Biological evaluation of azomethine-dihydroquinazolinone conjugates as cancer and cholinesterase inhibitors

Article abstract of DOI:10.2174/1573406411666150708111417

In an attempt to discover novel anti-cancer agents and potent cholinesterase inhibitors, 11 azomethine-dihydroquinazolinone conjugates were evaluated against lung carcinoma cells and cholinesterases. Most of the compounds exhibited significant cytotoxicity at low micromolar concentrations and were less toxic to normal cells. After 24 h incubation period, 2i showed maximum cytotoxicity. The 4-bromine substituted compounds showed higher acetylcholinesterase (AChE) inhibitory activity than other screened compounds. The most active compound 2c, among the series, had an IC50 value 209.8 μ M against AChE. The tested compounds showed less inhibition against butyrylcholinesterase. Molecular docking studies were performed in order to investigate the plausible binding modes of synthesized compounds. The compounds can be further optimized to treat cancer and Alzheimer's disease. These derivatives may open new pathways for introducing new therapies for curing cancer and senile dementia.

Full text of DOI:10.2174/1573406411666150708111417

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74
Medicinal Chemistry, 2016, 12, 74-82
Biological Evaluation of Azomethine-dihydroquinazolinone Conjugates as
Cancer and Cholinesterase Inhibitors
Jamshed Iqbala,b,*, Aamer Saeedc, Syed J.A. Shaha, Mariya al-Rashidad and
Shams-ul Mahmoodc
aCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
b
Abbottabad 22060, Pakistan; Department of Pharmaceutical Sciences, COMSATS Institute of
Information Technology, Abbottabad 22060, Pakistan; cDepartment of Chemistry, Quaid-I-Azam
University, Islamabad, Pakistan; dDepartment of Chemistry, Forman Christian College
(A Chartered University), Ferozepur Road, Lahore 54600, Pakistan
Abstract: In an attempt to discover novel anti-cancer agents and potent cholinesterase inhibitors, 11
azomethine-dihydroquinazolinone conjugates were evaluated against lung carcinoma cells and cho-
linesterases. Most of the compounds exhibited significant cytotoxicity at low micromolar concentra-
tions and were less toxic to normal cells. After 24 h incubation period, 2i showed maximum cytotoxicity. The 4-bromine
substituted compounds showed higher acetylcholinesterase (AChE) inhibitory activity than other screened compounds.
The most active compound 2c, among the series, had an IC50 value 209.8 ꢀM against AChE. The tested compounds
showed less inhibition against butyrylcholinesterase. Molecular docking studies were performed in order to investigate the
plausible binding modes of synthesized compounds. The compounds can be further optimized to treat cancer and Alz-
heimer’s disease. These derivatives may open new pathways for introducing new therapies for curing cancer and senile
dementia.
Keywords: Acetylcholinesterase, azomethine-dihydroquinazolinone, butyrylcholinesterase, cancer, lung carcinoma cells, senile
dementia.
INTRODUCTION
neurotransmission [14]. Acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) share extensive sequence ho-
mology, ~ 65%, however they both differ in inhibitors sensi-
tivity and substrate specificities [11]. AChE is most widely
distributed in brain with higher selectivity for hydrolyzing
ACh. On the contrary, BChE (known as pseudocho-
linesterase) hydrolyzes BCh more rapidly than ACh [15].
Quinazolinone derivatives demonstrate a broad range of
biological activities including anticancer [1-3], anticoccidial
[4], 5-HT7 receptor antagonist [5], analgesic and anti-
inflammatory properties [5]. In addition, 2-[4-(aminoalkoxy)
phenyl]-4(3H)-quinazolinone derivatives are potent human
H3 receptor inverse agonists [6] and 2-thiophen-5-yl-3H-
quinazolin-4-one analogues are inhibitors of transcription
factors NF-ꢁB and AP-1 mediated transcriptional activation
and thus find possible utilization as anti-inflammatory and
anti-cancer agents [7]. The azomethine derivatives of quina-
zolinyl acetohydrazide, in which the carbonyl group being
replaced by the imine or azomethine group [8], are found to
exhibit antimalarial [9], antibacterial and antifungal activities
[10].
