The effect of metal and substituent on DNA binding, cleavage activity, and cytotoxicity of new synthesized Schiff base ligands and Zn(II) complex

Article abstract of DOI:10.1016/j.molstruc.2017.06.137

New water soluble Schiff base ligands [N,N?-bis{5-[(triphenylphosphonium percholorate)-methyl]salicylidine}-1,3-diamino-2-propanol] (L₁) and [N,N?-bis(salicylidine)-1,3-diamino-2-propanol] (L₂) and zinc (II) complex of L₁: [N,N?-bis{5-[(triphenylphosphonium percholorate)-methyl]salicylidine}-1,3-diamino-2-propanol]Zn(II) were synthesized and characterized by elemental analysis, FT-IR, ¹HNMR and UV–Vis spectroscopy. In vitro DNA binding of the compounds were investigated by UV–Vis absorption spectroscopy, viscosity measurement, cyclic voltammetry, fluorescence spectroscopy, and gel electrophoresis. The present study aimed to investigate the effect of metal and substituent on DNA binding, cleavage activity and cytotoxicity of new synthesized Schiff base ligands and Zn(II) complex. The order of DNA binding affinity (K_b) calculated from the absorption spectroscopy was: ZnL₁ > L₂ > L₁. Molecular docking studies explore more details on the mode of binding and binding energies. Although the compounds revealed strong DNA binding affinity but electrophoresis studies don't show any effects on the DNA structure and single or double strand breaks. The cytotoxicity experiments against human Hepatoma (HepG2) showed the order: L₁ > ZnL₁ > L_2.

Full text of DOI:10.1016/j.molstruc.2017.06.137

Accepted Manuscript
The effect of metal and substituent on DNA binding, cleavage activity, and cytotoxicity
of new synthesized Schiff base ligands and Zn(II) complex
Zahra Asadi, Neda Nasrollahi
PII:
S0022-2860(17)30918-3
10.1016/j.molstruc.2017.06.137
MOLSTR 24023
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 October 2016
Revised Date: 2 June 2017
Accepted Date: 30 June 2017
Please cite this article as: Z. Asadi, N. Nasrollahi, The effect of metal and substituent on DNA binding,
cleavage activity, and cytotoxicity of new synthesized Schiff base ligands and Zn(II) complex, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.06.137.
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ACCEPTED MANUSCRIPT
The effect of metal and substituent on DNA binding, cleavage activity, and
cytotoxicity of new synthesized Schiff base ligands and Zn(II) complex
∗
Zahra Asadi , Neda Nasrollahi
Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz 71454, Iran
Abstract
New water soluble Schiff base ligands [N,Nʹ-bis{5-[(triphenylphosphonium percholorate)-
methyl]salicylidine}-1,3-diamino-2-propanol] (L ) and [N,Nʹ-bis(salicylidine)-1,3-diamino-2-
1
propanol] (L ) and zinc (II) complex of L : [N,Nʹ-bis{5-[(triphenylphosphonium percholorate)-
2
1
methyl]salicylidine}-1,3-diamino-2-propanol]Zn(II) were synthesized and characterized by
1
elemental analysis, FT-IR, HNMR and UV–Vis spectroscopy. In vitro DNA binding of the
compounds were investigated by UV-Vis absorption spectroscopy, viscosity measurement,
cyclic voltammetry, fluorescence spectroscopy, and gel electrophoresis. The present study aimed
to investigate the effect of metal and substituent on DNA binding, cleavage activity and
cytotoxicity of new synthesized Schiff base ligands and Zn(II) complex. The order of DNA
binding affinity (K ) calculated from the absorption spectroscopy was: ZnL > L > L .
b
1
2
1
Molecular docking studies explore more details on the mode of binding and binding energies.
Although the compounds revealed strong DNA binding affinity but electrophoresis studies don’t
show any effects on the DNA structure and single or double strand breaks. The cytotoxicity
experiments against human Hepatoma (HepG2) showed the order: L
1 1
> ZnL > L2.
Key words: Metal-based drugs, Zn(II), Schiff base, DNA.
1
. Introduction
Inorganic chemistry offers a wide area for the design of many metal-based drugs [1]. Existence
of the metal in many anticancer drugs makes them more effective in reacting with cancer cells.
To explore the medicinal application of metal-based anticancer drugs one important point is
minimization the unfavorable side effects of the drugs. Cisplatin (with many undesirable side
*
Corresponding author. E-mail address: zasadi@shirazu.ac.ir
Tel: +98 713 6137118; Fax: +98 713 646 0788
1

ACCEPTED MANUSCRIPT
effects) was an important anticancer drug for some decades thus designing other metal-based
drugs with lesser side effects is an active area of research [2]. One important step toward rational
drug design is studying the interaction of small molecules with DNA which is the subject of
interest in many research fields such as biochemistry, medicinal chemistry, life science, cancer
therapy and etc [2-5].
Schiff bases are important in biochemistry as antibacterial, antifungal and antitumor agents [6].
Transition metal Schiff base complexes can be studied to mimic the active sites of biologically
important molecules [7]. The interactions of Schiff bases metal complexes with
Deoxyribonucleic acid (DNA) have been studied extensively [8-11]. To study the interaction, it
is necessary that complexes be stable and inert in biological environment and be water-soluble
[12,13]. Usually, water solubility has been increased by functionalizing the ligand by polar or
charged groups. For example phosphonium group can increase the solubility of complexes in
water [14,15].
Metal-based drugs unlike the organic-based drugs can bind to DNA through both covalent and
non-covalent interactions. Non-covalent binding for both types of drugs contains electrostatic
interactions, hydrophobic interactions with minor and major grooves, hydrogen bonding, and
stacking interactions of aromatic heterocyclic groups and the base pairs [16-18]. Recognition the
type of interactions between drugs (metal complexes) and DNA is of great importance [19]. For
example two modes of actions are reported for the metal complexes containing aromatic ligands
in binding with DNA. I) Covalent binding contains coordination with the metal center, and II)
non-covalent interaction such as intercalation with the DNA base pairs or groove binding [20-
2
3].
In drug-DNA interactions, identifying the mechanism of interactions between drug molecules
and DNA is an important step specially to design new DNA-targeted drugs [24].
Taking into consideration the effect of metal and triphenylphosphine substituted groups on the
binding affinity and binding mechanism of the compounds with DNA, in this work, we report
synthesis of water-soluble Schiff base ligands, [N,Nʹ-bis{5-[(triphenylphosphonium
percholorate)-methyl]salicylidine}-1,3-diamino-2-propanol] (L ) and [N,Nʹ-bis(salicylidine)-1,3-
1
diamino-2-propanol] (L
2
) and Zn(II) complex of L
1
(ZnL
1
). These compounds were
1
characterized by various techniques, e.g., elemental analyses and spectral (FT-IR, HNMR, UV-
Vis) techniques. The binding property and mode of binding of the complexes with Fish DNA
2

