Highly selective and potent anti-cancer agents based on 2,9-substituted-1,10-phenanthroline derivatives

Article abstract of DOI:10.1016/j.inoche.2020.108085

A study concerning on in-vitro anticancer evaluation of structurally tuned 1,10-phenanthroline at 2,9 positions with different functional groups such as –CH₃ (S1), >C[dbnd]O (S2), –COOH (S3), –COOCH₃ (S4) and –CONHNH₂ (S5) were described. The solubility data revealed that all ligands were completely soluble in dimethyl sulphoxide (DMSO) and moderately soluble in water. The photo-physical properties of these ligands revealed that a common absorption peak appeared in the region of 270–300 nm and emission spectra in the region of 330–510 nm with a large Stokes shift of 85 nm. The binding constant of ligands (S1-S5) with calf-thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) were found to be 10⁵ M^?1 and 10⁴ M^?1 respectively. The fluorescence quenching of ethidium bromide (EtBr) from DNA upon addition of ligand was confirmed from binding affinity values K_SV (10⁴ M^?1) and K_app (10⁶ M^?1). The mode of interaction of ligand with DNA is either by intercalation or groove binding this is further supported by viscosity and in-silico studies. The gel electrophoresis studies exhibited that S4 and S5 have cleaved plasmid DNA completely within 60 min while rest of the ligands took more than 60 min. The cytotoxicity study of these ligands (S1-S5) were conducted with two different cancer cell lines (MDA-MB-231 and HeLa) and their performance were compared with normal HEK-293 cells. The study revealed that ligands S5 and S4 were found to be least inhibitory concentration (IC₅₀) and high selectivity factor values of 7.66 μM/12.97 and 13.35 μM/6.19 with respect to HeLa and MDA-MB-231 cell lines while ligands S1-S3 showed high IC₅₀ values compare to doxorubicin. However, both S5 and S4 ligands have been displayed higher cytotoxicity effect than doxorubicin and least effect on normal cell HEK-293.

Full text of DOI:10.1016/j.inoche.2020.108085

Inorganic Chemistry Communications 119 (2020) 108085
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Short communication
Highly selective and potent anti-cancer agents based on 2,9-substituted-1,
1
0-phenanthroline derivatives
⁎
Sourav De, S.K. Ashok Kumar
Department of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India
G R A P H I C A L A B S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords:
A study concerning on in-vitro anticancer evaluation of structurally tuned 1,10-phenanthroline at 2,9 positions
with different functional groups such as –CH (S1), > C]O (S2), –COOH (S3), –COOCH (S4) and –CONHNH
DNA and BSA binding
Gel electrophoresis
Cytotoxicity study
Molecular docking and DFT
3
3
2
(
(
S5) were described. The solubility data revealed that all ligands were completely soluble in dimethyl sulphoxide
DMSO) and moderately soluble in water. The photo-physical properties of these ligands revealed that a common
absorption peak appeared in the region of 270–300 nm and emission spectra in the region of 330–510 nm with a
large Stokes shift of 85 nm. The binding constant of ligands (S1-S5) with calf-thymus deoxyribonucleic acid (CT-
5
−1
4
−1
DNA) and bovine serum albumin (BSA) were found to be 10 M and 10 M respectively. The fluorescence
quenching of ethidium bromide (EtBr) from DNA upon addition of ligand was confirmed from binding affinity
4
−1
6
−1
values KSV (10 M ) and Kapp (10 M ). The mode of interaction of ligand with DNA is either by intercalation
or groove binding this is further supported by viscosity and in-silico studies. The gel electrophoresis studies
exhibited that S4 and S5 have cleaved plasmid DNA completely within 60 min while rest of the ligands took
more than 60 min. The cytotoxicity study of these ligands (S1-S5) were conducted with two different cancer cell
lines (MDA-MB-231 and HeLa) and their performance were compared with normal HEK-293 cells. The study
revealed that ligands S5 and S4 were found to be least inhibitory concentration (IC50) and high selectivity factor
values of 7.66 µM/12.97 and 13.35 µM/6.19 with respect to HeLa and MDA-MB-231 cell lines while ligands S1-
S3 showed high IC50 values compare to doxorubicin. However, both S5 and S4 ligands have been displayed
higher cytotoxicity effect than doxorubicin and least effect on normal cell HEK-293.
⁎
Corresponding author.
E-mail address: ashokkumar.sk@vit.ac.in (S.K. Ashok Kumar).
https://doi.org/10.1016/j.inoche.2020.108085
Received 26 April 2020; Received in revised form 26 June 2020; Accepted 29 June 2020
Available online 02 July 2020
1387-7003/ © 2020 Elsevier B.V. All rights reserved.

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
1
. Introduction
[17]. Kyziol et al. have reported copper(I) complexes (12 and 13) in-
duce mitochondrial dysfunctions due to reduction of mitochondrial
membrane potential leads to mitochondrial damage and causing
apoptotic pathways of cellular death by the activation of caspases with
IC50 value 6.7 and 7.1 μM [18].
Despite advances in chemotherapy, cancer is a key problem that
continues to struggle in our society. The scientific community is thor-
oughly studying new strategies of treatment for cancer by developing
new drugs. The main approach of designing an antineoplastic agent is
to destroy the DNA by various binding mechanisms such as hydro-
phobic binding, electrostatic binding to the minor groove and inter-
calation. The intercalation can cause deep modifications in the nu-
cleotide secondary structure, such as lengthening, stiffening and
unwinding of the DNA helix with major changes in DNA replication and
transcription [1]. The complete extent of their cellular process is vital to
balance potent and toxicity, is often not understood. In addition, the use
of many anticancer drugs is limited by dose-limiting toxicities as well as
the development of drug resistance. So, the novel anticancer agents are
constantly being prepared to address these issues.
As a part of our ongoing research in development of anti-cancer
agents, dipyridophenazine compounds (14) showed high cytoselectivity
and specificity against HeLa cells with IC50 of 9.65 μM [19]. Taking into
consideration of activity of phenanthroline derivatives reported in lit-
erature, recently we have synthesized phenanthroline dicarbohydrazide
derivative (15) to synthesise Schiff’s base for ion recognition studies
[20]. However, there is no report on anticancer studies by using these
derivatives (S1-S5).
