Discovery of novel orally active tetrahydro-naphthyl-N-acylhydrazones with in vivo anti-TNF-α effect and remarkable anti-inflammatory properties

Article abstract of DOI:10.1371/journal.pone.0156271

LASSBio-1524 was designed as inhibitor of the IKK-β (kappa β kinase inhibitor) enzyme, which participates in the activation of the nuclear factor κB (NF-κB) canonical pathway, and its three N-acylhydrazone new analogues, LASSBio-1760, LASSBio-1763 and LASSBio-1764 are now being tested on their anti-inflammatory potential. The activity of these compounds was evaluated with the subcutaneous air pouch induced by carrageenan and by subsequent measurement of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive oxygen species (ROS). In the acute inflammation model, the oral pretreatment with doses from 0.3 to 30 mg/kg of N-acylhydrazone derivatives was able to significantly reduce leukocyte migration to the cavity. Pretreatment with LASSBio-1524 and its analogues also decreased NO, TNF-α and ROS biosynthesis an events closely involved with NF-kB pathway. The tetrahydronaphthyl-N-acylhydrazone derivative LASSBio-1764 was the most promising compound from this series, surpassing even LASSBio-1524. Additionally, none of the compounds demonstrated myelotoxicity or cytotoxicity. Cell viability was assayed and these compounds demonstrated to be safe at different concentrations. Western blot analysis demonstrated that LASSBio-1524 and LASSBio-1760 inhibited NF-κB expression in RAW 264.7 cell lineage. Our data indicate that the tested compounds have anti-inflammatory activity, which may be related to inhibition of leukocyte migration, reducing the production of NO, TNF-α and ROS. LASSBio-1524 and LASSBio-1760, in addition to these features, also reduced p65 nuclear expression assessed by western blot in RAW 264.7 murine cells.

Full text of DOI:10.1371/journal.pone.0156271

RESEARCH ARTICLE
Discovery of Novel Orally Active Tetrahydro-
Naphthyl-N-Acylhydrazones with In Vivo
Anti-TNF-α Effect and Remarkable Anti-
Inflammatory Properties
1
,2
3,4
2,3,4
Natália M. Cordeiro , Rosana H. C. N. Freitas , Carlos A. M. Fraga
, Patricia
1,2
D. Fernandes
*
1
Federal University of Rio de Janeiro, Institute of Biomedical Science, Laboratory of Pharmacology of Pain
a11111
and Inflammation, Rio de Janeiro, Brazil, 2 Federal University of Rio de Janeiro, Institute of Biomedical
Science, Graduate Programm in Pharmacology and Medicinal Chemistry, Rio de Janeiro, Brazil, 3 Federal
University of Rio de Janeiro, Institute of Biomedical Science, Laboratory of Evaluation and Synthesis of
Bioactive Substances (LASSBio), Rio de Janeiro, Brazil, 4 Federal University of Rio de Janeiro, Chemistry
Institute, Graduate Programm in Chemistry, Rio de Janeiro, Brazil
*
patricia.dias@icb.ufrj.br
OPEN ACCESS
Citation: Cordeiro NM, Freitas RHCN, Fraga CAM,
Fernandes PD (2016) Discovery of Novel Orally
Active Tetrahydro-Naphthyl-N-Acylhydrazones with In
Vivo Anti-TNF-α Effect and Remarkable Anti-
Inflammatory Properties. PLoS ONE 11(5):
Abstract
LASSBio-1524 was designed as inhibitor of the IKK-β (kappa β kinase inhibitor) enzyme,
which participates in the activation of the nuclear factor κB (NF-κB) canonical pathway, and
its three N-acylhydrazone new analogues, LASSBio-1760, LASSBio-1763 and LASSBio-
e0156271. doi:10.1371/journal.pone.0156271
1
764 are now being tested on their anti-inflammatory potential. The activity of these com-
Editor: Silvana Allodi, Federal University of Rio de
pounds was evaluated with the subcutaneous air pouch induced by carrageenan and by
subsequent measurement of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive
oxygen species (ROS). In the acute inflammation model, the oral pretreatment with doses
from 0.3 to 30 mg/kg of N-acylhydrazone derivatives was able to significantly reduce leuko-
cyte migration to the cavity. Pretreatment with LASSBio-1524 and its analogues also
decreased NO, TNF-α and ROS biosynthesis an events closely involved with NF-kB path-
way. The tetrahydronaphthyl-N-acylhydrazone derivative LASSBio-1764 was the most
promising compound from this series, surpassing even LASSBio-1524. Additionally, none
of the compounds demonstrated myelotoxicity or cytotoxicity. Cell viability was assayed
and these compounds demonstrated to be safe at different concentrations. Western blot
analysis demonstrated that LASSBio-1524 and LASSBio-1760 inhibited NF-κB expression
in RAW 264.7 cell lineage. Our data indicate that the tested compounds have anti-inflamma-
tory activity, which may be related to inhibition of leukocyte migration, reducing the produc-
tion of NO, TNF-α and ROS. LASSBio-1524 and LASSBio-1760, in addition to these
features, also reduced p65 nuclear expression assessed by western blot in RAW 264.7
murine cells.
Janeiro, BRAZIL
Received: September 10, 2015
Accepted: May 11, 2016
Published: May 26, 2016
Copyright: © 2016 Cordeiro et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by Fundação
Carlos Chagas Filho de Amparo a Pesquisa do
Estado do Rio de Janeiro (FAPERJ): E-26/210.141/
2
0
014 (PDF); E-26/010.002566/2014 (PDF); E-26/
10.000908/2015 (PDF) Conselho Nacional de
Pesquisa (CNPq): 473106/2012-4 (PDF); 401683/
013-3 (PDF).
2
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
1 / 17

N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Introduction
There are several pathways and molecular mediators that modulate survival and death of leu-
kocytes at inflammation sites including the family of intracellular molecules of signaling path-
ways PI3K (phosphatidylinositol-3 kinase), cascade of MAPK (mitogen-activated protein
kinase) and NF-κB (nuclear factor kappa B) [1, 2], pro-inflammatory cytokines [3] and reactive
oxygen species (ROS) [4].
