Structure-activity relationships for binding of 4-substituted triazole-phenols to macrophage migration inhibitory factor (MIF)

Article abstract of DOI:10.1016/j.ejmech.2019.111849

Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable further exploration of MIF as a drug target. In this study, we investigated structure-activity relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding. Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with the MIF tautomerase activity under the assay conditions applied. EDTA was added to the assay buffer to avoid interference of residual heavy metals with tautomerase activity measurements. Using these assay conditions the structure-activity relationships for MIF binding of a series of triazole-phenols was explored. The most potent inhibitors in this series provided activities in the low micromolar range. Enzyme kinetic analysis indicates competitive binding that proved reversible. Binding to the enzyme was confirmed using a microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the observed structure-activity relationships. The most potent inhibitor 2d inhibited proliferation of A549 cells in a clonogenic assay. In addition, 2d attenuated MIF induced ERK phosphorylation in A549 cells. Altogether, this study provides insights in the structure-activity relationships for MIF binding of triazole-phenols and further validates this class of compounds as MIF binding agents in cell-based studies.

Full text of DOI:10.1016/j.ejmech.2019.111849

Journal Pre-proof
Structure-activity relationships for binding of 4-substituted triazole-phenols to
macrophage migration inhibitory factor (MIF)
Zhangping Xiao, Marieke Fokkens, Deng Chen, Tjie Kok, Giordano Proietti, Ronald
van Merkerk, Gerrit J. Poelarends, Frank J. Dekker
PII:
S0223-5234(19)31001-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111849
EJMECH 111849
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 July 2019
Revised Date: 18 October 2019
Accepted Date: 3 November 2019
Please cite this article as: Z. Xiao, M. Fokkens, D. Chen, T. Kok, G. Proietti, R. van Merkerk, G.J.
Poelarends, F.J. Dekker, Structure-activity relationships for binding of 4-substituted triazole-phenols
to macrophage migration inhibitory factor (MIF), European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.111849.
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Structure-activity relationships for binding of 4-substituted triazole-phenols
to macrophage migration inhibitory factor (MIF)
Zhangping Xiaoa, Marieke Fokkensa, Deng Chena, Tjie Koka,b, Giordano Proiettia, Ronald van Merkerka, Gerrit J.
Poelarendsa, Frank J. Dekkera,*
a Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of
Groningen, Groningen, The Netherlands
b Faculty of Biotechnology, University of Surabaya, Surabaya, Indonesia
* Corresponding author: f.j.dekker@rug.nl
Graphical abstract
Abstract
Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in
inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable
further exploration of MIF as a drug target. In this study, we investigated structure-activity
relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding.
Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with
the MIF tautomerase activity under the assay conditions applied. EDTA was added to the
assay buffer to avoid interference of residual heavy metals with tautomerase activity
measurements. Using these assay conditions the structure-activity relationships for MIF
binding of a series of triazole-phenols was explored. The most potent inhibitors in this series
provided activities in the low micromolar range. Enzyme kinetic analysis indicates
competitive binding that proved reversible. Binding to the enzyme was confirmed using a
microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the
observed structure-activity relationships. The most potent inhibitor 2d inhibited A549 cell
proliferation in a clonogenic assay. In addition, 2d attenuated rhMIF induced ERK
phosphorylation in A549 cell. Altogether, this study provides insight in the structure-activity

relationships for triazole-phenols and further validates this class of compounds as MIF
binding agents in cell-based studies.
Keywords: Microphage migration inhibitory factor (MIF), transition metals, triazole-phenols,
tautomerase activity, clonogenic assay

1. Introduction
Macrophage migration inhibitory factor (MIF) was discovered in 1966 by Bloom and
Bennett as a cytokine that is implicated in the inhibition of macrophage motility.[1]
Subsequently, MIF was also discovered to function as a hormone, a chemokine and as a
molecular chaperone. Thus, MIF proved to be involved in various physiological and
pathological processes.[2–4] Genetic deregulation of MIF (such as overexpression) has been
implicated in many inflammatory and immune diseases in humans, such as diabetes,
atherosclerosis and rheumatoid arthritis.[5] In addition, mounting evidence supports a role of
MIF in tumorigenesis and progression.[6] Its key roles in health and disease raised interest in
development of small molecule MIF modulators as potential therapeutics.
Many MIF functions are mediated by protein-protein interactions with membrane
receptors. The cluster of differentiation 74 (CD74) receptor is the best characterized
membrane receptor for MIF.[7] Recently, another membrane protein CD44 was reported as an
integral component of the CD74 receptor complex, which is essential for MIF signal
transduction.[8] By forming a complex with CD74 and CD44, MIF triggers activation of the
mitogen activated protein kinase (MAPK) pathway. Activation of this pathway is associated
with MIF mediated oncogenesis and inflammation. MIF functions in inflammation and
immune cell chemotaxis are also mediated by interactions with chemokine receptors such as
CXCR2 and CXCR4.[4] Besides extracellular functions, MIF is known to bind to intracellular
protein targets such as Jun-activated domain-binding protein 1 (JAB1), which results in
slowing down JAB1 mediated cell growth.[9] Therefore, development of molecules
interrupting with MIF protein-protein interaction with extra- or intracellular targets has been
emerging as an attractive strategy to block MIF signaling.
Prior evidence indicates that treatment with anti-MIF antibodies or small molecule
MIF modulators enables disruption of MIF mediated functions.[10] Besides discovering a
statistically significant up-regulation of MIF concentration in the blood of septic patients,
Calandra et al. reported that an anti-MIF antibody can protect TNFα-deficient mice from
fulminant septic shock.[11] Neutralization of MIF with antibodies also exhibited benefits on
autoimmune encephalomyelitis[12], endotoxic shock[13] and even cancer[14]. From the
perspective of drug discovery, development of small molecule MIF binders has advantages
compared to the development of antibodies.[15,16] Many small molecule MIF binders have
been discovered, but only a few of them have been tested for their biological activity.[17–19]
Progression along this line is needed to shed light on the utility of MIF binders as a potential
novel strategy for management of inflame diseases.

Normally, MIF exists in a homotrimeric form in which each monomer contains 115
amino acids and has a molecular mass of 12.4 kDa.[20] Apart from its functions in protein-
protein interactions, the MIF trimer also harbors keto-enol tautomerase activity. There are
three tautomerase active sites in the MIF homotrimer, each located at the interface between
two monomers, in which the residue Pro1 has a key role in catalysis.[21] Although, D-
dopachrome and 4-hydroxyphenylpyruvate (4-HPP) were identified as substrates for the keto-
enol tautomerase activity of MIF, the physiological role of this activity remains elusive.
Recent findings indicate that Tyr36, Lys66 and Asn109, which are located on the surface
surrounding the catalytic site pocket of MIF, are involved in activation of CD74.[22] Besides,
there is also a thiol-protein oxidoreductase (TPOR) active site located at the center of the
enzyme, which is conferred by its Cys57-Ala-Leu-Cys60 (CALC) motif.[23] Peptides
containing the CALC region were reported to retain some biochemical properties of the full-
length MIF protein.[24] The structural correlations between its enzymatic activity and its
physiological functions provide opportunities to develop small molecule inhibitors of the
enzymatic activity that also modulate the physiological functions in which MIF is involved.
Although, the MIF tautomerase activity shows no direct correlation with its biological
functions, the pocket that harbors this enzymatic activity has been employed for development
of inhibitors that should ultimately interfere with MIF protein-protein interactions.[25–27]
Inhibitors include the mostly investigated dopachrome analogue ISO-1, inhibitor Orita-13 and
others (Fig. 1).[28–31] Importantly, many previously described inhibitors have a non-
reversible binding or a slow tight binding mode,[26] which obscures estimation of the
equilibrium binding constants. Also direct measurement of the binding of compounds to the
enzyme active site proved to be important to gain a realistic insight in the binding capacities
of compounds that interfere with MIF tautomerase activity.[32] Recently, the Jorgensen group
reported a series of competitive inhibitors of MIF tautomerase activity with a biaryltriazole
structure for which they confirmed reversibility of binding. The Ki value of the most potent
compound, Jorgensen-3bb ((Fig. 1), is 57 nM.[33–35] An interesting feature in their inhibitor
collection is that adding a fluorine at the ortho-position of the phenolic hydroxyl group
strongly favors inhibition. The highest potency was observed upon substitution on the triazole
4-position with a quinoline. However, this provides molecules with almost exclusively sp2-
hybridized atoms and consequently a very flat molecular structure. This limits water solubility
and potentially inhibitor selectivity. The availability of structural information for binding of
this class of inhibitors to MIF provides a basis to explore the structure-activity relationships
(SAR) further.