2. Experimental
Among several class of compounds, phenanthroline derivatives are
important class of compounds due to their various structural and che-
mical properties; for example, rigidity, planarity, aromaticity, basicity,
and chelating capability, which makes them versatile starting materials
in many applications such as, luminescence, metal complexation and
biological activities. It is present in alkaloids, sterols, and hormones
which makes analogues a fair selection in the generation of character-
istic bioactive compounds [2–6]. In addition, metal complexes of these
ligands are used in molecular assemblies, DNA splitting, building blocks
for preparation of metallo-dendrimers, redox agents and catalysis
2.1. General procedure for spectral measurement, binding studies (DNA,
EtBr and BSA)
Both absorption and emission spectra were recorded on a UV–Vis
spectrophotometer (JASCO V-730) and spectrofluorometer (FP-8200)
using a 1 cm quartz cell. The DNA binding study was carried out by
using ligands (S1-S5) in 5 mM Tris-HCl-NaCl buffer at pH 7.4 in
phosphate buffer [21,22]. The intrinsic constant (K ) was calculated
b
using the equation (i).
DNA
DNA
1
[
7–10]. The capability of 1,10-phenanthroline to interact with nucleic
=
+
(
a
f )
( b
f )
Kb ( a
f )
(i)
acid base pairs have been clearly explained by using computational and
experimental [11,12]. In their studies, both adenine–thymine (AT) and
cytosine-guanine (GC) base pairs bind phenanthroline unit via hy-
drogen bonding. Therefore, 2.9 substituted phenanthroline compounds
have been reported recently by scientists for cellular applications
where ε , ε and ε are apparent extinction coefficient of ligand,
a
f
b
extinction coefficient of the ligand in its free form and completely
bound to DNA respectively.
The apparent association constant (K ) of ligand to CT-DNA was
app
(
Fig. 1). Wang et al. have reported the inhibitory effect of (1) on neck,
established from the emission response using EtBr as a spectral probe
head and lung cancer cell growth with IC50 in the range of 0.1–0.2 μM
13]. In another report, compounds (2–5) can effectively induce the
5 mM Tris-HCl-NaCl buffer at pH 7.4 [23]. The K was found by using
app
[
the equation (ii)
formation and stabilization of anti-parallel G-quadruplex structure of
human telomeric sequence (AG3(T2AG3)3, HTG22), and significantly
inhibit telomerase activity and HeLa cell with IC50 values 2.0–2.3 μM)
proliferation [14]. Neidle et al. have reported that a platinum-phe-
nanthroline square complexes (6 and 7) can persuade a high degree of
quadruplex DNA stabilization and inhibit telomerase activity having
IC50 value 3.1 and 8.9 μM) [15]. Komarnicka et al. have synthesized
copper complex (8) showed high activity against PANC-1 cell lines with
IC50 of 2.65 μM [16]. Ewelina et al. have prepared complexes (9–10)
and results revealed lower toxicity to normal human cell line with IC50
of 0.57 µM and 1.72 µM than A549 cell with IC50 value of 0.29 µM and
Kapp×[Complex]50 = KEtBr × [EtBr]
(ii)
where [ligand] represent the concentration of the complex at 50%
5
0
7
−1
quenching of DNA-bound EtBr emission intensity K
EtBr
(1.0 × 10 M
)
binding constant of EtBr, and concentration of EtBr was used 8 μM. The
Stern-Volmer quenching constant (K ) was calculated from equation
SV
(iii) [24].
I
I
0
= 1 + KSV [Q]
(iii)
where I
0
and I are emission intensities of EtBr-DNA in the absence and
0
.43 µM. The complex (11) reported to be more toxic to normal cells in
in the presence of complex of concentration [Q].
comparison to cancer cells studied (A549, HeLa, LoVo, and MCF-7)
The quantum yield (φ) was calculated according to comparative
William's method in MTT condition [25]. The quantum yield was
3
H C
O
N
P
N
N
CH
3
N
N
N
N
N
N
N
N
N
N
O
O
O
N
N
CH3
O
O
O
O
O
O
Pt
N
O
N
N
NH
HN
NH
HN
NH
HN
Cl
N
Pt
O
OH
NH
HN
Cu
Cl
N
N
P
O
N
O
Cu
N
O
I
9
N
O
1
2
3
4
5
6
CH
3
N
7
8
CH
3
O
CH3
OH
N
N
N
N
N
N
N
N
N
O
O
2
NH
F
2
O
N
OH
NH
O
OH
F
O
PF
6
CH3
CH
3
F
O
3
H C
N
N
O
O
H
3
C
O
N
H
3
C
N
N
N
N
N
CH
Cu
3
R
R
N
N
N
N
O
O
CH3
3
S1:R=-CH , S2:R= >C=O,
Cu
F
P
Cu
Ci
N
N
CH
3
N
S3:R= -COOH, S4:R=-COOCH3
S5:R=-COONHNH2
N
O
N
P
I
N
N
N
CH
3
NCS
Cu
N
OHC
CH
3
CH
3
N
15
14
H
3
C
11
12
CH3
13
10
CH
3
Fig. 1. . Molecular structure of phenanthroline derivatives previously reported in literature.
2

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
calculated according to the equation (iv)
plate. The entire cell lines was cultured in 100 μL of a growth medium
in 96-well plates and incubated at 37 °C under 5% CO overnight. After
2
I
I
R
S
OD
OD
S
R
S
=
×
×
×
24 h of incubation, the cultured cells were exposed to various con-
centrations of the ligands (S1-S5) (9–300 µM). After 24 h, the wells
were treated with 10 μL MTT reagent (1 mg/ml) and incubated for 3 h
at 37 °C. The suspension was retained on micro vibrator for 10 min and
afterward the absorbance was calculated by the ELISA reader at λmax
(570 nm). The testing was executed in triplicate. The growth inhibition
percentage was calculated according to the equation [33–35]: percen-
tage growth inhibition = 100-[(AD × 100)/AB], where AD, measured
absorbance in wells which contain samples and AB, measured absor-
bance for blank wells.
R
R
(iv)
where, φ, I, OD, and η represents quantum yield, peak area, absorbance
recorded at λmax, and refractive index of solvent respectively.
The interaction of ligand with BSA has been calculated from tryp-
tophan fluorescence quenching response [26]. Here, the ligand solution
was incrementally added to BSA in Tris-HCl-NaCl buffer at pH 7.2. The
SV quenching constant (KBSA) of the fluorescence at 340 nm was cal-
culated using equation (v and vi). Further, using Scatchard equation
(
vii), K and n values were calculated [27].
I
I
0
=
1 + KBSA [Q] = 1 + K
q 0
[Q]
2
.5. In-silico study and DFT theory
(v)
KBSA
In order to know molecular interactions, each ligand was in-
Kq =
0
(vi)
vestigated for their binding affinities by molecular docking study using
Autodock vina [36] along with the features of AutoDock Tools (ADT).