ACh released by the parasympathetic nervous system is a
neurotransmitter of the cholinergic innervation, stimulating
nicotinic as well as muscarinic receptors [16]. Muscarinic
receptor stimulation is associated with the smooth muscles
and heart, and detailed classical symptoms are previously
described. Nicotinic receptors stimulate the central nervous
system (CNS) and the skeletal muscles. Nicotinic receptor
stimulation causes contractions in the skeletal muscles and in
CNS, it primarily functions in memory and cognitive proc-
esses [17]. The cholinergic neurotransmission can be
strengthened by increasing ACh levels and it can be achieved
by cholinesterase inhibitors [11]. Cholinesterase inhibitors
used in the treatment of AD are rivastigmine, tacrine,
ensaculin [14], donepezil and galantamine [13, 14]. Com-
pounds such as organophosphates, coumarine, carbamates,
cinnamides [14] and dihydroquinazolinone [18] derivatives
have been reported to inhibit cholinesterase. Dihydroquina-
zolinone derivatives also possess antitumor [19], antidepres-
sant [20] and calcium channel blocking activity [21].
Alzheimer’s disease (AD) is an irreversible neurodegen-
erative disease characterized by loss of cholinergic neuro-
transmission in cerebral cortex and sub-cortical region [11].
ꢂ-Amyloid aggregation and diminished levels of acetylcho-
line (ACh) have been reported to cause Alzheimer’s disease
[12, 13]. ACh is rapidly degraded by cholinesterase (ChE)
into choline and acetic acid thus terminating the cholinergic
*Address correspondence to this author at the Centre for Advanced Drug
Research, COMSATS Institute of Information Technology, Abbottabad
22060, Pakistan; Tel: +92 992 383591 96; Fax: +92 992 383441;
E-mail: drjamshed@ciit.net.pk
1875-6638/16 $58.00+.00
© 2016 Bentham Science Publishers

Azomethine-dihydroquinazolinone Conjugates as Cancer and Cholinesterase Inhibitors
Medicinal Chemistry, 2016, Vol. 12, No. 1 75
Many remarkable biological functions in proteins and
DNA and their profound dynamic mechanisms, such as
switch between active and inactive states [22], cooperative
effects [23], allosteric transition [24-26] and intercalation of
drugs into DNA [27], can be revealed by studying their in-
ternal motions as summarized in a comprehensive review
[28]. Likewise, to fully understand the interaction of a pro-
tein receptor with its ligand and to in-depth reveal their bind-
ing mechanism, we should release the constraints on the re-
ceptor structure allowing it flexible as well during the dock-
ing process, and we shall make efforts in this regards in our
future work.
cells were used as normal cell and were treated like H157
throughout the assay [32].
Determination of AChE and BChE Inhibitory Activities
Commercially purified electric eel AChE and horse se-
rum BChE was used. Cholinesterases inhibition was meas-
ured by the standard procedure by Ellman [33] with slight
modification [34]. Synthesized compounds were dissolved in
DMSO and were screened at 0.2 mM per well concentration
for initial screening (for both AChE and BChE). Further di-
lutions were used for compounds having more than 50%
inhibition. The initial reaction mixture consisted of assay
buffer, test compound and enzyme (0.5 and 3.4 U/mg of
AChE or BChE, respectively). A 10 min preincubation of the
Keeping the above mentioned biological importance of
quinazolinone derivatives in mind, we evaluated the azome-
thine-dihydroquinazolinone conjugates for their anticancer
and cholinesterase inhibition studies.
o
reaction mixture at 25 C was carried out. After incubation
their respective substrates (1 mM) and coloring reagent, 5,5’-
Dithiobis-(2-Nitrobenzoic Acid) (3 mM) were added. The
mixtures were further incubated for 15 min at 25 °C. The
absorbance was determined at 405 nm using a microplate
reader (Bio-Tek ELx800TM, Instruments Inc., Winooski, VT,
USA). All experiments were repeated at least three times.
