Biological evaluation and molecular docking studies of nitro benzamide derivatives with respect to in vitro anti-inflammatory activity

Article abstract of DOI:10.1016/j.intimp.2016.12.009

A series of nitro substituted benzamide derivatives were synthesized and evaluated for their potential anti-inflammatory activities in vitro. Firstly, all compounds (1–6) were screened for their inhibitory capacity on LPS induced nitric oxide (NO) production in RAW264.7 macrophages. Compounds 5 and 6 demonstrated significantly high inhibition capacities in a dose-dependent manner with IC₅₀ values of 3.7 and 5.3 μM, respectively. These two compounds were also accompanied by no cytotoxicity at the studied concentrations (max 50 μM) in macrophages. Molecular docking analysis on iNOS revealed that compounds 5 and 6 bind to the enzyme more efficiently compared to other compounds due to having optimum number of nitro groups, orientations and polarizabilities. In addition, 5 and 6 demonstrated distinct regulatory mechanisms for the expression of the iNOS enzyme at the mRNA and protein levels. Specifically, both suppressed expressions of COX-2, IL-1β and TNF-α significantly, at 10 and 20 μM. However, only compound 6 significantly and considerably decreased LPS-induced secretion of IL-1β and TNF-α. These results suggest that compound 6 may be a multi-potent promising lead compound for further optimization in structure and as well as for in vivo validation studies.

Full text of DOI:10.1016/j.intimp.2016.12.009

International Immunopharmacology 43 (2017) 129–139
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
Biological evaluation and molecular docking studies of nitro benzamide
derivatives with respect to in vitro anti-inflammatory activity
b
a
b
a
c
Tugba B. Tumer a, , Ferah Comert Onder , Hande Ipek , Tugba Gungor , Seda Savranoglu , Tugba Taskin Tok ,
⁎
Ayhan Celik d,1, Mehmet Ay b,
⁎
a
Department of Molecular Biology & Genetics, Faculty of Art and Science, Çanakkale Onsekiz Mart University, Terzioğlu, 17020 Çanakkale, Turkey
Natural Products and Drug Research Laboratory, Department of Chemistry, Faculty of Art and Science, Çanakkale Onsekiz Mart University, Terzioğlu, 17020 Çanakkale, Turkey
Department of Chemistry, Faculty of Art and Science, Gaziantep University, Şehitkamil, 27310 Gaziantep, Turkey
b
c
d
Department of Chemistry, Faculty of Science, Gebze Technical University (GTU), Gebze, 41400 Kocaeli, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 June 2016
Received in revised form 30 November 2016
Accepted 6 December 2016
Available online xxxx
A series of nitro substituted benzamide derivatives were synthesized and evaluated for their potential anti-in-
flammatory activities in vitro. Firstly, all compounds (1–6) were screened for their inhibitory capacity on LPS in-
duced nitric oxide (NO) production in RAW264.7 macrophages. Compounds 5 and 6 demonstrated significantly
high inhibition capacities in a dose-dependent manner with IC50 values of 3.7 and 5.3 μM, respectively. These two
compounds were also accompanied by no cytotoxicity at the studied concentrations (max 50 μM) in macro-
phages. Molecular docking analysis on iNOS revealed that compounds 5 and 6 bind to the enzyme more efficient-
ly compared to other compounds due to having optimum number of nitro groups, orientations and
polarizabilities. In addition, 5 and 6 demonstrated distinct regulatory mechanisms for the expression of the
iNOS enzyme at the mRNA and protein levels. Specifically, both suppressed expressions of COX-2, IL-1β and
TNF-α significantly, at 10 and 20 μM. However, only compound 6 significantly and considerably decreased
LPS-induced secretion of IL-1β and TNF-α. These results suggest that compound 6 may be a multi-potent prom-
ising lead compound for further optimization in structure and as well as for in vivo validation studies.
© 2016 Published by Elsevier B.V.
Keywords:
Nitro substituted benzamides
Anti-inflammatory
Nitric oxide
iNOS
Molecular docking
1. Introduction
degeneration [6,7]. Therefore invention of new anti-inflammatory
agents with multifactorial mode of action could be a good strategy in
Inflammation is a general physiological process of the body for the
modulation of immune response against a diverse range of triggering
factors including infectious agents, allergens, free radicals, defective di-
etary habits rich in refined foods and sedentary lifestyle. A micro envi-
ronment activated persistently and dominated with low levels of
proinflammatory factors for a long time may predispose to chronic in-
flammation [1]. It is now well established that a state of low-grade
chronic inflammation is an underlying cause of various metabolic disor-
ders like obesity, insulin resistance, metabolic syndrome, type II diabe-
tes and even cancer at the long term period [2–5]. Excess production
of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and pros-
taglandins by cyclooxygenase 2 (COX-2) as well as increased release of
proinflammatory cytokines (e.g. TNF-α, IL-1β and IL-6) under inflam-
matory conditions, all contribute to intracellular cascades of molecular
fighting chronic inflammation and associated metabolic diseases.
