Synthesis of Ni(II) complexes bearing indole-based thiosemicarbazone ligands for interaction with biomolecules and some biological applications

Article abstract of DOI:10.1007/s00775-016-1424-1

Abstract: A series of new Ni(II) complexes containing indole-based thiosemicarbazone ligands was synthesized and characterized by elemental analyses, and UV–visible, FT-IR, ¹H & ¹³C NMR and mass spectroscopic techniques. The Ni(II) c

Full text of DOI:10.1007/s00775-016-1424-1

J Biol Inorg Chem
DOI 10.1007/s00775-016-1424-1
ORIGINAL PAPER
Synthesis of Ni(II) complexes bearing indole‑based
thiosemicarbazone ligands for interaction with biomolecules
and some biological applications
1
1
2
Jebiti Haribabu · Kumaramangalam Jeyalakshmi · Yuvaraj Arun ·
3
2
Nattamai S. P. Bhuvanesh · Paramasivan Thirumalai Perumal ·
Ramasamy Karvembu
1
Received: 4 June 2016 / Accepted: 26 November 2016
©
SBIC 2016
Abstract A series of new Ni(II) complexes containing
indole-based thiosemicarbazone ligands was synthesized
and characterized by elemental analyses, and UV–vis-
ible, FT-IR, H & C NMR and mass spectroscopic tech-
niques. The Ni(II) complexes (1–4) bear the general formula
complexes with the molecular target B-DNA, human DNA
topoisomerase I and BSA. All the Ni(II) complexes possess
high antioxidant activity against 2-2-diphenyl-1-picrylhy-
drazyl (DPPH) radical and antihaemolytic activity. In addi-
tion, in vitro cytotoxicity of the Ni(II) complexes against
lung cancer (A549), human breast cancer (MCF7) and
mouse embryonic fibroblasts (L929) cell lines was investi-
gated. Complex 4 has high cytotoxicity. The mode of cell
death effected by complex 4 has been explored using Hoe-
chst 33258 staining.
1
13
[
4
Ni{C H N NHCSNH(R)} ] where R = hydrogen (1),
1
0
9
2
2
-methyl (2), 4-phenyl (3) and 4-cyclohexyl (4). Molecular
structure of ligands (L3 and L4) and complexes (2, 3 and 4)
was confirmed by single crystal X-ray crystallography.
Four coordinated Ni(II) complexes showed square planar
geometry. The interaction of the Ni(II) complexes with calf
thymus DNA (CT-DNA) has been evaluated by absorp-
tion spectroscopic and ethidium bromide (EB) competitive
binding studies, which revealed the intercalative interac-
tion of the complexes with CT-DNA. Gel electrophoresis
experiments showed the cleavage of DNA by the complexes
without any external agent. Further, the interaction of the
complexes with bovine serum albumin (BSA) was inves-
tigated using UV–visible, fluorescence and synchronous
fluorescence spectroscopic methods, which showed that the
complexes could bind strongly with BSA. Molecular dock-
ing was employed to understand the binding of the Ni(II)
Graphical abstract Nickel(II) complexes of thiosemi-
carbazone ligands were synthesized and their DNA/pro-
tein binding, DNA cleavage and cytotoxicity abilities were
studied.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-016-1424-1) contains supplementary
material, which is available to authorized users.
*
Ramasamy Karvembu
kar@nitt.edu
1
2
3
Department of Chemistry, National Institute of Technology,
Tiruchirappalli 620015, India
Organic Chemistry Division, CSIR-Central Leather Research
Institute, Chennai 600020, India
Keywords Indole · Thiosemicarbazones · Nickel(II)
complexes · DNA/protein interactions · Cytotoxicity
Department of Chemistry, Texas A&M University, College
Station, TX 77842, USA
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J Biol Inorg Chem
Introduction
complexes containing indole-3-carbaldehyde-N-substituted
thiosemicarbazones.
Bioinorganic chemistry offers surplus opportunities
for the design and making of pharmaceutical drugs that
are not accessible to organic molecules [1–4]. Since the
approval of cisplatin for the treatment of cancer, a number
of other metal complexes has been investigated for their
therapeutic potential [5, 6]. Among them, transition metal
complexes of thiosemicarbazone ligands have received
considerable interest due to their inhibition of DNA syn-
thesis caused by a modification in the reductive conver-
sion of ribonucleotides to deoxyribonucleotides [7]. In
general, it has been observed that the biological activity
of the complexes of thiosemicarbazones is greater than
that of the corresponding free thiosemicarbazones [8–12].
Reactions of various thiosemicarbazones [7, 13–17] and
semicarbazones [18] with diverse transition metal ions
lead to the formation of metal complexes which have
shown valuable biological activities. In particular, het-
erocyclic thiosemicarbazone complexes have aroused
considerable interest in bioinorganic chemistry because
of their pharmacological properties, particularly as anti-
phrastic, antibacterial, antioxidant and antitumoral agents
Experimental
General methods
All the chemicals were purchased from Sigma-Aldrich/
Merck and used as received. Solvents were purified accord-
ing to the standard procedures.
Physical measurements
Elemental analyses were done using a Vario EL-III CHNS
analyser. FT-IR spectra were obtained as KBr pellets using
a Nicolet-iS5 spectrophotometer. UV–visible spectra were
recorded using a Shimadzu-2600 spectrophotometer. Emis-
sion spectral studies were carried out with a Jasco V-630
spectrophotometer using 5% DMF in buffer as the solvent.
1
13
H and C NMR spectra were recorded on a Bruker 400
and 100 MHz spectrometer, respectively. MS–ESI spectra
were recorded using a high-resolution JEOL and Q-Tof
mass spectrometer.
[
8, 19, 20]. For example, Ni(II) complexes with 6-meth-
oxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-based
N-substituted thiosemicarbazones showed significant anti-
oxidant and cytotoxicity [21].
Synthesis of thiosemicarbazones (L1–L4)
Indole skeleton is present in the structure of many nat-
ural products with high structural complexities and bio-
logically active molecules [22]. For this reason, indole and
indole derivatives have been used, continuously, in different
research areas such as pharmaceuticals, fragrances, agro-
chemicals, pigments and materials science [23]. One of the
most important indole derivatives is an essential amino acid,
tryptophan and it is one of the naturally occurring amino
acids. Tryptophan-containing proteins have reducing effect
on depression and insomnia related with hormonal fluc-
tuations [24]. Research on indole-3-carbinol has been con-
ducted using laboratory animals and cultured cells, which
showed that it can prevent the binding of aflatoxin to DNA
and hence it reduces the carcinogenic effects of aflatoxins
3-Indole aldehyde and the substituted thiosemicarbazide
were combined (1:1) in ethanol (15 mL) in the pres-
ence of acetic acid (1–2 drops). The mixture was refluxed
for 3 h, during which a white precipitate appeared. Then,
the mixture was allowed to cool to room temperature and
solid was filtered off. The product was washed with cold
ethanol and purified by recrystallization from a methanol–
dichloromethane mixture (1:2). The compounds were sub-
sequently crystallized from acetonitrile–methanol mixture
(1:1).
(E)‑2‑((1H‑indol‑3‑yl)methylene)
hydrazinecarbothioamide (L1)
[
25–27]. Indole-3-carbinol was also effective in the preven-
Thiosemicarbazide (0.091 g, 0.001 mol) and 3-indole alde-
hyde (0.145 g, 0.001 mol) were used. Yield: 91%. White.
m.p.: 162 °C. Anal. Calc. for C H N S (%): C, 55.02;
tion of breast and skin cancers [28, 29]. There are many
drugs whose structures contain the indole nucleus, including
sumatriptan, a tryptamine derivative [30] used in the treat-
ment of migraine headaches, indomethacin and etodolac
1
0
10
4
H, 4.62; N, 25.67; S, 14.69. Found: C, 55.27; H, 4.73; N,
25.26; S, 14.80. UV–Vis (CH OH): λmax, nm 264, 330.
