Phenolic hydrazones are potent inhibitors of macrophage migration inhibitory factor proinflammatory activity and survival improving agents in sepsis

Article abstract of DOI:10.1021/jm061477+

A series of phenolic hydrazones were synthesized and evaluated for their inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity. Compound 7 emerged as a potent inhibitor of MIF with an IC₅₀ of 130 nM. Compound 7 dose-de

Full text of DOI:10.1021/jm061477+

J. Med. Chem. 2007, 50, 1993-1997
1993
Phenolic Hydrazones Are Potent Inhibitors of Macrophage Migration Inhibitory Factor
Proinflammatory Activity and Survival Improving Agents in Sepsis
Darrin R. Dabideen,† Kai Fan Cheng,† Bayan Aljabari,† Edmund J. Miller,‡ Valentin A. Pavlov,§ and Yousef Al-Abed*,†,#
Laboratory of Medicinal Chemistry, Laboratory of Cardiopulmonary Research, and Laboratory of Biomedical Sciences,
The Feinstein Institute for Medical Research, 350 Community DriVe, Manhasset, New York 11030, and The New York School of Medicine,
New York, New York 10016
ReceiVed December 26, 2006
A series of phenolic hydrazones were synthesized and evaluated for their inhibition of macrophage migration
inhibitory factor (MIF) tautomerase activity. Compound 7 emerged as a potent inhibitor of MIF with an
IC50 of 130 nM. Compound 7 dose-dependently suppressed TNFR secretion from lipopolysaccharide
stimulated macrophages. The therapeutic importance of the MIF inhibition by 7 is demonstrated by the
significant protection from the lethality of sepsis when administration of the compound was initiated in a
clinically relevant time frame.
Scheme 1. Rational Design of MIF Inhibitors
Introduction
Macrophage migration inhibitory factor (MIFa) is a potent
proinflammatory cytokine critically involved in the pathogenesis
of sepsis and other inflammatory disorders.1,2 We and others
have demonstrated that MIF is an important late-acting mediator
of systemic inflammation and that inhibition of its activity in
vivo attenuates the lethal consequences of endotoxemia and
sepsis in rodents.3,4
MIF exists as a homotrimer5-8 with the unique ability to
catalyze the tautomerization of nonphysiological substrates such
as D-dopachrome and L-dopachrome methyl ester into their
respective indole derivatives (Scheme 1).9 While the physi-
ological role of the tautomerase activity is uncertain, we have
previously found that compounds that are structurally similar
to D- and L-dopachrome could bind to and thereby block the
MIF’s tautomerase active site.4,10-13 We have previously shown
that NAPQI forms a covalent complex with MIF at its active
site (Scheme 1) and is capable of irreversibly inhibiting the
adverse biological effect of MIF.13 However, the toxicity of
NAPQI precluded its use as a viable clinical inhibitor of MIF
and the development of nontoxic small-molecule inhibitors of
MIF tautomerase activity warranted further investigation.
The indole intermediate of MIF tautomerase catalysis pre-
sented itself as a suitable template for the development of
potential MIF inhibitors. We reasoned that compounds designed
around the phenylimine scaffold could act as potential MIF
antagonists. Indeed, from our rational design we developed the
amino acid Schiff bases and the isoxazoline compounds as MIF
tautomerase inhibitors.10,12 Among the amino acid Schiff bases
tested for their ability to inhibit the tautomerase activity of MIF,
it was found that 1 was the most potent (Scheme 1, Table 1).
Recently, we have reported an isoxazoline inhibitor of MIF, 2
(ISO-1), which blocks the tautomerase site, inhibits the ability
of MIF to overcome anti-inflammatory glucocorticoid activities
in vitro, and improves survival in animal models of experimental
sepsis (Scheme 1).4,12
In our quest to find more potent inhibitors of MIF, we
revisited the phenylimine scaffold and modified it by adding
nitrogen to afford the hydrazone type compound (Scheme 1).
Our previous studies on the amino acid Schiff bases and
compound 2 revealed that a para hydroxyl group, as exemplified
by a phenolic moiety, is a key structural feature and is required
for activity. We have shown that replacing the phenolic moieties
of 1 and 2 with phenyl or halide-substituted phenyl or
p-methoxyphenyl groups resulted in the decrease or complete
loss of their ability to inhibit MIF tautomerase activity.10,12 In
accordance with this result, the chemical structures of the new
pharmacophores must contain a phenolic ring (Scheme 1).
Utilizing this key structural feature and the observations of 1
and 2 complexed with MIF, we propose the synthesis of
phenolic hydrazones 3-15 as potential MIF inhibitors.
