Mechanistic evaluation of a novel cyclohexenone derivative's functionality against nociception and inflammation: An in-vitro, in-vivo and in-silico approach

Article abstract of DOI:10.1016/j.ejphar.2021.174091

The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharmacological evaluation as a putative ant

Full text of DOI:10.1016/j.ejphar.2021.174091

European Journal of Pharmacology 902 (2021) 174091
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European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
Mechanistic evaluation of a novel cyclohexenone derivative’s functionality
against nociception and inflammation: An in-vitro, in-vivo and
in-silico approach
,
Jawad Khana, Gowhar Alia *, Umer Rashidb, Rasool Khanc, Muhammad Saeed Jand,
Rahim Ullaha, Sajjad Ahmade, Sumra Wajid Abbasif, Atif Ali Khan Khalilf, RobertD.E. Sewellg
a Department of Pharmacy, University of Peshawar, Peshawar, 25120, Pakistan
b Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Pakistan
c Institute of Chemical Sciences, University of Peshawar, 25120, Pakistan
d Department of Pharmacy, Faculty of Biological Sciences, University of Malakand, Chakdara, 18000 Dir (L), Pakistan
e Department of Health and Biological Sciences, Abasyn University, Peshawar, 25000, Pakistan
f Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, 46000, Pakistan
g School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, CF10 3NB, UK. UK
A R T I C L E I N F O
A B S T R A C T
Keywords:
The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-
enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharma-
cological evaluation as a putative anti-nociceptive and anti-inflammatory agent in mice. Initial in vitro studies
revealed that CHD inhibited both cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes and it also
Cyclohexenone
Anti-nociceptive
Anti-inflammatory
Cyclooxygenase-2
5-Lipoxygenase
GABAA/Opioid receptors
reduced mRNA expression of COX-2 and the pro-inflammatory cytokines TNF-α and IL-1β. It was then shown that
CHD dose dependently inhibited chemically induced tonic nociception in the abdominal constriction assay and
also phasic thermal nociception (i.e. anti-nociception) in the hot plate and tail immersion tests in comparison
with aspirin and tramadol respectively. The thermal test outcomes indicated a possible moderate centrally
mediated anti-nociception which, in the case of the hot plate test, was pentylenetetrazole (PTZ) and naloxone
reversible, implicating GABAergic and opioidergic mechanisms. CHD was also effective against both the
neurogenic and inflammatory mediator phases induced in the formalin test and it also disclosed anti-
inflammatory activity against the phlogistic agents, carrageenan, serotonin, histamine and xylene compared
with standard drugs in edema volume tests. In silico studies indicated that CHD possessed preferential affinity for
GABAA, opioid and COX-2 target sites and this was supported by molecular dynamic simulations where
computation of free energy of binding also favored the formation of stable complexes with these sites. These
findings suggest that CHD has prospective anti-nociceptive and anti-inflammatory properties, probably mediated
through GABAergic and opioidergic interactions supplemented by COX-2 and 5-LOX enzyme inhibition in
addition to reducing pro-inflammatory cytokine expression. CHD may therefore possess potentially beneficial
therapeutic effectiveness in the management of inflammation and pain.
1. Introduction
patient quality of life (Ali et al., 2015; Chapman and Gavrin, 1999;
Shahid et al., 2017a, 2017b). Pathologically, pain may be categorized as
The process of drug discovery and development incorporating a
novel chemical moiety with a desirable therapeutic profile is a chal-
lenging task nowadays (DiMasi et al., 2010). Extensive research has
been carried out on pain and inflammation over a number of years,
particularly because these pathological conditions can greatly influence
nociceptive, neuropathic or inflammatory and if protracted, it may
progress into a chronic pain syndrome involving additional symptoms
such as anxiety and depression. Nociceptive pain is typically initiated by
stimulation of somatic sensory receptors designated as nociceptors,
which then transmit pain impulses to the central nervous system (CNS).
* Corresponding author. Department of Pharmacy University of Peshawar, Peshawar, 25120, KP, Pakistan.
E-mail addresses: gowhar_ali@uop.edu.pk, gohar.pharmacist@gmail.com (G. Ali).
https://doi.org/10.1016/j.ejphar.2021.174091
Received 31 October 2020; Received in revised form 25 March 2021; Accepted 31 March 2021
Available online 16 April 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.

J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
Alternatively, neuropathic pain arises from damage or lesions to the
nervous system (Van Hecke et al., 2014). Active inflammation is the
hallmark of inflammatory pain and is characterized by the presence of
2. Material and methods
2.1. Chemicals and drugs
inflammatory mediators such as interleukin, TNF-α, prostaglandins
(PGE2, PGI2, TXA2), histamine, serotonin, bradykinin and leukotrienes
(LTs) (Fernandes et al., 2015). These biochemical substances produce
changes in neuronal sensitivity and invoke the onset of tissue hyper-
sensitivity associated with inflammation (Kidd and Urban, 2001).
Currently, opioids and non-steroidal anti-inflammatory drugs (NSAIDs)
are the analgesic agents of choice often utilized in the management of
inflammatory pain. However, it is well documented that persistent use
of NSAIDs may well cause deleterious effects such as ulceration, hem-
orrhage or even perforation in the gastrointestinal tract, cardiovascular
system disorders and kidney damage (Gutthann et al., 1996; Jones et al.,
2008). Similarly, opioid analgesics are considered highly effective as
analgesics, but they are associated with dependence liability and other
side effects which may limit their usefulness (Laxmaiah Manchikanti
et al., 2010; Mayer et al., 1995; Shahid et al., 2016). Consequently, there
is a genuine need for substitute drugs that retain the analgesic and
anti-inflammatory effectiveness of conventional analgesic agents
without their untoward effects (Fawad et al., 2018; Islam et al., 2017,
2019).
Naloxone, serotonin, histamine, PTZ, xylene, indomethacin, lambda
carrageenan and aspirin were purchased from (Sigma-Aldrich, USA).
Formaldehyde was procured from Merck (Germany), glacial acetic acid
was obtained from Pancreac (Spain), tramadol (Tramal® 50 mg/ml) was
acquired from Searle Ltd (Pakistan). Fresh preparation of chalcone was
carried out in the laboratory of ICS (University of Peshawar, Pakistan).
Ethyl acetoacetate, ethyl acetate and potassium carbonate were pur-
chased from Merck (Pakistan). N-hexane and ethanol were procured
from Scharlau (Lahore, Pakistan). The cDNA synthesis kit, TRIzol re-
agent, master mix and primers were acquired from Thermofishcer Sci-
entific (USA).
2.2. Chemistry
2.2.1. General
A Gallenkamp melting point apparatus was used to determine
melting points. Purity was checked by thin layer chromatography (TLC).
A Shimadzu IR Prestige-21 FT-IR Spectrometer Instrument (Tokyo,
Japan) was utilized to record the Infrared spectra. 13C and 1H NMR
analyses (Agilent AV-300,400 and 500 Tokyo, Japan) were accom-
plished with D2O and DMSO-d6 as solvents. Mass spectra (ESI-MS) were
obtained on (Qp 2010 plus, Shimadzu, Tokyo, Japan). PerkinElmer 2400
CHN/O Analyzer was operated to determine Elemental analysis.
