Synthesis, characterization, molecular docking evaluation, antidepressant, and anti-Alzheimer effects of dibenzylidene ketone derivatives

Article abstract of DOI:10.1002/ddr.21537

Novel bioactive compounds as synthetic analogs of the potent herbal medicines can be optimized as potential drug candidates for various neurologic disorders. This study was performed to investigate the newly synthesized dibenzylidene ketone derivatives: (2E,6E)-2,6-dibenzylidene cyclohexanone (A1K1) and (1E,4E)-5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-2-methylpenta-1,4-diene-3-one (A2K2) and evaluate its potential anti-Alzheimer's and anti-depressant properties. Both the derivatives are chemically characterized by using HNMR and CNMR techniques. Auto Dock Vina program was used to investigate ligand-protein affinity. Forced swim test, tail suspension test, open field test, Y-maze test, and Morris water maze test (MWM) models were employed to evaluate anti-depressant and anti-Alzheimer's activity of dibenzylidene ketone derivatives in mice. Both A1K1 and A2K2 showed high binding affinities against various proteins involved in depression and Alzheimer's mechanisms like monoamine oxidase B, acetylcholinesterase, norepinephrine transporter 2, serotonin transporter, dopamine receptor, serotonin receptor modulator, and beta-amyloid targets. A1K1 and A2K2 dose-dependently (0.1–1 mg/kg) decreased immobility time, while increased swimming and climbing time of mice in forced swim test (FST). A1K1 and A2K2 decreased animal immobility time in TST. In the open field test, both A1K1 and A2K2 increased the number of ambulations and rearings. A1K1 and A2K2 dose-dependently (0.5–1.0 mg/kg) increased spontaneous alternation behavior (%) and the number of entries of mice in Y-maze test. In the MWM test, A1K1 and A2K2 decreased escape latency time. Overall, both in-silico and in-vivo investigations of A1K1 and A2K2, report their therapeutic potential for antidepressant and anti-Alzheimer properties. Hence, these compounds possess potent neuroprotective properties and may be further evaluated for their therapeutic potential in various neurological disorders.

Full text of DOI:10.1002/ddr.21537

Received: 6 November 2018
DOI: 10.1002/ddr.21537
Revised: 9 March 2019
Accepted: 24 March 2019
R E S E A R C H A R T I C L E
Synthesis, characterization, molecular docking evaluation,
antidepressant, and anti-Alzheimer effects of dibenzylidene
ketone derivatives
Muhammad Adnan Bashir1 | Arif-ullah Khan1 | Haroon Badshah1,2
Edson Rodrigues-Filho3 | Zia Ud Din3,4 | Aslam Khan5
|
1Riphah Institute of Pharmaceutical Sciences,
Abstract
Riphah International University, Islamabad,
Pakistan
Novel bioactive compounds as synthetic analogs of the potent herbal medicines can be
optimized as potential drug candidates for various neurologic disorders. This study was
performed to investigate the newly synthesized dibenzylidene ketone derivatives:
(2E,6E)-2,6-dibenzylidene cyclohexanone (A1K1) and (1E,4E)-5-(2,3-dichlorophenyl)-
1-(4-methoxyphenyl)-2-methylpenta-1,4-diene-3-one (A2K2) and evaluate its potential
anti-Alzheimer's and anti-depressant properties. Both the derivatives are chemically
characterized by using HNMR and CNMR techniques. Auto Dock Vina program was
used to investigate ligand-protein affinity. Forced swim test, tail suspension test, open
field test, Y-maze test, and Morris water maze test (MWM) models were employed to
evaluate anti-depressant and anti-Alzheimer's activity of dibenzylidene ketone deriva-
tives in mice. Both A1K1 and A2K2 showed high binding affinities against various pro-
teins involved in depression and Alzheimer's mechanisms like monoamine oxidase B,
acetylcholinesterase, norepinephrine transporter 2, serotonin transporter, dopamine
receptor, serotonin receptor modulator, and beta-amyloid targets. A1K1 and A2K2
dose-dependently (0.1–1 mg/kg) decreased immobility time, while increased swimming
and climbing time of mice in forced swim test (FST). A1K1 and A2K2 decreased animal
immobility time in TST. In the open field test, both A1K1 and A2K2 increased the num-
ber of ambulations and rearings. A1K1 and A2K2 dose-dependently (0.5–1.0 mg/kg)
increased spontaneous alternation behavior (%) and the number of entries of mice in
Y-maze test. In the MWM test, A1K1 and A2K2 decreased escape latency time. Over-
all, both in-silico and in-vivo investigations of A1K1 and A2K2, report their therapeutic
potential for antidepressant and anti-Alzheimer properties. Hence, these compounds
possess potent neuroprotective properties and may be further evaluated for their ther-
apeutic potential in various neurological disorders.
2Department of Pharmacy, Abdul Wali Khan
University, Mardan, Pakistan
3Departament of Chemistry, Federal
University of Sao Carlos, São Carlos,
São Paulo, Brazil
4Department of Chemistry, Woman University
Swabi, Swabi, Pakistan
5Basic Sciences Department, College of
Science and Health Professions, King Saud bin
Abdulaziz University for Health Sciences,
Ministry of National Guard Health Affairs,
Jeddah, Kingdom of Saudi Arabia
Correspondence
Arf-ullah Khan, Riphah Institute of
Pharmaceutical Sciences, Riphah International
University, Islamabad, Pakistan.
Email: arif.ullah@riphah.edu.pk
K E Y W O R D S
anti-Alzheimer, anti-depressant, dibenzylidene ketone derivatives
Drug Dev Res. 2019;1–11.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

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BASHIR ET AL.
derivatives. These derivatives have been determined as a synthetic
analogue of curcumin. A number of research bodies have reported
potential pharmacologic activities of curcumin including antitumor,
antiviral, anti-inflammatory, antibacterial, antifungal, and antioxidant
properties (Shen & Ji, 2007). Ishrat et al., 2009 reported that curcumin
inhibuits oxidative damage and improve cognitive dysfunction (Ishrat
et al., 2009). In view of previous literature, the dibenzylidene ketone
derivatives, that is, (2E,6E)-2,6-dibenzylidene cyclohexanone (A1K1)
and (1E, 4E)-5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-2-methylpenta-
1,4-diene-3-one (A2K2) were synthesized and chemically characterized
by the spectroscopic method. Furthermore, both A1K1 and A2K2 were
investigated for in-silico and in-vivo antidepressant and anti-Alzheimer's
potential, using different computational tools and pharmacological
assays. Chemical structures of A1K1 and A2K2 in the 2D format are
shown in Figure 1a.
