Design, synthesis, and protein crystallography of biaryltriazoles as potent tautomerase inhibitors of macrophage migration inhibitory factor

Article abstract of DOI:10.1021/ja512112j

Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl-aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

Full text of DOI:10.1021/ja512112j

Article
pubs.acs.org/JACS
Design, Synthesis, and Protein Crystallography of Biaryltriazoles as
Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory
Factor
Pawel Dziedzic,† Jose A. Cisneros,† Michael J. Robertson, Alissa A. Hare, Nadia E. Danford,
́
Richard H. G. Baxter, and William L. Jorgensen*
Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, United States
S
* Supporting Information
ABSTRACT: Optimization is reported for biaryltriazoles as
inhibitors of the tautomerase activity of human macrophage
migration inhibitory factor (MIF), a proinflammatory cytokine
associated with numerous inflammatory diseases and cancer. A
combined approach was taken featuring organic synthesis,
enzymatic assaying, crystallography, and modeling including
free-energy perturbation (FEP) calculations. X-ray crystal
structures for 3a and 3b bound to MIF are reported and
provided a basis for the modeling efforts. The accommodation
of the inhibitors in the binding site is striking with multiple
hydrogen bonds and aryl−aryl interactions. Additional
modeling encouraged pursuit of 5-phenoxyquinolinyl ana-
logues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the
phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties
of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most
potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-
ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.
MIF is a toroid-shaped, trimeric protein with a total of 342
amino acid residues. Besides its role as a cytokine, MIF is a keto−
enol tautomerase. Though the enzymatic activity appears to be
vestigial in humans, there are three tautomerase active sites at the
interfaces of the monomer units opening to the outside of the
toroid. Inhibition of protein−protein interactions is often
challenging; however, the presence of the tautomerase sites
presents an opportunity for complexation of a tautomerase
inhibitor that may also interfere with MIF/CD74 binding. This
notion has been supported by many studies that show correlation
between the inhibition of the enzymatic and biological activities
of MIF.7 For example, this has been demonstrated through assay
results for tautomerase activity, MIF/CD74 binding, and MIF-
induced phosphorylation of ERK1/2 in inflamed cells,
production of interleukins, glucocorticoid overriding ability,
and macrophage chemotactic migration.7−12 Nevertheless, the
discovery of potent MIF tautomerase inhibitors is not well
advanced as most inhibitors have arisen from screening exercises
with no lead optimization.8,11,12 In our efforts,8−10 lead
optimization has been limited by the lack of crystal structures
for our tautomerase inhibitors bound to MIF, associated
inconsistency between modeling results and activity data, and
INTRODUCTION
■
Macrophage migration inhibitory factor (MIF) is a cytokine that
plays a central role in numerous inflammatory diseases.1−3 MIF is
widely expressed in both immune and nonimmune cells
including macrophages, endothelial cells, and T-cells. Upon
activation, the cells release MIF, which promotes the release of
other inflammatory cytokines such as TNF-α and IL-1. Excessive
or chronic inflammatory response is associated with tissue
damage and autoimmune diseases such as rheumatoid arthritis,
Crohn’s disease, and lupus erythematosus. The connection
between inflammatory disease and cancer is also well-established,
and MIF has been shown to enhance cell proliferation by
inhibiting accumulation of the tumor suppressor p53 and by
promotion of angiogenesis.4 MIF is overexpressed in many
cancer cells and can serve as a marker for disease progression.
