Novel 2-amino-1,4-naphthoquinone hybrids: Design, synthesis, cytotoxicity evaluation and in silico studies

Article abstract of DOI:10.1016/j.bmc.2020.115718

In the present work, a novel series of 2-amino-1,4-naphthoquinones bearing oxyphenyl moiety (5a-5m) were designed and synthesized via a two-step route and evaluated for their in vitro cytotoxic activity against three different cancer cell lines (MCF-7, HL

Full text of DOI:10.1016/j.bmc.2020.115718

Bioorganic&MedicinalChemistry28(2020)115718
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 2-amino-1,4-naphthoquinone hybrids: Design, synthesis, cytotoxicity
evaluation and in silico studies
⁎
Maryam Gholampoura,b, Hassan Seradjc, Somayeh Pirhadid, Mehdi Khoshneviszadehb,d,
a Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
c Department of Pharmacognosy, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
d Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present work, a novel series of 2-amino-1,4-naphthoquinones bearing oxyphenyl moiety (5a-5m) were
designed and synthesized via a two-step route and evaluated for their in vitro cytotoxic activity against three
different cancer cell lines (MCF-7, HL-60 and U937) and normal human cell line (HEK-293) by MTT assay.
Compounds 5b (4-nitro-benzyl-) and 5k (4-bromo-benzyl-) were identified to possess the highest cytotoxic ac-
tivity against MCF-7 cancerous cells (IC50 values of 27.76 and 27.86 μM, respectively). At the same time, none of
the compounds exert significant toxicity against HEK-293 normal human kidney cells. Cell cycle analysis showed
that the selected derivatives increased the population of MCF-7 cells in the S phase at 25 and 50 μM con-
centrations. Annexin V-FITC/PI staining assay also confirmed that compounds 5b and 5k induced apoptosis in
the cell death pathway. Molecular docking and molecular dynamics studies were also performed to evaluate the
probable interactions between the hybrids and human ATP binding domain of topo IIα protein. Our findings may
provide new insight for further development of novel naphthoquinone-containing compounds.
1,4-naphthoquinone
Cytotoxic agent
Apoptosis
Cell cycle arrest
Molecular docking
MD simulations
1. Introduction
double strands cleavage. This mammalian enzyme has two isoforms, α
and β, with about 70% sequence similarity between them. Topo IIα is
Cancer incidence and mortality is one of the notable complications
of public health throughout the world.1,2 Chemotherapy is a well-
known and conventional approach in the treatment of many types of
cancer. However, accomplishment of therapeutic procedure in most
patients is affected by drug resistance and issues arising from the toxic
impact on normal cells. Indeed, the majority of cancer death cases are
due to the consequences of chemotherapy strategy failure.3–5 Thus,
there is an urgent demand to find novel chemotherapeutic agents with
high selectivity and minimum side effects.
overexpressed in different cancerous cells. Inhibition of topo IIα leads
to double-stranded DNA breaks or ATP hydrolysis blockage which in
most inhibitors cytotoxic activity could be through arrest in the G1, S
and G2 phases and/or via apoptosis induction.8–10
In view of such significant cytotoxic activity, identifying new che-
mical entities based on 1,4-naphthoquinone nucleus is one of attractive
research prospects in the field of anticancer medicinal chem-
istry.4,7,11,12
As a part of ongoing efforts in our lab aimed at the identification of
active small molecules with cytotoxic activity and following our pre-
vious report on naphthoquinone hybrids13, a series of novel 1,4-naph-
thoquinone derivatives was rationally designed and synthesized by
linking 1,4-naphthoquinone and 4-oxyphenylamino moiety in a single
molecular framework with the goal of obtaining enhanced anti-
proliferative activity. In vitro cytotoxicity, cell cycle analysis and
apoptosis studies on the most potent derivatives are also described.
Moreover, molecular docking and molecular dynamics studies were
performed to identify the interaction of human topoisomerase IIα
protein with the synthesized hybrids and physicochemical parameters,
Quinones are found in the structure of various biologically active
natural and synthetic molecules. 1,4-naphthoquinone (1,4-NQ) is pre-
sent in commonly used anticancer drugs such as doxorubicin and
daunorubicin as a major pharmacophore. 1,4-naphthoquinone core
induce cancer cell death by interaction of several molecular mechan-
isms, including inhibition of topoisomerase II (Top2), NAD[P]H-qui-
none oxidoreductase (NQO1), STAT3 and NF-κB signaling pathway,
regulation of tumor suppressor factor p53, induction of apoptosis via
ERS and DNA damage through generation of ROS.6,7
Human DNA topoisomerase II is a critical enzyme in process of DNA
⁎ Corresponding author at: Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
E-mail address: khoshnevim@sums.ac.ir (M. Khoshneviszadeh).
https://doi.org/10.1016/j.bmc.2020.115718
Received 31 March 2020; Received in revised form 4 August 2020; Accepted 16 August 2020
Availableonline28August2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Scheme 1. Rational design strategy for new cytotoxic candidates.
bioactivity scores and ADMETox properties were also predicted.
on potency.
Meanwhile, since in our previous study, the 2-(Prop-2-ynylamino)
naphthalene-1,4-dione derivative showed interestingly high cytotoxic
activity with the IC50 values in the range of 12.2–15.5 μM against se-
lected cancer cell lines, herein the introduction of propargyl moiety was
also considered.13
2. Results and discussion
2.1. Design strategy
Furthermore, we have been inspired by pharmacological im-
portance of phthalimide analogs; and designed hybrid of 1,4-naphtho-
quinone and N-propylphthalimide conjugated through an 4-amino-
phenoxy linkage.23
As evidenced by previous studies, there are several examples of
aminonaphthoquinones with considerable cytotoxic activity.14–18 It is
worth to mention that phenylaminonaphthoquinones displayed greater
antiproliferative activity than alkylamino analogs as reported by Pra-
chayasittikul et al. 19
2.2. Chemistry
In 2010, Benites et al. synthesized and evaluated a series of phe-
nylamino-1,4-naphthoquinones. Among the tested derivatives, com-
pound I (Scheme 1, I) exhibited high cytotoxic potential against MCF-7
and DU-145 cancerous cells. Considering the mechanism of cell death,
no activation of caspase-3 was observed. It was noted that, this might
also be explained by remarkably decrease in ATP level, which is es-
sential to form an apoptosome. The molecule was non-toxic towards
normal BALB/3T3 cells suggesting that it has an excellent safety pro-
file.20 (See Scheme 2.)
As illustrated in Scheme 2, 4-oxyphenylamino-1,4-naphthoquinone
hybrids (5a-m) were prepared.
1,4-Naphthoquinone (1) as the starting material was treated with 4-
aminophenol (2) in ethanol at room temperature for 1 h to produce
known compound 2-((4-hydroxyphenyl)amino)naphthalene-1,4-dione
(3). Based on reported method,20 herein, we synthesized compound (3)
by a simple modified procedure without any further purification.
In the next step, compounds 5a-k and 5m were synthesized via a
nucleophilic substitution reaction of different benzyl chlorides/bro-
mides (4a-k) or 3-bromopropylphthalimide (4m) as electrophile in the
presence of potassium carbonate.24 In the case of propargyl bromide
(4l), it was reacted with compound 3 in dimethylformamide for 48 h
under reflux to generate target molecule 2-((4-(prop-2-yn-1-yloxy)
phenyl)amino)naphthalene-1,4-dione (5l).
Lawsone, 2-hydroxy-1,4-naphthoquinone, (Scheme 1, II) is the ac-
tive dye component of Lawsonia Inermis L. which has been identified as
potent anticancer agent with topoisomerase II inhibition activity.21,22
Moreover, Kumar et al. developed a novel 2-benzyllawsone (Scheme 1,
III) as potential cytotoxic agent against FaDu cell line with IC50 value of
5.2 μM. It also arrested cell cycle at S phase in FaDu cells and induced
apoptosis through activation of caspase pathway. Noticeably, this
compound showed significant topoisomerase II inhibition activity
which was comparable to lawsone.22
2.3. In vitro cytotoxicity screening
The above-mentioned findings were applied to design and synthe-
size new cytotoxic hybrid molecules by combining the merits of a cy-
totoxic topo II inhibitor (Scheme 1, III) and 2-Anilino-1,4-naphtho-
quinone core (Scheme1, I) with the aim of producing synergistic effects
The cytotoxic effects of synthesized compounds were evaluated
against three human cancer cell lines: MCF-7, HL-60 and U937 and
normal human cells HEK-293 using 3-[4,5-dimethylthiazol-2-yl]-2,5-
2