In search of a smaller DNA section [d(CCGTCGACGG], an widely used
oligodeoxynucleotide [37] (pdb entry: 423D) was fetched procured
from Protein Data Bank (www.rcsb.org/pdb) [38]. 2D molecular ske-
leton of all five synthesized compounds (S1-S5) were developed using
ACD ChemSketch Freeware. Further, 3D coordinates structures were
built to make these fit to get into the subsequent stages of docking [39].
The binding site was mapped with a grid box with a spacing of 1 Å and
24 × 24 × 24 number of points was used in x y and z directions. Both
the target and the compounds were tuned to introduce their pdbqt
forms. The process yielded 9 poses for each ligand by considering the
exhaustiveness of 8. Two aspects, via the scoring functions and close
proximity between the conformer and active site residues were con-
sidered to assess the quality of all the bioactive conformers. Several
orientations of each compound within the active site were observed
using PyMOL (The PyMOL Molecular Graphics System, Version 1.3,
Schrodinger, LLC) molecular graphics program. In an attempt to de-
velop the virtual understanding of protein binding study and subse-
quently correlating it with the experimental study, molecular docking
study was conducted with the 3D structure of BSA with PDB ID: 4F5S
[40]. The protein structure was made free from water molecule so as to
avoid the probability of undesired interaction. The other process was
followed as similar as that of the earlier one. The grid box was placed
considering the relative positions of two essential active site residues,
via. Trp213 and Trp134 [41].
I0
I
log
= log k + n log[Q]
I
(vii)
where, I
0
, I , k
q
, τ , K and n represents emission intensities of BSA in the
0
absence and in the presence of quencher of concentration [Q],
−
8
quenching constant, average lifetime of tryptophan (1 × 10
binding constant and number of binding sites respectively [28].
s),
2
.2. Relative viscosity study
To find out the type of binding interaction of S1-S5 with DNA was
studied by viscosity measurements using Ostwald’s viscometer. Each
experiment was performed for three times and the average flow time
1
/3
was recorded. The data was plotted as (η/η
0
)
vs. [ligand]/[DNA],
where η and η
ligand, and viscosity of DNA alone respectively. The viscosity of DNA
was calculated using the formula η = (t-t )/t where t and t re-
presents the efflux time of DNA and PBS buffer solution respectively
29–31].
0
corresponds to viscosity of DNA in the presence of the
0
0
0
0
[
2
.3. DNA degradation study
Initially, 0.15 g of plasmid DNA (~1 kb) was dissolved in 1 mL of
Tris-HCl-NaCl buffer solution and further this solution was mixed with
an equal volume of ligand (containing 1, 2 and 3 mg/ml). This solution
mixture was incubated for 1 h at 37 °C and loaded on 1% agarose gel
containing EtBr (1.0 mg/ml) and 2 mL buffer solution (containing 25%
bromophenol blue, 30% glycerol and 0.25% xylene cyanol). Here,
plasmid DNA was used as a positive control. The degradation experi-
ment was carried out at 50 V for 60 min in buffer solution [32] and the
gel plate was visualized using a gel documentation instrument.
To calculate electronic properties of ligand, computational calcu-
lations was performed in the gas phase using density functional theory
(DFT) by applying the Becke three-parameter Lee-Yang-Parr (B3LYP)
exchange correlation functional. The basic set 6-311G(d,p) was used for
all atoms. All the geometrical optimizations were done with zero ne-
gative vibrational frequency. The calculations were carried out using
Gaussian 09 program [42].
2
.4. In-vitro cytotoxic study
3
. Results and discussion
Each ligand was dissolved in 0.1% DMSO and then serial dilution
with cell medium. Here, we have selected two different cancer cell lines
such as HeLa, MDA-MB-231 and normal cell lines HEK-293 were used
in this assay. The doxorubicin was used as a positive control with the
same volume of medium. Hemocytometer was used for counting of
3.1. Synthesis and photo-physical studies of ligands
The synthesis of ligand S5 follows four steps reaction via various
intermediates such as 1,10-phenanthroline-2,9-dicarbaldehyde (S2),
1,10-phenanthroline-2,9-dicarboxylic acid (S3), 1,10-phenanthroline-
4
cells. The amount of cell seeded was 1 × 10 cells per well in a 96 well
HNO3
MeOH
SeO2
NH -NH H O
2
2. 2
H SO
N
N
Dioxane
N
N
2
4
N
N
N
N
N
N
MeOH
HN
H N
NH
NH2
HO
OH
O
O
O
O
O
O
O
O
O
O
2
Scheme 1. Reagents and conditions to synthesize ligands S2-S5.
3

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
Fig. 2. . Absorption and emission spectral studies of S1-S5 compounds (3 × 10−5 M) in DMSO medium.
Fig. 3. . The ESP mapped on surface of ligands (S1-S5) by DFT-B3LYP method.
Fig. 4. . FMO’s of ligands (S1-S5) by using DFT-B3LYP method.
Table 1
.
Molecular electronic parameters of ligands (S1-S5) performed by DFT-B3LYP method.
Ligand
Energy (kJ/mol)
DM (Debay)
E
H
(eV)
E
L
(eV)
ΔE (eV)
χ (eV)
ɳ (eV)
μ (eV)
ω (eV)
S (eV)
S1
S2
S3
S4
S5
−650
−798
−949
−1028
−1020
2.27
8.85
6.73
6.02
4.25
−6.19
−6.90
−6.95
−6.81
−6.87
−1.53
−2.78
−2.61
−2.46
−2.49
4.65
4.12
4.34
4.36
4.39
3.86
4.84
4.78
4.64
4.68
−2.33
−2.06
−2.17
−2.18
−2.19
−3.86
−4.84
−4.78
−4.64
−4.68
−3.20
−5.69
−5.26
−4.94
−5.00
−1.17
−1.03
−1.09
−1.09
−1.10
4

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
Fig. 5. . Stability studies of S5 in natural physiological condition.
Fig. 6. . UV–visible spectral response of ligands (S1-S5) (3 × 10−5 M) in 5 mM tris-HCl-NaCl (pH, 7.2) with incremental addition of CT-DNA (3 × 10−5 M). Insert
Fig. Linear plot used for calculating binding constant of ligand.
