Aryl quinolinyl hydrazone derivatives as anti-inflammatory agents that inhibit TLR4 activation in the macrophages

Article abstract of DOI:10.1016/j.ejps.2019.04.016

A series of aryl 7-chloroquinolinyl hydrazone derivatives (3a-u)have been synthesized in 55–76% yield using simple reaction condition. The synthesized compounds were evaluated for their anti-inflammatory activities based on their ability to inhibit pro-inflammatory cytokine secretion from the macrophages after stimulation with lipopolysaccharide (LPS). Three compounds appeared as promising anti-inflammatory agents. The mechanism of inflammatory activity of the potent compound 3e was further investigated using a series of biochemical, molecular and microscopic techniques. Further structure activity relationship (SAR)study was carried out to validate the anti-inflammatory activities of the active compounds. Our experimental data revealed that the active moiety i.e. compound 3e majorly causes inhibition of TLR4 signaling pathway and this appears to be the novel functional attribute of this compound.

Full text of DOI:10.1016/j.ejps.2019.04.016

Accepted Manuscript
Aryl quinolinyl hydrazone derivatives as anti-inflammatory
agents that inhibit TLR4 activation in the macrophages
Utsab Debnath, Suprabhat Mukherjee, Nikhilesh Joardar, Santi P.
Sinha Babu, Kuladip Jana, Anup Kumar Misra
PII:
DOI:
Reference:
S0928-0987(19)30154-X
https://doi.org/10.1016/j.ejps.2019.04.016
PHASCI 4911
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
26 December 2018
12 April 2019
13 April 2019
Please cite this article as: U. Debnath, S. Mukherjee, N. Joardar, et al., Aryl quinolinyl
hydrazone derivatives as anti-inflammatory agents that inhibit TLR4 activation in the
macrophages, European Journal of Pharmaceutical Sciences, https://doi.org/10.1016/
j.ejps.2019.04.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Aryl quinolinyl hydrazone derivatives as anti-inflammatory
agents that inhibit TLR4 activation in the macrophages
Utsab Debnath,1. Suprabhat Mukherjee,2. Nikhilesh Joardar,3. Santi P. Sinha Babu,3*
Kuladip Jana,1 Anup Kumar Misra1*
1Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII M, Kolkata
700054, India. E-mail: akmisra69@gmail.com
2Department of Animal Science, Kazi Nazrul University, Asansol-713340, West Bengal,
India
3Parasitology Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan-
731235, West Bengal, India
. Contributed equally.
ABSTRACT
A series of aryl 7-chloroquinolinyl hydrazone derivatives (3a-u) have been synthesized in 55-
76% yield using simple reaction condition. The synthesized compounds were evaluated for
their anti-inflammatory activities based on their ability to inhibit pro-inflammatory cytokine
secretion from the macrophages after stimulation with lipopolysaccharide (LPS). Three
compounds appeared as promising anti-inflammatory agents. The mechanism of
inflammatory activity of the potent compound 3e was further investigated using a series of
biochemical, molecular and microscopic techniques. Further structure activity relationship
(SAR) study was carried out to validate the anti-inflammatory activities of the active
compounds. Our experimental data revealed that the active moiety i.e. compound 3e majorly
causes inhibition of TLR4 signaling pathway and this appears to be the novel functional
attribute of this compound.
Keyword: quinolinyl hydrazone; anti-inflammatory agents; toll-like receptor 4; structure-
activity relationtionship; macrophage.
INTRODUCTION
Inflammation and associated physiological responses play crucial role in the pathogenesis of
several human diseases that potentially include sepsis, cancer, type II diabetes, rheumatoid
arthritis, Crohn’s disease, systemic lupus erythematosus and infectious diseases viz. cerebral
malaria, leishmaniasis, trypanosomiasis, filariasis, enteric diseases etc. [1-5]. All the
aforementioned diseases are taking enormous toll from mankind by means of huge number of
morbidities, mortality and socioeconomic loss [1, 4]. Being the major determinant of
pathogenesis of several life-threatening diseases, targeting of the proinflammatory response
by chemotherapeutics is considered as an efficacious approach [1, 6]. Till date, several anti-
inflammatory drugs have been developed out of which most of the compounds possess a
1

ACCEPTED MANUSCRIPT
number of drawbacks like poor absorption, inadequate serum availability and toxic side
effects in non-targeted tissues and organs [7, 8]. Blockage of prostaglandin biosynthesis
through the suppression of the release of histamine [9, 10] and inhibition of cyclooxygenase
(COX) enzyme activity [11] are considered as the key targets of the majority of the anti-
inflammatory compounds available in the clinics. However, during chronic inflammation,
pro-inflammatory molecules, such as cytokines, inducible nitric oxide synthase (iNOS),
reactive oxygen species (ROS), and NF-κB are up-regulated [12, 13, 14] and hence COX
inhibitors are unable to show significant role as anti-inflammatory agents [15, 16].
Considering the alarming stipulation, development of novel anti-inflammatory compounds is
a thrust area in medicinal chemistry.
Over the last decade, especially after the discovery of TLR signaling pathways,
extensive research on the signal transduction of pro-inflammatory pathways and their
signaling cross-talks has uncovered novel target(s) for developing efficacious therapeutics
[17]. In this context, toll-like receptor 4 (TLR4) has been presented as the most influential
target due its extreme ability of regulating the immune-homeostasis in the human body
system [18, 19]. TLR4 has been demonstrated as the key mediator of immunopathogenesis in
most of the inflammatory and infectious diseases of human [18, 19]. TLR4 is an innate
immune receptor that recognizes and binds with lipopolysaccharide (LPS), an antigenic
component of the outer leaflet of gram-negative bacteria [20]. Out of the eleven mammalian
TLRs, TLR4 is considered as the prime receptor due to its ability of recognizing molecular
pattern from array of pathogens (viruses, bacteria, fungi, and parasites) and/or damage
associated molecular patterns (DAMPs) [21]. Upon binding with the ligand, TLR4 induces a
signaling cascade leading to the activation of the important intracellular transcription factors
like nuclear factor kappa B (NF-κB), activator protein-1 (AP-1) and/or interferon response
factor (IRF) [19]. TLR4 induced activation of the transcription factors result in the expression
of the pro-inflammatory cytokines like TNF-α, IL-6, and IL-1β [19] which activate different
types of immune cells present throughout the human body and trigger adaptive immune
responses through autocrine, paracrine, and endocrine signaling pathways [22]. TLR-
mediated over activation of the immune cells disrupts immune-homeostasis and induces
several pathological conditions [23]. In particular, TLR4 binds with free fatty acid and
induces inflammation of macrophages to secrete proinflammatory cytokines that results in
insulin resistance leading to type-II diabetes [24, 25]. On other side, TLR4 activation in
macrophages by tumor antigens and subsequent induction of inflammation is the cause of
2

