Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of macrophage migration inhibitory factor (MIF)

Article abstract of DOI:10.1016/j.bmcl.2010.07.129

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto-enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC₅₀ values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF-CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.

Full text of DOI:10.1016/j.bmcl.2010.07.129

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5811–5814
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of
macrophage migration inhibitory factor (MIF)
Alissa A. Hare a, Lin Leng b, Sunilkumar Gandavadi a, Xin Du b, Zoe Cournia a, Richard Bucala b,
,
*
William L. Jorgensen a,
*
a Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA
b Department of Medicine, Yale University School of Medicine, New Haven, CT 06520-8066, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding
to the transmembrane receptor CD74. MIF also exhibits keto–enol tautomerase activity, believed to be
vestigial in mammals. Starting from a 1 lM hit from virtual screening, substituted benzoxazol-2-ones
have been discovered as antagonists with IC50 values as low as 7.5 nM in a tautomerase assay and
80 nM in a MIF–CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that
it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in
human synovial fibroblasts.
Received 29 June 2010
Revised 28 July 2010
Accepted 29 July 2010
Available online 3 August 2010
Keywords:
MIF
Inhibitor design
Protein–protein interactions
Cytokine
Ó 2010 Elsevier Ltd. All rights reserved.
Inflammatory disease
MIF is a pro-inflammatory cytokine that is released by T-cells and
macrophages. It plays a key role in a wide range of inflammatory dis-
eases including rheumatoid arthritis, sepsis, and atherosclerosis.
MIF is also involved in cell proliferation and differentiation, and
anti-MIF antibodies suppress tumor growth and angiogenesis. The
biology of MIF and potential biomedical significance of MIF-inhibi-
tion are striking.1–3 Studies have shown that MIF signal transduction
is initiated by binding to a transmembrane protein, CD74.4,5 CD74
expression is required for MIF-mediated ERK1/2 phosphorylation
and cell proliferation, while inhibition of MIF–CD74 binding attenu-
ates tumor growth and angiogenesis.4–6
Besides being a cytokine, MIF is also a keto–enol tautomerase.
Though the catalytic activity of mammalian MIF is likely vestigial,7
there is evidence that the interaction of MIF with its receptor,
CD74, occurs in the vicinity of the tautomerase active site.8 Crystal
structures for MIF and MIF-tautomerase inhibitor complexes are
available,2,9,10 though no structure for a MIF–CD74 complex has
of substrates and it also serves as a locus for covalent inhibition.
For example, 4-iodo-6-phenylpyrimidine (4-IPP) was recently
demonstrated through crystallography to form a covalent complex
with MIF.13
O
N
N
O
N
I
H3C
1
4-IPP
The emerging role of MIF in hyperproliferative and inflamma-
tory diseases indicates that modulating the cytokine’s activity
can result in new therapies.1,2,14 To this end, we are seeking
small-molecule inhibitors of MIF–CD74 complexation through
structure-based design. In an initial report, virtual screening was
highly successful in leading to identification of 11 structurally di-
verse inhibitors with activities in the
zoxazol-2-one 1 was particularly compelling as it demonstrated
inhibitory potencies of 0.5 M in the tautomerase assay and
1.5 M in the MIF–CD74 binding assay. Optimization of this hit
l
M regime.15 N-Benzyl-ben-
been reported. The 114-residue MIF monomer has a b–a–b motif
and three monomers associate to form a symmetrical trimer. Each
MIF trimer has three tautomerase active sites at the interfaces of
the monomer subunits. The N-terminal proline resides in the
tautomerase binding pocket and has an unusually low pKa of
5.6–6.11,12 This nucleophilic proline can effect the tautomerization
l
l
has been pursued as summarized here.
A general route for the synthesis of analogues of 1 is shown in
Scheme 1. 2-Aminophenols were acylated with 4-nitrophenyl chlo-
roformate to yield benzoxazol-2-ones 2, which were alkylated with
substituted benzyl bromides to form the desired analogues 3. Alco-
hols were typically obtained from the corresponding methyl ethers
as in the conversion of 4 to 5. Most reactions proceeded in 70–80%
* Corresponding authors.
E-mail addresses: richard.bucala@yale.edu (R. Bucala), william.jorgensen@
yale.edu (W.L. Jorgensen).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.129