* To whom correspondence should be addressed. Phone: 516 562 3406.
Fax: 516 562 1022. E-mail: yalabed@nshs.edu.
† Laboratory of Medicinal Chemistry, The Feinstein Institute for Medical
Research.
‡ Laboratory of Cardiopulmonary Research, The Feinstein Institute for
Medical Research.
Results and Discussion
§ Laboratory of Biomedical Sciences, The Feinstein Institute for Medical
Research.
The phenolic hydrazones were obtained in high yields by
condensing p-hydroxybenzaldehyde and the appropriate hy-
drazide in an alcoholic solvent in the presence of an acid catalyst
at room temperature. The hydrazones obtained were assayed
# The New York School of Medicine.
a Abbreviations: MIF, macrophage migration inhibitory factor; NAPQI,
N-acetyl-p-benzoquinone imine; LPS, lipopolysaccharide; CLP, cecal liga-
tion and puncture.
10.1021/jm061477+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/27/2007

1994 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
Table 1. Phenolic Hydrazones and IC50 for Inhibition of MIF
Tautomerase Activity
Figure 1. Disubstituted hydrazone.
a result is a very poor inhibitor of MIF tautomerase activity
(Table 1). Upon replacement of the hydrogen with a methyl
group, a pronounced improvement in the ability of the meth-
ylhydrazone 4 to inhibit the tautomerase activity of MIF (IC50
) 43 µM) was observed. This suggests that there is a
hydrophobic interaction between the methyl group and the
surface of MIF. Replacing the methyl group of 4 with the more
hydrophobic phenyl ring affords the hydrazone 5 (IC50 ) 2.6
µM) that is 16 times more potent. Installation of a p-methoxy
group on the aromatic ring of 5 gives hydrazone 6 that is 5-fold
more potent as an inhibitor than the parent compound. This
increased binding of the p-methoxyphenylhydrazone may be
explained by hydrogen bond interactions between the ether
oxygen and the known amino acid residues and by the possibility
of π-π stacking and/or van der Waals interactions between
the p-methoxyphenyl ring and the second hydrophobic region
of the MIF active site. It has been reported by us and other
workers that the amino acids Pro-33, Tyr-36, Phe-49, Trp-108,
and Phe-113 make up the second hydrophobic surface at the
rim of the active site of MIF.10,14 These residues further
contribute to the hydrophobic and hydrogen bond interactions
between the pharmacophore and the active site of MIF.14 In
further support of these interactions, we have previously shown
that the L-amino acid Schiff base inhibitory effect was signifi-
cantly improved by 5-fold upon changing the amino acid residue
from L-phenylalanine (IC50 ) 50 µM) to L-tyrosine (IC50 ) 10
µM). This suggests that the increased potency was attributed
to a hydrogen bond interaction within residues at the rim of
MIF.10 Hydrazone 6 is 12 times more potent an inhibitor than
L-tryptophan Schiff base 1, so far the most potent inhibitor of
MIF described in the literature.4,10-12,14,15
Recently, we have discovered that monofluorination of 2
improved the inhibition of MIF activity.11 As a consequence,
we synthesized the 3-fluoro, 3-chloro, and 3-bromo-4-hydrox-
yphenyl derivatives of the more potent hydrazone, namely, the
p-methoxyphenylhydrazone (6). The synthesis of the monoha-
logenated hydrazone derivatives involved treatment of a suspen-
sion of the 4-methoxyphenylhydrazine hydrochloride and the
3-halogenated 4-hydroxybenzaldehyde in methanol with aqueous
sodium hydroxide (Table 1). A general increase in the inhibition
of the MIF tautomerase activity was observed for the 3-halo-
genated 4-hydroxyphenylhydrazone derivatives 7-9 (Table 1).
Among these hydrazones, 7 showed the most potent inhibition
with an IC50 of 130 nM while 8 and 9 gave values of 220 and
330 nM, respectively. The significant improvement in the
inhibitory effect of these halogenated hydrazones 7-9 may be
explained by the inductive effect that may lead to changes in
the polarization of the hydroxyl moiety, thereby making it a
stronger hydrogen bond donor/acceptor.
a Compounds were characterized by 1H NMR, 13C NMR, and MS.
b Spectrophotometric analysis of MIF tautomerase activity on L-dopachrome
methyl ester (see Experimental Section). c See Experimental Section for
general procedure for the synthesis of 6-9.
for their potency of inhibiting MIF tautomerase activity (Table
1).