The key role of the cyclohexenone ring is well established in the field
of biomedical research. It has been documented that this functionality is
an integral part of several interesting compounds and is of considerable
significance for the development of potentially valuable drugs (Das and
Manna, 2015; Fang et al., 2012). Chemically, the cyclohexenone nu-
cleus, serves as a convenient intermediate for synthesizing various het-
erocyclic compounds including fused pyrazoles, isoxazoles, quinazolines
(Senguttuvan and Nagarajan, 2010) and 2H-indazole (Gopalakrishnan
et al., 2008). Cyclohexenones are cyclohexane derivatives with a
carbonyl group at position-1 and a carbon-carbon double bond at
position-2 (Fig. 1). The enone functional group and substitution at a
carbon atom in the six membered ring have been used to synthesize
other substituted cyclohexenones (Johnson et al., 2016). The pharma-
2.2.2. Synthesis of ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-
enecarboxylate
The synthesis was conducted according to the synthetic protocol as
shown in Scheme 1. (E)-3-(-4-methoxyphenyl)-1-phenylprop-2-en-1-one
(10 mmol) was refluxed with ethyl acetoacetate (20 mmol) in the
presence of K2CO3 catalyst in 20 ml of ethanol for 3 h. The product
obtained was recrystallized from ethanol; a brownish yellow powder
was obtained having a yield of 85%. M.p = 92-95 0C; Rf = 0.51 n-
cological
properties
of
cyclohexenone
derivatives
include
anti-inflammatory and anti-nociceptive effects (Ahmadi et al., 2012;
Lednicer et al., 1981a, 1981b; Liu et al., 2013; Ming-Tatt et al., 2012,
2013; Sheorey et al., 2016; Wang et al., 2011) as well as
anti-neuropathic and antioxidant activity (Khan et al., 2019). The pre-
sent study was undertaken to evaluate a novel cyclohexenone derivative
Hexane/ethyl acetate (7:3); IR (KBr) υmax cm-1: 3077 (Ar–H), 1689
(ketone C O), 1735 (Ester C O) 2870 (Aliphatic C-H); 1H-NMR
–
–
–
–
(CDCl3, 400 MHz) δ:6.9–7.5 (m, Ar–H),3.05 (d, 2H, J = 2.3), 2.9 (t,1H J
= 5.0, C-3), 2.6–2.8 (q, 5H, CH2CH3, J = 7.0); 13C-NMR (100 MHz,
–
–
CDCl ) δ: 199.0 (C O), 125-130 (Ar-CH), 112 (C-6), 40.2 (OCH ), 159.0
3
3
(CHD;
Ethyl
6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohex-
(C-19), and 44.39 (C-3). EI-MS; m/z (rel. int. %) 351 (M+), CHN Anal.
e-3-enecarboxylate) as a possible inhibitor of cyclooxygenase-2 (COX-2)
and 5-LOX pro-inflammatory enzymes and subsequently examine its
effects against nociception using in vivo mouse models of pain and
inflammation. Additionally, the anti-nociceptive activity of CHD was
also investigated in the presence of pentylenetetrazole (PTZ) and
naloxone in order to probe any possible underlying mechanisms, which
might have been corroborated by in silico and in vitro studies.
Calcd for: C, 75.41; H, 6.33; O, 18.26. Found: C, 74.81; H, 6.38. Formula:
C
22H22O4, C = 22, H = 22, and O = 4.
2.3. In vitro activities
2.3.1. 5-LOX inhibition assay
The inhibitory potential of CHD was examined by utilizing human
recombinant 5-LOX. In this assessment, the enzyme inhibition was
determined through residual enzyme potential following 10 to 15 min
incubation at 25 ◦C in an incubator (Jan et al., 2020; Wisastra et al.,
2013). The activity was estimated through linoleic acid (lipoxygenase
substrate) conversion into hydroperoxy-octadecadienoate (HPOD). The
alteration rate was calculated in the form of absorbance at 234 nm with
UV–visible spectrophotometer. Ethylene diamine tetra acetic acid
(EDTA 2 mM) and CaCl2 (2 mM) containing Tris buffer (50 mM) of PH
7.5 was used as an assay buffer for this assay. The enzyme 5-LOX (20,
Fig. 1. Chemical structure of Ethyl 6-(-4-methoxyphenyl)-2-oxo-4-phenyl-
Scheme 1. Synthetic scheme of Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenyl-
cyclohex-3-enecarboxylate.
cyclohexe-3-enecarboxylate.
2

J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
000 U/ml) was diluted with buffer in a ratio of 1:4000. The assay buffer
was then diluted with 100 mM of inhibitor formerly blended with
DMSO. Linoleic acid was then diluted with ethyl alcohol to 20 mM.
Subsequently, various concentrations of CHD ranging from 31.25 to
2.4.2. Acute toxicity study of compound CHD
The acute toxicity profile of CHD was evaluated after intraperitoneal
(i.p) injection of selected doses (on
a sequential dose-doubling
increasing scale viz 15, 30, 60, 120 or 240 mg/kg (n = 6)). Animals
were observed at 30–60 min and 24–72 h for any abnormal behaviour
(Akbar et al., 2016).
1000
adenosine triphosphate (2 mM), 790
hibitor (1 mM) and then incubated for 10 min duration. Then to this
μg/ml and 1 ml of enzyme solution (1:4000) was mixed with 100 μl
μl of Tris buffer plus 100
μl in-
mixture was added 10
μ
l of substrate solution (20 mM) and after 10 s
2.4.3. Anti-nociceptive activity
mixing of the enzyme with substrate, the substrate conversion rate was
monitored. The reaction rate in the absence of inhibitor was employed as
positive control. The standard inhibitor agent used in this assay was
zileuton.
2.4.3.1. Anti-nociceptive activity of compound CHD and a standard drug in
the acetic acid abdominal constriction test. Food and water were with-
drawn 120 min prior to animal experiments. One percent acetic acid (10
ml/kg) i.p injection was used to induce abdominal constriction as a
reflection of tonic nociception. Five min after acetic acid i.p injection,
the incidence of abdominal constrictions was recorded over a 20 min
period (Abbas et al., 2011). The animals were randomly allocated to
different investigational groups (n = 6). Group I received normal saline
as vehicle, group II-IV received standard aspirin (15–45 mg/kg), group
V-VII received test compound (CHD) (15–45 mg/kg) via i.p injection, 30
min prior to 1% acetic acid injection. Percentage analgesia was calcu-
lated using the following formula:
2.3.2. COX-2 inhibition assay
The COX-2 inhibitory activity of the test compound was evaluated
according to a previously validated procedure (Burnett et al., 2007; Jan
et al., 2020). A COX-2 enzyme solution (300 U/ml) was prepared. For
activation, 10
and then mixed with 50
μ
l of enzyme solution was kept for 5 to 6 min on ice (4 ◦C)
μ
l of co-factor solution comprising 0.9 mM
glutathione, 1 mM hematin in 0.1 mM Tris buffer (pH 8.0) and 0.24 mM
tetramethyl-p-phenylenediaminedihydrochloride (TMPD). Then various
concentrations (31.25 to 1000
solution (60
followed by initiation of the reaction by adding 30 mM arachidonic acid
(20
l) and keeping this mixture at 37 ◦C for a duration of 15 min. Af-
μg/ml) of test sample (20 μl) plus enzyme
% protection = (1- m ean num ber of abdom inal constrictions of the treated drug
/ m ean num ber of abdom inal constrictions of the vehicle control) × 100
μl) were maintained at room temperature for 5 to 10 min,
μ
terwards, the reaction was terminated by addition of hydrochloric acid
(HCL) and absorbance was measured via a UV–visible spectrophotom-
eter at 570 nm. COX-2 percentage inhibition was calculated from the
absorbance value per unit time. In the study Celecoxib was utilized as
the standard inhibitor agent.
