Structure-based design and biological profile of (E)-N-(4-Nitrobenzylidene) -2-naphthohydrazide, a novel small molecule inhibitor of IκB kinase-β

Article abstract of DOI:10.1016/j.ejmech.2011.01.045

In this study, we describe the rational design, molecular modeling and pharmacological profile of a novel IKK-β inhibitor (E)-N-(4- nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524). The design based on the IKK-β active site, and a privileged structure template yielded a novel IKK-β inhibitor scaffold with significant selectivity over IKK-α and CHK2, as assessed by an in vitro kinase assay. For a better understanding of the structural requirements of IKK-β inhibition, molecular dynamics simulations of LASSBio-1524 (3) were performed. The NAH derivative LASSBio-1524 (3), was able to suppress arachidonic acid-induced edema formation in a dose-dependent manner, demonstrating an in vivo anti-inflammatory effect. The molecular architecture of this novel, low-molecular weight IKK-β inhibitor is encouraging for further lead optimization toward the development of innovative anti-inflammatory drug candidates.

Full text of DOI:10.1016/j.ejmech.2011.01.045

European Journal of Medicinal Chemistry 46 (2011) 1245e1253
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based design and biological profile of (E)-N-(4-Nitrobenzylidene)-
2-naphthohydrazide, a novel small molecule inhibitor of I B kinase-
k
b
,
,
,b
Carolina M. Avila a b, Alexandra B. Lopes a c, Arlan S. Gonçalves d, Leandro L. da Silva a
,
Nelilma C. Romeiro a e, Ana Luisa P. Miranda a b, Carlos M.R. Sant’Anna a
,
,
,
,f
,
,b,
*
Eliezer J. Barreiro a b, Carlos A.M. Fraga a
a Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBioÒ), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro (UFRJ), P.O. Box 68023,
21941-902 Rio de Janeiro, RJ, Brazil
b Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil
c Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Brazil
d Instituto Federal de Educação, Ciência e Tecnologia do Espírito Santo (IFES). Estrada da Tartaruga, S/N, Muquiçaba e Guarapari, Espírito Santo, ES 29.215-090, Brazil
e Universidade Federal do Rio de Janeiro (UFRJ)-Macaé-Rua Aluísio da Silva Gomes, 50, Granja dos Cavaleiros, 27930-560 Rio de Janeiro, RJ, Brazil
f Departamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropédica, RJ 23851-970, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we describe the rational design, molecular modeling and pharmacological profile of a novel
Received 2 November 2010
Received in revised form
14 January 2011
Accepted 25 January 2011
Available online 3 February 2011
IKK-
b
inhibitor (E)-N-(4-nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524). The design based on the
active site, and a privileged structure template yielded a novel IKK- inhibitor scaffold with signif-
and CHK2, as assessed by an in vitro kinase assay. For a better understanding of
the structural requirements of IKK- inhibition, molecular dynamics simulations of LASSBio-1524 (3) were
IKK-
b
b
icant selectivity over IKK-
a
b
performed. The NAH derivative LASSBio-1524 (3), was able to suppress arachidonic acid-induced edema
formation in a dose-dependent manner, demonstrating an in vivo anti-inflammatory effect. The molecular
Keywords:
anti-Inflammatory
IKK-b inhibitor
Molecular docking
N-Acylhydrazone
architecture of this novel, low-molecular weight IKK-
zation toward the development of innovative anti-inflammatory drug candidates.
Ó 2011 Elsevier Masson SAS. All rights reserved.
b inhibitor is encouraging for further lead optimi-
Privileged structure
Structure-based design
1. Introduction
through interaction with IkB inhibitory proteins. In response to
specific external stimuli, including proinflammatory cytokines,
NF-kB is a member of a family of cellular transcription factors
viral infection, oxidants, phorbol esters and ultraviolet irradiation,
that are implicated in the inducible expression of various genes
involved in immune responses, inflammation, cell survival and
cancer [1,2]. In unstimulated cells, NF-kB is kept in the cytoplasm
the I
resulting in translocation of NF-
induction of target gene transcription [3]. Two protein kinases,
IKK- and IKK- [3e5], phosphorylate I B proteins and represent
a convergence point for most signal transduction pathways leading
to NF- B activation [6].
IKK- and IKK- share 52% amino acid identity and are 65%
identical in the kinase domain [7]. In addition, these kinases have
similar biochemical activity: they both phosphorylate I B at serines
32 and 36 (Ser 32/36) to trigger degradation [3e5]. However, these
enzymes differ in their physiological functions. Whereas IKK- is
involved in the proinflammatory signal-induced phosphorylation
of the I B protein, IKK- is dispensable for these functions. On the
other hand, IKK- is a key regulator of keratinocyte and epidermal
differentiation and suppresses skin cancer [8,9], IKK- has been
implicated in some of the anti-inflammatory properties of
k
B component of the complex is phosphorylated and degraded,
k
B into the nucleus and the
a
b
k
k
Abbreviations: AA, arachidonic acid; ATP, adenosine-50-triphosphate; CA, capsid
protein; CB2, cannabinoid receptor type-2; DSSP, definition of secondary structure
a
b
of proteins; I
domain; MD, molecular dynamics; NAH, N-acylhydrazone; NF-
light-chain enhancer of activated cells; RMSD, root-mean-square deviation;
kB, inhibitor of NF-kB; IKK, IkB kinase; IKK-
b, Ik
B kinase-b; KD, kinase
kB, nuclear factor
k
k
B
RMSF, root-mean-square fluctuation; STA, staurosporine; TRPV-1, transient
receptor potential vanilloid subfamily type 1; VMD, visual molecular dynamics.