1
| INTRODUCTION
Depression is a neuro-psychological disease that affects interest,
thoughts, behavior, mental, and even physical health of a person.
Some common symptoms may include change in actions, loss of self-
confidence, loss of appetite, lack of interest in routine works, and
disturbed sleep (Rang, Dale, Ritter, & Moore, 2003). Depression can
be either develop primarily or secondarily to some serious conditions
like cancer, diabetes, heart diseases, and Alzheimer (Cassano & Fava,
2002). A number of mechanisms have been involved in depression,
like alteration in serotonin and dopamine levels, release of acetylcho-
line and increase concentration of monoamine oxidases. It has
been stated that most of the available pharmacological treatments
for depression work through altering monoaminergic transmission
(Berton & Nestler, 2006). Furthermore, according to Krishnan and
Nestler (2011), the understandings about the pathogenesis of altered
mood, impaired concentration, and neurovegetative symptoms in
major depression have come from animal models. Alzheimer's disease
(AD) is a neurodegenerative disorder that is characterized by progres-
sive loss of memory, deterioration of virtually all intellectual functions,
increased apathy, decreased speech function, disorientation, and gait
irregularities (Akina, Thati, & Puchchakayala, 2013). AD is associated
with neuronal loss and the deposition of abnormal proteins in the form
of amyloid plaques and neurofibrillary tangles. Research studies have
shown that deposition of the soluble oligomers of β-amyloid harm
excitatory synaptic functions which cause learning and memory
defects. In addition, it is also believed that amyloid β peptide stimulates
hyperphosphorylation of Tau protein and causes excessive neuronal
cell death (Kontsekova, Zilka, Kovacech, Skrabana, & Novak, 2014). Dif-
ferent studies suggest that β-amyloid induces oxidative stress in cells
which cause neurodegenerative disorders. RNA oxidation, a by-product
of glycation, oxidation of lipid and free carbonyls induce some changes
in the cell which are responsible for cognitive decline (Cioanca, Hritcu,
Mihasan, & Hancianu, 2013).
2
| MATERIALS AND METHODS
2.1 | Chemicals
Donepezil and fluoxetine hydrochloride were obtained from Sigma
Chemicals., St. Louis, MO. Benzaldehyde, cyclohexanone, dimethyl
sulfoxide (DMSO), and ethanol were purchased from Merck Millipore.,
Billerica, MA. All chemicals were of analytical grade.
2.2 | Animals
Balb-C mice of either sex (25–30 g) housed at controlled temperature
(22–25ꢀC) and maintained on a 12 hr light/dark cycle with standard
diet and water ad libitum. All the animals were randomly divided into
five groups: a saline-treated group, three test-drugs treated groups
(each receiving the various dose of the test compounds that is, A1K1
and A2K2) and a reference-drug treated group. Respective behavioral
studies were performed to different groups to observe the antidepres-
sant and anti-Alzheimer effects of A1K1 and A2K2. The experiments
were performed according to the rulings of Institute of Laboratory
The present study was conducted to investigate the potential
therapeutic properties of the newly synthesized dibenzylidene ketone
FIGURE 1 Chemical structures
of compound (a) (2E,6E)-
2,6-dibenzylidene cyclohexanone
(A1K1), (1E,4E)-5-
(2,3-dichlorophenyl)-1-
(4-methoxyphenyl)-2-methylpenta-
1,4-diene-3-one (A2K2),
(b) chemical structures of reference
drugs: Fluoxetine, selegiline,
tacrine and donepezil in 2D format

BASHIR ET AL.
3
Animal Resource, Commission on Life Sciences University, National
Research Council (1996), approved by Ethical Committee of Riphah
Institute of Pharmaceutical Sciences, Riphah International University
(Ref No. REC/RIPS/2016/11).
(NET3, PDB-ID: 4X8C), dopamine transporter (DAT, PDB-ID: 2Q73),
monoamine oxidase B (MAO-B, PDB-ID: 2 V61), N-methyl-D-aspartate
(NMDA, PDB-ID: 5VIH) receptor and serotonin receptor modulator
(SRM, PDB-ID: 4IAQ). The target proteins involved in Alzheimer
pathways include monoamine oxidase B (MAO-B, PDB-ID: 2V61),
N-methyl-D-aspartate (NMDA, PDB-ID: 5VIH) receptor, beta-secretase
(PDB-ID: 1M4H), butyrylcholinesterase (BChE, PDB-ID: 1P0M), beta-
amyloid (PDB-ID: 2Z5Y), acetylcholinesterase (AChE, PDB-ID: 4PQE),
and gamma-secretase (PDB-ID: 4R12). The structure of standard
drug molecules were downloaded from pubchem data base (https://
pubchem.ncbi.nlm.nih.gov/search/). Reference drugs used were fluoxe-
tine (PubChem CID: 62857), selegiline (PubChem CID: 26758), tacrine
(PubChem CID: 2723754), and donepezil (PubChem CID: 5741), as
shown in Figure 1b. All these structures were downloaded in.xml for-
mat and converted to PDB format via Open Babel JUI software. PDB
format of standard, a ligand as well as target proteins were converted
to PDBQT via AutoDockTools (Version1.5.6 Sep_17_14). A1K1 and
A2K2 along with protein targets were loaded in software named as
PyRx and then docked against respective targets. For post docking
interaction, Biovia Discovery Studio Visualizer (DSV) 2016 was used for
a number of hydrogen bonds (classical and nonclassical) and binding
amino acid residues: alanine (ALA), arginine (ARG), asparagine (ASN),
aspartic acid (ASP), cysteine (CYS), glutamic acid (GLU), glycine (GLY),
methionine (MET), serine (SER), threonine (THR), tyrosine (TYR), lysine
(LYS), phenylalanine (PHE), and proline (PRO).
2.3 | Synthesis of A1K1 and A2K2
Symmetrical diarylidene products (A1K1) was synthesized by the reac-
tion of benzaldehyde with cyclohexanone in a 50 mL two-necked
round-bottom flask in a ratio of 2:1 M. Dry HCl gas was passed from
the content of the flask till it was saturated and turns to red color. The
reaction mixture was stirred for 5–10 hr. The crude product was
diluted with toluene and washed with NaHSO3 solution. The organic
layer was separated, dried with anhydrous Na2SO4 and evaporated
under low pressure. The residue, when distilled under reduced pres-
sure yielded pure compounds A1K1, which was recrystallized from
ethanol as shown in Figure 2a. Chemical characterization was carried
out based on the analysis of spectroscopic and crystallographic data.