2
,9-dicarboxylate (S4), and 1,10-phenanthroline-2,9-dicarbohydrazide
Therefore, the solubility of the ligand was performed in selected sol-
vents from non-polar and polar solvents. The solubility reveals that all
ligands were insoluble in chloroform and moderately soluble in water
but good solubility was found in DMSO and DMF medium.
(
S5) were discussed in Scheme 1. Besides, S3 and S4 ligands are rich in
oxygen atoms while S5 rich in nitrogen atoms.
To work as a drug, given ligand has to acquire required lipophilicity.
5

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
Fig. 7. . Fluorescence quenching curves of EtBr bound to DNA by (S1-S5) in Tris-HCl buffer solution (1 × 10 mol/L, pH = 7.2) at 298 K ([EtBr] = 8 × 10−6 mol/
−3
−
4
−5
L; [DNA] = 1.2 × 10
mol/L; [S1-S5] = 3 × 10
mol/L; λex = 485 and λem = 598 nm). Insert Fig. SV plot of each ligand.
The photo physical properties of ligand was performed by mea-
suring absorbance and emission in DMSO and MTT environment. The
spectral studies revealed that high energy band appeared in the region
of 273–300 nm owing to ligand centered π → π* transitions shown in
Fig. 2. The emission ability of each ligand was studied at respective
wavelength maximum as their excitation energy. The maximum emis-
sion was in the range of 330–510 nm. Further, the quantum yield of all
ligands exhibit high in DMSO medium and the order of yield was found
to be S5 > S1 > S2 > S3 > S4.
S5, it is more at outside the semi cavity. The energies of the FMOs
describe the molecule’s ability to donate and accept an electron (ΔE)
shown in Fig. 4, electronegativity (χ), chemical potential (μ), global
hardness (ɳ), global softness (S) and global electrophilicity index (ω)
were listed in Table 1 [44–46]. The significant of these parameters is to
measure the molecular stability and reactivity of the ligands. The mo-
lecular electronic properties of ligands (S1-S5) reveals that these li-
gands acquired these properties which make suitable for nucleic acid
interactions [47]. However, the electrophilicity quantifies the biolo-
gical activity of ligand.
In order to support experimental results, theoretical studies were
performed using DFT, various quantum-chemical parameters were
calculated by applying B3LYP/6-311G** such as molecular energy,
electrostatic potential charges, and energy of HOMO/LUMO to calcu-
late energy gap [43]. The electrostatic potential mapped onto the
constant electron density surface. The 3D plot of molecular electrostatic
potential of S1-S5 were shown in Fig. 3. The maximum negative region
For unique therapeutics initiative, ligand has to be stable inside the
cells in internal physiological condition. Hence, in order to know the
stability in such environment, ligand S5 has selected for stability test
and it was performed in pure water, 0.1 mM GSH and MTT environment
by using UV–Vis spectrophotometer for about 24 h (Fig. 5). It reveals
that in all three medium, ligand S5 remain same in terms of absorbance
and wavelength maximum even after 24 h contact which establishes it’s
utility in biological system.
(
red colour) and positive region (blue colour) are preferred sites for
electrophilic and nucleophilic attack respectively. In S1-S4, the nega-
tive ESP is more on the phenanthroline semi cavity whereas in ligand
6

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
excitation energy 485 nm fluorescence emission was recorded at
5
98 nm (Fig. 7). The 50% quenching of DNA-EtBr occurred at con-
centration of 30–37.5 µM. The SV quenching constant of S1-S5 was
3
−1
3
−1
3
−1
4
found to be 5.2 × 10 M , 2.3 × 10 M , 8.0 × 10 M , 1.1 × 10
−1
4
−1
M
, 6.5 × 10
M
respectively which is low as compared to the
known intercalators. Among five ligands studied, S4 and S5 have
showed high order intercalation tendency compare to other ligands (S1-
S3) owing to more electrostatic and groove binding with DNA.
3.4. Viscosity study
To identify mode of interaction of S1-S5 with DNA, viscosity re-
sponses were carried out with CT-DNA by different concentration of S1
to S5 ligands. The ligands have to quandary with the DNA double helix
and separate the adjacent base pairs via intercalation mode thereby
consecutively an increase in the length and viscosity of DNA. As seen
from Fig. 8, there is an increase in relative viscosity with increasing in
concentration of ligand. The order of increasing in viscosity follows:
S5 > S4 > S3 > S2 > S1 this might be due to intercalation of the
ligand.
Fig. 8. Effect of increasing amounts of ligands (S1-S5) on the viscosity of CT-
−
6
−6
DNA at 298 K ([EtBr] = 1 × 10
mol/L; [DNA] = 1 × 10
mol/L;
−
3
[
Ligand] = 1 × 10
mol/L).
3.2. DNA binding study
3.5. DNA degradation study
The ligand-DNA interaction can be observed by change in absor-
The DNA degradation experiment of the ligands (S1-S5) was studied
by using agarose gel electrophoresis (Fig. 9). The cleavage studied was
done using three different concentration (containing 1, 2 and 3 mg/ml).
The control plasmid DNA was arranged in two conformations where the
upper and lower bands match to the nicked and supercoiled forms. The
study reveals that the plasmid DNA (≈1 kb) was degraded within 1.5 h
by all ligands at 3 mg/ml while ligands S4 and S5 were also found
degrade even at 2 and 1 mg/ml and even in 1 mg/ml. This degrading
efficiency of ligands (S4 and S5) may be due to disrupt the double and
triple hydrogen bonds formed between the nucleotide bases.
bance maxima owing to strong π-π stacking between DNA base pairs
and phenanthroline core. The absorption spectra of all ligands were
observed at 268 nm while neat CT-DNA seems a broad band
(
(
230–296 nm) with a wavelength maximum at 258 nm. DNA base pairs
adenine, guanine, cytosine and thymine) are mainly accountable for
this electronic transition. In ligands S1-S4, on increasing the CT-DNA,
hyperchromism effect was observed due to DNA duplex has denatured,
and ligand well intact with DNA which facilitates more light to be ab-
sorbed by the ligand. In case of S5, it exhibits hypochromic effect due to
intercalation in DNA helix and hence the amount of light absorbed by
ligand will be less. The intrinsic DNA binding constant (K
b
) of each
3.6. Protein binding study
ligand was calculated from the plot [DNA]/(ε
a
-ε ) vs. [DNA].