ACCEPTED MANUSCRIPT
tumor progression and metastasis in lung, breast and colorectal cancer [26, 27].
Immunopathogenesis of cerebral malaria has been reported to be associated with glycosyl
phosphatidyl inositol (GPI)-mediated TLR4 activation [19, 28]. Binding of ligands from
infectious pathogens is the cause behind the immunopathology of the major infectious
diseases of human [19]. Bestrophin homolog from the filarial parasite and LPS from the
Wolbachial endosymbiont has been found to act as a ligand for TLR4 and resulting
macrophage inflammation leading filarial lymphedema [29, 30, 31]. Binding of LPS from
gram-negative bacteria has been reported for inducing overt inflammatory pathology in post-
operational sepsis leading to death [32]. Therefore, disruption of LPS-TLR4 interaction with
chemically synthesized moiety is considered as one of key approaches of developing drug
against the inflammation induced pathological outcomes/death [33, 34]. Till date, a number
of anti-inflammatory compounds have been designed to trigger TLR-4-NF-κB pathway [35].
Unfortunately, most of the compounds were failed due to their high toxic effects on the
targeted and non-targeted cells and tissues [34].
Herein, a set of quinolinyl hydrazone derivatives were synthesized and their anti-
inflammatory activities have been investigated based on their ability to inhibit pro-
inflammatory cytokine secretion from macrophage after stimulation with lipopolysaccharide
(LPS). Earlier quinoline hydrazone derivatives have been evaluated for their anti-tuberculosis
[36], anti-leishmanial [37], trypanocidal [37], anti-cancer [36], and anti-microbial activities
[38]. However, their potential to act as anti-inflammatory agents has not been explored yet. In
this present communication, synthesis of aryl 7-chloroquinolinyl hydrazone (3a-u) and their
evaluation as anti-inflammatory agent is presented. A number of biochemical and
microscopic experiments have been carried out using significantly active compounds to
establish the mechanism of action of these molecules in controlling inflammation of
macrophages. Furthermore, molecular docking study was carried out to find out the structure
activity relationship (SAR) as well as most probable binding sites of the TLR4 enzyme for
anti-inflammatory activity of potent 7-chloroquinolinyl hydrazone derivatives.
RESULT & DISCUSSION
Chemistry
3

ACCEPTED MANUSCRIPT
Treatment of 4,7-dichloroquinoline (1) with hydrazine hydrate at elevated temperature
furnished 7-chloro-4-hydrazinyl-quinoline (2) in 97% yield [39]. Compound 2 was allowed to
react with a series of aromatic aldehydes (3a-u) under refluxing condition to furnish a library
of aryl 7-chloroquinolinyl hydrazone derivatives (3a-u) in satisfactory yield. All synthesized
compounds were characterized by satisfactory spectroscopic analysis (Scheme 1).
Ar
N
Cl
N
HN
N
NHNH2
.
NH2NH2 H2O
ArCHO
Neat, 90 oC,
12 h, 97%
EtOH, Reflux
2-6 h
Cl
Cl
Cl
N
3a-u
1
2
Ar = Aryl; Heteroaryl
3a
: Ar = p-methoxyphenyl [18] (67%)
3b: Ar = 3,5-dimethoxyphenyl (73%)
3c: Ar = 3,4-dimethoxyphenyl (76%)
3d: Ar = 3,4,5-trimethoxyphenyl [18] (81%)
3e: Ar = 3,5-dihydroxyphenyl [19] (60%)
3f
: Ar = 3-fluoro-4-methoxyphenyl (73%)
3g
3h
: Ar = 3,5-dibromophenyl (71%)
: Ar = 3,5-difluorophenyl (66%)
: Ar = 3,4-dihydroxyphenyl [20] (63%)
3i
3j: Ar = 4-nitrophenyl [21] (57%)
3k: Ar = 4-acetoxy phenyl (72%)
3l: Ar = 4-benzyloxyphenyl (73%)
3m: Ar = 3,5-dichlorophenyl (70%)
3n
3o
3p
: Ar = 3,5-dichloropyridinyl (55%)
: Ar = 3-benzyloxy-4-methoxyphenyl (71%)
: Ar = 2-thiophenyl [18] (76%)
3q: Ar = 3-coumarinyl (70%)
3r: Ar = 5-imidazolyl [22] (70%)
3s: Ar = 4-hydroxy-3-methoxyphenyl [19] (69%)
3t: Ar = 4-trifluoromethylphenyl (61%)
3u
: Ar = 2-pyrrolyl (57%)
Scheme 1: Preparation of aryl 7-chloroquinolinyl hydrazone derivatives (3a-u). Yields are
given in parentheses.
Biology
Anti-inflammatory activity of the synthesized compounds
The anti-inflammatory activities of the synthesized moieties were investigated, based on their
ability to inhibit pro-inflammatory cytokine secretion from macrophage after stimulation with
lipopolysaccharide (LPS) (Table 1). The initial screening study on the activity of the
synthesized 21 compounds employing a single dose (25 μM) revealed that compound 3e, 3f,
and 3l possess significant anti-inflammatory potential (Figure 1A and B). These three
compounds inhibit LPS induced secretion of the major pro-inflammatory cytokine, TNF-α
4

ACCEPTED MANUSCRIPT
from macrophages in vitro (Figure 1A). The inhibitory effect of the synthetic compounds (3a-
u) was compared with the standard anti-inflammatory drug dexamethasone (Figure 1A).
Moreover, these three compounds not only inhibit TNF-α secretion from macrophages but
also promote secretion of the anti-inflammatory cytokine, IL-10 which amplify the anti-
inflammatory response (Figure 1B). The activity of the compounds by using various
concentrations (0-50 μM) and IC50 values of all the compounds were determined based on
their potential in inducing 50% reduction in the secretion of TNF-α from macrophages
resulted by the action of LPS. The calculated IC50 for the three active compounds viz. 3e, 3f,
3l were 12.39±0.97 μM, 18.97±0.96 μM, and 15.92±0.96 μM respectively (Table 1).
Table 1: Inhibitory effects of the synthesized compounds (3a-3u) on the secretion of TNF-α
from LPS-treated RAW 264.7 macrophages.
Sl.
No.
1
2
3
4
5
6
7
Name of
compound
IC50 (μM)# CC50 (μM)
29.37±1.32 400.05±12.18
20.20±0.98 396.55±11.81
29.24±1.50 375.65±12.31
24.39±2.10 407.80±12.54
12.39±0.97 360.25±7.59
18.97±0.96 311.00±9.21
151.58±1.12 266.48±12.32
82.38±2.46 272.76±11.98
32.20±1.01 223.20±12.87
30.72±1.98 221.66±13.64
20.89±1.03 390.47±14.50
15.92±0.96 305.65±5.65
79.56±2.91 235.35±3.92
84.27±3.01 236.43±4.85
56.25±3.01 228.65±6.78
75.39±2.76 232.18±5.25
66.42±2.02 228.28±6.34
48.87±2.12 248.45±10.25
59.97±2.45 200.16±11.35
68.72±1.98 299.94±12.57
75.39±0.95 202.34±11.62
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
8
9
10
11
12
13
14
15
16
17
18
19
20
21
3t
3u
22 Dexamethasone 9.07±0.68
356.35±9.78
#IC50 value (μM) was calculated for the concentration of each compound potent enough to
reduce TNF-α secretion in the cell free supernatant up to 50% level. Level of TNF-α was
estimated from the spent culture supernatant from different compound treated macrophages
preincubated with LPS (100ng/mL). All the data were calculated from the experiments
conducted in triplicate and repeated for at least five times. Data represents mean±SD.
5

ACCEPTED MANUSCRIPT
Figure 1: Anti-inflammatory activity of the synthesized compounds (3a-u). (A) Stacked bar
diagram showing the comparative inhibitory activity of the synthesized compounds on LPS-
induced secretion of TNF-α in the culture medium. (B) Stacked bar diagram showing the
comparative inhibitory activity of the compounds (3a-u) on LPS-induced secretion of IL-10
from macrophages. The compounds were screened based on their potential in inhibiting LPS
induced secretion of TNF-α and promoting activity on the secretion of IL-10 from RAW
264.7 macrophages in vitro. The initial screening dose was set at 25 μM for all the
compounds and the standard; dexamethasone. LPS was used at a dose of 100 ng/mL. Levels
of TNF-α and IL-10 were determined in the spent culture media by sandwich ELISA. Each
data represents experiment conducted in triplicate and repeated for five times.
Furthermore, the toxic effects of these compounds on macrophages have also been checked.
It was found that, the three active compounds (viz. 3e, 3f and 3l) did not exert any notable
toxic alteration in the macrophages. The calculated CC50 values for the three active
compounds viz. 3e, 3f, 3l were 360.25±7.59 μM, 311.00±9.21 μM, and 305.65±5.65 μM
respectively (CC50, Table 1). Considering the initial screening studies of compound 3e was
found to be the most active compound in terms of its effect in inhibiting TNF-α and
promoting IL-10. Therefore, further biochemical and microscopic studies were conducted
with compound 3e to explore its mechanistic insights of the anti-inflammatory activity.
6