Figure 1. Reported inhibitors of MIF. ISO-1 is developed in 2002 by Al-Abed et al. and it is one of
the most investigated inhibitors of MIF.[28] Orita-13 was found in 2001 by a structure-based
computer-assisted search. Its Ki value was reported to be 38 nM.[29] Jorgensen-3bb is one of the most
potent inhibitors of MIF reported to date with a Ki value of 57 nM.[34]
Here we describe a SAR study for binding of the biaryltriazole-type of inhibitors to
MIF. Using the triazole-phenol unit as a core, we have explored alternatives for the quinoline
in the triazole 4-position in order to find inhibitors with a less planar structure. Firstly, we
identified the interference of transition metals such as copper(II) or zinc(II) with MIF
tautomerase activity, followed by optimizing the assay through addition of
ethylenediaminetetraacetic acid (EDTA) to the assay buffer. Subsequently, we investigated a
compound collection of 27 triazole-phenols that was assembled using the copper catalyzed
alkyne to azide cycloaddition (CuAAC) reaction as a key step. Using this approach, we were
able to identify novel triazole-phenols that reversibly inhibit MIF tautomerase activity and
also provide activity on the cellular level.
2. Results and discussion
2.1 MIF tautomerase activity assay optimization
Recombinant human MIF was expressed and purified following methods published
previously by our group [28] and others.[32] The MIF tautomerase activity assay using 4-HPP
as a substrate (Fig. 2A) was established using previously described assay conditions.[30] To
determine the IC50, the compounds were diluted from DMSO (20 µL) with water or water
with 20 mM EDTA (10 µL) into a a 96 well plate. Subsequently, the dilutions were pre-
incubated with the MIF enzyme solution (760 nM, 170 µL) for 15 minutes. The assay was
started by mixing 50 µL of the inhibitor enzyme pre-incubation mixture with 50 µL of an
aqeous 4-HPP (1.0 mM) solution. This provides a reaction mixture with 380 nM of the
enzyme, 0.5 mM 4-HPP and various concentration of the inhibitor. In the positive control,
MIF was incubated with a blanc DMSO dilution as a vehicle control before adding the 4-HPP
substrate. In the negative control, the substrate was mixed with a blanc DMSO dilution in

absence of MIF as an enzyme. In the negative control, the UV absorbance did not change over
time, which was set to 0%, whereas the positive control was set to 100%.
To test the stability of MIF, 3 samples from the same batch of enzyme were stored at
4 ℃ for 1, 12 and 25 days, respectively, after being taken from the -80 ℃ freezer. Different
storage times provide the same tautomerase activity levels (Fig. S1). In addition, unfolding
temperatures of fresh enzyme and enzyme stored for 2 weeks in at 4 ℃ were determined by
nanoDSF to be 77.9 ℃ and 78.9 ℃ (Fig. S1). These results together show that MIF is stable
upon prolonged storage at -80 ℃ or 4 ℃.
During our studies we noticed that irreproducible results from inhibitors synthesized
using the CuAAC “click reaction” were obtained. This raised the idea that transition metals
such as copper could influence the MIF tautomerase activity assay that we employed in our
studies. To our surprise, we found that copper(II) inhibits MIF tautomerase activity with an
IC50 of 1.0 µM (Fig. 2B). Expanding on this finding we also found that zinc(II) inhibits MIF
tautomerase activity with an IC50 of 1.0 µM (Fig. S3). This indicates that the observed
inhibition does not depend on the redox potential of the transition metal but rather suggests a
role for the metal ion as Lewis acid. These observations imply that presence of (traces of)
transition metals could contribute to irregularities in MIF tautomerase inhibition assays.[36]
Subsequently, we investigated the inhibition of MIF tautomerase activity by copper(II)
further. In the regular assays, we apply MIF with a C-terminal His-tag. To test the influence
of the His-tag, we used MIF without His-tag and found that the tautomerase activity of His-
tag free MIF was also completely blocked in presence of 20 µM copper(II) (Fig. S4). In
addition, we found that copper(II) also blocked the activity of a related enzyme, 4-
oxalocrotonate tautomerase (4-OT), in a Michael addition reaction (Fig. S4).[37] In the
literature, it has been reported that the copper(II)-containing protein ceruloplasmin (CP) can
suppress MIF enzymatic activity,[38] which is in line with our findings here. Altogether,
these findings indicate that the transition metals copper(II) and zinc(II) can interfere with the
4-HPP tautomerization reaction catalyzed by MIF under the assay conditions applied in this
study.
We adjusted the assay conditions to prevent interference of transition metals with MIF
tautomerase enzyme activity in inhibitor binding studies. Including ethylenediaminetetraacetic
acid (EDTA) in the assay buffer proved to be an effective strategy to prevent interference of
copper(II) with the MIF tautomerase activity. Our results indicate that 125 µM copper(II) has
no effect on 4-HPP tautomerization by MIF if 0.5 mM EDTA is added to the assay buffer (Fig.
2C). Addition of EDTA did not influence the Km value for 4-HPP conversion as both

conditions provided a Km of 1.1 mM in the Michealis-Menten enzyme kinetics (Fig. S2).
Based on these findings we included 0.5 mM EDTA in the MIF tautomerase assay buffer in
our studies. This is particularly important for activity measurements of 4-(1,2,3-
triazole)phenols, as studied here, because their synthesis requires significant amounts of
copper, which could result in the pollution of the final products.
We note that inhibition of MIF tautomerase activity by transition metals is the first
time reported here, but that EDTA has already been used before in the assay buffer in MIF
inhibitor development.[29,39] However, in other studies, including those using CuAAC for
inhibitor synthesis, did not apply EDTA in the assay buffer.[34,35] Here we provide a
rationale to include EDTA in the assay buffer in cases where the presence of traces of
transition metals in the final products can be expected.
Figure 2. MIF tautomerase activity using 4-HPP as a substrate and effect of copper(II) on MIF
tautomerase activity. A) MIF catalyzed conversion of 4-HPP. B) Concentration dependent inhibition
of MIF tautomerase activity by copper(II). C) Residual tautomerase activity of MIF in presence of 0.5
mM EDTA and different concentrations of copper(II).
2.2 Synthesis
A focused compound collection of 4-(1,2,3-triazole)phenol derivatives was obtained
by coupling 4-azidophenol to various terminal alkynes using CuAAC (Scheme 1). In this
study, the CuAAC reaction was performed by addition of a catalytic amount of CuSO4 and
sodium ascorbate in water to the alkyne and azide substrates dissolved in methanol. The
reaction was allowed to proceed at room temperature or 60 ℃ for 12 hours.[40] The terminal
alkyne precursors for compounds of group A were prepared using propiolic acid as a key
intermediate. Different amines were coupled to propiolic acid through a N,N'-
Dicyclohexylcarbodiimide (DCC) mediated amidation. The subsequent step provided the final
products in overall isolated yields between 30% to 86%. Compounds of group B were
synthesized by coupling various aliphatic terminal alkynes to 4-azidophenol at room
temperature to provide a series of 4-(1,2,3-triazole)phenol derivatives. For this series the

isolated yields varied between 12% and 96%. Compounds of group C were synthesized using
2-propargylamine as key intermediate. Different carboxylic acids were coupled to
propargylamine using DCC mediated amidation to provide substituted terminal alkynes that
were subjected to the “click” reaction. These two subsequent reactions were conducted at
room temperature with isolated yields between 15% and 98%. The variability of the yields
could be attributed to the low solubility of several products in combination with the filtration
in the final step of the synthesis.
Scheme 1. Synthesis of MIF inhibitors in four main groups. Group A represents compounds with
propiolic acid as key building block. Compounds of group B were synthesized using various aliphatic
terminal alkynes. Compounds of group C contain propargylamine as key precursor. Compounds of