2.4.3.2. Anti-nociceptive activity of compound CHD compared to a stan-
dard drug in the hot plate test. The hot plate analgesiometer, was kept at a
constant temperature of 54±0.10 C. After placement on the plate, animal
nociceptive reaction latencies (s) were determined to the following
escape end points: paw licking, flinching or jumping and a 30 s cut off
time was imposed after which mice were removed from the stimulus
(Ahmad et al., 2017; Rukh et al., 2020). Animals were randomly
assigned to groups (n = 6) and administered saline or drug treatment
intraperitoneally. Group I received normal saline as vehicle, group II
received standard drug (tramadol, 30 mg/kg, i.p), groups III-V received
the trial compound (CHD, 15–45 mg/kg, i.p).
2.3.3. Reverse transcription polymerase chain reaction (RT-PCR)
Post-mortem mouse paw sub plantar tissues were removed 5 h after
carrageenan administration and RNA was extracted using TRIzol re-
agent according to the manufacturer’s protocol. The total RNA was
reverse transcribed to cDNA following a standard protocol. The primers
for targeted genes use were;
COX-2: F-5’-GGAGAGACTATCAAGATAGTGATC -3’, R- 5’- ATGGT-
CAGTAGA-CTTT-TACAGCTC-3’. TNF-
α:
F-5’-CTTCTCCTTCCTGATC
2.4.3.3. Pharmacological antagonism study of CHD compared to a stan-
dard drug. In order to evaluate the possible involvement of GABAA or
opioid receptors in the anti-nociceptive activity of CHD, mice were
administered PTZ (15 mg/kg; i.p) or naloxone (1 mg/kg; subcutaneously
(s.c)) 10–20 min prior to i.p dosing with saline, CHD or standard drug.
Hot plate latencies were recorded 30,60 and 90 min after administration
of each drug (Muhammad et al., 2012). Percentage protection against
nociception was determined using the following formula:
GTGG-3’;
R-5’-GCTGGTTAT-CTCTCAGCTCCA-3’.
IL-1β:
F-5’-
AGAAGCTTCCACCAATACTC-3’, R-5’-AGCACCTAG-TTGTAAGGAAG-
3’. GAPDH: F-5’-TGCACCACCAACTGCTTAGC-3’; R- 5’-GGCATG-
GACTGTGGTCATGAG3’ was used as a housekeeping gene (Cheon et al.,
2009; Khalid et al., 2018). Amplified products were separated using
1.5% Agarose gel electrophoresis, analyzed with image J software
(Almeer et al., 2019; Ullah et al., 2021).
% protection = (Test latency
× 100
̶ baseline latency)/ (cut off tim e ̶ baseline latency)
2.4. In vivo pharmacological evaluation
2.4.1. Animals
Mice (Balb-C) of either sex weighing 18–30 g were used during the
investigation unless otherwise stated. Animals were maintained on
standard laboratory food and water ad libitum at an ambient temperature
of 22 ± 2 ◦C through a thermostatically controlled air conditioning
system on a 12/12 h light and dark cycle and they were habituated to
laboratory conditions for 2 h before experiments.
2.4.3.4. Anti-nociceptive activity of CHD compared to a standard drug in
the tail immersion test. Each animal was gently held in a vertical position
and half of the tail was immersed in a water bath maintained at a
temperature of 55 ± 0.5 ◦C. A nociceptive reaction latency (s) was
determined to a tail flick end point and a cut off time of 15 s imposed
after which animals were removed from the stimulus. Any non-
responders within the cut-off time were excluded from the study. The
vehicle, standard tramadol (30 mg/kg) and test compound (15–45 mg/
kg) were administered i.p to their respective groups. The readings were
taken at 30, 60, 90 and 120 min after drug administration (Sewell and
Spencer, 1976).
2.4.2. Ethical approval
The study and all in vivo protocols were conducted under a project
entitled “Studies on the nociceptive, inflammatory and neuropathic pain
relieving potential of a cyclohexenone derivative.” It was approved by
the Research Ethical Committee of the Department of Pharmacy, Uni-
versity of Peshawar, Pakistan which issued a certificate number of 01/
EC/18/Pharm. Furthermore, animal experiments were performed in
compliance with the Animals Scientific Procedure Act UK (1986).
2.4.3.5. Anti-nociceptive activity of CHD and standard drug in the formalin
induced biphasic pain model. Mice were administered a sub plantar
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J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
injection of 20 μl of freshly prepared 2% formalin in the right hind paw.
Table 1
Protein Data Bank (PDB) code numbers, names of their co-crystalized ligands
and resolution for the enzymes studied.
Thirty min prior to formalin injection, groups I-VI, received intraperi-
toneally normal saline as vehicle, standard drugs indomethacin (10 mg/
kg) or diclofenac (10 mg/kg), and CHD (15–45 mg/kg). The nociceptive
reaction time (s) (latency to biting, licking, paw lifting or flinching) was
measured in two phases: first phase (0 to 5 min) and second phase
(10–30 min) after the formalin injection (Silva et al., 2017; Мaione
et al., 2020).
Enzyme/
Receptor
PDB
code
Co-crystalized ligand
Resolution
(Å)
COX-1
1EQG
1CX2
4COF
4DKL
4EJ4
Ibuprofen
2.61
3.00
2.97
2.8
enzyme
COX-2
1-Phenylsulfonamide-3-trifluoromethyl-5-
parabromophenylpyrazole (SC-558)
Benzamidine
enzyme
GABAA
receptor
2.4.4. Anti-inflammatory activity
μ
-opioid
β-Funaltrexamine
receptor
(μ-opioid receptor antagonist)
2.4.4.1. Anti-inflammatory activity of compound CHD and standard in a
carrageenan induced paw edema model. Mice were treated with normal
saline, aspirin (50–150 mg/kg) or test compound (CHD, 15–45 mg/kg, i.
p) 30 min before s.c injection of 0.05 ml of freshly constituted carra-
geenan (1%) in the right hind paw. A digital Plethysmometer was uti-
lized to determine the inflammation in terms of paw edema volume (ml)
at hourly intervals up to 5 h post carrageenan injection (Ali et al., 2013).
δ-opioid
Naltrindole (δ-opioid receptor antagonist)
3.4
receptor
κ-opioid
receptor
4DJH
(3R)-7-Hydroxy-N-{(2S)-1-[(3R,4R)-4-(3-
hydroxyphenyl)-3,4-dimethylpiperidin-1-
yl]-3-methylbutan-2-yc-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide
(JDC)
2.9
2.4.4.2. Anti-inflammatory activity of compound CHD and standard drug
in a histamine induced paw edema model. Inflammation was induced in
mice (25–30 g) by sub plantar injection of 0.1 ml freshly constituted
histamine (1 mg/ml) in the right hind paw. Paw inflammation swelling
was measured by means of plethysmometer previously described in the
carrageenan test (Mequanint et al., 2011).
same protocol as explained in our previous study (Abbasi et al., 2016).
MD simulations facilitate understanding of the binding pattern and
determine the stability of selected receptor-CHD docked complexes.
Using AMBER 18 software, six different systems were prepared to run
MD simulations for 50 ns each (Case et al., 2010). In order to verify the
structural variations and convergence of the simulated systems, the
CPPTRAJ module of AmberTools18‘s was used to estimate the RMSDs
for all the studied systems.
2.4.4.3. Anti-edema activity of CHD and a standard drug in the serotonin
induced paw volume model. Mice were administered serotonin (0.001
mg/ml s.c) into the plantar surface of the right hind paw. The ensuing
inflammation and paw edema was measured by plethysmometer (Mas-
resha et al., 2012).
