerase assay are consistent among different labs43,47 and also in line with
K values measured by other assays35,37, which demonstrates that the MIF tautomerase assay itself
d
is also a reliable assay if handled properly. Irregularities as reported previously in the literature36
can be excluded by proper operation of the respective assay. We envision that the FID assay will
gain a role as a complementary assay to the 4-HPP tautomerase assay.
Besides its use as a fluorescent indicator for MIF binding, compound 7 was also analyzed for its
ability to interfere with MIF-induced signaling. The CD74 receptor is important for MIF-induced
signaling and can mediate the MIF-induced cell growth and proliferation.6,9 Interestingly,
compound 7 interfered with the MIF and MBP-sCD74 interaction in an ELISA assay in contrast
to previously described inhibitors ISO-1 and 8. This implies that molecules binding to the MIF
2
4
tautomerase active site also interfere with the MIF-CD74 PPI in line with the literature. We
anticipate that interference with the MIF-CD74 PPI is enabled by interactions between compound
2
4
7
with Tyr36 of MIF, which is a key residue for the MIF-sCD74 PPI. However, we also note that
a significant “drop-off” in potency is observed for inhibition of the MIF-CD74 interaction in
56
comparison to MIF binding. Similar inconsistencies were reported earlier literature. We have the
impression that the MIF-CD74 interaction has some unresolved issues with respect to binding
stoichiometry in connection to binding avidity. The ELISA assay format, as applied here, might
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influence the binding avidity of the MIF-CD74 interaction, which influences inhibitor binding
potency. Nevertheless, inhibitor 7 also inhibited MIF-induced ERK phosphorylation and colony
formation of A549 cells in the clonogenic assay. Taken together, our results provide further
evidence that MIF-induced signaling can be inhibited by small-molecule inhibitors that target the
MIF tautomerase active site.28
0
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The currently available inhibitors of MIF tautomerase activity suffer often from poor physico-
chemical properties such as poor water solubility and high ClogP values, which can, among others,
3
6
result in irregularities in the assay readout. Notably, the solubility of compound 7 in pH 7.4 PBS
buffer was 18.8±1.4 µg/mL (53±3.9 µM), which overcomes the solubility issue of the triazole
4
7
inhibitors. Furthermore, ligand efficiency and lipophilic ligand efficiency of 7 are calculated to
be 0.37 and 5.80, respectively, which are favorable for biological activity (>0.3 for LE, and >5.0
5
7
for LLE). This demonstrates that inhibitor 7 has favorable physico-chemical properties and good
efficiency and potency against MIF tautomerase activity (Table 3) as well as in a cell-based assays
on MIF-induced signaling.
Table 3. Potency, Clog P, solubility and ligand efficiency of MIF tautomerase inhibitors.
Ki
(µM)
Solubility
(µg/mL)
Compound
MW
Clog Pa
LEb LLEc
ISO-1
44±4.9
235.08
1.69
1.35
1.97
3.64
3.80
--
0.34 2.48
0.37 5.80
0.43 4.85
0.39 2.55
7
8
0.018±0.001 357.08
18.8±1.4
0.10±0.01
0.44±0.03
0.96±0.2
302.10
385.07
288.10
--
2.247
1
0
13
10.8±0.1035 0.30 2.05
a.
b.
c.
®
Calculated by ChemDraw Professional 18.1;
LE (ligand efficiency) =1.4(-logIC )/N, N is the number of non-hydrogen atoms;
5
0
LLE (lipophilic ligand efficiency) =pIC -Clog P, pIC =-logIC .
5
0
50
50
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In conclusion, we have developed a convenient and effective FID assay to evaluate the affinity
of MIF tautomerase active site binders. The fluorescent indicator 7 was designed based on the
SARs of a group of 7-hydroxylcoumarin derivatives. 7 displays clear fluorescence quenching upon
binding to the MIF tautomerase active site that is reversible in the presence of competing ligands.
Using fluorophore 7, an FID assay was developed that enabled quantification of MIF binding in a
competition assay. This assay system proved to be more sensitive as compared to the 4-HPP
tautomerase assay and can be performed in neutral pH in PBS buffer. These results demonstrate
that 7 is a convenient fluorescent probe for MIF binding studies in an FID assay format. The most
potent MIF enzyme inhibitor 7 provides inhibition of the MIF-CD74 PPI and interferes with MIF-
induced ERK phosphorylation as well as cell growth in a clonogenic assay with A549 cancer cells
at micromolar concentrations. Taken together, compound 7 provides a valuable novel tool to
advance MIF-oriented research.
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0
EXPERIMENTAL SECTION
General. All the reagents and solvents were purchased from Sigma-Aldrich, TCI, Fluorochem
or Acros and were used without further purification. Reactions were monitored by thin layer
chromatography (TLC) method, in which Merck silica gel 60 F254 plates were used and spots were
detected with UV light. MP Ecochrom silica 32-63, 60 Å was used for column chromatography.
Nuclear magnetic resonance spectra, H NMR (500 MHz) and 13C NMR (126 MHz), were
recorded on a Bruker Avance 500 spectrometer. Chemical shifts were reported in ppm. Chemical
shifts were referenced to the residual proton and carbon signals of the deuterated solvent, CDCl3:
1
1
13
1
13
δ = 7.26 ppm ( H) and 77.05 ppm ( C) or DMSO-d6: δ = 2.50 ppm ( H) and 39.52 ppm ( C).