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
O
O
OH
H2N
+
1
2
EtOH, rt
1 h
4m
O
4l
O
O
O
O
O
O
N
Br
H
H
H
Br
N
N
N
O
O
K2CO3, DMF
Reflux, 48 h
K2CO3, Acetone
Reflux, 48 h
OH
O
O
N
3
5l
5m
O
Ar-CH2X
X=Cl, Br
K2CO3, Acetone
Reflux, 24 h
4a-k
O
O
H
N
O
Ar
5a-k
Ar:
OCH3 Br
5j 5k
Cl
NO2
Cl
F
CH3
CF3
Cl
Cl
5b
5e
5f
5a
5c
5d
5g
5h
5i
Scheme 2. Synthesis of 4-oxyphenylamino-1,4-naphthoquinone derivatives.
Table 1
5d) reduced the potency against MCF-7 cell line.
Cytotoxic activities of compounds 5a-m and 3 against different cancer and
Insertion of a methyl group (5e) diminished the activity compared
to an unsubstituted one. Compound 5e (4-methyl-benzyl-) was com-
pletely inactive against all tested cell lines. Comparing to the other
compounds with larger lipophilic groups such as –Br (5k), –Cl (5c) and
–CF3 (5f), the enhancement of cytotoxic activity is probably a con-
sequence of electron withdrawing effect in this class of compounds.
Compound 5m (-propyl phthalimide) also exhibited good cytotoxicity
normal cell lines assessed by MTT assay.
Compound
IC50 values† (µM)
HEK-293
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
ND
U937
HL-60
MCF-7
> 100
36.02
27.76
46.67
33.70
> 100
61.38
54.97
58.06
38.91
40.61
27.86
69.98
32.15
2.99
3
> 100
63.84
63.37
6.84
6.83
5a
5b
5c
5d
5e
5f
4.61
> 100
> 100
> 100
68.92
6.29
3.20
3.38
2.86
> 100
83.13
against MCF-7 cells (IC50 = 32.15
2.67 μM).
0.045
> 100
> 100
> 100
89.36
Parent compound 3 was more active against HL-60 cancer cells
> 100
> 100
70.46
(IC50 = 63.37
6.84 μM) when compared to other analogs of the
5.88
0.20
4.68
3.38
8.49
1.88
1.45
2.67
series except for 5i. Moreover, the observed activity for 3-chloro-benzyl
derivative (5i) proved that the insertion of electron withdrawing group
in the meta position could enhance the cytotoxic activity especially
5g
5h
5i
7.98
3.1
> 100
> 100
57.77
> 100
50.08
4.83
5j
6.97
8.19
> 100
> 100
> 100
82.81
against HL-60 cell line (IC50 = 50.08
4.83 μM). It seems that the
5k
5l
> 100
60.12
presence of bulky group on para position resulted in diminished cyto-
toxicity activity.
5m
Dox.
> 100
1.01
8.10
Only derivatives 5a, 5c, 5g, 5j and 5l were active against the U937
histiocytic lymphoma cells with IC50 values in the range of
57.77–89.36 μM. The overall effectiveness of compounds revealed that,
in this study, the MCF-7 cell line is more sensitive in comparison with
other cell lines.
0.14
0.06
0.004
0.36
† 50% inhibitory concentrations after 72 h of drug treatment are represented as
mean
S.E.M. of 3 independent experiments. Dox: Doxorubicin. ND: Not
determined.
Furthermore, to investigate the selective cytotoxic activity, com-
pounds 5a-m and 3 were tested against HEK-293 cell line. As seen in
Table 1, all synthesized compounds showed low cytotoxicity
(IC50 > 100 μM) against normal HEK-293 cells. These results indicated
that the targeted compounds have a good in vitro safety profile.
diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as
positive control. The concentration (in μM) of the tested compounds
which induced 50% inhibition of cell growth is shown in Table 1.
As listed in Table 1, most of the naphthoquinone hybrids displayed
greater cytotoxic activity compared to the parent compound 3 against
all three cancerous cell lines.
2.4. Cell cycle analysis
Notably, compounds 5b and 5k with bulky electron withdrawing
substituents (–NO2 and –Br) showed the highest potency against MCF-7
In order to study the possible cellular cytotoxicity mechanism of
synthesized hybrids, two potent compounds 5k and 5b were selected to
be evaluated for their effects on the distribution of cells in different
phases of cell cycle using propidium iodide (PI) staining assay.
cells. The IC50 values of 5b and 5k were 27.76
3.20 and
27.86
1.88 μM, respectively. However, replacement of 4-Br with a
small more electron withdrawing group like Cl or F (compounds 5c and
3

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Table 2
assay was carried out to investigate whether cell death pathway is re-
lated to apoptosis or nonspecific necrosis induction. To bring out this
purpose, MCF-7 cells were treated with two potent compounds 5b and
5k at the concentration of 25 μM for 48 h.
Quantitative measurement of MCF-7 cells in different phases of cell cycle.
Compound
G0/G1 (%)
S (%)
G2/M (%)
Control
71.81 2.71
51.19 0.36
31.11 1.15
31.31 1.11
43.31 1.74
4.27
0.50
23.92 1.14
16.48 0.63
22.62 2.52
24.62 6.34
15.00 0.26
As illustrated in Fig. 2, the percentage of early apoptosis and late
apoptosis for 5b and 5k was determined to be 23.2%, 2.86%, and
38.9%, 2.59%, respectively, indicating the apoptotic pathway of cell
death.
5b (25 µM)
5b (50 µM)
5k (25 µM)
5k (50 µM)
32.32
46.27
44.06
41.68
0.59
3.67
5.22
2.02
2.6. In silico studies
As mentioned in Table 2, selected compounds significantly in-
creased the number of cells in the S phase from 4.27% in control to
32.32% (5b, 25 µM), 32.32% (5b, 50 µM), 46.27% (5k, 25 µM), and
41.68% (5k, 50 µM).
2.6.1. Molecular docking analyses
There are many previously reported small molecules with quinone
core, which proved to inhibit Topo II enzyme. Topo II is a homodimeric
enzyme with two functionally important domains, including the ATPase
domain, which catalyzes the hydrolysis of ATP molecules. The other
domain is located at the center of cleavage/religation part of the pro-
tein that is responsible for DNA breaking and resealing.27
1,4-naphthoquinone (1,4-NQ) containing compounds were found to
inhibit TopoIIα enzyme through the established binding site at the
ATPase domain interacting with important residues including Asn91,
Ile141, Ser148, Ser149, and Asn150.8,28
These findings clearly confirmed that both compounds induced an
accumulation of cells in the S phase compared to untreated cells
(Fig. 1). This is consistent with previous studies which showed that
naphthoquinone-based compounds could potentially arrest cell growth
in the S phase in FaDu, HT-29 and K562 cell lines.22,25,26
2.5. Apoptosis study
In the next step, annexin V-FITC/PI dual staining flow cytometry
In this study, molecular docking modelling was done to elucidate
Fig. 1. Effects of compounds 5b and 5k on the cell cycle distribution in MCF-7 cells. Cells treated with synthesized derivatives at 25 and 50 μM concentrations. The
control group was untreated cells.
4