A considerable number of drugs and other physiologically active
agents are in nitroaromatic structure, such as nilutamide (non-steroidal
antiandrogen), nitrendipine (antihypertensive), nitrazepam (anticon-
vulsant, hypnotic) and nitromide (antibacterial) [8–11]. In addition,
some nitro benzamides, such as 5-aziridinyl-2,4-dinitrobenzamide
(CB1954), its analog (nitrogen mustards SN23862), and another com-
pound ledakrin (1-nitro-9[(dimethylamino) propylamino]acridine),
have special attention as anti-tumor agents due to their remarkable ef-
fects obtained from in vitro/in vivo studies and also from clinical trials
during treatment of some cancers [12–14]. Although medicinal chem-
ists generally remove nitro containing compounds from their screening
collections since they are classified as “structural alert”, they cannot be
totally disregarding because of their high efficacy. For example
Nimesulide, the sole nitro group containing non-steroidal anti-inflam-
matory drug (NSAIDs), has been effectively utilized for N30 years due
to its multifactorial therapeutic actions and very good gastrointestinal
tolerability. Herein inspired by nitro aromatic compounds, a series of
benzamide derivatives (1–6) were synthesized and evaluated for their
⁎
Corresponding authors.
E-mail addresses: tumerb@gmail.com, tumertb@comu.edu.tr (T.B. Tumer),
mehmetay06@comu.edu.tr (M. Ay).
1
Left from GTU in September 2016.
http://dx.doi.org/10.1016/j.intimp.2016.12.009
1567-5769/© 2016 Published by Elsevier B.V.

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T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
potential anti-inflammatory activities. Additionally, interaction mecha-
nisms among these compounds and iNOS at the molecular level were
identified by molecular docking studies.
at reflux temperature for 10 h [20]. Similarly, compounds 2 and 4 were
obtained at reflux conditions for 1 h at pyridine/benzene mixture [21].
Moreover, compounds 3 and 6 were prepared from 4-nitrobenzoyl
chloride and 3-nitroaniline (for compound 3) or 3,5-dinitroaniline (for
compound 6) at the conditions that used triethylamine as base in ace-
tone at room temperature for 30 h [22]. In addition, compound 5 was
obtained using triethylamine in DMF at 125 °C for 40 h [23].
The general amidation reaction was applied to 4-nitro-N-(2-nitro-
phenyl) benzamide and 4-nitro-N-(4-nitrophenyl)benzamide (2 and
4) with moderate yields for the first time. While compound 3 was syn-
thesized according to the known procedure, this method was applied
for the first time for synthesis of compound 6. DMF was used for the
synthesis of 4-nitro-N-(2,4-dinitrophenyl)benzamide (5) as different
from the literature method.
We started to investigate the compounds for their activities with re-
spect to inhibiting NO release in lipopolysaccharide (LPS) induced RAW
264.7 macrophages. The excess production of NO by iNOS under inflam-
matory conditions is critical for the development of intracellular degen-
erative processes. It has been known for a long time that higher
concentration of NO synthesized by iNOS reacts with oxygen and gener-
ates various reactive nitrogen oxide intermediates (RNOIs) [15]. These
reactive compounds can attack to other biomolecules, especially pro-
teins result in conformational changes and lead to either inhibition or
inactivation through different mechanisms particularly, S-nitrosylation
(reviewed in [16]). These events trigger deleterious pathological cas-
cades of inflammatory processes. Recently, Yang et al., showed that in
the setting of obesity, which is characterized by chronic metabolic in-
flammation (termed as metaflammation), increased iNOS activity
causes S-nitrosylation of some key proteins such as UPR regulator and
IREα, and thus contributes to impaired ER function and prolonged ER
stress [17]. It was also reported that reversal of iNOS mediated S-
nitrosylation can promote ER homeostasis and result in substantial met-
abolic benefits. Therefore, design of new drugs acting on excessive NO
production through iNOS is a promising therapeutic intervention
against metabolic diseases originating from persistent inflammatory
conditions. In the present study; among six synthesized nitro-substitut-
ed benzamide derivatives, two molecules were selected as our lead
compounds (5 and 6) due to having considerably low half maximal in-
hibitory concentration (IC50; 3.7 vs 5.3 μM, respectively) for NO inhibi-
tion and compatible molecular docking studies for iNOS.
2.2.1. Compound 1
White solid, 64%, m.p. 214–215 °C (lit. m.p. 213–214 °C) [24], 1H
NMR (400 MHz, DMSO) δ 7.10 (1H, t, J = 6.85 Hz), 7.34 (2H, t, J =
7.96 Hz), 7.74 (2H, d, J = 8.65 Hz), 8.14 (2H, d, J = 8.95 Hz), 8.33 (2H,
d, J = 8.93 Hz), 10.53 (1H, s); 13C NMR (100 MHz, DMSO) δ 120.99,
124.09, 124.71, 129.26, 129.74, 139.21, 141.15, 149.66, 164.42.
2.2.2. Compound 2
Yellow solid, 63.5%, m.p. 222 °C (lit. m.p. 223 °C) [25], 1H NMR
(500 MHz, DMSO) δ 7.48 (1H, t, J = 8.08 Hz), 7.75 (2H, m, J =
8.12 Hz), 8.05 (1H, d, J = 6.79 Hz), 8.19 (2H, d, J = 9.71 Hz), 8.41 (2H,
d, J = 6.78 Hz), 11.04 (1H, s); 13C NMR (125 MHz, DMSO) δ 124.65,
125.62, 126.94, 129.91, 131.24, 134.51, 139.74, 144.04, 150.26, 164.38.