Furthermore, MIF in cancer cells is protected from degradation
by Hsp90, which has led to proposed targeting of Hsp90 as an
indirect way of inhibiting MIF function.5 Disruption of the
inflammatory cascade and restoration of normal p53 levels have
clear implications for the potential therapeutic value of inhibitors
of MIF signaling. Indeed, immunoneutralization of MIF or
deletion of the MIF gene is known to suppress inflammatory
response, tumor growth, and angiogenesis.1−4 At the molecular
level, what is needed is interference with the interaction between
MIF and its cell-surface receptor CD74.6
Received: November 26, 2014
Published: February 20, 2015
© 2015 American Chemical Society
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Article
sensitivity of assay results to the substrate and protein source. As
described here, these issues have been overcome for the series of
biaryltriazoles, 1−3. In particular, we report crystal structures for
two complexes of derivatives of 3 with MIF, extensive structure−
activity data that is consistent with both crystallography and
modeling, and reliable assay protocols. The most active
compounds are ca. 1000-fold more potent as MIF tautomerase
inhibitors than the known MIF inhibitor (R)-ISO-1,13,14 which
has shown efficacy in rodent models as an anti-inflammatory and
anticancer agent.2,4
EXPERIMENTAL SECTION
■
Chemistry. In a previous report, we described the synthesis of 27
1,2,3-triazole derivatives in four motifs with a 4-hydroxyphenyl group at
the 1-position and a substituted benzyl or aryl group at the 4-position or
vice versa.10 These constructs arose from de novo design using the
program BOMB, which builds and scores libraries of compounds that it
grows in a binding site.15 Only three compounds were synthesized with a
an aryl group at the 4-position, namely, the 3-pyridinyl analogue 1 (X =
H, Z = OH) and the corresponding 1-naphthyl and 4-isoquinolinyl
alternatives. The modeling indicated that the nitrogen atom in the 3-
pyridinyl and 4-isoquinolinyl compounds would coordinate with the
ammonium group of a lysine residue (Lys32A) and that this interaction
would likely be lost and replaced by a repulsive interaction with the
oxygen of Ile64A in 2-pyridinyl and 2-quinolinyl analogues, that is, 2 and
3. However, 1 (X = H, Z = OH) and the isoquinolinyl analogue
exhibited unexpected behavior as agonists in an assay for binding of MIF
with the ectodomain of CD74.10 They also showed inconclusive
behavior in a MIF tautomerase assay; they appeared to be inactive or
weak agonists lacking dose-dependent character. This eventually raised
questions: (a) would the 2-pyridinyl and 2-quinolinyl isomers of the
agonists be agonists, antagonists, or inactive, and (b) if an appendage
were added in the 6-position of the pyridine ring in 1, could this convert
the agonist into an antagonist? As a crystal structure was not available for
a
Table 1. Results for Inhibition of the Tautomerase Activity of Human MIF
b
compd
1a
X
Y
Z
Ki (μM)
H
OH
OH
OH
Cl
37
22
8.8
1b
MOEO
2a
H
2b
MOEO
ND (0%)
0.59
3a
H
H
OH
OH
OH
F
3b
MOEO
H
0.65
3c
AEOEO
H
0.77
3d
MOEO
H
8.9
3e
MOEO
H
Cl
ND (3%)
ND (13%)
ND (0%)
ND (8%)
ND (0%)
ND (15%)
ND (8%)
7.3
3f
MOEO
H
NH2
OMe
CN
3g
MOEO
H
3h
MOEO
H
3i
MOEO
H
CONH2
3j
MOEO
H
3-Me, 4-OH
3k
MOEO
H
3-OMe, 4-OH
3l
H
3-Me
4-Me
8-Cl
8-OMe
OH
3m
3n
H
OH
2.3
H
OH
ND (16%)
56
3o
H
F
3p
H
8-MOEO
F
64
3q
H
8-p-MeOPh
OH
ND (21%)
2.95
3r
H
8-OPh
OH
3s
H
5-OPh
OH
0.37
3t
Mr(CH2)3O
H
OH
1.95
3u
Mr(CH2)3O
3-Me
OH
3.12
3v
MrEOEO
H
OH
0.41
3w
3x
MrEOEO
H
3-F, 4-OH
F
0.15
MrEOEO
H
29.6
3y
H
H
H
H
H
H
5-p-MOEO-OPh
5-m-MOEO-OPh
5-p-COOH-OPh
5-m-COOH-OPh
H
OH
0.36
3z
3-F, 4-OH
3-F, 4-OH
3-F, 4-OH
OH
0.082
0.11
3aa
3bb
4a
0.057
1.48
4b
H
Cl
ND (9%)
17
Orita-13
(R)-ISO-1
120
a
b
MOEO = methoxyethoxy; AEOEO = aminoethoxyethoxy; Mr = N-morpholinyl. ND = Ki not determined; % inhibition at 10 μM in parentheses.
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any of our compounds bound to MIF, the structural analyses and
expectations for activity did not have firm footing. Thus, a scouting
mission was initiated for 1 (X = methoxyethoxy, Z = OH), 2 (X = H, Z =
OH), and 3 (X = H, Z = OH). As reported here, the surprising results led
to lead optimization of 2 and 3 for inhibition of MIF tautomerase
activity. The new compounds are listed as 1b−4b in Table 1; 4 is the 1,8-
naphthryidine analogue of 3, while (R)-ISO-1 and 3-(3,4-dihydrox-
yphenyl)-7-hydroxy-4H-chromen-4-one (Orita-13) are reference com-
pounds.13,16
either the 8-triflate or 8-pinacol boronic ester of 2-trimethylsilylethy-
nylquinolin-8-ol. Instead, starting with commercially available 4′-
methoxy-[1,1′-biphenyl]-2-amine, the desired 2-chloroquinoline was
built by acylation to the acrylamide, followed by cyclization to the
quinolone, and treatment with POCl3 at reflux (Scheme 2). 3q was then
completed via the Sonogashira-1,3-dipolar cyclization sequence.