ACCEPTED MANUSCRIPT
The effect of compound 3e in different doses of on LPS-induced inflammation on
macrophages was studies in dose dependent manner. It was observed that secretion of TNF-α
from LPS-sensitized macrophages were significantly decreased with the increase of treatment
dose from 6.25 μM-25 μM (Figure 2A). At the highest dose (25 μM) the inhibition of TNF-α
was so significant that the level of TNF-α is almost similar to control (Figure 2A). It is
intriguing to note that, compound 3e potentially up-regulates the secretion of IL-10 and the
effect exerted in a dose dependent manner (Figure 2B).
Figure 2: Kinetics of anti-inflammatory activity of compound 3e against LPS induced
inflammation in RAW 264.7 macrophages: Dose dependent action of compound 3e on the
secretion of (A) TNF-α and (B) IL-10 from RAW 264.7 macrophages. Time kinetics of the
effect of compound 3e on the secretion of (C) TNF-α and (D) IL-10 from the macrophages.
Each data represents experiment conducted in triplicate and repeated for five times.
It was also observed that the TNF-α inhibition activity of compound 3e is executed within an
hour of administration in macrophage culture (Figure 2C and 2D). In contrast, a rise in IL-10
level was noted up to 4 h of post administration of compound 3e in macrophage culture
7

ACCEPTED MANUSCRIPT
(Figure 2D). In order to investigate the possibility of compound 3e as therapeutic candidate,
its efficacy was checked in comparison to the standard anti-inflammatory drug,
dexamethasone (Figure 3 A-C). Although compound 3e was found as a non-toxic and
effective anti-inflammatory molecule, its effects were lesser than that of dexamethasone
(Figure 3A-C). Since compound 3e potentially subdued the secretion of TNF-α, we
investigated its effect on other two key pro-inflammatory cytokines viz. IL-6 and IL-1β
(Figure 3B and C). Levels of these two cytokines in the spent culture media were
significantly down-regulated after treatment with compound 3e. Data were represented as
mean±SD p<0.05 is considered as statistical significant.
Figure 3: Comparative efficacy of compound 3e as an anti-inflammatory compounds.
Comparative actions of compound 3e, dexamethasone, and CLI-095 on LPS-induced
secretion of (A) TNF-α; (B) IL-6, and (C) IL-1β. Each data represents experiment conducted
in triplicate and repeated for five times.
LPS is a classical ligand of TLR4 and intriguingly compound 3e blocks the inflammatory
responses induced by LPS in macrophages (Figure 1, 2, and 3). Therefore, it was necessary to
check whether compound 3e does acts like TLR4 antagonist, the action of compound 3e was
8

ACCEPTED MANUSCRIPT
compared withTLR4 inhibitor CLI-095 and it was found that the effect of compound 3e was
at par with CLI-095 (Figure 3A-C). The expression of TLR4 on macrophage membrane after
LPS stimulation was also down-regulated significantly after incubation with compound 3e
(Figure 4).
Figure 4: Immunofluorescence depicting the expression of TLR4 on the surface of
macrophages (RAW 264.7). All the confocal microscopic images are representative of the
experiments conducted in triplicate.
Since TLR4 is an immune receptor on macrophage and its expression rapidly increases
during stimulation with pathogenic ligand like LPS or pathogenic proteins [29, 30], down
9

ACCEPTED MANUSCRIPT
regulation of TLR4 in macrophage by compound 3e suggested that the effect of compound 3e
is majorly executed through inhibition of TLR4 (Figure 4). Although TLR is the key inducer
of inflammation, but its effect is executed through the activation of NF-κB, a transcription
factor for the proinflammatory cytokines [29, 30]. It was evident in the immunofluorescence
data that compound 3e inhibits activation of NF-κB in macrophages (Figure 5).
Figure 5: Immunofluorescence depicting intracellular activation of p65NF-κB in RAW 264.7
macrophages. Expression of p65NF-κB (phosphorylated at serine 536) was studied as it is the
active form of NF-κB that translocates towards the nucleus to induce the expression of the
proinflammatory cytokines in the macrophage. All the confocal microscopic images are
representative of the experiments conducted in triplicate.
10

ACCEPTED MANUSCRIPT
Figure 6: Compound 3e triggers TLR4 signaling pathways in macrophages. Immunoblotting
(upper) and corresponding densitometric analyses (lower) showing the expressions of various
signaling intermediates in RAW 264.7 macrophages. All data represent minimum of three
independent experiment and the bar graph shows ±SD (*p <0.05).
Inhibitory activity of compound 3e on TLR4 expression and NF-κB activation were also
noticed in immunoblotting experiments (Figure 6). Apart from TLR4 and NF-κB it has also
been found that compound 3e have a role in disrupting the activation of the downstream
signalling molecule, TAK1 (Figure 6). TAK1 in its phosphorylated form (pTAK1) transmit
the activation signal from TLR4 to NF-κB but compound 3e inhibits phosphorylation of
TAK1 (Figure 6) [29, 30]. Interestingly, an increment in the level of Arg-1, a mediator of
11

ACCEPTED MANUSCRIPT
anti-inflammatory activity on macrophages was also observed and this can be correlated with
the strong anti-inflammatory activity of compound 3e (Figure 6).
Figure 7: Possible mechanism of the anti-inflammatory activity of compound 3e in
macrophages.
Based on the above-mentioned experimental evidences it can be postulated that the mode of
anti-inflammatory activity of compound 3e is similar to that of the TLR4 inhibitor (CLI-095).
Since, TLR4 activation is generally determined by its expression in target cells as well as the
expression of downstream signalling molecules, a compound that inhibits TLR4 might have
different mode of action i.e. either it can interfere with ligand (LPS) binding or can suppress
the TLR4 expression or can inhibit downstream signalling. In this study, the active compound
(3e) was found to inhibit the TLR4 expression but the propagation of downstream signalling
was disrupted. It was therefore investigated whether compound 3e could interfere with LPS
binding and eventually it has been found that compound 3e does interfere with LPS binding.
Therefore, we presented a model that most likely represents the mode of action of compound
12