For the synthesis of A2K2, solution of intermediate compound 1 and
2,3-dichlorobenzaldehydes in ethanol (5 mL) was stirred for 5 min at
room temperature after which sodium hydroxide solution in ethanol
(4 mL, 50 mM) was added and stirring was continued until the reaction
was complete. The solvent ethanol was evaporated under reduced
pressure. The residue was dissolved in ethyl acetate extracted with
NaHSO3 solution and dried with Na2SO4, the solvent was evaporated
using a rotary evaporator and the crude product was collected as a
yellow precipitate which was further purified by column chromatogra-
phy and recrystallized from ethanol as shown in Figure 2b. Chemical
characterization was carried out based on the analysis of spectroscopic
and crystallographic data.
2.5 | Antidepressant activity
2.5.1 | Forced swim test (FST)
Antidepressant potential of A1K1 and A2K2 was determined by
forced swim test (FST) model (Cryan, Valentino, & Lucki, 2005;
Harquin Simplice, David Emery, & Hervé Hervé, 2014). The test model
consists of a transparent glass cylinder (30 cm high × 20 cm diameter)
filled with water up to 20 cm depth and maintained at 25 2ꢀC. Mice
were divided into five groups (n = 5 each group) and were treated a
single dose/day for seven days. Group I was used as negative control
which received an intraperitoneal (i.p.) dose of normal saline
(10 mL/kg). Group II, III, and IV were administered different doses of
test compound (0.1, 0.5, and 1 mg/kg, respectively), while Group V
2.4 | Docking studies
Auto Dock Vina program used to investigate ligand-protein affinity
(Trott & Olson, 2010). 3D-structures of target proteins were taken from
the protein data bank, RCSB PDB (http://www.rcsb.org/pdb/).The tar-
get proteins involved in depression pathways are serotonin transporter
2 (SERT2, PDB-ID: 5I6Z), serotonin transporter 3 (SERT3, PDB-ID:
5I6X), norepinephrine transporter 1 (NET1, PDB-ID: 5IPB), norepineph-
rine transporter 2 (NET2, PDB-ID: 4XJM), norepinephrine transporter 3
FIGURE 2 Synthesis of
(a) (2E,6E)-2,6-dibenzylidene
cyclohexanone (A1K1) and
(b) (1E,4E)-5-(2,3-dichlorophenyl)-
1-(4-methoxyphenyl)-
2-methylpenta-1,4-diene-
3-one (A2K2)

4
BASHIR ET AL.
was assigned for the positive control (fluoxetine, 20 mg/kg). On 7th
day, 30 min. Later to saline, test compounds and fluoxetine treatment,
each mouse was allowed to swim in the water tank for 5 min and
duration of immobility, swimming, and climbing were recorded via dig-
ital video camera. Mice were considered to be immobile when it
remained floating over water without struggling, making only little
movements to keep its head above water. Swimming is large horizon-
tal movements of mice with forepaws, displacing mice body around
the cylinder. Climbing is vigorous vertical movements of mice with
forepaws, directed against the wall of tank, leading to the displace-
ment of mice body around the cylinder. Decrease in immobility time
and an increase in swimming and/or climbing behaviors showed anti-
depressant activity.
percentage (%) was calculated as: (successive triplet sets [entries into
three different arms consecutively]/total number of arm entries − 2) ×
100 (Ali, Badshah, Kim, & Kim, 2015). Increase in the spontaneous alter-
nation behavior (%) showed anti-Alzheimer potential.
2.6.2 | Morris water maze test (MWM)
Mice were divided into four groups (n = 5) and were treated similar to
that of the Y-maze test except the treatment was prolonged to 5 days.
Mice were exposed to swimming training for 60 s in the absence of the
platform at day first. For four consecutive days the mouse was given
trial session with the platform in place. Once the mouse located
the platform, it was permitted to remain on it for 10 s. If the mice
were unable to locate the platform within 120 s, it was placed on the
platform for 10 s and then removed from the pool. On Day 5, mouse
was individually subjected to a probe trial session in which the platform
was removed from the pool. Mouse was allowed to swim for 120 s
and the escape latency time was determined (Morris, 1984; Vorhees &
Williams, 2006). Decrease in escape latency time showed anti-Alzheimer
potential.
2.5.2 | Tail suspension test
Mice were divided and treated similar to that of the FST. On 7th day,
30 min later to saline, test compounds and fluoxetine treatment, mice
were suspended 50 cm above floor on table's edge by adhesive tape
placed about 1 cm from tip of tail. Duration of immobility was
recorded for 5 min via digital video camera. Mouse was considered
immobile when it becomes completely motionless and hanged pas-
sively. Decrease in immobility time showed antidepressant activity
(Crowley et al., 2005).
2.7 | Acute toxicity
Acute toxicity test of the dibenzylidene ketone derivatives is car-
ried out according to the previously determined procedure (Adil
et al., 2018; Chen et al., 2009). Mice were kept on overnight
fasting and divided into four groups with each containing five ani-
mals. Group I was assigned as a saline group (10 mL/kg). Group II,
III, and IV were utilized to administer each test compound at doses
of 3, 5, and 10 mg/kg, respectively. All the animals were kept
under observation for 24 hr to determine the symptoms of toxicity
and mortality.
2.5.3 | Open field test
For open field test, the apparatus was made of wooden base divided
into 12 equal squares with glass walls of dimensions (50 × 25 × 50 cm).
All the animals were randomly divided in five groups and treated similar
to that of FST. On 7th day, 30 min later to saline, test compounds and
fluoxetine treatment, each mouse was placed in corner square to explore
test arena for 5 min and recorded number of ambulation's and rearings
(Brown, Corey, & Moore, 1999; Jo et al., 2019; Maurmann, Reolon,
Rech, Fett-Neto, & Roesler, 2011) via digital video camera. Increase in
number of ambulation's and rearings showed antidepressant potential.
2.8 | Statistical analysis
Data expressed as Mean standard error of mean (SEM). The results
were analyzed using one-way analysis of variance (ANOVA), followed
by post hoc Tukey's test. p < 0.05 was noted as significantly different.
The bar-graphs were analyzed using the Graph Pad Prism (Graph-Pad,
San-Diego, CA).