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Fig. 2. Flow cytometry analysis of apoptosis induction in MCF-7 cells. No treatment as a reference control, treated by compound 5b and 5k for 48 h (Q1: represents
live cells, Q2: late apoptotic cells, Q3: early apoptotic cells, and Q4: necrosis cells).
the differences between cytotoxic activity and molecular structure of
newly synthesized hybrids as these compounds were designed to act as
topo II inhibitors. Based on docking predicted binding affinities, potent
derivatives (5b and 5k) preferentially target the ATPase domain rather
than to the etoposide pocket and the central domain (Table 3).
The three-dimensional structure of ATP binding domain human
topoisomerase IIα complexed with an ATP analogue AMPPNP (PDB
code: 1ZXM) was chosen for molecular docking studies of all synthe-
sized compounds as well as doxorubicin. Root mean square deviation
(RMSD) value of 1.03 Å proved that the top-ranked conformer of ligand
superimposed well over the x-ray crystallographic one in to the binding
site of enzyme.
composed of various amino acids including Ser149, Asn150, Arg98,
Ser148, Lys157, and Tyr34 which are key residues. In pocket II essential
amino acids Asn91 and Asn120 play important roles and pocket III
contains Ala167, Gly166, Lys168, Tyr165, and Thr147 residues.
Mg2+ ion is also located in pocket III.8,30
Estimated free energies of binding and interactions of all com-
pounds in comparison with doxorubicin as a reference drug are sum-
marized in Table S1.
Results showed that the synthesized compounds have binding en-
ergy values between −9.6 to −11.2 kcal/mol. In comparison with
doxorubicin (ΔG = −8.2 kcal/mol), derivatives formed more stable
complexes with the target protein. Interestingly, the potent derivatives
5b, 4-nitro-benzyl- analog, and 5k, 4-bromo-benzyl- analog, possessed
the lowest free energy of binding (−11, −11.2 kcal/mol, respectively)
(Fig. 3).
The protein structure was determined with a resolution of 1.87 Å
using the X-ray diffraction method by Wei et al.29 T-shaped cavity of
ATP binding domain contains three binding pockets: Pocket I is
According to the obtained results, in all protein–ligand complexes,
1,4-naphthoquinone moiety was stabilized by at least a single hydrogen
bond interaction. Additionally, all synthesized ligands established pi-
cation interaction with magnesium ion implying that this might be one
of the crucial interactions with the underlying mechanism of TopoIIα
inhibition.31
Table 3
Binding energies of potent derivatives with different PDB codes of topo II en-
zymes.
Topoisomerase
Binding energies
(kcal/mol)
Two potent molecules, 5b and 5k formed six key hydrogen bonds
interaction between 2-amino-1,4-naphthoquinone core with amino
acids Lys168, Ser148, Asn150 and Ser149. Ether bond played an im-
portant role by establishing hydrogen bond with Arg98 at the distance
of 3.11 Å.
5b
5k
Human Topo II
1ZXM
1ZXN
-10.6 (ANP)
-10.3 (ADP)
-11
-11.2
-10.5
ATPase-noDNA
Human Topo II
-10.1
Other interactions of compound 5b bearing 4-bromo-benzyl moiety
included two pi-alkyl interaction with Ile141 and Ile125 and pi-sigma
interaction with Thr215 (Fig. 4).
ATPase-noDNA
Human Topo II -DNA
Human Topo II -DNA
Human Topo II -DNA
5GWK -8.5 (Etoposide)
-8.1
-8.1
-8.4
-8.4
-8.3
3QX3
4G0V
-8.5 (Etoposide)
The 4-bromo substituent changed the orientation of compound 5k
-6.3 (Mitoxantrone) -7.8
5

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Fig. 3. The alignment of docked compounds 5b (green) and 5k (yellow) in the binding site of topo IIα enzyme. (The protein key residues are shown in stick style).
Fig. 4. Binding mode and interactions of active compound 5b within the active site of topo IIα.
via additional pi-alkyl interactions with Ile88 and Ile217 residues
(Fig. 5).
landscapes of proteins and identification of their physiological con-
formations. MD is also useful in understanding structural refinements of
ligand and the receptor complexes.32,33
The binding position of the potent derivatives also revealed that
they make favorable hydrogen binding interactions with the key re-
sidues of the ATPase domain similar to ATP.27 Suggesting that these
molecules can potentially occupy the ATP-binding pocket
In order to further study the binding position of potent derivatives
and topo IIα enzyme and verify the accuracy of docking results, we
performed 50 ns MD simulations using the proposed binding modes of
5b and 5k to gain information about their dynamic behavior within the
active site of enzyme.
2.6.2. Molecular dynamics simulation analyses
RMSD was calculated to assess the conformational stability of topo
IIα enzyme throughout the simulation time. As shown in Fig 6, the
RMSDs of two systems remain stable in the last 40 ns for 5b and 35 ns
for 5k trajectories. The equilibrium time for further analyses was con-
sidered after 10 and 15 ns for 5b and 5k complexes, respectively.
It is worth mentioning that no structure of a small molecule in-
hibitor in complex with human topo IIα ATPase domain is reported up
to now. Molecular dynamics (MD) simulation is a valuable tool that can
predict the behavior of a biological molecular system during a definite
period. This method is widely used in the exploration of the free energy
6

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Fig. 5. Binding mode and interactions of active compound 5k within the active site of topo IIα.
Fig. 8. Occurrence of hydrogen bond as a function of time between compound
5b and amino acid residues Asn91, Asn150, Ser148, Ser149 and Arg98.
Fig. 6. RMSD evolution of interaction of compound 5b (green) and 5k (red)
with topo IIα enzyme during the simulation time.
Root mean square fluctuation (RMSF) was computed in order to
evaluate the flexibility of amino acid residues during the equilibrium
time, Fig. 7. Two complexes showed similar RMSF distributions. Ac-
cording to the results, there were no amino acid residues having RMSF
value > 0.4 nm and most of the peaks correspond to residues 341–351
and 409–422 which are not involved in active site of enzyme.
Hydrogen bond (H-bond) formation during equilibrium time of
compounds 5b and 5k and amino acid residues of active site was in-
vestigated in detail, Figs. 8 and 9. As summarized in Table 4, hydrogen
bond between Asn150 and compounds 5b and 5k had a life time of
32.60%, and 53.28% which suggested the relative stability during the
equilibrium time. These hydrogen bonds were also found in molecular
docking, indicating that the hydrogen bond between compounds and
Asn150 is essential for inhibitory activity of enzyme. Another stable
Fig. 9. Occurrence of hydrogen bond as a function of time between compound
5k and amino acid residues Ser149, Arg98 and Asn150.
Fig. 7. The root mean square fluctuation (RMSF) of the Cα atoms of residues of 1ZXM in complex with 5b (left) and 5k (right).
7