1
The identity of assayed compounds was confirmed by H and 13C
NMR and high-resolution mass spectrometry; HPLC analyses
established purity as >95%. Measurements of aqueous solubilities
were carried out using a shake-flask procedure.19 Saturated solutions
were made by stirring excesses of the compounds in Britton-Robinson
buffer for 48 h at 30 °C. The pH of the buffer solutions was 6.5 as
measured by using a Corning General Purpose pH Combination probe
(4136L21). The supernatant was collected using a Pall Life Sciences
Acrodisc syringe filter with a 0.2 μm pore size, and analyzed by UV−vis
spectrophotometry (Agilent 8453). Piroxicam was used as a control; our
values of 7−9 μg/mL are consistent with the literature value of 5.9 μg/
mL.19
Computer Modeling. All structure building was carried out using
the BOMB program starting from a previously reported crystal structure
of human MIF with 4-hydroxyphenylpyruvate (PDB code: 1CA7)20 or
from our structure of the complex with 3b. Subsequent calculations
included energy minimizations and free-energy perturbation (FEP)
calculations with the MCPRO program.21 Details of the calculations are
described elsewhere.22 Briefly, the OPLS-AA force field is used for the
protein, OPLS/CM1A for the ligands, and TIP4P for water molecules.23
For the FEP calculations, the unbound ligands and complexes were
solvated in water caps with a 25 Å radius, amounting to ca. 2000 and
1250 water molecules, respectively. The 218 amino acid residues nearest
to the ligand were included in the model for the complexes. A residue-
based cutoff for nonbonded interactions was invoked at 10 Å. After short
conjugate-gradient optimizations, the backbone atoms of the protein
were fixed. The ligand and side chains with any atom within ca. 15 Å of
the ligand were fully sampled. All water molecules were sampled using
translations and rigid rotations. The FEP calculations used 11 or 21
windows of simple overlap sampling. Each window covered at least 10
million configurations of equilibration and 10 million configurations of
averaging for the complexes and 30 million configurations of averaging
for the unbound inhibitors.
The synthetic approach was conceptually straightforward featuring a
1,3-dipolar cycloaddition between an azide and a substituted 2-ethynyl-
pyridine, quinoline, or naphthyridine; however, access to the
appropriately substituted heterocycles was not always easy. Though
full details are provided in the Supporting Information, Schemes 1 and 2
Scheme 1. Synthesis of 3s
Biology. Protein Expression and Purification. Recombinant human
MIF (rhMIF) was expressed as described previously.24 Escherichia coli
cells were pelleted by centrifugation and stored at −80 °C. The
purification followed published protocols24,25 with slight modifications.
Cell pellets were resuspended in a lysis buffer containing 20 mM Tris-
HCl pH 7.5, 20 mM sodium chloride, 10% glycerol, 2 mM magnesium
chloride, and 0.2× cOmplete EDTA-free protease inhibitor cocktail
(Roche), lysed by sonication and centrifuged at 27 000g for 30 min. The
supernatant was filtered through a 0.22 μm syringe filter and applied to
Hi-Trap SP HP and Hi-Trap Q SP columns (GE Healthcare) in tandem.
As rhMIF did not bind to either ion-exchange resin, the flow-through
was collected, being sufficiently pure (∼90%) for crystallography.
Higher purity was achieved by size-exclusion chromatography on a
Superdex 200 16/60 column (GE Healthcare). The resulting rhMIF was
assessed by SDS gel electrophoresis to be of sufficiently high purity
(>95%) for tautomerase assays. Pure protein was concentrated to 30.6
mg/mL in 20% glycerol and stored at −80 °C.