5812
A. A. Hare et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5811–5814
Table 1
OH
O
4-O2NPhOCOCl
Activities for analogues of benzoxazol-2-one 3 from the tautomerase (4-HPP) and
X
O
X
MIF–CD74 assays, and GLIDE XP scoresa
Et3N, DCM,
1 h, rt
N
H
NH2
Compd
X
R
IC50 or max % inhib.
XP
2
4-HPP
CD74
Score
O
N
RPhCH2Br
1
3
4
5
6
7
8
9
5-Me
—
—
—
3-OMe
3-OH
0.5
21
2.9
1.5
À9.0
À8.8
À8.5
À9.0
À8.3
À8.8
À9.0
À8.8
À8.6
À9.1
À8.4
À8.7
À9.0
À8.5
À8.9
À9.3
À9.4
À9.1
À9.4
À9.2
À9.1
À9.0
À8.6
À8.5
À8.3
À8.9
À8.4
À8.8
À8.6
À8.8
À9.0
À8.9
À8.5
À9.1
À8.5
À9.6
À9.5
X
O
18%
42%
40%
0.090
45%
30%
32%
7.0
11%
22%
35%
27%
14%
10%
25%
24%
10%
0.115
32%
15%
19%
200
0.080
23%
27%
30
2
K2CO3, DMF,
3 h, 40 oC
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
6-Me
6-Me
6-Me
5,6-DiMe
5,6-DiMe
5-Et
R
0.010
35%
21%
20%
NA
32%
17%
12%
30%
31%
NA
3
2-OMe
2-OEt
3-OEt
4-OMe
2,3-DiOMe
3,5-DiOMe
3-OH,5-OMe
3-OH,5-OEt
3-NH2
3-F
4-OH
—
3-OMe
3-OH
3-OMe
3-OH
O
O
N
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
BBr3, DCM
-78 oC, 71%
O
OCH3
H3C
OH
N
H3C
4
5
Scheme 1. Synthesis of N-benzyl-benzoxazol-2-ones.
NA
3.4
16%
0.0075
25%
33%
13%
2.5
41%
10%
NA
30
26
2.1
25%
28%
44%
NA
1.0
NA
yield. The identity of all assayed compounds was confirmed by 1H
(Bruker DRX-500) and 13C NMR and HRMS; purity was normally
>95% as determined by reverse-phase HPLC. The choices of ana-
logues that were synthesized and assayed were guided by compu-
tational modeling of the corresponding complexes with MIF, as
described more below.
The tautomerase and MIF–CD74 assays were performed as
previously presented.15 The former used 4-hydroxyphenylpyruvate
(4-HPP) as the substrate, while the latter features biotinylated MIF
and immobilized CD74 ectodomain (CD7473–232) with streptavidin
conjugated alkaline phosphatase processing p-nitrophenylphos-
phate as the reporter. Compounds were tested at concentrations
over 500- to 5000-fold ranges. Human MIF, prepared recombinantly,
was used throughout.16
The assay results are summarized in Table 1. In considering the
data, a complicating issue is that MIF is a trimer with three active
sites. It is likely that this contributes to the common behavior in both
assays where the dose–response curves show initial linearity, but
may not reach 50% inhibition. In these cases, the maximum percent
inhibition is reported since the IC50 is not defined. For example, the
results for 5 in the two assays are shown in Figure 1. Also, a simple
correlation between the results for the two assays is not expected
since one measures the inhibition of MIF’s tautomerase activity
and the other the inhibition of the protein–protein recognition be-
tween MIF and CD74. For example, a small, but potent tautomerase
inhibitor might have little structural effect on the surface of MIF and,
therefore, little effect on MIF–CD74 binding. The equilibria may also
be such that a weak tautomerase inhibitor has negligible influence
on CD74 binding uncomplexed MIF. The activity data will be consid-
ered after discussion of computed results for the MIF–3 complexes.
Docking calculations for all complexes were carried out using
GLIDE 5.517 in the extra precision (XP) mode18 and the 1CA7 crystal
structure for MIF.9 Such computations yield predicted structures
for the protein–ligand complexes and a score that is proportional
to the expected free energy of binding. The XP scoring is the most
accurate with GLIDE; it considers the details of the protein–ligand
binding, internal energy of the ligand, desolvation, and hydropho-
bic interactions.18 Additional model building and energy minimi-
zations were performed with BOMB and MCPRO using the OPLS/
CM1A force field.19–21 In the previous study, it was demonstrated
that GLIDE 4.0 XP scores correlated well with experimental log Ki
or log IC50 values for complexes of 10 known MIF tautomerase
inhibitors.15 The calculations were repeated here with GLIDE 5.5
with similar outcome; the correlation coefficient r2 improved a lit-
tle from 0.75 to 0.78, while the range of XP scores was compressed
from 7 to 1.5 units. The most favorable XP score, À9.4, for the
known inhibitors is for a coumarin derivative with a reported Ki
3-OMe
3-OH
3-OMe
3-OH
5-Et
5-OMe
5-OMe
5-OH
5-OH
5-OH
5-OH
5-OH
6-OH
5-CH2OH
5-CH2OH
5-F
—
2-OMe
3-OMe
2,3-DiOMe
3-OH
—
3-OMe
3-OH
—
3-OMe
3-OH
3-OMe
3-OH
300
36%
22%
27%
30
25
5-F
5-F
6-F
6-F
17%
18%
15%
NA
32%
35%
NA
a
IC50 in lM. NA = inactive. XP scores from GLIDE v. 5.5.
of 38 nM,2 and inhibitors with Ki values near 1
lM gave XP scores
near À9.0. These values can be compared to the results obtained
for 1–38 in Table 1. Clearly, some of the compounds are expected
to be MIF tautomerase inhibitors, though the correlation between
the XP scores and tautomerase log IC50 values is weak with an r2
of ca. 0.3.
The preferred binding mode for 1–38 with MIF from XP GLIDE
features the benzoxazol-2-one fragment inserted into the active-
site cavity and the benzyl substructure facing outwards near the
protein’s surface. This was noted previously for 1,15 and the corre-
sponding ‘in’ complex is illustrated for 5 in Figure 2. The structure
allows favorable hydrogen bonding with the NH of Ile64, electro-
static interactions with the side chain of Lys32, and aryl–aryl inter-
actions with Tyr36, Tyr95, and Phe113. Furthermore, it is
consistent with overlaying the chromen-2-one fragment from the
inhibitor in the 1GCZ crystal structure2 and the benzoxazol-2-
one ring system (Scheme 2). The calculations with GLIDE also find
a reversed ‘out’ MIF binding mode with the benzyl ring inserted
into the cavity towards Asn97. However, the XP scores for the re-
versed mode are generally several units less favorable; such struc-
tures generated with BOMB and MCPRO were also not competitive.
Nevertheless, in the absence of experimental structural data, the
binding mode or modes of analogues of 3 are uncertain. Indeed,
an alternative conformation has been found recently for Tyr36 that
leads to binding for some inhibitors on the protein’s surface be-
tween Tyr36 and Phe113.10