ACCEPTED MANUSCRIPT
under physiological conditions were studied using UV-Vis spectrophotometry, viscosity
measurements, fluorescence spectroscopy, and molecular docking. Also, agarose gel
electrophoresis to study the mode of binding of the complexes with plasmid DNA and MTT
assay for anticancer activity measurements on HepG2 cell line was performed. The ligand and
+
Zn complex which contain PPh groups can interact by electrostatic attraction of positive charge
3
with the phosphate backbone of DNA. Also, phenylene rings and diamines which were used in
designing the ligands accelerate intercalation of the compounds and also caused hydrogen
binding between N and O atoms of the compounds and DNA interior. Zn(II) complex revealed
the more DNA binding propensity than free ligands which confirmed the effectiveness of metal-
based drugs. The gel electrophoresis study revealed that different concentrations of compounds
2 2
in the absence and presence of H O cannot cleave single or double strand DNA. The cell
viability of the cancerous cell lines in various concentrations of compounds showed cytotoxic
activities of them.
2
2
. Experimental
.1. Analytical instruments
All of the scanning UV-Vis measurements were carried out on a Perkin-Elmer (Lambda 2)
1
UV-Vis spectrophotometer equipped with a LAUDA ecoline RE 104 thermostat. The HNMR
spectra were recorded by Bruker Avance DPX 250 MHz spectrometer. FT-IR spectra were
recorded using Shimadzu FT-IR 8300 infrared spectrophotometer. Elemental microanalyses
(C.H.N.) were obtained using a CHN Thermo-Finnigan Flash EA1112. Fluorescence spectra
were carried out on a Perkin Elmer (LS-45). BUCHI 535 instrument was used to obtain the
melting point of the compounds. Viscosity measurements were carried out using an Ostwald
microviscometer. Electrochemistry studies were obtained using Auto lab 302N instrument.
2
.2. Materials
Zinc(II) nitrate tetrahydrate, 1,3-diamino-2-propanol, salicylaldehyde, tri-phenylphosphine,
sodium perchlorate, paraformaldehyde, conc. HCl, 0.5% NaHCO solution, acetonitrile, ethanol,
3
6
methanol, dueterated-dimethylsulfoxide (DMSO-d ), ethylene diamine tetra acetic acid (EDTA),
and potassium bromide (KBr) were purchased from Merck, Fluka, Acros and Aldrich. Agarose
gel was obtained from Sangon (Shanghai) Biotechnology. Fish Deoxyribonucleic acid obtained
from New England Biolabs (Beijing) Ltd. Tris-HCl-NaCl buffer solution (TBS, 1 mM Tris, 5
mM NaCl, pH 7.4) was used for FS-DNA binding experiments and Tris buffer (1 mM Tris-HCl,
3

ACCEPTED MANUSCRIPT
pH 8.2, and 1 mM EDTA) for gel-electrophoresis experiments. All reagents were used as
received and solvents were purified by the standard methods.
2
2
.3. Synthesis and characterization
.3.1. Preparation of 3-formyl-4-hydroxybenzyl-triphenylphosphoniumchloride
A mixture of salicylaldehyde (8.5 mL, 80 mmol), paraformaldehyde (1.5 gr, 50 mmol) and 50
ml of conc.HCl was stirred at room temperature for 48h. The resulting dark red powder
precipitate was collected by filtration, washed with 0.5% NaHCO solution then water, till
3
neutral, and dried in vacuum. The product was crystallized from acetonitrile and petroleum ether.
A mixture of 5-chloromethyl salicylaldehyde (10.2 g, 60 mmol) and triphenylphosphine (15.7 g,
6
0 mmol) in 200ml acetonitrile was refluxed for 6 h and cooled. The precipitated phosphonium
salt was filtered off and washed with ether (Scheme 1).
ο
Yield > 70%; m.p. >250 C; F.W: 432.69, Color: white, Anal. calcd for C H PO Cl : C, 72.22;
26
22
2
–
1
H, 5.12%. Found: C, 72.57; H, 5.24%. FT-IR (KBr, cm ): 3446 (ν ), 2873 (νC-H), 1672 (νC=O).
OH
1
HNMR (250MHz, DMSO-d
6
, room temperature): δ(ppm) = 11.60 (s, 1H, OH), 10.63(s,
1
H,CHO), 8.37-7.41 (m, 18H, ArH) 5.66 (d, 2H, H C-P),.
2
2
.3.2.
Preparation
of
[N,Nʹ-bis{5-[(triphenylphosphonium
percholorate)-
methyl]salicylidine}-1,3-diamino-2-propanol] (L1)
To a vigorously stirred solution of 3-formyl-4-hydroxybenzyl-triphenylphosphoniumchloride
0.433g, 1mmol) in 20 mL ethanol, an ethanolic solution (20mL) of 1,3-diamino-2-propanol
0.046g, 0.5mmol) was added dropwisely. The solution turned to yellow and the mixture refluxed
(
(
4 2
for 4 h, then NaClO .H O (0.240g, 1mmol) was dissolved in a minimum amount of water (5 mL)
and added. The resulting yellow powder was collected by filtration after evaporate of solvent to
the volume of 10 mL, washed with cold ethanol and ether and dried in the air (Scheme 1).
Yield > 70%.; m.p >180°C; F.W: 1047.85, Color: yellow. Anal. calcd for C55
H
50
N
2
Cl
2
O
11
P
2
: C,
–
1
6
3.04; H, 4.80; N, 2.67%. Found: C, 62.69; H, 4.39; N, 2.43%. FT-IR (KBr, cm ): 3447 (ν ),
OH
-
2
904 (νC-H), 1632, (νC=N), 1494 (νC=C), 1095, 622 (νClO4 ). UV–Vis (H O): λmax(nm) = 390, 327.
2
1
HNMR (250 MHz, DMSO-d6, room temperature): δ (ppm) = 13.62 (s, 2H, OH), 8.19(s, 2H,
HC=N), 7.85-6.70 (m, 36H, ArH), 5.21(s, 1H, CHOH), 5.14, 5.08 (d, 4H, CH P), 3.90(s, 1H,
2
CHOH), 3.69(d, 2H, CH ), 3.51(d, 2H, CH ).
2
2
2
.3.3. Preparation of [N,Nʹ-bis(salicylidine)-1,3-diamino-2-propanol]
(L )
2
4