group D take advantage of ortho-fluor substitution of the phenol for enhancing potency. Reagents and
conditions: i. a) NaNO2, HCl, H2O, rt, 2h; b) NaN3, rt,1h, 99%; ii. DCC, CH3CN, rt, 2h, 95%; iii.
CuSO4 (0.1 eq), sodium ascorbate (0.2 eq), MeOH, rt, overnight, 12-90%; iv. DCC, CH3CN, rt, 2h; v.
Pd, H2, rt, 4h, EtOH, 99%.
2.3 Enzyme inhibition study
The focused compound collection was screened for inhibition of MIF tautomerase
activity by an assay employing 4-HPP as a substrate. The assay conditions, as described
above, include 0.5 mM EDTA to avoid interference of residual copper(II) with the
tautomerase activity. The inhibitors were screened for MIF binding by measurement of the
residual MIF tautomerase activity in presence of 50 µM of the respective inhibitor. MIF
tautomerase inhibitors ISO-1 and Jorgensen-2b were tested as a reference to literature values
(Table 1). These inhibitors reduced the activity of MIF with percentages of 20% and 90%
respectively at 50 µM, which is in line with their reported potency for MIF inhibition in
literature.[34]
Screening of the residual enzyme activity upon preincubation with 50 µM of the
respective inhibitor provided clear structure-activity relationships for the three groups of 4-
(1,2,3-triazole)phenol inhibitors as shown in Fig. 3. For compounds of group A the residual
enzyme activity is around 75% of the positive control for all inhibitors, which indicates a
potency similar to ISO-1 without a clear structure-activity dependence. For group B the
residual enzyme activity varies between 90% and 10% of the positive control, thus indicating
a clear structure-activity dependence. Extending the 4-substitution of the 4-(1,2,3-
triazole)phenol scaffold from propyl to pentyl and 5-hexynyl reduces the residual enzyme
activity from 90% to 10% of the control, whereas octyl substitution provides increases
residual enzyme activity. This indicates that an aliphatic tail with 5 or 6 carbon atoms is
optimal in this series of inhibitors. The structure-activity relationships for the compounds in
group C are less clear with residual enzyme activities between 40 and 10% of the control with
compound 7c as the most active one in this series.

100
75
50
25
0
Figure 3. Screening of the inhibitory potency of the triazole-phenol collection at 50 µM inhibitor
concentration. The enzyme activity in absence of inhibitor was set to 100%. Data are presented as
mean ± standard deviation (n = 3).
All the compounds that inhibited MIF tautomerase activity by more than 50% of the
positive control were subjected for IC50 determination. The IC50 values are shown in Table 1.
Ki values are calculated by the Cheng-Prusoff equation: Ki=IC50/(1+[S]/KM)[41] in which KM
= 1.1 mM. The Ki value of ISO-1 was measured to be 44 ± 4.9 µM, which is in line with
values reported in literature.[30][36] To further confirm the validity of our assays and to
enable direct comparison, inhibitor Jorgensen-2b and 3b were synthesized according to
literature.[42] Using Jorgensen-2b we investigated the effect of addition of EDTA to the
assay buffer on the IC50 and Ki values in the MIF inhibition assay. For Jorgensen-2b, a Ki of
5.0±0.6 µM was determined in presence of 0.5 mM EDTA, whereas a Ki value of 2.9±0.3 µM
was determined in absence of 0.5 mM EDTA. This demonstrates that EDTA can influence the
Ki values to a certain extend but that the observed differences in this case a limited to less
than a two-fold change in potency. Ki value of Jorgensen-3b was determined to be 0.42±0.03
µM with 0.5 mM EDTA presence. The Ki values observed in our assay are in line with the Ki
of 8.8 and 0.59 µM for Jorgensen-2b and 3b, respectively, reported before by Jorgensen.[42]
Compounds in group A were not subjected to IC50 determinations due to a lack of inhibitory
potency at concentrations of 50 µM. Group B provided two inhibitors with potencies in the
low micromolar range. Compounds 5b and 7b had a Ki of 6.9±0.3 µM and 6.5±0.8 µM,
respectively. The clear activity dependence on the length of the aliphatic tail indicates a role
for lipophilic interactions in binding. For group C, inhibitors with variant monocyclic

aromatic groups exhibit Ki values ranging from 19 to 35 µM. However, the bycyclic indole
functionality provided stronger inhibition with a Ki value of 6.9±0.5 µM for 7c. This could be
due to both stacking and/or lipophilic interactions between the indole functionality of 7c and
MIF. Extending the single carbon atom spacer between the triazole and the indole in 7c to a
two-carbon atom spacer in 8c caused a loss of potency with a Ki of 43±2.8 µM, which
indicates a clear structure dependence of the activity.Table 1. Inhibition of MIF tautomerase
activity by 4-substituted triazole phenols.
%residual
IC50 (µM)
Comp.
ISO-1
R1, R2, R3 or R4
Ki
activity*
--
--
64±7.1
44±4.9
Jorgensen
--
--
--
--
7.2±0.9
5.0±0.6
-2b
Jorgensen
0.64±0.05
0.44±0.03
-3b
3,4-OMe-phenyl
4-F-phenyl
2-F-phenyl
2-amine-phenyl
phenyl
77 %
74 %
80 %
77 %
71 %
74 %
81 %
91 %
73 %
62 %
50 %
--
--
--
1a
2a
3a
4a
5a
6a
7a
1b
2b
3b
4b
5b
6b
7b
8b
--
--
--
--
--
--
--
--
cyclopropyl
2-ethoxy-2-oxoethyl
hydroxymethyl
1-hydroxyethyl
isopropyl
--
--
--
--
--
--
--
--
--
--
propyl
--
--
pentyl
10±0.5
26±2.2
9.5±1.1
--
6.9±0.3
17±1.5
6.5±0.8
--
cyclohexyl
5-hexynyl
--
--
octyl
53 %
4-methoxy-4-
oxobutyl
--
--
33±2.0
47±5.0
23±1.4
32±3.4
9b
3-oxo-3-
10b
(phenylamino)propyl

phenyl
--
--
--
--
33±1.4
41±4.0
43±4.2
38±4.1
23±1.0
28±2.7
30±2.9
26±2.8
1c
2c
3c
4c
thiophen-2-yl
1H-pyrrol-2-yl
5-bromofuran-2-yl
5-methylthiophen-2-
yl
--
28±2.0
19±1.4
5c
4-F-phenyl
--
--
53±3.0
10±0.7
36±2.0
6.9±0.5
6c
7c
1H-indol-2-yl
(1H-indol-3-
yl)methyl
--
--
--
62±4.0
4.8±0.5
1.4±0.3
43±2.8
8c
1d
2d
H
3.3±0.3
0.96±0.2
Cl
* Residual MIF tautomerase activityin presence of 50 µM inhibitor.
2.4 Inhibitor optimization and enzyme kinetic study
To further improve the inhibitory potency of the inhibitors identified against MIF, we
substituted the phenolic ortho-position with a fluorine.[34] Therefore, two new inhibitors
were synthesized as shown in Scheme 1D. The resulting compound 1d provided a Ki of
3.2±0.3 µM, which is two times enhanced compared to its equivalent without fluorine 7c (Fig.
4A). This proves again that an ortho-fluoro substitution is a favorable modification for
triazole-phenol MIF inhibitors. By substitution of the 5-position of the indole in 1d with
chlorine, inhibitor 2d was obtained, which proved to me more potent with a Ki of 0.96 ± 0.2
µM. The solubility of 1d and 2d were respectively 24±0.19 (41.41±0.51 µM) and 10.8±0.10
µg/mL (28.01±0.26 µM) in pH 7.4 PBS buffer as measured by the shake-flask method (Fig.
S10). Both 1d and 2d have significantly improved aqueous solubility compared toinhibitor
Jorgensen-3b, which is 2.2 µg/mL.[34] This demonstrates that reducing the planar character
of inhibitors indeed improved water solubility. We note, however, that 2d did not dissolve
completely at concentrations of 50 µM and higher, which prohibits measuring full inhibition
in the enzyme inhibition study.
To investigate the mechanism of inhibition and to avoid artefacts as described
previously,[25] the binding behavior of inhibitor 1d was characterized further. A pre-
incubation and dilution assay was performed to study reversibility of 1d binding to MIF.