3
−1
[31], which are used as non-steroidal anti-inflammatory
FT-IR (KBr): ʋ, cm
3448 (N–H), 3312, 3230 (H–N–
1
drugs, and pindolol, a β-adrenoceptor antagonist [32]. These
stem a great interest to develop indole-based thiosemicar-
bazone complexes for biological applications. Herein, we
present the synthesis, structure, DNA binding, DNA cleav-
age, protein binding, DNA/protein molecular docking,
antioxidant, antihaemolytic and cytotoxic studies of Ni(II)
C=S), 1548 (C=N), 1295 (C=S). H NMR (400 MHz,
DMSO-d ): δ, ppm 11.61 (s, 1H), 11.19 (s, 1H), 8.30 (s,
6
1H), 8.22 (d, J = 7.6 Hz, 1H), 8.02 (s, 1H), 7.80 (s, 1H),
7.43 (d, J = 7.2 Hz, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.13 (t,
1
3
J = 7.6 Hz, 1H). C NMR (100 MHz, DMSO-d ): δ, ppm
6
176.8 (C=S), 141.4 (C=N), 137.4, 131.4, 124.3, 123.1,
1
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J Biol Inorg Chem
1
22.5, 121.1, 112.2, 111.5 (aromatic C). TOF–MS–ES+:
(d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.32–7.24
(m, 2H), 4.33 (t, J = 4.0 Hz, 1H), 2.14 (t, J = 12.0 Hz,
+
m/z [Found (Calcd)] 219.0713 (218.0626) (M + H) .
1
3
2
H), 1.80–1.63 (m, 4H), 1.51–1.28 (m, 4H). C NMR
(
E)‑2‑((1H‑indol‑3‑yl)
(100 MHz, CDCl ): δ, ppm 174.9 (C=S), 139.3 (C=N),
3
methylene)‑N‑methylhydrazinecarbothioamide (L2)
136.9, 129.1, 124.2, 123.7, 121.6, 121.5, 112.2, 111.7 (aro-
matic C), 52.6, 32.8, 29.7, 25.5, 24.6, 14.1 (aliphatic C).
TOF–MS–ES+: m/z [Found (Calcd)] 301.1478 (300.1409)
4
3
8
-Methyl thiosemicarbazide (0.105 g, 0.001 mol) and
-indole aldehyde (0.145 g, 0.001 mol) were used. Yield:
5%. White. m.p.: 154 °C. Anal. Calc. for C H N S (%):
+
(M + H) .
1
1
12
4
C, 56.87; H, 5.21; N, 24.12; S, 13.80. Found: C, 56.94; H,
Synthesis of Ni(II) complexes (1–4)
5
3
.32; N, 24.0; S, 13.91. UV–Vis (CH OH): λmax, nm 262,
3
−
1
29. FT-IR (KBr): ʋ, cm 3440 (N–H), 3325, 3238 (H–N–
The ethanolic solution of NiCl ·6H O (1 mmol) was added
2
2
1
C=S), 1551 (C=N), 1299 (C=S). H NMR (400 MHz,
into the solution of an appropriate indole-based thiosemi-
carbazone ligand (1 mmol) in ethanol and a few drops of
triethyl amine were added. The reaction mixture was stirred
for 3 h under reflux, and then the precipitate formed was
filtered and washed with ethanol. The suitable crystals for
X-ray diffraction were grown from CH Cl –DMF mixture
DMSO-d ): δ, ppm 11.61 (s, 1H), 11.18 (s, 1H), 8.29 (d,
6
J = 11.6 Hz, 2H), 7.92 (d, J = 4.0 Hz, 1H), 7.80 (s, 1H),
7
.43 (d, J = 7.6 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.15
13
(
(
1
t, J = 7.2 Hz, 1H), 3.08 (d, J = 4.0 Hz, 1H). C NMR
100 MHz, DMSO-d ): δ, ppm 177.1 (C=S), 140.9 (C=N),
6
2
2
37.4, 131.3, 124.3, 123.1, 122.7, 121.0, 112.2, 111.6 (aro-
(2:1).
matic C), 31.5 (aliphatic C). TOF–MS–ES+: m/z [Found
+
(
Calcd)] 233.0862 (232.0783) (M + H) .
Bis[(E)‑2‑((1H‑indol‑3‑yl)methylene)
hydrazinecarbothioamide]Ni(II) (1)
(
E)‑2‑((1H‑indol‑3‑yl)
methylene)‑N‑phenylhydrazinecarbothioamide (L3)
NiCl ·6H O (0.237 g, 0.001 mol) and L1 (0.218 g,
2
2
0
.001 mol) were used. Yield: 87%. Light green solid.
4
3
8
-Phenyl thiosemicarbazide (0.167 g, 0.001 mol) and
-indole aldehyde (0.145 g, 0.001 mol) were used. Yield:
9%. White. m.p.: 170 °C. Anal. Calc. for C H N S (%):
m.p.: 216 °C. Anal. Calcd. For C H N NiS : C, 48.70;
H, 3.68; N, 22.72; S, 13.00. Found: C, 48.53; H, 3.40; N,
2
0
18
8
2
22.96; S, 13.14. UV–Vis (DMF): λmax, nm 271, 334, 408.
1
6
14
4
−1
C, 65.28; H, 4.79; N, 19.03; S, 10.89. Found: C, 65.41; H,
4
3
FT-IR (KBr): ʋ, cm 3439, 3304 (N–H), 1502 (C=N),
1
.65; N, 19.37; S, 10.94. UV–Vis (CH OH): λmax, nm 265,
1263 (C–S). H NMR (400 MHz, DMSO-d ): δ, ppm 11.99
3
6
−
1
41. FT-IR (KBr): ʋ, cm 3453 (N–H), 3300, 3169 (H–N–
(s, 1H), 8.64 (s, 1H), 7.61 (s, 1H), 7.51 (s, 1H), 7.46 (d,
1
13
C=S), 1549 (C=N), 1287 (C=S). H NMR (400 MHz,
J = 8.0 Hz, 1H), 7.19 (t, J = 7.6 Hz, 2H), 6.92 (s, 1H).
C
DMSO-d ): δ, ppm 11.72 (s, 1H), 11.63 (s, 1H), 9.65 (s,
NMR (100 MHz, DMSO-d ): δ, ppm 173.2 (C–S), 147.2
6
6
1
H), 8.46 (s, 1H), 8.27 (d, J = 7.6 Hz, 1H), 7.92 (s, 1H),
(C=N), 135.6, 134.4, 126.8, 122.9, 121.3, 117.1, 112.7,
7
.67 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 7.39 (d,
109.6 (aromatic C). FT–MS–ESI+: m/z [Found (Calcd)]
1
3
+
J = 7.6 Hz, 2H), 7.25–7.16 (m, 3H). C NMR (100 MHz,
493.0522 (492.0447) (M + H) .
DMSO-d ): δ, ppm 174.9 (C=S), 141.8 (C=N), 139.7,
6
1
1
37.5, 131.7, 128.6, 125.5, 125.4, 124.5, 123.2, 122.2,
Bis[(E)‑2‑((1H‑indol‑3‑yl)
methylene)‑N‑methylhydrazinecarbothioamide]Ni(II)
(2)
21.2, 112.4, 111.4 (aromatic C). TOF–MS–ES+: m/z
+
[
Found (Calcd)] 295.1005 (294.0936) (M + H) .
(
E)‑2‑((1H‑indol‑3‑yl)
NiCl ·6H O (0.237 g, 0.001 mol) and L2 (0.232 g,
2
2
methylene)‑N‑cyclohexylhydrazinecarbothioamide (L4)
0.001 mol) were used. Yield: 80%. Light green solid. m.p.:
2
04 °C. Anal. Calcd. For C H N NiS : C, 50.69; H, 4.25;
22 22 8 2
4
3
8
-Cyclohexyl thiosemicarbazide (0.173 g, 0.001 mol) and
-indole aldehyde (0.145 g, 0.001 mol) were used. Yield:
9%. White. m.p.: 179 °C. Anal. Calc. for C H N S (%):
N, 21.50; S, 12.30. Found: C, 50.75; H, 4.08; N, 21.21;
S, 12.48. UV–Vis (DMF): λmax, nm 276, 380, 410. FT-IR
−1
(KBr): ʋ, cm 3436, 3321 (N–H), 1509 (C=N), 1261
1
6
20
4
1
C, 63.97; H, 6.71; N, 18.65; S, 10.67. Found: C, 64.37; H,
(C–S). H NMR (400 MHz, DMSO-d ): δ, ppm 11.92 (s,
6
6
3
.43; N, 18.86; S, 10.90. UV–Vis (CH OH): λmax, nm 264,
1H), 8.40 (d, J = 9.2 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H),
3
−
1
31. FT-IR (KBr): ʋ, cm 3425 (N–H), 3343, 3241 (H–N–
7.83 (s, 1H), 7.45 (d, J = 10.0 Hz, 1H), 7.30–7.18 (m, 2H),
1
13
C=S), 1567 (C=N), 1291 (C=S). H NMR (400 MHz,
2.82 (s, 3H). C NMR (100 MHz, DMSO-d ): δ, ppm
6
CDCl ): δ, ppm 11.64 (s, 1H), 11.21 (s, 1H), 9.72 (s, 1H),
173.6 (C–S), 141.6 (C=N), 135.4, 133.9, 126.8, 123.1,
3
8
.76 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H), 7.44
121.4, 117.2, 112.8, 109.7 (aromatic C), 32.3 (aliphatic C).