Tautomerase Assay, Ki Determination. Inhibition of the tautomer-
ase activity of MIF was measured using 4-hydroxyphenyl pyruvic acid
(HPP) as substrate, largely following previously reported protocols.26
HPP was dissolved in 0.5 M acetate buffer, pH 6.0 to a final
concentration of 10 mM and incubated overnight at room temperature
to allow equilibration of the keto and enol forms. MIF (6 μL) was
premixed in 500 mM boric acid, pH 6.2 (142 μL) and transferred to a
transparent U bottom 96-well plate to a final concentration of 200 nM
MIF. It was important to optimize the protein concentration; this was
performed by analysis of progress curves for enol production at protein
concentrations of 50−800 nM. High signal-to-noise and linearity were
observed for 200 and 400 nM MIF; below these levels, weaker signal
limited accuracy of the results. Inhibitors were dissolved in DMSO to 10
mM and an initial screen was performed. For compounds that showed
Scheme 2. Synthesis of 3q
illustrate the synthetic routes for two cases, 3s and 3q. Typically, 2-
chloroquinolinols are available commercially at low cost; however, this is
not the situation for 2-chloroquinolin-5-ol, which would have facilitated
the synthesis of 3s. Thus, starting with quinolin-5-ol, arylation was
followed by oxidation to the N-oxide, which was chlorinated17 to yield 2-
chloro-5-phenoxyquinoline. A standard sequence10 featuring Sonoga-
shira coupling with TMS-protected acetylene followed by one-pot,
Cu(I)-catalyzed reaction of the azide formed from an aryl bromide or
iodide then yielded 3s.18
With 3q (Scheme 2), a substituted phenyl group was desired in the 8-
position of the quinoline. This turned out to be challenging as numerous
attempts at Suzuki couplings failed. For example, couplings with the
triflate of 2-chloro-quinolin-8-ol gave at best 1:1 mixtures of 2- and 8-
arylation, and no desired product arose from attempted couplings of
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ca. 25% or greater inhibition at 10 μM, an inhibition constant, Ki, was
measured. Compounds were placed into wells (2 μL) at six different
concentrations and incubated for 30 min until the assay was started by
addition of HPP (50 μL) at two concentrations (1.0 and 2.5 mM). The
negative control was MIF incubated with DMSO vehicle, which in all
assays was 1% and did not influence tautomerase activity. MIF activity
was monitored at 305 nm for formation of the borate−enol complex
using an Infinite F500 plate reader (TECAN, Morrisville, NC) for 175 s.
Calculation of initial velocities and the nonlinear regression analyses for
the enzyme kinetics were repeated three times with the program Prism6
(GraphPad, La Jolla, CA). The results always fit better to the
competitive inhibition mode rather than the noncompetitive one,
which is consistent with the crystallographic results.
Previously, we had attempted assays using rhMIF purchased from
external vendors. This proved unsatisfactory as different vials of protein
showed great variation in activity, including totally inactive. Even
different vials received at the same time from the same vendor showed
different activities. The present assay results were all obtained with
protein prepared on only two occasions; when more protein was
needed, another aliquot was thawed, and never refrozen. Also, Orita-13
and/or 3b were used as control compounds when new rounds of assays
were conducted. The Ki results all fell in the range 13−22 μM for Orita-
13, and nine independent measurements for 3b yielded results of 0.55−
0.85 μM. Samples of Orita-13 and (R)-ISO-1 were purchased both from
Alfa Aesar and Santa Cruz Biotechnology; consistent spectra and Ki
results were obtained.
We also investigated use of L-dopachrome methyl ester (DOPA)
rather than HPP as the substrate in the manner of Orita et al.16 An
advantage in principle is that the absorbance is evaluated at 492 nm,
which may experience less interference from the inhibitors than the 305
nm detection with HPP. However, DOPA is photosensitive, which
limits the data collection to 25 s versus 175 s with HPP. The shorter
linear range for calculation of the initial velocities results in poorer fits for
the results at different concentrations and much less reliable Ki values.
Protein Crystallography. To obtain cocrystals of MIF in complex
with 3a, 100 μM 3a in DMSO was added to rhMIF (24 μg/mL) to
achieve a 3:1 molar ratio and incubated for 1 h at 5 °C. The solution was
centrifuged at 13 000g to remove precipitated compound and used to set
up hanging-drop crystallization experiments. A reservoir of 2.0 M
ammonium sulfate, 0.1 M Tris pH 7, and 3% isopropanol was added to
the protein solution in a 1:1 ratio and stored at 20 °C. Diffraction-quality
crystals with a rod morphology grew within 2 weeks. The crystals were
cryo-protected in 25% glycerol, 2.0 M ammonium sulfate, 0.1 M Tris pH
7, and 3% isopropanol and shipped to the Advanced Photon Source for
remote data collection on the NE-CAT 24-ID-E beamline.