Towards this aim, MIF was pre-incubated with 83 µM of 1d for 10 minutes before 100-fold
dilution and testing of the residual enzyme activity. Upon pre-incubation with 1d the MIF
tautomerase activity could be fully recovered as compared to the positive control without
inhibitor (Fig. 4B). This indicates reversible binding for 1d.
The influence of inhibitor 1d on the enzyme kinetics of MIF-catalyzed conversion of
4-HPP was analyzed (Fig. 4C, 4D). In presence of 2 or 5 µM 1d the Km increases from 1.14
to 1.60 and 1.95 µM respectively, whereas the Vmax remains constant between 0.24 and 0.29
(absorbance/min) compared to 0.27 for the control (Table 2). Thus, the enzyme kinetics
demonstrate that 1d is a competitive inhibitor, which is consistent with previous reports for
MIF inhibition by inhibitors with a triazole-phenol core.[34] Similar results were obtained for
2d as shown in the supporting information.
Figure 4. Inhibition of MIF tautomerase activity by 1d. A) Dose-response curve for inhibition of MIF
tautomerase activity by 1d. B) Pre-incubation and dilution assay of 1d and MIF. After 10 minutes pre-
incubation at 83 µM 1d for 10 minutes the MIF solution was diluted 100-fold and employed for 4-
HHP conversion. In the control group no enzyme was added. C) Michaelis-Menten plots of MIF
activity at concentration of 0, 1.0 and 2.5 µM of inhibitor 1d. D) Lineweaver-Burk plots of MIF at
concentration of 0, 1.0 and 2.5 µM of 1d (n is 3 for all results shown in this figure.).
Table 2. Enzyme kinetic parameters for inhibition of MIF by 1d.
ꢀꢂꢁꢃꢃ (mM)
ꢄꢂꢁꢃꢂꢃꢅ (absorbance/min)
1d (µM)

0
1
1.14±0.17
1.60±0.24
1.95±0.33
0.27±0.02
0.29±0.02
0.24±0.02
2.5
The p-values show that the slopes are significantly non-zero (n=3, p-value < 0.05).
2.5 Binding affinity study
To confirm binding of 1d to MIF, we performed a microscale thermophoresis (MST)
assay. MST is a newly emerging technology for analysis of binding to proteins. This
technology exploits the ligand-induced changes in the molecular movement of fluorescently
labeled proteins in a temperature gradient.[43] We determined the thermophoresis shift upon
titration of different concentration of 1d to 50 nM MIF. The KD value was determined from
the changes in thermal shifts upon titration of 1d, which provides an sigmoidal curve from
which the binding affinity (KD) was calculated to be 3.63 µM. Thus, the KD observed in the
MST assay is comparable to the Ki value of 3.2 µM calculated from the enzyme kinetic
experiments (Fig. 5). Altogether, we conclude that the 4-(1,2,3-triazole)phenol inhibitor 1d
binds reversibly to MIF with a potency in the low micromolar range.
Figure 5. Microscale thermophoresis measurements of the binding affinity of 1d for MIF. Fnorm
(Normalized fluorescence)=Fhot/Fcold. n=3.
2.6 Molecular modeling
Docking studies were performed to rationalize the structure-activity relationships
observed for MIF inhibition. Structural information for the MIF interaction of the analogous
biaryltriazoles was used as a basis for the molecular modeling (PDB code:4wrb and 5hvs ).[34]
Modelling was performed using the software Discovery Studio 3.0. The compounds were
docked into the crystal structure of MIF and energy minimized. The highest scoring poses
were analyzed and compared with reference inhibitor Jorgensen-3bb.[34] The 2-fluoro-4-
(1,2,3-triazole)phenol part of 1d occupies the same position as observed in Jorgensen-3bb

(Fig. 6A, 6B, 6C). A main difference is that the quinolone in Jorgensen-3bb occupies a
position different from the indole 2-carboxamide functionality in 1d. The difference in
potency could originate from the loss of stacking interactions between quinoline ring and
residues Pro33 and Lys32 that convey a high potency to Jorgensen-3bb. Instead, 1d has a
hydrogen bond with Lys32 and a hydrophobic interaction with Ile64. Although 1d does not
reach the same potency as reported for Jorgensen-3bb, the interactions observed in the
modelling appear to be useful because they enable targeting of a different part of the binding
pocket.
The structure-activity relationship was further rationalized by investigating details
from docking poses of 1d, 7c and 3c. The binding difference between 1d and 7c was a
hydrogen bond could be found between the fluoro of 1d and residue Asn97, which was not
existing for 7c. The extra hydrogen bond can contribute to the two-time enhancement of
potency from 7c to 1d. Comparison was also made between 7c with the indole 2-carboxamide
that provided a Ki of 6.7 µM and 3c with a pyrrole 2-carboxamide that provided a Ki of 29
µM (Fig. 6D, 6E). The 4-fold change in potency between 3c and 7c could be attributed to
hydrophobic interaction from Ile64.
Figure 6. Interactions between inhibitors and MIF. A) Binding of 1d (yellow) and Jorgensen-3bb
(cyan) to the MIF active site. The protein is shown in cartoon. Inhibitors and the residues involved in
binding are displayed as sticks. B) Binding mode of compound 1d. Compound 1d is shown as sticks.
Some residues are removed for clarity. Key residues are highlighted. C) Binding modes of compound
1d. D) Binding modes of compound 7c. E) Binding modes of compound 3c. Inhibitors are shown as
thick sticks and residues involved are represented by thin sticks with carbon in gray, oxygen in red and
nitrogen in blue. Key interaction differences are highlighted.

2.7 A549 cell colony formation assay and inhibition of ERK phosphorylation
MIF inhibitors have been shown to have inhibit the growth of A549 non-small cell
lung cancer cells , in which MIF plays an essential role for anchorage-independent growth and
invasive behavior.[44,45] The growth inhibitory potency of 2d against A549 cells was
measured by a colony formation assays. A549 cells were seeded in 6-well plates with 200
cells per well. The cells were incubated for 10 days in medium contain various concentrations
of the respective inhibitor. ISO-1 was applied as a positive control. The results are shown in
Fig. 7A. Inhibitor ISO-1 inhibits colony formation at 20 and 100 µM Also treatment of A549
cell with inhibitor 2d at 2.5, 10 and 20 µM decreased the number of colonies in a
concentration–dependent manner. This demonstrates that compound 2d inhibits cell
proliferation in the clonogenic assay at 10-fold lower concentrations compared to MIF
inhibitor ISO-1.
To gain more insight into the effect of MIF inhibitors to tumor cell proliferation,
phosphorylation of ERK is investigated in A549 cells. Towards this aim the cells are
incubated with 2d or ISO-1 before treatment with rhMIF. Both 2d, as well as ISO-1,
attenuate rhMIF induced ERK phosphorylation in A549 cell (Fig. 7B). Thus inhibitor 2d is
able to inhibit ERK phosphorylation and cell proliferation in A549 cells.
Figure 7.MIF inhibition in A549 cells. A) A549 cells were treated with varying concentration of ISO-
1 or 2d and stained with crystal violet. Bar chart showing the decreased number of colonies after

incubation with ISO-1 or 2d. Colonies were counted by by ImageG and confirmed by manually
counting. One colony was defined to be an aggregate of >50 cells. Data was shown as mean±SD of
three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs control group. B) Effect of
MIF inhibitors on activated ERK signaling pathway. The cells were treated with the respective MIF
inhibitors for 10 min, followed by the stimulation of 50 ng/µl MIF for 15 min at 37°C. pERK:GAPDH
ratio was applied to quantify pERK level (n=1, duplicates will be provided).
3. Conclusion
In this study, we investigated the structure-activity relationships for inhibition of MIF
tautomerase activity by inhibitors with a 4-(1,2,3-triazole)-phenol core. During our studies we
discovered that the transition metals copper(II) and zinc(II) inhibit MIF tautomerase activity
with potencies in the low micromolar range. Addition of 0.5 mM EDTA to the assay buffer
proved to be effective to avoid potential interferences from residual metal ions in inhibitor
preparates. We aimed to replace the previously employed quinolone functionality by
screening of a focused compound collection of 4-(1,2,3-triazole)-phenols. Several novel
inhibitors were identified from which 7c with an indole 2-carboxamide functionality proved
to be the most potent one. Further improvement of potency was achieved by ortho-
fluorination of the phenol functionality to provide 1d. Compound 1d proved to be a reversible
and competitive inhibitor of MIF tautomerase activity with a Ki value of 3.2 µM. Binding was
confirmed using an MST assay, which provided a KD of 3.6 µM. In addition, we
demonstrated addition of a chlorine in the indole 5-position of 1c provided compounds 2d
with further enhance potency. This compound proved also effective in the inhibition of colony
formation and attenuation of ERK signaling in A549 cells. Altogether, we present novel
insights in the MIF tautomerase activity assay and we provide a novel triazole-phenol
inhibitor 2d with cellular activity, which provides a basis for further development of MIF
inhibitors.
4. Experimental section
4.1. Chemistry
4.1.1. General
All the reagents and solvents were purchased from Sigma-Aldrich, AK Scientific,
Fluorochem or Acros and were used without further purification. Reactions were monitored
by thin layer chromatography (TLC). 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