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J Biol Inorg Chem
FT–MS–ESI+: m/z [Found (Calcd)] 521.0835 (520.0762)
pinholes) for the latter. Sixty data frames were taken at
widths of 0.5°. These reflections were used in the auto-
indexing procedure to determine the unit cell. A suitable
cell was found and refined by nonlinear least squares and
Bravais lattice procedures. Integrated intensity informa-
tion for each reflection was obtained by reduction of data
frames with APEX2 [33]. SADABS was employed to
correct the data for absorption effects [34]. The struc-
tures were solved by direct methods SHELXTL (XS), and
refined (SHELX-97, weighted least squares refinement on
+
(
M + H) .
Bis[(E)‑2‑((1H‑indol‑3‑yl)
methylene)‑N‑phenylhydrazinecarbothioamide]Ni(II)
(
3)
NiCl ·6H O (0.237 g, 0.001 mol) and L3 (0.294 g,
2
2
0
.001 mol) were used. Yield: 83%. Light green solid.
m.p.: 237 °C. Anal. Calcd. For C H N NiS : C, 59.55;
3
2
26
8
2
2
H, 4.06; N, 17.36; S, 9.94. Found: C, 59.32; H, 4.19; N,
7.11; S, 10.03. UV–Vis (DMF): λmax, nm 284, 392, 416.
F ) using APEX2 and OLEX2 [35, 36]. Hydrogen atoms
1
were placed in idealized positions and were set riding on
the respective parent atoms. All non-hydrogen atoms were
refined with anisotropic thermal parameters. The structures
−
1
FT-IR (KBr): ʋ, cm 3437, 3304 (N–H), 1500 (C=N),
1
1
243(C–S). H NMR (400 MHz, DMSO-d ): δ, ppm 12.17
6
2
(
7
1
s, 1H), 9.56 (s, 1H), 8.33 (s, 1H), 8.13 (d, J = 7.2 Hz, 1H)
were refined (weighted least squares on F ) to convergence
.81 (s, 1H), 7.55 (d, J = 6.4 Hz, 2H), 7.36 (t, J = 6.4 Hz,
H), 7.27 (d, J = 6.4 Hz, 2H), 7.10–7.05 (m, 2H), 6.96 (t,
[35]. Olex2 was employed for the final data presentation
and structure plots [36]. The crystallographic data and the
results of refinements are summarized in Tables 1, 2, S1
and S2.
1
3
J = 6.4 Hz, 1H). C NMR (100 MHz, DMSO-d ): δ, ppm
6
1
1
74.7 (C–S), 141.7 (C=N), 139.6, 137.4, 131.7, 128.5,
26.8, 125.4, 124.4, 123.0, 121.1, 120.4, 112.3, 110.2 (aro-
matic C). FT–MS–ESI+: m/z [Found (Calcd)] 645.1148
DNA binding experiments
+
(
644.1075) (M + H) .
For DNA absorption spectral studies, DNA samples were
dissolved in 50 mM NaCl/5 mM Tris–HCl (pH 7.2) solu-
tion. CT-DNA solution displayed a UV absorbance ratio
at 260 and 280 nm (A260/A280) of ca. 1.9:1, indicating
that the CT-DNA was sufficiently in protein-free form
[37]. The concentration of the nucleic acid solutions was
determined by UV absorbance at 260 nm after 1:100 dilu-
tions. The extinction coefficient at 260 nm was taken
as 6600 M cm [38]. Stock solutions were stored at
4 °C and used within 4 days. Concentrated stock solu-
tions were prepared by dissolving calculated amounts of
the complexes (1, 2, 3 and 4) in a 5% DMF/5 mM Tris–
HCl/50 mM NaCl buffer to required concentrations for all
the experiments. Absorption spectral titration experiments
were performed by maintaining a constant concentration
of the complex and varying the nucleic acid concentra-
tion (0–35 μM). The spectra were recorded after equili-
bration for 3 min, allowing the compounds to bind to the
CT-DNA.
Bis[(E)‑2‑((1H‑indol‑3‑yl)
methylene)‑N‑cyclohexylhydrazinecarbothioamide]
Ni(II) (4)
NiCl ·6H O (0.237 g, 0.001 mol) and L4 (0.300 g,
2
2
0
.001 mol) were used. Yield: 86%. Light green solid.
m.p.: 230 °C. Anal. Calcd. For C H N NiS : C, 58.45;
3
2
38
8
2
−
1
−1
H, 5.83; N, 17.04; S, 9.75. Found: C, 58.19; H, 5.92; N,
7.35; S, 9.61. UV–Vis (DMF): λmax, nm 274, 328, 413.
1
−
1
FT-IR (KBr): ʋ, cm 3419, 3336 (N–H), 1511 (C=N),
1
1
254 (C–S). H NMR (400 MHz, DMSO-d ): δ, ppm 12.05
6
(
s, 1H), 8.39 (s, 1H), 7.68 (s, 1H), 7.54 (s, 1H), 7.47 (d,
J = 8.0 Hz, 2H), 7.34 (s, 1H), 7.20 (t, J = 6.8 Hz, 2H),
2
1
.01 (d, J = 10.4 Hz, 2H), 1.77 (d, J = 11.6 Hz, 2H),
.62 (d, J = 11.6 Hz, 1H), 1.40–1.28 (m, 2H), 1.26–1.15
1
3
(
(
m, 4H). C NMR (100 MHz, DMSO-d ): δ, ppm 174.0
C–S), 141.1 (C=N), 135.5, 133.1, 126.8, 121.3, 117.2,
6
1
12.7, 109.8 (aromatic C), 79.6, 55.0, 32.5, 25.8, 25.4 (ali-
phatic C). FT–MS–ESI+: m/z [Found (Calcd)] 657.2087
The competitive studies of each complex with ethidium
bromide (EB) have been investigated using fluorescence
spectroscopic technique. The excitation wavelength was
fixed, and emission range was adjusted before measure-
ments. DNA was pretreated with EB in the 1:1 ratio for
15 min. The complexes were then added to this mixture,
and their effect on the emission intensity was measured.
The Stern–Volmer equation was used to evaluate the appar-
ent binding constant (Kapp) and quenching constant (Kq)
values of the complexes. The changes in fluorescence inten-
sities at 596 nm (510 nm excitation) of EB bound to DNA
were recorded with an increasing amount of the complexes
+
(
656.2014) (M + H) .
Single crystal X‑ray crystallography
Single crystal X-ray diffraction data were collected using
Bruker APEX2 (L4, 2, 3 and 4) and Bruker GADDS (L3)
X-ray (three-circle) diffractometer. The X-ray radiation
employed was generated from a Mo sealed X-ray tube
(
K = 0.70173 Å) for the former and Cu sealed X-ray
α
tube (K = 1.5418 Å) fitted with a graphite monochroma-
α
tor in the parallel mode (175 mm collimator with 0.5 mm
1
3

J Biol Inorg Chem
Table 1 Crystallographic data and refinement parameters for complexes (2, 3 and 4)
2
3
4
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C H N NiO S
C H N NiO S
C H N NiO S
2 2
2
8
36 10
2
2
44 54 12
4
2
38 52 10
667.50
110.15
0.71073
Triclinic
P-1
937.82
803.72
110.15
0.71073
Triclinic
P-1
110.15
0.71073
Triclinic
P-1
8.4986(17)
10.336(2)
10.454(2)
62.048(2)
70.741(2)
78.337(2)
764.5(3)
1
8.985(2)
10.067(3)
13.855(4)
108.319(3)
91.914(3)
104.849(3)
1141.0(5)
1
8.187(3)
12.458(4)
19.638(7)
88.627(4)
78.777(4)
83.653(4)
1952.5(12)
2
b (Å)
c (Å)
α (°)
β (°)
γ (°)
Volume (Å3)
Z
Density (calculated) Mg/m3
Absorption coefficient (mm−1)
F(000)
1.450
1.365
1.367
0.816
0.573
0.652
350
494
852
Crystal size (mm3)
0.57 × 0.56 × 0.53
0.48 × 0.2 × 0.17
0.54 × 0.52 × 0.2
Theta range for data collection (°) 2.234–27.509
2.221–27.613
1.057–27.768
Index ranges
−11 ≤ h ≤ 10, −13 ≤ k ≤ 13,
−11 ≤ h ≤ 11, −13 ≤ k ≤ 13,
−10 ≤ h ≤ 10, −16 ≤ k ≤ 16,
−
13 ≤ l ≤ 13
−18 ≤ l ≤ 17
0 ≤ l ≤ 25
Reflections collected
8713
13209
8859
8859
99.5%
Independent reflections [R(int)]
3453 [0.0260]
99.5%
5195 [0.0314]
99.5%
Completeness to
theta = 25.24°/25.24°/25.24°
Absorption correction
Max. and min. transmission
Refinement method
Semi-empirical from equivalents
0.7456 and 0.4507
Full-matrix least squares on F2
Semi-empirical from equivalents
0.7456 and 0.6258
Full-matrix least squares on F2
5195/0/290
Semi-empirical from equivalents
0.746 and 0.456
Full-matrix least squares on F2
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2 sigma(I)]
R indices (all data)
3453/0/200
8859/8/501
1.053
1.031
1.046
R1 = 0.0256, wR2 = 0.0644
R1 = 0.0268, wR2 = 0.0651
0.108(4)
R1 = 0.0391, wR2 = 0.0923
R1 = 0.0539, wR2 = 0.0996
n/a
R1 = 0.0519, wR2 = 0.1291
R1 = 0.0733, wR2 = 0.1421
n/a
Extinction coefficient
Largest diff. peak and hole (e.Å−3) 0.501 and −0.325
0.608 and −0.301
0.633 and −0.636
until maximum reduction in the intensity of fluorescence
occurred.