ACCEPTED MANUSCRIPT
To a vigorously stirred ethanolic solution of salicylaldehyde (0.106 mL, 1 mmol) an ethanolic
solution (20 mL) of 1,3-diamino-2-propanol (0.046 g, 0.5 mmol) was added dropwisely. The
solution turned to light yellow and the mixture refluxed for 4 h. The resulting yellow powder was
collected by filtration, washed with cold ethanol and ether and dried in the air (Scheme 1).
L was synthesized in order to compare with L from the point of view of the effect of
2
1
substituent on DNA-binding measurements, cleavage activity, and cytotoxicity.
ο
Yield >70%; m.p. >250 C; F.W: 298.20, Color: yellow. Anal. calcd for C H N O : C, 68.44;
17
18
2
3
–
1
H, 6.08; N,9.39%. Found: C, 68.04; H, 5.73, N, 9.56%. FT-IR (KBr, cm ): 3394 (ν ), 2893 (ν
OH
C-
1
H
), 1635 (νC=N). UV–Vis (H
2
O): λmax (nm) = 386, 324. HNMR (250 MHz, DMSO
-
d
6
, room
temperature): δ(ppm) = 13.52 (s, 2H, OH), 8.50 (s, 2H, HC=N), 7.42-6.85 (m, 8H, ArH), 5.17(s,
H, CHOH), 3.97(s, 1H, CHOH), 3.76(d, 2H, CH ), 3.57(d, 2H, CH
.3.4. Preparation of
[N,Nʹ-bis{5-[(triphenylphosphonium
1
2
2
)
2
percholorate)-
methyl]salicylidine}-1,3-diamino-2-propanol]Zn(II)
a. Template method
To a vigorously stirred solution of zinc(II)nitrate-tetrahydrate (0.261g, 1 mmol) in 20 mL
ethanol, a solution of (3-formyl-4-hydroxybenzyl)triphenylphosphoniumchloride (0.866 g, 2
mmol) in l0 mL ethanol was added and then an ethanolic solution (5 mL) of 1,3-diamino-2-
propanol (0.046 g, 0.5 mmol) was added dropwisely. The solution turned to yellow and the
4 2
mixture refluxed for 3h then NaClO .H O (0.240 g, 2 mmol) was dissolved in a minimum
amount of water (5 mL) and added. The resulting light yellow powder was collected by filtration
after evaporate of solvent to the volume of 10 mL, washed with cold ethanol and ether and dried
in the air (Scheme 1).
b. Non template method
To a vigorously stirred solution of zinc(II)nitrate-tetrahydrate (0.261g, 1 mmol) in 20 mL
ethanol, a solution of L (0.109 g, 2 mmol) in l0 mL ethanol was added. The solution turned to
1
4 2
yellow and the mixture refluxed for 3h then NaClO .H O (0.240 g, 2 mmol) was dissolved in a
minimum amount of water (5 mL) and added. The resulting light yellow powder was collected
by filtration, washed with cold ethanol and ether and dried in the air (Scheme 1).
Yield:75%; m.p>180°C; F.W:1147.25, Color: light yellow; Anal. calcd for
C
55
H
48
N
2
Cl
2
O11ZnP
2
.2H
2
O: C, 57.58; H, 4.57; N, 2.44%. Found: C, 57.09; H, 4.39; N, 2.04%.
–
1
-
FT-IR (KBr, cm ): 3433 (ν ), 2895 (νC-H), 1626 (νC=N), 1479 (νC=C), 1110, 689 (νClO4 ), 623
OH
5

ACCEPTED MANUSCRIPT
1
(ν
M-O), 569 (νM-N). UV–Vis (H
2
O): λmax(nm)=364. HNMR (250 MHz, DMSO-d
6
, room
temperature): δ (ppm) =7.92, (s, 2H, HC=N), 7.89-6.33 (m, 36H, ArH), 5.10(s, 1H, CHOH),
4
.93, 4.88 (d, 4H, CH P), 3.93(s, 1H, CHOH), 3.85(d, 2H, CH ), 3.48(d, 2H, CH ).
2
2
2
a)
H
Cl
O
OH
HCl(l)
OH
PPh3
+
OH(CH O)nH
Cl-
OH
2
stirre/48h
acetonitrile
+
3
Ph P
O
O
b)
ClO -
4
ClO -
4
OH
H
OH
H
O
H
O
Cl-
+
Etoh/ref
+
+
PPh3
Ph3P
+
3
NaClO4
N
Ph P
NH2 NH2
N
O
L1
HO
H
c)
H
H
O
OH
H
O
H
O
+
Etoh
ref
OH
NH2 NH2
L2
HO
H
d)
H
OH
OH
ClO4-
ClO4-
Cl-
O
O
N
NH2 NH2
ref /Na
+
+
+
3
PPh3
Zn(NO ) .4H O
Ph3P
Zn
Ph P
+
3 2
2
ClO4-
N
O
Etoh
ZnL1
HO
H
e)
ClO4-
-
ClO
4
Etoh
L1
+
Zn(NO
3
)
2
.4H
2
O
O
O
+
ref /Na ClO-
+
4
PPh
3
Ph3P
Zn
N
N
ZnL1
HO
H
Scheme 1 synthetic route of a) (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride b) L
1
C) L and d) & e) ZnL1
2
2
.4. DNA-binding measurements
6

ACCEPTED MANUSCRIPT
The interaction of Zn(II) complex and Schiff base ligands with FS-DNA was studied by
absorption spectroscopy, fluorescence spectroscopy, gel electrophoresis, viscosity measurement,
cyclic voltammetry (CV). Absorption titration was performed using Perkin-Elmer (LAMBDA 2)
UV-Vis spectrophotometer. All absorption spectrophotometric measurements were performed in
ο
thermostated quartz sample cells at 25 C. FS-DNA dissolved in buffer solution and the
concentration of FS-DNA was determined from its absorption intensity at 260 nm with a molar
−
1
−1
extinction coefficient of 6600M cm . The ratio of UV absorbance, A /A was 1.9, indicated
2
60 280
that the DNA was sufficiently free from protein [25]. The stock solutions of complex and ligands
3
(
1.0×10 µM) were prepared by dissolving an appropriate amount of the compounds in 10 mL
DMSO/Tris–HCl buffer (1:9 v/v) solution. These solutions were diluted to 200µM with Tris–
HCl buffer for UV-Vis spectroscopy. No precipitation or turbidity was observed in solutions
during a week and the UV-Vis spectra of the compounds did not change. The solutions of Zn(II)
complex and Schiff base ligands(L and L ) with the fixed concentration were titrated by FS-
1
2
1 1 2
DNA in Tris–HCl buffer (pH=7.4). Fluorescence spectra of ZnL , L and L were recorded at
ο
2
5 C. The stock solutions of compounds were diluted to 50µM with Tris–HCl buffer for
fluorescence spectra and titrated with FS-DNA (0 to 458 µM). The concentration of compounds
was fixed and the changes in fluorescence intensities at 450- 494 nm (excited at 350-390 nm)
were studied.
ο
Viscosity measurements were carried out at constant temperature (25.0±0.5 C). The
concentration of FS-DNA was 100 µM, and flow time was measured with a digital stopwatch.
The mean values were used to evaluate the viscosity η of the samples. The values for relative
specific viscosity (η/η ), where η and η are the specific viscosity contributions of DNA in the
o
o
absence (η ) and in the presence of the complex (η), were plotted against r (r =
o
i
i
[complex]/[DNA] = 0, 0.2, 0.4, 0.6, 0.8 and 1).
Cyclic voltammetry of ZnL , L and L were carried out in the potential range from -1.5V to
1
1
2
1
.5V. 10 mL DMSO/Tris–HCl buffer (1:9 v/v) solution of compounds with fixed concentration
3
4
(
1×10 µM) in the cell were titrated by FS-DNA and in each injection 10µL DNA (1.0×10 µM)
was injected. A three-electrode cell, the glassy carbon electrode (GCE, Φ=3 mm) as working
electrode, an Ag/AgCl (KCl) as reference electrode and a Pt wire as counter electrode was used.
2
.5. DNA cleavage studies
7