Although non-steroidal anti-inflammatory drugs (NSAIDs) have less
serious side effects compared to steroidal drugs, the latter have been
recognized as the most effective treatment in the inflammatory diseases
for N50 years [18]. The potency of steroidal is derived from their non-
specific inhibition capacity for the production of many proinflammatory
cytokines [19]. For this reason, the elucidation of new non-steroidal
molecules having multi-factorial inhibitory effects on major proinflam-
matory factors is still desirable. Considering this fact, in the current
work, the above-mentioned two compounds (5 and 6) were additional-
ly investigated for their multi-inhibitory effects on the LPS-induced re-
lease of IL-1β and TNF-α, expression level of iNOS mRNA and protein
as well as expression level of COX-2, IL-1β and TNF-α mRNAs. The cyto-
toxic effects of these lead compounds were also evaluated in RAW 264.7
macrophages.
2.2.3. Compound 3
Yellow solid, 68%, m.p. 227–228 °C (lit. m.p. 227–228 °C) [26]; 1H
NMR (500 MHz, DMSO) δ 7.67 (1H, t, J = 8.2 Hz), 7.98 (1H, d, J =
5.92 Hz), 8.21 (3H, m, J = 8.52 Hz), 8.37 (2H, d, J = 8.64 Hz), 8.77
(1H, s), 10.97 (1H, s); 13C NMR (125 MHz, DMSO) δ 115.12, 119.22,
124.26, 126.74, 129.94, 130.85, 140.26, 148.14, 149.77, 164.85.
2.2.4. Compound 4
Yellow solid, 68.8%, m.p. 270 °C (lit. m.p. 274 °C) [26], 1H NMR
(500 MHz, DMSO) δ 8.06 (2H, d, J = 7.49 Hz), 8.23 (2H, d, J =
7.08 Hz), 8.27 (2H, d, J = 7.49 Hz), 8.38 (2H, d, J = 7.09 Hz); 13C NMR
(125 MHz, DMSO) δ 120.43, 123.72, 125.44, 130.08, 140.71, 143.32,
149.98, 165.92.
2.2.5. Compound 5
2. Experimental section
Yellow solid, 35%, m.p. 194–195 °C (lit. m.p.199–200 °C) [23], 1H
NMR (500 MHz, DMSO) δ 8.06 (1H, d, J = 8.99 Hz), 8.22 (2H, d, J =
8.85 Hz), 8.43 (2H, d, J = 8.84 Hz), 8.61 (1H, dd, J = 7.97 Hz), 8.77
(1H, d, J = 7.97 Hz), 11.48 (1H, s); 13C NMR (125 MHz, DMSO) δ
121.64, 124.41, 126.73, 129.06, 129.96, 136.91, 138.76, 142.07, 143.86,
150.31, 164.59.
2.1. General
All reagents were obtained from various sources at highest purity
possible. Reagents for synthesis from Merck, Fluka and Sigma–Aldrich
were used as supplied without prior purification. Melting points were
determined with X-4 Melting Point Apparatus and are uncorrected.
Synthesis reactions were monitored by TLC on 0.25 mm silica gel plates
(60 GF254) and visualized with ultraviolet light. Infrared spectra were
measured using ATR techniques on a Perkin Elmer Spectrum 100 FTIR
spectrophotometer. The 1H NMR and 13C NMR spectra were recorded
at Varian Mercury 500 MHz High Performance Digital FT-NMR spec-
trometer and Jeol NMR-400 MHz using TMS as an internal standard.
2.2.6. Compound 6
Yellow powder solid, 55%, m.p. N250 °C; 1H NMR (500 MHz, DMSO)
δ 8.24 (2H, d, J = 8.71 Hz), 8.40 (2H, d, J = 9.69 Hz), 8.57 (1H, s), 9.08
(2H, s), 11.32 (1H, s); 13C NMR (125 MHz, DMSO) δ 113.61, 120.09,
124.22, 129.87, 139.45, 141.29, 148.60, 150.11, 165.13.
All spectroscopic data of compounds were consistent with literature
values (seen in supp. part).
2.2. Preparation of compounds (1–6)
2.3. Effects of synthesized compounds on inflammatory mediators
Nitro substituted aromatic amides (1–6) were prepared by sub-
stitution reaction of 4-nitrobenzoyl chloride with nitro containing
aniline derivatives (2-nitroaniline, 3-nitroaniline, 4-nitroaniline,
2,4-dinitroaniline, 3,5-dinitroaniline) in the different reaction conditions.