ACCEPTED MANUSCRIPT
3e (Figure 7). This postulation was verified by confocal microscopic analyses as well as
immunoblotting that revealed the compound 3e is a putative inhibitor of TLR4. Studies on
the interactions between TLR4 and compound 3e also indicated that it preferentially interacts
with TLR4 through the formation of noncovalent binding, majorly hydrogen bonding.
Therefore, compound 3e disrupt the initial route of inflammation by inhibiting LPS-TLR4
interaction. The possible mechanism of action of 3e in LPS-induced inflammatory responses
in macrophages has been presented in Figure 7.
Structure activity relationship (SAR) studies
Targeting LPS binding site is a convincing approach to stop the dimerization of TLR4. There
are no allosteric binding sites present in TLR4. There is no earlier report demonstrating direct
competition of the synthesized compounds with LPS binding. Therefore, it was decided to
develop new and potential competitive inhibitors of LPS. After biological evaluation of the
synthesized compounds, docking procedure was carried out to get a probable/hypothetical
binding area of our synthesized compounds in TLR4 to explain structure activity relationship
of active as well as non active compounds. This information may further be used to validate
the specific mechanism of action in LPS binding sites of TLR4. It is already reported that Phe
440, Phe 463, Leu 444, Gln 436, Ser 416, Asn 417, Glu 439, Lys 388, Arg 264, Lys 341 and
Lys 362 are the favorable interacting amino acids into the conserved domain of TLR4. Also,
it has been seen that LPS binding area covers all these above mentioned residues. Therefore,
we took all these amino acids into the grid volume during docking study and accomplished
two different options for docking experiment i.e. residue based docking and ligand based
docking methods. Since, CLI-095 is a well established TLR4 inhibitor; we believed the
binding energy as a selection parameter of this inhibitor (CLI-095) to choose the best option
for docking study of all synthesized compounds. Based on this study, it was confirmed that
ligand (LPS) based docking approach was more suitable for all synthesized compounds (3a-
u) in comparison with residue-based docking approach. Additionally, it was also cleared from
the TLR4 crystal structure that binding area of lipopolysaccharide (LPS) with TLR4 is more
electrostatic in nature than its hydrophobic characteristic due to the presence of less aromatic
amino acids / residues in the pocket [42]. Therefore, the formation of π-π stacking interaction
is negligible for any ligands. Docked conformation of CLI-095 identified two important
binding residues (Thr 413 and Ser 415) of the binding domain for its h-bond interaction sites
(Figure 8). In addition, it also showed hydrophobic interactions with surrounding amino acids
including Phe 387, Lys 388, Gly 389, Ile 412, Met 414 and Lys 435 (Table S1, supporting
13

ACCEPTED MANUSCRIPT
information). By considering this information docking analysis were made for all hydrazone
derivatives. After docking experiment, interestingly three different binding sites were
identified for active, less active and almost inactive molecules (Figure 8). This evidence
clearly represented the reason of inhibitory and non-inhibitory activity of synthesized
hydrazone analogues against TLR4. It has been seen that compound 3e, 3f and 3l showed
their binding interactions site nearly similar to the reference molecule, whereas other
remaining compounds exhibited different locations of binding. For example, compound 3e, 3f
and 3l revealed strong h-bond and hydrophobic interactions with active binding residues like
Lys 362, Gly 384, Lys 388, Thr 413 etc. Binding energies (B.E.) of these molecules showed
good correlation with their biological activity (Table 2, Figure 9). Compound 3e showed
most potent activity due to the presence of 3,5-dihydroxyl group which was used to make a
strong h-bond interaction with two active amino acids (Gly 384 and Lys 388). However, less
active compounds (Compound 3a, 3b, 3c, 3d, 3k, 3j etc.) binds to a unlike hydrophobic
binding pocket with reduced binding energy which includes Tyr 65, Phe 121, Tyr 131, Phe
147, Phe 151 etc. amino acids (Table S1, supporting information). Thus, the presence of
methoxy, nitro and phenyl acetate groups were less effective for showing anti-inflammatory
activity via TLR4 binding. Similarly, halogen group containing compounds (Compound 3g,
3h and 3n) bind to a different area of the LPS binding pocket including Ile 52, Leu 54, Leu
78 and Ile 80 (Figure 8). As a consequence, it showed inactivity to TLR4 along with very
poor binding energies (Table S1, supporting information). Hence, the presence of diverse
aromatic groups plays a crucial role to optimize the activity of hydrazone analogous.
Furthermore, 2D and 3D interactions in Figure 9 pointed out the existence of 7-
chloroquinoline moiety has no significant role for its inhibitory activity against TLR4.
Superimposed structures (Figure 9) of CLI-095 with all three active compounds (Compounds
3e, 3f and 3l) also provided the same facts in this regard.
Table 2: Table of binding interactions of active compounds with TLR4.
Compound
3e
∆G (-Kcal/mol)
H-bond interaction Hydrophobic interaction
sites
sites
Ser 360, Asn 361, Lys 362,
Gly 364, Arg 382, Asn 383,
Thr 413
Lys 362, Leu 385,
Lys 388
7.95
Ser 360, Asn 361, Lys 362,
Asn 365, Arg 382, Phe 387,
Val 411, Thr 413
Ser 360, Asn 361, Lys 362,
Asn 365, Arg 382, Gly 384,
3f
3l
7.74
7.81
Asn 361
Thr 413
14

ACCEPTED MANUSCRIPT
Phe 387, Val 411, Lys 435
Figure 8: In this figure, TLR4 inhibitor CLI-095 based docking result of hydrazone
derivatives is illustrated. Here, (A) indicates two different docked conformations of CLI-095.
Green colored conformation was identified through ligand based docking study, whereas
cyan colored docked conformation was recognized by using active residue based docking
method. Red color (---) specify h-bond interaction between CLI-095 and binding pocket
residues. (B) Shows the alignment of two different docked poses of CLI-095. (C) Explain the
diverse binding areas of all synthesized molecules related to their binding affinity towards the
pocket residues. (D) Shows a graphical representation indicating a correlation between IC50
and B.E. (Binding energy).
15

ACCEPTED MANUSCRIPT
Figure 9: Illustration of binding interactions of active hydrazone analogues (Compounds 3e,
3f and 3l) along with the binding pocket of toll-like receptor 4 (TLR4) protein (PDB ID:
3FXI). Figure (A), (B) and (C) shows the 3D binding poses of compounds 3e, 3f and 3l
respectively. Red colored dotted line (---) indicate the h-bond interactions between active
molecules and the amino acids of the binding pocket. Additionally, figure (D), (E) and (F)
shows 2D view of compounds 3e, 3f and 3l correspondingly in conjunction with binding
residues. Figure (G), (H) and (I) showed the superimposed docked conformation of
compound 3e, 3f and 3l with CLI-095 respectively.
CONCLUSION
A set of aryl 7-chloroquinolinyl hydrazone derivatives (3a-u) were prepared in excellent
yield using simple reaction conditions. The synthesized compounds were evaluated for their
anti-inflammatory activities. This study is a maiden report on the anti-inflammatory potential
of the 7-chloroquinolinyl hydrazone derivatives that triggers TLR4-NF-κB pathway in
macrophage by acting as antagonist of TLR4 located on macrophage membrane. Among 21
16

ACCEPTED MANUSCRIPT
synthetic compounds, three compounds (3e, 3f and 3l) were found as potent inhibitors of
TLR4 pathway and compound 3e was found as most promising lead. Molecular docking
study provides additional information regarding the binding affinity of the synthesized
compounds to TLR4-LPS binding site, which reveals that intermolecular polar interactions
are more useful than hydrophobic interactions. Therefore, this study is a platform to present
compound 3e as a promising lead for the therapeutic intervention of the diseases those are
primarily resulted from inflammation.
EXPERIMENTAL
General methods: All reactions were monitored by thin layer chromatography over silica gel
1
coated TLC plates. Silica gel 230-400 mesh was used for column chromatography. H and
13C NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer using DMSO-d6
as solvent and TMS as the internal reference unless stated otherwise. Chemical shift values
are expressed in  ppm. ESI-MS were recorded on a Micromass mass spectrometer.
Elementary analysis was carried out on a Carlo Erba analyzer. All the chemicals and
antibodies used in the study were of analytical grade and commercially available in India.
Dimethyl sulfoxide (DMSO), methanol, ethanol, tris-HCl buffer, glycine, Tween-20, sodium
dodecyl sulfate (SDS), sodium azide etc. were purchased from Merck, India. Anti mouse
TLR4, NF-κBp65, pTAK1, Arg-1 were purchased from Santa Cruz Biotechnology (USA).
Alkaline phosphatase conjugated anti-rabbit IgG, FITC conjugated anti-rabbit IgG, and
Hoechst (H6024) were purchased from Sigma Aldrich (USA). PVDF membrane was
obtained from Milipore (USA). BCIP-NBT was obtained from Amresco (USA). Cell culture
media and fetal bovine serum (FBS) obtained from Gibco (USA). ELISA kits for TNF-α and
IL-10 were obtained from R&D systems (USA). Dexamethasone, CLI-095 and Eschereria
coli lipopolysaccharide (LPS) were purchased from Sigma Aldrich (USA). Cell culture plates
were obtained from Tarson, India. Bovine serum albumin (BSA) was procured from Genei
(India), and DPX was procured from Merck, India.
RAW 264.7, a mouse leukemic macrophage cell lines were obtained from ATCC, USA. Cells
were cultured in DMEM supplemented with 10% FBS and antibiotics (10,000 Units
Penicillin and 10mg/mL Streptomycin) with regular passages.
17