f
4
−1
Accordingly, the K
b
value of S1-S5 were found to be 2.1 × 10 M ,
The binding interaction of ligands (S1-S5) with BSA was studied by
using intrinsic tryptophan emission quenching of BSA. Upon increase in
concentration of ligand, the quenching of BSA was decreased gradually
(Fig. 10). This may be due to several molecular interactions and per-
turbation in the secondary structure of the protein upon interaction
with ligand. In case of ligands (S1-S4), a minor red shift of 10–50 nm of
emission maximum was observed. This could be due to an increase in
hydrophobicity of the microenvironment around the tryptophan re-
sidues leads [48]. These variations happened owing to ligand-protein
interaction leads to disruption of protein tertiary structure and hence
losses its activity [49,50]. Therefore, ligands (S1-S4) showing destruc-
tion of tertiary structure of protein due to damage of charged amino
acid side chain interaction while S5 ligand exhibits secondary structure
damage due to destruction of hydrogen bonding (carbonyl oxygen of
amino acid and the amino hydrogen of ligand S5) which is an important
factor. In order to quantify these interactions, the SV quenching con-
4
−1
5
−1
5
−1
5
−1
8
.6 × 10
M
, 3.1 × 10
M
, 3.5 × 10
M
, 5.9 × 10 M
respectively (Fig. 6). The obtained K value revealed that ligand S5
b
showed higher binding affinity towards DNA in comparison with rest of
the ligands.
3
.3. EtBr displacement study
Competitive binding studies of S1-S5 with CT-DNA was calculated
by using spectrofluorimetric in the presence of EtBr. It is a highly
sensitive emissive sensor which interrelates with DNA via π-π* inter-
calation. The introduction of new ligand in the EtBr-DNA molecular
assembly results in decrease of emission intensity due to displacement
of EtBr from the DNA double helix. The ability of fluorescence
quenching of EtBr pre-treated DNA might be used to determine the
apparent binding constant (Kapp) of the ligand with CT-DNA by using
stant (KBSA), quenching rate constant (K ) were calculated from slope of
q
Fig. 9. . Gel electrophoresis diagram showing chemical nuclease activity of S1 to S5: Lane B: PBS Buffer solution; Lane D: pure DMSO; Lane S1-S5: containing (a)
mg/ml (b) 2 mg/ml and (c) 1 mg/ml.
3
7

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
Fig. 10. . Fluorescence quenching of BSA on addition of ligands (a-e, S1-S5) in 5 mM Tris HCl/NaCl buffer at pH 7.2 (λex = 295 nm; λem = 350 nm). (e) Inserted
figure, SV plot of I
0
/I vs. [S5] (f) Scatchard plot of log ([I -I]/I) vs. log [S5].
0
Table 2
.
Docking studies of ligands (S1-S5) with DNA and BSA.
Ligand Binding free energy
Vdw_hb_desolv energy
Electrostatic energy
Total internal energy Torsional free energy
Unbound system’s energy
α
(
ΔGbinding
)
(ΔGvdW+hb+desolv
)
(ΔGelec
)
(ΔGtotal
)
(ΔGtor
)
(ΔGunb)
(
a) DNA
S1
S2
S3
S4
S5
−7.2
−6.9
−7.3
−7.5
−7.9
−6.7
−6.4
−6.9
−6.9
−7.4
−0.83
−0.62
−0.57
−0.88
−0.71
0.73
0.33
0.12
0.17
0.28
−0.73
−0.94
−1.12
−1.01
−1.03
−0.94
−1.12
−1.01
−1.03
−0.21
(
b) BSA
S1
S2
S3
S4
S5
−7.1
−7.5
−7.7
−8.4
−8.6
−6.7
−6.9
−7.2
−7.9
−8.1
−0.58
−0.67
−0.72
−0.75
−0.69
−0.89
−1.09
−0.99
−1.12
−1.04
0.28
0.07
0.22
0.25
0.19
−0.89
−1.09
−0.99
−1.12
−1.04
α
ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor - ΔGunb (expressed in terms of Kcal/mol).
8

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
Fig. 11. . Docking pose of S1 to S5 within the minor groove of DNA.
Fig. 12. . Docking pose of S1-S5 within the active site of BSA protein.
the linear plot of I
0
/I vs. [ligand]. The order of increasing in KBSA fol-
for transportation of protein-bound complexes in biological systems.
lows: S5 > S4 > S3 > S2 > S1 this might be due to intercalation of
the ligand. This may be due to more electrostatic interaction is expected
3.7. In-silico study
4
−1
4
−1
4
−1
in S5 (3.9 × 10 M ), S4 (1.7 × 10 M ) and S3 (1.5 × 10 M
)
3
3
−1
compare to corresponding S2 (7.9 × 10
M
) and S1 (5.6 × 10
Molecular docking studies of synthesized ligands (S1-S5) were
carried out with the DNA duplex consisting of d(CGCGAATTCGCG)
dodecamer sequence. Scoring functions/binding energy, one of the
important parameter for prioritizing the efficiency of all the docked
compounds was given in Table 2(a). DNA binding study reveals that
ligand S5 ranked to be the 1st owing to its highest binding energy
−
1
M
). The binding affinity (K) and number of binding sites (n) of ligand
5
-1
S5 was found to be 1.4 × 10 M and 0.79 respectively. The obtained
K
BSA value revealed that ligand S5 showed higher binding affinity to-
wards BSA in comparison with rest of the ligands. These binding
characteristics showing a binding tendency with BSA which is a crucial
9

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
Table 3
respectively. This high potency of S4 and S5 were caused by more rapid
diffusion into cell as a result there is enhanced intracellular ligand
concentration, which eventually led to the stronger cytotoxicity. Be-
sides, all these ligands exhibits least effect to normal cells compare to
standard drug used in this study. The cytotoxicity performance of or-
ganometallic complex based on 2,9 substituted phenanthroline (16–18)
have shown high anticancer agent properties compare to 2,9 sub-
stituted phenanthroline ligands (13,14). However, the selectivity factor
of ligands S4 and S5 are found to be high compare to reported work.
.
MTT cytotoxicity screening of ligands (S1-S5) at 24 h of exposure.