* Corresponding author. Laboratório de Avaliação e Síntese de Substâncias Bio-
ativas (LASSBioÒ), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro
(UFRJ), P.O. Box 68023, 21941-902 Rio de Janeiro, RJ, Brazil. Tel.: þ55 21 2562 6503;
fax: þ55 21 2562 6478.
b
k
a
a
b
E-mail address: cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga).
URL: http://www.farmacia.ufrj.br/lassbio
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.045

1246
C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
commercial drugs, such as aspirin and salicylates [10]. These
pharmacological effects indicate that an inhibitor of IKK- could
effectively treat autoimmune and inflammatory disorders such as
rheumatoid arthritis, lupus, Crohn’s disease and multiple sclerosis.
Thus, there is a need for the development of novel drug candidates.
derivatives on distinct pharmacological targets, such as transient
receptor potential vanilloid subfamily type 1 (TRPV-1) [16],
cannabinoid receptor type-2 (CB2) [17] and HIV-1 capsid protein
(CA) [18]. Thus, we used a structure-based approach based on the
b
IKK-b active site with the N-acylhydrazone scaffold as a starting
The discovery and development of small molecule IKK-
b
point; we used this structure as a hinge-binding group and
introduced molecular changes to provide additional interactions
with the target kinase. This has previously been achieved by
docking studies using the ChemScore fitness function of the GOLD
suite [19].
The GOLD score function was optimized for the prediction of
ligand-binding positions rather than the prediction of binding
affinities. However, the ChemScore fitness function estimates the
change in total free energy that occurs upon ligand binding and is
determined by linear regression of the binding affinity data [19]. For
these reasons, we used the structural design approach based mainly
modulators is a significant area of research, and several classes of
inhibitors have been previously reported [11,12]. The majority of
these inhibitors share a core heterocycle that mimics the adenosine
ring of adenosine-50-triphosphate (ATP); this modification allows
for additional interactions with the protein.
Our research group has recently reported molecular insights
into the inhibition of IKK-b by natural products, such as the ATP
competitive inhibitors staurosporine (1) and quercetin (2). In this
manuscript we describe the construction of a novel 3D homology
model of the IKK-b
KD with the inclusion of two Mg2þ ions in the
phosphate-binding site, which emphasizes the importance of these
ions in theoretical calculations. In addition, we propose a model of
ATP binding in its fully ionized form [13].
In the present work we describe the development of (E)-N-(4-
nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524, 3) (Chart 1),
on compounds with potential complementarity to the IKK-
b binding
site. In addition, the binding energy ( Gbind) was analyzed. In order
D
to gain insight into the contribution of the lipophilic and hydrogen
bonding properties of the free energy, the terms Slipo (score for
lipophilic interactions) and Shbond (score for hydrogen-bonding)
were analyzed separately. This strategy is attractive because the
values are limited to the prediction of binding affinity, helping to
prioritize sites and molecular substituents for further experimental
modification.
Structural analysis of the conserved kinase two-domain fold
showed that the active site is located at the cleft between these
lobes and includes a hydrophobic pocket bound by the hinge that
interacts with the adenine unit of the ATP molecule. This pocket is
commonly targeted as a binding subsite for drug candidates [20]. In
order to occupy the hydrophobic pocket, two phenyl groups were
a novel IKK-
core, using a structure-based approach. For a better understanding of
the structural requirements of IKK- inhibition, molecular dynamics
b inhibitor scaffold that does not contain the heterocycle
b
(MD) simulations of the ligandebioreceptor complexes were per-
formed. This low-molecular weight inhibitor is an encouraging step
toward further lead optimization and drug development.
Chart 1. IKK-
b inhibitors staurosporine (1), quercetin (2) and the novel N-acylhy-
drazone derivative LASSBio-1524 (3).
2. Results and discussion
2.1. Structure-based design
The concept of privileged structures, in its strict definition,
represents an important tool for the identification of novel struc-
tural subunits that provide pharmacophoric points for interaction
with different bioreceptors to promote various pharmacological
activities. Studies at LASSBio with N-acylhydrazone (NAH) deriv-
atives, as reviewed by Fraga and Barreiro [14], evidenced the
privileged structure character of this bioactive framework [15]. In
a recent report, various examples illustrated the action of these
Fig. 1. (a) Initial structural design strategy. (b) 3D hydrophobicity surface of the IKK-
b
active site and compound 4 shown in stereo. The surface was generated using Chimera
software [21]. The color code represents highly hydrophobic (brown) to highly
hydrophilic (blue) regions. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
1247
Fig. 2. (a) The top-scoring binding pose of compound 4 (carbon atoms in yellow) with IKK-b. (b) The top-scoring binding poses of compounds 5 (carbon atoms in yellow) and 6 (by
atom type) with IKK- . The hydrogen atoms were removed for ease of visualization. The figure was generated using Pymol [26]. (For interpretation of the references to colour in this
b
figure legend, the reader is referred to the web version of this article.)
added to the NAH central core, yielding (E)-N-benzylidenebenzo-
hydrazide (4, Fig. 1a). This compound was docked in the 3D-
homology model, and the hydrophobic surface of the active site
was generated in order to observe the molecular interactions, as
illustrated in Fig. 1.