2.6. Binding free energy calculations
The MMPB/GBSA methods, integrated in AMBER 18, were employed
to calculate the binding free energies for all six systems (Miller III et al.,
2012). Binding free energy calculations were performed on 100 snap-
shots taken from the MD trajectories as described previously by Abro
and Azam, . The binding free energy can be expressed as:
2.4.4.4. Anti-inflammatory action of compound CHD and standard drug in
the xylene provoked ear edema model. In mice weighing 25–35 g, ear
edema was evoked by topical application of 0.03 ml of xylene to the
internal and outer surface of the right ear while the left ear was used as
control. Thirty min before induction of xylene edema, saline vehicle was
administered i.p to group I, standard indomethacin (10 mg/kg) or
diclofenac (15 mg/kg) to groups II-III and test compound CHD (15–45
mg/kg) to groups IV-VI respectively. Subsequently, 15 min after xylene
application, animals were killed and the ears were amputated then
weighed. The mean weight difference between right and left ears was
then determined (Manouze et al., 2017).
ΔGbind = ΔGcom plex – [ ΔGreceptor + ΔGligand
]
where ΔG is the Gibb’s free energy calculated by MMGB/PBSA.
2.7. Statistical analysis
The data were analyzed statistically utilizing Graph Pad Prism
Software, version 5, for manifold assessments via one-way analysis of
variance (ANOVA) with Post-hoc Dunnett’s test. Outcomes were regar-
ded as statistically significant at P < 0.05.
2.5. In silico activity
2.5.1. Docking studies
3. Results
Docking studies were executed through the Molecular Operating
Environment (MOE) version 2016.08 docking program. Three-
dimensional (3D) structures of the enzymes, GABAA and opioid re-
ceptors with their co-crystalized ligands were obtained from the Protein
Data Bank as listed in Table 1. The docking algorithm was validated by
re-docking of native ligands as shown in Table 1. The computed root
mean square deviation (RMSD) between the experimental and re-docked
poses was found within a threshold limit < 2 Å. The 3D structures of the
compound were constructed in MOE by utilizing Builder Module. Energy
minimization of the ligand, preparation of structures of the downloaded
enzymes and active site identification was carried out according to our
earlier validated methods (Iftikhar et al., 2017, 2018; Rashid et al.,
2016). Assessment of docking outcomes and scrutiny of their surface
with graphical demonstrations were accomplished with discovery studio
visualizer and MOE (Systemes, 2015).
3.1. In vitro activities
All enzyme suppression results are presented as the mean of triplicate
determinations for each concentration studied and an IC50 value was
extrapolated from the overall inhibitory concentration relationships.
3.1.1. 5-LOX inhibitory activity
The 5-LOX inhibitory activity of CHD was examined at various
concentrations ranging from 31.25 to 1000 μg/ml and the compound
displayed a potent inhibition of 5-LOX with an extrapolated IC50 value of
10. 27 μg/ml as compared to the standard 5-LOX inhibitor drug zileuton
(extrapolated IC50 = 5.50 μg/ml) over the same tested concentration
range (Table 2).
3.1.2. COX-2 inhibitory activity
2.5.2. Molecular dynamic simulation of complexes
CHD disclosed a potent inhibitory action on the COX-2 enzyme as
shown in Table 3. It was also evident from the outcomes that CHD
Molecular Dynamic (MD) simulations were performed using the
4

J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
the maximum tolerated dose (MTD) which was devoid of unacceptable
Table 2
5-LOX enzyme inhibitory activity of CHD in comparison with zileuton as a
standard 5-LOX inhibitor drug.
toxicity for CHD was >240 mg/kg.
3.2.2. CHD attenuation of chemically induced tonic nociceptive behaviour
Injection of acetic acid (1%) was accompanied by a significant rise in
the nociceptive response perceived as an onset increase in the incidence
of abdominal constriction. The percentage protection against this
chemically induced tonic nociception in the group of animals treated
with CHD at a lower dose (15 mg/kg) decreased the nociceptive
response as evidenced by an increase in the percentage protection
(44.66%, P < 0.05). Likewise, the mid-range CHD dose (30 mg/kg) also
protected against acetic acid evoked abdominal constriction (49.78%,
P< 0.01). and the higher dose (45 mg/kg) had an even greater anti-
nociceptive effect (59.81%) reflecting dose dependent activity relative
to the saline treated animals. The aspirin positive control also yielded a
dose dependant anti-nociceptive response (15–45 mg/kg) versus the
saline controls (Fig. 3).
Compound
Conc. (
μ
g/
% 5-LOX inhibition
Extrapolated
IC50 g/ml
ml)
(Mean ± S.E.M)
μ
Cyclohexenone
1000
500
89.44 ± 0.55b
83.17 ± 0.72c
78.30 ± 0.64c
73.34 ± 0.63c
68.30 ± 0.64c
61.93 ± 1.13c
93.55 ± 0.40
89.37 ± 1.65
85.50 ± 0.40
79.60 ± 0.90
74.17 ± 0.72
70.35 ± 0.45
10.27
derivative (CHD)
250
125
62.5
31.25
1000
500
Zileuton
5.50
250
125
62.5
31.25
Data is represented as mean ± S.E.M; Values were significantly different as
compared to the positive control (zileuton); n = 3, b = P < 0.01, c = P < 0.001.
3.2.3. CHD attenuation of phasic thermal nociception
In the hot plate test, the saline treated animal group displayed a
control escape response from the thermal nociceptive stimulus of 7.3%,
7.5% and 7.7% after 30, 60 and 90 min respectively. The lower dose of
CHD was ineffective in producing any detectable anti-nociception be-
tween 30 and 90 min (19.3%–17.6%). However, CHD at 30 mg/kg did
produce an anti-nociceptive effect at 30 min (24.5%) and 60 min
(20.8%) but this was not evident after 90 min (19.5%). A greater anti-
nociceptive response was noted at the 45 mg/kg dose (27.3%, 24.0%
and 20.3% at 30, 60 and 90 min respectively) while the tramadol (30
mg/kg) positive control produced an even bigger response 77.6%,
72.0% and 69.7% at 30, 60 and 90 min respectively (Fig. 4).
Table 3
COX-2 enzyme inhibitory assay of CHD in comparison with celecoxib as a
standard COX-2 inhibitor drug.
Compound
Conc. (
μg/
% COX-2
Extrapolated IC50
g/ml
ml)
inhibition
μ
(Mean ± S.E.M)
Cyclohexenone derivative
(CHD)
1000
500
88.91 ± 1.30c
85.00 ± 0.30c
78.76 ± 0.58c
73.67 ± 0.61c
67.74 ± 0.61c
63.47 ± 0.56c
95.20 ± 0.15
91.17 ± 0.53
86.98 ± 0.85
81.20 ± 0.65
77.80 ± 0.37
73.11 ± 1.20
8.94
250
125
62.5
31.25
1000
500
Celecoxib
4.30
3.2.4. CHD attenuation of phasic nociception in the tail immersion test
CHD produced a measurable anti-nociceptive response in the tail
immersion test at the 30 mg/kg dose (30 min). However, the 45 mg/kg
dose produced a peak response at 60 min which subsided by 120 min.
Treatment with the positive tramadol control (30 mg/kg), produced an
intense long-acting anti-nociceptive effect which lasted up to 120 min
(Fig. 5).