All of the hydrazones prepared in this study 3-15 have one
common key structural feature; they all possess a “phenolic
head” in the form of a 4-hydroxyphenyl ring. We and other
workers have shown that the phenolic ring forms key hydrogen
bond interactions between the amino acid residue asparagine-
97C of the hydrophobic surface within the MIF active site.10,12,14
In addition to this important hydrogen bond interaction, there
is a hydrophobic interaction that exists between the aromatic
ring of the phenol and the side chains of the amino acid residues
Pro-1, Met-2, Ile-64, Tyr-95, Val-106, and Phe-113 of the
hydrophobic pocket that further contributes to the binding of
the inhibitor.14 Supporting evidence for the key interaction
between the hydroxyl functionality and the amino acid residue
asparagine-97C was obtained by (1) modifying its position and
(2) replacing the hydroxyl group with other functional groups.
The position of the hydroxyl group is a critical feature of the
hydrazones in that changing its position from para (IC50 ) 2.5
µM) to meta (IC50 ) 150 µM) resulted in a dramatic loss of its
ability to inhibit MIF tautomerase activity. Furthermore, replac-
ing the hydroxyl group with hydrogen, fluoro, amino, methoxy,
and nitro functionalities afforded hydrazones that were inactive
(data not shown). In developing a structure-activity relationship
between the phenolic hydrazones and MIF, we synthesized
hydrazones 3-6 from the simple hydrazine, methylhydrazine,
phenylhydrazine, and p-methoxyphenylhydrazine. One further
prerequisite for activity of our phenolic hydrazones is that they
require a hydrophobic tail. This is evident from 3 (IC50 g 500
µM). The hydrogen substituent of the hydrazone in this
compound does not offer any hydrophobic interactions and as
To determine if the activity would be affected by disubsti-
tution on the nitrogen, 10 was synthesized (Figure 1). Methy-
lation of phenylhydrazine (5) with methyl iodide and sodium
amide afforded N-methyl-N-phenylhydrazine. Hydrazone forma-
tion under typical conditions gave 10 in 86% yield. To our
disappointment 10 was a very weak inhibitor of MIF activity
and displayed an IC50 of 300 µM. This observation could be
accounted for by increased steric hindrance or by the reduced
number of hydrogen bonding interactions between the disub-
stituted hydrazone derivative 10 and the active site of MIF.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1995
Table 2. Phenolic Hydrazone Carbamates and IC50 for Inhibition of
MIF Tautomerase Activity
Figure 2. Compound 7 inhibits TNF secretion from LPS-treated
macrophages. RAW 267.4 macrophages (105) were treated with various
concentrations of hydrazone 7 (0.01-100 µM) 30 min prior to LPS
addition or with 10 µg/mL mouse monoclonal antibody against MIF
(XIV.15.5, R-MIF). After 16 h of incubation, cell culture supernatants
were collected for determination of TNFR concentration by ELISA.
Data are presented as the mean ( SD (n ) 3, (/) p < 0.01).
1
a Compounds were characterized by H NMR, 13C NMR, and MS (see
Supporting Information). b Spectrophotometric analysis of MIF tautomerase
activity on L-dopachrome methyl ester.
To further investigate the structure-activity relationship
between the hydrazones and MIF, we looked at another class
of compounds, namely, the phenolic hydrazone carbamates. The
phenolic hydrazone carbamates 11-15 were chosen because
they displayed key functionalities to 1 and 2 (Table 2). These
functionalities are the hydroxyl group, phenyl ring, and the
carboxylate moiety. It has been reported by us that a secondary
hydrogen bond interaction exists between the carboxylate moiety
of 2 and lysine-32A of the MIF active site.10 Significant
improvement of approximately 10-fold in the inhibition of MIF
tautomerase activity was observed on changing the methyl group
of 11 (IC50 ) 55 µM) to the more lipophilic tert-butyl moiety
13 (IC50 ) 5.5 µM).11 To determine if phenyl rings improve
the inhibition of MIF activity, we replaced the tert-butyl
carbamate with a benzyl carbamate 14 and p-methoxybenzyl
carbamate 15. This change from a bulky alkyl 13 to aromatic
14 and 15 (IC50 ) 10 and 8 µM, respectively) resulted in a
slight decrease in the inhibitory effect.
Figure 3. Compound 7 is protective after 24 h of late treatment in a
CLP model. Mice were injected intraperitoneally with 7 (4 mg/kg) (n
) 13, p < 0.01) or vehicle 24 h after CLP (n ) 13). A single injection
is composed of 100 µg of 7 (equivalent to 4 mg/kg) in 200 µL of 20%
DMSO and 80% saline solution. Additional administrations of 7
(bidaily) were given on days 2 and 3.