The transcription factor NF-κB has described action on several cells that make up the com-
plex organisms, presenting a range of action exceeding all transcription factors previously char-
acterized [5]. NF-κB has served as a model system for inducible transcription and due to their
broad physiological and clinical effects, attracts great research interest. However, the role of
NF-κB even raises questions about how a limited set of signaling mediator is able to integrate
different stimuli to reach a specific cell type and response [6]. Various types of stimuli can acti-
vate the transcription factor NF-κB, such as viral, bacterial, inflammatory cytokines and anti-
gen-receptor engagement, as well as mechanical and chemical stimuli, including the oxidative
stress [7].
Under resting conditions, NF-κB dimers are linked to IκBs (κB inhibitory protein) seques-
tering the complexes of inactive NF-κB in the cytoplasm. An induced stimulus triggers the
degradation of IκB proteins through phosphorylation by IκB kinase (IKK) complex, which
consists of two catalytically active kinases, IKKα and IKKβ, and a regulatory subunit IKKγ
(
NEMO). Phosphorylated IκB is targeted for ubiquitination and proteosomal degradation,
which leads to the release of NF-κB dimers that can translocate to the nucleus. Since activation
depends on IκB degradation, the IKK complex is like the guardian of the NF-κB signaling and
is critical for interaction with parallel signaling pathways [8].
There are some pathological conditions that characterize the involvement of NF-κB expres-
sion, i.e., inflammatory bowel disease (Chron’s disease and colitis), endometriosis and arthritis
rheumatoid. These conditions are of difficult treatment and with several adverse effects. In this
regard, the search of new drugs, more potent and with fewer toxic effects continues to be a
goal.
Previous work from our research group reported the discovery of N-acylhydrazone deriva-
tive LASSBio-1524 (substance 1, Fig 1) through structure-based drug design studies, as an
IKKβ inhibitor with potent anti-inflammatory properties [9]. However, LASSBio-1524 needed
to be administered intraperitoneally, since its poor aqueous solubility limited its administration
by oral route and its development as drug candidate.
Fig 1. LASSBio-1524 and the design concept of the novel N-acylhydrazone derivatives (2a-c).
doi:10.1371/journal.pone.0156271.g001
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
2 / 17

N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
In order to optimize the pharmacological activity of LASSBio-1524, we proposed three new
N-acylhydrazone derivatives based on their structure. The N-acylhydrazone (NAH) framework
was maintained in all compounds since it is recognized as a privileged structure [10] and
changes were made in the aryl groups attached to acyl and imine subunits at NAH moiety. In
LASSBio-1764 (substance 2a, Fig 1) and LASSBio-1763 (substance 2b, Fig 2) naphthyl ring
of LASSBio-1524 was partially reduced in order to diminish the extension of planarity of the
aromatic system and therefore try to improve the aqueous solubility profile, as previously
described in literature [11]. LASSBio-1760 (2c) and LASSBio-1763 (2b) were proposed by isos-
teric exchange of the nitro group of LASSBio-1524 by a boronic acid, since this latter is able to
perform more interactions with water, as H-bond acceptor and donor, and thus increase the
aqueous solubility.
Results and Discussion
Chemistry
The synthesis of the new N-acylhydrazone derivatives (2a-c) was carried out as outlined in Fig
2. LASSBio-1763 (2a) and LASSBio-1764 (2c) were obtained by linear three steps synthesis.
Commercially available 5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (3) was submitted to
reacted by Fischer esterification condition by treatment with methanol and catalytic sulfuric
acid at reflux to furnish the corresponding methyl ester (4a). Hydrazides (5a or 5b) were
obtained by reaction from methyl ether (4a or 4b) and hydrazine hydrate 80% in ethanol at
reflux [12]. Finally, hydrazide (5a) is condensed with the appropriate aromatic aldehydes
under acid catalysis at room temperature to generate LASSBio-1763 (2a) and LASSBio-1764
(
2c) in high yields [12]. For the synthesis of LASSBio-1760 (2c), the starting material was the
commercially available 2-naphthoyl chloride (6), which was treated with methanol at room
temperature to give the corresponding methyl ester (4b). Acid-catalyzed condensation of
the 2-naphthohydrazide (5b) and 4-formylphenylboronic acid resulted in LASSBio-1760 (2c)
[12]. Yields, purity and physical properties of N-acylhydrazone derivatives 2a-c are shown in
Table 1.
1
The synthesis of LASSBio-1760 (2c) was confirmed by analysis of H-NMR spectra, where
was observed the presence of characteristic signals of the NAH function, the singlet at 12.08 ppm,
referring to the amide hydrogen and the singlet at 8.55 ppm (Fig 3), which corresponds to the
imine hydrogen. The absence of duplicity on the signals related to imine and amide hydrogens of
NAH subunit is a strong indicative of the presence of only one diastereomer, probably presenting
Fig 2. Synthesis of N-acylhydrazone derivatives 2a-c. Reagents and conditions: a) MeOH, H
0°C, 6 h, 90%; b) NH NH O 80%, EtOH, 80°C, 4 h, 84–88%; c) ArCHO, EtOH, HCl 10%, 3 h, 75–94%; d)
MeOH, r.t., 2 h, 86%.
2
SO4 cat.
7
2
2 2
H
doi:10.1371/journal.pone.0156271.g002
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Table 1. Yields and physical properties of N-acylhydrazone derivatives 2a-c.
a
b
Compound
Molecular Formula
Purity (%)
Yield (%)
m.p. (°C)
LASSBio-1764 (2a)
LASSBio-1763 (2b)
LASSBio-1760 (2c)
C
18
H
17
N
3
O
3
99.252
95.917
98.187
94
91
75
226–228
222–224
240–242
C
18
H19BN
2
O
O
3
3
C
18
H15BN
2
a
Obtained by high performance liquid chromatography (HLPC)
b
Yields refer to the condensation step of hydrazides 4a–b with the corresponding aromatic aldehydes.
doi:10.1371/journal.pone.0156271.t001
the relative (E)-configuration. In addition, these results are consistent with other published works
of our research group describing the relative configuration of N-acylhydrazones [13, 14]. How-
ever, in order to obtain a further indication that the diastereomer obtained exhibits the relative
(E)-configuration, we carried out the NOESY (spectroscopy nuclear Overhauser effect) experi-
ment on derivative LASSBio-1760. Thus, the amide hydrogen was irradiated and the influence
was observed on the hydrogens spatially closer to it, such as the imino hydrogen of the (E)-
diastereomer. In fact, the NOESY experiment confirmed that the amide hydrogen is spatially
near to the imine hydrogen, since we were able to detect nuclear Overhauser effect (NOE) (Fig 3)
between them. This finding is an additional evidence that only the (E)-diastereomer was formed,
once that the (Z)-diastereomer does not have the same spatial proximity between the amide
and imine hydrogens. LASSBio-1763 and LASSBio-1764 also had their structure confirmed by
1
H-NMR analysis, which also indicated the presence of the characteristic signals of the hydrogens
at NAH moiety in approximately 12 and 8.5 ppm. Both signals showed no duplicity, probably
indicating the presence only of the (E)-diastereomer.