Compound 1 was synthesized in pyridine that used as base and solvent
2.3.1. Cell culture and sample treatment
All reagents used in cell culture experiments were supplied from
Sigma–Aldrich Co. (St. Louis, MO) unless otherwise noted. Murine
RAW macrophage cell line (RAW 264.7, ATCC®TIB-71™) was routinely
maintained in Dulbecco's modified Eagle's medium (DMEM) (Caisson,

T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
131
North Logan, UT) supplemented with 100 U/mL penicillin, 100 μg/mL
streptomycin, and 10% fetal bovine serum (FBS). Cells were incubated
at 37 °C in humidified air containing 5% CO2 and sub-cultured by cell
scraping. Fortreatments, cellswereplatedata densityof4 ×105 cells/mL
in 24-well plates in phenol red free DMEM supplemented with 10% FBS
and then incubated with or without LPS (1 μg/mL) and synthesized
compounds (1–6) or with positive control (1400 W, N-[[3-
(aminomethyl)phenyl]methyl]-ethanimidamide, dihydrochloride) as
triplicate for 24 h. Compounds were dissolved in 10% dimethyl sulfoxide
(DMSO) and final concentration of DMSO was 0.1% in each well. All bi-
ological experiments were repeated at least three times, and the data
were represented as mean values (means SD).
Biosystems, Foster City, CA). The thermal cycle program was set as fol-
lows: 25 °C for 10 min; 37 °C for 120 min; 37 °C; 85 °C for 5 s, and
final hold at 4 °C. Synthesized cDNAs were diluted 20-fold and the dilut-
ed sample (5 μL) was used for quantitative PCR with specific TaqMan®
Gene Expression Assay for each gene (Life Technologies, Thermo Fisher
Scientific). Quantitative PCR amplifications were performed on Step
One Plus® Real-Time PCR Systems (Applied Biosystems, Thermo Fisher
Scientific). β-Actin was used to normalize target gene expression. The
effects of compounds on gene expression levels of inflammatory
markers were evaluated by the comparative ΔΔCt method.
2.3.6. Western blot analysis
RAW 264.7 macrophages were plated at a density of 1.5 × 106 cells/mL
in 6-well plates in phenol red free DMEM supplemented with 10% FBS
and then incubated without LPS (vehicle) and with 1 μg/mL LPS including
compounds 5 and 6 as triplicate for 6 h. After incubation period, cells were
washed with ice-cold phosphate buffered saline (PBS, Caisson Lab) and
collected and lysed by using radio-immuno assay buffer (RIPA) contain-
ing (150 mM sodium chloride, 1.0% NP-40 or Triton X-100 0.5%, sodium
deoxycholate, 0.1% Sodium dodecyl sulphate (SDS), 50 mM Tris,
pH 8.0), 1× protease (Amresco) and phosphatase inhibitor (Cell Signal-
ing) cocktails. The cell lysate centrifuged at 14.000 g at 4 °C for 15 min.
The protein content of supernatant was quantified with the BCA protein
assay kit (Pierce). Supernatants (60 μg) were separated on 4% stacking
gel and 8.5% SDS-PAGE in a discontinuous buffer system as described by
[29] and subjected to Western blotting. Nitrocellulose membrane was
incubated with iNOS and β-actin primary antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA) overnight while shaking at 4 °C. After
washing with TBST buffer (500 mM NaCl, 20 mM Tris pH 7.4, 0.05%
Tween 20). The membrane incubated with HRP-conjugated secondary
antibody for 1 h at room temperature and visualized with western
blotting luminol reagents (Santa Cruz Biotechnology, Santa Cruz, CA)
on C-DiGit Chemiluminescence Western Blot Scanner (Licor, NE, USA).
The abundance of protein was determined by the densitometric analy-
sis of bands by using Image Studio Lite Software 5.0.
2.3.2. Nitric oxide production assay
At the end of treatment period, as described above, media was col-
lected from each well and centrifuged at 14,000g at 4 °C for 10 min. Su-
pernatant was assayed immediately or stored at −80 °C. Nitric oxide
production was determined in duplicate by using Griess reagent [27].
Briefly, 100 μL cell supernatant was transferred onto a 96-well plate
and incubated with 100 μL of Griess reagent containing 1%
sulphanilamide and 0.1% naphthylethylenediamine in 5% phosphoric
acid solution for 10 min under light protected conditions. The nitrite
standard reference curve was prepared by a serial dilution (0 to
100 μM) from 0.1 M NaNO2. Absorbance was read at 540 nm by using
a microplate reader (Tecan Infinite® 200 PRO, Switzerland).
2.3.3. IL-1β and TNF-α secretion
For IL-1β and TNF-α secretion, the supernatant was assayed sepa-
rately in duplicate with Mouse IL-1β and TNF-α Platinum ELISA kit (e-
Bioscience, Affymetrix) according to the procedure described by manu-
facturer. To determine the concentration of IL-1β and TNF-α, a refer-
ence curve was prepared by using standards provided with the kit.
Absorbance was read at 450 nm. Concentrations of NO, IL-1β and TNF-α
were normalized to the cellular protein content as quantified by the
Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).
2.3.4. Cell viability assay
2.4. Molecular docking studies
The effect of treatments on cell viability was examined by using MTT
[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide] (TCI,
Portland, OR) [28]. MTT working solution (5 mg/mL) was dissolved in
PBS (1×, Cayman Chemical, Ann Arbor, MI), filtered through a
0.22 μm sterile syringe filter (Corning, NY, USA) and added to treated
cells during the last 2 h of treatment. Media were carefully aspirated
cells were dissolved in DMSO and the absorbance was read at 570 nm.