Viscosity experiments were carried out using a semi-
microviscometer maintained at 27 °C in a thermostatic water
bath. DNA samples (0.5 mM) were prepared by sonica-
tion to minimize complexities arising from DNA flexibility
solution (t) corrected for the flow time of the buffer alone
(t ), using the expression η = (t − t )/t [40–42].
0
0
0
0
CD spectroscopic technique is useful in monitoring the
conformational changes induced by the interaction of drugs
with the DNA helix. The B-form conformation of DNA
showed two consecutive CD bands in the UV region, a posi-
tive band at 275 nm is due to base stacking and a negative
band at 245 nm is due to polynucleotide helicity. CD spectra
of DNA in the absence and presence of the complexes was
recorded with a J-715 spectropolarimeter (Jasco) at 25 °C
with a 0.1 cm path length cuvette. The spectra were recorded
for 20 μM of DNA in the absence and presence of 5 μM of
the Ni(II) complexes (1–4) in the region 220–300 nm [43].
[39]. Flow time was measured three times for each sample
and an average flow time was calculated. The values of rela-
0
tive specific viscosity (η/η ), where η is the relative viscos-
0
ity of DNA in the presence of the complex and η is the
relative viscosity of DNA alone, were plotted against 1/R
0
(
1/R = [compound]/[DNA]). Relative viscosity (η ) values
were calculated from the observed flow time of the DNA
1
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J Biol Inorg Chem
Table 2 Selected bond lengths (Å), angles (°) of 2, 3 and 4
excitation and emission wavelengths of BSA) values such
as 15 and 60 nm [44].
2
3
4
Ni(1)–S(1)#1
2.1683(5)
2.1683(5)
1.9087(11)
1.9087(11)
180.0
2.1764(6)
2.1764(6)
1.9068(16)
1.9068(16)
180.0
2.1618(10)
2.1618(10)
1.911(2)
1.911(2)
180.0
Molecular docking studies
Ni(1)–S(1)
Ni(1)–N(1)
The X-ray crystal structure of B-DNA (PDB ID: 1BNA),
dodecamer d(CGCGAATTCGCG) human DNA Topo-
Ni(1)–N(1)#1
2
S(1)–Ni(1)–S(1)#1
N(1)#1–Ni(1)–S(1)#1
N(1)–Ni(1)–S(1)#1
N(1)–Ni(1)–S(1)
N(1)#1–Ni(1)–S(1)
N(1)#1–Ni(1)–N(1)
C(10)–S(1)–Ni(1)
N(2)–N(1)–Ni(1)
C(1)–N(1)–Ni(1)
I complex (PDB ID: 1SC7) and BSA (PDB ID: 3V03)
was obtained from the Protein Data Bank (http://www.
rcsb.org/pdb). 2D structure of the Ni(II) complexes
was drawn using Chem Draw Ultra 12.0 (ChemOf-
fice 2010). Chem3D Ultra 12.0 was used to convert 2D
structure into 3D and the energy was minimized using
semi-empirical AM1 method. Molecular docking studies
have been done using the AutoDock Tools (ADT) ver-
sion 1.5.6 and Auto Dock version 4.2.5.1 docking pro-
gram [45]. The energy calculations were made using
genetic algorithms. The outputs were exported to PyMol
for visual inspection of the binding modes and for possi-
ble polar and hydrophobic interactions of the complexes
with DNA and BSA.
85.58(3)
94.42(3)
85.58(3)
94.42(3)
180.0
85.46(5)
94.54(5)
85.46(5)
94.54(5)
180.00(9)
95.24(7)
120.69(12)
125.33(13)
85.86(7)
94.14(7)
85.86(7)
94.14(7)
180.0
95.11(5)
120.45(8)
125.28(9)
96.75(10)
122.01(18)
125.39(19)
Symmetry transformations used to generate equivalent atoms:
1 − x + 1, −y, −z + 1
#
DNA cleavage experiment
A mixture of Tris buffer (5 mM Tris–HCl/50 mM NaCl
buffer, pH 7.2), pUC19 plasmid DNA (150 μg/mL) and
different amounts of the complexes was incubated for 4 h
at 37 °C. A dye solution (0.05% bromophenol blue and
Antioxidant assay
The antioxidant activity of the Ni(II) complexes was evalu-
ated using the DPPH free radical scavenging assay [46,
47]. 5 mg of the each compound was dissolved in 1 mL of
DMSO, which was maintained as a stock solution. From
the stock, 25, 50, 75, 100, 250 and 500 µg of solutions were
prepared. For the positive control, ascorbic acid at different
concentrations (25, 50, 75, 100, 250 and 500 µg) was used.
100 µL of DPPH (at 10 mM concentration) and 900 µL
of methanol were added to different concentrations of the
compounds and also to the positive control, to screen the
antioxidant potential. 100 µL of DPPH was added to 100
µL of DMSO and 900 µL of methanol to reveal the scav-
enging effect of DMSO. 1 mL of methanol was added to
100 µL of DPPH, which was used as a negative control.
The mixtures were shaken vigorously and allowed to stand
for 30 min in dark, which were analysed for the absorbance
at 517 nm by UV spectrometer.
5
% glycerol) was added to the reaction mixture prior to
electrophoresis. The samples were then analysed by 1.5%
agarose gel electrophoresis [Tris HCl/Boric acid/EDTA
(
TBE) buffer, pH 8.0] for 3 h at 60 mV. The gel was stained
−1
with 0.5 μg mL EB, visualized by UV light, and photo-
graphed. The extent of cleavage of the pUC19 DNA was
determined by measuring the intensities of the bands using
Alpha Imager HP instrument.
Protein binding experiment
Quenching of the emission of tryptophan residues of
BSA was performed using the complexes as quenchers.
To a solution of BSA in phosphate buffer (pH 7.2), incre-
ments of the quenchers were added, and the emission sig-
nal at 341 nm (280 nm excitation) was recorded after each
addition of the quencher. The excitation and emission slit
widths and scan rates were constantly maintained for all
the experiments. Concentrated stock solutions of the test
compounds were prepared by dissolving them in DMF and
diluted suitably with phosphate buffer to get required con-
centrations. 2.5 mL of BSA solution (1 μM) was titrated by
successive additions of a 5 mL of stock solution of the test
Haemolysis assay
Human red blood cells (RBC) were isolated using the
standard procedure [48] and diluted with 0.1 M phosphate
buffer saline (PBS) (pH 7.2). To 0.5 mL of RBC solu-
tion, six different concentrations (25, 50, 75, 100, 250 and
500 µg/mL) of the Ni(II) complexes were added to test the
toxicity of the complexes. 100% lysis was initiated by add-
−4
compounds (10 M) using a micropipette. Synchronous
fluorescence spectra were also recorded using the same
concentration of BSA and the test compounds as men-
tioned above with two different Δλ (difference between the
ing 500 µL of H O, which acted as a negative control. RBC
2
samples without H O and the Ni(II) complexes acted as a
2
positive control. All the samples were incubated at 37 °C
1
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J Biol Inorg Chem
for 30 min and centrifuged at 3000 rpm. The supernatant
was used to analyse the percentage of lysis based on the
absorbance at 560 nm.