A. A. Hare et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5811–5814
5813
Figure 1. Results of assays performed in triplicate for 5.
Some additional studies have been carried out to further char-
acterize the MIF inhibition. First, Michaelis–Menten analyzes pre-
viously confirmed that 1 is a competitive inhibitor of MIF’s
tautomerase activity.15 As many MIF inhibitors have been reported
to function by covalent attachment to Pro1 or Cys residues,13,22
mass spectrometry (Bruker 9.4T APEX-Qe FTICR) was used to
examine this possibility for the potent 5 and for 4-IPP as a control.
Ten microlitres of rh-MIF (30
lM) were added to 10 ll of 4-IPP
(30 M) or 5 and 1 l of 1% FA buffer. In the case of 4-IPP, these
l
l
conditions result in 90% tautomerase inhibition and 50% inhibition
of MIF receptor binding,13 while for 5, 50% inhibition is observed
for both MIF activities. The samples were incubated for 1 h at room
temperature in the dark and desalted using ZipTipC18, eluting into
10
l
l 60% acetonitrile/0.1% FA.
The expected mass spectrometric behavior was observed for
4-IPP with appearance of the covalent adduct at the mass of
MIF + ca. 155 Da (Fig. 3), while for 5 no additional peaks emerged,
though the scan was analyzed beyond the expected combined
mass of MIF + 5 (255 Da). 4-IPP is a potent MIF inhibitor by virtue
of its covalent interaction with Pro1.13 However, not all of the pro-
tein was modified, and covalent reaction may not fully account for
the MIF-inhibitory activity of 4-IPP. In fact, the covalent inhibitor
NAPQI, which also decreases MIF tautomerase and receptor bind-
ing activities,8 was recently reported to additionally bind to MIF
in a non-covalent manner.23
Finally, inhibition of MIF signal transduction by 5 in human
target cells was explored. The extracellular-signal-regulated kinase
(ERK) pathway is of particular interest as aberrations in it are asso-
ciated with hyperproliferative diseases as well as autoimmune dis-
orders including rheumatoid arthritis (RA). Thus, inhibition of ERK
phosphorylation has promising therapeutic potential.14,24 Specifi-
cally, synovial fibroblasts from a patient with RA were incubated
with MIF (1.3 nM trimer) together with vehicle control (DMSO)
or 5 (300 nM). The cells were lysed after 30 min and the intracellu-
Figure 2. Computed structure for 5 bound to MIF after energy minimization with
MCPRO. Carbon atoms of 5 are in green.
O
O
N
O
O
O
O
OH
Scheme 2. Alignment of 1 and the inhibitor in the 1GCZ structure.
Returning to Table 1, potent inhibitors were identified in both
assays. Notably, 5 and 19 give IC50 values in the 5–10 nM range
in the tautomerase assay, while 6, 20, and 25 fall in the
80–120 nM range in the MIF–CD74 binding assay. For the tautom-
erase inhibitors, it is best to have a 3-OH group in the benzyl ring
and a 5- or 6-CH3 in the heterocycle. Larger groups on the hetero-
cycle, as in 23, 25, and 33, lead to diminished activity. In the ‘in’
geometry, there appear to be steric clashes for groups larger than
methyl at the 5- and 6-positions, and energy minimizations sug-
gest that the 3-OH may benefit from hydrogen bonding to the
C@O of Tyr36 (Fig. 2) or via a water bridge to the C@O of
Phe113. In the ‘out’ mode, the 3-OH might form a hydrogen bond
with Asn97; however, the 5- and 6-substituents are then largely
solvent exposed, and the size restriction does not make sense.
The results for the MIF–CD74 binding are harder to interpret owing
to the even greater structural uncertainties. Many compounds do
show significant inhibitory power, though many did not reach
the 50% level. The most active compounds have an OH or OCH3
group at the 2- and/or 3-position in the benzyl ring and a CH3 or
OCH3 group at the 5-position in the heterocycle.
Figure 3. Mass spectrometry results showing covalent modification of MIF by 4-IPP
and absence of MIF + 5 adducts.