the 3-pyridinyl (1a) to 2-pyridinyl (2a) to 2-quinolinyl (3a)
derivatives shows strong enhancement in activity from 37 to 8.8
to 0.59 μM. For comparison, (R)-ISO-1, which is reported14 to
have an IC50 of 7 μM in a MIF tautomerase assay with DOPA as
the substrate, has a Ki of 120 μM in our assay. Issues with IC50
measurements are well-known; they depend on the concen-
tration and Michaelis constant (Km) of the substrate, while Ki is
an intrinsic measure of the binding of the protein and inhibitor.27
In an independent assay of ISO-1 using DOPA, an IC50 of >100
μM was reported; the discrepancy with the 7 μM value was
suggested to arise from use of different concentrations of
rhMIF.28 We also find Orita-13 with a Ki averaging 17 μM to be
much less active than from the previously reported Ki of 0.038
μM, again in a dopachrome assay.16 Orita-13 would seem to be
the most potent MIF inhibitor in the journal literature.11 It arose
from a screening study that reported Ki values for 14 compounds
with the next most potent compound at 0.28 μM.16 There have
been no follow-up reports with the compound except for a crystal
structure29 that found it rotated 180° in the binding site from the
original X-ray study,16 and it appears not to have been assayed
again until now. Though we find Orita-13 to be 7-fold more
active than ISO-1, it is 30-fold less active than 3a.
Crystallography. To progress from this point, a recurrent
conformational issue complicated modeling efforts.10 For 3a, as
an example, four principal geometries could be constructed in the
binding site, which can be labeled 3a-ud, 3a-dd, 3a-du, and 3a-
uu. These represent two conformers that can both be rotated
180° about the long axis in the binding site. The structure
building and energy minimizations with BOMB and MCPRO
could rule out 3a-ud and 3a-uu, but the preference between 3a-
dd and 3a-du was uncertain. 3a-dd is the higher-energy
conformer in the gas-phase than 3a-du, by 6 kcal/mol according
to OPLS/CM1A calculations, owing to the added quinoline N−
triazole N3 repulsion; however, 3a-dd appeared to make better
hydrogen-bonding interactions in the binding site.
Cocrystals of MIF in complex with 3b were obtained by soaking
crystals of apo MIF. Crystals were obtained by the hanging-drop method
at 20 °C. A reservoir of 2.2 M ammonium sulfate, 0.1 M Tris pH 7, and
3% isopropanol mixed in a 1:1 ratio with rhMIF (16 mg/mL) was used
to produce 2 μL drops. Once crystals formed, 0.5 μL of a suspension of
10 mM 3b in 10% DMSO, 2.0 M ammonium sulfate, 90 mM Tris pH 7,
and 2.7% isopropanol was added to the drop and allowed to incubate for
14 days. Crystals were cryo-protected with 25% glycerol, 2.2 M
ammonium sulfate, 0.1 M Tris pH 7, and 3% isopropanol and diffracted
on a Rigaku 007 HF+ X-ray source equipped with a Saturn 944+ CCD
detector at Yale. Full details of the data collection and refinement for 3a
and 3b are provided in the Supporting Information.
This issue was resolved by obtaining the crystal structures for
3a and 3b in complex with MIF at resolutions of 2.60 and 1.81 Å,
respectively. As illustrated in Figure 1 for 3b, the crystal
structures of both complexes showed that 3a-dd is the preferred
geometry. The inhibitor is inserted such that the phenolic
hydroxyl group is hydrogen-bonded with Asn97C (r(OO) = 2.52
Å); there is also a hydrogen bond between N2 of the triazole and
the backbone NH of Ile64A (r(NN) = 2.90 Å), and there is the
striking complexation of the ammonium group of Lys32A by the
quinoline N, triazole N3, and O of Ile64A (r(NN) = 3.33, 2.95;
r(NO) = 2.81 Å), which requires the higher-energy 3a-dd
geometry. There are also aryl−aryl interactions between the
phenolic and quinolinyl fragments of the inhibitor and Tyr95C
RESULTS AND DISCUSSION
■
In presenting the results, a sense of the progression of events will
be given. The previous report was for work through mid-2010
and included 1a.10 The triazole series were not pursued again
until early 2012, when 1b, 2a, and 3a were synthesized. However,
the assay issues and protein access discussed above, which were
needed to allow report of the carefully controlled results in Table
1, were not fully worked out until 2013.