Results and discussion
Synthesis
In vitro cytotoxicity evaluation by MTT assay
The thiosemicarbazone ligands (L1–L4) were synthesized
via the typical condensation route from 3-indole aldehyde
and appropriate thiosemicarbazides in the presence of gla-
cial acetic acid (Scheme 1). Subsequently, the Ni(II) com-
plexes (1–4) were prepared by heating the corresponding
ligand with NiCl ·6H O (Scheme 2). The ligands and their
Cytotoxicity of the Ni(II) complexes was carried out on
lung cancer (A549), human breast cancer (MCF7) and
mouse fibroblasts (L929) cell lines. Cell viability was car-
ried out using the MTT assay [49]. The lung adenocarci-
noma (A549), human breast (MCF7) cells and mouse
fibroblasts (L929) were plated separately in 96-well plates
2
2
Ni(II) complexes were characterized by elemental analyses
and various spectroscopic techniques. The molecular struc-
ture of L3, L4, 2, 3 and 4 was confirmed by single crystal
X-ray diffraction study.
5
at a concentration of 1 × 10 cells/well. Complexes (1–4)
of concentration ranging from 1 to 500 µM dissolved in
DMSO were seeded to the wells. DMSO was used as the
control. After 24 h, the wells were treated with 20 μL of
MTT [5 mg/mL PBS] and incubated at 37 °C for 4 h. The
purple formazan crystals formed were dissolved in 200 μL
of DMSO. The absorbance of the solution was measured at
a wavelength of 570 nm using a Beckmann Coulter Elisa
plate. Triplicate samples were analysed for each experi-
ment. The percentage inhibition was calculated using the
formula
Spectroscopy
The electronic spectra of the ligands showed two bands
around 262–265 and 329–341 nm, which correspond to
π–π* and n–π* transitions, respectively. The spectra of
the complexes exhibited three bands in DMF solvent. Two
bands appeared around 271–288 and 328–392 nm cor-
respond to intraligand transitions. The band in the region
4
08–416 nm was attributed to ligand-to-metal charge-trans-
Mean OD of the untreated cells control − Mean OD of the treated cells
Mean OD of the untreated cells (control)
fer (LMCT) transitions which indicated the formation of
the complexes [17].
×
100.
In the FT-IR spectra of the ligands, three bands were
observed for indole N–H, N–H (attached to azomethine)
and N–H (attached to thiocarbonyl) in the range of 3448–
Hoechst 33258 staining
−
1
Cell pathology was detected by staining the nuclear
3409, 3343–3300 and 3238–3169 cm , respectively. The
azomethine (C=N) and thiocarbonyl (C=S) stretching
frequencies of the ligands were observed at 1567–1548
4
−1
chromatin of trypsinized cells (4.0 × 10 mL ) with
1
μL of Hoechst 33258 (1 mg/mL) for 10 min at 37 °C.
−
1
Staining of suspension cells with Hoechst 33258 was
used to view apoptotic changes. A drop of cell suspen-
sion was placed on a glass slide and a coverslip was laid
over to reduce light diffraction. At random, 300 cells
were observed in a fluorescent microscope fitted with
a 377–355-nm filter and observed at 400× magnifica-
tion, and the percentage of cells reflecting pathological
changes was calculated [50].
and 1299–1261 cm , respectively. On complexation,
−
1
there was a decrease in azomethine (1511–1500 cm )
−
1
and thiocarbonyl (1263–1243 cm ) stretching frequen-
cies, which suggested the coordination of the ligands to
Ni ion through azomethine nitrogen and thiocarbonyl sul-
phur [51].
1
In the H NMR spectra of the ligands, H–N–C=S pro-
ton resonated in the region 11.63–11.18 ppm. However, in
Scheme 1 Synthetic route of indole-based N-substituted thiosemicarbazone ligands
1
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J Biol Inorg Chem
Scheme 2 Synthetic scheme of Ni(II) complexes
the spectra of the complexes (1–4), there was no resonance
attributable to H–N–C=S, indicating the coordination of
the ligands in the anionic form after deprotonation. The ter-
minal N–H proton signal in the spectra of the ligands (L2–
L4) and complexes was observed at 9.65–7.92 and 9.56–
by slow evaporation of acetonitrile–methanol (1:1) solu-
tions of the ligands. Red or brown crystals of the com-
plexes suitable for X-ray diffraction were obtained by slow
evaporation of their dichloromethane–dimethyl formamide
(2:1) solutions. The important torsional angles N(2)–N(3)–
C(10)–S(1), N(2)–N(3)–C(10)–N(4), N(3)–N(2)–C(9)–
C(7) and C(9)–N(2)–N(3)–C(10) in L3 are 176.72(11),
3.5(2), −177.02(13) and −162.50(14)°, and those in L4 are
174.65(11), −6.5(2), −177.53(13) and 174.37(14)° [53].
The S(1)–C(10), N(2)–C(9) and N(2)–N(3) bond lengths
of L3/L4 are 1.6945/1.6935(16), 1.2890(2)/1.2835(19) and
1.3842(17)/1.3927(18) Å, respectively.
7
.89 ppm, respectively. Two broad singlets were observed
in the spectrum of L1 at 7.80 and 8.02 ppm corresponding
to NH protons. In the spectrum of 1, one broad singlet was
2
observed at 6.92 ppm corresponding to NH protons of the
2
coordinated ligand. The signal of indole N–H proton in the
ligands and complexes appeared at 11.72–11.61 and 12.17–
1
7
1.92 ppm, respectively. The signals around 8.31–8.05 and
.81–7.61 ppm in the spectra of the ligands correspond to
Complexes 2, 3 and 4 crystallized in triclinic P-1 space
group with Z of 1 (2 and 3) or 2 (4). The X-ray structure
of 2, 3 and 4 showed a perfect square planar geometry
with nitrogen and sulphur atoms coordinate to Ni ion in a
trans fashion. One molecule of dimethyl formamide was
found in the asymmetric unit of 2 and four molecules of
the same solvent were found in the symmetric unit of 3.
Two intramolecular hydrogen bonds were present between
the azomethine H and S, which was rarely observed.
The perfect square planar geometry was also reflected
by τ parameter [54, 55]. τ can be calculated using the
azomethine CH and indole CH protons. In the spectra of
the complexes, the azomethine CH and indole CH pro-
tons showed signals around 8.04–7.80 and 7.57–7.51 ppm,
respectively. Further, a doublet was observed at 3.09 and
2
.84 ppm in the spectra of L2 and 2, respectively, which
was attributed to the presence of terminal CH protons.
3
1
3
C NMR spectra of the ligands showed resonances due
to C=S and C=N carbons in the regions 177.1–174.1 and
40.9–139.3 ppm, respectively. However, in the spectra of
1
the complexes (1–4), C–S (after enolization and deproto-
nation) and C=N chemical shifts were observed at 174.7–
4
4
formula τ = [360° − (α + ß)]/141°. The value of τ
4
4
1
73.2 and 147.2–141.1 ppm, respectively. Chemical shift of
ranges from 1.00 for a perfect tetrahedral geometry, since
360 − 2(109.5) = 141, to zero for a perfect square planar
geometry, since 360 − 2(180) = 0. For the intermediate
all other protons and carbons was observed in the expected
regions [52].
structure like trigonal pyramidal, τ falls within the range
4
Single crystal X‑ray crystallography
of 0–1.00. In complexes 2, 3 and 4, the angles S(1)–Ni(1)–
S(1)#1 and N(1)#1–Ni(1)–N(1) were 180° which revealed
perfect square planar geometry. There was an increase in
the C–N and C–S bond lengths and decrease in the N(2)–
N(3) bond length in 2, 3 and 4 compared to L1, L3 and L4,
respectively, which clearly attested the coordination of the
ligand to Ni ion through azomethine N and thiocarbonyl
Thermal ellipsoid plot of the ligands (L3 and L4) and
complexes (2, 3 and 4) with the atomic labelling scheme
is shown in Figs. 1 and 2. Crystal data and selected inter
atomic bond lengths and angles are given in Tables 1, 2,
S1 and S2. Colourless crystals of the ligands were grown
1
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J Biol Inorg Chem
Fig. 1 Thermal ellipsoid plot of L3 and L4
Fig. 2 Thermal ellipsoid plot of 2, 3 and 4
1
3

J Biol Inorg Chem
S after enolization and deprotonation. The Ni(1)–S(1)
2.161–2.176 Å) bond length is higher than Ni(1)–N(1)
straight line with a slope of 1/(ε − ε ) and an y-intercept
b
f
(
of 1/K (ε − ε ) and the value of K was determined from
b
b
f
b
bond length (1.906–1.911 Å) in 2, 3 and 4. The torsional
angles Ni(1)–S(1)–C(10/1)–N(2), Ni(1)–S(1)–C(10/1)–
N(4), Ni(1)–N(1)–N(2)–C(10) and Ni(1)–N(1)–C(1/2)–
C(2/3) in 2 are −13.30(12), 166.62(11), 16.17(14) and
the ratio of slope to intercept (Fig. 4a). The magnitudes of
intrinsic binding constants (K ) are given in Table 3. The
b
4
K values were found to be in the range of 3.87 × 10 –
b
5
−1
2.29 × 10 M . The high binding ability of complex 4
compared to the other complexes might be due to the pres-
ence of cyclohexyl ring at the terminal nitrogen atom of the
coordinated ligand [21].