Visual inspection of compound 4 in the 3D hydrophobic surface
map (Fig. 1b) showed that the phenyl ring attached to the carbonyl
group of NAH (phenyl ring A, Fig. 1a) is located inside the hydro-
phobic region.
phosphate-binding site of IKK-b. In addition, the binding pose of
compound 4 in the ATP-binding site (Fig. 2a) identifies areas that
may be explored for structural modification in order to improve the
interaction with the IKK-b active site. The amino acid residues Ile41,
Ile43, Lys44, Asn150, and Try169, as well as the metal ions, coor-
dinate interaction with the water molecules around phenyl ring B
(Fig. 1a). Interaction with some of these amino acid residues is
important for IKK-
when mutated at Lys44, failed to activate NF-
b
activity. Mercurio et al. [4] showed that IKK-
B, suggesting that
this residue is important for IKK-b activity. Asn150 is conserved
b,
k
Putative binding of compound 4 (Fig. 2a) is consistent with our
original hypothesis, in which the NAH core provides ligand points,
making hydrogen bonds with Glu39 (N/O ¼ 2.94 Å) in the
throughout the kinase family and interacts with the Mg2þ ion in the
phosphate-binding site [22]. In our previous studies, Lys44, Ile43
and Try169 participated in ATP recognition through H-bonding
Table 1
with the oxygen of the b- and g-phosphates [13]. Based on this
N-Acylhydrazone compounds docked in the IKK-
b
active site.
information, we added polar groups at the 4-position of phenyl ring
B (Fig. 1a) to derive compounds 5 and 6 (Table 1). The nitro and
carboxyl moieties were chosen because they are hydrogen bond
acceptors and are able to form ion-dipole and ioneion interactions
with the IKK-b site, respectively, representing an isosteric rela-
shionship [23,24]. In addition, it has previously been reported that
these functional groups are capable of coordinating the metal ions
in the active site of enzymes [25].
The interactions of compounds 5 and 6 with the IKK-b catalytic
domain obtained in the docking study are illustrated in Fig. 2b.
a,b
a,c
Compound
R1
R2
Shbond
Slipo
DGbind
(kJ/mol)a
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
ePhenyl
ePhenyl
ePhenyl
eH
1.87
4.96
4.94
2.67
1.60
1.63
1.30
2.86
5.08
4.97
4.98
4.64
5.06
4.85
4.94
4.86
5.01
4.86
143.11
147.09
144.41
136.67
131.52
146.64
142.66
115.13
127.95
128.27
130.85
128.43
137.17
134.71
137.95
136.17
136.96
136.73
ꢀ30.59
ꢀ46.94
ꢀ46.76
ꢀ38.74
ꢀ33.63
ꢀ36.18
ꢀ34.19
ꢀ30.82
ꢀ44.80
ꢀ44.97
ꢀ44.93
ꢀ44.27
ꢀ46.14
ꢀ45.12
ꢀ45.69
ꢀ45.61
ꢀ45.88
ꢀ45.57
Compounds 5 and 6 make identical interactions with the amino
acid residues in the IKK-b active site; this observation is in accor-
eNO2
eCOꢀ2
eH
eH
eH
-2-Furanyl
-3-Furanyl
-2-Pyridinyl
-3-Pyridinyl
-4-Pyridinyl
-2-Furanyl
-2-Furanyl
-3-Furanyl
-3-Furanyl
-2-Pyridinyl
-2-Pyridinyl
-3-Pyridinyl
-3-Pyridinyl
-4-Pyridinyl
-4-Pyridinyl
dance with the isosteric character of these functional groups, as
stated in the design approach. The NAH cores of both molecules
interact by hydrogen bonding with Glu39 (N/O ¼ 2.75e2.87 Å),
Lys44 (O/N ¼ 3.16e3.19 Å), Tyr169 (O/O ¼ 3.10e3.14 Å), and Ile43
(O/N ¼ 2.61e2.64; O/N ¼ 2.77e2.81 Å). Furthermore, there was
eH
eH
-NO2
eCOꢀ2
eNO2
eCOꢀ2
eNO2
eCOꢀ2
eNO2
-COꢀ2
eNO2
eCOꢀ2
a considerable reduction in the
DGbind of these compounds in
comparison to compound 4, which was associated with the incre-
ment of hydrogen bonding and corroborated the design strategy
(Table 1). The distance from the Mgþ2(1) ion to the oxygen atoms of
the nitro (average distance Mg2þ/O ¼ 3.34 Å) and carboxylate
(average distance Mg2þ/O ¼ 3.30 Å) moieties suggests ion-dipole
and ioneion interactions, respectively.