Unfortunately, despite the design concept of the new N-acylhydrazone derivatives 2a-c are
focused in the optimization of some drug-like properties, the evaluation of its aqueous solubil-
ity profile indicated that this parameter did not change significantly. LASSBio-1763 (2b) and
LASSBio-1764 (2a) showed minimal improvement in the solubility profile (Table 2), while
LASSBio-1760 (2c) showed a worsening in the aqueous solubility when compared to LASSBio-
Fig 3. NOESY experiment of N-acylhydrazone derivative LASSBio-1760 (2c).
doi:10.1371/journal.pone.0156271.g003
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Table 2. Aqueous solubility and other drug-like properties of N-acylhydrazone derivatives (2a-c).
a
b
2 c
Compound
Aqueous Solubility(mg/mL)
cLog P
PSA (Å )
−
−
−
−
3
3
3
4
LASSBio-1524 (1)
LASSBio-1764 (2a)
LASSBio-1763 (2b)
LASSBio-1760 (2c)
1.2 x 10
1.0 x 10
1.5 x 10
4.4 x 10
3.56
4.04
3.27
2.98
73.090
73.119
71.768
71.340
a
Solubility was determined by ultraviolet spectroscopy as described by Schneider and co-workers [15].
cLog P was calculated using program ACD Percepta.
b
c
PSA (polar surface area) was calculated using program Spartan, Wavefunction, Inc.
doi:10.1371/journal.pone.0156271.t002
1
524 (1). Thus, one way to minimize the problem of low solubility of compounds was used
polysorbate 80 as vehicle. This allowed to administer all N-acylhydrazone derivatives orally,
including LASSBio-1524.
Pharmacology
Anti-inflammatory effect in the subcutaneous air pouch model (SAP). Acute inflamma-
tion induced by carrageenan is characterized by accentuated cell migration, predominance of
polymorphonuclear cells, increased vascular permeability, extravasation of plasma proteins
and increased levels of inflammatory mediators, such as nitric oxide and cytokines [15–18].
The first model used to investigate the anti-inflammatory potential of new NAH derivatives
was the subcutaneous air pouch model (SAP). This model can be used for the wide quantitative
analysis of the mediators involved in the inflammatory process, which makes it an excellent
tool to study and screening new anti-inflammatory agents [19]. Additionally, it is worth noting
that Ellis and colleagues [20] reported that this model involves the activation of NF-κB, thus
highlighting the role of this transcription factor in induced inflammation.
The IKK inhibitor SC-514 (S1 Fig) and dexamethasone were chosen as standards for this
model. Our results indicate that all new compounds (2a-c) and LASSBio-1524 (1) reduced sig-
nificantly influx of leukocytes that migrate to the cavity at all doses tested, showing a dose-
response (Fig 4).
LASSBio-1764 (2a) and LASSBio-1763 (2b) were the most promising compounds in this
model, since they were able to decrease dramatically leukocytes into the air pouch at 30 mg/kg,
9
8% and 96%, respectively. This is an indicative that the strategy of reducing partially the
naphthyl subunit of LASSBio-1524 (1) was great to increase the anti-inflammatory activity.
At the highest concentration tested, all new compounds (2a-c) were better than the proto-
type LASSBio-1524 (1), being more effective in reducing cell migration. Complementarily, at
the concentration 10 mg/kg, all new compounds (2a-c) and LASSBio-1524 (1) exhibited an
activity similar to that of IKKβ inhibitor at the same concentration. This may be an indication
that the anti-inflammatory action of the new compounds is mediated by blocking IKKβ-NF-
κB pathway.
Interestingly, the reduction of the aromatic ring of naphthyl proved to be better for anti-
inflammatory activity, as LASSBio-1763 (2b) and LASSBio-1764 (2a) were better than the cor-
responding analogues with the naphthyl group, LASSBio-1524 (1) and LASSBio-1760 (2c).
Additionally, the nitro group demonstrated contributes more to the anti-inflammatory action
than the boronic acid group. However, since the compounds exhibit ED50 nearby for this activ-
ity, it can be said that 4-nitrophenyl subunit has bioisosteric relationship with the 4-phenyl
boronic acid subunit.
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Fig 4. Effect of N-acylhydrazone derivatives in leukocyte migration induced by carrageenan in
subcutaneous air pouch (SAP). The animals were pretreated orally 60 minutes before carrageenan
injection in the SAP, with vehicle (Polysorbate 80) or compounds at doses of 0.3, 3, 10 or 30 mg/kg, IKK-β
inhibitor SC-514 (10 mg/kg) or intraperitoneally pretreated with dexamethasone (2.5 mg/kg). Results were
3
expressed as mean ± standard deviation of the number of total leukocytes (x 10 /μL). Statistical significance
#
(
p <0.05) was calculated by analysis of variance (ANOVA) followed by Bonferroni post-test. p<0.05 when
compared to the vehicle-treated animals in the group of PBS in SAP; *p<0.05 compared to the vehicle-
treated animals in the group of carrageenan in the SAP.
doi:10.1371/journal.pone.0156271.g004
LASSBio-1764 (2a) was the compound that showed the best activity and, consequently
showed the best ED50 among the tested compounds, i.e. 1.5 mg/kg (Table 3). Furthermore, it is
important to note that again LASSBio-1764 was better than LASSBio-1524 prototype (1), who
presented ED50 2.7 mg/kg. LASSBio-1763 (2b) also showed similar ED50 value to LASSBio-
1
764, i.e. 2.1 mg/kg.
The active form of NF-κB is already found in the SAP model from the first day of the exper-
iment [20]. Thus, the action of NAH compounds by blocking mediators that control NF-κB
activation pathway or the nuclear factor itself would explain the significant anti-inflammatory
activity and evidenced a reduction of cytokines precisely whose expression is regulated by this
transcription factor.