ACCEPTED MANUSCRIPT
DNA cleavage activity of the compounds was studied using gel electrophoresis technique.
Briefly, circular pBluescript II KS (+) plasmid DNA was incubated with different concentrations
of the compounds in PBS buffer pH~7.4 for 1 h at 37 °C, followed by addition of the loading
buffer containing 0.25% bromophenol blue, 0.25% xylene xyanol FF and 60% glycerol (3 ꢀL).
Experiments were done under both physiological condition and oxidative condition by adding
H
2
O
2
(50 µM). The samples were then analyzed by 1% agarose gel electrophoresis applying
Tris-acetic acid-EDTA (TAE) buffer, pH = 8.2 for 50 min, 50 V. The gel was stained with 0.5
g/mL ethidium bromide and visualized by UV light and photographed for analysis. The
cleavage was measured by determining the ability of the compounds to convert the supercoiled
SC) DNA to the nicked circular (NC) form and/or the linear circular (LC) form (cleavage), or
change in speed of electrophoretic movement, or lack of staining by ethidium bromide
substitution by the compounds).
ꢀ
(
(
2
.6. Cell viability assay
2
The cancerous cell line were cultured in a humidified atmosphere containing 5% CO at 37 °C
−
1
−1
in DMEM high medium supplemented with 100 units mL penicillin, 100 ꢀg mL streptomycin
and 10% fetal bovine serum. Cell viability assay on HepG2 (heptoma cell line) performed by
MTT method (Riss et al. 2013). Briefly, cells seeded in 96 well plates and incubated for 24 hours
o
at 37 C in the presence of 5% CO , then a defined number of cells exposed to the synthesized
2
compounds in different concentrations from 10 to 200 ꢀM in a period of 48 and 72 hours.
Afterward, 10 ꢀL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution
per well was added to achieve a final concentration of 0.45 mg/ml and incubated for another 4
hours at 37°C. Finally, 100 ꢀL solubilization solution (40% (vol/vol) dimethylformamide (DMF)
in 2% (vol/vol) glacial acetic acid and 16 % sodium dodecyl sulfate (SDS), pH ̴ 4.7) was added
to each well to dissolve formazan crystals and absorbance was recorded at 570 nm using Biotech
ELX808 96 well reader. Cell viability percentage calculated as follow:
Cell viability % = [A570 sample / A570 control] * 100 %
2
.7. Theoretical calculations and molecular docking
8

ACCEPTED MANUSCRIPT
The Gaussian 03 software package was used to all geometry optimization of compounds. The
complexes were characterized by means of density functional theory (DFT) calculations
(B3LYP) model and all structures were fully optimized in the gas phase. LANL2DZ was used
for the zinc metal centre while for rest of the atoms the 6-311G* standard basis set was
employed [26,27].
Molecular docking studies were carried out using the Molegro Virtual Docking (MVD)
software. This software generates the best DNA-ligand configurations according to several
scoring criteria such as Moldock and Rerank scores. In MVD the units are arbitrary, but an ideal
hydrogen bond contributes to the overall energy [28]. The crystal structure of DNA, PDB code:
4
HC9 d(TTTTTGGCCCCCAAAAAAAA) was taken from the protein Data bank
www.rsbs.org/pdb) [29]. Water and protein molecules in this structure were removed before
performing docking calculations. Then, the optimized structure of ZnL , L , L and DNA were
(
1
1
2
imported to Molegro Virtual Docker (MVD) [30] to carry out the docking simulations. Bonding
site was chosen by (x=8, y=-107, z=50, and radius=28). We have selected score as Moldock
score [GRID], with GRID resolution of 0.3 Å. Algorithm selected for docking was Moldock
along with number of runs as 20 for each docking calculation, 10 different poses were requested.
All other parameters were kept at their default values. Parameter settings, pose generation and
simplex were evolution selected as default settings.
3
. Results and discussion
3
.1. General information
The complex and ligands were stable at ambient temperature and were not light sensitive and
their solutions were stable for a long period of time in the air. The complex was soluble in water,
DMSO (dimethylsulfoxide) and DMF (dimethylformamide). The ligands were soluble in water,
DMSO (dimethylsulfoxide), DMF (dimethylformamide), ethanol and methanol. In the FT-IR
-1
spectrum of the aldehyde a broad strong intensity band at 3446 cm was assigned to O-H
vibration. The complex, L and L were characterized by a strong band due to ν at 1626,
1
2
(C=N)
-
1
-1
1
6
3
632 and 1635cm , respectively. The bands around 1110, 689cm for the complex and 1095,
-1
-
22cm for L were assigned to the ClO groups. The broad bands of the O-H were observed at
1
4
-1
433, 3447 and 3394 cm for the complex, L and L , respectively. The medium–weak bands at
1
2
-1
the lower frequencies (623 and 569cm ) have been assigned to the (M–N) and (M–O) vibrations
1
of the complex, respectively [31-33]. In the HNMR spectra of L and L , bands of the O-H were
1
2
9

ACCEPTED MANUSCRIPT
1
observed at13.62 and 13.52 ppm respectively. In the HNMR spectra of the complex, L
1
and L
2
,
the signal of aromatic hydrogens were observed in the 7.89-6.33, 8.37-7.41 and 7.42-6.85 ppm
range, respectively. Signals of the imine protons for all the compounds were observed in 7.92-
8
.50 ppm range. A doublet due to CH
2
P hydrogens appeared at 4.88-5.14 ppm region in the
complex and L The aliphatic alcoholic proton appeared in 5.10-5.21 ppm range for the
1
.
complex, L
1
and L2. The singlet appearing at 3.93, 3.90 and 3.97 ppm were assigned to CHOH of
the complex, L and L , respectively [34-36]. Electronic spectral data of the compounds in water
1
2
are given in the experimental part (Fig. 1). The bands around 300-400 nm of the ligands were
attributed to intramolecular π-π* and n-π* transition in aromatic rings or imines groups. Due to
the coordination of nitrogen of imines to the metal, the n-π*band was emitted for the complex.
The band around 390 nm of complex was attributed to intramolecular π-π* in aromatic rings
[37,38].
Fig. 1 A comparison of the electronic spectrum of ZnL , L and L
2
1
1
3
.2. Electronic absorption titration
1
0

ACCEPTED MANUSCRIPT
Electronic absorption spectroscopy is one of the most useful techniques to examine the
binding mode and binding affinity of metal complexes with DNA [39,40]. In this regard, by
monitoring the change in absorption intensity, the binding constant (K ) and binding site size (s)
b
of the complex and ligands were determined. Spectrophotometric titration of the complex and
ligands with FS-DNA in Tris-HCl buffer is shown in Fig. 2. With increasing the concentration of
1
DNA, the bands around 386 nm and 327 nm in L spectra exhibit both hypochromism and
hyperchromism, respectively. An isosbestic point around 375 nm was appeared and the band
around 386 nm indicated blue shift (4nm). Spectral changes for L were the same as L it means
2
1
that hypochromism with a slight red shift (1nm) and hyperchromism were observed. With
increasing the concentration of DNA in the solution of ZnL , a new band was emerged, and two
1
isosbestic points were appeared about 342 and 436 nm. Furthermore, a strong hypochromism
with red-shift and hyperchromism with blue shift was revealed in the absorption spectra. This
observations suggested that new species was generated after interaction of complex with DNA
[41].The overall spectral changes with isosbestic points also suggested a strong interaction.
Hypochromism along with hyperchromism describe the mixed modes of interaction for the
complex and ligands with FS-DNA. Hypochromism with red or blue shift indicated that the
mode of binding to DNA was intercalation, involving partial insertion of the aromatic ring
between the base pairs of DNA [42]. By decreasing the distance between the intercalated
compound and DNA bases (after binding) hypochromism was observed because of the strong
π→π* stacking interaction between aromatic chromophore of the ligand and the base pair of
DNA [43]. Hyperchromism may be indicative of groove binding and hydrogen binding between
N and O of the ligand and DNA interior or the electrostatic attraction between the positive
charge of the compound and the backbone phosphate groups which reflect the changes of
2
conformation and structure of DNA after the interaction [19]. Lower hypochromism of L can be
+
attributed to the absence of electrostatic attraction between the positive charge of PPh and the
3
backbone phosphate groups in comparing with L and ZnL . Metal center in Zn complex caused
1
1
more planer structure and increase the interaction between aromatic chromophore of ZnL
the base pair of DNA
1
and
.
1
1