To increase interaction with critical amino acid residues in the
a
Values are the mean of five consecutive docking experiments using the Chem-
Score function of the GOLD software, (version 3.1.0; see Tables S5eS7, supple-
mentary material).
hinge region of the IKK-b heterocyclic core, hydrogen bonding
acceptors such as 2-furanyl (7), 3-furanyl (8), 2-pyridinyl (9), 3-
pyridinyl (10) and 4-pyridinyl (11) (Table 1) were introduced into
compound 4, replacing aromatic ring A (Fig. 1a).
b
Scores for hydrogen-bonding.
Scores for lipophilic interactions.
c

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C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
Fig. 3. (a) The top-scoring binding poses of compounds 7 (carbon atoms in yellow) and 8 (by atom type) with IKK-b. (b) The top-scoring binding poses of compounds 9 (carbon
atoms in yellow), 10 (carbon atoms in blue) and 11 (by atom type). The hydrogen atoms were removed for ease of visualization. The figure was generated using Pymol [26]. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The observed binding mode for 2-furanyl derivative (7) and IKK-
in the ATP-binding site showed hydrogen bond interactions with
Compounds 12e21 were developed with 4-carboxyphenyl
and 4-nitrophenyl rings attached to the imine unit of the NAH
backbone, replacing the unsubstituted phenyl ring in compounds 5,
6 with the 2-furanyl, 3-furanyl, 2-pyridinyl, 3-pyridinyl and 4-
pyridinyl rings in an effort to mimic adenosine interactions in the
hinge region. Molecular modification of the NAH core improved
b
Glu61 (N/O ¼ 3.05 Å) and coordination of the water molecule (1)
(O/O ¼ 2.90 Å); this was in opposition to 3-furanyl derivative (8),
which formed a hydrogen bond with Lys44 (O/N ¼ 3.20 Å).
Compounds with 2-pyridinyl and 4-pyridinyl groups (compounds 9
and 11, respectively) interacted similarly with the water molecule
(1) (O/O ¼ 2.27 Å), and the 3-pyridinyl derivative (10) formed
a hydrogen bond with Cys46 (O/S ¼ 2.61 Å) (Fig. 3).
interaction with the IKK-b active site. In addition, these modifica-
tions demonstrated the effect of docking molecules that combine
the strong interacting 4-carboxy and 4-nitrophenyl groups with
extra hydrogen bonding acceptor heteroatoms within the hetero-
cyclic rings (Table 1).
Molecular visualization of the top docking poses obtained
for compounds 12e21 revealed a common binding pattern (see
Figs. S1eS8, supplementary material), similar to the interactions
between the 4-carboxy and 4-nitro groups of compounds 5, 6,
and residues close to the metal binding site. Compounds 14 and
15 (with 3-furanyl group) and compounds 18 and 21 (with the 3-
and 4-pyridinyl groups) formed hydrogen bonds with the
nitrogen atom of Gly102. The putative binding modes of these
compounds are illustrated by compounds 15 and 18 in Fig. 4. It is
Interactions with the hinge region of IKK-b were not observed
for the new heterocyclic compounds 7e11, in contrast to the
prediction of the molecular model. This discrepancy can be
explained by the existence of a strong ion-dipole interaction
between the heteroatoms and the Mg2þ(1) ion (average
distance ¼ 2.48 Å) and hydrogen bonding with the charged Glu61
and Lys44 residues. In addition to hydrogen bonding with Cys46,
these interactions may prevent hydrophobic interactions or
hydrogen bonding with amino acid residues in the hinge region
(Fig. 3). Furthermore, in comparison to the nitro and carboxyl
derivatives of compounds 5 and 6, which form strong interactions
in this region, these compounds had an elevated
a reduced Shbond (Table 1) (Fig. 2b).
DGbind and
worth noting that the
(Table 1) was similar, indicating a lack of significant interaction
DGbind of each of these compounds
Fig. 4. The top-scoring binding poses of compounds 15 and 18 (carbon atoms in yellow) with IKK-
b. The hydrogen atoms were removed for ease of visualization. The figure was
generated using Pymol [26]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
1249
Table 2
Docking values and the IKK-
3, 22, 23.
consideration (Table 2). This is also important because the 3D
homology model of IKK- was constructed using CHK2 as
a template [13] and there is considerable sequence identity
between IKK- and IKK-
a
, IKK-b and CHK2 inhibitory profiles of NAH derivatives
b
a
b.
We developed the methyl ester (23) (see Fig. S10, supplementary
material) to avoid possible pharmacokinetic effects of compound 22
in vivo. However, an increase in the
Shbond were observed (Table 2).
DGbind and a decrease in the
Interestingly, compound 22 (see Fig. S9, supplementary material)
wasinactiveagainstalltestedenzymesinvitro, whereascompound3
was selective for IKK-b (Table 2). These data suggest that the 4-
nitrophenyl- and 4-carboxyphenyl groups are not bioisosteric;
however, these moieties interact similarly with amino acid residues
a,b
a,c
a
Compound R2
Shbond
Slipo
D
Gbind IKK-ad IKK-bd CHK2d
(kJ/mol)
IC50 (nH)e or % I at 30
mM
in theIKK-b active site, as illustrated bycompound 3 (Fig. 5). This type
3
22
23
eNO2
eCOꢀ2
eCO2CH3 3.54
4.90
4.80
180.69 ꢀ50.55 0% 20.0
171.97 ꢀ49.43 0% 0%
184.61 ꢀ44,21 0% 0%
m
M (1.2)
0%
0%
0%
of behavior has been reported for other isosteric compounds [25,28].