250
125
62.5
31.25
Data is represented as mean ± S.E.M; Values were significantly different as
compared to the positive control (celecoxib); n = 3, c = P < 0.001.
possessed valuable COX-2 inhibitory activity in comparison with the
standard COX-2 inhibitor drug celecoxib. Thus, the IC50 value for CHD
3.2.5. CHD attenuation of the formalin induced biphasic nociceptive
response
was extrapolated as 8.94
4.30 g/ml) (Table 3).
μg/ml in contrast to that of celecoxib (IC50 =
Administration of formalin in the sub-plantar mouse hind paw
initiated a marked nociceptive response as indicated by an increase in
the duration of biting, licking, lifting and flinching of the affected paw.
This was observed throughout the first phase (0–5 min) and also the
second phase (15–30 min) following formalin administration in the sa-
line treated animals. Treatment with CHD (15 mg/kg) only diminished
the second phase of formalin induced nociception. Conversely, the 30
mg/kg CHD dose was more effective in that it markedly reduced the
formalin nocifensive response in both the second and first phases (P<
0.05). Similarly, treatment with the higher CHD dose (45 mg/kg) did
induce an anti-nociceptive response in the first phase, but a more sta-
tistically significant response in the second phase. The indomethacin
and diclofenac positive controls both at 10 mg/kg generated comparable
anti-nociception to CHD in both phases (Fig. 6).
μ
3.1.3. RT- PCR
To further investigate the anti-inflammatory potential of CHD, RT-
PCR was utilized to assess the mRNA levels of COX-2 enzyme and the
pro-inflammatory cytokines TNF-α and IL-1β in the carrageenan induced
paw edema test in mice. The outcomes of this assessment revealed that
CHD (45 mg/kg) significantly reduced the mRNA expression of COX-2
(P<0.001), while in the case of TNF-α and IL-1β, CHD also produced a
reduction (P<0.01) compared to the carrageenan treated vehicle group.
Aspirin (150 mg/kg) as the standard positive control decreased
(P<0.001) the expression of COX-2, TNF-α and IL-1β as presented in
(Fig. 2).
3.2.6. Opioidergic and GABAergic mediation of CHD anti-nociception
Any possibility of GABAergic or opioidergic mechanisms underlying
the anti-nociceptive effect of CHD in the hot-plate test were probed
using pentylenetetrazole (PTZ) and naloxone as respective antagonists.
Hence, the anti-nociceptive effect of CHD (30 and 40 mg/kg), was
significantly antagonized (P< 0.001) by naloxone (1 mg/kg) implicating
3.2. In vivo pharmacological activity
3.2.1. Acute toxicity of CHD
After i.p. injection of selected doses of CHD (15–240 mg/kg; n = 6),
there was no acute toxicity observed in gross animal behaviour, neither
was any incidence of mortality recorded up to the highest dose. Thus,
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European Journal of Pharmacology 902 (2021) 174091
Fig. 2. Agarose gel electrophoresis (A) quantification of CHD activity on the mRNA level of COX-2 (B), TNF-
α
(C), and IL-1β (D) in carrageenan induced hind paw
edema in mice. The results are shown in relative arbitrary units (A.U). Bars represent mean expression in A.U ± S.E.M. ###P < 0.001 compared to the saline group.
**P< 0.01, ***P< 0.001 compared to the vehicle group.
Fig. 3. Anti-nociceptive activity of (A) CHD and (B) the positive control, aspirin in the acetic acid (1%) induced abdominal constriction test. Each bar represents
mean percentage protection ±S.E.M). *P< 0.05, **P< 0.01, ***P< 0.001 as compared to the saline treated group (one-way ANOVA followed by post hoc Dunnett’s
test), (n = 6 mice per group).
the involvement of an opioidergic mechanism. Likewise, in animals
treated with the opioid agonist, tramadol (30 mg/kg) as a positive
control, naloxone also blocked the anti-nociceptive response (Fig. 7A).
Administration of PTZ (15 mg/kg) did not modify the ani-nociceptive
action of tramadol (30 mg/kg), but it did markedly decrease the anti-
nociceptive response of CHD (30 and 45 mg/kg) in the hot plate para-
digm. This would tend to suggest an involvement of a GABAergic
mechanism in the anti-nociceptive action of CHD but not tramadol
(Fig. 7B).
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European Journal of Pharmacology 902 (2021) 174091
Fig. 6. Anti-nociceptive activity of CHD and the positive controls, indometh-
acin (Indo), and diclofenac (Diclo) in the formalin induced paw nociceptive
test. Each bar represents mean nociceptive response in s ± S.E.M). *P< 0.05,
**P< 0.01, ***P< 0.001 as compared to the saline treated group (one-way
ANOVA followed by post hoc Dunnett’s test), (n = 6 mice per group).
throughout the advanced stages of inflammation i.e. up to 5 h of the
study duration. A dose dependent anti-inflammatory effect was pro-
duced by CHD in the three paradigms of paw edema. Treatment with
CHD (15, 30 and 45 mg/kg) reduced the inflammatory response evoked
up to 5 h after administration of carrageenan (Fig. 8A), histamine
(Fig. 9A), and serotonin (Fig. 10A), Treatment with the aspirin positive
control, (50–150 mg/kg) consistently displayed an anti-inflammatory
effect up to 5 h after injection of carrageenan, serotonin or histamine
in the inflammatory paradigms (Figs. 8B, 9B and 10B).
Fig. 4. Anti-nociceptive activity of CHD and the positive control, tramadol, in
the hot-plate test. Each bar represents mean percentage protection ±S.E.M).
*P< 0.05, **P< 0.01, ***P< 0.001 as compared to saline treated group (one-
way ANOVA followed by post hoc Dunnett’s test), (n = 6 mice per group).
In the xylene provoked ear inflammatory edema paradigm, appli-
cation of xylene produced a marked inflammatory response as observed
by the increased ear weight recorded in the saline treated control ani-
mals (Fig. 11). This marked oedematous change was significantly
countered by treatment with CHD (30 and 45 mg/kg). The positive anti-
inflammatory control drugs, indomethacin (10 mg/kg) and diclofenac
(15 mg/kg) both produced a noteworthy decline in the augmented ear
weight edema induced by xylene, as compared to the saline treated
controls (Fig. 11).
3.3. In silico studies
3.3.1. Molecular docking
Docking studies were performed to explore any possible underlying
mechanism(s) of CHD anti-nociception and anti-inflammatory activity.
Accordingly, simulations were carried out on: (1) cyclooxygenase-2
enzyme (COX-2), (2) GABA receptors and (3) opioid μ-, δ- and κ-re-
ceptors using Molecular Operating Environment (MOE 2016.08,
Chemical Computing Group, Canada). Data concerning three-
dimensional (3D) structures of enzymes with their co-crystalized li-
gands were downloaded from the Protein Data Bank (PDB) listed in
Table 1 and the docking algorithm was validated by re-docking native
co-crystalized ligands (Table 1). The computed root mean square devi-
ation (RMSD) between experimental and re-docked poses was found to
be within a threshold limit < 2 Å.
Fig. 5. Anti-nociceptive activity of CHD and the positive control, tramadol in
the thermal tail immersion test. Each bar represents mean withdrawal latency
time in s ± S.E.M). *P< 0.05, **P< 0.01, ***P< 0.001 as compared to saline
treated group (one-way ANOVA followed by post hoc Dunnett’s test), (n = 6
mice per group).
The binding orientation of CHD and the native ligand into the
binding site of the COX-2 isoform is shown in Fig. 12A. The three-
dimensional (3-D) interaction plot of CHD showed that the methoxy
group formed a hydrogen bond interaction with His90, an important
residue of a selectivity pocket. The carbonyl oxygen formed hydrogen
bond interactions with Ala527 (Fig. 12B). The computed binding energy
for the CHD-COX-2 complex was ꢀ 8.1050 kcal/mol and the docking
score was ꢀ 12.0458.