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A. A. Hare et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5811–5814
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U.S.A. 2002, 99, 144.
Figure 4. Effect of 5 on MIF-dependent ERK phosphorylation in human synovial
fibroblasts.
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lar content of phospho-ERK1/2 and total ERK1/2 were detected by
specific antibodies and western blotting (Fig. 4). Addition of MIF
enhances ERK1/2 phosphorylation,4 while co-administration of 5
strongly inhibits the process. The observations are consistent with
the observed inhibitory effect of 5 on MIF–CD74 binding (Fig. 1).
In summary, optimization of MIF inhibitor 1, which arose from
virtual screening,15 has been pursued. Multiple highly potent inhib-
itors of MIF’s keto–enol tautomerase activity and of the binding of
MIF to its functional receptor, CD74, have been discovered. Compu-
tational work has suggested structures for the MIF-inhibitor com-
plexes, though improved understanding of the activity data awaits
experimentalstructural results. Additionalinvestigations for the po-
tent inhibitor 5 were carried out that demonstrated that it is not a
covalent inhibitor of MIF and that it is active in suppressing MIF-
dependent ERK phosphorylation in human synovial fibroblasts.
Acknowledgments
Gratitude is expressed to T. Lam and E. Voss of the Keck Biotech-
nology Facility and the National Institutes of Health (AIO42310,
AR049610, AR050498, GM032136) for support.
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