Decrease of Nitric oxide (NO) production in vivo. In order to access inflammatory
mediators that are produced during the inflammatory process and a possible effect of the
substances, the amount of nitric oxide (NO) accumulated in the SAP exudate was quantified.
As shown in Fig 5, animals pretreated orally with vehicle and injected with PBS in SAP, had a
concentration of 13.4 ± 8.6 μM of NO, while in animals pretreated orally with vehicle and
that received carrageenan injection in SAP the NO production increased almost 19-fold
Table 3. ED50 values of the N-acylhydrazone derivatives LASSBio-1760 (2c), LASSBio-1763 (2b) and
LASSBio-1764 (2a) and the LASSBio-1524 (1) in the subcutaneous air pouch (SAP) model in mice,
after oral administration.
N-acylhydrazone derivative
ED50
LASSBio-1524 (1)
LASSBio-1764 (2A)
LASSBio-1763 (2b)
LASSBio-1760 (2C)
2.7 mg/kg
1.5 mg/kg
2.1 mg/kg
4.8 mg/kg
doi:10.1371/journal.pone.0156271.t003
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Fig 5. Effect of N-acylhydrazone derivatives in nitric oxide production induced by carrageenan at
SAP. The animals were pretreated orally 60 minutes before carrageenan injection in the SAP, with vehicle
(
Polysorbate 80) or N-acylhydrazone derivatives at doses of 0.3, 3, 10 or 30 mg/kg, IKK-β inhibitor SC-514
(
10 mg /kg) or intraperitoneally pretreated with dexamethasone (2.5 mg /kg). Results were expressed as
mean ± standard deviation of the number of total leukocytes (x 10³/μL). Statistical significance (p <0.05) was
#
calculated by analysis of variance (ANOVA) followed by Bonferroni post-test. p<0.05 when compared to the
vehicle-treated animals in the group of PBS in SAP; *p<0.05 compared to the vehicle-treated animals in the
group of carrageenan in the SAP.
doi:10.1371/journal.pone.0156271.g005
(
243.3 ± 56.5 μM). Pretreatment with SC-514 or dexamethasone reduced in 70% and 58%
NO levels (74.8 ± 44.8 μM and 102.4 ± 6.3 μM, respectively). Our results also indicate that
pretreatment with all N-acylhydrazone derivatives reduced by more than 65% NO produc-
tion. At the lowest dose, 0.3 mg/kg, the reduction was almost equal to the SC-514 treated
group (Fig 5). This result is another evidence that the compounds are perhaps acting by
blocking the IKKβ-NF-κB pathway.
In addition, LASSBio-1760 (2c) almost completely blocked NO production in doses of 10
and 30 mg/kg, 94% and 93%, respectively. This suggests that these substances could be acting
through direct inhibition of nitric oxide synthase (iNOS), reducing its enzymatic activity or
perhaps inhibiting the biosynthesis/iNOS expression in cells [21–23].
We also confirmed that compounds affects chemotaxis through the inhibition of NO pro-
duction and pro-inflammatory cytokines levels resulting in the suppression of inflammatory
process. It is interesting to note that these inhibitory properties were similar with the anti-
inflammatory effects produced by dexamethasone [19]. This steroidal anti-inflammatory drug
is effective in inhibiting cell migration, NO production and cytokines levels, as well as leuko-
cytes influx.
Blockade of NO has the potential to deliver therapeutic benefit in inflammation, once its
overproduction is present in a number of diseases such as arthritis, inflammatory bowel dis-
ease, Alzheimer's, Parkinson's and others [24, 25]. It has been observed that blockade of NO in
acute models of inflammation contributes to the improved reduction of the inflammatory phe-
nomena [26, 27].
Extensive inhibition of TNF-α production. TNF-α level was quantified to evaluate if the
reduction in cell migration would reflect only the number of cells or if there was interference of
the transcription factor after treatment with the compounds. An increase in the production of
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Fig 6. Effect of N-acylhydrazone derivatives in TNF-α production induced by carrageenan at SAP. The
animals were pretreated orally 60 minutes before carrageenan injection in the SAP, with vehicle (Polysorbate
80) or N-acylhydrazone derivatives at doses of 0.3, 3, 10 or 30 mg/kg, IKK-β inhibitor SC-514 (10 mg /kg) or
intraperitoneally pretreated with dexamethasone (2.5 mg /kg). Results were expressed as mean ± standard
3
deviation of the number of total leukocytes (x 10 /μL). Statistical significance (p <0.05) was calculated by
#
analysis of variance (ANOVA) followed by Bonferroni post-test. p<0.05 when compared to the vehicle-
treated animals in the group of PBS in SAP; *p<0.05 compared to the vehicle-treated animals in the group of
carrageenan in the SAP.
doi:10.1371/journal.pone.0156271.g006
this cytokine was evident in animals that received oral pretreatment with vehicle and SAP
injection of carrageenan. The N-acylhydrazone derivatives 2a-c were able to significantly
reduce levels of TNF-α. In all doses, new N-acylhydrazone derivatives (2a-c) almost abolished
TNF-α levels, while LASSBio-1524 (1) reduced with less effectively the cytokine levels (Fig 6).
Again, LASSBio-1763 (2b) and LASSBio-1764 (2a) were the most promising compounds and
show a bioisosteric relationship for this pharmacological action.
TNF-α levels were reduced in, at least, 95%, at dose of 30 mg/kg, to all N-acylhydrazone
derivatives. These data, together with those obtained by the inhibition of NO indicates that
TNF-α and NO pathways are interconnected. Pro-inflammatory cytokines as TNF-α, trigger a
series of signaling pathways, including the pathway of NF-κB and its level is known to be a
direct correlation to the iNOS enzyme [28–30].
Decrease in production of reactive oxygen species (ROS). Fig 7 shows that only LASS-
Bio-1763 (2b) and LASSBio-1764 (2a) significantly reduced the production of reactive oxygen
species (ROS) by PMA-stimulated leukocytes. And the reduction caused by LASSBio-1764 (2a)
almost abolished levels of ROS. Unfortunately, LASSBio-1760 (2c) and LASSBio-1524 (1) were
not significantly active in this test, as the IKK inhibitor. However, it is possible to look again
the bioisosteric relationship between boronic acid group and nitro group viewed by similar
activity of LASSBio-1763 and LASSBio-1764 in reducing ROS biosynthesis.
