Optimization of Pyrazoles as Phenol Surrogates to Yield Potent Inhibitors of Macrophage Migration Inhibitory Factor

Article abstract of DOI:10.1002/cmdc.201800158

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is implicated in the regulation of inflammation, cell proliferation, and neurological disorders. MIF is also an enzyme that functions as a keto–enol tautomerase. Most potent MIF tautomerase inhibitors incorporate a phenol, which hydrogen bonds to Asn97 in the active site. Starting from a 113-μm docking hit, we report results of structure-based and computer-aided design that have provided substituted pyrazoles as phenol alternatives with potencies of 60–70 nm. Crystal structures of complexes of MIF with the pyrazoles highlight the contributions of hydrogen bonding with Lys32 and Asn97, and aryl–aryl interactions with Tyr36, Tyr95, and Phe113 to the binding.

Full text of DOI:10.1002/cmdc.201800158

Accepted Article
Title: Optimization of Pyrazoles as Phenol Surrogates to Yield Potent
Inhibitors of Macrophage Migration Inhibitory Factor
Authors: Vinay Trivedi-Parmar, Michael J. Robertson, Jose A.
Cisneros, Stefan G. Krimmer, and William L. Jorgensen
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To be cited as: ChemMedChem 10.1002/cmdc.201800158
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10.1002/cmdc.201800158
ChemMedChem
COMMUNICATION
Optimization of Pyrazoles as Phenol Surrogates to Yield Potent
Inhibitors of Macrophage Migration Inhibitory Factor
Vinay Trivedi-Parmar, Michael J. Robertson, José A. Cisneros, Stefan G. Krimmer, and William L.
Jorgensen*
Dedicated to Prof. E. J. Corey on the occasion of his 90th birthday.
Abstract: Macrophage migration inhibitory factor (MIF) is
a
of the protein in the vicinity of Pro1, which serves as the catalytic
base. The resultant strategy for interference with the binding of
MIF to its receptor CD74 is then to find tautomerase inhibitors that
change the surface characteristics of MIF.[6] Indeed, numerous
studies have shown a correlation between inhibition of the
enzymatic and biological activities of MIF by measuring
tautomerase activity, and, for example, MIF/CD74 binding, protein
phosphorylation in inflamed cells, production of interleukins, and
glucocorticoid overriding ability.[6,7] Though many MIF
tautomerase inhibitors have been discovered through screening
of compound libraries,[6,8] lead optimization to give inhibitors with
nanomolar potency has been limited. In fact we have tested the
most promising compounds from the literature in a tautomerase
inhibition assay[9] and only found compounds from one patent[10]
and our biaryltriazole series[11] to have sub-micromolar Ki values.
The results were confirmed by measurement of Kd values in a
fluorescence polarization assay.[12] Exemplary potent compounds
are 1 (NVS-2[10]) and 2[11] with Ki values of ca. 0.03 μM, which are
ca. 1000-fold lower than for well-known MIF inhibitors such as 3
((R)-ISO-1[13]) and the chromen-4-one 4[6a] (Scheme 1).
proinflammatory cytokine that is implicated in the regulation of
inflammation, cell proliferation, and neurological disorders. MIF is also
an enzyme that functions as a keto-enol tautomerase. Most potent
MIF tautomerase inhibitors incorporate a phenol, which hydrogen
bonds to Asn97 in the active site. Starting from a 113-μM docking hit,
we report results of structure-based and computer-aided design that
have provided substituted pyrazoles as phenol alternatives with
potencies of 60-70 nM. Crystal structures of complexes of MIF with
the pyrazoles highlight the contributions of hydrogen bonding with
Lys32 and Asn97, and aryl-aryl interactions with Tyr36, Tyr95, and
Phe113 to the binding.