Biological Activity. Intracellular MIF plays a critical role in
mediating the cellular responses to pathways activated by LPS
endotoxin,16 and MIF deficient cells are hyporesponsive to
endotoxin.17 We and others have shown that MIF null mac-
rophages can produce 50-60% less TNF compared to wild
type.4,18 Previously, we have shown that anti-MIF antibody (10
µg/mL) inhibits 50% of TNF release from LPS-treated mac-
rophages.4 We also have found that 2 dose-dependently inhibit
LPS-induced TNF release from wild type and not MIF null
macrophages, suggesting that the chemical inhibitor of MIF is
specific. Accordingly, we reasoned that 7 binding to MIF would
suppress LPS responses in macrophages. Compound 7 dose-
dependently inhibited LPS-induced TNF release (Figure 2).
Thus, 7 recapitulates the phenotype of the MIF deficient
macrophages and is associated with decreased TNF production
in response to LPS.
The importance of MIF as a molecular therapeutic target in
sepsis has been confirmed by our recent observation that
treatment with anti-MIF antibodies (4 mg/kg of monoclonal
antibody against MIF (XIV.15.5) daily for 3 days) or 2 (40 mg/
kg bidaily for 3 days) significantly improves survival in septic
mice.4 We also recently discovered that serum MIF levels
increased to 70% of maximum levels within 24 h after CLP
and peaked at 36 h.4 This identified MIF as a late mediator in
sepsis and indicated the therapeutic potential of inhibiting MIF
in a clinically relevant time frame. Therefore, we reasoned that
a delayed treatment with 7, consistent with the kinetics of MIF
release, could be successfully applied to improve survival in
sepsis. We tested the ability of 7 to improve the survival rate
in CLP-induced peritonitis, a widely used preclinical model of
sepsis. Intraperitoneal treatment of 7 (4 mg/kg) initiated 24 h
after CLP surgery and continued for 3 days resulted in a survival
rate of 65% (p < 0.01) compared to 28% in the control (vehicle-
treated) group (Figure 3). Thus, 7 treatment provided significant
protection against sepsis lethality, comparable to the effect of
anti-MIF antibody and 2.4 Remarkably, a dose of 7, 10-fold
less than 2, achieved similar protection. This finding indicates
an association between the potency of 7 in inhibiting the MIF
tautomerase active site and its beneficial effect of improving
survival in experimental sepsis.
In summary, we have designed and synthesized phenolic
hydrazones as nontoxic, potent MIF antagonists. Our structure-
activity relationship study suggests that minor changes in
functionalization of the hydrazones affect the binding of these
compounds to the MIF active site. Notably, 7 exhibits the
greatest activity of all the compounds tested and is so far the
most potent inhibitor of MIF described in the literature.4,10-12,14,15
Compound 7 exhibited a potent anti-inflammatory activity in
vitro as demonstrated by suppression of LPS-induced macroph-

1996 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
supernatants were collected for determination of TNF concentration
by ELISA. Data are presented as the mean ( SD (n ) 3, p <
0.01).
age activation. Moreover, a relatively low concentration of this
small-molecule MIF inhibitor improved survival in sepsis when
treatment was initiated at 24 h after the onset of the disease.
Sepsis is a complex inflammatory disorder, and its clinical
management is a challenging health issue. Therefore, our finding
that 7 is effective at 24 h after the onset of the disorder could
be of considerable clinical interest.
Animal Studies. All animal experiments were approved by the
Institutional Animal Care and Use Committee of the North Shore-
Long Island Jewish Research Institute. Male Balb/C mice, ∼8 weeks
old, were subjected to cecal ligation and puncture. Details of the
CLP procedure has been carried out as follows: In anesthetized
male BALB/c mice (100 mg/kg ketamine and 8 mg/kg xylazine
administered intramuscularly) the cecum was ligated and given a
single puncture. Abdominal access was gained via a midline
incision. The cecum was isolated and ligated with a 6-0 silk ligature
below the ileocecal valve, and the cecum was punctured once with
a 22G needle. Stool (approximately 1 mm) extruded from the hole,
and the cecum was placed back into the abdominal cavity. The
abdomen was closed with two layers of 6-0 Ethilon sutures.
Antibiotics were administered immediately after CLP (0.5 mg/kg
Premaxin, subcutaneously, in a total volume of 0.5 mL/mouse),
and a single dose of resuscitative fluid (normal saline solution) was
administered subcutaneously (20 mL/kg body weight) immediately
after CLP surgery.19 Mice were injected intraperitoneally with 4
mg/kg (n ) 13, p < 0.01) or vehicle 24 h after CLP (n ) 13).