1
chromatography. Nuclear magnetic resonance spectra, H NMR (500 MHz) and 13C NMR
(126 MHz), were recorded on a Bruker Avance 500 spectrometers. Chemical shifts were
reported in ppm relative to the solvent. 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. Melting points
were measured by Electrothermal IA9100 Melting Point Apparatus.
4.1.2. Azidophenol synthesis
Concentrated HCl (3.5 mL) was added dropwise to a solution of 4-aminophenol (1.5 g,
13.7 mmol) in water (20 mL) over a period of 5 minutes. After cooling the resulting solution
to 0℃, NaNO2 (1.9 g, 27.5 mmol) was added portion-wise. The mixture was left stirring for 1
hour at room temperature. A freshly made solution of NaN3 (1.8 g, 27.5 mmol) in a few mL
of demi-water was added dropwise to the reaction mixture and left stirring for 1h at room
temperature. The reaction mixture was extracted with ethyl acetate (3x50 mL). The combined
organic layers were extracted with brine and dried with MgSO4, filtered and concentrated
under reduced pressure. The product was obtained as a dark liquid and used without further
purification.
4-azido-2-fluorophenol was prepared analogously to 4-azidophenol using 4-amino-2-
fluorophenol as starting material. The product was obtained as a dark liquid and used without
further purification.
4.1.3. Synthetic procedure of compounds in group A
Compounds of group A were synthesized by coupling of propiolic acid to the
corresponding amines followed by CuAAC coupling to azidophenol. Towards this aim,
propiolic acid (1 eq.) and N,N’-dicyclohexylcarbodiimide (DCC, 1.0 eq.) were dissolved in
dry acetonitrile (MeCN) and cooled in an ice bath for 15 minutes. The corresponding amines
were added into the respective mixture and stirred at room temperature for 2h. The resulting
precipitate was removed by filtration. The solvent was removed under reduced pressure and
the products were directly used for the next step. 4-azidophenol (1.0 eq) and the appropriate
alkyne (1.0 eq) were dissolved in MeOH (4 mL) in a round bottom flask equipped with
stirring bar. Fresh prepared solutions of CuSO4 (0.1 eq) in demi-water (0.20 mL) and sodium
ascorbate (0.20 eq) in demi-water (0.20 mL) were added sequentially to the reaction mixture.
The reaction mixture was left stirring overnight for at 60 ℃. The reaction mixture was diluted
with AcOEt and then filtered. The residue was washed with a small amount of methanol. The

filtrate was extracted with brine and then concentrated under reduced pressure. The residue
was purified using column chromatography with CH2Cl2: MeOH 10:1 to yield the desired
products.
N-(3,4-dimethoxyphenyl)-1-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (1a, MZ038)
1
Yield 77%. m.p. 250.4-253.0 ℃. H NMR (500 MHz, DMSO-d6) δ 10.36 (s, 1H),
10.03 (s, 1H), 9.20 (s, 1H), 7.76 (d, J = 8.9 Hz, 2H), 7.54 (d, J = 2.4 Hz, 1H), 7.45 (dd, J = 8.7,
2.4 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.8 Hz, 1H), 3.75 (s, 3H), 3.74 (s, 3H). 13C
NMR (126 MHz, DMSO) δ 158.19, 157.84, 148.44, 145.26, 143.63, 132.05, 128.34, 125.01,
122.42, 116.09, 112.34, 111.84, 105.61, 55.74, 55.43. HRMS, calculated for C17H17O4N4
[M+H]+: 341,1244, found 341,1247.
N-(4-fluorophenyl)-1-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (2a, MZ044)
1
Yield 30%. m.p. 252.5-254.0 ℃. H NMR (500 MHz, DMSO-d6) δ 10.65 (s, 1H),
10.05 (s, 1H), 9.26 (s, 1H), 7.89 (dd, J = 9.1, 5.1 Hz, 2H), 7.78 (d, J = 8.9 Hz, 2H), 7.21 (t, J =
8.9 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 158.66, 158.86 (d, J =
240.3 Hz), 143.82, 135.38 (d, J = 2.5 Hz), 128.77, 125.77, 122.85, 122.73, 122.80, 116.54,
115.68 (d, J = 22.2 Hz). 19F NMR (376 MHz, DMSO-d6) δ -118.70 (tt, J = 9.3, 5.1 Hz).
HRMS, calculated for C15H12O2N4F [M+H]+: 299,0939, found 299,0939.
N-(2-fluorophenyl)-1-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (3a, ZP034)
1
Yield 53%. m.p. 224.8-226.3 ℃. H NMR (500 MHz, DMSO-d6) δ 10.16 (s, 1H),
10.05 (s, 1H), 9.28 (s, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 5.4 Hz, 1H), 7.36 – 7.20 (m,
3H), 6.96 (d, J = 8.7 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 158.65, 156.64, 154.68, 143.24,
128.77, 127.17, 126.53, 125.77, 125.61, 124.88, 122.87, 116.55, 116.22. 19F NMR (376 MHz,
DMSO-d6) δ -122.20 (ddd, J = 9.3, 7.1, 4.2 Hz). HRMS, calculated for C15H12O2N4F
[M+H]+: 299,0939, found 299,0936.
N-(2-aminophenyl)-1-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (4a, ZP039)
1
Yield 86%. Decomposed at 250 ℃. H NMR (500 MHz, DMSO-d6) δ 10.04 (s, 1H),
9.82 (s, 1H), 9.22 (s, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 7.6 Hz, 1H), 6.97 (m,3H),
6.80 (d, J = 7.8 Hz, 1H), 6.62 (t, J = 7.4 Hz, 1H), 4.93 (s, 2H). 13C NMR (126 MHz, DMSO)
δ 158.67, 143.89, 143.08, 128.85, 126.86, 126.53, 125.44, 123.50, 122.87, 122.72, 117.10,
116.94, 116.57. HRMS, calculated for C15H14O2N5 [M+H]+: 296,1142, found 296,1140.
1-(4-hydroxyphenyl)-N-phenyl-1H-1,2,3-triazole-4-carboxamide (5a, ZP042)
1
Yield 54%. m.p. 232.2-232.6 ℃. H NMR (500 MHz, DMSO-d6) δ 10.53 (s, 1H),
10.06 (s, 1H), 9.26 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.37 (t, J = 7.8
13
Hz, 2H), 7.12 (t, J = 7.3 Hz, 1H), 6.97 (d, J = 8.4 Hz, 2H). C NMR (126 MHz, DMSO) δ

158.23, 158.21, 143.48, 138.52, 128.63, 128.32, 125.26, 123.84, 122.38, 120.45, 116.08.
HRMS, calculated for C15H13O2N4 [M+H]+: 281,1033, found 281,1031.
N-cyclopropyl-1-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide(6a, MZ049)
1
Yield 42%. m.p. 233.2-233.5 ℃. H NMR (500 MHz, DMSO-d6) δ 10.02 (s, 1H),
9.07 (s, 1H), 8.65 (d, J = 4.3 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 2.88
(dt, J = 10.1, 4.9 Hz, 1H), 0.72 – 0.63 (m, 4H). 13C NMR (126 MHz, DMSO) δ 161.18,
158.54, 143.90, 128.84, 124.68, 122.73, 116.51, 23.01, 6.18. HRMS, calculated for
C12H13O2N4 [M+H]+: 245,1033, found 245,1031.
Ethyl (1-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carbonyl)glycinate(7a, MZ053)
1
Yield 78%. Decomposed at 220 ℃. H NMR (500 MHz, DMSO-d6) δ 10.04 (s, 1H),
9.14 (s, 1H), 8.95 (t, J = 6.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 4.13
13
(q, J = 7.1 Hz, 2H), 4.02 (d, J = 6.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). C NMR (126 MHz,
DMSO-d6) δ 170.09, 160.42, 158.62, 143.32, 128.80, 125.11, 122.74, 116.51, 60.99, 41.19,
14.54. HRMS, calculated for C13H15O4N4 [M+H]+: 291,1088, found 291,1086.
4.1.4. Synthetic procedure of compounds in group B
For compounds of group B, 0.5 mmol of 4-azidophenol (1.0 eq) and the 0.5 mmol
appropriate alkyne (1.0 eq) were dissolved in MeOH (4 mL) in a round bottom flask. Fresh
prepared solutions of CuSO4 (0.1 eq) in demi-water (0.20 mL) and sodium ascorbate (0.20 eq)
in demi-water (0.20 mL) were added sequentially to the reaction mixture. The reaction
mixture was left stirring overnight at room temperature. The reaction mixture was diluted
with AcOEt and precipitate was removed by filtration. The residue was washed with methanol.
The filtrate was extracted with brine and then concentrated under reduced pressure. The
residue was purified using column chromatography with CH2Cl2: MeOH 50:1 to yield the
desired products.
4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenol (1b, MZ028)
Yield 21%. m.p. 212.5-213.8 ℃. 1H NMR (500 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.49
(s, 1H), 7.66 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 5.29 (t, J = 5.6 Hz, 1H), 4.59 (d, J
= 5.5 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 158.04, 149.19, 129.41, 122.26, 121.34,
116.47, 55.44. HRMS, calculated for C9H10O2N3 [M+H]+: 192,0768, found 192,0769.
4-(4-(1-hydroxyethyl)-1H-1,2,3-triazol-1-yl)phenol (2b, MZ043)
Yield 96%. m.p. 89.9-90.3 ℃. 1H NMR (500 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.44 (s,
1H), 7.66 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 5.34 (d, J = 4.0 Hz, 1H), 4.93 – 4.85