ACCEPTED MANUSCRIPT
Synthetic organic compounds were dissolved in DMSO to prepare the stock and thereafter
diluted using the culture media to achieve the desired treatment concentration. DMSO
concentrations of each compound were maintained at the same level like 1% DMSO control.
Preparation of 7-chloroquinolinyl hydrazone (2): A mixture of 4,7-dichloroquinoline (1;
20 g, 100.9 mmol) and hydrazine monohydrate (5 mL, 103 mmol) was stirred at 90 C for 12
h. The solvents were removed under reduced pressure and co-evaporated with toluene (2 x 20
mL). The solid product was crystallized from EtOH to give pure compound 2 (19 g, 97%).
General Synthesis of aryl 7-chloroquinolinyl hydrazone (3a-u): To a solution of
Compound 2 (194 mg, 1.0 mmol) in EtOH (5 mL) was added aromatic aldehyde (1.5 mmol)
and the reaction mixture was stirred under reflux condition for 3-6 hours (till the starting
material consumed) (Scheme 1). The solvents were removed under reduced pressure and the
product was crystallized from EtOH to give compounds 3a-3i.
Analytical data of the synthesized compounds which were not reported earlier:
(E)-7-Chloro-4-(2-(3,5-dimethoxybenzylidene)hydrazinyl)quinoline (3b): Yield: 73%;
m.p. 151-153 C; 1HNMR (500 MHz, DMSO-d6): δ 8.40 (2H, d, J = 9.0Hz, ArH), 8.33 (1H,
s, ArH), 7.93 (1H, s, ArH), 7.90 (1H, s, ArH), 7.56 (1H, d, J = 7.5 Hz, -N=CHAr), 7.36 (1H,
13
d, J = 5.0 Hz, ArH), 6.95 (2H, s, ArH), 6.56 (1H, s, ArH), 3.82 (6H, s, 2 OCH3); C NMR
(125MHz, DMSO-d6): δ 161.3(2 C), 151.7, 146.2(2 C), 144.6, 137.2, 134.6, 130.4, 129.2,
128.2, 127.5, 125.6, 124.2, 118.7, 107.5, 102.3, 56.4(2 C); ESI-MS (C18H16ClN3O2): 342.1
[M+H]+.
(E)-7-Chloro-4-(2-(3,4-dimethoxybenzylidene)hydrazinyl)quinoline (3c): Yield: 76%;
m.p. 152154 C; 1HNMR (500 MHz, DMSO-d6): δ 8.40 (2H, d, J = 9.0Hz, ArH), 8.33 (1H,
s, ArH), 7.93 (1H, s, ArH), 7.90 (1H, s, ArH), 7.56 (1H, d, J = 7.5 Hz, -N=CHAr), 7.36 (1H,
d, J = 5.0 Hz, ArH), 6.95 (2H, s, ArH), 6.56 (1H, s, ArH), 3.79 (6H, s, di-OCH3); 13C NMR
(125MHz, DMSO-d6): δ 151.3, 150.4, 146.2 (2 C), 137.2, 134.6, 130.4, 129.2, 128.2, 127.5,
125.6, 124.2, 122.3, 118.7, 116.3, 114.5. 56.2(2 C); ESI-MS (C18H16ClN3O2): 342.1 [M+H]+.
(E)-7-Chloro-4-(2-(3-fluoro-4-methoxybenzylidene)hydrazinyl)quinoline (3f): Yield:
73%; m.p. 151-153 C; 1HNMR (500 MHz, DMSO-d6): δ 8.50 (1H, d, J = 9.0Hz, ArH), 8.45
(1H, d, J = 5.5Hz, ArH), 8.41 (1H, s, ArH), 7.87 (1H, s, ArH), 7.61 (1H, d, J = 1.5 Hz, -
N=CHAr), 7.43-7.36 (2H, m, ArH), 6.95 (2H, s, ArH), 6.56 (1H, s, ArH), 3.81 (3H, s,
18

ACCEPTED MANUSCRIPT
13
OCH3); C NMR (125MHz, DMSO-d6): δ 152.4, 151.2, 150.5, 147.3, 145.2, 135.6, 134.6,
131.4, 128.4, 128.1, 127.5, 125.6, 125.2, 117.3, 115.7, 115.1, 60.1; ESI-MS:
(C17H13ClFN3O): 330.0 [M+H]+.
(E)-7-Chloro-4-(2-(3,5-dibromobenzylidene) hydrazinyl)quinoline (3g): Yield: 71%; m.p.
1
144-146 C; HNMR (500 MHz, DMSO-d6): δ 8.31 (2H, d, J = 9.0Hz, ArH), 8.18 (1H, s,
ArH), 7.88 (1H, s, -N=CHAr), 7.52 (1H, d, J = 8.5Hz, ArH), 7.24 (2H, d, J = 1.5Hz, ArH),
7.17 (1H, d, J = 2Hz, ArH), 6.74 (1H, d, J = 8.0Hz, ArH); 13C NMR (125MHz, DMSO-d6): δ
155.3, 151.2(2 C), 147.3, 136.7, 135.4, 131.5, 129.2(2 C), 128.5, 126.3, 124.5(2 C), 118.2,
116.3, 112.4. ESI-MS (C16H10Br2ClN3): 437.9 [M+H]+.
(E)-7-Chloro-4-(2-(3,5-diflurobenzylidene)hydrazinyl)quinoline (3h): Yield: 66%; m.p.
1
142-144 C; HNMR (500 MHz, DMSO-d6): δ 8.70-8.62 (13H, m, ArH), 8.03 (1H, s, -
N=CHAr), 9.05 (1H, s, ArH), 7.83 (1H, s, ArH), 7.70-7.60 (3H, m, ArH), 8.25 (1H, s, ArH),
7.89 (1H, d, J = 4.5Hz, ArH), 7.80-7.76 (2H, m, ArH), 7.53-7.49 (1H, m, ArH), 7.46 (1H, s,
ArH); 13C NMR (125MHz, DMSO-d6): δ 160.3, 151.2, 147.5(2 C), 141.7, 135.6, 133.5,
128.2, 124.3, 120.6, 118.4, 112.6, 106.4(2 C), 104.3; ESI-MS (C16H10ClF2N3): 318.0
[M+H]+.
(E)-4-((2-(7-Chloroquinolin-4-yl)hydrazono)methyl)phenyl acetate (3k): Yield: 72%;
1
m.p. 144-146 C; HNMR (500 MHz, DMSO-d6): δ 8.72 (1H, s, -N=CHAr), 8.42-8.38 (1H,
m, ArH), 7.99 (1H, d, J = 8.5Hz, ArH), 7.85 (2H, d, J = 8.5Hz, ArH), 7.65 (1H, d, J = 8.5Hz,
13
-ArH), 7.29-7.22 (4H, m, ArH), 2.30 (3H, s, -COCH3); C NMR (125MHz, DMSO-d6): δ
169.5, 167.06, 161.1(2 C), 154.3, 153.1, 151.7, 131.8, 131.3, 130.0, 128.7(2 C), 128.2,
122.9(2 C), 121.5, 120.3, 21.3; ESI-MS (C18H14ClN3O2): 340.1 [M+H]+.
(E)-4-(2-(4-(Benzyloxy)benzylidene)hydrazinyl)-7-chloroquinoline (3l): Yield: 73%; m.p.
1
161-163 C; HNMR (500 MHz, DMSO-d6): δ 8.48 (1H, s, ArH), 8.46 (2H, d, J = 4.0Hz,
ArH), 7.93 (1H, s, -N=CHAr), 7.87 (2H, d, J = 8.6 Hz, ArH), 7.80 (1H, d, J = 8.5 Hz, ArH),
7.33-7.59 (6H, m, ArH), 7.11 (2H, d, J = 9.0 Hz, ArH), 5.14 (2H, s, CH2); 13C NMR
(125MHz, DMSO-d6): δ 160.2, 150.4, 149.4(2 C), 146.3, 137.2, 135.2, 129.0(2 C), 128.9,
128.4, 128.2 (2 C), 127.8 (2 C), 125.1, 122.4, 118.7, 116.0, 115.7 (2 C), 101.1, 69.8; ESI-MS
(C23H18ClN3O): 418.1 [M+H]+.
(E)-7-Chloro-4-(2-(3,5-dichlorobenzylidene)hydrazinyl)quinoline (3m): Yield: 70%; m.p.
1
154-156 C; HNMR (500 MHz, DMSO-d6): δ 8.34 (2H, d, J = 9.0Hz, ArH), 8.22 (1H, s,
19