Human macrophage migration inhibitory factor (MIF) is a
proinflammatory cytokine that is implicated in the pathogenesis of
numerous inflammatory diseases,[1] neurological disorders,[2] and
cancer.[3] MIF is expressed in many cell types and its tissue
distribution is wide-spread. Upon activation of cells such as
macrophages, monocytes and T-cells, expression of MIF in turn
activates release of inflammatory cytokines including interleukins,
interferon, and TNFα. Complex signaling pathways are invoked
when MIF binds to its membrane-bound receptors CD74 and
CXCR4, leading to leukocyte chemotaxis, inflammatory response,
and potential tissue damage.[3] Strong correlation is observed
between MIF expression and the severity of many inflammatory
and autoimmune diseases including asthma, sepsis, lupus, and
rheumatoid arthritis.[4] For cancer, the AKT pathway may be
activated by MIF binding causing suppression of apoptosis by
inhibition of the normal action of BAD, BAX, and p53.[3] However,
MIF’s role in cancer is multifaceted with undesirable effects also
on cell proliferation, angiogenesis, and metastasis;[3,4] MIF is over-
expressed in most human cancer cells.[5]
Scheme 1. Examples of MIF tautomerase inhibitors with Ki data
from Ref. 12.
Interestingly, MIF also shows enzymatic activity as a keto-
enol tautomerase. MIF is a toroid-shaped, trimeric protein
consisting of 342 amino acid residues with three identical active
sites occurring at the interfaces of the monomer subunits.[6] The
active sites are small, relatively cylindrical and open to the surface
A feature, which is addressed here, is that 1 - 4 and many
other non-covalent MIF tautomerase inhibitors and substrates
contain a phenol subunit, which lodges in the back of the active
site and forms hydrogen bonds with the sidechain of Asn97
(Figure 1).[6,11,12] Though there are more than 125 approved drugs
that contain
a
phenolic group including, for example,
acetaminophen, albuterol, amoxicillin, raloxifene, and doxycycline,
the oral bioavailability of phenols is well-known to often be
unacceptably low owing to metabolic glucoronidation[14] and/or
sulfation.[15] Thus, we set out to find a phenol-free series of MIF
tautomerase inhibitors with low-nanomolar potencies.
[*]
V. Trivedi-Parmar, Dr. M. J. Robertson, Dr. J. A. Cisneros, Dr. S. G.
Krimmer, Prof. Dr. W. L. Jorgensen
Department of Chemistry
Yale University
New Haven, CT 06520-8107 (USA)
Success in the past has come from exchange of the phenol
for a 6:5 fused heteroaromatic incorporating a pyrrole or pyrazole
that retains the hydrogen-bond donating character of phenol.[16]
However, the MIF active site is too constricted near Asn97 for this
approach to be viable; addition of a methyl group ortho to the
Prof. Dr. W. L. Jorgensen
E-mail: william.jorgensen@yale.edu
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. Rendering from an 1.8-Å crystal structure of an analog
of 2 bound to MIF.[11a] Carbon atoms of the inhibitor are colored
yellow. Hydrogen bonds are indicated with dashed lines.
Figure 2. Rendering from the 2.0-Å crystal structure of 5 bound
to MIF. Details as in Figure 1.
hydroxyl group for the compound in Figure 1 leads to a ca. 100-
fold loss in activity.[11a] Instead, our interest has focused on
replacement of the phenol by a pyrazole. Owing to the
geometrical differences, this requires exploration of new series
with a pyrazole core. Fortunately, in the initial virtual screening
study[8a] 11 compounds were found to be active in an assay that
measured interference of binding between MIF and immobilized
CD74 ectodomain; and, one contained a pyrazole with the
expected hydrogen bonds to Asn97 in the docked structure. This
compound, 5, gave an IC50 of 15 μM in the binding assay; how-
substitution para to the pyrazolyl group seemed likely to yield
beneficial interactions with Tyr36 and possibly Phe113. Thus,
constructs 6 - 8 were pursued where R1 was mostly an aryl group.
Scheme 3. Designs for pyrazole-based MIF inhibitors.
The syntheses of 6 – 8 are detailed in the Supplementary
Information. As summarized in Scheme 4, the key steps started
from the commercially available phenyl iodide 9, which underwent
Pd- or Cu-mediated coupling to yield phenylaryl, arylanilinyl, or
biaryl ether derivatives 10 – 12. Installation of the pyrazole was
then achieved by a Suzuki coupling to yield esters 13 – 15, which
were hydrolyzed under mild conditions to provide the desired
carboxylic acids.