Results reported are mean of three independent experiments
(± SEM) and expressed as percent inhibitions calculated by
the formula:
MATERIALS AND METHODS
A general procedure for the synthesis of azomethine di-
hydroquinazolinone conjugates (2a-k) has been reported in
our recently published paper [29].
Cell Lines and Cell Cultures
Lung carcinoma (H157) cell lines (ATCC CRL-5802)
and human corneal epithelial cells (HCEC) (obtained from
RIKEN Bio Resource Center, Japan) were used. Cell lines
were maintained in medium and cultured at 37 ºC in a 5%
CO2 incubator using the previous method [30]. Monolayers
obtained were used for experimental purpose.
Percent inhibition = 100 – (Sample absorbance / control
absorbance) ꢁ 100
Neostigmine and donepezil were used as reference com-
pounds. The buffer for enzyme dilution contained 50 mM
Tris-HCl and 0.1% (w/v) BSA having pH 8.0. The analysis
of each concentration was done in triplicate, IC50 values of
potential inhibitors (ꢂ50%) were determined with the help of
the Graph Pad prism 5.0 Software Inc., San Diego, Califor-
nia, USA [35].
Cytotoxicity Analysis by Sulforhodamine B (SRB) Assays
Cytotoxicity assays were performed using the standard
SRB method of Skehan et al., [31]. Cells were cultured in 96
well plates and test compounds were added to wells at final
concentration of 100 ꢀM, and incubated for 24 h. The wells
containing culture media with cells being treated as control,
whereas the wells with culture medium without cells were
taken as blank. After 24 h incubation, cells were fixed by
using 50% trichloroacetic acid (TCA) solution and incubated
for 1 h at 4 °C. Plates were washed five times with phos-
phate-buffered saline (PBS) and left for air drying. After
drying of TCA fixed cells, staining was done by 0.4% SRB
solution for 30 min at room temperature. The unbound dye
was removed by rinsing four times with acetic acid solution
(1%) and left for air drying. The protein bound dye was
solubilized, after air drying, by the addition of Tris-base so-
lution (100 ꢀL/well, 10 mM). The optical densities (OD)
were measured at 490 nm subtracting the background meas-
urement at 630 nm using 96-well microplate reader (Bio-Tek
ELx 800TM, Instruments, Inc. Winooski, VT, USA). All ex-
periments were repeated at least three times. Results reported
are mean of three independent experiments (± SEM) and
expressed as percent inhibitions calculated by the formula:
Molecular Docking
The X-ray crystal structure of human AChE was down-
loaded from the RCSB (PDB ID: 4BDT) [36]. AutoDock 4.2
[37] was used for docking studies. Prior to docking, the en-
zyme or receptor was prepared by removing all water and
other heteroatoms from it, hydrogen atoms and Gasteiger
charges were added using MGL Tools [37]. AutoDock uses
AutoGrid to calculate affinity maps for interaction energy of
various atom types with the enzyme which is then used by
the AutoDock docking to calculate the total interaction en-
ergy for a ligand with receptor (enzyme). In AutoDock the
receptor is kept rigid, whereas the ligand is treated as flexi-
ble. The grid-box (80 x 80 x 80) was centered on the active
site gorge was large enough to allow free movement of
ligand. Method validation was carried out by re-docking the
ligand (extracted from 4BDT) into the same enzyme. The
docking methodology was able to reproduce the experimen-
tally determined binding conformation for the ligand with an
rmsd of less than 0.5 Å. For ligands, pdb files were created,
Gasteiger charges were added using ANTECHAMBER [38].
Before docking the energies of the ligands were minimized
using Chimera, through 100 steepest descents and 100 con-
jugate gradient steps using a step size of 0.02. Lamarckian
Genetic Algorithm (LGA) was used for docking, the number
of GA runs was set to 20 and the number of maximum
evaluation was 5x106 for Genetic Algorithm parameter and
Percent inhibition = 100 – (Sample absorbance / control
absorbance) ꢁ 100
Blank background optical density was measured in wells
incubated with growth medium without cells. Control values
were obtained from H157 incubated in RPMI medium alone
without test compounds. HCEC cells were used as normal

76 Medicinal Chemistry, 2016, Vol. 12, No. 1
Iqbal et al.