ACCEPTED MANUSCRIPT
Fig. 2 Spectrophotometric titration L
0 to 458 µM of DNA)
1 2 1
(a), L (b) and ZnL (c) with FS-DNA in Tris–HCl buffer,
(
Investigation of the binding strength, the intrinsic binding constant, K , and the binding site
b
size (s) were determined by using equation (1a ,1b) [44,45]:
ꢇ
ꢌ
ꢌ
ꢀꢁ
ꢁ ꢅꢈꢀꢁ ꢁ ꢇꢅ = ꢉꢊ − ꢋꢀꢊ − 2ꢍ ꢎ ꢐꢑꢒꢓꢔ⁄ꢕꢅꢖꢈ2ꢍ ꢎ
ꢆꢇ ꢏ
(1a)
(1b)
ꢂꢃ ꢄ
ꢆꢃ ꢄ
ꢆ
ꢏ
ꢊꢇ = 1 + ꢍ ꢎ + ꢍ ꢐꢑꢒꢓꢔ ∕ 2ꢕ
ꢆꢇ
ꢏ
ꢆ
Where ε , ε and ε are apparent absorption coefficient, ε is the extinction coefficient
a
b
f
a
f
observed for the absorption band at a given DNA concentration, ε is the extinction coefficient of
the complexes in its free form and ε of the complex in the fully bound form, respectively. C is
b
t
1
2

ACCEPTED MANUSCRIPT
the total concentration of the metal complexes and s is the binding site size. Plots of
(
ε −ε )/(ε −ε ) versus [DNA] are shown in Fig. 3.The intrinsic binding constant, K , obtained for
a
f
b
f
b
5
5
5
-1
L L and ZnL were, 3.0×10 , 4.2×10 and 9×10 M respectively. It has been found that
1
,
2
1
complex strongly bound with DNA relative to ligands and the order of binding affinity is ZnL
1
>
L > L For Zn complex more planar structure than the free ligand, increased the intercalation
2
1.
+
b 3
propensity and consequently increased the K value. The absence of PPh substituted groups on
L ligand decrease the steric hindrance of L thus it can insert between DNA base pairs more
2
2
deeply and effectively.
Fig. 3 Plot of (ε −ε )/(ε −ε ) versus [DNA] for the titration of L (a), L (b) and ZnL (c) with FS-
a
f
b
f
1
2
1
DNA.
1
3

ACCEPTED MANUSCRIPT
3
.2. Fluorescence spectral studies
Fluorescence spectroscopy is probably one of the techniques which most commonly used to
study the interaction between small molecules and DNA [46]. Two ligands and complex exhibit
strong emission bands with a maximum at 450-500 nm (excited at 350-400 nm) in Tris–HCl
buffer at room temperature. By increasing the concentration of DNA, emission band at about 494
nm obviously quenched and at about 390 nm slightly enhanced but the fluorescence spectra of L
2
showed only enhancement (Fig. 4). The fluorescence quenching and enhancing for ZnL and L
1
1
suggested the mixed modes of electrostatic and intercalation interaction [44]. The enhancement
of the fluorescence spectrum of L can be due to the penetrating of compound into the
2
hydrophobic environment inside the FS-DNA which prevents the quenching of spectrum by the
solvent water molecules [19].
Fig. 4 The effect of FS-DNA (0 to 458 µM) on the emission intensity of L (a), L (b) and ZnL
1
1
2
(c), in Tris–HCl buffer (excited at 350-400 nm)
1
4

ACCEPTED MANUSCRIPT
The Stern-Volmer plots of L
2
obtained by using the equation (2):
1 + ꢍ ꢐꢛꢔ = 1 + ꢍ ꢇꢝ ꢐꢛꢔ
(2)
ꢙꢚꢇ ꢜ ꢞꢇ
ꢗꢘ
ꢗ
=
Where
respectively. ꢍꢙꢚꢇ is the Stern−Volmer enhancing constant, [E] is the concentration of enhancer,
ꢍꢜ is the bimolecular enhancing constant and τ was the average lifetime of biomolecule in the
ꢟ and ꢟꢞ are the fluorescence intensities in the presence and the absence of enhancer,
0
−
8
absence of enhancer, and its value is 10 s [47,48]. The fluorescence enhancing curve of L by
2
FS-DNA (Fig. 5) illustrates a good agreement with the linear curve of the Stern–Volmer
−
1
equation and ꢍꢙꢚꢇ is calculated by the ratio of the slope to the intercept (ꢍꢙꢚꢇ =ꢇ404.55
M ). The
10
−1
-
bimolecular enhancing constant ( ꢜ) is calculated from ꢍꢙꢚꢇ= ꢍ ꢝ and for L is 4.04 ×10 M s
ꢍ
ꢜ ꢞꢇ
2
1
10
-1
.
q
According to equation (2), k is greater than the limiting diffusion rate constant (2.0×10 M
-1
s ) for a biomacromolecule, indicating the existence of a static enhancing mechanism for L . In
2
a static enhancing mechanism, the fluorophore and the enhancer form a complex in the ground
state [47].
Like to the enhancing process, the quenching constant can be obtained by the Stern–Volmer
equation (3):
ꢗꢘ
=
1 + ꢍ ꢐꢠꢔ = 1 + ꢍ ꢇꢝ ꢐꢠꢔꢇ
(3)
ꢙꢚꢇ
ꢡ
ꢞꢇ
ꢗ
Where [ꢠ] is the concentration of quencher, ꢍ is the bimolecular quenching constant. The
ꢡ
fluorescence quenching curve of L by FS-DNA (Fig. 5) illustrates a good agreement with the
1
linear curve of the Stern–Volmer equation and ꢍꢙꢚꢇ is calculated by the ratio of the slope to the
−
1
intercept (ꢍꢙꢚꢇ =ꢇ2157.53 M ). The bimolecular quenching constant (ꢍ ) is calculated from
ꢡ
1
1
−1 -1
ꢍ = ꢍ ꢝ and for L
1
is 2.16×10 M s indicating the existence of a static quenching
ꢙꢚꢇ
ꢡ ꢞꢇ
mechanism for L
1
. In a static enhancing mechanism, the fluorophore and the quencher form a
complex in the ground state [47].
1
The obtained Stern−Volmer plot for ZnL was nonlinear (Fig. 5). These results revealed the
existence of two modes of interaction, static and dynamic, between ZnL and DNA. Calculating
1
the Stern−Volmer constant (ꢍ ) and enhancing rate constant (ꢍ ) were not possible for the
ꢙꢚꢇ
ꢜ
nonlinear Stern−Volmer plots [49,50].
1
5