(m, 1H), 1.47 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 157.99, 129.47, 119.94,
116.45, 62.02. HRMS, calculated for C10H12O2N3 [M+H]+: 206,0924, found 206,0925.
4-(4-isopropyl-1H-1,2,3-triazol-1-yl)phenol (3b, ZP180)
Yield 52%. 1H NMR (500 MHz, Methanol-d4) δ 8.15 (s, 1H), 7.62 (d, J = 8.9 Hz, 2H),
6.96 (d, J = 8.9 Hz, 2H), 3.14 (m, 1H), 1.38 (d, J = 7.0 Hz, 6H). 13C NMR (126 MHz,
Methanol-d4) δ 158.03, 154.60, 129.51, 121.90, 118.74, 115.70, 25.66, 21.49. HRMS,
calculated for C11H13ON3 [M+H]+: 204.1131, found 204.1131.
4-(4-propyl-1H-1,2,3-triazol-1-yl)phenol (4b, MZ018)
Yield 12%. 1H NMR (500 MHz, Chloroform-d) δ 9.89 (s, 1H), 8.37 (s, 1H), 7.63 (d, J
= 8.9 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 1.66 (m, 2H), 0.94 (t, J = 7.4
Hz, 3H). 13C NMR (126 MHz, DMSO) δ 157.45, 147.58, 129.05, 121.63, 120.03, 115.98,
27.10, 22.19, 13.67. HRMS, calculated for C11H14ON3 [M+H]+: 204,1131, found 204,1129.
4-(4-pentyl-1H-1,2,3-triazol-1-yl)phenol (5b, ZP085)
Yield 67%. m.p. 93.2-94.0 ℃. 1H NMR (500 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.38 (s,
1H), 7.64 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 1.66 (m, 2H),
1.33 (m, 4H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 157.90, 148.24, 129.51,
122.01, 120.42, 116.42, 31.31, 29.05, 25.46, 22.36, 14.38. HRMS, calculated for C13H18ON3
[M+H]+: 232,1444, found 232,1442.
4-(4-cyclohexyl-1H-1,2,3-triazol-1-yl)phenol (6b, ZP179)
Yield 46%. 1H NMR (500 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.36 (s, 1H), 7.64 (d, J = 8.8 Hz,
2H), 6.92 (d, J = 8.8 Hz, 2H), 2.72 (t, J = 10.9 Hz, 1H), 2.01 (d, J = 11.3 Hz, 2H), 1.77 (d, J =
13
12.0 Hz, 2H), 1.69 (d, J = 12.7 Hz, 1H), 1.41 (m, 4H), 1.24 (m, 1H). C NMR (126 MHz,
DMSO) δ 157.88, 153.42, 129.57, 122.12, 119.29, 116.42, 35.13, 33.03, 25.95. HRMS,
calculated for C14H17ON3 [M+H]+: 244.1444, found 244.1443.4-(4-(hex-5-yn-1-yl)-1H-1,2,3-
triazol-1-yl)phenol (7b, MZ014)
1
Yield 21%. Decomposed at 220 ℃. H NMR (500 MHz, DMSO-d6) δ 9.88 (s, 1H),
8.38 (s, 1H), 7.63 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 2.77 (t, J = 2.6 Hz, 1H), 2.69
(t, J = 7.6 Hz, 2H), 2.21 (td, J = 7.1, 2.6 Hz, 2H), 1.73 (m, 2H), 1.52 (m, 2H). 13C NMR (126
MHz, DMSO) δ 157.92, 147.92, 129.49, 122.12, 120.48, 116.44, 84.87, 71.80, 28.41, 27.93,
24.93, 17.91. HRMS, calculated for C14H16ON3 [M+H]+: 242,1288, found 242,1298.
4-(4-octyl-1H-1,2,3-triazol-1-yl)phenol (8b, ZP087)
Yield 30%. m.p. 103.8-105.2 ℃. 1H NMR (500 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.37
(s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H), 1.64 (m,
13
2H), 1.35 – 1.22 (m, 10H), 0.86 (t, J = 6.7 Hz, 3H). C NMR (126 MHz, DMSO) δ 157.89,

148.27, 129.51, 122.04, 120.39, 116.43, 31.76, 29.35, 29.24, 29.10, 25.49, 25.46, 22.57, 14.43.
HRMS, calculated for C16H24ON3 [M+H]+: 274,1914, found 274,1911.
Methyl 4-(1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)butanoate (9b, MZ056)
Yield 17%. m.p. 89.9-90.3 ℃. 1H NMR (500 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.40 (s,
1H), 7.63 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 3.59 (s, 3H), 2.70 (t, J = 7.6 Hz, 2H),
2.40 (t, J = 7.4 Hz, 2H), 1.91 (m, 2H). 13C NMR (126 MHz, DMSO) δ 173.75, 157.91, 129.42,
122.20, 120.78, 116.48, 51.76, 33.08, 24.69, 24.58. HRMS, calculated for C13H16O3N3
[M+H]+: 262,1186, found 262,1184.
3-(1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)-N-phenylpropanamide (10b, ZP078)
1
Yield 27%. m.p. 190.9-192.0 ℃. H NMR (500 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.90
(s, 1H), 8.38 (s, 1H), 7.60 (m, 4H), 7.28 (t, J = 7.9 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 6.91 (d, J
= 8.9 Hz, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H). 13C NMR (126 MHz, DMSO)
δ 170.62, 157.99, 147.18, 139.67, 129.41, 129.14, 123.52, 122.16, 120.68, 119.52, 116.48,
36.09, 21.47. HRMS, calculated for C17H17O2N4 [M+H]+: 309,1346, found 309,1343.
4.1.5. Synthetic procedure of compounds in group C
For compounds of group C, 0.5 mmol 2-propynylamine was used to form amides with
corresponding carboxylic acid derivatives mediated by N,N’-dicyclohexylcarbodiimide (DCC,
1.0 eq.) in dry acetonitrile (MeCN). Conditions and procedures used here were same as these
in group A. Afterwards, different amides were mixed with 4-azidophenol (1.0 eq), fresh
prepared solutions of CuSO4 (0.1 eq) in demi-water (0.20 mL), sodium ascorbate (0.20 eq) in
demi-water (0.20 mL) in MeOH (4 mL). The reaction mixture was left stirring overnight for
at 60 ℃. The CuAAC reaction and purification were also done in the same way as for group
A described above.
N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (1c, ZP049)
1
Yield 28%. m.p. 229.1-230.6 ℃. H NMR (500 MHz, DMSO-d6) δ 9.90 (s, 1H), 9.04
(t, J = 5.2 Hz, 1H), 8.48 (s, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 7.52 (t, J =
7.1 Hz, 1H), 7.46 (m, 2H), 6.91 (d, J = 8.5 Hz, 2H), 4.59 (d, J = 4.8 Hz, 2H). 13C NMR (126
MHz, DMSO) δ 166.22, 157.64, 134.14, 131.34, 131.29, 128.88, 128.28, 127.36, 121.80,
121.16, 116.02, 34.89. HRMS, calculated for C16H15O2N4 [M+H]+: 295,1189, found
295,1189.
N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)thiophene-2-carboxamide (2c, ZP054)
1
Yield 40%. m.p. 235.2-237.1 ℃. H NMR (500 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.07
(bs, 1H), 8.49 (s, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.76 (d, J = 3.9 Hz, 1H), 7.66 (d, J = 7.9 Hz,