1
74.64°(11), and those in 3 are 11.94(18), −169.99(14),
−
15.9(2) and 174.23°(16). 0.8(3), 180.0(2), 0.9(3) and
−
175.1°(2) are the values of the same torsional angles in 4.
DNA binding studies
EB displacement study
DNA binding is the predominant property looked for in
pharmacology when evaluating the potential of new anti-
cancer complexes, and hence, the interaction between DNA
and the complexes needs to be investigated. The mode and
tendency of the binding of complexes 1–4 with CT-DNA
were studied with different methods.
Fluorescence property has not been observed for the com-
plexes at room temperature in solution or in the presence of
CT-DNA. So the binding of the complexes with DNA could
not be directly predicted through the emission spectra.
Hence, competitive binding study was done to understand
the mode of DNA interaction with the complexes [59–61].
EB is one of the most sensitive fluorescence probes that
can bind with DNA [62]. The fluorescence of EB increases
after intercalating into DNA. If the metal complex interca-
lates into DNA, it leads to a decrease in the binding sites of
DNA available for EB, resulting in decrease in the fluores-
cence intensity of the CT-DNA–EB system [63]. The extent
of decrease in the fluorescence intensity of CT-DNA–EB
reflects the extent of interaction of the complex with CT-
DNA. On adding the Ni(II) complexes (0–50 µM) to CT-
DNA–EB, the quenching in the emission of DNA-bound
EB took place (Figs. 3, S2) due to intercalation of the com-
plexes with DNA. Fluorescence quenching is explained by
Electronic absorption titration
Before performing DNA interaction studies, the stability
of the complexes was verified in physiological medium
(
Tris–HCl buffer solution with a pH value of 7.2, contain-
ing 5% DMF) by UV–visible spectroscopic technique.
There were no obvious changes in either the intensity or
the position of the absorption bands in Tris–HCl buffer
solution compared to those in DMF alone, which sug-
gested that the complexes are stable under physiologi-
cal conditions. The electronic spectra of Ni(II) complexes
(
1–4) in Tris–HCl buffer/5% DMF exhibited two bands
the Stern–Volmer equation [64] F°/F = 1 + K [Q] where
q
around 273–284 and 333–391 nm, which correspond to
intraligand transitions. The observation of changes in this
band upon incremental addition of CT-DNA was used to
find out the binding constant. Complexes binding to DNA
through intercalation usually results in hypochromism with
or without a small red or blue shift, since the intercalative
mode involves a strong stacking interaction between the
planar aromatic chromophore and the base pairs of DNA
F° and F are the fluorescence intensities in the absence and
presence of complex, respectively, K is a linear Stern–Vol-
q
mer quenching constant, and [Q] is the concentration of
complex. The slope of the plot of F°/F versus [Q] gave Kq
(Fig. 4a). The apparent DNA binding constant (Kapp) val-
ues were calculated using the equation KEB [EB] = K
app
[complex] where [complex] is the complex concentra-
tion at 50% reduction in the fluorescence intensity of EB,
7
−1
[
56, 57]. The interaction of complexes 1–4 was confirmed
KEB = 1.0 × 10 M and [EB] = 5 µM. K and K val-
q
app
by the observed hypochromism with red shift (Figs. 3,
S1). The magnitude of hypochromism was in the order of
ues are listed in Table 3.
4
> 3 > 1 > 2, which reflected the DNA binding affinities
Viscosity measurements
of the complexes. The intrinsic binding constant K was
b
obtained from the ratio of slope to intercept in the plot of
Lengthening of DNA helix occurs on intercalation as base
pairs are separated to accommodate the binding com-
pound leading to increase in DNA viscosity. The values of
relative specific viscosities of CT-DNA in the absence and
presence of the compounds are plotted against [complex]/
[DNA]. The relative viscosities of CT-DNA bound to the
complexes (1–4) increased with increasing the compound
concentration (Fig. 4b) similar to some known intercalators
[65], indicative of a classical intercalation. The ability of
[
[
DNA]/(ε − ε ) versus [DNA] according to the equation
a
f
58] [DNA]/(ε − ε ) = [DNA]/(ε − ε ) + 1/K (ε − ε )
a
f
b
f
b
b
f
where [DNA] is the concentration of DNA in base pairs,
εa is the apparent extinction coefficient value found by cal-
culating A(observed)/[complex], ε is the extinction coeffi-
f
cient for the free compound and ε is the extinction coef-
b
ficient for the compound in the fully bound form. Each
set of data, when fitted into the above equation, gave a
1
3

J Biol Inorg Chem
Fig. 3 a Absorption spectra of complex 4 in Tris–HCl buffer upon
addition of CT-DNA. [Complex] = 20 µM, [DNA] = 0–35 µM.
Arrow shows that the absorption intensity decreases upon increasing
DNA concentration. b Fluorescence quenching curves of EB bound
to DNA in the presence of 4. [DNA] = 5 µM, [EB] = 5 µM and
[complex] = 0–50 µM
the complexes to increase the viscosity of DNA followed
the order 4 > 3 > 1 > 2.
partial intercalation of the cyclohexyl ring at the terminal
nitrogen atom [70].
CD spectra
Protein binding studies
The CD spectrum of CT-DNA exhibited a positive band at
2
75 nm due to base stacking and a negative band at 245 nm
Fluorescence and absorbance quenching of BSA by the
Ni(II) complexes
due to helicity of B-DNA [66]. After the addition of 1–4,
the spectrum showed an increase in positive and a decrease
in negative ellipticity with a red shift of 6–4 nm. These
alterations in the CD spectrum of DNA indicated strong
conformational changes by the complexes [67]. It was
observed that complex 4 showed significant changes in the
CD spectrum compared to the other complexes (Fig. 4c).
Furthermore, the decrease in negative band is probably due
to the unwinding of the DNA helix upon interaction with
the complexes followed by transformation into A-like con-
formations [43, 68]. The CD results supported the order
of binding ability of the complexes, obtained from other
experiments.
Fluorescence spectra are useful in the qualitative analysis
of the binding of chemical compounds to BSA. Generally,
the fluorescence of BSA is caused by two intrinsic char-
acteristics of the protein, namely tryptophan and tyrosine.
The intrinsic fluorescence of BSA will provide consider-
able information on their structure and dynamics and is
often utilized in the study of protein folding and association
reactions. The interaction of BSA with the complexes was
studied by fluorescence measurement at room temperature.
A solution of BSA (1 µM) was titrated with various con-
centrations of the complexes 1–4 (0–20 µM). Fluorescence
spectra were recorded in the range of 290–500 nm upon
excitation at 280 nm. The changes observed in the fluores-
cence emission spectra of BSA on addition of increasing
concentration of the Ni(II) complexes are shown in Figs. 5a
and S3. On the addition of complexes 1–4 to BSA, there
was a significant decrease in the fluorescence intensity
of BSA at 345 nm up to 75.23, 71.47, 85.62 and 78.01%,
respectively, from the initial fluorescence intensity of BSA
accompanied by a hypsochromic shift of 10–15 nm. The
observed blue shift is mainly due to the fact that the active
site in protein is buried in a hydrophobic environment.
These results indicated a definite interaction of all of the
complexes with the BSA protein [71, 72]. The fluorescence
Nuclease activity
To explore the DNA cleavage ability of the complexes (1–
4
), supercoiled (SC) pUC19 plasmid DNA (40 μM in base
pairs) was incubated at 37 °C with the complexes (100 µM)
in a 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH
7
.2 for 4 h in the absence of external agent [69]. Complexes
1
–4 cleaved SC (Form I) DNA into nicked circular (NC)
(
Form II) DNA (Fig. 4d), and the DNA cleavage strength
follows the order 4 (92.3%) > 3 (52.6%) > 1 (38.2%) > 2
1.7%). The study revealed that 4 cleaved DNA more effi-
ciently than the other complexes, because of the strong
(
1
3

J Biol Inorg Chem
Fig. 4 a Plot of [DNA]/(ε − ε ) versus [DNA] and Stern–Volmer
Tris HCl/50 mM NaCl at pH = 7.2 and 37 °C with an incubation
time of 4 h. Lane 1 DNA control; lane 2 DNA + 1 (100 μM); lane 3
DNA + 2 (100 μM); lane 4, DNA + 3 (100 μM); lane 5, DNA + 4
(100 μM); forms SC and NC are supercoiled and nicked circular
DNA, respectively
a
f
plot of fluorescence titrations of the complexes with CT-DNA. b
Effect of the complexes (1, 2, 3 and 4) on the viscosity of CT-DNA.
c CD spectra of CT-DNA (20 μM) in the absence and presence of
5
μM of the complexes. d Cleavage of supercoiled pUC19 DNA
(
40 μM) by complexes 1–4 in a buffer containing 5% DMF:5 mM
1
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J Biol Inorg Chem
Table 3 DNA binding constant (K ), quenching constant (K ) and
apparent binding constant (Kapp) values
equilibrium between free and bound molecules is repre-
sented by the Scatchard equation log[(F° − F)/F] = log
K + n log [Q] where K is the binding constant of the
b
q
−1
K (M )
q
−1
Complex
K (M−1)
Kapp (M
)
b
b
b
complex with BSA and n is the number of binding site.