2.6 | Anti-Alzheimer's activity
2.6.1 | Y-maze test
In Y-maze test apparatus, each arm of the maze was 50 cm long, 20 cm
high, and 10 cm wide at the bottom and top. Mice were divided into
four groups, having five mice in each group and were administered a
single dose/day for three days. Group I was used as negative control
which received an i.p. dose of normal saline (10 mL/kg). Group II and III
were treated with 0.5 and 1 mg/kg doses, respectively, of test com-
pounds, while Group IV was assigned for positive control (donepezil,
3 mg/kg). Each mouse was placed at the center of the apparatus and
allowed to move freely through the maze for three sessions (8 min each).
The series of arm entries was observed via digital video camera. Sponta-
neous alternation was defined as the successive entry of the mice into
the three arms in overlapping triplet sets. Alternation behavior
3
| RESULTS
3.1 | Spectral analysis of A1K1
Mp: 118–119ꢀC; 117–118.1H NMR (400 MHz, CDCl3) δ 7.82 (s, 2H),
7.47 (d, J = 7.4 Hz, 4H), 7.45–7.38 (m, 4H), 7.38–7.31 (m, 2H), 2.94
(td, J = 6.6, 2.0 Hz, 4H), 1.84–1.74 (m, 2H);13C NMR (400 MHz,
CDCl3) δ 190.47(1C), 137.04(2C), 136.03(2C), 136.09(2C), 130.48(2C),
128.70(2C), 128.49(2C), 28.56(6C), 23.12 (2C).

BASHIR ET AL.
5
Selegiline showed E-value of −7.4 kcal/mol and formed no hydrogen
bond. The hydrogen bonding of A1K1, A2K2, and selegiline with
MAO-B are shown in Figure S7. Against NMDA, A1K1 showed E-value
of −7.9 kcal/mol and formed no hydrogen bond. A2K2 showed E-value
of −8.3 kcal/mol and formed one-hydrogen bond with TYR-214.
Tacrine showed E-value of −7.4 kcal/mol and formed no hydrogen
bond. The hydrogen bonding of A1K1, A2K2, and tacrine with NMDA
are shown in Figure S8. Against SRM, A1K1 showed E-value of
−9.8 kcal/mol and formed no hydrogen bond. A2K2 showed E-value
of −8.9 kcal/mol and formed one-hydrogen bond with TYR-359. Fluox-
etine showed E-value of −7.9 kcal/mol and formed one-hydrogen bond
with SER-212 and THR-209. The hydrogen bonding of A1K1, A2K2,
and fluoxetine with SRM are shown in Figure S9. Against beta-secre-
tase, A1K1 showed E-value of −8.4 kcal/mol and formed one-hydrogen
bond with ASP-62. A2K2 showed E-value of −7.7 kcal/mol and formed
one-hydrogen bond with ASP-62. Donepezil showed E-value of
−8.3 kcal/mol and formed one-hydrogen bond with TYR-124.
The hydrogen bonding of A1K1, A2K2, and donepezil with beta-
secretase are shown in Figure S10. Against BChE, A1K1 showed
E-value −9.1 kcal/mol and formed no hydrogen bond. A2K2 showed
E-value of −8.6 kcal/mol and formed no hydrogen bond. Donepezil
showed E-value of −9.5 kcal/mol and formed no hydrogen bond. The
hydrogen bonding of A1K1, A2K2, and tacrine with BChE are shown in
Figure S11. Against beta amyloid, A1K1 showed E-value of −9.1 kcal/mol
and formed no hydrogen bond. A2K2 showed E-value of −7.9 kcal/mol
and formed no hydrogen bond. Donepezil showed E-value of
−11.3 kcal/mol and formed one-hydrogen bond with CYS-406. The
hydrogen bonding of A1K1, A2K2, and donepezil with beta amyloid
are shown in Figure S12. Against AChE, A1K1 showed E-value of
−9.3 kcal/mol and formed no hydrogen bond. A2K2 showed E-value
of −10.2 kcal/mol and formed one-hydrogen bond with TYR-341.
Donepezil showed E-value of −8.3 and formed one-hydrogen bond
with TYR-124. The hydrogen bonding of A1K1, A2K2, and donepezil
with AChE are shown in Figure S13. Against gamma secretase, A1K1
showed E-value of −8.4 kcal/mol and formed no hydrogen bond. A2K2
showed E-value of −7.3 kcal/mol and formed one-hydrogen bond with
ASN-132. Donepezil showed E-value of −8.2 kcal/mol and formed
two-hydrogen bonds with ASN-132 and ASP-127. The hydrogen bond-
ing of A1K1, A2K2, and donepezil with gamma secretase are shown in
Figure S14. The results of E-value, hydrogen bonds and binding resi-
dues of A1K1 and A2K2 with target proteins involved in depression
pathways (SERT2, SERT3, NET1, NET2, NET3, DAT, MAO-B, NMDA,
and SRM) along with standard drugs are shown in Table 1. The results
of E-value, hydrogen bonds and binding residues of A1K1 and A2K2
with target proteins involved in Alzheimer's process (MAO-B, NMDA,
beta secretase, BChE, beta amyloid, AChE, and gamma secretase) along
with standard drugs are shown in Table 2.
3.2 | Spectral analysis of A2K2
Percent yield: 79. m.p.: 119–121ꢀC. 1H NMR (400 MHz, CDCl3) δ ppm:
8.02 (d, J = 15.7 Hz, 1H); 7.61 (dd, J = 7.9, 1.1 Hz, 1H); 7.55 (s, 1H),
7.51–7.42 (m, 3H); 7.35 (d, J = 15.7 Hz, 1H); 7.24 (d, J = 8.0 Hz, 1H);
6.96 (d, J = 8.8 Hz, 2H); 3.86 (s, 3H); 2.21 (d, J = 1.2 Hz, 3H). 13C NMR
(400 MHz, CDCl3) δ ppm: 192.42 (1C, CO); 160.26 (1C, ArC); 139.91
(1C, C); 138.89 (1C, C); 136.49 (1C, C); 136.30 (C, ArC); 134.15
(1C, ArC); 131.88 (2C, ArC); 131.34 (1C, C); 128.47 (1C, ArC); 127.56
(1C, ArC); 127.47 (1C, ArC); 126.27 (1C, ArC); 125.96 (C, ArC); 114.23
(2C, ArC); 55.51 (1C, OCH3); 13.89 (1C, CH3). HRMS ESI(+): calcd for
C19H16Cl2O2Na (M + Na) 369.0527, found 369.0381.