Ligand
Cell Line (*IC50 μM )
Selectivity
**
factor
HeLa
MDA-MB-231 HEK-293
HeLa
MDA-
MB-231
S1
S2
S3
S4
S5
37.47 ± 1.2 57.03 ± 1.3 97.12 ± 1.6
2.59
1.70
1.83
1.37
6.19
31.69 ± 1.8 66.12 ± 1.9 121.38 ± 2.1 3.83
35.87 ± 1.3 68.75 ± 1.6 94.62 ± 1.5
64.66 ± 2.2 13.35 ± 1.1 82.75 ± 1.9
2.63
1.27
3.9. Structure activity relationship
7.66 ± 1.1
19.23 ± 1.4 99.42 ± 1.4
12.97 5.17
Doxorubicin 19.71 ± 1.2 20.22 ± 1.6 74.21 ± 1.1
3.76
3.67
SAR was performed according to the results of cytotoxicity of the
*
IC50 is the concentration at which 50% of cells undergoes cytotoxic cell
evaluated compounds. Among five compounds S5 and S4 were exhibits
an excellent IC50 values in the studied ligands including standard drug
doxorubicin. Further, on comparing selectivity factor, ligand S5 and S4
exhibits highest selectivity while remaining three ligands (S1-S3) and
standard drug shows almost same factor. One of the key future of li-
gands (S1-S5) is that phenanthroline core will provide rigidity, pla-
narity, aromaticity, basicity, rotatable bonds, low molecular weight and
hydrophobic nature enrich the cellular accumulation. Hence, these li-
gands are reasonably a worthy to use as cytotoxic agents (Fig. 13).
death under treatment.
*
* Selectivity factor: ratio of IC50 for HEK-293 and IC50 for all the cancer cell
lines. HEK-293 fibroblasts are generally selected as the model for healthy cells
in the evaluation of chemotherapeutic drug selectivity.
Lipinski's Rule
log P< 5
Good in-vivo
drug absorption
and permeation
Molecular weight < 500 DA
H-Bond accepter <10
H-Bond donors < 5
4
. Conclusion
In summary, we have designed and synthesized four 2,9 substituted
,10 phenanthroline as rigid derivatives and to evaluated as cytotoxi-
1
Tunable metal binding site
π−acidic properties
Rigid planar structure
N
N
city against two cancer cell lines. The stability test revealed that ligand
is stable in all three (water, GSH and MTT) selected media. Both ex-
perimental and computation studies shows that all ligands have good
binding affinity with nucleic acids. The gel electrophoresis studies ex-
hibited that S4 and S5 have cleaved plasmid DNA completely within
R
R
S1:R=-CH , S2:R= >C=O,
S3:R= -COOH, S4:R=-COOCH3
S5:R=-COONHNH2
3
Intercalation property
6
0 min. Ligands S5 and S4 showed least IC50 values and high selectivity
Fig. 13. . SAR of phenanthroline derivatives (S1-S5).
factor with selected HeLa and MDA-MB-231 cell lines respectively.
Therefore, in conclusion, we believe that ligands S5 and S4 have an
important role for displaying the cytotoxic effects.
(
−8.6 kcal/mol). The binding energy can be well correlated with the
docking pose obtained for the same ligand as shown in Fig. 11. The
cluster of conformers are lying well within the base pairs, thus could be
a better candidate as DNA intercalator. The other ligands in the same
series also possess near equal poses with the target DNA. On the other
hand, Table 2(b) consists of the binding energy of the same series of
ligands employed for the BSA binding study. From an overall perusal of
literature, it is observed that the important amino acids constitute ac-
tive site of bovine serum albumin are Tyr137, Tyr149, Tyr156, Phe205,
Arg208, Arg217, Ala290, Ala341 etc. Ligand S5 being the highly active
one as DNA intercalator, scores highest (binding energy: −7.9 kcal/
mol) against the protein as well. The docking poses as shown in Fig. 12
implied that respective conformer of each compound are held strongly
by the active site residues. Thus, it is inferred that docking study serves
as an important linker between ideation of the proposal and execution
of the work.
CRediT authorship contribution statement
Sourav De: Conceptualization, Methodology. S.K. Ashok Kumar:
Investigation, Writing - original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
Authors are grateful to Department of Science and Technology,
Government of India for supporting the work through the project grant
3
.8. MTT cytotoxicity test
(
EMR/2017/000816). Authors are also thankful to DST-VIT-FIST for
NMR and GC-MS for research facilities. The authors are grateful to VIT
for providing VIT SEED funding.
In this assay, living cells reduce the yellow MTT to insoluble purple
formazan crystals. All ligands were dissolved in DMSO. The cytotoxicity
studies of ligands S1-S5 were examined by using MTT method using cell
lines such as MDA-MB-231, HeLa and HEK-293. Cells were well-main-
tained with ligands along with doxorubicin as a standard positive
control with various concentrations from 9 μM −300 μM for 24 h.
DMSO was used as a control and it didn’t disturb any inhibition of
cancer cell growth. The anti-proliferative activity of the ligands based
on the half maximal inhibitory concentrations (IC50 values) is shown in
Table 3. The IC50 values of ligands with HeLa and MDA-MB-231 cells
follows an order: S5 < doxorubicin < S4 < S3 < S2 < S1 and
S4 < S5 < doxorubicin < S3 < S2 < S1 were observed
References
[
[
1] D. Richards, A. Rodger, Synthetic metallomolecules as agents for the control of DNA
structure, Chem. Soc. Rev. 36 (2007) 471–483.
2] A. Bencini, V. Lippolis, 1, 10-Phenanthroline: a versatile building block for the
construction of ligands for various purposes, Coord. Chem. Rev. 254 (2010)
2
096–2180.
[3] G. Accorsi, A. Listorti, K. Yoosaf, N. Armaroli, 1, 10-Phenanthrolines: versatile
building blocks for luminescent molecules, materials and metal complexes, Chem.
Soc. Rev. 38 (2009) 1690–1700.
[
4] L. Viganor, O. Howe, P. McCarron, M. McCann, M. Devereux, The antibacterial
10

S. De and S.K. Ashok Kumar
Inorganic Chemistry Communications 119 (2020) 108085
activity of metal complexes containing 1, 10-phenanthroline: potential as alter-
native therapeutics in the era of antibiotic resistance, Curr. Top. Med. Chem. 17
[26] V.D. Suryawanshi, L.S. Walekar, A.H. Gore, P.V. Anbhule, G.B. Kolekar,
Spectroscopic analysis on the binding interaction of biologically active pyrimidine
derivative with bovine serum albumin, J. Pharm. Anal. 6 (2016) 56–63.
[27] J. Liu, J. Tian, W. He, J. Xie, Z. Hu, X. Chen, Spectrofluorimetric study of the
binding of daphnetin to bovine serum albumin, J. Pharm. Biomed. Anal. 35 (2004)
671–677.
(
2017) 1280–1302.
[
[
5] A. Airinei, R. Tigoianu, R. Danac, C.M. Al Matarneh, D.L. Isac, Steady state and time
resolved fluorescence studies of new indolizine derivatives with phenanthroline
skeleton, J. Lumin. 199 (2018) 6–12.