Since oxidative stress may be harmful in the resolution of inflammation and the overpro-
duction of reactive oxygen species (ROS) is the mechanism behind many neuropathologies,
any impaired induction of antioxidant enzymes or antioxidants that are induced by NF-κB
under normal conditions might be responsible for ROS accumulation. Studies demonstrated
that antioxidant enzyme genes including manganese-dependent SOD (MnSOD), glutathione
S-transferase, and ferritin heavy chain (FHC), are induced by TNF-α activation of NF-κB [31].
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Fig 7. Oxidative metabolism of leukocytes collected from the SAP 24 hours after carrageenan
injection, stimulated with PMA. Intracellular ROS levels were quantified and reading was performed by flow
cytometry counting 10,000 events. DCF expression is presented in values of the geometric mean
fluorescence intensity in FL1.
doi:10.1371/journal.pone.0156271.g007
Our results indicate that inhibition in the production of ROS also appears to be part of the
mechanism of action of tested N-acylhydrazone derivatives (1, 2a-c).
Since LASSBio-1524 is an IKKβ inhibitor, in order to check the inhibitory profile of its new
analogs in the target enzyme, we performed an in vitro test in the CEREP Company. However,
the new analogues, LASSBio-1760, LASSBio-1763 and LASSBio-1764 did not inhibit the
enzyme activity in that protocol [32] until the concentration of 30 μM (Data not shown).
LASSBio-1524 and LASSBio-1760 attenuates the LPS-induced NF-kB expression in
RAW 264.7. Since the analogues were not active in CEREP’s protocol, in order to determine
a possible target for the anti-inflammatory action observed, we evaluated NF-κB p65 expres-
sion by western blot. NF-kB expression in RAW 264.7 murine cells was decreased with concen-
trations of LASSBio-1524 and LASSBio-1760 ranging from 1 μM to 30 μM (Fig 8). The
compounds were also tested in the cells without LPS stimulation and demonstrated no activity
per se (data not shown). However, did not significantly affect activation of NF-kb as assessed
by kinase activity or western blot analysis, suggesting that they are not specific inhibitors of
pospho p65 although in vivo LASSBio-1764 had a significant anti-inflammatory effect, the
phosphorylation occurred normally and its potencial activity may be due to its effect on other
mediators, as ROS, NO and TNF-α.
Myelotoxicity and Cytotoxicity Evaluation. Finally, to make sure that the inhibitory
effect on cell migration was due to a direct effect on cellularity and not a nonspecific toxic
effect, the leukocyte counting was performed in the blood and bone marrow of animals that
were pre-treated with the higher dose (30 mg/kg) of each N-acylhydrazone derivatives. The
results did not show any significant differences regarding the number of total leukocytes from
the blood and bone marrow between the pretreated groups with the N-acylhydrazone deriva-
tives (1, 2a-c) or control groups (S2 Fig). Cell viability was measured by the MTT assay [33]
and the compounds did not reduce the cell viability of RAW 264.7 mouse macrophages at con-
centrations of up to 30 μM (S3 Fig). These data are a strong indication of the low toxicity of the
compounds, showing their potential as drug candidates.
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Fig 8. Cell lysates were prepared and Western blots were probed for phospho-NFkB p65 antibody. Western
blot and densitometric analysis of NF-κB activation in LPS stimulated RAW 264.7 cells. Cells were stimulated with
LPS (1 μg/mL) for 2 hours. β-actin protein levels were used as loading control. Representative Western blot of β-actin
and phospho-NF-κB p65 (Ser536) expression. Equal amounts of nuclear proteins were loaded onto each lane.
Graphical densitometry representation of ratio between β-actin and phospho-NF-κB p65 levels after incubation with
1, 10 or 30 μM for 2 hours. Negative control without LPS treatment. Each value is expressed as mean± SD of
triplicate experiments. Statistical significance (*p <0.05) was calculated by analysis of variance (ANOVA) followed
by Bonferroni post-test. Each Fig is representative of 3 independent experiments.
doi:10.1371/journal.pone.0156271.g008
Conclusions
We described the design and synthesis of novel N-acylhydrazone derivatives structurally ana-
logues to LASSBio-1524 (1). Our results indicate that all compounds (2a-c) demonstrated a
potential and significant anti-inflammatory activity, reducing leukocytes migration, TNF-α
levels, NO production, inhibition of NF-kB enzyme expression and provided strong inhibition
of free radical activities in in vivo assays. And it is worth pointing out that these N-acylhydra-
zone compounds were administered orally and show no myelotoxicity toxicity. In addition, a
bioisosteric relationship between boronic acid and nitro subunits is clearly. The partial reduc-
tion of the naphthyl group of LASSBio-1524 (1) proved to be an extremely beneficial modifica-
tion to the anti-inflammatory activity. LASSBio-1764 (2a) demonstrated to be the most
promising compounds from this series, surpassing even LASSBio-1524 (1).
It seems that LASSBio-1760 inhibits NF-κB enzyme expression thus reducing production of
final mediators dependent of this pathway. The mechanism of action remains to be elucidated,
but a possible target may be the NF-κB since this is present in second messenger signaling
pathways of NO, ROS and TNF-α, whose levels have been reduced. These findings provide evi-
dence that supports these compounds as candidates to the treatment of several inflammatory
diseases.
Materials and Methods
Chemistry
Reagents and solvents were purchased from commercial suppliers and used as received. The
1
13
H and C NMR spectra were recorded on a Bruker AC-200 NMR, operating at 200 MHz for
1
13
H and 50 MHz for C NMR. Thin layer chromatography (TLC) was performed on Merck
KieselGel 60 F254 (0.2 mm thickness) using as the mobile phase n-hexane and ethyl acetate or
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
10 / 17

N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
dichloromethane and methanol at various concentrations. The TLC plates with the reactants
and products were visualized with ultraviolet light at wavelengths of 254 and 365 nm.
Infrared spectra (IR) were carried out in the spectrophotometer apparatus Fourier trans-
form IR Nicolet 6700 FT-IR using tablets of potassium bromide (KBr). Melting points were
determined in Quimis Q340.23 apparatus and are uncorrected.