3.3 | Docking evaluation
The 3D pattern of the derivative compounds showing the maximum
binding interactions formed between ligands and amino acid residues
are shown in Figures S1–S14. Docking against SERT2, A1K1 showed
E-value −10.1 kcal/mol and formed two-hydrogen bonds with
THR-497 and TYR-175. A2K2 showed E-value of −7.3 kcal/mol and
formed 1 hydrogen bond with ALA-150 and SER-142. Fluoxetine
showed E-value of −6.8 kcal/mol and formed one-hydrogen bond
with ARG-104. The hydrogen bonding of A1K1, A2K2, and fluoxetine
with SERT2 are shown in Figure S1. Against SERT3, A1K1 showed
E-value of −9.5 kcal/mol and formed no hydrogen bond. A2K2 showed
E-value of −9.1 kcal/mol and formed two hydrogen bonds with
THR-497, TYR-175, and GLU-493. Fluoxetine showed E-value
of −7.1 kcal/mol and formed no hydrogen bond. The hydrogen
bonding of A1K1, A2K2, and fluoxetine with SERT3 are shown in
Figure S2. Against NET1, A1K1 showed E-value of −8.4 kcal/mol and
formed no hydrogen bond. A2K2 showed E-value −7.0 kcal/mol and
formed one-hydrogen bond with SER-107. Reboxetine showed E-value
of −6.5 kcal/mol and formed no hydrogen bond. The hydrogen bonding
of A1K1, A2K2, and fluoxetine with NET1 are shown in Figure S3.
Against NET2, A1K1 showed E-value of −10.9 kcal/mol and formed no
hydrogen bond. A2K2 showed E-value of −9.8 kcal/mol and formed no
hydrogen bond. Reboxetine showed E-value of −9.2 kcal/mol and
formed three-hydrogen bonds with GLY-93, PHE-92, and PRO-401.
The hydrogen bonding of A1K1, A2K2, and fluoxetine with NET2
are shown in Figure S4. Against NET3, A1K1 showed E-value of
−8.5 kcal/mol and formed one-hydrogen bond. A2K2 showed E-value
of −8.2 kcal/mol and formed one hydrogen bond with ASN-585 and
ALA-581. Fluoxetine showed E-value of −7.3 kcal/mol and formed
one-hydrogen bond with ALA-581. The hydrogen bonding of A1K1,
A2K2, and fluoxetine with NET3 are shown in Figure S5. Against DAT,
A1K1 showed E-value of −8.9 kcal/mol and formed no hydrogen bond.
A2K2 showed E-value of −8.9 kcal/mol and formed two-hydrogen
bonds with TYR-6 and TYR-65. Fluoxetine showed E-value of −8.5 and
formed two-hydrogen bonds with LYS-84 and TYR-60. The hydrogen
bonding of A1K1, A2K2, and fluoxetine with DAT are shown in
Figure S6. Against MAO-B, A1K1 showed E-value of −10.8 kcal/mol
and formed one-hydrogen bond with GLY-58 and MET-436. A2K2
showed E-value of −10.4 kcal/mol and formed no hydrogen bond.
3.4 | Effect on immobility, swimming, and
climbing time
A1K1 dose-dependently (0.1–1 mg/kg) decreased immobility time,
while increased swimming and climbing time in FST. The immobility

6
BASHIR ET AL.
time noted for saline (10 mL/kg), A1K1 (0.1 mg/kg), A1K1 (0.5 mg/kg),
A1K1 (0.1 mg/kg) and fluoxetine (20 mg/kg) treated groups were
150.4 4.10 s, 64.80 6.36 s (p < 0.001), 47.60 3.26 s (p < 0.001),
23.80 2.81 s (p < 0.001) and 63.60 5.19 s (p < 0.001), respectively.
The swimming time recorded for saline (10 mL/kg), A1K1 (0.1 mg/kg),
A1K1 (0.5 mg/kg), A1K1 (1 mg/kg), and fluoxetine (20 mg/kg) treated
groups were 90.0 3.58 s, 170.2 8.43 s (p < 0.001), 180.4 4.96 s
(p < 0.001), 194.6 3.07 s (p < 0.001), and 168.60 5.27 s (p < 0.001),
respectively. Similarly the climbing time noted for saline (10 mL/kg),
A1K1 (0.1 mg/kg), A1K1 (0.5 mg/kg), A1K1 (1 mg/kg), and fluoxetine
(20 mg/kg) treated groups were 59.6 4.90 s, 67.2 4.46 s (p > 0.05),
72.0 3.61 s (p > 0.05), 82.40 4.17 s (p < 0.01), and 68.4 3.15 s
(p > 0.05), respectively (Figure 3a).
swimming and climbing time were 63.60 5.19, 181.60 4.19, and
54.8 3.87 s (p < 0.001 vs. saline group), respectively (Figure 3b).
3.5 | Effect on immobility time
Similar to FST, our tail suspension test (TST) results also showed
dose-dependent decrease in immobility time. The immobility time
noted for saline (10 mL/kg), A1K1 (0.1 mg/kg), A1K1 (0.5 mg/kg),
A1K1 (0.1 mg/kg), and fluoxetine (20 mg/kg) treated groups were
154.8 7.86 s, 72.6 3.50 s, 42.60 1.16 s, 30.40 1.66 s, and
62.40 3.12 s, respectively (Figure 4a). The immobility time recorded for
saline (10 mL/kg), A2K2 (0.1 mg/kg), A2K2 (0.5 mg/kg), A2K2 (0.1 mg/kg),
and fluoxetine (20 mg/kg) treated groups were 166.0 6.30 s,
84.4 3.97 s, 62.80 4.38 s, 53.0 8.03 s, and 60.60 3.40 s,
respectively (Figure 4b). All the results were highly significant (p < 0.001
vs. saline group).
Similarly, A2K2 dose-dependently (0.1–1 mg/kg) decreased immo-
bility time, while increased swimming and climbing time. In saline
(10 mL/kg) treated group, immobility, swimming and climbing time
was 172.0 4.37, 87.8 4.00, and 40.2 4.32 s, respectively. In
A2K2 (0.1 mg/kg) treated group, immobility time was 99.20 4.87 s
(p < 0.001), swimming time was 154.2 6.01 s (p < 0.001), and
climbing time was 46.6 4.92 s (p > 0.05 vs. saline group). In A2K2
(0.5 mg/kg) treated group, immobility time reduced to 54.60 7.46 s
(p < 0.001 vs. saline group), while swimming and climbing time increased
to 181.6 4.66 s (p < 0.001 vs. saline group) and 61.8 6.82 s
(p < 0.001 vs. saline group), respectively. In A2K2 (1 mg/kg) treated
group, immobility time reduced to 39.80 7.65 s (p < 0.001 vs. saline
group), while swimming and climbing time increased to 191.0 7.12 s
(p < 0.001 vs. saline group), and 69.20 5.48 s (p < 0.001 vs. saline
group), respectively. In fluoxetine (20 mg/kg) treated group, immobility,
3.6 | Effect on the number of ambulations and
rearings
Both A1K1 and A2K2 showed a significant increase in mice locomotor
activity that is, number of ambulations and rearings in open field test.