With the present assay protocols, the parent compounds 1a,
2a, and 3a are all MIF tautomerase inhibitors. Progression from
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Table 2. MC/FEP Results for Changes in Free Energy of
a
Binding to Human MIF
b
b
H to X
ΔΔGb
H to Y
ΔΔGb
H to Y
ΔΔGb
F
−2.27
−1.77
−1.06
−1.16
−0.62
2.73
3-F
0.15
−0.82
−0.32
−2.69
−1.56
0.23
4-OMe
4-OH
8-F
0.02
1.64
Cl
3-Cl
3-Clx
3-Me
3-Et
Clx
Br
−0.79
−1.49
−2.06
0.61
8-Cl
Brx
Me
Et
8-Clx
3-OMe
4-F
8-Me
6.26
0.46
8-Et
−0.11
−0.51
−0.16
0.27
NH2
OMe
OH
1.89
4-Cl
0.32
8-OMe
8-OEt
8-MOM
8-CH2F
0.14
4-Clx
4-Me
4-Et
0.18
Figure 1. Rendering of 3b bound to human MIF from the 1.81 Å crystal
structure (PDP ID: 4WRB). Carbon atoms of 3b are shown in yellow;
some residues have been removed for clarity. Hydrogen bonds are
highlighted with dashed lines.
−2.70
−1.00
−0.34
0.40
a
ΔΔGb is the computed change in free energy of binding (kcal/mol);
Clx and Brx include X-sites to allow halogen bonding; the statistical
uncertainty in the results ( 1σ) is 0.2 kcal/mol. With X = OH.
b
and Tyr36A, respectively, and Phe113A has contacts at the
junction of the quinoline and triazole. The structure appears very
well packed with a large number of favorable intermolecular
features given the size of the inhibitor. The crystal structures for
3a and 3b are essentially the same, and it was pleasing that the
methoxyethoxy substituent of 3b was well-resolved (Figure S2).
Though the substituent would be solvent-exposed in dilute
solution, in the crystal it is in contact with an adjacent MIF
trimer. The fact that 3a and 3b show the same activity (Table 1)
is fully consistent with the crystal structures. It may also be noted
that both crystal structures only have one of the three
tautomerase sites occupied by 3a or 3b, the remaining sites
being occupied by solvent and/or glycerol.
In previously reported crystal structures, hydrogen bonding
interactions for an inhibitor with the side chains of Asn97 and
Lys32 and with the backbone NH of Ile64 have frequently been
observed along with aryl−aryl contacts involving Tyr95 and
Phe113.16,20,29 The interactions with Lys32 have typically come
from a carbonyl or carboxylate group of the inhibitor. For
example, in the 3L5R structure for Orita-13, the hydroxyl group
of the chromen-4-one is hydrogen bonded with Asn97, and the
carbonyl oxygen atom is hydrogen-bonded to both Lys32 and the
NH of Ile64.29 The present structures are particularly striking
because the triazolylquinoline substructure provides three
separate hydrogen bonds with Lys32A and Ile64A as well as an
additional aryl−aryl interaction with Tyr36A (Figure 1). It may
also be noted that, in the present structures Pro1A, the catalytic
base for the tautomerase reaction and nucleophile for formation
of covalent inhibitors11 is probably protonated. One hydrogen
atom on the nitrogen is likely pointing toward the carbonyl
oxygen atom of Tyr36A with an N−O distance of 3.22 Å, while
there is a clear hydrogen bond between this carbonyl oxygen
atom and the hydroxyl group of Tyr95C at an O−O distance of
2.51 Å. If there is a second hydrogen atom on the Pro1A nitrogen
atom, it would be pointing toward N1 of the triazole (r(NN) =
3.25 Å), and there would be favorable cation−π interactions with
the electron-rich ring.
halogens with an extra partial positive charge, which allows
representation of halogen bonding.30 There is little empty space
in the vicinity of the phenolic hydroxyl group. The FEP results
indicated that all replacements of the hydroxyl group would be
unfavorable, though the penalty for switching to fluorine should
not be severe. There is a trade-off between loss of hydrogen-
bonding with Asn97C and a diminished dehydration penalty. An
amino group is not favorable since only one of the two amino
hydrogens can form a hydrogen bond to Asn97C. The activity
data for 3b and 3d−3i in Table 1 are consistent with the
predictions. Only the fluoro analogue 3d showed significant
activity with a Ki of 8.9 μM, ca. 15-fold higher than that for 3b.
For 3j and 3k, a methyl or methoxy substituent adjacent to the
hydroxyl group was also considered, but as expected from the
tight packing, these additions were unfavorable.