Zakharian et al. [25] found that the isosteric replacement of the nitro
moieties with a carboxylate group in human arginase I inhibitors
improved chemical interactions with Mn2þ in the arginase catalytic
site, causing a dramatic 500-fold decrease in binding affinity. The
authors suggested that, whereas the carboxylate moiety ought to
serve as a stronger metal ligand due to its negative charge, it
appeared that the binuclear manganese cluster of arginase is better
suitedtoaccommodatetheneutralnitromoiety. Wehypothesizethat
a
Values are the mean of five consecutive docking experiments using the Chem-
Score function in the GOLD software (version 3.1.0; see Tables S5eS7, supplemen-
tary material).
b
Scores for hydrogen-bonding.
Scores for lipophilic interactions.
See Experimental section for a description of the assay conditions.
Hill coefficient.
c
d
e
between the heterocycle core and amino acid residues within the
hinge region.
this phenomenon is also occurring in the ATP-binding site of IKK-b.
Compound 3 formed hydrogen bonds with Glu39 (N/O ¼
3.0 Å), Lys44 (O/N ¼ 3.12 Å), Tyr169 (O/O ¼ 3.16 Å) and Ile43
(O/N ¼ 2.61; O/N ¼ 2.79 Å) (Fig. 5).
The introduction of extra hydrogen acceptor heteroatoms in the
NAH derivatives did not improve binding interactions in the hinge
region. Therefore, we introduced a 2-naphthyl group instead of the
heterocyclic core, yielding compounds 3, 22, 23 (Table 2). The
volume of the 2-naphthyl group mimics the volume of the aden-
osine ring of the ATP molecule. In addition, this moiety makes
hydrophobic interactions with non-polar amino acid residues in
the anterior portion of the hinge region, such as Cys99 and Met83. A
similar approach has been utilized recently in the design and
synthesis of a series of diarylbenzamide IKK-
The docking results (Table 2) showed considerable improve-
ment in the Gbind, with increased lipophilicity; this is in agree-
ment with the design strategy of compounds 3 and 22, which
possess the naphthyl core and the 4-nitrophenyl or 4-carbox-
yphenyl rings, respectively (Table 2). Due to their optimal fit in the
2.2. Chemistry
The synthetic route used to construct the NAH derivatives 3, 22,
and 23 is depicted in Scheme 1. Commercially available b-naphthoic
acid (24) was used as the starting material; it was converted to the
hydrazide intermediate (26) after Fischer esterification and nucle-
ophilic substitution at the carbonyl group of methyl ester (25)
with hydrazine hydrate. The desired NAH derivatives were then
obtained from acid-catalyzed condensation of hydrazide (26) with
appropriate aromatic aldehydes (Scheme 1). In general, these
reactions were generated under mild conditions and provided
products with high yields. This is the advantage of working with
NAH derivatives.
b inhibitors [27].
D
IKK-b active site, we submitted these two compounds to pharma-
cological evaluation. In addition, we evaluated the efficacy of
The structural characterization of the NAH compounds 3, 22 and
23 dissolved in DMSO-d6 by 1H NMR spectroscopy indicated that
each derivative was obtained as a single geometric isomer; a single
signal for NH and N]CH protons was identified. We assume that
the E isomer was obtained after comparison of the 1H NMR results
with findings in the literature [29]; NAH compounds, except for 2-
pyridinylcarbaldehyde, are formed preferentially in the E isomer
when derived from aromatic aldehydes [30]. In addition, the
chemical shift of the imine hydrogen of the NAH derivatives 3, 22,
23 (i.e. 8.54 and 8.56 ppm; see supplementary material) fell
between 8.47 and 8.65 ppm. This is in agreement with studies
performed by Palla and colleagues on the E isomer in DMSO-d6 [29].
inhibition because compound selectivity is an important
2.3. Molecular dynamics simulation
To determine the IKK-
a series of MD simulations in a water solution at 310 K. To deter-
mine the structural stability of the IKK- KD homology model in the
MD simulation [13], 5000-ps MD simulations were performed
using an uncomplexed IKK- KD. In addition, a 10,000-ps MD
simulation of LASSBio-1524 (3, Table 2) (Chart 1) in complex with
IKK- was performed, and the interactions that were crucial for
b interaction pattern, we performed
b
b
Fig. 5. The top-scoring binding pose of compound 3 (carbon atoms in yellow) with
IKK-b. The hydrogen atoms were removed for ease of visualization. The figure was
generated using Pymol [26]. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
b
stability of the complex were determined. The convergence of the
trajectories was monitored by two-dimensional root-mean-square

1250
C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
R2
O
O
O
N
c
a
N
H
OH
R
R2 = NO2 (3)
24
R2 = CO2H (22)
R2 = CO2CH3 (23)
R = OCH3 (25)
b
R = NHNH2 (26)
Scheme 1. Reagents and conditions: (a) 5% HCleMeOH, 55 ꢁC, 16 h, 85%; (b) NH2NH2$H2O 98%, EtOH, 55 ꢁC, 40 min, 94%; (c) aromatic aldehyde derivatives (commercially available),
EtOH, HCl, rt, 30 min, 76e91%.
deviation (2D-RMSD) plots in combination with plots of the energy
variation. Dynamic descriptions of these systems should replace
our predominantly static view of both target and ligand and can aid
in future drug development [31].