In saline (10 mL/kg) treated group, number of ambulations and rearings
recorded were 57.4 5.91 and 18.4 0.81, respectively. Treatment
with A1K1 (0.1 mg/kg), A1K1 (0.5 mg/kg), A1K1 (1 mg/kg), and fluoxe-
tine (20 mg/kg) increase the numbers of ambulations to 83.40 3.93,
102.80 3.98, 115.6 4.95, and 83.4 3.47, respectively, while
increase the number of rearings to 28.60 2.76, 31.8 2.67,
TABLE 1 E-value (kcal/Mol) and post-docking analysis of best pose of (2E,6E)-2,6-dibenzylidene cyclohexanone (A1K1), (1E,
4E)-5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-2-methylpenta-1,4-diene-3-one (A2K2) and standard drugs against serotonin transporter 2
(SERT2), serotonin transporter 3 (SERT3), norepinephrine transporter 1 (NET1), norepinephrine transporter 2 (NET2), norepinephrine transporter
3 (NET3), dopamine transporter (DAT), monoamine oxidase B (MAO-B), N-methyl-D-Aspartate (NMDA) receptor, and serotonin receptor
modulator (SRM)
A1K1
A2K2
Standard drugs
No of
Binding
No of
E-value H-bonds Binding residues
No of
E-value H-bonds Binding residues
Target
E-value H-bonds residues
Standard
Fluoxetine −6.8
SERT2
−10.1
−9.5
2
0
THR-197,
TYR-175
−7.3
1
ALA-150, SER-142
1
ARG-104
SERT3
0
−9.1
2
THR-497, TYR-175, Fluoxetine −7.1
0
0
0
GLU-493
NET1
NET2
−8.4
0
0
0
0
−7.0
−9.8
1
0
SER-107
0
Fluoxetine −6.5
Fluoxetine −9.2
0
3
−10.9
GLY-93, PHE-92,
PRO-401
NET3
DAT
−8.5
−8.9
1
0
1
0
0
−8.2
−8.9
1
2
0
ASN-585, ALA-581
TYR-6, TYR-65
0
Fluoxetine −7.3
Fluoxetine −8.5
1
2
0
ALA-581
LYS-84, TYR-60
0
MAO-B −10.8
GLY-58,
−10.4
Selegiline
−7.4
MET-436
NMDA
SRM
−7.9
−9.8
0
0
0
0
−8.3
−8.9
1
1
TYR-214
Tacrine
−7.4
0
1
0
TYR-359, PHE-330
Fluoxetine −7.9
SER-212, THR-209
Abbreviations: ALA, alanine; ARG, arginine; ASN, asparagine; GLU, glutamic acid; GLY, glycine; LYS, lysine; MET, methionine; PHE, phenylalanine;
PRO, proline; SER, serine; THR, threonine; TYR, tyrosine.

BASHIR ET AL.
7
TABLE 2 E-value (kcal/mol) and post-docking analysis of best pose of (2E,6E)-2,6-dibenzylidene cyclohexanone (A1K1), (1E,
4E)-5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-2-methylpenta-1,4-diene-3-one (A2K2) and standard drugs against monoamine oxidase B
(MAO-B), N-methyl-D-aspartate (NMDA) receptor, beta secretase, butyrylcholinestrase (BChE), beta amyloid, acetylcholinestrase (AChE), and
gamma secretase
A1K1
A2K2
Standard drugs
No of
Binding
No of
E-value H-bonds Binding residues Standard
No of
Target
E-value H-bonds residues
E-value H-bond Binding residues
MAO-B
−10.8
1
GLY-58
−10.4
0
0
Selegiline
−7.4
0
0
MET-436
NMDA
−7.9
−8.4
−9.1
−9.1
−9.3
−8.4
0
1
0
0
0
0
0
−8.3
−7.7
−8.6
−7.9
−10.2
−7.3
01
01
0
TYR-314
ASP-62
0
Tacrine
−7.4
−8.3
−9.5
0
0
Beta secretase
BChE
ASP-62
Donepezil
Donepezil
02
0
ARG-61, TYR-320
0
0
0
0
0
Beta amyloid
AChE
0
0
Donepezil −11.3
1
CYS-406
01
01
TYR-341
ASN-132
Donepezil
Donepezil
−8.3
−8.2
1
TYR-124
Gamma secretase
02
ASN-132, ASP-127
Abbreviations: ARG, arginine; ASN, asparagine; ASP, aspartic acid; CYS, cysteine; MET, methionine; TYR, tyrosine.
39.0 1.41, and 29.0 1.81, respectively (Figure 5a). In accordance
with A1K1, treatment with A2K2 (0.1 mg/kg), A2K2 (0.5 mg/kg), A2K2
(1 mg/kg), and fluoxetine (20 mg/kg) increase the numbers of ambula-
tions to 85.40 13.4, 101.4 8.28, 123.4 4.61, and 88.2 3.33,
respectively, while increase the number of rearings to 26.4 3.14,
32.4 3.09, 42.6 3.07, and 30.2 3.44, respectively (Figure 5b). All
the results were highly significant (p < 0.001 vs. saline group).
78.6 3.77% (p < 0.001 vs. saline group), respectively. Donepezil
(3 mg/kg) treated group showed alternation behavior of 64.8 1.90%
(p < 0.01 vs. saline group; Figure 6b).
3.8 | Effect on number of entries
A1K1 dose-dependently (0.5–1 mg/kg) increased number of entries in
Y-maze test. In saline (10 mL/kg) treated group, number of entries on
Day 1, 2, and 3 were 21.4 1.43, 18.4 1.12, and 17.0 0.70,
respectively. In A1K1 (0.5 mg/kg) treated group, number of entries on
Day 1, 2, and 3 increased to 28.2 1.35, 25.8 1.24, and 24.2 1.39
(p < 0.01 vs. saline group), respectively. In A1K1 (1 mg/kg) treated
group, number of entries on Day 1, 2, and 3 increased to 31.2 1.02,
28.2 1.39, and 26.4 1.43 (p < 0.001 vs. saline group), respectively.