3.2.7. Anti-inflammatory action of CHD against phlogistic agents
(carrageenan, histamine, and serotonin) in the paw volume inflammation
and xylene in the ear inflammation test
Intraplantar administration of the phlogistic agents, carrageenan,
histamine, and serotonin was associated with a pronounced inflamma-
tory response manifested by a substantial increase in the paw volume.
The increased edema formation followed a temporal pattern and was
first expressed during the initial h of the paradigm and maintained
For the GABA receptor, the docking study was carried out on PDB
code 4COF (benzamidine). The computed binding energy for the ligand-
GABAA complex was obtained as ꢀ 5.4853 kcal/mol with a docking score
of ꢀ 8.4314. The superimposed three-dimensional ribbon model of the
CHD, (purple), methaqualone (orange) (a positive allosteric GABAA
7

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European Journal of Pharmacology 902 (2021) 174091
Fig. 7. (A) Effect of naloxone at 1 mg/kg (NLX-1) and (B) PTZ at 15 mg/kg (PTZ-15) on the anti-nociceptive activity of CHD (30 mg/kg, CHD-30 and 45 mg/kg, CHD-
45) or tramadol (30 mg/kg, TRD-30) in the mouse hot-plate test. Each bar represents mean percentage protection ± S.E.M. ***P ˂ 0.001 compared to saline control
(SAL). (two sample t-test), (n = 6 mice per group).
Fig. 8. Anti-inflammatory activity of (A) CHD and (B) the positive control, aspirin in the carrageenan induced paw edema test. Each bar represents paw volume in ml
± S.E.M. *P< 0.05, **P< 0.01, ***P< 0.001 as compared to saline treated group (one-way ANOVA followed by post hoc Dunnett’s test), (n = 6 mice per group).
Fig. 9. Anti-inflammatory activity of (A) CHD and (B) the positive control, aspirin in the histamine induced paw edema test. Each bar represents paw volume in ml ±
S.E.M. *P< 0.05, **P< 0.01, ***P< 0.001 as compared to saline treated group (one-way ANOVA followed by post hoc Dunnett’s test), (n = 6 mice per group).
Fig. 10. Anti-inflammatory activity of (A) CHD and (B) the positive control, aspirin in the serotonin induced paw edema test. Each bar represents paw volume in ml
± S.E.M. *P< 0.05, **P< 0.01, ***P< 0.001 as compared to saline treated group (one-way ANOVA followed by post hoc Dunnett’s test), (n = 6 mice per group).
8

J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
receptor modulator) and native ligand benzamidine (yellow) is shown in
(Fig. 13). The 2D interaction plot showed that the phenyl ring of the
synthesized compound creates
For -opioid receptors (
ligand-receptor complex was ꢀ 7.0501 kcal/mol and the docking score
π-π assembling interactions with Tyr62.
μ
μ
OR), the computed binding affinity for the
was computed as ꢀ 11.4240 (Fig. 14).
μ
OR are important opioid re-
ceptors for pain perception and are currently the target of various potent
centrally-acting analgesic drugs. The binding pose of CHD (purple)
overlaid with β-funaltrexamine is shown in (Fig. 14). The ligand enzyme
complex was stabilized by hydrophobic and
phenyl ring formed
methoxyphenyl group formed
π
-sulfur interactions. The
stacking interactions with Tyr326, while the 4-
-sulfur interactions with Met151.
π-π
π
The binding affinity and docking score in the case of the κ-opioid
ligand-receptor complex was calculated as ꢀ 8.0501 kcal/mol and
ꢀ 12.0240, respectively. The binding pose of the synthesized compound
(purple) into the κ-opioid receptor active site (PDB code 4DJH) is shown
in Fig. 14. CHD exhibited a binding pose similar to that of the co-
crystalized ligand (JDC). The 3D interaction plot showed that the
Fig. 11. Anti-inflammatory activity of CHD and the positive controls, indo-
methacin (Indo), and diclofenac (Diclo) in the xylene induced ear edema test.
Each bar represents ear weight in mg ± S.E.M. *P< 0.05, **P< 0.01, ***P<
0.001 compared to the saline treated group (one-way ANOVA followed by post
hoc Dunnett’s test), (n = 6 mice per group).
ligand-enzyme complex was stabilized by
a
hydrogen bond,
Fig. 12. (A) Ribbon diagram of overlaid binding orientation of CHD and native ligand into the binding site of the COX-2 enzyme. (B) Three-dimensional ligand-
enzyme interaction plots of the cyclohexenone derivative (CHD) into the binding site of COX-2 enzyme.
Fig. 13. (A) Three-dimensional superimposed binding pose of the native ligand benzamidine (yellow), cyclohexenone derivative (CHD; purple) and methaqualone
(orange) into the binding site of the GABAA receptor (PDB code 4COF) and (B) Two-dimensional interaction plot for CHD.
Fig. 14. Three and Two dimensional models of CHD
binding with opioid receptors. (A) Three-dimensional
and (B) Two-dimensional modeled superimposed
binding pose of native ligand and CHD (purple) into
the binding site of δ-opioid receptors (PDB code =
4EJ4). (C) Three-dimensional and (D) Two-
dimensional model superimposed binding pose of
native ligand and CHD (purple) into the binding site
of κ-opioid receptor (PDB code = 4DJH). (E) Three-
dimensional and (F) Two-dimensional model super-
imposed binding pose of the native ligand and
selected compound CHD (purple) into the binding site
of μ-opioid receptors (4DKL).
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J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
hydrophobic,
π
-sulfur as well as
π
-CH type interactions. Met142 formed
The mean RMSDs of these were 2.0 Å, 2.4 Å, and 2.9 Å, respectively.
These sites in the presence of CHD at the docked position revealed very
constant RMSD patterns throughout the simulaiton time, indicating a
good intermolecular strength of affinity and stability pattern. 4DJH
(mean RMSD = 6.8 Å), 4DKL (mean RMSD = 5.4 Å), and 4EJ4 (mean
RMSD = 4.6 Å) showed major fluctuations in the receptor structures,
however, these changes do not affect the binding and conformation of
the compound with the receptors. In essence, these RMSD receptor
fluctuations correspond to local protein structure movments which are
normal to their function. To substantiate compound conformation sta-
bility, we additionally computed compound RMSDs in all complexes and
plotted them versus time. As can be seen in (Fig. 15B), the compounds
were significantly stable with all receptor RMSDs <1 Å in all frames of
the MD simulation.
π
-sulfur interactions with the 4-methoxyphenyl ring. The phenyl ring of
CHD engages in
π-π stacking interactions with Trp287. A hydrogen
bonding interaction was found between the carbonyl oxygen of the ring
with Tyr312. Similarly, Val108, Val230, Val290, Ile294 and Ile316
formed some π-alkyl interactions.
For the δ-opioid receptor, the binding affinity for the ligand-enzyme
complex was calculated as ꢀ 7.4000 kcal/mol and the docking score was
noted as ꢀ 11.0903. In the case of δ-opioid receptors, the 3D structure
with naltrindole as co-crystalized ligand was retrieved (PDB code =
4EJ4). The superimposed 3D binding pose of CHD (purple) with nal-
trindole (yellow) is shown in (Fig. 14). The two-dimensional interaction
plot showed that it interacted with Met132 via hydrogen bond donor
interactions.