The purity of the final product was determined by High Performance Liquid Chromatogra-
phy (HPLC) on Shimadzu LC-20AD with Kromasil 100–5 C18 column (4.6 mm x 250 mm)
and Detector SPD-M20A (Diode Array). The quantification of analyte was performed using a
standardized wave length, 308 nm. The mobile phase used was acetonitrile and water 60–80%,
running time (R ) varying between 10 and 20 minutes.
t
The determination of the solubility in water was performed using UV-VIS spectrophotome-
ter with Femto Scan Model 800XI.
Methyl 5,6,7,8-tetrahydronaphthalene-2-carboxylate (4b). A solution of 0.5 g (2.83
mmol) of 5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (3) in 30 mL of methanol was
refluxed at 70°C for 6 hours. Afterwards, the solvent was partially concentrated at reduced
pressure and the resulting mixture was extracted with dichloromethane (80 mL x 2) and brine.
The organic layer was dried with anhydrous sodium sulfate and the solvent was evaporated to
1
give 0,460 g of colorless oil. H-NMR (200 MHz, DMSO-d ): δ = 7.63 (2H, m); 7.16 (1H, d,
6
1
3
J = 6 Hz); 3.84 (3H, s); 2.76 (4H, ls); 1.73 (4H, ls) [S4 Fig]. C- NMR (50 MHz, DMSO-d ): δ
6
-
1
:
66.3; 142.6; 137.1; 129.7; 129.2; 126.7; 126.0; 51.8; 28.8; 28.5; 22.3; 22.2 (S5 Fig). IR (KBr) cm
1741 (ʋ C = O).
1
Methyl 2-naphthoate (4a). A solution of 0.2 g (1.05 mmol) of 2-naphthoyl chloride (6), in
2
0 mL of methanol was stirred at room temperature for 2 hours. Afterwards, the solvent was
partially concentrated at reduced pressure and the resulting mixture was poured into ice and
cold water. The precipitate formed was filtered and dried under vacuum, producing the solid
1
white powder in 86% of yield. H (400 MHz, CDCl ): δ 3.99 (3H, s); 7.53–7.61 (2H, m); 7.88
(
3
13
2H, m); 7.95 (1H, d, J = 8 Hz); 8.05 (1H, d, J = 8 Hz); 8.62 (1H, d). C (50 MHz, DMSO-d ): δ
6
5
3.08; 125.44; 127.53; 127.87; 128.41; 129.16; 129.48; 129.95; 131.21; 132.69; 135.75; 167.55. IR
-
1
(
KBr): cm 1713 (ʋ C = O).
General procedure for the preparation of 2-naphthohydrazide (5a) and 5,6,7,8-tetrahy-
dronaphthalene-2-carbohydrazide (5b). To a solution of 2.47 mmol (1 eq.) of methyl
-naphthoate (4b) or methyl 5,6,7,8-tetrahydronaphthalene-2-carboxylate (4a) in absolute
2
EtOH (20 mL) was added 7.73 mL (0.15 mmol, 40 eq.) hydrazine hydrate 80%. The mixture
was refluxed at 80°C for 4 hours. Afterwards, solvent was partially concentrated at reduced
pressure and the resulting mixture was poured into ice. The precipitate formed was filtered out
and dried under vacuum, producing the desired product.
1
5,6,7,8-tetrahydronaphthalene-2-carbohydrazide (5a). White powder, 84%, H-NMR
(
(
1
200 MHz, DMSO-d ): δ = 1.77 (4H, ls); 2.76 (4H, ls,); 3.74 (2H, s); 7.06 (1H, d, J = 6 Hz); 7.42
6
13
2H, m); 7.99 (1H, s) [S6 Fig]. C-NMR (50 MHz, DMSO-d ): δ 22.5; 28.7; 123.9; 127.6; 128.8;
6
-
1
30.4; 136.5; 140.0; 166.1 (S7 Fig). IR (KBr) cm : 1662 (ʋ C = O); 3200 e 3300 (ʋ N-H).
1
2
-naphthohydrazide (5b). White powder, 88%, H-NMR (200 MHz, DMSO-d ): δ 4.59
6
13
(
2H, s); 7.56–7.60 (2H, m); 7.94–8.00 (4H, m); 8.44 (1H, s); 9.94 (1H, s). C-NMR (50 MHz,
DMSO-d ): δ 123.8; 126.6; 127.2; 127.4; 127.5; 127.8; 128.7; 130.6; 132.1; 134.0; 165.9. DEPT-
6
-
1
1
35 (50 MHz, DMSO-d6): 124.3; 127.1; 127.7; 127.9; 128.0; 128.3; 129.2. IR (KBr) cm : 1619
(ʋ C = O); 3182 e 3314 (ʋ N-H).
General procedure for the preparation of N-acylhydrazone derivatives (2a-c). To a solu-
tion of 1.61 mmol (1 eq.) of 2-naphthohydrazide (8) or 5,6,7,8-tetrahydronaphthalene-2-car-
bohydrazide (5) in absolute EtOH (20 mL) containing two drops of solution hydrochloric acid
1
0%, was added 1.7 mmol (1 eq.) of the corresponding aromatic aldehyde. The mixture was
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
11 / 17

N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
stirred at room temperature for 3 hours. Afterwards, the solvent was partially concentrated at
reduced pressure and the resulting mixture was poured into ice. The precipitate formed was fil-
tered out and dried under vacuum, producing the desired N-acylhydrazone in high yield.