ACCEPTED MANUSCRIPT
ArH), 7.86 (1H, s, -N=CHAr), 7.52 (1H, d, J = 8.5Hz, ArH), 7.26 (2H, d, J = 1.5Hz, ArH),
13
7.10 (1H, d, J = 2.0 Hz, ArH), 6.79 (1H, d, J = 8.0Hz, ArH); C NMR (125MHz, DMSO-
d6): δ 155.3, 151.2(2 C), 147.3, 136.7, 135.4, 131.5, 129.2 (2 C), 128.5, 126.3, 124.5 (2 C),
118.2, 116.3, 112.4; ESI-MS (C16H10Cl3N3): 349.9 [M+H]+.
(E)-7-Chloro-4-(2-(2,6-dichloropyridin-4-yl)methylene)hydrazinyl)
quinoline
(3n):
Yield: 55%; m.p. 148-150 C; 1HNMR (500 MHz, DMSO-d6): δ 8.44 (2H, s, ArH), 8.32 (1H,
s, -N=CHAr), 7.79 (2H, d, J = 8.6Hz, ArH), 7.22 (2H, d, J = 15.0 Hz, ArH), 7.00 (1H, d, J =
13
9.0 Hz, ArH); C NMR (125MHz, DMSO-d6): δ 164.5 (2 C), 160.3, 153.6, 152.1, 148.7 (2
C), 133.6, 129.2, 126.6 (2 C), 122.5, 117.6, 111.2. ESI-MS (C15H9Cl3N4): 350.9 [M+H]+.
(E)-4-(2-(3-(Benzyloxy)-4-methoxybenzylidene)hydrazinyl)-7-chloroquinoline
(3o):
Yield: 71%; m.p. 151-153 C; 1HNMR (500 MHz, DMSO-d6): δ 8.48 (1H, s, ArH), 8.46 (2H,
d, J = 4.0Hz, ArH), 7.93 (1H, s, -N=CHAr), 7.87 (2H, d, J = 8.6 Hz,ArH), 7.80 (1H, d, J =
8.5 Hz, ArH), 7.33-7.59 (6H, m, ArH), 7.11 (2H, d, J = 9.0 Hz, ArH), 5.14 (2H, s, CH2), 3.45
13
(3H, s, -OCH3); C NMR (125MHz, DMSO-d6): δ 157.2, 150.7, 149.1(2 C), 145.4, 137.3,
133.8, 129.4(2 C), 128.7, 128.0, 127.5(2 C), 127.2 (2 C), 125.2, 121.6, 118.3, 117.1, 116.4(2
C), 105.9, 69.7, 55.1; ESI-MS (C24H20ClN3O2): 418.1 [M+H]+.
(E)-3-((2-(7-Chloroquinolin-4-yl)hydrazono)methyl)-2H-chromen-2-one (3q): Yield:
70%; m.p. 148-150 C; 1HNMR(500 MHz, DMSO-d6): δ 11.05 (1H, s, -NH), 9.10 (1H, d, J =
5.0 Hz, ArH), 9.05 (1H, s, ArH), 8.41 (1H, s, ArH), 8.33 (1H, d, J = 9.0 Hz, -N=CHAr), 8.25
(1H, s, ArH), 7.89 (1H, d, J = 4.5Hz, ArH), 7.80-7.76 (2H, m, ArH), 7.53-7.49 (1H, m, ArH),
7.02-6.97 (2H, m, ArH); 13C NMR (125MHz, DMSO-d6): δ 159.3, 153.2, 152.7(2 C),
148.5(2 C), 134.8, 134.2, 129.2, 128.2, 127.5, 126.5, 125.6, 124.4, 122.3, 119.7, 117.5, 115.3,
112.5; ESI-MS (C19H12ClN3O2): 350.0 [M+H]+.
(E)-7-Chloro-4-(2-(4-(trifluoromethyl)benzylidene) hydrazinyl)quinoline (3t): Yield:
61%; m.p. 148-150 C; 1HNMR (500 MHz, DMSO-d6): δ 10.63 (1H, s, -NH), 8.43 (1H, d, J
= 3.7Hz, ArH), 7.94 (1H, s, ArH), 7.72 (5H, d, J = 8.6 Hz,ArH), 7.47 (1H, d, J = 6.1 Hz, -
13
N=CHAr), 7.14 (2H, d, J = 5.6 Hz,ArH); C NMR (125MHz, DMSO-d6): δ 152.3, 146.2,
137.2, 135.7, 134.6, 130.4, 129.1(2 C), 128.2, 127.5, 126.5, 125.6, 124.4, 123.3, 118.7, 116.3,
111.7; ESI-MS (C17H11ClF3N3): 350.0 [M+H]+.
(E)-4-(2-((1H-Pyrrol-2-yl)methylene)hydrazinyl)-7-chloroquinoline (3u): Yield: 57%;
m.p. 153-154 C; 1HNMR (500 MHz, DMSO-d6): δ 10.15 (2H, d, J = 6.4Hz, -NH), 8.49 (2H,
20