ACCEPTED MANUSCRIPT
Fig.5 The Stern–Volmer plots for L (a), L (b) and ZnL (c) at room temperature
1
2
1
To determine the strength of the interaction of ZnL and L with DNA, the values of the
1
1
binding constants (ꢍ ) were resulted from Scatchard equation (4) [51]:
ꢄ
ꢗ ꢃꢗ
ꢘ
log ꢢ ꢗ ꢣ = log ꢍ + ꢤ logꢐꢑꢒꢓꢔ
(4)
ꢄ
ꢗꢘꢃꢗ
The plot of log ꢢ ꢗ ꢣ versus logꢐꢑꢒꢓꢔꢇfor ZnL and L are given in Fig. 6. The values of ꢍ
1
1
ꢄ
5
-1
3
-1
for ZnL
1 1
and L were found to be 1.37×10 M , 7.44×10 M and n=1.56, 1.15 respectively. Due
to different fluorescence behavior of L comparing between ꢍ ofꢇꢇZnL and L with L is not
2
ꢄꢇ
1
1
2
evaluable. For some typical intercalators such as ethidium bromide and acridine orange the
6
-1
5
-1
binding constant has been reported 2.6 ×10 M and 4×10 M , respectively [52].ꢇꢍ for ZnL
ꢄ
1
comparable to that of acridine orange, therefore ZnL can have intercalation mode.
1
1
6

ACCEPTED MANUSCRIPT
ꢗꢘꢃꢗ
Fig.6 The plot of log ꢢ ꢗ
ꢣ
versus logꢐꢑꢒꢓꢔꢇfor L (a) and ZnL (b) at room temperature
1
1
3
.3. Cyclic voltammetry studies
In evaluating the mode of action and the binding strength of drug–DNA interaction cyclic
voltammetry (CV) is widely used. Cyclic voltammetry of ZnL , L and L in DMSO/Tris–HCl
1
1
2
buffer (1:9 v/v) solution showed oxidation waves in the sweep range from -1.5 V to +1.5 V (Fig.
). In the absence of DNA, all three compounds show anodic peaks around 0.6 V and 1.00 V and
7
cathodic peaks around -0.5 V. With increasing the concentration of DNA, decreasing in both the
anodic and cathodic peak currents was observed which suggested the binding of compounds to
DNA. For L and ZnL the anodic peak potential E revealed a positive shift while the cathodic
1
1
pc
peak potentials E showed a negative shift. The anodic peak potential of L showed a positive
pa
2
shift. This result revealed that ZnL and L bind to DNA by both intercalation and electrostatic
1
1
interaction and L bind to DNA by intercalation mode [19].
2
1
7

ACCEPTED MANUSCRIPT
Fig.7 Cyclic voltammograms of L (a), L (b) and ZnL (c) at room temperature
1
2
1
3
.4. Viscosity measurement
To further investigate the mode of binding of compounds with DNA, viscosity measurement
was carried out. Spectroscopic methods provide necessary evidence to the binding mode but they
are not sufficient. Hydrodynamic measurements which are sensitive to the change in length of
DNA must confirm the spectral evidences. Viscosity measurements are the most critical and least
ambiguous tests to evaluate the binding mode in solution [53]. A classical intercalation mode,
increase the viscosity of DNA, due to the increasing in separation of base pairs at the
1 2
intercalation sites and consequently an increasing in overall DNA length. Thus ZnL and L
indicated an intercalation mode of binding. In contrast the electrostatic binding, grooves binding,
or non-classical intercalation affect slightly on the viscosity of DNA [54,55]. According to the
1
results for L , the relative viscosity increased in low ratio but in high ratios no significant change
1
8

ACCEPTED MANUSCRIPT
1 0
was observed suggested both electrostatic and intercalation mode for L . The values of (ɳ/ɳ )
were plotted against [complex]/[DNA] for both ligands and complex as shown in Fig. 8.
2 2 1
Fig. 8 Effect of increasing amounts of L , L and ZnL on the viscosity of HS-DNA (100 µM) in
°
1
mM tris-HCl buffer (r = 0.0-0.3) at 25 C
3
.5. Results of theoretical calculations and molecular docking
The GAUSSIAN 03 program was used for optimization of ligands and complex. DFT method
with B3LYP functional, LANL2DZ basis set for metal centers and 6-311g* for other atoms were
used. The optimized geometries of the ligands and complex are shown in Fig. 9. Selected
geometrical parameters including bond lengths and bond angles of ligands and complex are
listed in Table 1. Results showed that all of the bond lengths and bond angles are in the normal
range.
Using the molecular docking technique is important in predicting the mode and affinity of
drug-DNA interactions [56]. Molecular docking studies of the complex and two ligands with
DNA were carried out in order to preliminarily predict the DNA-binding affinity as well as the
preferred orientation of the docking pose. The lower binding free energy predicted the more
binding affinity of the complexes to DNA. The negative values of binding free energy (Table 2)
of the docked complexes indicated that the binding of the complex and two ligands to the DNA
were spontaneous. The DNA binding affinity of complex is stronger than that of two ligands
1
9

ACCEPTED MANUSCRIPT
1 2 1
according to the binding energy: ZnL > L > L and as shown in Fig.10, the mode of interaction
was minor groove binding. The values of energy for different pose of compounds were collected in
Table 2.
a)
b)
Fig. 9 The optimized geometrical structures of compounds L (a), L (b) and ZnL (c)
1
2
1
2
0

ACCEPTED MANUSCRIPT
Table 1 Selected bond lengths in Aº and angle (º) by theoretical calculation at B3LYP method
for compounds
ZnL1
L2
L1
Bond length
1.26
Zn-N
Zn-N
Zn-O
2
2.103
2.085
1.952
1.954
C
C
C
C
C
24=N
2
1
N
1
2
= C
6
5
2.26
2.27
1.44
1.45
1.35
3
4
13=N
1.27
1.44
1.44
1.41
N
= C
29-N
28-N
2
C
C
C
7
-N
1
2
Zn-O15
2
8
-N
29-O35
22-O
3
Bond angles
N
2
-Zn-N
3
88.296
118.38
90.85
C
13-N
1
-C27
118.39
117.41
123.59
121.26
C
5
- C15-N
2
122.48
118.96
121.23
121.02
O -Zn-O
C -N -C
C -N - C
6 1 7
4
15
17
2
24
N -Zn-O
C -C -C
C - C -C
16 15 5
2
15
20
14
24
N -Zn-O4
89.87
C -C -C
C -C -C
23 6 1
3
13
7
6
a)
b)
c)
Fig. 10 Computational docking models (using the UCSF Chimera software) illustrating the mode
of interaction between DNA and compound a) model of docking of L b) L c) ZnL
2
1
1
2
1

ACCEPTED MANUSCRIPT
1 2 1
Table 2 The values of binding energy for L , L and ZnL
Pose
Binding Energy
Binding Energy
Binding Energy
(
Rerank Score)
L1
(Rerank Score)
(Rerank Score)
L2
ZnL1
-92.0976
-
101.512
-111.146
1
2
3
4
5
6
7
8
9
-81.2682
-66.1541
-77.3645
-61.1758
-64.2672
-53.8503
-44.8293
-67.36
-
100.83
100.463
101.706
95.2193
96.5064
96.0838
-90.2352
-87.6727
-73.3327
-80.2388
-78.7554
-78.8049
-75.5211
-75.2371
-76.6015
-
-
-
-
-
-
89.909
-90.2121
-46.135
-
90.4271
1
0
3
.6. Results of DNA cleavage study
The chemical nuclease activities of the compounds have been studied using supercoiled
pBluescript plasmid DNA in phosphate buffer saline (PBS), pH 7.4 in the absence and present
of oxidant agent like H O under physiological conditions. As shown in Fig. 11, although all
̴
2
2
compounds have strong DNA binding affinity based on spectroscopy studies, electrochemical
studies and molecular docking, different concentrations of compounds(100 and 200 ꢀM) alone
and in presence of H O doesn’t show any effects on the DNA structure and single or double
2
2
strand breaks.
2
2