Two additional injections were given on days 2 and 3. Vehicle
(aqueous 20% DMSO) or 7 (4 mg/kg, intraperitoneally) treatment
was started 24 h after the induction of sepsis and repeated twice
daily on days 2 and 3. Animal survival was monitored for 14 days.
Experimental Section
General Procedure. All chemicals were obtained from com-
mercial suppliers and used without further purification. Aluminum-
backed silica gel 60 with 254 nm fluorescent indicator TLC plates
were used. Developed TLC plates were visualized under a short-
wave UV lamp, stained with an I2-SiO2 mixture. Flash column
chromatography (FCC) was performed using flash silica gel (32-
63 µm) and usually employed a stepwise solvent polarity gradient
correlating with TLC mobility. Melting points were determined in
a Gallenkamp melting point apparatus in open capillaries and are
uncorrected. IR spectra were obtained on a Thermo Nicolet IR 100
FT-IR spectrometer. All 1H spectra were recorded on a Joel
spectrometer or a GE QE 300 spectrometer at 270 or 300 MHz.
The 13C spectra were recorded on a GE QE 300 spectrometer at 75
MHz. Chemical shifts are relative to the deuterated solvent peak
and are in ppm. The coupling constants (J) are measured in Hz.
1
The H signals are described as s (singlet), d (doublet), t (triplet),
q (quartet), m (multipet), or br s (broad singlet). Low- and high-
resolution mass spectrometry was carried out at the Mass Spec-
trometry Facility at the University of Illinois at Urbanas
Champaign.
Acknowledgment. This research was supported by an
Institutional grant and the NIH (Grant HL081655) awarded to
Dr. E. J. Miller and Dr. Y. Al-Abed.
General Procedure for Compounds 3-5 and 10-15. 4-Hy-
droxybenzaldehyde (122 mg, 1 mmol) or 3-fluoro-4-hydroxyben-
zaldehyde (140 mg, 1 mmol) and the hydrazide (2 mmol) were
dissolved in ethanol (10 mL). To this was added acetic acid (1
mmol), and the mixture was stirred overnight at room temperature.
Removal of the ethanol in vacuo afforded an oily residue. The
residue was taken up with ethyl acetate and washed with water.
The organic layer was separated and dried with anhydrous Na2-
SO4. Concentration in vacuo afforded a residue that was subse-
quently purified by FCC using hexane and ethyl acetate as eluent
(4:1) to give 3-5 and 10-15.
General Procedure for Compounds 6-9. 4-Hydroxybenzal-
dehyde (122 mg, 1 mmol) or 3-fluoro-4-hydroxybenzaldehyde (140
mg, 1 mmol) or 3-chloro-4-hydroxydroxybenzaldehyde (156 mg,
1 mmol) or 3-bromo-4-hydroxybenzaldehyde (201 mg, 1 mmol)
and 4-methoxyphenylhydrazine hydrochloride (350 mg, 2 mmol)
were suspended in methanol (10 mL). To this suspension was added
a 2 M aqueous solution of sodium hydroxide (60 mg, 1.5 mmol),
and the mixture was stirred overnight at room temperature. Upon
completion of the reaction, the solution was then acidified to pH 4
by the addition of 1 M HCl. Removal of the methanol in vacuo
afforded an oily residue. The residue was taken up with ethyl acetate
and washed with water. The organic layer was separated and dried
with anhydrous Na2SO4. Concentration in vacuo afforded a residue
that was subsequently purified by FCC using hexane and ethyl
acetate as eluent (4:1) to give 6-9.
Spectrophotometric Assay for Enzymatic Activity. A fresh
stock solution of L-dopachrome methyl ester (2.4 nM) was generated
by oxidation of L-3,4-dihydroxyphenylalanine methyl ester with
sodium periodate, producing an orange solution. Activity was
determined at room temperature by adding dopachrome methyl ester
(0.3 mL) to a cuvette containing 1 µL of MIF solution (850 ng/
mL) in 50 mM potassium phosphate buffer, pH 6, and measuring
the decrease in absorbance from 2 to 20 s at 475 nm spectropho-
tometrically. Inhibitors 3-15 were dissolved in DMSO at various
concentrations (0.1-100 µM), and 1 µL was added to the cuvette
with the MIF prior to the addition of the dopachrome.
Supporting Information Available: Physical and spectral data
for 3-15. This material is available free of charge via the Internet
at http://pubs.acs.org.
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