Scheme 2. Docking hit 5.[8a]
ever, it showed little activity in a tautomerase assay using 4-
hydroxyphenylpyruvate (HPP) as the substrate, with a maximum
of 30% inhibition at 50 μM.[8a] Thus, we pursued alternative series
from the virtual screening and from de novo design, which
provided the biaryltriazoles including 2.[11] However, our interest
in 5 was renewed since in another phenol-containing inhibitor
series[8b] rapid metabolic glucoronidation and sulfation were
observed. It was decided to retest 5 in an HPP tautomerase assay
using optimized protocols in our laboratory.[9] Though the Ki for 5
from this assay was only 113 μM, in view of its low molecular
weight and possibilities for substitution in the phenyl ring, we were
encouraged to perform structure-based, computer-aided lead
optimization.[17] As detailed here, this has been successful in
providing pyrazole derivatives with ca. 2000-fold greater potency.
In working with 5, it was noted that it had high solubility in
polar media. This motivated successful pursuit of a crystal
structure with MIF in spite of the modest Ki (Figure 2). There are
two copies of 5 in each MIF trimer. The expected hydrogen bonds
with Asn97 have average N-O and N-N lengths of 3.0 and 3.1 Å,
while Lys32 has hydrogen bonds with the carboxylate group of 5
(3.0 and 2.7 Å) and the oxygen atom of Ile64 (2.7 Å). The NH of
Ile64 also forms one with the carboxylate (2.9 Å), and the phenyl
ring of 5 is well packed between Pro1, Tyr95, and Phe113. From
this structure and model building with the BOMB program,[17b]
Scheme 4. Synthesis of pyrazole-based MIF inhibitors.
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COMMUNICATION
The compounds reported here are listed in Table 1 along with
the results from the tautomerase assay. The identity of assayed
compounds was confirmed by 1H and 13C NMR and high-
resolution mass spectrometry; HPLC analyses established purity
as >95%. As in prior studies, the inhibition constants Ki were
determined using HPP as the substrate.[9,11] Inhibitory activity is
Table 1. Experimental inhibition constants, Ki
Cmpd
R1[a]
R2
Z
X
Ki (μM)
5
H
-
-
-
113
20.6
19.5
5.4
6a
6b
6c
7a
7b
8a
8b
8c
8d
8e
8f
Ph
-
-
-
1-Np
-
-
-
2-Np
-
-
-
Ph
H
Me
-
-
-
12.7
4.2
Figure 3. Rendering from the 2.3-Å crystal structure of 8a bound
to MIF. Details as in Figure 1.
2-Np
-
-
Ph
COOH
COOH
COOH
COOH
COOH
COOH
COOH
SO2Me
SO2NH2
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
F
6.8
measured from formation of the borate complex of the enol
product at 305 nm using a plate reader. Absorbance is measured
in triplicate on two occasions. The average Ki results are reported;
the standard error is typically 10-20% of the Ki value. In addition,
the aqueous solubilities of several compounds were determined
with a shake-flask procedure.[11,12,18] Saturated solutions are
filtered and analyzed by UV-vis spectroscopy.
o-MePh
m-MePh
p-MePh
m-FPh
-
4.3
-
3.8
-
7.0
-
1.7
Consistent with the modeling, addition of an aryl group in 6
did provide a significant boost over 5, bringing the Ki values down
to ca. 20 μM for a phenyl or 1-naphthyl group and to 5 μM for 2-
naphthyl (6c). The analogous anilinyl and phenoxy compounds,
7a and 8a, were prepared, and the greater activity and
pharmacological desirability of diarylethers placed the
subsequent focus on the latter series. A 2.3-Å crystal structure for
the complex of 8a with MIF was also obtained (Figure 3), which
does show aryl-aryl contacts between the phenoxy phenyl group
and both Tyr36 and Phe113. A basic SAR (structure-activity
relationship) study was then carried out with 8b – 8f, which
revealed a small activity range for addition of a methyl or fluoro
substituent, with para-substitution the least favored. Consistent
with this guidance, the 2-naphthyl analog 8g was found to show
good activity at 4.3 μM; the BOMB modeling indicated increased
contact with Phe113 projecting to the right in Figure 3. Modeling
further indicated that still larger hydrophobic groups could be
accommodated in this region at the entrance of the MIF active site.