Turning to the quinoline ring, the 3-, 4-, and 8-positions were
first considered for possible additions of small groups. Structure-
building with BOMB suggested that at least a small alkyl group
might be accommodated at C3 or C4 in the space near Tyr36A.
However, the FEP results indicated that about the only hope
would be for a methyl group at C3 (Table 2), since we have
generally observed that the computed enhancement needs to be
beyond −2 kcal/mol to have good confidence in an observed
activity increase.15 Indeed, the assay results for 3l and 3m (7.3
and 2.3 μM) showed weaker inhibition than for the parent 3a
(0.59 μM), while 3t and its 3-methyl analogue 3u showed
essentially the same activity. In view of the FEP results for the
other options, further exploration at these sites was not pursued.
Structure building at C8 then suggested that addition of an
alkoxy group or substituted phenyl might provide additional
coordination with Lys32A (Figure 1). Enticing images could be
generated as in Figure 2 showing a possible cation−π interaction
in the latter case. Such interactions require representation of
explicit polarization effects in the force fields,23 so a confident
FEP result could not be obtained with OPLS/CM1A. The FEP
calculations were executed for nine options at C8, as shown in
Table 2 with the result that chlorine might be most promising.
However, the predicted ΔΔGb values of −1.49 and −2.06 kcal/
mol are on the fringe of the −2 kcal/mol threshold. In fact, the 8-
Cl analogue 3n was found to have diminished activity. The
Phenyl and Quinolinyl Substituents. Having resolved the
structural issue, FEP calculations were performed to seek
substituent modifications in the phenyl and quinolinyl fragments
that could improve potency. The results are summarized in Table
2. It should be noted that Clx and Brx refer to modeling the
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Figure 3. Computed structure for 5-phenoxy-containing 3s bound to
human MIF illustrating surface contact and a possible aryl−aryl
interaction with Tyr36A. Carbon atoms of the ligand are in yellow. Some
residues have been removed for clarity.
Figure 2. Computed structure for an analogue of 3a with a phenyl
substituent on C8 of the quinoline highlighting a possible cation−π
interaction with Lys32A. Carbon atoms of the ligand are in yellow; the
ammonium group of Lys32A is shown as a blue sphere. Some residues
have been removed for clarity.
(Figures 1−3). For illustration, aqueous solubility was
considered. Thus, polyether-containing groups were appended
at C6 with the 6-hydroxy compounds as precursors. Aqueous
solubilities were measured for a series of four compounds as
summarized in Scheme 3. The solubilities of 3a (2.2 μg/mL or
ΔΔGb for an 8-methoxy group of −0.51 kcal/mol made it likely
that this modification would also be unproductive. This was
tested with 3o that has a fluorine replacing the phenolic hydroxyl
group; its Ki of 56 μM suggests that the phenol would be 4 or 0.8
μM, if the 3d/3b or 3x/3v ratio is transferable. Though the
oxygen of the methoxy group of 3o can form a hydrogen bond
with the ammonium group of Lys32, it is replacing a water
molecule that is fulfilling this role and it requires orientation of
the oxygen toward the quinoline N, which promotes lone pair−
lone pair repulsion. In the balance, consistent with the FEP
prediction, the 8-methoxy group in 3o and the methoxyethoxy
(MOEO) group in 3p did not provide a notable activity boost.
Returning to the possibility of a phenyl substituent at C8, as
noted in Scheme 2, there were challenges in the synthesis of such
compounds. Nevertheless, there was success with the p-methoxy
analogue 3q. In spite of the attractive graphics for 8-phenyl
analogues (Figure 2), 3q only showed 21% inhibition of MIF’s
tautomerase activity at 10 μM (Table 1). Lys32 is on the surface
of MIF, and it appears difficult to substitute favorably for the
water molecules on its solvent-exposed side. The possibility of
additional coordination from the ligand with Lys32A via the 1,8-
naphthyridine 4a was also considered; the result was a
respectable Ki of 1.48 μM, but not an improvement over 3a.
Still not relenting, structures were built with BOMB with a
phenoxy group at C5, C6, C7, and C8 of the quinoline ring. A
conformational search on the 8-phenoxy analogue indicated that
it would be better preorganized for binding and interaction with
Lys32A than the 8-methoxy alternative, and perhaps the phenoxy
groups might interact with other surface residues. In fact, energy
minimizations showed that this could be the case for the C5
(Figure 3) and C8 phenoxy compounds. Thus, this notion was
pursued yielding 3r and 3s. The 8-phenoxy analogue 3r is a 3.0
μM inhibitor, while the 5-phenoxy 3s at 0.37 μM did provide an
advance over 3a and 3b (Table 1). The presumption is that the
added intermolecular contacts illustrated in Figure 3 are
beneficial for binding.