O
CHO
NH2
N
H
R
N
H
N
O
S
N
CH3
O
N
S
N
R
N
C2H5OH / reflux
CH3
O
(1)
(2a-k)
2a: R= 2-Br
2c: R= 4-Br
2e: R= 3-Cl
2g: R= 3-NO2
2i: R= 2-OMe
2k: R= 2-OBz
2b: R= 3-Br
2d: R= 2-Cl
2f: R= 4-Cl
2h: R= 4-NO2
2j: R= 3,4,5-(OMe)3
Scheme 1. Synthetic pathway to azomethine dihydroquinazolinone conjugates.
Table 1. Cytotoxicity activity (%) in vitro of the 2a-k against lung carcinoma cells (H157) and human corneal epithelial cells (HCEC).
N
H
N
O
N
S
N
R
CH3
O
(2a-k)
Compounds
H157
HCEC
% Inhibition ± SEM (100 ꢀM)
2a
35.2 ± 6.41
36.3 ± 7.46
38.4 ± 4.91
34.2 ± 4.92
53.0 ± 2.51
40.1 ± 2.58
52.1 ± 7.05
56.3 ± 5.27
62.3 ± 7.82
42.1 ± 1.44
53.7 ± 3.74
22.3 ± 1.42
6.21 ± 0.13
5.13 ± 1.21
5.70 ± 0.12
7.33 ± 2.11
7.42 ± 2.12
5.81 ± 1.91
4.42 ± 1.42
7.21 ± 3.5
2b
2c
2d
2e
2f
2g
2h
2i
7.35 ± 2.65
7.51 ± 2.18
4.58 ± 1.12
6.48 ± 3.18
2j
2k
Methotrexate
docking generations were 27000. For visualization of docked
results, Discovery Studio Visualizer 3.5 [39] was used.
Biological Assays
In vitro Evaluation of Cytotoxic Activity
Cytotoxic properties of compounds (2a-k) were evaluated
in vitro in lung carcinoma cells H157 and human corneal
epithelial cells HCEC. Cytotoxicity data is shown in Table 1.
A dose-dependent decrease of cell density in H157 cells was
observed after 24 h exposure to all azomethine derivatives.
The tested compounds were non toxic to normal cells, HCEC
(Table 1). At 100 ꢀM, compounds presented the percent in-
hibition against H157 ranging from 62% (2i) to 34% (2d).
The compound 2i was found to be the most active in the se-
RESULTS AND DISCUSSION
Chemistry
The synthetic pathway leading to the synthesis of title
compounds is shown in Scheme 1. Thus, eleven azomethine
dihydroquinazolinone conjugates (2a-k) were obtained by
treating acetohydrazide (1) with suitably substituted aromatic
aldehydes in ethanol [29].

Azomethine-dihydroquinazolinone Conjugates as Cancer and Cholinesterase Inhibitors
Medicinal Chemistry, 2016, Vol. 12, No. 1 77
Fig. (1). Percent inhibition of derivatives and methotrexate against H157 and HCEC cell line at 100 ꢀM.
from meta to para position as in 2h decreases the potency. A
series with 62.3% inhibition against cancer cells, whereas
same compound represents 7.35% inhibition against normal
cells. It was indicated that the compound has shown inhibi-
tory potential against cancer cells, in contrast, the same com-
pound was non-toxic to normal cells. The responses of the
tested cell lines to new drugs were compared to the effect of
methotrexate, a drug approved for use in lung cancer. To test
the toxicity of derivatives against normal cells, HCEC cells
were treated in the same way as cancer cell line, H-157. The
results of cytotoxicity investigation proved azomethine de-
rivatives as good anticancer agents having significant cyto-
toxic property against lung carcinoma cells. Whereas the
compounds were non-toxic towards normal cell lines. The
toxicity against normal cell lines was less than 10% for all
the compounds and reference drug at 100 ꢀM. The percent
inhibition of screened compounds against cancer cells and
normal cells has been shown in (Fig. 1). Based on the results,
further derivatives of most active compounds may be synthe-
sized, which after in vivo studies may be used as a lead
molecule for cancer treatment. These derivatives showed
relatively low toxicity against normal cells which could be a
positive aspect of this study in the design of more active and
safer analogues.