6] C. Sall, A.D. Yapi, N. Desbois, S. Chevalley, J.M. Chezal, K. Tan, J.C. Teulade,
A. Valentin, Y. Blache, Design, synthesis, and biological activities of con-
formationally restricted analogs of primaquine with a 1, 10-phenanthroline fra-
mework, Bioorg. Med. Chem. Lett. 18 (2008) 4666–4669.
[28] S. De, S.K. Subran, S.K. Ramasamy, S. Banerjee, P. Paira, S.K. Ashok Kumar,
Luminescent anticancer acenaphtho[1, 2-b]quinoxaline: green synthesis, DFT and
molecular docking studies, live-cell imaging and reactivity towards nucleic acid and
protein BSA, ChemistrySelect. 3 (2018) 5421–5430.
[
[
[
7] A. Rezvani, H.S. Bazzi, B. Chen, F. Rakotondradany, H.F. Sleiman, Ruthenium (II)
dipyridoquinoxaline-norbornene: synthesis, properties, crystal structure, and use as
a ROMP monomer, Inorg. Chem. 43 (2004) 5112–5119.
[29] S. Banerjee, I. Pant, I. Khan, P. Prasad, A. Hussain, P. Kondaiah, A.R. Chakravarty,
Remarkable enhancement in photocytotoxicity and hydrolytic stability of curcumin
on binding to an oxovanadium (IV) moiety, Dalton Trans. 44 (2015) 4108–4122.
[30] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Neither. DELTA.-nor. LAMBDA.-tris
(phenanthroline) ruthenium (II) binds to DNA by classical intercalation,
Biochemistry 31 (1992) 9319–9324.
8] K. Binnemans, P. Lenaerts, K. Driesen, C. Gorller-Walrand, A luminescent tris (2-
thenoyltrifluoroacetonato) europium (III) complex covalently linked to a 1, 10-
phenanthroline-functionalised sol–gel glass, J. Mater. Chem. 14 (2004) 191–195.
9] P. Lenaerts, A. Storms, J. Mullens, J. D’Haen, C. Gorller-Walrand, K. Binnemans,
K. Driesen, Thin films of highly luminescent lanthanide cosmplexes covalently
linked to an organic-inorganic hybrid material via 2-substituted imidazo [4, 5-f]-1,
[31] R.K. Vuradi, K. Dandu, P.K. Yata, R.R. Mallepally, N. Chintakuntla, R. Ch,
S.S. Thakur, C.M. Rao, S. Satyanarayana, Studies on the DNA binding and antic-
ancer activity of Ru (ii) polypyridyl complexes by using a (2-(4-(diethoxymethyl)-1
H-imidazo [4, 5-f][1, 10] phenanthroline)) intercalative ligand, New J. Chem. 42
(2018) 846–859.
1
0-phenanthroline groups, Chem. Mater. 17 (2005) 5194–5201.
[
[
[
[
[
10] A.M. Thomas, A.D. Naik, M. Nethaji, A.R. Chakravarty, Synthesis, crystal structure
and photo-induced DNA cleavage activity of ternary copper (II)-thiosemicarbazone
complexes having heterocyclic bases, Inorg. Chim. Acta. 357 (2004) 2315–2323.
11] P. Hazarika, B. Bezbaruah, P. Das, O.K. Medhi, C. Medhi, A model study on the
stacking interaction of phenanthroline ligand with nucleic acid base pairs: an ab
initio, MP2 and DFT studies, J. Biophys. Chem. 2 (2011) 152–157.
[32] A. Chandra, K. Singh, S. Singh, S. Sivakumar, A.K. Patra, A luminescent europium
(III)–platinum (II) heterometallic complex as a theranostic agent: a proof-of-concept
study, Dalton Trans. 45 (2016) 494–497.
[33] B.A. Lakshmi, J.Y. Bae, J.H. An, S. Kim, Facile design and spectroscopic char-
acterization of novel bio-inspired Quercetin-conjugated tetrakis (dimethylsulfoxide)
dichlororuthenium (II) complex for enhanced anticancer properties, Inorg. Chim.
Acta 495 (2019) 118989.
12] S.V. Kumar, T.D. Sakore, H.M. Sobell, Structure of a novel drug-nucleic acid crys-
talline complex: 1,10-phenanthroline-platinum(II) ethylenediamine-5′-phosphoryl-
thymidylyl (3′-5′) deoxyadenosine, J. Biomol. Struct. Dyn. 2 (1984) 333–344.
13] D. Wang, S. Peng, A.R.M.R. Amin, M.A. Rahman, S. Nannapaneni, Y. Liu, D.M. Shin,
N.F. Saba, J.F. Eichler, Z.G. Chen, Antitumor activity of 2, 9-Di-Sec-Butyl-1, 10-
Phenanthroline, PLoS ONE 11 (2016) e0168450.
[34] A. Mondal, S. De, S. Maiti, B. Sarkar, S.K. Ashok Kumar, R. Jacob, A. Moorthy,
P. Paira, Amberlite IR-120 (H) mediated “on water” synthesis of fluorescent
Ruthenium(II)-arene 8-hydroxyquinoline complexes for cancer therapy and live cell
imaging, J Photochem. Photobiol. 178 (2018) 380–394.
14] L. Wang, Y. Wen, J. Liu, J. Zhou, C. Li, C. Wei, Promoting the formation and sta-
bilization of human telomeric G-quadruplex DNA, inhibition of telomerase and
cytotoxicity by phenanthroline derivatives, Org. Biomol. Chem. 9 (2011)
[35] D. Smilowicz, N. Metzler-Nolte, Bioconjugates of Co(III) complexes with Schiff base
ligands and cell penetrating peptides: Solid phase synthesis, characterization and
antiproliferative activity, J. Inorg. Biochem. 206 (2020) 111041.
2
648–2653.
[36] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J. Comput.
Chem. 31 (2010) 455–461.
[
[
15] J.E. Reed, S. Neidle, R. Vilar, Stabilisation of human telomeric quadruplex DNA and
inhibition of telomerase by a platinum–phenanthroline complex, Chem. Commun.
4
366–4368 (2007).
[37] H. Rozenberg, D. Rabinovich, F. Frolow, R.S. Hegde, Z. Shakked, Structural code for
DNA recognition revealed in crystal structures of papillomavirus E2-DNA targets,
Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 15194–15199.