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Table 4
no interaction was observed in all cluster representatives with magne-
The hydrogen bond life time between potent derivatives and key residues in
PDB code 1ZXM of topo IIα enzyme.
sium ion.
These observations are in good agreement with the results of
docking studies and previous reported naphthoquinone-based small
molecules with topo IIα inhibitory activity.28,34–37 These obtained re-
sults provide invaluable initial data on ligand dynamics, which can also
be employed in further optimization and development.
Ligand
Donor part (atom)
Acceptor part
Occupancy (%)
5b
Ser148 (O)
Asn150 (N)
Amine (H)
Arg98 (N)
Asn91 (N)
Amine (H)
Asn150 (N)
Arg98 (N)
C4-Carbonyl (O)
C4-Carbonyl (O)
Ser149 (O)
3.07
32.60
15.09
30.55
11.41
11.39
53.28
6.29
Ether (O)
2.6.3. Chemoinformatics prediction
Nitro (O)
5k
Ser149 (O)
To support biological data, we further examined in silico drug-like-
ness, bioactivity, pharmacokinetic and toxicology properties of the
newly synthesized hybrids. To determine in silico parameters, 2D
structure molecules were subjected to the various online servers.
C4-Carbonyl (O)
Ether (O)
hydrogen bond between 5b and Arg98 with 30.55% occupancy was
identified. In addition, 5b formed three less stable H-bonds with Ser149
(15.09%), Asn91 (11.41%) and Ser148 (3.07%) and derivative 5k
formed H-bonds with Ser149 (11.39%) and Arg98 (6.29%).
2.6.3.1. Physicochemical properties of synthetic compounds and drug-
likeness prediction. Drug-likeness properties and molecular descriptors
were calculated using SwissADME.38 Theoretical calculations of
Lipinski’s rule of five involves the following items:
A cluster analysis using gromos method and cut off value 0.15 was
done on trajectories to identify the most representative frames. For
compound 5b, the first representative frame of the first cluster with the
highest population showed three hydrogen bonds between Asn150 and
C4-Carbonyl, Arg98 and ether bond, Asn91 and nitro group (Fig 10).
Besides this, 5b also established pi-alkyl interactions with Ile125 and
Ala167 and one pi-sulfur interaction with Lys168. Furthermore, pi-ca-
tion interactions between magnesium ion and 1,4-naphthoquinone
moiety was observed in all cluster representatives. Compound 5k,
showed one hydrogen bonding interaction between amine group and
Ser149 in the first representative frame. Besides, as depicted in Fig 10,
several pi-alkyl interactions with the residues of Ala92, Ile217, Ile118,
Ile88 and Arg98 were formed as well as one pi-pi interaction with
Gln97 and two pi-sulfur interaction with Arg98 and Lys157. However,
- Molecular weight ⩽500;
- Hydrogen bond donors ⩽5 (OH and NH groups);
- Hydrogen bond acceptors ⩽10 (N and O atoms);
- Lipophilicity (Log P value) ⩽5.
Violation of more than one of these rules may lead to bioavailability
related issues.39 The calculated parameters are given in Table 5. All of
the compounds exhibited desirable molecular properties with no Li-
pinski property violations. Along with Lipinski parameters the total
polar surface area (TPSA) was in the range of 55.40–101.22 Å2 sug-
gesting the high oral bioavailability (expected for compounds with
TPSA ≤ 140 Å2) and the calculated number of rotatable bonds present
in the molecules were in the range of 4–7 which indicates that the
synthesized compounds obey Veber rule.40 Molar refractivity (MR) was
also predicted which is an important factor for steric measurement,
ranging from 87.34 to 128.98.
The calculated drug-likeness values confirmed their compliance
with Ghose, Egan and Mugge rules with no more than one violation.
2.6.3.2. Bioactivity score. The bioactivity scores including G protein-
coupled receptor ligand (GPCRL), ion channel modulator (ICM), kinase
inhibitor (KI), protease inhibitor (PI), nuclear receptor ligand (NRL)
and enzyme inhibitor (EI) properties were calculated for the title
compounds (Table 6) by Molinspiration online server.
The bioactive score interpretation is summarized as below:
- > 0 active;
- −5.0 to 0.0 moderately active;
- < −5.0 inactive
The G protein-coupled receptor ligand score of the compounds was
calculated in the limits of −0.17 to 0.00, ion channel modulator
property was identified ranging from −0.44 to −0.15, nuclear receptor
ligand score was predicted in the range of −0.17 to 0.06 and protease
inhibitor score was within −0.26 to −0.12.
All of the synthesized compounds were active enzyme inhibitors.
However, compounds 5b and 5c scored less and kinase inhibitor
property of new hybrids were superior compared to doxorubicin.
2.6.3.3. Pharmacokinetic properties. All phases of pharmacokinetic
profile inclusive of absorption, distribution, metabolism, excretion
and toxicity of compounds were predicted using pkCSM online server41
.
All of the compounds were predicted to have good Caco-2 perme-
ability > 0.9 (logarithm of the apparent permeability coefficient, log
Papp, in 10−6 cm/s) except compound 5b. Human intestinal absorption
of compounds estimated to be in the high range of 90.15–99.77 percent,
much higher than the threshold value of 30%. VDss (volume of
Fig. 10. Interaction pattern of compound 5b (green) and 5k (red) in the cluster
representative 1. (H-bond and pi-interactions are shown in green and pink
dashed lines, respectively).
8

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Table 5
Physicochemical properties and drug-likeness prediction of the synthesized compounds.
Compounds
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
Physicochemical
Properties
MW
355.39 400.38 389.83 373.38 369.41 423.38
424.28
411.49
389.83 385.41 433.28 303.31 452.46
Num. rotatable
bonds
5
6
5
5
5
6
5
6
5
6
5
4
7
Num. H-bond
acceptors
3
5
3
4
3
6
3
3
3
4
3
3
5
Num. H-bond
donors
1
1
1
1
1
1
1
1
1
1
1
1
1
Log P o/w
(average)*
MR
3.76
3.18
4.23
4.00
4.01
4.80
4.81
4.97
4.30
3.68
4.32
2.75
3.49
104.05 112.87 109.06 104.01 109.01 109.05
114.07
55.40
Yes
123.32
55.40
Yes
109.06 110.54 111.75 87.34
128.98
92.78
Yes
TPSA (Å2)
55.40
Yes
101.22 55.40
55.40
Yes
55.40
Yes
55.40
Yes
55.40
Yes
64.63
Yes
55.40
Yes
55.40
Yes
Drug-likeness rules
Lipinski (Pfizer)
Ghose (Amgen)
Veber (GSK)
Yes
Yes
Yes
Yes
Yes
0.55
Yes
Yes
Yes
Yes
Yes
0.55
Yes
Yes
Yes
1 violation 1 violation Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Egane (Pharmacia) Yes
Yes
Yes
1 violation Yes
Yes
Yes
Yes
Yes
Yes
Mugge (Bayer)
Bioavailability
score
Yes
Yes
Yes
1 violation 1 violation 1 violation Yes
Yes
Yes
Yes
Yes
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
MW: Molecular weight (g/mol), MR: Molar Refractivity, TPSA: Topological polar surface area
*Lipophilicity, expressed as average of four log P amounts based on different methods.
compounds 5a, 5e and 5l may also interact with estrogen receptor
alpha (ER) (Table 8).
Table 6
Bioactivity score of the synthesized compounds.
Toxicity risks of naphthoquinone hybrids were calculated by Osiris
property explorer (Table 9). Results suggested that all of the synthe-
sized compounds were estimated to show no significant toxicity with
regard to tumorigenic effect, reproductive effect and irritancy. Com-
pounds were predicted to be moderately mutagenic.
GPCRL
ICM
KI
NRL
PI
EI
5a
5b
5c
5d
5e
5f
−0.05
−0.17
−0.05
−0.04
−0.09
0.00
−0.26
−0.27
−0.25
−0.26
−0.31
−0.15
−0.23
−0.18
−0.24
−0.28
−0.31
−0.44
−0.38
−0.20
0.30
0.14
0.27
0.31
0.25
0.30
0.25
0.25
0.26
0.26
0.24
0.31
0.20
−0.07
−0.06
−0.14
−0.08
−0.04
−0.09
0.06
−0.15
−0.26
−0.19
−0.17
−0.20
−0.12
−0.18
−0.14
−0.19
−0.18
−0.25
−0.24
−0.21
0.67
0.08
−0.02
−0.04
0.06
0.02
0.07
0.04
0.07
0.04
0.04
0.01
0.10
0.02
0.66
3. Conclusion
5g
5h
5i
−0.04
−0.02
−0.05
−0.07
−0.14
−0.03
−0.10
0.20
−0.08
0.03
In summary, molecular design based on hybridization strategy led
to the synthesis, cytotoxic evaluation and flow cytometry-based as-
sessment of novel molecules with 2-phenylamino-1,4-naphthoquinone
backbone containing oxyphenyl moiety. Among the tested analogs, 4-
nitro-benzyl- (compound 5b) and 4-bromo-benzyl- (compound 5k)
derivatives displayed the highest cytotoxic activity against MCF-7
cancer cells. Based on the obtained results, the most active compounds
were investigated for their mechanism of action through cell cycle and
apoptosis studies. Flow cytometric analysis revealed that compounds
5b and 5k arrested the cell cycle in the S phase and potentially induced
apoptosis in MCF-7 cells.
−0.09
−0.09
−0.16
−0.02
−0.17
0.32
5j
5k
5l
5m
Dox.
GPCRL: G protein-coupled receptor Ligand; ICM: Ion channel modulator; KI:
Kinase; NRL: Nuclear receptor ligand; PI: Protease inhibitor; EI: Enzyme in-
hibitor.
distribution at steady state) indicates drug distribution in tissues, the
tested compounds presented an average of −0.38 to 0.195. Regarding
BBB and CNS permeability, derivatives were predicted to be CNS in-
active.
Molecular docking modelling and molecular dynamics simulations
studies of potent derivatives into the ATPase domain of topo IIα sug-
gested their mode of action, plausibly via blocking the ATP-binding site
of enzyme. However, further investigation especially in vitro inhibition
of the topo IIα ATPase activity is required in order to obtain compre-
hensive insight for development of potentially cytotoxic agents with 2-
amino-1,4-naphthoquinone pharmacophore.
Various isoforms of cytochrome P450 enzymes are involved in drug
metabolism. Compounds were predicted to be either substrate or/and
inhibitors of cytochrome P450 enzyme CYP3A4, CYP2C9 isoforms. The
maximum tolerated dose for human was estimated higher than doxor-
ubicin except for compounds 5b and 5l. Inhibition of potassium chan-
nels are encoded by hERG I and II gene which is related to Long QT
syndrome. All compounds predicted to show inhibitory activity against
hERG II and LD50 value of derivatives calculated to be less than dox-
orubicin (Table 7).
4. Experimental
4.1. Chemistry
4.1.1. General
2.6.3.4. Toxicity assessment. Lastly, toxicity evaluation of compounds
was carried out using ProTox-II.42 This online sever gives an in silico
analysis whether the compounds bind to the known receptors to
produce adverse reactions. Two major toxicological pathways were
considered: nuclear receptor signaling and stress response.
All reagents and chemicals were commercially available and were
used without further purification. 1H and 13C NMR spectra were re-
corded on a Bruker Avance 300 spectrometer (300 and 75 MHz for 1
H
and 13C NMR, respectively) in CDCl3 or DMSO‑d6. Chemical shifts (δ)
are given in ppm and coupling constants (J) in Hertz (Hz). The MS
spectra were recorded using an Agilent spectrometer (9575c inert MSD;
Agilent Technologies, Santa Clara, CA, USA). IR spectra were recorded
on a Perkin-Elmer spectrometer (KBr disk) (PerkinElmer, Waltham, MA,
All the synthesized ligands showed probable affinity to aryl hy-
drocarbon receptor (AhR) and mitochondrial membrane potential
(MMP) related targets except compounds 5h and 5m. Furthermore,
9