In donepezil (3 mg/kg) treated group, number of entries on Day 1, 2,
and 3 recorded were 26.0 1.00, 23.0 0.70, and 20.6 0.51
(p > 0.05 vs. saline group), respectively (Figure 7a). A2K2 dose-
dependently (0.5–1 mg/kg) increased number of entries. In saline
(10 mL/kg) treated group, numbers of entries on Day 1, 2, and 3 were
20.4 0.87, 18.0 0.89, and 16.8 0.73, respectively. In A2K2
3.7 | Effect on alternation behavior
The Y-maze task was performed to analyze the spatial working memory
using spontaneous alteration behavior (%). Both A1K1 and A2K2 dose-
dependently (0.5-1 mg/kg) increased spontaneous alternation behavior
(%) of mice in the Y-maze test. Percent spontaneous alternation behav-
ior was recorded for saline (10 mL/kg), A1K1 (0.5 mg/kg), A1K1
(1 mg/kg), and Donepezil (3 mg/kg) treated groups as 48.0 2.34%,
63.40 1.86%, 72.4 1.63, 65.0 2.38%, respectively (Figure 6a). All
the results were highly significant (p < 0.001 vs. saline group). Similarly
A2K2 (0.5 mg/kg) and A2K2 (1 mg/kg) treated group showed increased
alternation behavior of 57.0 1.58% (p > 0.05 vs. saline group) and
FIGURE 3 Bar-graph showing anti-
depressant effect of (a) (2E,6E)-
2,6-dibenzylidene cyclohexanone (A1K1),
(b) (1E,4E)-5-(2,3-dichlorophenyl)-
1-(4-methoxyphenyl)-2-methylpenta-
1,4-diene-3-one (A2K2) and fluoxetine on
immobility, swimming and climbing time of
mice in forced swim test. Data expressed
as mean SEM, n = 5. **p < 0.01,
***p < 0.001 versus saline group, one-way
ANOVA followed by posthoc Tukey's test.
ANOVA, analysis of variance

8
BASHIR ET AL.
FIGURE 4 Bar-graph showing anti-depressant
effect of (a) (2E,6E)-2,6-dibenzylidene
cyclohexanone (A1K1), (b) (1E,4E)-
5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-
2-methylpenta-1,4-diene-3-one (A2K2) and
fluoxetine on immobility time of mice in tail
suspension test. Data expressed as mean SEM,
n = 5. ***p < 0.001 versus saline group, one-way
ANOVA followed by posthoc Tukey's test. ANOVA,
analysis of variance
(0.5 mg/kg) treated group, number of entries on Day 1, 2, and
3 increased to 24.8 1.15, 21.8 0.86, and 20.2 0.89 (p > 0.05
vs. saline group), respectively. In A2K2 (1 mg/kg) treated group, number
of entries on Day 1, 2, and 3 increased to 33.2 1.28, 30.0 1.04, and
28.0 1.43 (p < 0.001 vs. saline group), respectively. In donepezil
(3 mg/kg) treated group, number of entries on Day 1, 2, and 3 recorded
were 25.6 1.07, 23.0 0.89 and 21.2 1.02 (p < 0.05 vs. saline
group), respectively (Figure 7b).
A1K1 in the final trial in saline (10 mL/kg), A1K1 (0.5 mg/kg), A1K1
(1 mg/kg) and donepezil (3 mg/kg) treated groups was, 19.9 1.10,
13.15 1.14, 8.3 0.63, and 15.2 0.54, respectively (Figure 8a).
Similarly the escape latency time recorded for A2K2 in the final trial in
saline (10 mL/kg), A1K1 (0.5 mg/kg), A1K1 (1 mg/kg), and donepezil
(3 mg/kg) treated groups was 20.8 0.66, 14.4 1.40, 07.8 0.97,
and 13.6 0.81, respectively (Figure 8b). All the results were highly
significant (p < 0.01 vs. saline group).
3.9 | Effect on escape latency
3.10 | Acute toxicity analysis
In MWM, escape latency time for A1K1 and A2K2 was performed in
three trials. Our results showed that both A1K1 and A2K2 dose-
dependently (0.5–1 mg/kg) decreased escape latency time as com-
pared to the saline-treated group. The escape latency time noted for
Compound A1K1 and A2K2 were found safe up to the dose of
10 mg/kg, as no mortality was caused at the given doses. Furthermore,
there was not any kind of detrimental effect observed on overall health
during the experimental studies.
FIGURE 5 Bar-graph showing anti-depressant
effect of (a) (2E,6E)-2,6-dibenzylidene
cyclohexanone (A1K1), (b) (1E,4E)-
5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-
2-methylpenta-1,4-diene-3-one (A2K2) and
fluoxetine on ambulations and rearings of mice in
open field test. Data expressed as mean SEM,
n = 5. *p < 0.05, **p < 0.01, ***p < 0.001 versus
saline group, one-way ANOVA followed by posthoc
Tukey's test. ANOVA, analysis of variance

BASHIR ET AL.
9
FIGURE 6 Bar-graph showing anti-Alzheimer's
effect of (a) (2E,6E)-2,6-dibenzylidene
cyclohexanone (A1K1), (b) (1E,4E)-
5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-
2-methylpenta-1,4-diene-3-one (A2K2) and
donepezil on alternations behavior of mice in
Y-maze test in mice. Data expressed as
mean SEM, n = 5. **p < 0.01, ***p < 0.001 versus
saline group, one-way ANOVA followed by
posthoc Tukey's test. ANOVA, analysis of variance
test models were used for screening of antidepressant potential (Brown
et al., 1999; Maurmann et al., 2011; Simplice et al., 2014). Stressful
condition plays a key role in depression. Substances that decreased
immobility time in FST and TST, while increased swimming and
climbing behavior in FST, showed antidepressant effects (Borsini &
Meli, 1988). A1K1and A2K2, dose-dependently decreased immobility
time in FST and TST, while increased swimming and climbing time in
FST. Mice locomotor activity was also increased in the open field test.
Swimming behavior is sensitive to serotonergic, while climbing behav-
ior is sensitive to noradrenergic neurotransmissions (Detke, Rickels, &
Lucki, 1995). So A1K1 and A2K2 have serotonergic as well as norad-
renergic effects that reveal their antidepressant potential. Depression
can result from reduction of monoamine neurotransmitters (norepi-
nephrine, dopamine, and serotonin) in brain (Delgado, 2000). MAO-B
is involved in catalyzing the oxidation of monoamines. Norepinephrine
involved in attentiveness, learning, emotions, dreaming, and sleeping.