further decrease in potency was observed when chlorine was
added at either of the three positions (i.e. o-, m- or p-) in 2d,
2e and 2f, respectively, and decreased activity was observed
for 2j containing tri-substituted methoxy groups. 2k showed
least activity owing to the presence of the electron donating
benzyloxy group at ortho-position.
For the determination of potential interest of synthesized
compounds in the treatment of AD, their AChE and BChE
inhibitory activities were assayed. Compounds displayed
inhibitory abilities in micromolar range for AChE, but they
were not active against BChE. Recent studies have shown
that in AD patients with severe pathology, BChE increases
while AChE is reduced in specific brain regions [40], while
another recent study shows a decrease of BChE activity in
vivo [41]. The results presented herein showed that these
derivatives may be used as an important starting point for
further investigation of the possible use of these compounds
in the therapy of neurodegenerative diseases.
Docking Studies
Molecular docking studies were carried out in order to
gain insight into important binding site interactions that
could aid in the design of more potent inhibitors. Com-
pounds 2a-k, were found to have similar binding modes. All
compounds occupied the same position inside the active site
(Fig. 2). AutoDock calculated binding free energies of com-
pounds 2a-k are given in Table 3. In compound 2c, the aro-
matic dihydroquinazlinone ring was found to be involved in
a ꢁ-stacking interaction with the hydrophobic residue
Tyr337. The NH of azomethine group is making a hydrogen
bond with oxygen atom of Tyr124 (2.05 Å). The amino acid
residues Phe338 and Phe297 are in close contact with the
azomethine group (Fig. 3). Predicted binding site interac-
tions of compound 2g are given in (Fig. 4). The dihydro-
quinazlinone ring occupies the same position as compound
2c. The NH of azomethine group is making a hydrogen bond
with oxygen atom of Tyr124 (1.82 Å). This hydrogen bond
interaction was also found for compound 2c, and seems to be
an important contact. An additional hydrogen bond interac-
Anticholinesterase Activity
The synthetic dihydroquinazolin derivatives were evalu-
ated in vitro as inhibitors of electric eel acetylcholinesterase
(AChE) and horse serum butyrylcholinesterase (BChE). The
inhibitory activities of these derivatives were compared to
those of neostigmine and donepezil. Inhibition potencies for
compound showing greater than 50% were expressed as IC50
values and compounds with less than 50% were shown in
percentage values (as in case of BChE) in Table 2.
Bromine substituted derivative 2c showed potent AChE
inhibitory activity because of the presence of electron with-
drawing bromine at para-position, substituting the same
group at ortho-position (2a) decreases potency and substitut-
ing the same group at meta-position (2b) further declines the
potency. However, adding electron donating groups such as
nitro (2g) or methoxy group (2i) at meta-position results in
potencies comparable to 2a. Substituting the nitrite group

78 Medicinal Chemistry, 2016, Vol. 12, No. 1
Iqbal et al.
Table 2. Inhibitory activities of azomethine derivatives (2a-k) against cholinesterase enzymes (AChE and BChE).