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
USA). Melting points were taken on
a hot stage apparatus
(Electrothermal, Essex, UK) and were reported uncorrected. Elemental
analysis was performed by Microanalytical Department, Central
Laboratory for Research, Shiraz University of Medical Sciences and was
within 0.4% of the calculated value. Merck silica gel 60 F254 plates
were used for analytical TLC. Preparative thin-layer chromatography
was done with prepared glass-backed plates (20*20 cm2) using silica gel
(Merk Kieselgel 60 HF254). Preparative column chromatography was
carried out on silica gel (230–400 mesh, G60 Merck).
4.1.2. General synthetic procedure for 2-((4-hydroxyphenyl)amino)
naphthalene-1,4-dione (3):
1,4-naphthoquinone (210 mg, 1.33 mmol) was dissolved in EtOH
(15 mL). The mixture was allowed to warm up until became a clear
solution, then 4-aminophenol (109 mg, 1 mmol) was slowly added
while stirring. The reaction was stirred at room temperature for 1 h.
The progress of the reaction was monitored by TLC using hexane–ethyl
acetate (3:2) as solvent system. After completion, the precipitated
product was filtered off, washed with cold ethanol, dried and re-
crystallized from ethanol to give a dark red crystalline solid.
Dark red crystals, isolated: 210 mg; yield: 79%; mp: 243–245 °C; Rf:
0.4 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1): 3304, 3030, 1671,
1625, 1569, 1365, 1269, 796. 724. 1H NMR (300 MHz, DMSO‑d6) δ
(ppm): 9.59 (s, 1H, OH), 9.09 (s, 1H, NH), 8.04 (d, J = 7.6 Hz, 1H,
naphthoquinone-H), 7.94 (d, J = 7.7 Hz, 1H, naphthoquinone-H), 7.85
(t, 7.5 Hz, 1H, naphthoquinone-H), 7.76 (t, J = 7.5 Hz, 1H, naphtho-
quinone-H), 7.17 (d, J = 8.3 Hz, 2H, Ar-H), 6.83 (d, J = 8.7 Hz, 2H, Ar-
H), 5.87 (s, 1H, naphthoquinone-H). MS (EI) m/z (%): 265.1 (M+, 100),
236 (26), 209 (11), 180 (8), 105 (21), 76 (23). Anal. Calcd. for
C16H11NO3: C, 72.45; H, 4.18; N, 5.28; O, 18.09; found: C, 72.39; H,
4.20; N, 5.29; O, 18.05.
4.1.3. General synthetic procedure for 2-((4-(benzyloxy)phenyl)amino)
naphthalene-1,4-dione derivatives (5a-k):
2-((4-hydroxyphenyl)amino)naphthalene-1,4-dione (3) (265 mg,
1 mmol) and K2CO3 (414.6 mg, 3 mmol) were dissolved in acetone
(15 mL) and refluxed for 30 min. The reaction was then cooled to room
temperature, and various types of benzyl halides were added dropwise.
Then the reaction refluxed for 48–72 h. Water was then added to the
mixture and extracted with ethyl acetate (3×50 mL), washed with
water, brine, then dried over Na2SO4 and solvent was evaporated under
vacuum. Final product was purified by preparative thin/column chro-
matography using petroleum ether-ethyl acetate mixtures to give
compounds 5a-k.
4.1.3.1. 2-((4-(benzyloxy)phenyl)amino)naphthalene-1,4-dione
(5a):. Red solid, isolated: 150 mg; yield: 42%; mp: 169–171 °C; Rf: 0.6
(Hex/EtOAc 2:3). FT-IR (KBr) υmax (cm−1): 3259, 1685, 1514, 1343,
1286, 1156, 1006, 747, 698. 1H NMR (300 MHz, DMSO‑d6) δ (ppm):
9.16 (s, 1H, NH), 8.04 (d, J = 6.1 Hz, 1H, naphthoquinone-H), 7.94 (d,
J = 6.1 Hz, 1H, naphthoquinone-H), 7.84 (t, J = 6.7 Hz, 1H,
naphthoquinone-H), 7.76 (t, J = 6.7 Hz, 1H, naphthoquinone-H),
7.5–7.3 (m, 5H, Ar-H), 7.3 (d, J = 9 Hz, 2H, Ar-H), 7.09 (d, J = 9 Hz.
2H, Ar-H), 5.93 (s, 1H, naphthoquinone-H), 5.13 (s, 2H, CH2). MS (EI)
m/z (%): 355.1 (M+, 37), 264.1 (100), 236 (6), 208 (6), 180 (5), 152
10