(
E)-N-(4-nitrobenzylidene)-5,6,7,8-tetrahydronaphthalene-2-carbohydrazide (2a, LASS-
1
Bio-1764). Beige powder purified by recrystallized from ethanol, 94%, H-NMR (200 MHz,
DMSO-d ): δ 1.76 (4H, sl); 2.78 (4H, sl); 7.21 (1H, d, J = 8 Hz); 7.64 (2H, sl); 8.00 (2H, m); 8.33
6
13
(
2H, d, J = 10 Hz); 8.53 (1H, s); 12.04 (1H, s) (S8 Fig). C-NMR (50 MHz, DMSO-d ) δ 22.5;
6
2
8.8; 124.1; 124.8; 128.0; 128.4; 129.2; 130.1; 137.0; 140.8; 141.4; 145.0; 147.8; 163.5 (S9 Fig). IR
-
1
(
KBr) cm : 1314 e 1519 (ʋ N-O); 1652 (ʋ C = O), 3231 (ʋ N-H). % of purity >99% by HPLC
E)-N-(4-boronic acid benzylidene)-5,6,7,8-tetrahydronaphthalene-2-carbohydrazide
(
(
2b, LASSBio-1763). White yellow powder purified by recrystallization from ethanol, 91%;
1
H-NMR (200 MHz, DMSO-d ): δ 1.74 (4H, sl); 2.76 (4H, sl); 7.20 (1H, d, J = 10 Hz); 7.69 (4H,
6
13
m); 7.87 (2H, m); 8.44 (1H, s); 11.79 (1H, s) [S10 Fig]. C-NMR (50 MHz, DMSO-d ): δ 22.5;
6
2
8.8; 124.7; 126.0; 128.2; 129.0; 130.4; 134.5; 135.8; 136.8; 141.0; 147.5; 163.2 (S11 Fig). IR
-
1
(
KBr) cm : 1641 (ʋ C = O); 3231 (ʋ N-H); 3432 (ʋ O-H). % of purity >95% by HPLC
E)-N-(4-boronic acid benzylidene)-naphthalene-2-carbohydrazide (2c, LASSBio-
(
1
1
760). White powder purified by recrystallized from ethanol, 75%; H (200 MHz, DMSO-d ):
6
δ 7.61–7.64 (2H, m); 7.73 (2H, d, J = 8 Hz); 7.90 (2H, d J = 8 Hz); 7.98–8.03 (4H, m); 8.20 (2H,
13
s); 8.51 (1H, s); 8.56 (1H, s); 12.08 (1H, s) (S12 Fig). C-NMR (50 MHz, DMSO-d ): δ 124.3;
6
1
26.1; 126.9; 127.7; 127.9; 128.0; 128.1; 128.9; 130.7; 132.1; 134.3; 134.5; 135.7; 147.9; 163.3
-
1
[S13 Fig]. IR (KBr) cm : 3232–3311 (ʋ O-H), 1615 (ʋ C = O). % of purity >98% by HPLC
Determination of aqueous solubility
The solubility test was made on the basis of ultraviolet (UV) absorbance obtained. The sweep
wavelength was determined by greater absorbance characteristic of each compound. One satu-
rated aqueous solution was stirred for 2 hours at 37°C, then filtered and the supernatant was
transferred to a quartz cuvette to perform the reading. The solubility was determined by linear
regression equation of the line. To obtain the line of points (known concentrations), it was nec-
essary to use the absorbance obtained with progressive concentrations obtained by the dilution
of a stock solution (1 mg/50 mL) in a solvent in which the analyzed compound present better
profile solubility. Obviously, the choice of solvent can’t be absorbed by UV. For all compounds
was used methanol. Then, it was made the chart with the dilutions and the absorbance, and
was extracted from the line graph of the equation. Then, the straight line equation was solved
where "Y" is the absorbance of the saturated solution in water, and the value "X" to be found
will be the solubility of the compound [15].
Pharmacology
Animals. The experimental groups were composed of 5–8 Swiss Webster mice (20–25 g)
donated by the Animal Production Centre of the Instituto Vital Brazil (Niteroi, Rio de Janeiro).
Animals (in a total of 208 mice) were maintained with a 12-h light/dark cycle and controlled
temperature, ad libitum access to water and food. To avoid interference of food on absorption
of substances administered to animals, they were fasted for 3 hours before the experiments.
Animals were acclimatized to the laboratory for at least 1 hour before testing and were used
only once throughout the experiments. After assays animals were euthanized with an overdose
of choral hydrate. The experimental protocols used in this work followed the rules advocated
by Law 11,794, of October 8, 2008 by the National Council of Animal Experimentation Control
(
CONCEA) and were approved by the Ethics Committee of Animal Use (CEUA), Science Cen-
ter Health/UFRJ and received the number DFBCICB015-04/16.
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
Preparation and administration of compounds. All compounds tested were prepared in
a stock solution at 100 μmol/mL of dimethyl sulfoxide (DMSO) and stored at -20°C until
assays. The compounds were given orally at doses of 0.3, 3, 10 and 30 mg/kg, in a final volume
of 100 μL of vehicle (Polysorbate 80). The selective and reversible inhibitor of IKKβ inhibitor,
SC-514 [34] was given orally at a single dose of 10 mg/kg. The standard anti-inflammatory
drug used was dexamethasone (2.5 mg/kg, i.p.).
Subcutaneous Air Pouch (SAP) model. The procedure used was similar to the first
described method [35] with some modifications [36]. The SAP was formed on the back of the
animals by injecting 10 mL of sterile air. After 3 days the cavity was injected over 7 mL of sterile
air. At day 6, animals were orally treated with compounds and 60 minutes later, a sterile 1%
carrageenan injection was performed into the formed cavity. A negative control group was
treated with vehicle (Polysorbate 80) 60 minutes before receiving the injection of sterile carra-
geenan solution at SAP and a positive control groups received dexamethasone (2.5 mg/kg, i.p.)
or SC-514 (10 mg/kg, p.o.). After 24 hours of carrageenan injection, animals were euthanized
by an overdose of ketamine/xylazine, SAP was washed with 1 ml sterile phosphate buffer saline
(
PBS) and exudate was collected. Total leukocyte counts were determined in an automatic cell
counter (CellPoch-100iV Diff, Sysmex). The exudates were centrifuged at 1,000 rpm, 10 min-
utes, at 4°C and aliquots of supernatant was stored at -20°C for subsequent measurements.
For the white blood cells counting, the mice were anesthetized with anesthetic ketamine/
xylazine and 100 μL of blood were collected and placed into tubes with 15 μL of ethylenedia-
mine tetraacetic acid (EDTA). For leukocyte count in the bone marrow, the femur was
removed; its ends were cut and washed with 1 mL of sterile PBS. The white blood cells determi-
nation was performed in the automatic cell counter (Poch-100iV Diff, Sysmex).
Measurement of nitric oxide (NO). The NO produced in the SAP supernatant was quan-
tified according to the technique of the conversion of nitrate to nitrite [31]. The SAP samples
were deproteinized and then admixed to a sample of sodium phosphate (0.5 M, pH 7.2),
ammonium formate (2.4 M, pH 7.2), and E. coli. After incubation for 2 hours at 37°C, centrifu-
gation was performed at 10,000 rpm for 10 minutes. Equal portions of the supernatant and
Griess reagent were incubated for 10 minutes [37] and the absorbance was measured spectro-
photometrically at 540 nm. Nitrate concentration values are expressed in μM, calculated from
a standard curve of sodium nitrate done previously.