Molecular docking was performed with Discovery Studio [30] (DS)
2017 to provide an insight into the benzamide derivatives (1–6). Firstly,
ligand(s) and enzyme were prepared using Gaussian 09 (G09) [31] and
DS 2017 software for molecular docking study. The nitro functional
group containing aromatic amide compounds (Fig. 1, Scheme 1) were
constructed and geometrically optimized using DFT/B3LYP/6-31G*
basis set as implemented in G09. Their conformational analyses were
performed using DS 2017.
2.3.5. Gene expression analyses by quantitative PCR
Total RNA was isolated from RAW 264.7 macrophage cells for quan-
titative determination of iNOS (NOS2), TNF-α, IL-1β and COX-2 gene
expressions by using PureLink® RNA mini kit plus on-column DNase
treatment (Applied Biosystems, Foster City, CA) according to
manufacturer's specifications. The protocol has been tailored for the iso-
lation and purification of RNA from RAW264.7 cell line using TRIzol.
Briefly, 180 μL chloroform was added to TRIzol harvested samples
(900 μL). Samples were vigorously mixed for 15 s, incubated at room
temperature for 3 min, and centrifuged at 12,000g for 15 min at 4 °C.
After that, the upper phase (colorless, 400 μL) was collected and contin-
ued with the instructions written in kit's protocol. Samples were
allowed to dry and re-suspended in RNAase/DNAase free-water. The in-
tegrity of the RNA was evaluated by running RNA on a 1% agarose gel.
RNA was then treated with Deoxyribonuclease I Amplification grade
(Life Technologies), following the manufacturer's guidelines. RNA qual-
ity was checked on the Tecan's Nano Quant Plate (Tecan Infinite® 200
PRO, Switzerland). A ratio of OD 260/280 ≥ 2.0 and OD 260/230 ≥ 1.8
was considered to be good quality. First-strand cDNA was synthetized
from 3000 ng total RNA using the high capacity cDNA reverse transcrip-
tion kit (with oligo-d(T)s as primers) plus RNase inhibitor (Applied
The protein crystal structures of iNOS (PDB codes: 3NW2) was se-
lected for this study. Hydrogen and missing heavy atoms were added
to the protein structure, and atom types and partial charges were
assigned. Hem group and the tetrahydrobiopterin cofactor were kept.
The protein binding sites were defined using those occupied by the
co-crystallized ligand MPW1 for iNOS. Additionally, 1400 W was used
as reference ligand to examine its interactions with iNOS deeply. Their
positions of iNOS were subsequently optimized using CHARMm force
field and the adopted-basis Newton-Raphson (ABNR) method [32]
available in the DS 2017 protocol until the root mean square deviation
(RMSD) gradient was b0.05 kcal/mol Å2. The binding site was defined
from literatures about iNOS [33] as given detailed information about
the binding site of iNOS on Figs. S1 and S2 in supplementary part.
During docking analysis, the ligands were flexible and iNOS enzyme
was held rigid. Dock Ligands (CDOCKER) was performed using the de-
fault settings. The docking parameters were as follows: Top Hits: 50;
Random Conformations: 10; Random Conformations Dynamics Step:
1000; Grid Extension: 8.0; Random Dynamics Time Step: 0.002. After
this step, all docked poses were scored by applying Analyze Ligand
Poses sub-protocol in Discovery Studio 2017. Finally, binding energies

132
T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
Fig. 1. Synthesized nitro-substituted aromatic amide compounds (1–6).
were also calculated by applying Calculate Binding Energy sub-protocol
in DS 2017 using in situ ligand minimization step in the ABNR method.
The binding between ligands and iNOS were evaluated with Binding En-
ergy, CDOCKER energy, CDOCKER interaction energy values.
3.1. Identification of lead compounds
We have identified two lead compounds among six nitro substituted
benzamide derivatives as promising anti-inflammatory agents accord-
ing to results obtained from NO inhibition assay in LPS induced RAW
264.7 macrophages and molecular docking studies performed on iNOS
enzyme. For each assay, a control (vehicle alone), and an induction con-
trol (1 μg/mL LPS) were used to set a baseline data. Previous works dem-
onstrated that LPS, a component of the gram-negative bacterial cell
walls, can trigger the inflammatory process by binding to the CD14
toll-like receptor-4 (TLR-4) complex at the surface of innate immune
cells and subcutaneous infusion of LPS to animals resulted in the same
metabolic abnormalities induced by the high-fat diet [34]. For this rea-
son, in recent works LPS have been successfully used and generally pre-
ferred due to its dual role in inducing both acute and low-level chronic
inflammatory responses [34,35].
2.5. Statistical analysis
Statistical analyses were performed using the SPSS Windows Ver-
sion release 16.0. Equality of variances was analyzed by Levene's test.
Group means were compared by a Student's t-test for the independent
samples. P-values b 0.05 were considered indicative of significance. All
experiments and analyses were repeated at least three times.
3. Results and discussion
In this study, we synthesized aromatic amide compounds containing
nitro functional group(s) (1–6, Fig. 1) by using different reaction condi-
tions. Structures of the compounds were characterized by melting point,
FT-IR, 1H NMR and 13C NMR spectral analysis. A convenient method for
synthesizing nitro-substituted aromatic amide compounds was given in
Scheme 1. FT-IR data of the compounds generally show a band at ~3410
and/or 3324 cm−1 for N\\H stretching of amide and a band at
~1670 cm−1 corresponds to C _O stretching of amide linkage. Two in-
tensive bands were observed at ~1530 and 1350 cm−1 correspond to
asymmetrical and symmetrical stretching of –NO2 groups, respectively.