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
Table 8
Toxicity pathway prediction of the synthesized compounds.
Tox21 Nuclear receptor signaling pathways
Tox21 Stress response pathways
AhR
AR
AR-LBD
Aromatase
ER
ER-LBD
PPAR-Gamma
nrf2/ARE
HSE
MMP
p53
ATAD5
5a
5b
5c
5d
5e
5f
Active
Active
Active
Active
Active
Active
Active
Inactive
Active
Active
Active
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Active
Active
Active
Active
Active
Active
Inactive
Active
Active
Active
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
5g
5h
5i
5j
5k
5l
5m
Dox.
Inactive
Inactive
AhR: Aryl hydrocarbon Receptor; AR: Androgen Receptor; AR-LBD: Androgen Receptor Ligand Binding Domain; ER: Estrogen Receptor Alpha; ER-LBD: Estrogen
Receptor Ligand Binding Domain; PPAR-Gamma: Peroxisome Proliferator Activated Receptor Gamma; nrf2/ARE: Nuclear factor (erythroid-derived 2)-like 2/anti-
oxidant responsive element; HSE: Heat shock factor response element; MMP: Mitochondrial Membrane Potential; p53: Phosphoprotein (Tumor Suppressor); ATAD5:
ATPase family AAA domain containing protein 5.
Table 9
Toxicity risk parameters calculated by Osiris property explorer.
Mutagenic
Tumorigenic
Irritant
Reproductive effect
5a
5b
5c
5d
5e
5f
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Red
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
4.1.3.3. 2-((4-((4-chlorobenzyl)oxy)phenyl)amino)naphthalene-1,4-dione
(5c):. Red crystals, isolated: 90 mg; yield: 23%; mp: 168–170 °C; Rf:
0.62 (Hex/EtOAc 2:3). FT-IR (KBr) υmax (cm−1): 3302, 1719, 1677,
1514, 1351, 1231, 1107, 1035, 824, 700. 1H NMR (300 MHz, DMSO‑d6)
δ (ppm): 8.11 (s, 1H, NH), 7.8–7.71 (m, 1H, naphthoquinone-H),
7.7–7.62 (m, 4H, naphthoquinone-H, Ar-H), 7.61–7.54 (m, 3H, Ar-H),
7.25–7.2 (m, 2H, Ar-H), 7.05–6.95 (m, 2H, Ar-H), 6.24 (s, 1H,
5g
5h
5i
5j
5k
5l
5m
Dox.
naphthoquinone-H), 5.15 (s, 2H, CH2). MS (EI) m/z (%): 389.1 (M+
,
15), 338 (5), 264.1 (100), 236 (6), 208.1 (8), 180.1 (4), 152 (6), 125
(34), 104.9 (13), 89 (13). Anal. Calcd. for C23H16ClNO3: C, 70.86; H,
4.14; Cl, 9.09; N, 3.59; O, 12.31; found: C, 70.90; H, 4.01; Cl, 9.16; N,
3.62; O, 12.39.
Green: no risk or low risk; Yellow: medium risk; Red: more toxicity risk.
(8), 129 (7), 104.9 (12), 91 (44), 76.1 (10). Anal. Calcd. for C23H17NO3:
C, 77.73; H, 4.82; N, 3.94; O, 13.51; found: C, 77.75; H, 4.95; N, 3.98;
O, 13.38.
4.1.3.4. 2-((4-((4-fluorobenzyl)oxy)phenyl)amino)naphthalene-1,4-dione
(5d):. Dark amber crystals, isolated: 45 mg; yield: 12%; mp:
149–151 °C; Rf: 0.58 (Hex/EtOAc 2:3) .FT-IR (KBr) υmax (cm−1):
3241, 1684, 1572, 1351, 1234, 1121, 1008, 821, 7776. 1H NMR
(300 MHz, DMSO‑d6) δ (ppm): 9.18 (s, 1H, NH), 8.05 (d, J = 7.5 Hz,
1H, naphthoquinone-H), 7.93 (d, J = 7.8 Hz, 1H, naphthoquinone-H),
7.87–7.74 (m, 2H, naphthoquinone-H), 7.6–7.42 (m, 2H, Ar-H),
7.36–7.2 (m, 4H, Ar-H), 7.1–7.03 (m, 2H, Ar-H), 5.93 (s, 1H,
naphthoquinone-H), 5.11 (s, 2H, CH2). 13C NMR (75 MHz, DMSO‑d6)
δ (ppm): 182.69, 182.09, 156.33, 147.25, 135.31, 133.61, 133.19,
132.92, 131.34, 130.87, 130.51, 130.39, 126.50, 125.99, 125.70,
115.87, 115.58, 101.56, 69.18. MS (EI) m/z (%): 373.1 (M+, 30),
264.1 (100), 236.1 (4), 208 (5), 152 (7), 129 (5), 109 (10). Anal. Calcd.
for C23H16FNO3: C, 73.99; H, 4.32; F, 5.09; N, 3.75; O, 12.85; found: C,
73.91; H, 4.49; F, 5.04; N, 3.80; O, 12.85.
4.1.3.2. 2-((4-((4-nitrobenzyl)oxy)phenyl)amino)naphthalene-1,4-dione
(5b):. Dark brown solid, isolated: 42 mg; yield: 11%; mp: 171–173 °C;
Rf: 0.45 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1): 3315, 1628, 1571,
1509, 1343, 1293, 1109, 989, 824, 725. 1H NMR (300 MHz, CDCl3) δ
(ppm): 8.3–8.18 (m, 2H, naphthoquinone-H), 8.1–7.98 (m, 2H,
naphthoquinone-H), 7.71–7.54 (m, 4H, Ar-H), 7.19–7.1 (m, 2H, Ar-
H), 7.02–6.89 (m, 2H, Ar-H), 6.17 (s, 1H, naphthoquinone-H), 5.13 (s,
2H, CH2). 13C NMR (75 MHz, CDCl3) δ (ppm): 183.78, 182.06, 156.04,
147.67, 145.50, 144.04, 134.93, 133.30, 132.30, 130.91, 130.36,
127.62, 126.48, 126.16, 124.92, 123.92, 115.86, 102.71, 99.98,
68.98. MS (EI) m/z (%): 400.1 (M+, 11), 370 (2), 285.1 (4), 264
(100), 236 (22), 180.1 (9), 120.9 (21), 105 (36), 89 (25), 76.9 (35).
Anal. Calcd. for C23H16N2O5: C, 69.00; H, 4.03; N, 7.00; O, 19.98;
found: C, 69.14; H, 4.05; N, 7.00; O, 19.92.
11