13
2H), 7.19 – 7.11 (m, 1H), 6.91 (d, J = 7.8 Hz, 2H), 4.55 (d, J = 3.5 Hz, 2H). C NMR (126
MHz, DMSO) δ 161.16, 157.67, 139.70, 130.95, 128.87, 128.44, 128.29, 127.97, 121.86,
121.30, 116.05, 34.66. HRMS, calculated for C14H13O2N4S [M+H]+: 301,0754, found
301,0753.
N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrole-2-carboxamide
(3c,
ZP063)
Yield 15%. m.p. 244.1-246.2 ℃. 1H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 9.92
(s, 1H), 8.52 (s, 1H), 8.44 (s, 1H), 7.65 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 6.86 (d, J
= 2.7 Hz, 1H), 6.81 (d, J = 3.8 Hz, 1H), 6.07 (m, 1H), 4.53 (d, J = 3.5 Hz, 2H). 13C NMR (126
MHz, DMSO) δ 160.59, 157.63, 146.10, 128.88, 126.03, 121.83, 121.45, 121.01, 116.02,
110.29, 108.60, 34.09. HRMS, calculated for C14H14O2N5 [M+H]+: 284,1142, found
284,1140.
5-bromo-N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)furan-2-carboxamide
(4c,
ZP064)
1
Yield 22%. m.p. 204.7-205.0 ℃. H NMR (500 MHz, DMSO-d6) δ 9.93 (s, 1H), 9.02
(bs, 1H), 8.47 (s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 3.6 Hz, 1H), 6.91 (d, J = 8.0 Hz,
2H), 6.77 (d, J = 3.9 Hz, 1H), 4.53 (d, J = 4.5 Hz, 2H). 13C NMR (126 MHz, DMSO) δ
158.10, 157.14, 150.01, 145.90, 129.30, 124.98, 122.27, 121.60, 121.53, 116.48, 114.42,
34.62. HRMS, calculated for C14H12O3N4Br [M+H]+: 363,0087, found 363,0086.
N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methylthiophene-2-carboxamide
(5c, ZP065)
1
Yield 38%. m.p. 225.9-227.7 ℃. H NMR (500 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.95
(s, 1H), 8.49 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 3.0 Hz, 1H), 6.91 (d, J = 8.4 Hz,
13
2H), 6.83 (d, J = 3.3 H, 1H), 4.53 (d, J = 4.5 Hz, 2H), 2.45 (s, 3H). C NMR (126 MHz,
DMSO) δ 161.09, 157.63, 145.83, 144.70, 137.11, 128.84, 128.50, 126.39, 121.79, 121.23,
116.00, 34.55, 15.20. HRMS, calculated for C15H15O2N4S [M+H]+: 315,0910, found
315,0907.
4-fluoro-N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (6c, ZP069)
1
Yield 31%. m.p. 248.0-249.4 ℃. H NMR (500 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.08
(t, J = 5.6 Hz, 1H), 8.49 (s, 1H), 7.97 (dd, J = 8.7, 5.6 Hz, 2H), 7.65 (d, J = 8.9 Hz, 2H), 7.30
13
(t, J = 8.8 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 4.58 (d, J = 5.6 Hz, 2H). C NMR (126 MHz,
DMSO) δ 165.16, 163.94 (d, J = 248.4 Hz), 157.62, 145.77, 130.64 (d, J = 2.9 Hz), 130.03 (d,
J = 14.2 Hz), 130.08, 129.97, 128.87, 121.81, 121.08, 115.98, 115.21 (d, J = 21.8 Hz) 39.85,

39.69, 34.92. 19F NMR (376 MHz, DMSO-d6) δ -119.43 (tt, J = 9.0, 5.1 Hz). HRMS,
calculated for C16H14O2N4F [M+H]+: 313,1095, found 313,1093.
N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole-2-carboxamide (7c, ZP071)
Yield 22%. m.p. 246.8-250.0 ℃. 1H NMR (500 MHz, DMSO-d6) δ 11.59 (s, 1H), 9.92
(s, 1H), 9.04 (t, J = 5.7 Hz, 1H), 8.50 (s, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.61 (d, J = 8.0 Hz,
1H), 7.43 (d, J = 8.2 Hz, 1H), 7.20 – 7.15 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 8.9
13
Hz, 2H), 4.62 (d, J = 5.6 Hz, 2H). C NMR (126 MHz, DMSO) δ 161.09, 157.66, 145.73,
136.47, 131.49, 128.87, 127.09, 123.35, 121.85, 121.54, 121.13, 119.73, 116.03, 112.33,
102.86, 34.41. HRMS, calculated for C18H16O2N5 [M+H]+: 334,1299, found 334,1297.
N-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(1H-indol-2-yl)acetamide
(8c,
ZP075)
Yield 47%. m.p. 225.9-226.8 ℃. 1H NMR (500 MHz, DMSO-d6) δ 10.88 (s, 1H), 9.93
(s, 1H), 8.46 (t, J = 5.6 Hz, 1H), 8.23 (s, 1H), 7.55 (m, 3H), 7.34 (d, J = 8.1 Hz, 1H), 7.21 (d,
J = 2.2 Hz, 1H), 7.06 (s, 1H), 6.96 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 8.9 Hz, 2H), 4.36 (d, J =
5.6 Hz, 2H), 3.56 (s, 2H). 13C NMR (126 MHz, DMSO) δ 170.87, 157.67, 145.88, 136.14,
128.80, 127.22, 123.94, 121.76, 120.98, 120.80, 118.77, 118.35, 116.07, 111.38, 108.80,
34.40, 32.66. HRMS, calculated for C19H18O2N5 [M+H]+: 348,1455, found 348,1453.
4.1.6. Synthetic procedure of compounds in group D
2-fluoro-4-nitrophenol (1.15 g, 7.3 mmol) was dissolved in ethanol (10 mL) and added
to Pd/C (66 mg). Subsequently the flask was charged with hydrogen (H2) gas and the black
suspension was stirred at room temperature for 4 hours. After 4 hours the starting material
disappeared as analyzed by TLC. The resulting mixture was filtered and the solvent was
evaporated under reduced pressure to provide the crude product that was used directly for the
next reaction step. The crude product wasdissolve in water (10 mL) with concentrated HCl (2
mL). Subsequently, NaNO2 (1.0 g, 15 mmol) and NaN3 (0.95 g, 15 mmol) were added
1
sequentially to yield 4-azido-2-fluorophenol. H NMR (500 MHz, DMSO-d6) δ 9.94 (s, 1H),
7.06 – 6.90 (m, 2H), 6.79 (d, J = 6.3 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 151.67 (d, J
= 242.8 Hz), 142.89 (d, J = 12.1 Hz), 130.75 (d, J = 8.6 Hz), 119.09, 115.70, 108.26 (d, J =
22.0 Hz).
Subsequently, 0.25 mmol of the required alkynes was synthesized as described above
by dissolving 2-propynylamine (0.25 mmol) with the corresponding carboxylic acids (0.25
mmol) in dry acetonitrile (MeCN). Subsequently, N,N’-dicyclohexylcarbodiimide (DCC, 1.0
eq.) was added as a coupling reagent. The reactants mixtures were stirred at 60 ℃ for

overnight. The products were purified follow the same methods as methods used to purify
compounds in group C.
N-((1-(3-fluoro-4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole-2-carboxamide
(1d, ZP086)
1
Yield 41%. m.p. 253.6-254.3 ℃. H NMR (500 MHz, DMSO-d6) δ 11.59 (s, 1H),
10.38 (s, 1H), 9.05 (t, J = 5.7 Hz, 1H), 8.59 (s, 1H), 7.77 (dd, J = 11.9, 2.5 Hz, 1H), 7.60 (d, J
= 8.0 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.19 – 7.15 (m, 2H), 7.10 (t,
J = 9.0 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.62 (d, J = 5.6 Hz, 2H). 13C NMR (126 MHz,
DMSO) δ 161.56, 151.18 (d, J = 242.7 Hz), 146.45, 145.68 (d, J = 11.9 Hz), 136.92, 131.90,
δ 128.99 (d, J = 8.7 Hz), 127.53, 123.82, 122.00 (d, J = 19.1 Hz), 121.67 (d, J = 6.1 Hz),
120.20, 118.69, 117.04 (d, J = 16.3 Hz), 112.77, 109.50, 103.32, 34.82. 19F NMR (376 MHz,
DMSO-d6) δ -133.75 – -133.81 (m). HRMS, calculated for C18H15O2N5F [M+H]+: 352,1204,
found 352,1205.
5-chloro-N-((1-(3-fluoro-4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole-2-
carboxamide (2d, ZP094)
Yield 35%. 1H NMR (500 MHz, DMSO-d6) δ 11.59 (s, 1H), 10.38 (s, 1H), 9.05 (t, J =
5.7 Hz, 1H), 8.59 (s, 1H), 7.77 (dd, J = 11.9, 2.5 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.56 (d, J
= 8.8 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.19 – 7.15 (m, 2H), 7.10 (t, J = 9.0 Hz, 1H), 7.03 (t,
J = 7.5 Hz, 1H), 4.62 (d, J = 5.6 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 161.18, 151.17 (d, J
= 242.6 Hz), 146.31, 145.68 (d, J = 11.9 Hz), 135.31, 133.42, 128.98 (d, J = 8.9 Hz), 128.59,
124.69, 123.92, 121.68 (d, J = 4.5 Hz), 121.07 (d, J = 18.3 Hz), 118.68, 117.03(d, J = 16.3
Hz), 114.38, 109.49, 102.89, 34.86. HRMS, calculated for C18H14O2N5ClF [M+H]+:
386.0820, found 386.0823.
4.2. Enzyme activity study
4.2.1. Protein Expression and Purification.
C-terminal His-tagged recombinant human MIF was expressed with pET-20b(+)
plasmid and Escherichia coli BL21 according to literature procedures.[46] After culturing
Escherichia coli cells were pelleted by centrifugation at 4000 rpm for 20 minutes. Cell pellets
were resuspended in a lysis buffer containing 20 mM Tris- HCl (titrated to pH 7.5 with an
aqueous concentrated NaOH solution), 20 mM sodium chloride, 10% glycerol, 2 mM
magnesium chloride, and 0.2× complete EDTA-free protease inhibitor cocktail (Roche).
Subsequently, the cells were lysed by sonication and centrifuged at 17,000 g for 20 minutes.
The supernatant was purified by medium pressure chromatography system (Biologic Duoflow)