3.3.2. Molecular dynamics (MD) simulations
3.3.3. MMPB/GBSA binding energy calculation
MD simulations were performed in order to understand the dynamics
of all complexes and check the stability of the CHD conformation at the
docked site with respect to the backbone atoms for each receptor.
Among the complexes, 4COF, 1CX2, and 1EQG showed good stabilty in
the presence of CHD compared to the other three receptors (Fig. 15A).
The free energy of binding was computed for all complexes to eval-
uate and revalidate the affinity of intermolecular interactions and
discover which type of interaction energy was dominant in contributing
to complex stability. All the complexes divulged robust interaction en-
ergies and were dominated by gas phase energy in both MMGBSA and
MMPBSA methodologies. Solvation energy appeared to play less of a
role in molecular interactions and was therefore non-favorable. More
specifically, the van der Waals energy of the gas phase disclosed by both
methods played a key role in complex stability whereas a minor
contribution from electrostatic energy was also evident except in the
case of ICX2.The non-polar energy of solvation also favored docked
molecules as opposed to a highly unfavorable contribution from polar
solvation energy. Overall, the 4DKL receptor in complex with the CHD
compound was highly stable with a MMGBSA energy of ꢀ 55.4492 kcal/
mol and ꢀ 47.9865 kcal/mol in MMPBSA. Details of MMGBSA and
MMPBSA energies of the complexes can be viewed in Table 4.
4. Discussion
Cyclohexenone derivatives have received considerable attention
over recent years not only preclinically, but also clinically because of
their extensive pharmacological possibilities. These include: analgesic
(Said et al., 2009), anti-inflammatory (Yaouba et al., 2018),
anti-neuropathic (Khan et al., 2019), antipyretic (Mousavi, 2016),
antibacterial (Saranya and Ravi, 2012), antioxidant (Okoth et al., 2016),
antifungal (Kanagarajan et al., 2013), antimalarial (Ledoux et al., 2017),
anti-tubercular (Monga et al., 2014), anti-leishmanial (Das and Manna,
2015), anticonvulsant (Said et al., 2009) tyrosine kinase inhibitory
(Nazar et al., 2015) cytotoxic (Ayyad et al., 1998) and anticancer (Okoth
and Koorbanally, 2015) activities. Bearing in mind these wide-ranging
potential capacities of cyclohexenone functionality, this study was
designed to examine a selected cyclohexanone derivative (CHD) exem-
plar
(Ethyl
6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohex-
e-3-enecarboxylate). This was done firstly for its safety profile; secondly,
to investigate any feasible in vivo effects in standard animal models of
nociceptive and inflammatory pain; thirdly, to perform molecular
docking and molecular dynamic (MD) simulation studies to facilitate
interpretation of targeted drug-receptor interactions to corroborate the
in vivo findings. In parallel with this research approach, in vitro assays
were conducted to examine any possibility of COX-2 or 5-LOX enzyme
inhibition and/or suppression of mRNA expression of TNF-α, IL-1β and
COX-2 that might underlie anti-nociceptive and anti-inflammatory
effects.
Four nociceptive and inflammatory, highly reproducible standard
models were used to generate the results. The findings clearly indicated
that CHD possessed a noteworthy degree of safety with a maximum
tolerated dose above 240 mg/kg. Statistically significant anti-
nociceptive and anti-inflammatory activity was found in the rodent
models. These effects were comparable to those of aspirin, tramadol,
indomethacin and diclofenac used as positive controls (Figs. 3–11).
Fig. 15. (A) Root Mean Square Devitaions of backbone atoms for each receptor
of docked complexes. (B) CHD Root Mean Square Devitaions over 50-ns of MD
simulation in complex with receptors.
10

J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
Table 4
MGBSA and MMPBSA binding energies of the complexes.
Method
Energy Component
1EQG
1CX2
4COF
4DKL
4EJ4
4DJH
MMGBSA
VDWAALS
EEL
ꢀ 40.4511
ꢀ 6.2952
18.2375
ꢀ 4.3177
ꢀ 46.7463
13.9198
ꢀ 32.8265
ꢀ 40.4511
ꢀ 6.2952
22.4009
ꢀ 2.9491
0.0000
ꢀ 52.1322
3.4110
ꢀ 47.2564
ꢀ 25.3784
35.5743
ꢀ 62.2517
ꢀ 8.2760
21.3416
ꢀ 6.2631
ꢀ 70.5277
15.0785
ꢀ 55.4492
ꢀ 62.2517
ꢀ 8.2760
26.2948
ꢀ 3.7537
0.0000
ꢀ 61.1760
ꢀ 10.7373
24.9894
ꢀ 53.3142
ꢀ 9.9464
19.8313
ꢀ 5.5521
ꢀ 63.2606
14.2792
ꢀ 48.9814
ꢀ 53.3142
ꢀ 9.9464
26.3717
ꢀ 3.6908
0.0000
EGB
8.9329
ESURF
ꢀ 5.6008
ꢀ 48.7212
3.3320
ꢀ 4.8037
ꢀ 72.6348
30.7706
ꢀ 6.2177
ꢀ 71.9133
18.7716
DELTA G gas
DELTA G solv
DELTA TOTAL
VDWAALS
EEL
ꢀ 45.3892
ꢀ 52.1322
3.4110
ꢀ 41.8641
ꢀ 47.2564
ꢀ 25.3784
40.4885
ꢀ 53.1417
ꢀ 61.1760
ꢀ 10.7373
32.7712
MMPBSA
EPB
14.6505
ꢀ 3.5859
0.0000
ENPOLAR
EDISPER
DELTA G gas
DELTA G solv
DELTA TOTAL
ꢀ 3.3982
0.0000
ꢀ 3.7336
0.0000
ꢀ 46.7463
19.4518
ꢀ 27.2945
ꢀ 48.7212
11.0646
ꢀ 37.6567
ꢀ 72.6348
37.0904
ꢀ 70.5277
22.5411
ꢀ 47.9865
ꢀ 71.9133
29.0376
ꢀ 63.2606
22.6810
ꢀ 40.5796
ꢀ 35.5444
ꢀ 42.8757
Moreover, in silico docking analysis demonstrated that CHD manifested
favorable interactions with common pain targets i.e. COX-1/2 enzymes
in addition to opioid and GABAA receptors (Figs. 12–14.) substantiating
the in-vivo results. Equally, CHD produced marked inhibition of COX-2
and 5-LOX in the enzyme assays while in the case of RT-PCR, CHD
action of a drug depends on several factors including biological half-life,
first pass effect, plasma protein binding and other pharmacokinetic
factors, nature of formulation, co-morbid conditions such as renal
impairment or liver disfunction. Any of the above cited factors, may be a
potential contributor to the loss of CHD effectiveness at the doses of 30
and 45 mg/kg in the thermal nociception tests within 90 min (hot plate
test) and 120 min (tail immersion test), respectively. The formalin
induced nociceptive paradigm comprises of a binary phased nociceptive
reaction and neuropathic pain (Salinas-Abarca et al., 2017). A neuro-
genic or first phase (0–5 min) in which class C fibres are stimulated and
an inflammatory or second phase (10 to 30 min) which involves the
release of inflammatory mediators (Hunskaar and Hole, 1987; Tjølsen
et al., 1992). Interestingly, CHD was effective in both the neurogenic
and inflammatory mediator phases (Fig. 6), further reinforcing the
concept of a possible centrally acting anti-nociceptive component
mechanism in the activity of this compound. Moreover, in experiments
involving pharmacological antagonism of CHD anti-nociception with
PTZ and naloxone, it was divulged that an apparent participation of both
GABAergic and opioidergic mechanisms was implicated (Fig. 7A and B).