ACCEPTED MANUSCRIPT
Fig. 11 Gel electrophoresis of plasmid DNA treated with the compounds. a) with
concentration 100ꢀM of compounds in physiological conditions or in presence of H O , Lane
2
2
1
+
7
(DNA + ZL ), lane 2 (DNA + ZL ) in presence of H O , lane 3 (DNA + L ), lane 4 (DNA
1
1
2
2
1
L
1
) in presence of H
2
O
2
, lane 5 (DNA + L
2
), lane 6 (DNA + L
2
2 2
) in presence of H O , lane
(DNA alone), b) with concentration 200ꢀM of compounds in physiological conditions or in
, lane 1 (DNA + ZL ), lane 2 (DNA + L ) lane 3 (DNA + L ) in presence of
H O , lane 4 ((DNA + L ), lane 5 (DNA + L ) in presence of H O and lane 6 (DNA alone).
presence of H
2
O
2
1
1
1
2
2
2
2
2
2
3
.7. Anticancer activity studies
According to Tables 3-5, the anticancer activities of compounds toward human HepG2 cancer
cell line have been examined by using MTT assay. It is found that the compounds exhibit
cytotoxic activities in a time-dependent experiment in 48 and 72 hours of incubation. The order
of cytotoxic activities of compounds was L > ZnL > L . Based on anticancer activity studies, L
1
1
2
2
had no significant effect on HepG2 cancerous.
Table 3 HepG2 cell viability treated with different concentrations of ZL
1
and
Cell viability (%)L1
48 hours 72 hours
L
1
Ligand
Cell viability(%)ZL1
48 hours 72 hours
Concentration
(ꢀM)
1
1
50
20
4
6
6
7
7
8
6.64 ± 1.14 22.84 ± 2.15 38.77 ± 1.28 32.385 ± 0.27
0.87 ± 0.94 44.71 ± 0.94 41.88 ± 0.88 35.64 ± 2.24
90
60
30
10
6.58 ± 4.24 50.85 ± 0.95
44.51 ± 2 40.29 ± 2.37
0.17 ± 0.97 55.86 ± 2.12 54.54 ± 1.85
1.74 ± 0.94 61.65 ± 2.54 68.08 ± 1.92
55 ± 4.67
70.29 ± 8
5.27 ± 4.04 75.86 ± 3.24 77.83 ± 2.69 80.16 ± 6.39
2
3

ACCEPTED MANUSCRIPT
Table 4 HepG2 cell viability treated with different concentrations of
2
L .
Ligand
Concentration
Cell viability (%)L2
8 hours
4
72 hours
(ꢀM)
2
1
1
00
60
20
88.65 ± 2.3
87.43 ± 4.56
83.66 ± 1.04
86.3 ± 5.67
80.72 ± 1.5
90.4 ± 2.54
51.02 ± 1.37
55 ± 2
78.33 ± 1.93
84.14 ±1.58
95.25 ± 2.65
88.59 ± 7.41
80
40
10
Table 5 Comparative IC50 Values of ZL1, L and L when tested on HepG2 and Cell Line after
1
2
4
8 and 72 hours.
IC ZL (µM)
72 hours
68.75 ± 3.23
IC50 L1(µM)
72 hours
65.75 ± 8.3
IC50 L2 (µM)
72 hours
198.1 ± 2.1
Not Valid!!
5
0
1
4
8 hours
48 hours
74.92 ± 0.06
48 hours
No
cytotoxicity
effect in the
range
1
26.8 ± 1.3
4
.
Conclusions
New water soluble Schiff base ligands (L &L ) and zinc (II) complex of L were synthesized
1
2
1
1
and characterized by elemental analysis, FT-IR, HNMR and UV–Vis spectroscopy. The DNA
binding propensities of ZnL , L and L were examined by UV-Vis absorption spectra, emission
1
1
2
spectra, viscosity, voltammetric techniques, molecular docking, and gelelectrophoresis. The
order of binding affinity was ZnL > L and L interacted by electrostatic attraction
and intercalation. By omitting phenyl rings in L the steric hindrances of the ligand decreased
1
2
> L1. ZnL
1
1
2
2
and intercalation of L between DNA base pairs occurred more effectively. The DNA docking
studies suggested that the compounds interacted in the minor groove of DNA. Agarose gel
electrophoresis study revealed that different concentrations of compounds alone and in presence
of H O didn’t effect on the DNA structure. Finally, the anticancer experiments for the treated
2
2
cancerous cell line with synthesized compounds showed that the order of cytotoxic activities of
compounds was L > ZnL
1
1
> L2.
The main results of the present study confirmed the metal and substituent effect of the
compounds on DNA binding affinity and anticancer activities which are useful for design and
synthesis of new metal based drugs
.
2
4

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Acknowledgement
We are grateful to Shiraz University Research Council for its financial support.
References
[1] C. Rajarajeswari, R. Loganathan, M. Palaniandavar, E. Suresh, A. Riyasdeen, M. A. Akbarsha,
Dalton Trans 42 (2013) 8347-8363.
[2] G. Barone, A. Terenzi, A. Lauria, A. M. Almerico, J. M. Leal, N. Busto, B. García, C. Chem.
Rev 257 (2013) 2848-2862.
[
[
3] B.-d. Wang, Z.-Y. Yang, T.-r. Li, Bioorg. Med. Chem 14 (2006) 6012-6021.
4] E. Meggers, Exploring biologically relevant chemical space with metal complexes. Curr. Opin.
Chem. Biol. 11 (2007) 287-292.
[5] A. A. Abu-Hussen, J. Coord. Chem. 59 (2006) 157-176.
[6] C. M. da Silva, D. L. da Silva, L. V. Modolo, R. B. Alves, M. A. de Resende, C. V. Martins, Â.
de Fátima, J. Adv. Res. 2 (2011) 1-8.
[
[
[
7] R. Atkins, G. Brewer, E. Kokot, G. M. Mockler, E. Sinn, Inorg. Chem. 24 (1985) 127-134.
8] M. B. Halli, R. Sumathi, J. Mol. Struct. 1022 (2012) 130-138.
9] A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E. Lederer, S. Wölfl, N. Metzler-
Nolte, S. Schäfer, W. S. Sheldrick, J. Biol. Inorg. Chem. 14 (2009) 711-725.
10] A. Silvestri, G. Barone, G. Ruisi, D. Anselmo, S. Riela, V. T. Liveri, J. Inorg. Biochem. 101
[
(2007) 841-848.
[
[
11] V. Uma, V. Vaidyanathan, B. U. Nair, Bull. Chem. Soc. Jpn. 78 (2005) 845-850.
12] S.-Y. Lee, A. Hille, C. Frias, B. Kater, B. Bonitzki, S. Wölfl, H. Scheffler, A. Prokop, R.
Gust, J. Med. Chem. 53 (2010) 6064-6070.
[13] S. Pradeepa, H. B. Naik, B. V. Kumar, K. I. Priyadarsini, A. Barik, T. R. Naik, Spectrochim
Acta A Mol Biomol Spectrosc. 101 (2013) 132-139.
[
14]. M. Sathisha, U. N. Shetti, V. Revankar, K. Pai, Eur. J. Med. Chem 43 (2008) 2338-2346.
15] W. A. Herrmann, C. W. Kohlpaintner, Water-Soluble Ligands, Angew. Chem., Int. Ed. Engl.
[
3
2 (1993) 1524-1544.
[
[
[
16] N. e Iram, M. S. Khan, R. Jolly, M. Arshad, M. Alam, P. Alam, R. H. Khan, F. Firdaus, J.
Photochem. Photobiol. B, Biol. 153 (2015) 20-32.
17] P. Kumar, B. Baidya, S. K. Chaturvedi, R. H. Khan, D. Manna, B. Mondal, Inorg. Chim. Acta
3
76 (2011) 264-270.
18] C. Icsel, V. T. Yilmaz, Y. Kaya, H. Samli, W. T. Harrison, O. Buyukgungor, Dalton Trans 44
2015) 6880-6895.
(
[
[
19] M. Sirajuddin, S. Ali, A. Badshah, J. Photochem. Photobiol. B, Biol. 124 (2013) 1-19.
20] H. Baruah, C. G. Barry, U. Bierbach, Curr. Top. Med. Chem. 4 (2004) 1537-1549.
2
1.
J. Med. Chem. 51 (2008) 7574-7580.
22] H.-K. Liu, P. J. Sadler, Acc. Chem. Res. 44 (2011) 349-359.
Z. Ma, J. R. Choudhury, M. W. Wright, C. S. Day, G. Saluta, G. L. Kucera, U. Bierbach,
[
2
5