The following abbreviations were used for spin multiplicity: s (singlet), d (doublet), t (triplet), q
(quartet), dd (double of doublets), and m (multiplet). Coupling constants were reported in Hertz
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(
Hz). High-resolution mass spectra were recorded using Fourier Transform Mass Spectrometry
FTMS) and electrospray ionization (ESI) on an Applied Biosystems/SCIEX API3000-triple
(
quadrupole mass spectrometer. Purity of the compounds was determined by reverse-phase high-
performance liquid chromatography (HPLC) analysis to be >95%.
0
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Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (2).58 2,4-dihydroxybenzaldehyde (569
mg, 4.12 mmol) was dissolved in diethyl malonate (1.5 mL) and piperidine (0.5 mL). The mixture
was stirred at room temperature overnight. The resulting solution was acidified with an aqueous
solution of HCl (1 N, 5 mL). The precipitate was filtrated and washed with cold water (10 mL×2).
8
50 mg of desired product was obtained as yellow solid. Purity was 97% determined by HPLC.
1
Yield 76%. H NMR (500 MHz, DMSO-d ) δ 11.11 (s, 1H), 8.68 (s, 1H), 7.76 (d, J = 8.6 Hz, 1H),
6
6
.85 (dd, J = 8.6, 2.2 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1
1
3
Hz, 3H). C NMR (126 MHz, DMSO) δ 164.5, 163.4, 157.6, 156.9, 149.93, 132.6, 114.46, 112.5,
+
110.9, 102.3, 61.3, 14.6. HRMS, calculated for C H O [M+H] : 235.0601, found 235.0598.
1
2
11
5
59
7
-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (3). Malonic acid (1.35g, 13 mmol) was
dissolved in pyridine (4 mL), followed by addition of 2,4-dihydroxybenzaldehyde (1.0 g, 7.3
mmol) and aniline (0.1 mL). After stirring at room temperature overnight, the mixture was
acidified with 1 N HCl to pH 4.0. Precipitate was isolated by filtration and recrystallization in
methanol to provide 1.3 g product as yellow solid, yield 89%, Rf value 0.60 (CH Cl /MeOH,
2
2
1
1
0:1). H NMR (500 MHz, DMSO-d ) δ 12.89 (s, 1H), 11.07 (s, 1H), 8.68 (s, 1H), 7.75 (d, J = 8.6
6
1
3
Hz, 1H), 6.84 (dd, J = 8.6, 2.2 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H). C NMR (126 MHz, DMSO) δ
1
64.7, 164.4, 158.00, 157.5, 149.9, 132.5, 114.5, 113.0, 111.1, 102.3.
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7
-hydroxy-2-oxo-N-phenyl-2H-chromene-3-carboxamide (4a). 3 (80 mg, 0.4 mmol) was
mixed with aniline (60 µL) in dry DMF (2 mL), followed by adding EDCI (120 mg, 0.8 mmol)
and HOBt (70 mg, 0.5 mmol). The yellow solution was stirred under argon atmosphere at room
temperature overnight. Subsequently, the reaction mixture was diluted with CH Cl (20 mL) and
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0
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7
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7
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0
2
2
washed with a saturated aqueous NaCl solution (20 mL). The organic layer was concentrated under
reduced pressure to obtain the crude product, which was purified by column chromatography using
CH Cl as eluent. 80 mg pale yellow powder was obtained with yield 54%, Rf value 0.30
2
2
1
(
CH Cl /MeOH, 20:1). H NMR (500 MHz, DMSO-d ) δ 11.15 (s, 1H), 10.65 (s, 1H), 8.87 (s,
2
2
6
1
H), 7.86 (d, J = 8.6 Hz, 1H), 7.71 (d, J = 7.9 Hz, 2H), 7.37 (t, J = 7.8 Hz, 2H), 7.13 (t, J = 7.4 Hz,
1
3
1H), 6.91 (dd, J = 8.6, 2.1 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H). C NMR (126 MHz, DMSO) δ 164.4,
1
61.8, 160.6, 156.9, 148.88, 138.5, 132.69, 129.5, 124.6, 120.3, 115.0, 114.6, 111.7, 102.4.
+
HRMS, calculated for C H O N [M+H] : 282.0761, found 282.0757.
1
6
12
4
N-Benzyl-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4b). 3 (70 mg, 0.3 mmol) was
mixed with benzylamine(100 µL, 1.0 mmol) in dry DMF (1 mL), followed by addition of EDCI
(
120 mg, 0.8 mmol) and HOBt (70 mg, 0.5 mmol). The yellow solution formed was stirred at room
temperature with argon for overnight. Afterwards, reaction mixture was diluted with CH Cl (10
2
2
mL) and washed with water (10 mL×2). Aqueous layer was washed with CH Cl (10 mL×3). The
2
2
organic layer was collected and dried with MgSO , filtrated and concentrated by reduced pressure
4
evaporation. The product was purified by chromatography with Petroleum ether and ethyl acetate
1
1:1. 80 mg white solid was obtained with yield 54%, Rf value 0.40 (CH Cl /MeOH, 20:1). H
2
2
NMR (500 MHz, DMSO-d ) δ 11.06 (s, 1H), 9.05 (t, J = 6.0 Hz, 1H), 8.81 (s, 1H), 7.82 (d, J = 8.7
6
Hz, 1H), 7.33 (m, 4H), 7.25 (m, 1H), 6.88 (dd, J = 8.6, 2.2 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 4.53
1
3
(d, J = 6.0 Hz, 2H). C NMR (126 MHz, DMSO) δ 164.1, 162.2, 161.5, 156.8, 148.7, 139.6,
ACS Paragon Plus Environment
29