equipped with a His trap HP (5 mL) column with detection at 280 nm for the eluent. The
binding buffer contained 50 mM Tris and 10 % glycerol that was titrated to pH 7.4 using 1 M
NaOH or 1 M HCl. The elution buffer contained 500 mM imidazole, 50 mM Tris, 10 %
glycerol that is also titrated to pH 7.4 using 1 M NaOH or 1 M HCl. The collected protein was
purified again by PD-10 column (GE healthcare) to remove the high concentration of
imidazole. The resulting MIF was assessed by SDS gel electrophoresis and no impurities were
observed (>95%). The concentration of MIF was determined by Bradford protein assay to be
1.83 mg/mL (135 µM). The purified protein was aliquoted and stored at −80 °C. The stability
of the protein was tested by the tautomerase assays (Synergy H1 Hybrid Reader, BioTek) and
thermal stability assays (nanoDSF, Prometheus NT.48).
4.2.2. Tautomerase Assay
Inhibition of the tautomerase activity and kinetics of MIF was measured using 4-
hydroxyphenyl pyruvic acid (4-HPP) as substrate. A stock solution was prepared by
dissolution of 4-HPP in 50 mM ammonium acetate buffer that was titrated to pH 6.0 using 1.0
M NaOH or 1.0 M HCl. 4-HPP was dissolved to provide a concentration of 10 mM and this
solution was incubated overnight at room temperature to allow equilibration of the keto and
enol forms. Subsequently this 4-HPP stock solution was stored at 4 °C. The MIF stock
solution (80 µL, 135 µM MIF) was diluted in the boric acid buffer to provide a MIF solution
(12 mL, 0.9 µM) in boric acid buffer (435 mM, pH 6.2). The enzyme activity was determined
by premixing 170 µL of the MIF dilution with 10 µL EDTA (20 mM in demiwater) and
DMSO (20 µL). This mixture was pre-incubated for 15 min. Next, 50 µL of this mixture was
mixed with 50 µL of 1 mM 4-HPP solution in 50 mM pH 6.0 ammonium acetate buffer.
Subsequently, MIF tautomerase activity was monitored by formation of the borate−enol
complex, which was measured by the increase in UV absorbance at 305 nm.The increase in
UV absorbance was monitored over the first 10 minutes of incubation using a BioTek
Synergy H1 Hybrid plate reader. In experiments where EDTA was excluded the 10 µL of the
20 mM EDTA solution is replaced for demi water. Inhibitors were added to the experiment as
DMSO solutions by replacing the blank DMSO for a DMSO solution with a corresponding
inhibitors concentration that provides the final inhibitors concentration after dilution. Initially,
the inhibitors were dissolved in DMSO at 10 mM, which was diluted further in DMSO to
provide 1 mM from which 20 µL was added to the enzyme activity assay to provide a final
concentration of 50 µM in the screening. For compounds that showed ca. 50 % or greater

inhibition at 50 µM, an IC50 was measured. Towards this aim the compounds were stepwise
diluted in DMSO and subsequently the MIF tautomerase activity was measured using the
same protocol. The DMSO concentration in all assays was kept constant at 5% and control
experiments demonstrated that this DMSO concentration did not influence the tautomerase
activity. MIF tautomerase activity in the presence of blank DMSO was set to 100% enzyme
activity. In the negative control the enzyme was excluded to monitor non-catalyzed
conversion of the substrate, which did not show a change in absorbance at 305 nm. Data from
the first three minutes were used to calculate the initial velocities and the nonlinear regression
analyses for the enzyme kinetics were repeated three times with the program Prism6
(GraphPad).
4.3. Docking study
Docking studies were performed to gain insight in the structure-activity relationships.
All molecular modelings were done with the program Discovery studio (Dassault systems)
version 2018 and the crystal structures of human recombinant MIF (PDB-code:4wrb,[42]
5hvs[32]) were used. The CDOCKER protocol was used for docking which is a CHARMM
based algorithm. Docking was verified by use of the ligand 3-((6-(1-(3-fluoro-4-
hydroxyphenyl)-1H-1,2,3-triazol-4-yl)naphthalen-1-yl)oxy)benzoic acid (Jorgensen-3bb)
from the crystal structure 5hvs. This ligand contains the 4-(1,2,3-triazole)phenol functionality,
which is also present in our molecules. First, the ligand was removed from the protein and
subsequently docked back in the crystal structure. All 10 highest ranked poses show a
comparable position compared to the original pose from the crystal structure in the 4-(1,2,3-
triazole)phenol functionality (Fig. S8). Poses with the lowest CDOCKER energies were
chosen for representation.
4.4. MST
MST experiments were performed on a Monolith NT.115 system (NanoTemper
Technologies) using 100% LED and 20% IR-laser power. Laser on and off times were set at
30s and 5s, respectively. Recombinant His-tagged MIF was labeled with RED-tris-NTA for
30 minutes (NanoTemper Technologies) and applied at a final concentration of 50 nM. A
two-fold dilution series was prepared for compound 1d in PBS-T with 5% DMSO.
Subsequently, 10 µL of labeled MIF was mixed with 10 µL samples with different
concentration of compound 1d. Samples were filled into hydrophilic capillaries (Monolith
NT.115 capillary, standard treated) for measurement.

4.5. Colony formation assay
A549 cells were seeded in 6-well plates, each well contained 2 mL medium with 200
A549 cells and incubated for 24 h. 10 mM ISO-1 or 2d were prepared by dissolving in
DMSO. The inhibitors were diluted to different concentration in fresh medium before addition
into the corresponding well upon which the cells were treated continuously for 10 days.
Finally, the cells were fixed with paraformaldehyde for 20 minutes and stained with crystal
violet for 20 minutes. After washing, the image of each well was photographed and analyzed
with ImageG. We defined one colony as an aggregate of >50 cells. The numbers of colonies
was analyzed as the ratio of the numbers found in inhibitor treated samples compared to
untreated samples.
4.6 ERK signaling pathway study
Cells were seeded into a 6-well plate at a density of 2x105 cells per well with RPMI-
1640 medium containing 10% fetal bovine serum (FBS) (Costar Europe, Badhoevedorp, The
Netherlands), and 1% penicillin/streptomycin. After overnight culturing, the cells were treated
with the respective MIF inhibitors for 10 min, followed by the stimulation of 50 ng/µl MIF
for 15 min. After that, cells were lysed by RIPA buffer. The BCA Protein Assay Kit (Pierce,
Rockford IL, USA) was using to determine the protein concentration according to the
manufacturing instruction. Thirty-microgram protein was separated by a pre-cast 10%
NuPAGE Bis-Tris gel (Invitrogen, USA). The separated proteins were transferred to a
polyvinylidene difluoride (PVDF) membrane. Five percent skimmed milk was used to block
the membrane for 1 hour at RT. The blocked membrane was incubated with appropriate
primary antibody (phosphor-ERK, pERK, #9101, 1:1000, Cell Signaling; GAPDH, #97166,
1:10000, Cell Signaling) overnight at 4°C, followed by the treatment of an HRP-conjugated
secondary goat anti-rabbit antibody (#P0448, 1:2000) or rabbit anti-mouse antibody (#P0260,
1:2000) (Dako Cytomation, Glostrup, Denmark) at RT for 1 hr. The protein bands were
visualized with enhanced chemiluminescence (ECL) solution (GE Healthcare, Amersham,
UK). The figures were quantified with imageJ software (National Institutes of Health, USA)
based on greyscale.
Acknowledgements
Z. X. acknowledges funding from the China Scholarship Council. We thanks prof. M.R.
Groves and G. Kai for advice and help on MST assays.

Supporting information
Supplementary data associated with this article can be found in appendix. These data include
computational experiments, NMR spectra of compounds, and IC50 measurements.
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