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
(300 MHz, CDCl3) δ (ppm): 8.08–7.98 (m, 2H, naphthoquinone-H),
7.74–7.55 (m, 2H, naphthoquinone-H), 7.44–7.07 (m, 6H, Ar-H), 7–6.9
(m, 2H, Ar-H), 6.17 (s, 1H, naphthoquinone-H), 2.1 (s, 2H, CH2), 1.26
(s, 9H, t-Butyl). 13C NMR (75 MHz, CDCl3) δ (ppm): 183.75, 182.15,
156.93, 151.23, 145.58, 134.90, 133.39, 132.21, 130.41, 127.45,
126.46, 126.15, 125.64, 124.78, 115.83, 102.53, 70.21, 34.62, 31.35.
MS (EI) m/z (%): 411.2 (M+, 9), 389 (3), 264.1 (31), 236.1 (5), 208.1
(4), 180 (3), 147.1 (100), 117 (19), 105 (15). Anal. Calcd. for
4.1.3.5. 2-((4-((4-methylbenzyl)oxy)phenyl)amino)naphthalene-1,4-
dione (5e):. Red-brown solid, isolated: 22 mg; yield: 6%; mp:
157–159 °C; Rf: 0.75 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1):
3295, 1720, 1595, 1352, 1236, 1121, 1089, 844, 726. 1H NMR
(300 MHz, CDCl3) δ (ppm): 8.1–7.9 (m, 2H, naphthoquinone-H),
7.74–7.64 (m, 1H, naphthoquinone-H), 7.64–7.54 (m, 1H,
naphthoquinone-H), 7.41–7.3 (m, 3H, Ar-H), 7.16–7.08 (m, 3H, Ar-
H), 7–6.9 (m, 2H, Ar-H), 6.96 (s, 1H, naphthoquinone-H), 5.01 (s, 2H,
CH2), 3.05 (br s, 3H, CH3). MS (EI) m/z (%): 369.1 (M+, 5), 355.1 (26),
264.1 (100), 236.1 (6), 208 (8), 180 (4), 152 (7), 129.9 (6), 105 (65),
91 (57). Anal. Calcd. for C24H19NO3: C, 78.03; H, 5.18; N, 3.79; O,
12.99; found: C, 78.00; H, 5.24; N, 3.82; O, 13.05.
C27H25NO3: C, 78.81; H, 6.12; N, 3.40; O, 11.66; found: C, 78.75; H,
6.10; N, 3.58; O, 11.65.
4.1.3.9. 2-((4-((3-chlorobenzyl)oxy)phenyl)amino)naphthalene-1,4-dione
(5i):. Red crystals, isolated: 51 mg; yield: 13%; mp: 157–159 °C; Rf: 0.6
(Hex/EtOAc 2:3). FT-IR (KBr) υmax (cm−1): 3324, 3218, 1679, 1509,
1353, 1255, 1179, 1035, 860, 721, 680. 1H NMR (300 MHz, CDCl3) δ
(ppm): 8.07–8.00 (m, 2H, naphthoquinone-H), 7.71–7.64 (d, 1H,
J = 7.1 Hz, naphthoquinone-H), 7.62–7.54 (d, 1H, J = 7.8 Hz,
naphthoquinone-H), 7.41–7.34 (m, 3H, Ar-H), 7.20–7.08 (m, 3H, Ar-
H), 6.97–6.90 (m, 2H, Ar-H), 6.16 (1H, s, naphthoquinone-H), 4.98
(2H, s, CH2). 13C NMR (75 MHz, CDCl3) δ (ppm): 183.72, 182.10,
156.43, 145.52, 138.68, 134.88, 134.60, 133.35, 132.22, 130.56,
130.40, 129.94, 128.25, 127.41, 126.44, 126.15, 125.33, 124.81,
115.88, 102.64, 69.49. MS (EI) m/z (%): 389.1 (M+, 15), 355.2 (2),
264.1 (100), 236.1 (5), 208.1 (6), 108.1 (4), 152 (6), 125 (16), 105
(12), 89.1 (10). Anal. Calcd. for C23H16ClNO3: C, 70.86; H, 4.14; Cl,
9.09; N, 3.59; O, 12.31; found: C, 70.80; H, 4.18; Cl, 9.10; N, 3.45; O,
12.47.
4.1.3.6. 2-((4-((4-(trifluoromethyl)benzyl)oxy)phenyl)amino)
naphthalene-1,4-dione (5f):. Ochre solid, isolated: 111 mg; yield: 26%;
mp: 161–163 °C; Rf: 0.64 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1):
3198, 1682, 1571, 1333, 1240, 1160, 1068, 827, 777. 1H NMR
(300 MHz, DMSO‑d6) δ (ppm): 8.64 (s, 1H, NH), 8.07–7.86 (m, 2H,
naphthoquinone-H), 7.8–7.56 (m, 4H, naphthoquinone-H, Ar-H),
7.42–7.12 (m, 4H, Ar-H), 7.03–6.88 (m, 2H, Ar-H), 6.02 (s, 1H,
naphthoquinone-H), 5.02 (s, 2H, CH2). MS (EI) m/z (%): 423.1 (M+
,
21), 338 (5), 264.1 (100), 236 (4), 208.1 (6), 159 (38), 129 (5), 109
(19), 89 (7). Anal. Calcd. for C24H16F3NO3: C, 68.08; H, 3.81; F, 13.46;
N, 3.31; O, 11.34; found: C, 68.14; H, 3.88; F, 13.42; N, 3.33; O, 11.40.
4.1.3.10. 2-((4-((4-methoxybenzyl)oxy)phenyl)amino)naphthalene-1,4-
dione (5j):. Red-brown solid, isolated: 18 mg; yield: 5%; mp:
189–191 °C; Rf: 0.7 (Hex/EtOAc 3:2) .FT-IR (KBr) υmax (cm−1): 3291,
1678, 1566, 1237, 1170, 1004, 832, 727. 1H NMR (300 MHz, CDCl3) δ
(ppm): 8.17–8.06 (m, 2H, naphthoquinone-H), 7.82–7.72 (m, 1H,
naphthoquinone-H), 7.72–7.6 (m, 1H, naphthoquinone-H), 7.5–7.31
(m, 2H, Ar-H), 7.25–7.15 (m, 2H, Ar-H), 7.07–6.86 (m, 4H, Ar-H), 6.25
4.1.3.7. 2-((4-((3,4-dichlorobenzyl)oxy)phenyl)amino)naphthalene-1,4-
dione (5g):. Brownish-purple crystals, isolated: 125 mg; yield: 30%; mp:
179–180 °C; Rf: 0.55 (Hex/EtOAc 2:3) .FT-IR (KBr) υmax (cm−1): 3196,
1679, 1509, 1334, 1228, 1032, 894, 734, 670. 1H NMR (300 MHz,
(s, 1H, naphthoquinone-H), 5.03 (s, 2H, CH2), 3.84 (s, 3H, OCH3). 13
C
DMSO‑d6)
δ
(ppm): 9.19 (s, 1H, NH), 8.1–7.99 (m, 1H,
NMR (75 MHz, CDCl3) δ (ppm): 183.72, 182.09, 159.57, 156.87,
145.60, 134.86, 132.19, 130.17, 129.22, 128.59, 126.43, 126.13,
124.76, 115.93, 114.08, 102.56, 77.20, 76.94, 76.52, 70.16, 55.31.
MS (EI) m/z (%): 385.1 (M+, 5), 338 (2), 264 (9), 236 (4), 208 (3), 121
(100), 104.9 (9), 90.9 (11), 76.9 (21). Anal. Calcd. for C24H19NO4: C,
74.79; H, 4.97; N, 3.63; O, 16.60; found: C, 74.70; H, 5.00; N, 3.70; O,
16.56.
naphthoquinone-H), 8–7.9 (m, 1H, naphthoquinone-H), 7.89–7.62 (m,
4H, naphthoquinone-H, Ar-H), 7.57–7.4 (m, 1H, Ar-H), 7.36–7.24 (m,
2H, Ar-H), 7.15–7 (m, 2H, Ar-H), 5.94 (s, 1H, naphthoquinone-H), 5.16
(s, 2H, CH2). MS (EI) m/z (%): 423 ((M−1)+, 9), 264.2 (100), 236.2
(5), 208.2 (7), 180.1 (3), 130 (5), 105 (11), 76.1 (6). Anal. Calcd. for
C
23H15Cl2NO3: C, 65.11; H, 3.56; Cl, 16.71; N, 3.30; O, 11.31; found: C,
65.05; H, 3.50; Cl, 16.82; N, 3.30; O, 11.38.
4.1.3.11. 2-((4-((4-bromobenzyl)oxy)phenyl)amino)naphthalene-1,4-
dione (5k):. Red solid, isolated: 260 mg; yield: 60%; mp: 197–199 °C;
Rf: 0.65 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1): 3229, 1736, 1676,
1514, 1352, 1230, 1172, 1090, 842, 726. 1H NMR (300 MHz, DMSO‑d6)
δ (ppm): 9.15 (br s, 1H, NH), 8.06–8.00 (d, J = 7.6 Hz, 1H,
4.1.3.8. 2-((4-((4-(tert-butyl)benzyl)oxy)phenyl)amino)naphthalene-1,4-
dione (5h):. Dark brown solid, isolated: 23 mg; yield: 6%; mp:
130–131 °C; Rf: 0.71 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1):
3273, 1682, 1571, 1350, 1238, 1174, 1120, 989, 825, 719. 1H NMR
12