Thermodynamic properties, such as total energy and tempera-
ture (see Figs S12 and S13, supplementary material), were moni-
tored during MD simulations, and each parameter converged to
a stable value. Time-dependent secondary structure fluctuations
were analyzed using the DSSP program [32], and most of the
The 2D-RMSD plot versus time (simulation time) obtained by
the 5000-ps MD simulation of the IKK-
b
homology model showed
secondary structures (such as the b-sheet and a-helix) from the
that the system stabilized after 3000 ps (see Fig. S11, supplemen-
tary material) and remained relatively constant thereafter.
initial structure remained stable (see Fig. S14, supplementary
material).
Fig. 6. Conformational changes of LASSBio-1524 (3) in the IKK-b site in the MD simulation (a) in the initial conformation; (b) after 100 ps; (c) after 4500 ps; (d) and at the end of the MD
simulation. The carbon atoms of STA are in yellow. The hydrogen atoms and the solvation waters were removed for ease of visualization. The figure was generated using Pymol [26]. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
1251
The complex of LASSBio-1524 (3) with IKK-
b
(IKK-
b
/LASSBio-
we assumed that the binding sites for the magnesium ions were
highly stable in this system. However, we verified that an ion-
dipole interaction between the nitro group of LASSBio-1524 (3) and
the Mg2þ (1) ion was not maintained throughout the simulation.
1524 system) obtained by docking experiments was used for the
initial MD simulation (Fig. 5). The 2D-RMSD plot showed that this
system equilibrated after 5000 ps and remained relatively constant
thereafter (see Figs. S15 and S16, supplementary material). Ther-
modynamic properties, such as total energy and temperature (see
Figs. S17 and S18, supplementary material), were monitored
during the MD simulations, and each parameter converged to
stable values. Our results suggest that LASSBio-1524 interaction
The starting conformation of IKK-b/LASSBio-1524 (3) in the MD
simulation showed loss of H-bonding with Tyr169, indicating
conformational adjustments in the free minimization steps
(Fig. 6a). In the MD simulation, this residue was very mobile (RMSD
3.2 nm). Since Tyr169 is in the T-loop, we suppose that the increase
in the RMSF of this region was caused by this event (see Figs. S19
and S20, supplementary material). The distance between Tyr169
and the oxygens of the nitro group was greater than 4.5 Å.
with the IKK-
b active site is stable; a stable RMSD value was
obtained and the total energy of the complex decreased during the
simulation.
IKK-
b
is rapidly activated through phosphorylation in its activa-
After the energy minimization step, we determined that
conformational changes in the 2-naphthyl ring probably improved
interactions with the ATP-binding site, resulting in an approxima-
tion of the non-polar residues Cys59, Cys46 and Cys99. These
residues are around the naphthyl ring of LASSBio-1524 (3), sug-
gesting a hydrophobic interaction (Fig. 6aed). These results are in
accordance with the structure-based model. The role of the
tion loop (T-loop), which may be mediated both by upstream kinases
and by trans-autophosphorylation. Phosphopeptide mapping of
IKK-
the T-loop (Ser177 and Ser 181) [4,33] (for the T-loop sequence and
its position in the IKK- binding site see Fig. S19, supplementary
b indicates that phosphorylation occurs at serine residues in
b
material). In order to determine the mechanical stabilization caused
by molecular interactions, we analyzed the root-mean-square
fluctuation (RMSF) of the T-loop upon interaction with IKK-
5000 ps using MD simulation. The RMSF increased in the T-loop
region from 0.6 nm (IKK- uncomplexed protein) to 1.0 nm (com-
plexed form) (see Figs. S19 and S20, supplementary material). These
results suggest that weak interactions resulted in an increase in
the mobility of the T-loop after complexing with LASSBio-1524
(3, Chart 1). In addition, as expected, the ligand alone displayed
greater fluctuation than when complexed; however, very few
fluctuations exceeded 0.4 nm throughout the other regions of the
protein.
cysteine residues in IKK-
the literature [34] and could be exploited by novel IKK-b
b
activity has been previously reported in
inhibitors.
b
at
MD simulation analysis of LASSBio-1524 (3) showed that the
NAH core provides ligand points through hydrogen bonds with
Gln48 and Glu39. Additional interactions between NAH and Gln48
were observed after 100 ps and were maintained during the
majority of the simulation time (Fig. 6b). In addition, transient
H-bonding was observed between the oxygen atom of the NAH
moiety and Gly102 after 4500 ps (Fig. 6c). At the end of the simu-
lation (Fig. 6d), the nitro group formed a hydrogen bond with Lys44
and Glu39.