TNF-α measurement. The quantification of TNF-α was held at the SAP exudate. Specific
ELISA kits (B&D ELISA OptEIA™) were used and TNF-α and their concentrations were deter-
mined according to the manufacturer's recommendations (B&D Biosciences).
Determination of reactive oxygen species (ROS) production. In order to study whether
N-acylhydrazone were able to contain oxidative metabolism of neutrophils and investigate fur-
ther if this is one of their mechanisms of action, leukocytes collected in SAP were placed in
6
tubes (10 cells) in a volume of 1 mL. Then incubation was performed at 37°C and 5% CO for
2
one hour. Then the compounds were added at concentrations of 10 or 30 μM and incubated
for 30 minutes at 37°C and 5% CO . Cells were treated with 10 nM phorbol myristate acetate
2
(
PMA) and incubated for 45 minutes at 37°C and 5% CO . 2'-7'diclorodihidrofluoescein diace-
2
tate (DCF-DA, 2 mM) was added to follow by further incubation for 30 minutes at 37°C. The
emitted fluorescence was captured in the FL-1 channel flow cytometer (BD Accuri™) and was
expressed as DCF-DA fluorescence [38].
Western blot analysis. Expression of the phosphorylated NF-κB p65 subunit was assessed
by Western blot analysis. The monoclonal anti phospho-NFkB p65 (Ser536) and anti-β-actin
antibodies (Cell Signaling, MA, USA) were used at 1:1,000 dilution for detecting the phosphor-
ylated form of NF-kB and β-actin by Western blot analysis. Murine RAW 264.7 macrophages
6
were seeded at 4 x 10 cells/mL in 12 well plates. Cell lysates were prepared after 2 h incubation
PLOS ONE | DOI:10.1371/journal.pone.0156271 May 26, 2016
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
with 1 μg/ml of LPS (Lipopolysaccharide) and 1, 10 or 30 μM of LASSBio-1524, LASSBio-
1
760, LASSBio-1763 and LASSBio-1764. A control-unstimulated group was incubated with the
compounds. Protein concentration was determined using a BCA Protein Assay Reagent kit
Thermo Fisher Scientific, Inc.). Proteins (30 μg) were separated by electrophoresis on 10%
(
SDS-PAGE gel and then electrophoretically transferred to a PVDF membrane (Bio-Rad). The
membrane was incubated with the antibodies during overnight, at 10°C. Immunoreactive
bands were visualized using an enhanced chemiluminiscence system (ECL) substrate in a Che-
miDoc Imaging system (Bio-Rad) [39].
Statistical analysis
In vivo experiments were composed of 6–8 animals per group, randomly selected. For in vitro
assays, each experimental group was done in triplicate. Each protocol was repeated at least 3
times. ED50 doses were calculated using GraphPad Prism 5.0 software and the following for-
mula "Dose-response curves—Inhibition" and 95% confidence intervals. Y = 100/(1+10^
Ã
(
(LogIC50-X) HillSlope))).
Results are presented as mean ± standard deviation (SD). Statistical significance was calcu-
lated by analysis of variance (ANOVA) followed by Bonferroni post-test. P values less than
Ã
0
.05 ( p <0.05) were considered significant.
Supporting Information
S1 Fig. Structure of IKK inhibitor SC-514.
(
TIF)
S2 Fig. Total leukocytes on blood and bone marrow. The animals were pretreated orally 60
minutes before carrageenan injection in the SAP, with vehicle (Polysorbate 80) or compounds
at doses of 30 mg/kg. Results expressed as mean ± standard deviation of the number of total
leukocytes (x 10³/ μL). Statistical significance (p <0.05) was calculated by analysis of variance
(
(
ANOVA) followed by Bonferroni post-test.
TIF)
S3 Fig. Effect of the compounds cytotoxicity in Raw 264.7 cells. Cells were incubated in
5
9
6-well plates (10 cells/well) were first incubated with and without indicated concentrations
of the compounds for 2 hours, and then incubated with LPS (1 μg/mL) for 24 hours. Negative
control without LPS treatment. Each value is expressed as mean± SD in triplicate experiments.
Statistical significance (p <0.05) was calculated by analysis of variance (ANOVA) followed by
Bonferroni post-test.
(
TIF)
1
S4 Fig. H-NMR spectrum of methyl 5,6,7,8-tetrahydronaphthalene-2-carboxylate (4a)
(
200 MHz, DMSO-d6).
(
TIF)
1
3
S5 Fig. C-NMR spectrum of methyl 5,6,7,8-tetrahydronaphthalene-2-carboxylate (4a) (50
MHz, DMSO-d6).
(
TIF)
1
S6 Fig. H-NMR spectrum of 5,6,7,8-tetrahydronaphthalene-2-carbohydrazide (5b) (200
MHz, CDCl3).
(
TIF)
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N-Acylhydrazone Derivatives as New Anti-Inflammatory Drugs
1
3
S7 Fig. C-NMR spectrum of 5,6,7,8-tetrahydronaphthalene-2-carbohydrazide (5b) (50
MHz, CDCl3).
(
TIF)
1
S8 Fig. H-NMR spectrum of LASSBio-1764 (2a) (200 MHz, DMSO-d ).
6
(
TIF)
1
3
S9 Fig. C-NMR spectrum of LASSBio-1764 (2a) (50 MHz, DMSO-d6).
TIF)
(
1
S10 Fig. H-NMR spectrum of LASSBio-1763 (2b) (200 MHz, DMSO-d ).
6
(
TIF)
S11 Fig. C-NMR spectrum of LASSBio-1763 (2b) (50 MHz, DMSO-d6).
TIF)
S12 Fig. H-NMR spectrum of LASSBio-1760 (2c) (200 MHz, DMSO-d ).
1
3
(
1
6
(
TIF)
S13 Fig. C-NMR spectrum of LASSBio-1760 (2c) (50 MHz, DMSO-d6).
TIF)
1
3
(
Acknowledgments
The authors thank Institute Vital Brazil for animal donation and Mr. Alan Minho for technical
support.
Author Contributions
Conceived and designed the experiments: CAMF PDF. Performed the experiments: NMC
RHCNF. Analyzed the data: NMC RHCNF PDF. Contributed reagents/materials/analysis
tools: CAMF PDF. Wrote the paper: NMC RHCNF CAMF PDF.
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