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
naphthoquinone-H), 7.96–7.90 (d, J = 7.7 Hz, 1H, naphthoquinone-H),
7.89–7.68 (m, 2H, naphthoquinone-H), 7.64–7.56 (m, 2H, Ar-H),
7.46–7.39 (m, 2H, Ar-H), 7.31–7.24 (m, 2H, Ar-H), 7.12–7.04 (m, 2H,
Ar-H), 5.9 (s, 1H, naphthoquinone-H), 5.12 (s, 2H, CH2). 13C NMR
(75 MHz, DMSO‑d6) δ (ppm): 182.29, 156.11, 147.64, 136.94, 135.24,
133.41, 132.77, 132.10, 131.83, 130.96, 130.25, 126.46, 125.87,
125.67, 121.41, 115.92, 101.39, 79.63, 69.12. MS (EI) m/z (%): 435
((M+2)+, 10), 355 (2), 264.1 (100), 236.1 (5), 208.2 (6), 180.1 (3),
152.1 (5), 130 (5), 105 (11), 90 (15). Anal. Calcd. for C23H16BrNO3: C,
63.61; H, 3.71; Br, 18.40; N, 3.23; O, 11.05; found: C, 63.68; H, 3.72;
Br, 18.45; N, 3.20; O, 11.09.
H), 6.14 (s, 1H, naphthoquinone), 3.97 (t, J = 6.1 Hz, 2H,
NCH2CH2CH2-phthalimide), 3.86 (t, J = 6.9 Hz, 2H, NCH2CH2CH2-
phthalimide), 2.13 (t, J = 6.3 Hz, 2H, NCH2CH2CH2-phthalimide). 13
C
NMR (75 MHz, CDCl3) δ (ppm): 183.68, 182.13, 168.36, 156.76,
145.57, 134.85, 133.97, 133.38, 132.17, 132.14, 130.41, 130.12,
126.42, 126.12, 124.69, 123.26, 115.47, 102.52, 66.04, 35.42, 28.25.
MS (EI) m/z (%): 452.1 (M+, 3), 389.2 (21), 355.1 (5), 264.1 (100),
236.2 (8), 208 (8), 125 (21), 105.2 (21), 89.1 (10). Anal. Calcd. for
C27H20N2O5: C, 71.67; H, 4.46; N, 6.19; O, 17.68; found: C, 71.77; H,
4.50; N, 6.22; O, 17.64.
4.2. Biological evaluation
4.1.4. General synthetic procedure for 2-((4-(prop-2-yn-1-yloxy)phenyl)
amino)naphthalene-1,4-dione (5l):
4.2.1. Cell culture
A
mixture of 2-((4-hydroxyphenyl)amino)naphthalene-1,4-dione
Four cell lines including MCF-7 (breast adenocarcinoma), HL-60
(acute promyelocytic leukemia), U937 (histiocytic lymphoma) and
HEK-293 (embryonic kidney) were used in this study. Cell lines were
obtained from the National Cell Bank, Pasteur Institute, Tehran, Iran.
MCF-7 and HEK-293 was grown in monolayer cultures, HL-60 and
U937 cells were grown in suspension. The cells were cultured in RPMI
1640 supplemented with L-glutamine 2 mM (for MCF-7), 10% FBS, 1%
penicillin/streptomycin and were kept at 37 °C in humidified air con-
taining 5% CO2.
(3) (265, 1 mmol) and K2CO3 (414.6 mg, 3 mmol) in DMF (15 mL) was
refluxed for 30 min and then allowed to cool down to room tempera-
ture. Propargyl bromide (4 l) was added dropwise, then the reaction
was refluxed for 48 h.
Water was then added to the mixture and extracted with ethyl
acetate (3×50 mL), washed with water, brine, then dried over Na2SO4
and solvent was evaporated under vacuum. The crude product was
purified by flash column chromatography using a mixture of petroleum
ether-ethyl acetate (7:3) as mobile phase.
4.2.2. Cytotoxicity screening using MTT assay
The cytotoxic activities of 2-amino-1,4-naphthoquinone hybrids 3
and 5a-m were evaluated in comparison with reference drug doxor-
ubicin using MTT assay.13,43 MCF-7, HL-60, U937 and HEK-293 cells
were seeded into 96-well plates and incubated for 24 h at 37 °C. Syn-
thesized compounds or doxorubicin was added in triplicates at 4 dif-
ferent concentrations in the ranges of 5–100 μM and the cells were
incubated at 37 °C for further 72 h. After that, 80 μl of medium was
removed and 80 μl MTT solution at a final concentration of 0.5 mg/ml
was added. Plates were incubated for 4 h at 37 °C to allow formazan to
be formed and then 200 μl DMSO was added to each well to dissolve the
formazan crystals. Absorbance was determined at 570 nm with back-
ground correction at 655 nm using a Bio-Rad microplate reader (Model
680). The percentage of inhibition of viability compared to control was
measured for each concentration of tested compounds and IC50 values
were calculated with CurveExpert software version 1.40 for Windows.
Brown solid, isolated: 122 mg; yield: 40%; mp: 205–207 °C; Rf: 0.62
(Hex/EtOAc 7:3). FT-IR (KBr) υmax (cm−1): 3318, 3074, 1687, 1625,
1531, 1353, 1305, 1201, 1036, 827, 773, 625. 1H NMR (300 MHz,
DMSO‑d6) δ (ppm): 9.2 (s, 1H, NH), 8.10–8.01 (m, 1H, naphthoqui-
none-H), 7.97–7.92 (m, 1H, naphthoquinone-H), 7.89–7.67 (m, 2H,
naphthoquinone-H), 7.34–7.25 (m, 2H, Ar-H), 7.10–7.01 (m, 2H, Ar-H),
5.95 (s, 1H, naphthoquinone-H), 4.83 (s, 2H, CH2), 3.61 (m, 1H,
C CH). 13C NMR (75 MHz, DMSO‑d6) δ (ppm): 182.72, 182.09, 155.25,
147.26, 135.33, 133.18, 132.96, 132.04, 131.77, 130.89, 129.11,
126.52, 125.95, 125.71, 116.00, 101.62, 79.64, 78.82, 56.12. MS (EI)
m/z (%): 303.1 (M+, 45), 264.1 (100), 236 (6), 208.1 (8), 152 (6), 130
(6), 105 (11), 76 (10). Anal. Calcd. for C19H13NO3: C, 75.24; H, 4.32; N,
4.62; O, 15.82; found: C, 75.32; H, 4.18; N, 4.60; O, 15.67.
4.2.3. Cell cycle analysis
Cell cycle analysis was done using propidium iodide (PI)-based flow
cytometry assay.13 MCF-7 cells were plated in 12-well microplates with
a density of 2×105 cells/ml and after being incubated at 37 °C for 48 h,
microplates were treated with compounds 5b and 5k at 25 and 50 μM
concentrations for 24 h. Then, control and treated cells were trypsinized
and collected, washed with PBS and fixed in 70% cold ethanol over-
night at − 20 °C. After 24 h, the fixed cells were washed twice with PBS
and subsequently stained with DNA staining solution (PI 20 μg/mL and
RNase 200 μg/mL) for 30 min at 37 °C in the dark (at least 1 × 106
cell). Twenty-thousand events were analyzed by a FACS Calibur flow
cytometer (BD Pharmingen, San Diego, CA, USA) and the percentage of
the cells in G0/G1, S, and G2/M phases were calculated using CellQuest
software (BD, USA).
4.1.5. General synthetic procedure for 2-(3-(4-((1,4-dioxo-1,4-
dihydronaphthalen-2-yl)
(5m):
amino)phenoxy)propyl)isoindoline-1,3-dione
This compound was prepared from 2-((4-hydroxyphenyl)amino)
naphthalene-1,4-dione (3) (265, 1 mmol) and 2-(3-bromopropyl)iso-
indoline-1,3-dione (5m) in the presence of K2CO3 (414.6 mg, 3 mmol)
using a similar procedure as described for compounds (5a-k). The crude
product was purified by column chromatography using petroleum
ether-ethyl acetate (3:2) to afford pure compound.
4.2.4. Apoptosis study
The Apoptosis induction effect was determined by FACS analysis
with annexin V-FITC/PI staining.44 MCF-7 cells were cultured and
treated with compounds 5b and 5k at the concentration of 25 μM. After
48 h of incubation, the cells were harvested, washed with PBS and
stained with annexin V-FITC (5 μl) and propidium iodide (5 μl), fol-
lowed by flow cytometry analysis using FACS Calibur (BD, USA). The
effect on the apoptosis rate was evaluated based on the fluorescence of
10,000 events.
Brownish-purple crystals, isolated: 95 mg; yield: 21%; mp:
184–186 °C; Rf: 0.68 (Hex/EtOAc 3:2). FT-IR (KBr) υmax (cm−1): 3255,
1716, 1676, 1573, 1370, 1297, 1151, 1036, 823, 722. 1H NMR
(300 MHz, CDCl3) δ (ppm): 8.03 (d, J = 7.6 Hz, 2H, naphthoquinone-
H), 7.83–7.73 (m, 2H, naphthoquinone-H), 7.73–7.52 (m, 4H, Ar-H),
7.12–7.02 (d, J = 8.2 Hz, 2H, Ar-H), 6.82–6.72 (d, J = 8.4 Hz, 2H, Ar-
13

M. Gholampour, et al.
Bioorganic&MedicinalChemistry28(2020)115718
4.3. In silico studies
some toxicity risks were predicted using Osiris property explorer.
(Available online http://www.organic-chemistry.org/prog/peo/.)
4.3.1. Molecular docking
Three dimensional (3D) structures of ligands were sketched and
minimized under Molecular Mechanics MM+ and then semi-empirical
AM1 methods using HyperChem software. The 3D X-ray crystal struc-
ture of different human topoisomerase II proteins (PDB ID: 1ZXM,
1ZXN, 5GWK, 3QX3, 4G0V) were downloaded from protein data bank
(http://www.rcsb.org). Preparation of protein was performed with
AutoDockTools-1.5.6. and molecular docking study was performed
using AutoDock Vina program 1.1.2.45 The grid box with the size of
50×50×50 Å was determined and the box was centered on co-crys-
talized ligand. The center coordinates of grid box was attained at x:
41.996, y: 2.482, and z: 33.507 and exhaustiveness was set to be 100
and other docking parameters were set as default. Validity of docking
procedure was verified using co-crystallized ligand and the above-
mentioned protocol. The results were visualized with Discovery Studio
Client 2017 software.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgment
The authors wish to thank the support of the Vice-Chancellor for
Research, Shiraz University of Medical Sciences (Grant No. 97-01-05-
17463). This study was part of the Pharm.D thesis of Maryam
Gholampour.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115718.
4.3.2. Molecular dynamics simulation
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filed of Amber99sb was applied to set the atom types and parameters of
the simulation. A dodecahedral box type with a distance of 1.0 nm
between the complex and the box edge was defined to set the minimum
distance of at least 2 nm between the solute's two periodic images.
Then, TIP3P water types filled the box, and the system was neutralized
with sodium chloride counter ions in a concentration of 0.15 mol/lit.
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