b
Hydrogen bonding with Lys44, Ile43, Tyr169, and Glu39 and an
ion-dipole interaction with Mg2þ (1) ion were observed in the
binding complex of LASSBio-1524 (3) suggested by the docking
experiments. Because neither direct bonds nor distance constraints
were set between the Mg2þ ions and the surrounding amino acids,
2.4. In vivo anti-inflammatory efficacy of LASSBio-1524 (3)
In vivo efficacy of LASSBio-1524 (3) was characterized in an
acute inflammation model. Arachidonic acid-induced ear edema
depends mechanistically on COX-2 expression, which is regulated
by NF-
model of inflammation for the evaluation of IKK-
LASSBio-1524 (3) showed a dose-dependent effect, significantly
inhibiting ear edema at a dose of 10 mol/kg, with nearly complete
inhibition observed at a dose of 100 mol/kg (Fig. 7). The effect of
10 mol/kg LASSBio-1524 (3) was similar to that of the IKK-
inhibitor SC514 [36,37], which was used as a positive control.
k
B activation [35]. Thus, this model is considered a suitable
b
inhibitors.
m
m
m
b
3. Conclusions
In this study, the structure design strategy based on the IKK-
b
b
active site and a privileged structure template yielded a novel IKK-
inhibitor scaffold with significant selectivity over IKK- and CHK2,
a
as assessed by in vitro kinase assays. We observed that the NAH
core provides ligand points for hydrogen bonding with the IKK-
b
site.
The NAH derivative LASSBio-1524 (3) suppressed arachidonic
acid-induced edema formation in a dose-dependent manner,
showing an in vivo anti-inflammatory effect. The molecular archi-
tecture of this novel low-molecular weight IKK-b inhibitor is
encouraging for further lead optimization toward the development
of innovative anti-inflammatory drug candidates.
4. Experimental section
Fig. 7. Effects of LASSBio-1524 on ear edema induced by topical application of
arachidonic acid (AA) in mice. LASSBio-1524, SC514 or vehicle was administered i.p.
60 min before AA (500 mg/ear) application. Ear thickness was measured 0, 1, 3, and 6 h
4.1. General information
after AA application. Data show the mean ꢂ S.E.M. for six to nine animals. Statistical
differences between vehicle (10% DMSO in saline) and treatment groups were analyzed
using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
For details about chemicals and equipment see the Supple-
mentary Material section. The synthetic route of (E)-N-

1252
C.M. Avila et al. / European Journal of Medicinal Chemistry 46 (2011) 1245e1253
benzylidene-2-naphthohydrazide (3, 22, 23) is outlined in Scheme
1. The synthesis of compound 3 is described here, and the synthetic
procedures for intermediates 25 and 26 and target NAH derivatives
22 and 23 are reported in the Supplementary Material section.
Compound structures were built manually with Spartan for Linux v.
O8 software (Wavefunction Inc.). MMFF94 [41] implemented in
Spartan was used for the conformational search. Geometry optimi-
zation was performed using the semi-empiric method AM1 [42]. The
ChemScore fitness function in the GOLD docking software [19]
(version 3.1.0) was used to score the docked compounds because
of the program’s ability to accurately predict the binding modes of
known ligands, being particularly sensitive to metal parameters. The
active site was defined as the atoms within 10 Å of Asn150, which
was used as the reference residue. This residue is conserved
throughout the kinase family and coordinates with Mg2þ in the
phosphate-binding site [22]. The coordination geometries of the
metal ions were assigned as octahedral, and the water orientation
was set to Spin. In the GOLD software, consecutive dockings using
the same input files will not give identical solutions or fitness scores;
this is a fundamental aspect of the GOLD software algorithm. In
order to solve this problem, five consecutive docking runs using the
same input files were performed, and the best-ranking energy value
from each run was collected. The binding energy represented the
mean of these values. The protein-ligand complex with the most
4.2. Chemistry
4.2.1. (E)-N-(4-nitrobenzylidene)-2-naphthohydrazide, (3)
4-Nitrobenzaldehyde (0.17 g; 1.13 mmol) was added to a solu-
tion of 2-naphthohydrazide (26) (0.20 g, 1.07 mmol) in absolute
ethanol (5 mL) supplemented with two drops of 37% hydrochloric
acid. The mixture was stirred at room temperature for 30 min and
a precipitate was formed. After the reaction was complete (as
assessed by TLC), the mixture was poured into ice, and the
precipitate was filtered and dried under vacuum. The desired title
compound formed as a pale yellow amorphous solid (0.310 g 90%;
Mp 244e246 ꢁC; 1H NMR (300 MHz, DMSO-d6, TMS)
d: 12.24 (s, 1H,
NH); 8.58 (s, 1H, Naph-1-H); 8.54 (s, 1H, N]CH); 8.28 (d,
3J ¼ 9.0 Hz, 2H, Ph-3-H, Ph-5-H); 8.08e7.97 (m, 6H, Naph-3-H),
(-4H, -5H, -8H), Ph-2-H, Ph-6-H); 7.67e7.58 (m, 2H, Naph-6-H,
Nap-7-H). 13C NMR (75 MHz, DMSO-d6, TMS)
d: 164.18 (C]O);
favorable free energy of binding (DGbind) among the top-scoring
148.58 (Ph-4-C); 145.99 (N]CH); 141.36 (Ph-1-C); 135.12 (Naph-9-
C); 132.74 (Naph-2-C); 131.13 (Naph-10-C); 129.62 (Naph-8-C);
128.67 (Naph-1-C, -4C, -6C); 128.37 (Naph-5-C); 127.61 (Naph-7-
C); 125.01 (Naph-3-C); 124.72 (Ph-2-C, -3C, -5C, -6C). IR (cmꢀ1, KBr)
3433, 3235 (NH); 1653 (C]O); 1536, 1350 (NO2). Anal. Calcd for
C18H13N3O3: C: 67.71; H: 4.10; N: 13.16. Found: C: 67.63; H: 4.03; N:
13.26. % purity >99% by HPLC (tR ¼ 5.179 min).