From the plot of log [(F° − F)/F] versus log [Q] (Fig. 6a),
the number of binding site (n) and the binding constant
4
4
5
5
4
4
4
4
6
6
6
6
1
2
3
4
4.25 × 10
3.87 × 10
1.95 × 10
2.29 × 10
8.62 × 10
7.16 × 10
9.20 × 10
9.82 × 10
4.31 × 10
3.58 × 10
4.60 × 10
4.91 × 10
(
K ) values have been obtained. K , K and n values for the
b q b
interaction of the Ni(II) complexes with BSA are provided
in Table 4. The calculated value of n is around 1 for all of
the compounds, indicating the existence of just a single
binding site in BSA for all of the complexes. From the val-
ues of K and K , it was inferred that complex 4 interacted
quenching is described by the Stern–Volmer relation
o
F°/F = 1 + K [Q] where F and F demonstrate the fluores-
q
q
b
cence intensities in the absence and presence of quencher,
with BSA more strongly than the rest of the complexes.
Here again, complex 4 showed better activity.
respectively, K is a linear Stern–Volmer quenching con-
q
stant, and [Q] is the quencher concentration. The quench-
Quenching usually occurs either by dynamic or static
mode. Dynamic quenching is a process in which the fluo-
rophore and the quencher come into contact during the
transient existence of the excited state. On the other hand,
ing constant (K ) can be calculated from the plot of F°/F
q
versus [Q] (Fig. 6a). When small molecules bind indepen-
dently to a set of equivalent site, on a macromolecule, the
Fig. 5 a Fluorescence quenching curves of BSA in the absence and presence of 4. [BSA] = 1 µM and [complex] = 0–20 µM. b Synchronous
spectra of BSA (1 µM) as a function of concentration of 4 (0–20 µM) with Δλ = 15 and 60 nm
1
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J Biol Inorg Chem
Fig. 6 a Stern–Volmer and Scatchard plots of the fluorescence titrations of the complexes with BSA. b The absorption spectra of BSA (10 µM)
and BSA with 1–4 (5 µM)
static quenching refers to the formation of a fluorophore–
quencher complex in the ground state [73]. A simple
method to determine the type of quenching is UV–vis-
ible absorption spectroscopy. Addition of the complexes
to BSA leads to an increase in BSA absorption intensity
without affecting the position of absorption band (Fig. 6b).
It showed the existence of static interaction between BSA
and the complexes due to the formation of the ground-state
complex as reported earlier [74].
before and after the addition of the test compounds. The
results provided reasonable information on the molecular
microenvironment, particularly in the vicinity of the fluo-
rophore functional groups [74]. The difference between
the excitation and emission wavelengths (Δλ) reflected
the nature of the chromophore [62]. The large Δλ value
(60 nm) is characteristic of tryptophan residue and a small
Δλ value (15 nm) is characteristic of tyrosine. The syn-
chronous fluorescence spectra of BSA with various con-
centrations of the complexes (1–4) were recorded at
Δλ = 15 and 60 nm. On addition of the complexes, the
fluorescence intensity of tryptophan residue at 340 nm
decreased in the magnitude of 76.9, 81.3, 79.7 and 71.6%
for complexes 1–4, respectively (Figs. 5b and S4). Simi-
larly, there was also decrease in the intensity of tyrosine
Characteristics of synchronous fluorescence spectra
To investigate in detail the structural changes which
occurred in BSA upon the addition of the new compounds,
synchronous fluorescence spectra of BSA were measured
1
3

J Biol Inorg Chem
Table 4 Protein binding constant (K ), quenching constant (K ) and
number of binding site (n) values
In designing chemotherapeutic drugs, their interac-
tion with DNA is vital to arrest the replication step in the
cell cycle. DNA function can be suppressed in the physi-
ological processes by the specific host–guest interaction
between DNA and drugs. The metal complexes are widely
used as the chemotherapeutic agents by destabilizing the
DNA structure to block the cell functions.
b
q
−1
K (M )
q
Complex
K (M−1)
n
b
5
5
6
6
5
5
5
5
1
2
3
4
9.98 × 10
9.05 × 10
1.81 × 10
2.57 × 10
3.93 × 10
2.43 × 10
4.56 × 10
4.92 × 10
1.19
1.13
1.03
1.10
We employed docking studies with the B-DNA (PDB
ID: 1BNA) to predict the DNA binding affinity of the
nickel(II) complexes. Ni(II) complexes 1, 2, 3 and 4
showed the binding energy values of −10.31, −9.72,
−11.28, and −10.84 kcal/mol with two, four, three and
three hydrogen bond interactions, respectively. Among all
the complexes docked, 3 showed the lowest binding energy
value of −11.28 kcal/mol with three hydrogen bonds with
the docked DNA receptor. One of the phenyl attached N–H
in 3 interacted with the oxygen of C=O of DT-20 and
nitrogen of DA-6 to form two hydrogen bonds. Further-
more, indole N–H interacted with the oxygen of C=O of
DT-8 and formed a hydrogen bond [78]. Interactions of all
the Ni(II) complexes with the B-DNA receptor are shown
in Fig S6. Docking studies revealed the efficient binding of
the Ni(II) complexes with the DNA. This might disturb the
stability of the DNA, which could trigger the DNA cleav-
age. Notably, molecular docking study was performed to
predict the binding affinity and the sterically favourable
conformations of the Ni(II) complexes towards DNA with-
out considering the intercalation [79]. As molecular dock-
ing study considered only minor groove binding and exper-
imental findings proposed intercalation of the complexes
with DNA, no attempt was made to correlate the results of
docking and experimental studies.
residue at 300 nm. The magnitude of decrease was 70.1,
5.0, 80.6 and 87.9% for complexes 1–4, respectively
Figs. 5b and S5). The synchronous fluorescence spectral
studies clearly suggested that the fluorescence intensities
of both the tryptophan and tyrosine were affected with
increasing concentration of the complexes. The results
indicated that the interaction of the complexes with BSA
affected the conformation of both tryptophan and tyrosine
micro-region [56].
8
(
Molecular docking with DNA and BSA
To gain insights into the possible reasons for the biologi-
cal activity, the synthesized Ni(II) complexes were sub-
jected to molecular docking studies using the AutoDock
Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1
docking [75, 76]. In silico molecular docking studies were
performed to simulate the interaction of the Ni complexes
with the receptor binding site of B-DNA (PDB ID: 1BNA)
dodecamer d(CGCGAATTCGCG) , human DNA topoi-
2
somerase I complex (DNA-T) (PDB ID: 1SC7) and BSA
(
PDB ID: 3V03) [77]. Docked conformation was analysed
in terms of energy, hydrogen bonding and hydrophobic
interactions between the Ni(II) complexes and receptors
to find the binding nature of the potent inhibitors. The
compound–DNA interactions were carried out with the
detailed analysis, and final coordinates of the compound
and receptor were saved. To display the interaction of
the complexes with the receptor binding site, output was
exported to PyMol software and detailed analysis was
done, the free energy of binding (FEB) of the compounds
was calculated from the docking scores and details are
shown in Table 5.