16] U.K. Komarnicka, S. Kozieł, P. Zabierowski, R. Kruszyński, M.K. Lesiów, F. Tisato,
M. Porchia, A. Kyzioł, Copper (I) complexes with phosphines P (p-OCH3-Ph)
2
CH2OH and P (p-OCH3-Ph) 2CH2SarGly. synthesis, multimodal DNA interactions,
[38] P.W. Rose, A. Prlic, C. Bi, W.F. Bluhm, C.H. Christie, S. Dutta, R.K. Green,
D.S. Goodsell, J.D. Westbrook, J. Woo, The RCSB Protein Data Bank: views of
structural biology for basic and applied research and education, Nucleic Acids Res.
43 (2015) D345–D356.
and prooxidative and in vitro antiproliferative activity, J. Inorg. Biochem. 203
2020) 110926.
(
[
[
[
[
[
17] E.I. Sliwa, U. Sliwinska-Hill, B. Bazanow, M. Siczek, J. Klak, P. Smolenski,
Synthesis, structural, and cytotoxic properties of new water-soluble copper (II)
complexes based on 2, 9-dimethyl-1, 10-phenanthroline and their one derivative
containing 1, 3, 5-Triaza-7-Phosphaadamantane-7-Oxide, Molecules 25 (2020) 741.
18] A. Kyziol, A. Cierniak, J. Gubernator, A. Markowski, M. Jezowska-Bojczuk,
U.K. Komarnicka, Copper (I) complexes with phosphine derived from sparfloxacin.
Part III: multifaceted cell death and preliminary study of liposomal formulation of
selected copper (I) complexes, Dalton Trans. 47 (2018) 1981–1992.
[39] N.M. O’Boyle, M. Banck, C.A. James, C. Morley, T. Vandermeersch, G.R. Hutchison,
Open Babel: An open chemical toolbox, J. Cheminf. 3 (2011) 33.
[40] A. Bujacz, Structures of bovine, equine and leporine serum albumin, Acta
Crystallogr, Sect. D: Biol. Crystallogr. 68 (2012) 1278–1289.
[41] G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug
binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824–832.
[42] M.J. Frisch, G.W. Trucks, H.B. Schlegel, Gaussian, lnc, Wallingford CT, 2009.
[43] E. Scrocco, J. Tomasi, Electrostatic molecular potential analysis of electron density
distribution in (ClAlMe2) 2 and (AlCl3) 2, Adv Quantum Chem. 11 (1978) 115–121.
[44] M. Govindarajan, S. Periandy, K. Carthigayen, FT-IR and FT-Raman spectra, thermo
dynamical behavior, HOMO and LUMO, UV, NLO properties, computed frequency
estimation analysis and electronic structure calculations on α-bromotoluene,
Spectrochim Acta, Part A 97 (2012) 411–422.
19] S. De, B. Sarkar, G.R. Jadhav, S.K. Ramasamy, S. Banerjee, A. Moorthy, P. Paira,
S.K. Ashok Kumar, Experimental and Theoretical Study on the Biomolecular
Interaction of Novel Acenaphtho Quinoxaline and Dipyridophenazine Analogues,
ChemistrySelect 3 (2018) 10593–10602.
20] R. Selva Kumar, S.K. Ashok Kumar, K. Vijayakrishna, A. Sivaramakrishna,
C.V.S.R. Brahmmananda, N. Sivaraman, S.K. Sahoo, Development of the smart-
phone-assisted colorimetric detection of thorium by using New Schiff’s base and its
applications to real time samples, Inorg. Chem. 57 (2018) 15270–15279.
21] S. De, S. R. Chaudhuri, A. Panda, G. R. Jadhav, R. S. Kumar, P. Manohar, N.
Ramesh, A. Mondal, A. Moorthy, S. Banerjee, S. K. Ashok Kumar, P. Paira,
Synthesis, characterisation, molecular docking, biomolecular interaction and cy-
totoxicity studies of novel ruthenium (II)–arene-2-heteroarylbenzoxazole com-
plexes, New J. Chem., 43 (2019) 3291-3302.
[45] R.G. Pearson, Absolute electronegativity and hardness: applications to organic
chemistry, J. Org. Chem. 54 (1989) 1423–1430.
[46] J. Padmanabhan, R. Parthasarathi, V. Subramanian, P.K. Chattaraj, Electrophilicity-
based charge transfer descriptor, J. Phys. Chem. A 111 (2007) 1358–1361.
[47] R. Parthasarathi, J. Padmanabhan, V. Subramanian, U. Sarkar, B. Maiti,
P. Chattaraj, Toxicity analysis of benzidine through chemical reactivity and se-
lectivity profiles: a DFT approach, Internet Electronic J. Molecular Des. 2 (2003)
798–813.
[
[
22] K. Singh, S. Banerjee, A.K. Patra, Photocytotoxic luminescent lanthanide complexes
of DTPA–bisamide using quinoline as photosensitizer, RSC Adv. 5 (2015)
[48] S. Deep, J.C. Ahluwalia, Interaction of bovine serum albumin with anionic surfac-
tants, Phys. Chem. Chem. Phys. 3 (2001) 4583–4591.
1
07503–107513.
23] S. Dasari, A.K. Patra, Luminescent europium and terbium complexes of dipyr-
idoquinoxaline and dipyridophenazine ligands as photosensitizing antennae:
structures and biological perspectives, Dalton Trans. 44 (2015) 19844–19855.
24] J. Keizer, Nonlinear fluorescence quenching and the origin of positive curvature in
Stern-Volmer plots, JACS 105 (1983) 1494–1498.
[49] S. Sonu, S. Halder, S. Kumari, R. Aggrawal, V.K. Aswal, S.K. Saha, Study on inter-
actions of cationic gemini surfactants with folded and unfolded bovine serum al-
bumin: effect of spacer group of surfactants, J. Mol. Liq. 243 (2017) 369–379.
[50] J.M. Khan, A. Ahmed, S.F. Alamery, M.A. Farah, T. Hussain, M.I. Khan, R.H. Khan,
A. Malik, S. Fatima, P. Sen, Millimolar concentration of sodium dodecyl sulfate
inhibit thermal aggregation in hen egg white lysozyme via increased α-helicity,
Colloids Surf. 572 (2019) 167–173.
[
[
25] M. Shamsi-Sani, F. Shirini, M. Abedini, M. Seddighi, Synthesis of benzimidazole and
quinoxaline derivatives using reusable sulfonated rice husk ash (RHA-SO 3 H) as a
green and efficient solid acid catalyst, Res. Chem. Intermed. 42 (2016) 1091–1099.
11