complexes over the five runs was used for further visual inspection.
4.4.2. Molecular dynamics simulation
For the MD simulation, the ligand was parameterized using the
Dundee PRODRG Server [43,44] (beta version), and charge calcula-
tions were performed using the GAMESS US software package
[45,46]; Hartree Fock with a basis set 6-31G(d) and CHELPG [47e49]
were used to develop an optimal charge fit. The two systems (the
uncomplexed protein and the protein-ligand complex chosen with
the optimal docking result) were immersed into a dodecahedral box
with SPC water molecules [50,51] and three Clꢀ counter-ions to
neutralize the total charge of the solvated protein. Four Clꢀ counter-
ions were also used to neutralize the protein-ligand complex. The
energy of the solvated systems was minimized using Steepest
Descent with position restraints of the heavy atoms followed by
Steepest Descent without position restraints. Thereafter, energy
minimization was performed using the Conjugate Gradient function
followed by the quasi Newton method. In total, 2000 steps were used
to minimize energy. Particle Mesh Ewald [52,53] (PME, Fourier
spacing 1.2 Å, 4th order and tolerance 10ꢀ5) and 6e12 Lennard-Jones
potentials were applied to account for Coulomb and van der Waals
interactions, using a cut-off of 1.0 nm for both interactions. The LINCS
algorithm [54] was applied to each covalent bondin order to preserve
the character of the interaction. The systems were equilibrated after
500 ps ofMD simulation (i.e. the temperature reached 310 K and 1 bar
of pressure). After the equilibration step, MD simulations were per-
formed without position restraints for 5000 ps of uncomplexed
protein and 10 000 ps of protein-ligand using the GROMACS software
package (version 4.0) [55,56]. Both the minimization step and the MD
simulations were performed using GROMOS96 force field [57]. MD
analysis was performed using the GROMACS software package
(version 4.0) [55,56], VMD [58]. Analysis of the secondary structure
was performed using the DSSP program [32].
4.3. Pharmacology
4.3.1. Human kinase assays
The effects of these compounds on IKK-a and IKK-b kinase
activity were determined by measuring the phosphorylation of the
substrate phospho-Ulight-IkappaB-alpha using a human recombi-
nant enzyme, which was expressed in Sf21 cells [18]. CHK2 kinase
assays were performed by measuring the phosphorylation of the
substrate phospho-CREBtide (CKRREILSRRPSYRK) using a human
recombinant enzyme, which was expressed in insect cells [38]. In
both assays, the LANCE detection method was used in combination
with the Cerep IKK kinase assay [39]. The compounds were tested at
concentrations of 0.3, 1.0, 3.0 and 30 mM. We used staurosporine as
a reference compound (see Tables S4, supplementary material). Data
are expressed as a percent of control specific activity (observed
specific activity/control specific activity ꢃ 100) obtained in the
presence of the test compounds. The IC50 value is the concentration
causing a half-maximal inhibition of control specific activity.
4.3.2. Ear edema in mice
Arachidonic acid (100 mg, AA; Sigma) was dissolved in 4 mL of
acetone. This solution (20 mL; 500 mg/ear) was applied to the inner
and outer surfaces of the right ear of each mouse (Swiss, female and
male, seven to eight weeks old). Ear thickness was measured at 0, 1,
3, and 6 h after AA application using an electronic digital caliper. Ear
edema was expressed as a difference in ear thickness at 1, 3 or 6 h
compared to time 0. The reference compound 2-amino-4-(thio-
phen-2-yl)thiophene-3-carboxamide (SC514) was obtained from
Tocris Bioscience (Missouri, USA). The vehicle was 10% dimethyl
sulfoxide (DMSO) in saline.
Acknowledgments
The authors are thankful for financial support from INCT-INO-
FAR (573.564/2008-6), CNPq, FAPERJ, and fellowships (to EJB,
CAMF, CMRS, NCR, ABL, ASG and CMA).
4.4. Molecular modeling
Appendix. Supplementary material
4.4.1. Docking studies
The IKK-
b
KD structure was obtained by homology modeling
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.01.045.
using an active kinase conformation of CHK2 [40] as a template [13].

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