In 1H NMR spectrum, all singlet protons for -NH groups were observed
between ~11.48 and 7.10 ppm. Characteristic aromatic ring protons
were observed between 9.08 and 7.10 ppm. The spectral values of the
compounds 1–6 are given in the Experimental section and supplemen-
tary data (Figs. S4–S15). Compounds 1–6 have poor solubility in water.
Their solubility is below 5 mg/mL at 25 °C.
3.2. Screening of compound by NO inhibition
As shown in Fig. 2 all compounds (1–6) were first screened for their
effect on LPS induced NO production in RAW 264.7 macrophage cells at
20 μM. 1400 W was used as a positive control. Tested compounds exhib-
ited no cytotoxic effect (N85 cell viability) on cells at 20 μM, except com-
pound 1. The relative cell viability for compound 1 was 52% (data not
shown). Among synthesized six compounds, 5 and 6 demonstrated con-
siderably high effects -N50% inhibition- against NO production at 20 μM
with inhibition rates of 62% and 54%, respectively (p b 0.05 for both).
When cells were simultaneously treated with different concentra-
tions of these two molecules (5–50 μM) and LPS (1 μg/mL), as seen in
Fig. 3, NO production was significantly inhibited in a dose-dependent
manner with N70% and 90% inhibitions at 50 μM for compounds 5 and
6, respectively. At all tested doses including the highest 50 μM, the
Scheme 1. Synthesis of 4-Nitro-N-(4-phenyl) benzamide (1) and its derivatives.

T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
133
Fig. 2. Screening of all synthesized compounds (1–6) at 20 μM for inhibiting LPS-induced NO production in RAW 264.7 macrophage cells. Ctrl: Control (vehicle alone). 1400 W: positive
control at 5 μM. Values show relative change of NO (%) in production of NO compared to vehicle with LPS. Each bar represents the mean SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * =
p b 0.05.
cellular viability were N90% for both 5 and 6 eliminating the doubt that
the observed considerable decrease in LPS induced NO production could
be related to cytotoxic effects (Fig. 3).
imidazo[4,5-b]pyridine (MPW1). The docking results on iNOS
exhibited that obtained conformation of MPW2 was superimposed
with MPW1 that were nearly identical for the crystallographic
crystal complex. A potent, selective inhibitor of iNOS, N-(3-
(Aminomethyl)benzyl)acetamidine (1400 W) was used to describe an
insight into the iNOS-1400 W complex in molecular docking studies.
As mentioned in upper part, two molecules (5 and 6) have remark-
ably low half maximal inhibitory concentration for LPS induced NO pro-
duction. Based on this finding, docking studies were performed to
determine why compounds 5 and 6 demonstrated relatively efficient
inhibition capacity compared to others. All six models were flexibly
docked with iNOS by using CDOCKER. Of the ten poses produced, the
best pose of each ligand was selected based on CDOCKER top score
and the target structure was selected for further analysis. The ligand
poses were analyzed and a heat map was produced to count H bonds
made by the poses to the enzyme molecule using Analyze Ligand
Poses sub-protocol in DS 2017 (data were shown in supplementary
part, Tables S1–S2). For comparison of orientations of compounds 1–6,
we superimposed each compound's best pose, which was obtained by
locating the lowest binding energy, the largest minus CDOCKER energy
and the lowest minus CDOCKER interaction energy, (see Table S2 in
supp. part). Additionally, the synthesized compounds (1–6) including
The IC50 values of these two molecules were investigated for NO pro-
duction at nine different doses ranging between 1 and 20 μM, resulted
in 3.7 μM for 5 and 5.7 μM for 6. In recent years, it has been shown
that increased production of NO by iNOS is responsible for some acute
pathological conditions such as stroke and septic shock, as well as for
a diverse array of metabolic disorders originating from chronic low
grade inflammation [17,36]. Therefore, the inhibition capacities of 5
and 6 on inducible NO production at quite low micro molar ranges
(3.7 and 5.7 μM) compared to reported IC50 values of 1) L-NIL (L-N6-
(1-iminoethyl)-lysine [37]: 27.1 μM, a selective inhibitor of iNOS, and
2) Indomethacin [37] (65.3 μM, a prescribed NSAID, make these two
molecules promising as lead anti-inflammatory compounds.
3.3. Molecular docking studies of lead compounds on iNOS
Molecular docking calculations were implemented with one crystal-
lographic complex obtained from the Protein Data Bank (PDB) [36]
which is the structure of human inducible nitric oxide synthase
(iNOS) complexed with 2-[2-(4-methoxypyridin-2-yl)ethyl]-3H–
Fig. 3. Screening of compounds 5 and 6 at different doses 5–50 μM for inhibiting LPS-induced NO production in RAW 264.7 macrophage cells. Ctrl: Control (vehicle alone). Each bar
represents relative change of NO (%) as compared to vehicle with LPS. Each dot on the line shows relative survival rate of cells in case of different treatments as compared to vehicle
with LPS. Values represent the mean SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * = p b 0.05.