ACCEPTED MANUSCRIPT
d, J = 4.4Hz, ArH), 7.93 (1H, s, ArH), 7.55 (1H, d, J = 8.2 Hz,ArH), 7.43 (1H, d, J = 6.6 Hz,
13
-N=CHAr), 7.11-6.95 (4H, m, ArH); C NMR (125MHz, DMSO-d6): δ 151.3, 146.2(2 C),
137.2, 134.6, 130.4, 129.2, 128.2, 127.5, 125.6, 124.2, 118.7, 116.3, 114.5; ESI-MS
(C14H11ClN4): 271.1 [M+H]+.
Determination of anti-inflammatory activity
RAW 264.7 macrophages (1×106) were cultured in six well plates using 10% FBS-
supplemented DMEM and treated with 100 ng/mL LPS for 1 h. Variable concentration of
synthetic compounds (0-50μM) were added in the culture and incubated for 1 h. After
completion of pretreatment, LPS (100 ng) was added accordingly to each culture and
ο
incubated for 6 h at 37 C in a CO2 incubator. Upon completion of treatment, cells were
harvested by scrapping and centrifuged at 2500×g for 3 mins. Cell free culture supernatants
were collected for studying the levels of pro-and anti-inflammatory cytokines. Cell pellets
were subjected to viability determination experiment by MTT assay.
MTT assay
MTT assay was performed following our earlier report [45]. MTT solution (200 μL; prepared
in 100 mM PBS) was added to all the cell pellets, mixed thoroughly and incubated at 37οC
for 1 h in dark. After incubation, the mixture was centrifuged at 4000 rpm for 5 min,
supernatant was discarded and DMSO (200 μL) was added to each pellet. Intensity of the
purple colored formazan crystal was determined at 595 nm in a plate reader (Bio-Rad, USA).
Percentage (%) cell viability was calculated by the formula as mentioned in earlier report
[45].
ELISA Assay
Levels of TNF-α and IL-10 in the cell free culture supernatants were determined by sandwich
ELISA kits (R&D systems, Cat no DY210 (TNF-α), and DY217B (IL-10)) following
manufacturer’s guidelines.
Immunofluorescence Assay
RAW 264.7 cells were prepared for immunofluorescence analysis following Mukherjee et al.
[29]. Briefly, cells were cultured on sterilized cover slips, treated with different compounds
and/or LPS, washed with PBS, and blocked with 1% BSA in PBST (PBS containing 0.1%
Tween 20) for 2 h. After blocking, cells were subjected to incubation with primary antibodies
21

ACCEPTED MANUSCRIPT
ο
(TLR4 and p65NF-κB) overnight in a humidified chamber at 4 C. Cells were washed with
PBST for three times and incubated for FITC-conjugated secondary antibody for 2 h. After
incubation, cells were washed thrice with PBS, counter stained with Hoechst (1μg/mL) and
mounted on glass slides with DPX. Slides were observed and analyzed using a confocal
microscope (Leica).
Immunoblotting Assay
Control and treated RAW 264.7 macrophages from the different experimental
conditions were lysed with CellLyticTM buffer (Sigma), supplemented with protease inhibitor
cocktail (Thermofisher) and centrifuged at 10000×g for 20 mins at 4 C. Supernatants were
collected and protein quality was estimated by Lowry’s method [46]. Macrophage protein (70
μg) from each experimental conditions were resolved in 12.5% SDS-PAGE, immunoblotted
on to methanol charged PVDF membrane and blocked with 1% BSA in PBST. After
blocking membranes were incubated with primary antibodies for TLR4, pNF-κB, pTAK1,
Arg-1, and GAPDH for overnight at 4 C. Membranes were washed twice with PBST, and
incubated with ALP-conjugated rabbit IgG for 6 h at room temperature. After incubation
membranes were washed twice with PBST and developed by adding BCIP-NBT solution at
dark. The colored bands were examined and micrographed using Bio-Rad Chemidoc XR+
(Bio-Rad, USA). Densitometry was performed using Quantity One software.
Determination of toxicity
Toxicity of the test compounds was determined in vitro using RAW 264.7 (ATCC, USA)
macrophage cell line following our earlier report [47]. In brief, cells were cultured in 24 well
plates in DMEM supplemented with 10% foetal bovine serum and treated with different
concentrations (10 μM to 1 mM) of each synthesized compound including 1% DMSO as
control. Each experiment was conducted for 24 h at 37 C in a humidified CO2 incubator.
After completion of treatment, cell viability in each set of experiment was determined by
MTT assay as cited in the earlier section. The toxicity of the compounds was determined in
terms of CC50 which designates the 50% cytotoxic concentration obtained from the dose-
response curve.
Statistical analysis
22

ACCEPTED MANUSCRIPT
All the experiments were conducted in triplicate and repeated for at least five times. Data
were represented as mean±SD. Data were subjected to One-way ANOVA analysis following
Tukey’s multiple comparison test using GraphPad Prism 6.0 (USA). p<0.05 is considered as
statistical significant.
Molecular Modeling study
Molecular docking study was carried out through Autodock 4.2 software [48]. Before
docking, all the structures (Compound 3a-3u) of TLR4 inhibitors were made in Chemdraw 10
and optimize their geometry using the default minimization algorithm. The protein
coordinates of 3FXI [49] was prepared in Autodock. For method standardization, at first
TLR4 inhibitor CLI-095 (Resatorvid) was located into the grid of the binding site of 3FXI in
two different ways. Two different grids were generated on the basis of ‘fit atom method’ with
ligand (LPS) and active / interacting residues of TLR4 [50]. The docking was made in
Autodock by setting the grid size for the search as 68×64×68Å and 60×60×60Å dispersed
around the ligand binding site and active residues in that order along with default grid
spacing (0.375Å). The molecules were allowed for flexible docking in the protein. 100
docking runs were performed for each molecule and the best docked poses were isolated by
identifying their Gibbs free energies (ΔG) of the population. The interactions between protein
and docked molecules were visualized in Autodock, Pymol and Discover studio visualize [48,
51, and 52]. After getting the best docked conformation of CLI-095, it was treated as a new
ligand and extracted the docked conformation. Finally, all synthesized molecules were
aligned to the docked CLI-095 via ‘fit-atom method’ and placed into the protein coordinate
using same grid volume for docking study. To validate docking experiments, the extracted
ligand (LPS) of the co-crystals was docked individually with same protein to compare docked
conformation of LPS along with the crystal forms and also relate the binding interaction sites.
Acknowledgements
U.D. thanks SERB, New Delhi for providing national post-doctoral fellowship (NPDF).
Grateful thanks are due to Department of Science and Technology (DST), Govt. of India for
funding this study with a grant (SR/SO/AS-006/2014). DST-FIST program is thankfully
acknowledged for the Confocal Microscopy facility at Department of Zoology, Visva-
Bharati.
References
23