The anti-inflammatory activity of CHD was investigated by
employing four standard models of inflammation i.e., the carrageenan,
serotonin, histamine and xylene mediated edema tests (Figs. 8–11). The
carrageenan incited paw volume model is most extensively employed for
evaluating the anti-edematous potential of drugs (Mazzanti and Bra-
ghiroli, 1994). Localised paw injection of carrageenan in mice initiates a
three-phased inflammatory process. The primary phase (0 to 1.5 h), is
caused by the release of serotonin and histamine whereas the secondary
phase (1.5 to 2.5 h) is mediated via bradykinin and the tertiary phase
(2.5 to 5 h) is elicited mainly by the generation of prostaglandins (Suba
et al., 2005).
reduced the mRNA expression of TNF-α, IL-1β and COX-2.
Administration of GABA receptor agonists either supraspinally,
spinally or peripherally, has been reported to reduce the nociceptive
index in models of neuropathic and inflammatory pain (Malan et al.,
2002; Patel et al., 2001). In our study, it is postulated that CHD alleviates
centrally mediated nociception via GABAergic and opioidergic mecha-
nisms (Fig. 7) alongside a capability of interaction with the COX-1/2
target (Fig. 12). An involvement of GABAergic and opioidergic sys-
tems was further reinforced by computational studies whereby CHD
exhibited favorable binding affinity for the GABAA (Fig. 13) and opioid
receptor subtypes (μ, κ and δ) (Fig. 14). It has been reported that
GABAergic agonists may augment the anti-nociceptive effect of a cen-
trally acting analgesic such as morphine (Sawynok, 1984), hence, it is
conceivable that GABA receptor agonist administration may represent a
therapeutic option for the management of both chronic and acute pain
(McCarson and Enna, 2014) or as a combination of GABA with opioid
receptor related therapies.
The acetic acid induced abdominal constriction assay is a tonic
visceral pain model frequently utilized for monitoring the anti-
nociceptive action of drugs (Utsunomiya et al., 1998). Although it is a
very sensitive test, it cannot distinguish whether the nociceptive activity
is peripherally or centrally mediated (Chen et al., 1995). It entails
stimulation of visceral receptors followed by the release of bradykinin,
serotonin, cyclooxygenase, prostaglandins and interleukins which
induce pain and inflammation (Olonode et al., 2015; Rodrigues et al.,
2012). It also implicates an enhanced activation of peripheral receptors
(Bentley et al., 1983) and innervated nociceptive nerve terminals
(Duarte et al., 1988). In the current study, CHD induced a significant
reduction in abdominal constrictions in a dose-dependent manner
comparable to standard aspirin (Fig. 3A and B).
CHD (15–45 mg/kg) substantially reduced the elevated paw edema
in all three phases of the carrageenan-induced paw volume assay and
this was comparable to the response yielded by the standard drug,
aspirin (Fig. 8A and B). In order to authenticate the finding from the
carrageenan paw edema model, the anti-edematous effect of CHD was
further investigated in the three other standard models (histamine and
serotonin induced paw volume and xylene induced ear edema). Hista-
mine and serotonin can increase vascular permeability and both are
effective vasodilators (Skidmore and Whitehouse, 1967) which are
conducive to an ensuing edema. CHD not only suppressed the edema
mediated by histamine and serotonin but also that of xylene at doses
corresponding to standard anti-inflammatory drugs (Fig. 9A and B,
10A-B and 11). The xylene induced ear edema model is extensively
utilized to determine the anti-inflammatory action of steroidal and
non-steroidal anti-phlogistic agents (Zanini Jr et al., 1992). Studies re-
ported in the literature have revealed that xylene also promotes vascular
permeability causing skin edema owing to the release of inflammatory
Hot plate and tail immersion nociceptive tests were employed to
determine the central anti-nociceptive potential of CHD. These models
can specifically evaluate possible central nociception (Eddy and Leim-
bach, 1953), where there is a non-inflammatory and acute nociceptive
reaction developed upon exposure to heat via spinal receptors which is
evidence of centrally mediated anti-nociception (Amabeoku and Kaba-
tende, 2012; Pini et al., 1997). CHD moderately enhanced the hot plate
latencies of mice compared to standard tramadol, suggesting it to be a
centrally acting analgesic (Fig. 4). In the tail immersion test, the
behavioural response is predominantly controlled by supraspinal and
spinal entities (Danneman et al., 1994). At the doses studied, CHD
presented a modest increase in tail withdrawal latency, but tramadol
produced a more pronounced latency elevation (Fig. 5). The duration of
´
mediators leading to acute neurogenic inflammation (Banki et al.,
11

J. Khan et al.
European Journal of Pharmacology 902 (2021) 174091
2014). CHD markedly reduced ear edema induced by xylene comparable
to the standard agents (Fig. 11).
reading the final draft. Rahim Ullah: Methodology, Investigation,
Formal analysis, Writing – original draft, reading the final draft. Sajjad
Ahmad: Methodology, Investigation, Formal analysis, Writing – original
draft, reading the final draft. Sumra Wajid Abbasi: Methodology,
Investigation, Formal analysis, Writing – original draft, reading the final
draft. Atif Ali Khan Khalil: Methodology, Investigation, Formal anal-
ysis, Writing – original draft, reading the final draft. RobertD.E. Sewell:
Conceptualization, Writing – original draft, Writing – review & editing,
preparation and reading the final draft.
To further corroborate the anti-nociceptive and anti-inflammatory
potential of CHD, it was subjected to in vitro studies involving 5-LOX
and COX-2 enzyme inhibition assays along with RT-PCR studies. Thus,
CHD substantially inhibited 5-LOX and COX-2 enzymes in comparison
with the standard inhibitors zileuton and celecoxib respectively as
shown in (Table 2 and 3). In the case of RT-PCR studies, CHD decreased
the mRNA expression of COX-2, TNF-
α and IL-1β compared to the
carrageenan treated control group as presented in Fig. 2. This in vitro
study therefore endorsed the promising anti-nociceptive and anti-
inflammatory findings with CHD in both the in vivo and in silico
studies which further strengthens a potential for application in pain and
inflammation.
Declaration of competing interest
The authors have no conflict of interest.
In summary, in silico docking analysis demonstrated that the syn-
thesized cyclohexanone derivative has shown favorable interactions
with common pain targets i.e. COX 1/2, GABAA and opioid receptors
(Figs. 12–14). The binding affinity study revealed that the intensity of
interactions of the CHD ligand with the COX-2 isozyme was more than
that with COX-1 and this was supported by the degree of 5-LOX and
COX-2 enzyme inhibition observed (Table 2 and 3). In addition, MD
simulations of the complexes revealed that CHD was a highly stable
molecule at the docked site and generated robust chemical interactions
underlying strong intermolecular affinity (Table 4). Interactions with
other pharmacological targets suggest that CHD may act as a novel
nociceptive and inflammatory pain reliever supported by in vivo studies
(Figs. 3–11).
Acknowledgments
Dr. Umer Rashid is grateful to the Higher Education Commission of
Pakistan for budgetary support for purchasing an MOE license under
HEC-NRPU project 5291/Federal/NRPU/R&D/HEC/2016. The selected
compound under study has been synthesized as a part of series of
compounds with confirmed structures by Dr. Rasool Khan, Associate
Professor, Institute of Chemical Science, University of Peshawar. We are
grateful to him for providing a series of compounds and after pre-
liminary study, we selected the cited compound (CHD) for our study.
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