This was borne out by Ki values of 1 - 3 μM obtained for
phenanthryl, adamantyl, and acenaphthyl analogs, 8j – 8l
(Scheme 5). However, the project seemed stalled at this point
without reaching the desired low-nanomolar range and with
increasing concerns about solubility.
p-FPh
-
4.6
8g
8h
8i
2-Np
-
4.3
2-Np
-
6.4
2-Np
-
5.6
8j
9-Phenanthryl
2-Adamantyl
4-Acen
1-Np
-
2.3
8k
8l
-
2.6
-
1.1
8m
8n
8o
8p
8q
8r
-
0.48
0.51
0.15
0.17
0.14
0.11
0.066
0.35
0.13
0.24
0.075
0.067
2-Np
-
4-Et-2-Np
5-Et-2-Np
7-Et-2-Np
4-Cp-2-Np
4-Cp,7-Et-2-Np
p-Bp
-
-
-
-
8s
8t
-
-
8u
8v
8w
8x
m-Bp
-
For the biaryltriazoles series, it was recalled that placement
of a fluorine adjacent to the hydroxyl group in compounds like 2
provided a ca. 3-fold increase in activity.[11] The effect was
attributed to enhancing the acidity of the phenol, which increases
the strength of the hydrogen bond with Asn97, and also to
hydrophobic contact of the fluorine with the side chain of Met 101
(Figure 1). For the pyrazoles, the enhanced hydrogen bonding
could be envisioned for a fluorine at the 3-position; however, the
3,5-diMe-m-Bp
4-OEt-m-Bp
4-MrPrO-m-Bp
-
-
-
[a] Np = naphthyl; Acen = 1,2-dihydroacenaphthyl; Cp = cyclopropyl; Bp =
biphenyl; MrPrO = N-morpholinylpropoxy.
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COMMUNICATION
Figure 4. Rendering from the 2.0-Å crystal structure of 8n bound
Scheme 5. Some pyrazole-based MIF inhibitors reported here.
to MIF. Details as in Figure 1.
fluorine would project more towards the side chain of Ile64 rather
than Met101 with uncertain outcome (Figure 2). Still, a potential
additional benefit might arise from the influence of the fluorine on
the tautomeric equilibrium for the pyrazole. Reliable quantum
mechanical calculations (MP2/6-311++G**) show that the N1-H
tautomer is favored by 3.6 kcal/mol over the N2-H tautomer with
a fluorine in the 3-position (Scheme 6).[19] From the present crystal
structures the hydrogen bonds are expected to be more linear for
the N1-H tautomer as implied by the alignment of the side-chain
oxygen atom of Asn97 and N1 in Figures 2 and 3.
aryl-aryl interactions between the naphthyl group of 8n and Tyr36
and Phe113.
Though the exact positioning of the naphthyl group may be
influenced by crystal packing, the structure and BOMB modeling
indicated that additional gains in activity could arise from alkyl-
substitution at the 4-, and 5-positions of the naphthyl group to
achieve further contact with Phe113 or at the 7-position for
contact with Ile64. This was shown to be correct with the ethyl
analogs 8o, 8p, and 8q, which each provided a 3-fold lowering of
the Ki relative to 8n. Addition of a cyclopropyl group at the 4-
position also appeared promising for interaction with the front
edge of Phe113; this was realized with 8r bringing the Ki to 0.11
μM. Combining this with the 7-ethyl substitution provided the very
potent 8s with a Ki of 0.066 μM. From the structures for 8a and 8n
(Figures 3 and 4) and modeling, it was also clear that it should be
possible to expand to a biphenyl at either the para or meta
position of 8a. Thus, 8t and 8u were synthesized and provided
significantly lower Ki values (0.35 and 0.13 μM) than the
unsubstituted naphthyl analogs, 8m and 8n. Substantial activity
gains could be expected by judicious substitution for the
biphenyls; however, only a few derivatives were prepared with 8w
and 8x (Scheme 4) demonstrating ca. 0.07 μM potency and that
large groups can be extended into the solvent from the terminal
4-position.
Scheme 6. Shift in the tautomeric equilibrium with a fluorine.[19]
Preparation of the fluorinated pyrazole for the Suzuki
coupling in Scheme 3 proved difficult. Multiple routes were
attempted, but success was only achieved using a SEM [2-
(trimethylsilyl)ethoxymethyl] protecting group; the yield was still
low, but sufficient to proceed (Scheme 7).