Dopamine involved in pleasure, emotions, and locomotion regulation.
Serotonin plays a major role in memory, learning, mood, and behavior.
A1K1 and A2K2 modulate the levels of these neurotransmitters and
reduce serotonin metabolism in mice brain, demonstrating their antide-
pressant potential. Antidepressant activity of A1K1 and A2K2 might be
possible due to the involvement of these transporters as well as the
involvement of different other pathways.
4
| DISCUSSION
In this study, we first assessed the affinity of dibenzylidene ketone
derivatives (A1K1 and A2K2) through E-value and number of hydro-
gen bonds against protein targets that influence antidepressant and
anti-Alzheimer effects. Docking tool was preliminary used to check
the affinity of ligands to their respective protein targets. Hydrogen
bonding is reported to be significant in formation of ligand protein
complex (Wang, Lai, & Wang, 2002). The docking of the selected
ligands was carried out by using Auto Dock Vina program through
PyRx (Dallakyan & Olson, 2015). A1K1 order of binding affinity
against target proteins was found as: NET2 > MAO-B > SERT-Ts2 >
SRM > SERT-Ts3 > AChE > BChE > Beta amyloid > DAT > NET3 >
NET1 > beta secretase > gamma secretase > NMDA. Similarly the
order of binding affinity of A2K2 against target proteins was found as:
MAO-B > AChE > NET2 > SERT-Ts3 > DAT > SRM > BChE > NMDA >
NET3 > beta amyloid > beta secretase > SERT-Ts2 > gamma secre-
tase > NET1. A1K1 and A2K2 showed highest binding affinity against
NET2 and MAO-B, which can be correlated to its highest in-vivo anti-
depressant and anti-Alzheimer activities, respectively. Behavioral phar-
macological techniques were employed to investigate the antidepressant
and anti-Alzheimer effects of A1K1 and A2K2. FST, TST, and open field
FIGURE 7 Bar-graph showing anti-Alzheimer's
effect of (a) (2E,6E)-2,6-dibenzylidene
cyclohexanone (A1K1), (b) (1E,4E)-
5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-
2-methylpenta-1,4-diene-3-one (A2K2) and
donepezil on number of mice entries in Y-maze test
on day 1, 2 and 3. Data expressed as mean SEM,
n = 5. *p < 0.05, **p < 0.01, ***p < 0.001 versus
saline group, one-way ANOVA followed by posthoc
Tukey's test. ANOVA, analysis of variance

10
BASHIR ET AL.
FIGURE 8 Bar-graph showing anti-Alzheimer's
effect of (a) (2E,6E)-2,6-dibenzylidene
cyclohexanone (A1K1), (b) (1E,4E)-
5-(2,3-dichlorophenyl)-1-(4-methoxyphenyl)-
2-methylpenta-1,4-diene-3-one (A2K2) and
donepezil on escape latency time of mice in trial
1, 2 and 3 in Morris water maze test. Data
expressed as mean SEM, n = 5. *p < 0.05,
**p < 0.01, ***p < 0.001 versus saline group,
one-way ANOVA followed by posthoc Tukey's
test. ANOVA, analysis of variance
Next, in this study Y-maze test and MWM test models were used
involved in depression and AD-like SERT2, SERT3, NET1, NET2,
NET3, DAT, SRM, MAO-B, NMDA, beta-secretase, BChE, beta-amy-
loid, AChE, and gamma-secretase. Furthermore, it was also noted that
A1K1 has more binding sites for targets involved in depression mech-
anism while A2K2 has more therapeutic potential to target
Alzheimer's related proteins. Both A1K1 and A2K2 exhibited protec-
tive effect against Alzheimer's and depressive like behavior. Hence,
our studies suggest that these dibenzylidene ketone derivatives could
be intriguing therapeutic approaches for the treatment of various neu-
rological disorders.
for the screening of anti-Alzheimer potential (Ali et al., 2015; Moris,
1984). Accumulation of amyloid plaques increased oxidative stress,
neurofibrillary tangles in neurons and acetylcholine deficiency play role
in AD (Hiremathad, 2017). The molecular docking results showed that
these compounds possess good binding affinity against MAO-B, AChE,
BChE, and beta amyloid targets, so it might be possible that the phar-
macological potential of A1K1 and A2K2 is due to modulation of these
targets. It has also been stated that AD is associated with progressive
cognitive deterioration along with behavioral changes (Mishra
&
Palanivelu, 2008). Spontaneous alteration behavior (%) was tested to
assess spatial working memory that is dependent upon the hippocam-
pus (Holcomb et al., 1998; Lelong, Lhonneur, Dauphin, & Boulouard,
2003; Ohno et al., 2004). A higher percentage of spontaneous alter-
ation behavior represents enhanced cognitive performance (Ali et al.,
2015). A1K1 and A2K2 dose-dependently increased spontaneous alter-
ation behavior (%) and a number of entries of mice in the Y-maze test.
Learning and memory were also evaluated using the spatial reference
version of the MWM, which is highly dependent on the hippocampus
(Billings, Oddo, Green, McGaugh, & LaFerla, 2005). The MWM results
showed that A1K1 and A2K2 dose-dependently decreased escape
latency time which reveals their anti-Alzheimer's potential.
ACKNOWLEDGMENT
The authors are thankful to Riphah Academy of Research and Education,
Riphah International University, for facilitation and support.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Overall, we can infer that our compounds have potential antide-
pressant and anti-Alzheimer's actions. The dibenzylidene ketone
derivative A1K1 has been studied to possess lipophilic character and
that they can cross the blood brain barrier (Sasaki et al., 2011). By
improving bioavailability, A1K1 and A2K2 must have improved action
against AD. It was observed that even at high doses all the compounds
were safe, producing no mortality after 24 hr of administration. Further
investigation is needed in order to confirm its permeability, stability,
suitability for oral administration, pharmacodynamics and pharmacoki-
netic confirmation before launching these new moieties in the market.
All authors listed have made a substantial, direct and intellectual contri-
bution to the work, and approved it for publication. M.A. carried
out the computational studies, experimental work, analyzed the data
and documentation. A-U.K and H.B. supervised the research project.
E.F. and Z.D. synthesized dibenzylidene ketone derivatives. A.K. revised
the final manuscript.
ORCID
Haroon Badshah
https://orcid.org/0000-0003-2959-0414
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SUPPORTING INFORMATION
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