134
T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
ortho, meta, para and two nitrophenyl combination were evaluated
based on N-(phenyl)-4-nitrobenzamide (1) with binding site of the en-
zyme, as given Figs. S1 and S2 in supplementary part. Hydrogen bonding
and lipophilic interactions have great importance in any biological sys-
tem. Numerical values demonstrating these interactions among
benzamide derivatives 5–6 and iNOS were presented in Table S6 (for
H-bonds and lipophilic interactions) and Table S4 (Non-bonding inter-
actions data of the 1–4 ligands).
Specifically for compound 5, as can be seen in Fig. 4A, while the
Arg260 residue formed two hydrogen bonds with the 4-nitrophenyl
moiety, the ortho-nitro group made single hydrogen bond (2.471 Å)
with Gln486 residue of the enzyme. Single hydrogen bond (2.0296 Å)
is also observed with Asn115 residue. Among compound 5 and iNOS
five lipophilic interactions can be described: one of them is between
2.4-dinitrophenyl ring and NH1 moiety of Arg382 residue, the other
four interactions are observed between Hem group of the enzyme and
N7 atom of the compound 5 (Fig. 4A and Table S4). The orientation of
compound 6 is quite different from the orientation of compound 5,
therefore hydrogen bonds and lipophilic interactions are different. The
hydrogen bonds for compound 6 were observed with Arg260, Asn348
and Glu488 residues (1.981 Å, 2.193 Å, 2.222 Å, respectively). In case
of lipophilic interactions, compound 6 formed eight different interac-
tions between Hem group and Phe280, Tyr367 and Arg382 residues of
the enzyme as shown in Fig. 4B.
Based on docking results and relative orientation of ligands for crys-
tallographic complex (seen in Fig. 5A, B), compounds 5 and 6 seem to be
good inhibitors of iNOS as compared to other compounds (1–4). When
two lead molecules are superimposed with the reference ligand
(1400 W), the results showed that they are quite similar (Fig. 5C). The
phenyl moieties are almost parallel to Hem group, so that a series of
π-π and π-cation interactions can be observed as shown in Fig. 4 and
Table S6. Fig. 6A-C also represent that reference ligand (1400 W), com-
pounds 5 and 6 are all at the same conformation on the protein. Howev-
er, other compounds (1–4) are in perpendicular conformation to Hem
group at the binding site of iNOS, thus they cannot make lipophilic inter-
actions with this prosthetic group on the protein.
NO production, the optimum number of nitro group, determined as
three, in the binding site of the enzyme has great importance rather
than the position of these groups. For this reason, compounds 5 and 6
showed remarkable results. Furthermore, orientations and polarizabil-
ities of lead compounds have very dominant properties for binding will-
ingness on the enzyme. Gray labelled two rows on Table 1 indicate these
statuses with numerical values. The compound 5 have higher numerical
polarizability than compound 6 (351.811 vs 319.119, respectively). It
means that compound 5 exhibits suitable polarizability and orientation
for active site of the enzyme as compared to compound 6. This differ-
ence might explain slightly lower IC50 value obtained for compound 5
according to compound 6.
3.4. Effects of lead compounds on iNOS mRNA/protein expression
The molecular docking studies gave us detailed information about
the inhibitory potential of two lead compounds on iNOS enzyme. How-
ever, to elucidate the action mechanism of compounds 5 and 6 on the
inhibition of NO production in a detailed way, we carried out further
studies. To understand whether these two lead molecules inhibit NO
production at the level of transcription or protein expression, we deter-
mined the relative level of iNOS gene by qPCR and iNOS protein by
Western blot in LPS activated macrophages. In both qPCR and Western
blot analyses β-actin was used as housekeeping gene/protein to nor-
malize the expression data. Firstly, as shown in Fig. 7 (A-C), the treat-
ment of the macrophages with LPS (1 μg/mL) markedly increased the
expression of both iNOS protein and mRNA levels compared to control
group (−LPS). In the qPCR analyses, co-treatment with 5 did not result
in a decrease in the mRNA expression level at both 10 and 20 μM. Al-
though a significant decrease in protein expression was observed by
co-treatment of compound 5 at a ratio of 19% for 10 μM and 24% for
20 μM as seen on Fig. 7A and B, these decreases may not be biologically
relevant due to relatively low ratios. Taken together; the present data
showed that compound 5 inhibits NO production without effecting
mRNA or protein level of iNOS. The very low IC50 value of 5 for NO pro-
duction may be explained by direct inhibitory action of this compound
on catalytic activity of iNOS enzyme. Some of the iNOS inhibitors such
as aminoguanidine and S,S′-1.4-phenylene-bis(1.2-ethanediyl)bis-
isothiourea (PBIT) do not influence the synthesis of iNOS [38,39]. Im-
portantly, the administration of PBIT, a selective iNOS inhibitor that
These results suggest that our lead compounds might be potential
iNOS inhibitors as determined experimentally by their IC50 values
which resulted in 3.7 μM for 5 and 5.7 μM for 6. Considering all com-
pounds, the results demonstrated that for the efficient inhibition of
A
B
Fig. 4. The orientations of compounds A) 5 and B) 6 including hydrogen bonds (left side) and lipophilic interactions (right side) with Hem and residues of iNOS enzyme. 5 and 6 are shown
as orange and pink color, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
135
A
B
C
Fig. 5. A) The relative orientation of ligands MPW1 (blue color), MPW2 (gray color) and 1400 W (green color) in relation to the Hem and H4B groups. B) Docking poses of 1, 2, 3, 4 (stick)
and 5, 6 (ball and stick, pink and orange) in iNOS. C) The compounds 5, 6 and ligand 1400 W in iNOS. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
decreased NO generation without affecting iNOS expression significant-
ly suppressed carcinogen-induced tumor development in several cancer
models [39,40].