ACCEPTED MANUSCRIPT
[1] D. Okin, R. Medzhitov, Curr. Biol. 22 (2012) 733-740.
[2] A. Cekici, A. Kantarci, H. Hasturk, T.E. Van Dyke, Periodontol. 2000, 64 (2014) 57-
80.
[3] G.M. Slavich, M.R. Irwin, Psychol. Bull. 140 (2014) 774-815.
[4] P. Braveman, L, Gottlieb, Public Health Rep. 129 (2014) 19-31.
[5] S. Mukherjee, N. Mukherjee, P. Gayen, P. Roy, S. P. Sinha Babu, Curr. Drug Metab.
17 (2016) 937-970.
[6] R. Medzhitov, Cell 140 (2010) 771-776.
[7] C. A. Dinarello, Cell 140 (2010) 935-950.
[8] K. Brune, P. Patrignani, J. Pain Res. 8 (2015) 105-118.
[9] X. S. Wang, H. Y. Lau, Allergy 61 (2006) 503-506.
[10] P. Kalinski, J. Immunol. (Baltimore, Md.: 1950), 188 (2012) 21-28.
[11] M. Barrios-Rodiles, K. Keller, A. Belley, K. Chadee, Biochem. Biophys. Res.
Commun. 225 (1996) 896-900.
[12] D. Sarkar, P. B. Fisher, Cancer Lett. 236 (2006) 13-23.
[13] S. Reuter, S. C. Gupta, M. M. Chaturvedi, B. B. Aggarwal, Free Radic. Biol. Med. 49
(2010) 1603-1616.
[14] T. Liu, L. Zhang, D. Joo, S. C. Sun, Signal Transduct. Target Ther. 2 (2017) e17023.
[15] J. L. Wallace, K. Chapman, W. McKnight, Br. J. Pharmacol. 126 (1999) 1200-1204.
[16] J. Martel-Pelletier, D. Lajeunesse, P. Reboul, J Pelletier, Ann. Rheum. Dis. 62 (2003),
501-509.
[17] S. Maeda, Gastroenterol. Res. Pract. 2010 (2010) 367694; doi: 10.1155/2010/367694.
[18] E. J. Mifsud, A. C. Tan, D. C. Jackson, Front. Immunol. 5 (2014) 79.
[19] S. Mukherjee, S. Karmakar, S.P. Sinha Babu, Braz. J. Infect. Dis. 20 (2016) 193-204.
[20] B. A. Beutler, Blood 113 (2009) 1399-1407.
[21] T. H. Mogensen, Clin. Microbiol. Rev. 22 (2009), 240-273.
[22] G. A. Duque, A. Descoteaux, Front. Immunol. 5 (2014) 491.
[23] W. Gao , Y. Xiong , Q. Li, H. Yang, Front. Physiol. 8 (2017) 508.
[24] M.
M.
Rogero,
P.
C.
Calder,
Nutrients
10
(2018)
432;
https://doi.org/10.3390/nu10040432.
[25] D. Pal, S. Dasgupta, R. Kundu, S. Maitra, G. Das, S. Mukhopadhyay, S. Ray, S. S.
Majumdar, S. Bhattacharya, Nat. Med. 18 (2012) 1279-1285.
[26] A. Oblak, R. Jerala, Clin. Dev. Immunol. 2011 (2011) 609579.
24

ACCEPTED MANUSCRIPT
[27] J. Li, F. Yang, F. Wei, X. Ren, Oncotarget, 8 (2017) 66656-66667.
[28] C. Coban, K. J. Ishii, S. Uematsu, N. Arisue, S. Sato, M. Yamamoto, T. Kawai, O.
Takeuchi, H. Hisaeda, T.o Horii, S. Akira. Int. Immunol. 19 (2006) 67-79.
[29] S. Mukherjee, S. Mukherjee, S. Bhattacharya, S. P. Sinha Babu, Parasite Immunol. 39
(2017). DOI: 10.1111/pim.12389.
[30] S. Mukherjee, S. Mukherjee, T.K. Maiti, S. Bhattacharya, S. P. Sinha Babu, J. Infect.
Dis. 215 (2017) 954-965.
[31] S. Mukherjee, N. Joardar, S.P. Sinha Babu, J. Helminthol 2018,
https://doi.org/10.1017/S0022149X18001050.
[32] R. Nau, H. Eiffert, Clin. Microbiol. Rev. 15 (2002) 95-110.
[33] G. Malgorzata-Miller, L. Heinbockel, K. Brandenburg, J. W. M. van der Meer, M. G.
Netea, L. A. B. Joosten, Sci. Rep. 6 (2016) 34221.
[34] N. N. Kuzmich, K. V. Sivak, V. N. Chubarev, Y. B. Porozov, T. N. Savateeva-
Lyubimova, F. Peri, Vaccines 5 (2017) 34.
[35] M. H. Park, J. T. Hong, Cells 5 (2016) E15. DOI: 10.3390/cells5020015.
[36] M. C. Mandewale, U. C. Patil, S. V. Shedge, U. R. Dappadwad, R. S. Yamgar, Beni-
Suef University J. Basic Applied Sci. 6 (2017) 354-361.
[37] J. C. Coa, W. Castrillon, W. Cardona, M. Carda, V. Ospina, J. A. Munoz, I.D. Velez,
S. M. Robledo, Eur. J. Med. Chem. 101 (2015) 746-753.
[38] O. O. Ajani, K. T. Iyaye, O. Y. Audu, S. J. Olorunshola, A. O. Kuye, I. O.
Olanrewaju, J. Heterocyclic Chem. 55 (2018) 302-312.
[39] V. Murugesan, N. Makwana, R. Suryawanshi, R. Saxena, R. Tripathi, R. Paranjape, S.
Kulkarni, S. B. Katti, Bioorg. Med. Chem. 22 (2014) 3159-3170.
[40] S. G. Alegaon, P. Parchure, L. D. Araujo, P. S. Salve, K. R. Alagawadi, S. S.
Jalalpure, V. M. Kumbar, Bioorg. Med. Chem. Lett. 27 (2017) 1566-1571.
[41] A. L. M. Ribeiro; S. I. de Oliveira; G. Nicolas; A. A. da Chagas; P. G. Nascimento; V.
E. Gomes; da Silva, A. David; C. E. Soares, Exptl. Parasitol. 171 (2016) 10-16.
[42] M. R. Carvalho, L. L. Veras, O. de M. Manoel, P. C. do O., R. F. A. Rocha; Bispo,
Marcelle de Lima Ferreira; B. A. Freire, C. R. Kaiser, V. Marcus, Lett. Drug Design
Discov. 9 (2012) 251-256.
[43] R. C. Montenegro, L. V. Lotufo, M. M. de Odorico; C. do O. Pessoa, F. A. R.
Rodrigues, Bispo L. F. de Marcelle, L. N. de F. Cardoso, C. R. Kaiser, S. de Nora, V.
Marcus, Medicinal Chem. 7 (2011) 599-604.
25

ACCEPTED MANUSCRIPT
[44] C. Fattorusso, G. Campiani, G. Kukreja, M. Persico, S. Butini, M. P. Romano, M.
Altarelli, S. Ros,; M. Brindisi, L. Savini, J. Med. Chem. 51 (2008) 1333-1343.
[45] B. Roy, S. Mukherjee, N. Mukherjee, P. Chowdhury, S. P. Sinha Babu, RSC
Advances 4 (2014) 34487-34499.
[46] O. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, J. Biol. Chem. 193 (1951) 265-
275.
[47] A. S. Ray, N. Joardar, S. Mukherjee, C. H. Rahaman, S. P. Sinha Babu, PLOS One 13
(2018) e0208201. https://doi.org/10.1371/journal.pone.0208201.
[48] G.M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell, A.
J. Olson, J. Comput. Chem. 30 (2009) 2785-2791.
[49] B. S. Park, D. H. Song, H. M. Kim, B. S. Choi, H. Lee, J. O. Lee, Nature 458 (2009)
1191-1195.
[50] U. Debnath, S. Verma, P. Singh, K. Rawat, S. K. Gupta, R. K. Tripathi, H. H.
Siddiqui, S. B. Katti, Y. S. Prabhakar, Chem. Biol. Drug Des. 86 (2015) 1285-1291.
[51] D. Seeliger, De B. L. Groot, J.Comput. Aided Mol. Des. 24 (2010) 417-422.
[52] Discovery studio 2017 R2 BIOVIA, San Diego: Dassault Systèmes, 2017.
http://accelrys.com/products/collaborative-science/biovia-discoverystudio/
visualization.html.
26

ACCEPTED MANUSCRIPT
Graphical Abstract
Ar
N
Cl
N
HN
Cl
Cl
N
1
3a-u
Ar = phenyl moieties substituted with methoxy
helogen, nitro, hydroxy groups and heteroaryl
The anti-inflammatory agents based on their ability to inhibit
pro-inflammatory cytokine secretion from macrophage after
stimulation with lipopolysaccharide (LPS)
27