AChE
BChE
Codes
Compounds
IC50 (ꢀM ± SEM)
% Inhibition
H3C
O
Br
2a
281.1 ± 1.08
23.2
24.6
N
O
N
N
S
N
H
H3C
O
Br
2b
642.9 ± 1.97
N
O
N
N
S
N
H
H3C
O
2c
2d
2e
209.8 ± 1.11
690.5 ± 1.58
582.3 ± 1.28
6.8
N
O
N
Br
N
S
N
H
H3C
O
Cl
33.3
26.5
N
O
N
N
S
N
H
H3C
O
Cl
N
O
N
N
S
N
H
H3C
O
N
2f
627.2 ± 1.24
276.1 ± 2.31
36.5
25.9
N
O
N
Cl
S
N
H
H3C
O
NO2
2g
N
O
N
N
S
N
H
H3C
O
2h
2i
765.7 ± 2.86
281.5 ± 2.13
538.6 ± 1.12
26.6
21.7
27.5
N
O
N
NO2
N
S
N
H
H3C
O
MeO
N
O
N
N
S
N
H
H3C
O
OMe
OMe
OMe
N
O
2j
N
N
S
N
H

Azomethine-dihydroquinazolinone Conjugates as Cancer and Cholinesterase Inhibitors
Medicinal Chemistry, 2016, Vol. 12, No. 1 79
Table 2. contd…
AChE
BChE
Codes
2k
Compounds
IC50 (ꢀM ± SEM)
% Inhibition
H3C
O
926.1 ± 2.08
34.9
O
N
O
N
N
S
N
H
Standard
Standard
Neostigmine
Donepezil
24.3 ± 4.11
0.02 ± 0.01
IC50 = 37.1 ± 7.23 ꢀM
IC50 = 6.72 ± 0.48 ꢀM
IC50 = Concentration of inhibitor required for half-maximal enzyme inhibition and are reported as means of three independent experiments, each performed in triplicate (SEM, stan-
dard error of mean).
Table 3. AutoDock 4.2 calculated binding free energies for
docked inhibitors.
The information of a binding pocket of a receptor for its
ligand is very important for drug design, particularly for
conducting mutagenesis studies [42]. In the literature, the
binding pocket of a protein receptor to a ligand is usually
defined by those residues that have at least one heavy atom
(i.e., an atom other than hydrogen) within a distance of 5Å
from a heavy atom of the ligand. Such a criterion was origi-
nally used to define the binding pocket of ATP in the Cdk5-
Nck5a* complex [43] that has later proved quite useful in
identifying functional domains and stimulating the relevant
truncation experiments. The similar approach has also been
used to define the binding pockets of many other receptor-
ligand interactions important for drug design [25, 44-49].
Codes
Binding Free Energy (ꢁG) kcal/mol
2a
2b
2c
2d
2e
2f
-12.2
-11.76
-12.89
-11.5
-11.55
-11.72
-11.93
-11.55
-11.69
-11.45
-11.44
2g
2h
2i
CONCLUSION
Azomethine-dihydroquinazolinone derivatives (2a-k)
were evaluated for cytotoxicity and cholinesterase inhibitory
activities. The investigated compounds possess anticancer
activity. Compounds 2i and 2h were the most active com-
pounds against lung carcinoma cells H157 and were less
toxic to normal cells. The compounds were further tested
against cholinesterases such as acetylcholinesterase and bu-
2j
2k
tion was found to exist between the oxygen of NO2 group
and NH of Arg296 (1.74 Å).
Fig. (2). Overlap of compounds 2a-k into the active site gorge of AChE. All inhibitors were found to have similar binding orientations.

80 Medicinal Chemistry, 2016, Vol. 12, No. 1
Iqbal et al.
Fig. (3). Binding site interacions of compound 2c (pink) inside active site gorge of AChE and its 2D interaction diagram. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this paper).
Fig. (4). Binding site interacions of compound 2g (green) inside active site gorge of AChE and its 2D interaction diagram. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this paper).
tyrylcholinestrease. Most of the evaluated compounds were
significant inhibitors of the AChE. The azomethine deriva-
tives can be considered as anti-acetylcholinesterase and
therefore, can be used in the search of new therapies for Alz-
heimer’s disease. Furthermore, the results suggest that these
derivatives may be used as preventive and chemotherapeutic
agents for cancer. Hence it is recommended to find the corre-
lation of AChE inhibitors and their role in the therapy of
cancer.
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Received: February 21, 2015
Revised: June 29, 2015
Accepted: July 03, 2015