Scheme 3. Experimental Solubilites (S, μg/mL) and
Computed log P
7.6 μM) and 3b (3.6 μg/mL or 9.9 μM) are surprisingly low
given the small size, percentage of heteroatoms, and the
computed octanol/water partition coefficients of 3.3 to 4.0
from ChemDraw and QikProp.31,32 When experimental data for
log S and log Po/w are analyzed,33 a rule-of-thumb is log S = −log
Po/w − 0.2 with r2 = 0.70 and rms = 1.0 for 271 compounds with
log Po/w > 0. Thus, log S ≈ 10−4 or S ≈ 100 μM for 3a and 3b
could be expected within a factor of 10. The observed results are
at the very bottom of this range. Addition of the amino group in
3c has the expected qualitative effect, raising the solubility to 13.9
μg/mL. A further 3−4-fold boost is obtained upon replacement
of the amino group with N-morpholinyl, a well-known
solubilizing group.34,35 3v is potent with a Ki of 0.41 μM, and
its solubility of 48.5 μg/mL (1.05 × 10−4 M) is well inside the
range normally observed for oral drugs, 4−4000 μg/mL.33 The
ClogP appears to be high in this case; the pattern for the four
compounds from QikProp seems more reasonable. The predicted
Aqueous Solubility. From the crystal structures and
modeling, it was expected that physical properties of the
compounds could be modulated by variation of substituents
that would be solvent exposed. Attachments at the 6- or 7-
positions in the quinoline ring seemed likely to be appropriate
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solubility from QikProp of 0.2 × 10−4 M for 3v is also within
hydrogen bonding with Asn97C yielded additional gains in
potency with 3w, 3z, 3aa, and 3bb. With Ki values below 100 nM,
3z and 3bb are likely the most potent known inhibitors of the
tautomerase activity of human MIF; they are more than 1000-
fold more active than the well-studied (R)-ISO-1 and more than
200-fold more active than the chromen-4-one Orita-13.
normal error bounds.32
Final Compounds. Given the good results for 3s and 3v,
additional analogues were synthesized to again test the impact of
replacing the phenolic hydroxyl group with fluorine (3x) and for
addition of a fluorine adjacent to the hydroxyl group (3w, 3z, 3aa,
3bb). Though methyl and methoxy groups could not be
accommodated at the meta position (3j and 3k), fluorine is
smaller and it is expected to enhance the hydrogen-bond donor
character of the hydroxyl group and strengthen the hydrogen
bond with Asn97C. This idea was fruitful, as 3w with a Ki of 0.15
μM is 3-fold more potent than 3v. The added fluorine makes 3w
somewhat less soluble with S = 27.2 μg/mL. The same idea was
then applied to the 5-phenoxy compounds. Addition of a para-
methoxyethoxy group in 3y did not change the potency from that
of 3s, since the added group is expected to be largely solvent-
exposed (Figure 3). However, again several-fold enhancements
of the potency accompanied introduction of a meta solubilizing
group along with a fluorine adjacent to the hydroxyl group
yielding the most potent compounds, 3z and 3bb, with Ki values
of 0.082 and 0.057 μM. The solubility of 3bb was also measured;
the result of 47.2 μg/mL further supports the potential value of
this compound.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic details, NMR and HRMS spectral data for compounds
in Tables 1, and crystallographic details. The crystal structure
data for the complexes of 3a and 3b with MIF have been
deposited in the RCSB Protein Data Bank with the codes 4WR8
and 4WRB. These materials are available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*william.jorgensen@yale.edu
Author Contributions
†P.D. and J.A.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Gratitude is expressed to the National Institutes of Health
(GM32136), Yale University, and Debiopharm SA for research
support, to the National Science Foundation (DGE-1122492)
for a fellowship to M.J.R., to Drs. Richard Bucala and Lin Leng for
the MIF expression vector and interactions on the tautomerase
assay, and to Dr. Binh Le for assistance with crystallizations and
X-ray data collection. The data collection for the structure of 3a
with human MIF was conducted at the Advanced Photon Source
(APS) on the Northeastern Collaborative Access Team
beamlines which are supported by the National Institutes of
Health (P41 GM103403); the APS is operated for the U.S.
Department of Energy Office of Science by Argonne National
Laboratory under Contract No. DE-AC02-06CH11357.
CONCLUSION
REFERENCES
■
■
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