Two additional items are worth noting. First, the results for
8g, 8h, and 8i show that the carboxylic acid group may be
replaced by a methylsulfone or sulfonamide with little impact on
potency. This is relevant if one wished to explore these
compounds as potential neurological agents,[2] since sulfones are
expected to exhibit better penetration of the blood-brain barrier
than carboxylic acids or sulfonamides.[20] Secondly, it is always
important to monitor aqueous solubilities for compounds of
interest for oral administration.[11,17,21] Most oral drugs are
observed to have aqueous solubilities of 4 to 4000 μg/mL, which
translates to 10 μM to 10 mM for a drug with a molecular weight
of 400.[21] The solubilities of several of the present compounds
were measured in Britton-Robinson buffer at pH 6.5.[18] As noted,
the solubility of the starting compound 5 is very high (927 ± 88
μg/mL). The solubility of the parent 2-naphthyl analog 8g is also
high (739 ± 32 μg/mL); it is affected little by addition of the fluorine
in 8n (681 ± 59 μg/mL), while switch to the sulfonamide 8i yields
Scheme 7. Synthesis of fluorinated pyrazoles.
The effort was highly fruitful yielding a nearly 10-fold
increase in potency in progressing from the parent 2-naphthyl
inhibitor 8g (4.3 μM) to its fluorinated analog 8n (0.51 μM). It was
also possible to obtain a crystal structure for this compound in
complex with MIF at 2.0-Å resolution (Figure 4). The structure
confirmed the positioning of the fluorine between the sidechains
of Ile64 and Met101. There is one copy of the inhibitor in each
MIF trimer in this case; the N-O and N-N hydrogen bond lengths
with Asn97 are 2.87 and 3.12 Å. There are also close-packed
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a significant reduction (55.2 ± 4.8 μg/mL). Given these results, it
was surprising to find in the biphenyl series that the solubility of
8w is only 1.7 ± 0.7 μg/mL. However, this is readily remedied by
attachment of solvent-exposed, solubilizing groups[11b] as in 8x
(34.6 ± 4.8 μg/mL, or 67 μM).
In order to facilitate further study of the in vitro and in vivo
biology of MIF, series of potent MIF tautomerase inhibitors have
been pursued. Starting from a 113-μM docking hit, a novel series,
which features a pyrazole instead of a phenol, was optimized to
yield compounds with Ki values as low as 60-70 nM. The
optimization was greatly facilitated by molecular modeling and the
ability to obtain multiple high-resolution crystal structures, which
guided the effective selection and placement of substituents.
Recognition of the potential benefit of addition of a fluorine in the
pyrazole ring also provided an essential boost along with a
synthetic challenge. Current efforts are being directed at testing
the influence of the inhibitors on suppressing MIF-stimulated cell
proliferation and at preclinical studies for off-target activity and
metabolism.
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Experimental Section
Recombinant expression and purification of human MIF was carried out as
previously reported.[11] Crystallization of MIF in complex with 5 and 8g was
achieved by soaking with apo-MIF crystals, while for the complexes of 8a
and 8n, co-crystallization was performed via sitting drop vapor diffusion at
20 °C. The structures were determined in-house using a Rigaku 007 HF+
diffractometer and Saturn 944+ CCD detector at T = 100 K. The crystal
structures have been deposited in the RSC Protein Data Bank with IDs
6CBG (5), 6CBF (8a), 6CB5 (8g), and 6CBH (8n). The Supporting
Information contains the synthetic procedures, NMR and HRMS spectral
data for all new compounds, and details for the crystallography and
solubility measurements (72 pages).
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Acknowledgements
Gratitude is expressed to the U. S. National Institutes of Health
(GM32136) for research support, to the National Science
Foundation for a fellowship for MJR (DGE-1122492), and to Drs.
Thomas Steitz, Michael Strickler, and Yong Xiong for assistance at
the Yale Richards Center.
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Conflict of interest
The authors declare no conflict of interest.
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10.1002/cmdc.201800158
ChemMedChem
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
A novel inhibitor series has been
pursued for macrophage
Vinay Trivedi-Parmar, Michael J.
Robertson, José A. Cisneros,
Stefan G. Krimmer, and William L.
Jorgensen*
migration inhibitory factor (MIF).
Starting from a 113-μM docking
hit, results of structure-based
and computer-aided design are
reported to yield substituted
pyrazoles as alternatives to
metabolically labile phenols with
potencies of 60-70 nM.
Page No. – Page No.
Optimization of Pyrazoles as
Phenol Surrogates to Yield
Potent Inhibitors of Macrophage
Migration Inhibitory Factor
This article is protected by copyright. All rights reserved.