ACCEPTED MANUSCRIPT
[23] S. Komeda, T. Moulaei, K. K. Woods, M. Chikuma, N. P. Farrell, L. D. Williams, J. Am.
Chem. Soc. 128 (2006) 16092-16103.
[
[
24] R. Hajian, N. Shams, M. Mohagheghian, J. Braz. Chem. Soc. 20 (2009) 1399-1405.
25] G.-J. Chen, Z.-G. Wang, X. Qiao, J.-Y. Xu, J.-L. Tian, S.-P. Yan, J. Inorg. Biochem. 127
(2013) 39-45.
[26] R. Parr. (Oxford, 1989).
[27] M. Frisch, G. Trucks, H. Schlegel, G. Scuseria, M. Robb, J. Cheeseman, J. Montgomery Jr, T.
Vreven, K. Kudin, J. Burant, Gaussian 03, revision c. 02; Gaussian. Inc., Wallingford, CT 4
(
2004).
28] M. A. Lie, R. Thomsen, C. N. Pedersen, B. Schiøtt, M. H. Christensen, J. Chem. Inf. Model.
1 (2011) 909-917.
[
5
[
[
[
[
29] F. Keshavarz, D. Mohammad-Aghaie, Phys. Chem. Res.3 (2015) 125-143.
30] R. Thomsen, M. H. Christensen, MolDock: J. Med. Chem. 49 (2006) 3315-3321.
31] H.-D. Yin, Q.-B. Wang, S.-C. Xue, J. Organomet. Chem. 689 (2004) 2480-2485.
32] Y.-L. Zhang, W.-J. Ruan, X.-J. Zhao, H.-G. Wang, Z.-A. Zhu, Polyhedron 22 (2003) 1535-
1
545.
[33] G. Deacon, R. Phillips, C. Chem. Rev 33 (1980) 227-250.
[34] R. Prabhakaran, R. Karvembu, T. Hashimoto, K. Shimizu, K. Natarajan, Inorg. Chim. Acta
3
58 (2005) 2093-2096.
3
5.
P. Kalaivani, R. Prabhakaran, F. Dallemer, P. Poornima, E. Vaishnavi, E. Ramachandran,
V. V. Padma, R. Renganathan, K. Natarajan, Metallomics 4 (2012) 101-113.
[
[
36] J. H. Cameron, J. A. McEwan, Inorg. Chim. Acta 211 (1993) 111-116.
37] M. Asadi, S. B. Sadi, Z. Asadi, R. Yousefi, A. R. Barzegar Sadi, H. Khalili-Hezarjaribi, J.
Coord. Chem. 65 (2012) 722-739.
[
[
[
38] J. Ribas, C. Diaz, R. Costa, Y. Journaux, C. Mathoniere, O. Kahn, A. Gleizes, Inorg. Chem.
2
9 (1990) 2042-2047.
39] S. Roy, R. Sadhukhan, U. Ghosh, T. K. Das, Spectrochim. Acta Mol. Biomol. 141 (2015)
76-184.
40] M. A. Ragheb, M. A. Eldesouki, M. S. Mohamed, Spectrochim. Acta Mol. Biomol. 138
2015) 585-595.
1
(
[41] K. Ghosh, P. Kumar, N. Tyagi, Inorg. Chim. Acta 375 (2011) 77-83.
[42] X.-W. Liu, Y.-M. Shen, Z.-X. Li, X. Zhong, Y.-D. Chen, S.-B. Zhang, Spectrochim. Acta
Mol. Biomol.149 (2015) 150-156.
[43] J. Liu, H. Zhang, C. Chen, H. Deng, T. Lu, L. Ji, Dalton Trans (2003) 114-119.
[44] N. Shahabadi, S. Kashanian, F. Darabi, Eur. J. Med. Chem 45 (2010) 4239-4245.
[45] M. T. Carter, M. Rodriguez, A. J. Bard, J. Am. Chem. Soc. 111 (1989) 8901-8911.
[46] M. F. Hassan, A. Rauf, Spectrochim. Acta Mol. Biomol. 153 (2016) 510-516.
[47] N. Shahabadi, S. Kashanian, F. Darabi, . Dna cell biol. 28 (2009) 589-596.
[48] F. Samari, B. Hemmateenejad, M. Shamsipur, M. Rashidi, H. Samouei, Inorg. Chem. 51
(2012) 3454-3464.
2
6

ACCEPTED MANUSCRIPT
[49] A. Moodi, M. Khorasani-Motlagh, M. Noroozifar, S. Niroomand, J. Biomol. Struct. Dyn. 31
(2013) 937-950.
[50] M. Anjomshoa, H. Hadadzadeh, M. Torkzadeh-Mahani, S. J. Fatemi, M. Adeli-Sardou, H. A.
Rudbari, V. M. Nardo, Eur. J. Med. Chem 96 (2015) 66-82.
[
[
51] X.-Z. Feng, Z. Lin, L.-J. Yang, C. Wang, C.-l. Bai, Talanta 47 (1998) 1223-1229.
52] M. Khorasani-Motlagh, M. Noroozifar, A. Moodi, S. Niroomand, J. Photochem. Photobiol. B,
Biol. 127 (2013) 192-201.
[53] S. S. Bhat, A. A. Kumbhar, H. Heptullah, A. A. Khan, V. V. Gobre, S. P. Gejji, V. G. Puranik,
Inorg. Chem. 50 (2010) 545-558.
[54] A. Manna, S. Chakravorti, Spectrochim. Acta Mol. Biomol. 150 (2015) 120-126.
[55] S. Ramakrishnan, M. Palaniandavar, Dalton Trans (2008) 3866-3878.
[56] K. Zheng, L. Zhu, Y.-T. Li, Z.-Y. Wu, C.-W. Yan, J. Photochem. Photobiol. B, Biol. 149
(2015) 129-142.
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ACCEPTED MANUSCRIPT
Highlights
•
Novel water soluble Schiff bases and zinc(II) complex were synthesized and
characterized.
•
•
•
The interaction with DNA was investigated.
In vitro cytotoxicity was studied.
Theoretical docking studies was performed.