Co-treatment of cells with compound 6 reduced LPS induced iNOS
mRNA expression in a dose dependent manner such that while 10 μM
decreased the expression 28%, 20 μM resulted in 51% suppression
(p b 0.05 for both, Fig. 7C). Furthermore, co-treatment of cells with 6
at both 10 and 20 μM markedly suppressed the LPS induced protein
level at nearly same levels 41% vs 45%, respectively (Fig. 7A and B).
These results were found to be consistent with the NO generation data
and suggest that in contrast to compound 5, the mechanism for the
NO inhibitory activity of compound 6 might be correlated to the sup-
pression of both iNOS gene and protein expression.
3.5. Effects of lead compounds on TNF-α and IL-1β release
We investigated the multi-potent inhibitory activity of lead com-
pounds 5 and 6 on secretion of some major proinflammatory factors

136
T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
A)
B)
C)
Fig. 6. A, B) Binding site of iNOS with the surface occupied by ligand MPW1and 1400 W is shown in light pink with compounds 5 and 6 inside it. C) The compounds 5 and 6 in the iNOS
cavity, showing the aliphatic moiety coming out. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
under inflammatory response. Both IL-1β and TNF-α secretion have
been identified decades ago as hallmarks of proinflammatory response
in obesity-associated chronic metabolic diseases like insulin resistance
and type II diabetes [41,42]. In the current work, treatment of macro-
phage cells with compound 5 did not result in any change in the amount
of LPS induced IL-1β secretion at both 10 and 20 μM doses (Fig. 8A).
However, treatment with compound 6 at 10 μM and 20 μM resulted in
57% and 65% inhibition, respectively. Similar trends were also observed
for LPS-induced TNF-α secretion. Compound 5 did not present any ef-
fect on the secretion of LPS-induced TNF-α at both 10 and 20 μM
doses. However, treatment with compound 6 exhibited 46% and 55% in-
hibition at 10 and 20 μM (Fig. 8B).
44]. In the present study, to assess the effects of compounds 5 and 6
on COX-2, IL-1β and TNF-α expression by LPS-inflamed macrophages,
we measured their gene expression level by qPCR. As seen on Fig. 8, in
LPS induced macrophages the expression of these three markers were
markedly increased compared to control cells. However, co-treatment
of cells both by compounds 5 and 6 with LPS resulted in a significant
downregulation in the mRNA expression of all genes. The magnitude
of suppression for compound 5 was changing between 37 and 50% at
10 and 20 μM in three different genes (Fig. 9). In case of compound 6,
the more effective downregulation (around 60%, at 10 μM) has been ob-
served, particularly in the expression of IL-1β. This value increased up to
88% at 20 μM in a dose dependent manner (Fig. 9).
3.6. Effects of lead compounds on IL1β, TNF-α and COX-2 mRNA expression
4. Conclusion
Unregulated overexpression of IL1β, TNF-α and COX-2 along with
various other proinflammatory factors have been connected to metasta-
tic cells and contribute to development and progression of cancer [43,
In the current study, six (1–6) nitro substituted benzamide deriva-
tives have been synthesized and addressed for their potential anti-in-
flammatory activities for the first time. All compounds were screened
Table 1
Orientations and polarizabilities of the compounds (1–6).
Index
Ligand names
Pose numbers
Exact polarizabilities
96
7
22
56
42
62
1
2
3
4
5
6
5
1
1
5
1
1
280.866
301.144
206.112
300.322
351.811
319.119
−3.880
4.718
−60.517
5.319
3.731
158.001
194.257
309.624
181.637
205.421
205.102
4.059
−7.216
0.000
8.906
−6.062
−5.009
−2.235
7.771
0.000
−9.506
8.830
15.199
61.726
63.345
57.842
68.935
68.801
75.274
0.123

T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
137
Fig. 7. The effects of 5 and 6 on the LPS induced iNOS protein and gene expression level. (A) Western blot analysis for iNOS and β-actin (endogenous control) protein expression (B) Bands
were quantified by densitometry and expressed by relative iNOS/β-actin ratio compared to vehicle with LPS. (C) iNOS mRNA expression was determined by qPCR using probe. Values show
relative gene expression compared to vehicle with LPS evaluated by comparative ΔΔCt analysis. Each bar represents the mean SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * = p b 0.05.
Fig. 8. Relative change of A) IL-1β (%) and B) TNF-α secretion as compared to vehicle with LPS. Each bar represents the mean SEM (n = 3). *** = p b 0.001, ** = p b 0.01.

138
T.B. Tumer et al. / International Immunopharmacology 43 (2017) 129–139
and spectral analyses of the compounds. Supplementary data associated
with this article can be found in the online version, at http://dx.doi.org/
10.1016/j.intimp.2016.12.009.
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