In the human DNA topoisomerase I complex (PDB ID:
1SC7), Topo-I was bound to the oligonucleotide sequence
50-AAAAAGACTTsX-GAAAATTTTT-30 in which ‘s’
was 50-bridging phosphorothioate of the cleaved strand and
‘X’ represented any of the four bases A, G, C or T. The SH
of G11 on the scissile strand was changed to OH and phos-
phoester bond of G12 in 1SC7 was rebuilt [80]. Molecular
docking studies with DNA topoisomerase I clearly showed
that the Ni(II) complexes approached towards the DNA
cleavage site to form a stable complex which resulted in
the binding energy between −9.29 and −12.80 kcal/mol
Table 5 Molecular docking
Free energy of binding (kcal/mol)a
Complex
parameters of complexes 1–4
with B-DNA (1BNA), DNA
topoisomerase I (1SC7) and
BSA (3V03)
B-DNA (PDB ID: 1BNA) DNA topoisomerase I (PDB ID: 1SC7) BSA (PDB ID: 3V03)
1
2
3
4
−10.31
−9.72
−9.29
−9.65
−7.24
−7.36
−7.45
−8.07
−11.28
−10.84
−12.15
−12.80
a
Calculated by Autodock
1
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J Biol Inorg Chem
and subsequently leading to inhibitory effect. Among all
the Ni(II) complexes docked, 4 showed the highest bind-
ing energy value of −12.80 kcal/mol with three hydrogen
bonds with the docked 1SC7. In complex 4, one of the
indole N–H interacted through hydrogen bond with the
pentose oxygen of DA-113 and C=O oxygen of ASN-722
amino acid. Binding interaction of the complexes with the
DNA topoisomerase I receptor is shown in Fig. S7.
Docking studies of the complexes with 3V03 receptor
showed that all the docked compounds bound efficiently
with the receptor and exhibited free energy of binding value
from −7.24 to −8.07 kcal/mol. The previously reported
binding site in BSA was taken for this study [81]. All the
Ni(II) complexes effectively bound in the 22 active site
amino acids, namely PHE-205, ARG-208, ALA-209, LYS-
In vitro cytotoxicity
The positive results obtained from the DNA and protein
binding studies exhilarated us to test the cytotoxicity of the
complexes (1–4). It was carried out against lung (A549)
cancer, human breast (MCF7) cancer and mouse fibro-
blasts (L929) cell lines using MTT assay [84]. Figure S11
shows the cytotoxicity of the complexes (1–4) after 24 h
incubation on A549, MCF7 and L929 cell lines, respec-
tively. Complexes were dissolved in DMSO and a blank
sample containing the same volume of DMSO was taken
as a control to identify the activity of the solvent in this
experiment. Cisplatin was used as a positive control to
assess the cytotoxicity of the tested complexes. The results
were analysed by means of cell inhibition expressed as
IC50 values and are shown in Table 6. Figures S12 and S13
show digital images of the cancer cells (A549 and MCF7)
and the cancer cells treated with the complexes (1–4).
The IC50 values showed that complexes 4 and 1 exhibited
higher inhibitory effect than that of the other complexes
and the IC50 was close to that of cisplatin. Fortunately,
all the complexes were less toxic towards the normal cell
(L929) as it was evident from the higher IC50 values (600–
750 µM) (Table 6). The cytotoxicity results are in good
agreement with DNA binding ability of the complexes.
Cytotoxicity of the nickel(II) complexes was compared
with that of previously reported square planar nickel(II)
complexes (Fig. 7). The present Ni(II) complexes showed
higher activity than some of the other reported nickel(II)
complexes. The cytotoxicity of nickel(II) complexes con-
taining l-proline- and l-homoproline-4-N-pyrrolidine-
3-thiosemicarbazone hybrid ligand showed IC50 value
around 222.6 ± 17.1 μM against the A549 (96 h of incu-
bation) cancer cell line [85]. The ionic square planar
nickel complex bearing 4-chloro-5-methyl-salicylaldehyde
thiosemicarbazone ligand showed higher cytotoxicity with
lower IC50 (<10 μM) value against A549 (48 h of incuba-
tion) cancer cell line [16]. In vitro cytotoxicity of the Ni(II)
complexes with substituted thiosemicarbazone ligands was
good (IC50 = 25–30 μM) against human lung adenocar-
cinoma (A549) cell line, which was comparable with the
2
11, TRP-213, VAL-215, PHE-227, THR-231, VAL-234,
THR-235, ASP-323, LEU-326, GLY-327, LEU-330, SER-
43, LEU-346, ALA-349, LYS-350, GLU-353, SER-479,
3
LEU-480 and VAL-481. Among all the compounds docked
with 3V03 receptor, compound 4 exhibited high binding;
cyclohexyl attached N–H formed one hydrogen bond with
ARG-208 amino acid, resulted in the binding energy of
−
8.07 kcal/mol. Binding interaction of the complexes with
the BSA receptor is shown in Fig. S8.
Antioxidant activity
Reactive oxygen species cause oxidative damage to the
cellular tissues and initiates various disorders. Due to this,
there is an increasing interest in antioxidants, particularly
in those intended to prevent the adverse effects of free radi-
cals in the human body. In this regard, the Ni(II) complexes
were studied for their antioxidant potential using DPPH
scavenging assay. DPPH reacts with antioxidant compound
which donates hydrogen and reduces the DPPH radical.
The change in colour was measured at 517 nm. From the
assay it was found that the IC50 value of 1 and 2 was about
7
5
5 µg/mL. The IC50 value of 3 and 4 was found to be 50 and
00 µg/mL, respectively. The results clearly showed that 3
was more potent radical scavenger compared to the other
three complexes (1, 2 and 4), which might be due to the
electron withdrawing effect of the phenyl group on the ter-
minal nitrogen atom [82]. The results are shown in Fig. S9.
Table 6 In vitro cytotoxicity of the Ni(II) complexes in A549, MCF7
and L929 cell lines
Haemolysis assay
Complex
IC50 (μM)
Ni(II) complexes (1–4) were tested for their toxicity against
human RBC. From the results it was clearly inferred that the
Ni(II) complexes (1–4) did not induce any haemolysis (Fig.
S10). The results were compared to the control cells treated
with water which produced 100% lysis. The results sug-
gested that the Ni(II) complexes (1–4) could be used further
as they did not show any toxicity against RBC [83].
A549
MCF7
L929
1
52.4
56.1
57.2
45.1
18.0
125.1
106.5
62.0
>600
>700
>700
>750
6.0
2
3
4
59.6
Cisplatin
12.0
1
3

J Biol Inorg Chem
Fig. 7 IC50 values of reported square planar Ni(II) complexes
present results [86]. The complexes containing 2,3-dihy-
droxybenzaldehyde N4-substituted thiosemicarbazone
ligands showed IC50 value around 14.7–46.2 μM against
the MCF7 cell line [87]. The cytotoxicity of [Ni(MTSali)L]
L = imidazole or benzimidazole) type complexes against
MCF7 cancer cell line is provided in Fig. 7 [88]. It is well
evident from the comparison that our complexes showed
high cytotoxicity. However, our complexes are less effec-
tive compared to cisplatin.
(
MTSali = salicylaldehyde-4-methylthiosemicarbazone;
1
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J Biol Inorg Chem
Fig. 8 Photomicrograph show-
ing the features of Hoechst
3
3258 staining of A549 and
MCF7 cancer cells. Cancer cells
were treated with IC50 con-
centration of 4. The cells were
stained with Hoechst 33258
fluorescent dye: a untreated
A549 cancer cells (control), b
A549 cancer cells treated with 4
after 24 h. c MCF7 cancer cells
(
control), d MCF7 cancer cells
treated with 4 after 24 h. Arrow
marks indicate the apoptotic
cells
Hoechst staining
significant and the complexes bound to BSA in both tyros-
ine and tryptophan residues. The docked models also con-
firmed the binding affinity of the Ni(II) complexes with
targeted DNA/protein. In addition, the complexes possess
significant antioxidant activity. The haemolysis results
revealed that the Ni(II) complexes (1–4) could be used for
further pharmacological studies. The complexes showed
high cytotoxicity against human lung (A549) and human
breast (MCF7) cancer cell lines. DNA/protein binding
and DNA cleavage ability of the complexes followed the
order 4 > 3 > 1 > 2. Cytotoxic property of the complexes
was in different order towards A549 and MCF7, but 4
exhibited pronounced activity against both the cell lines.
The high cytotoxicity of 4 might be due to the presence
of bulky substitution (cyclohexyl) at the terminal nitrogen
atom of the ligand. But the activity of the complexes was
less compared to cisplatin. It is a convincing factor that
there are reports on complexes having higher IC50 values
than cisplatin (in vitro) showed significant activity in vivo
[92, 93]. Apoptosis mechanism for the cell death was also
confirmed.
Many antitumor agents used in chemotherapy are based on
their ability to induce apoptosis in cancer cells [89, 90]. So
the molecular mechanism of cell death has been studied by
treating the complexes with the lung (A549) and human
breast (MCF7) cancer cells. IC50 concentration of the Ni(II)
complex (4) was used for the staining assay. The complex
was incubated for 24 h and it was observed for cytological
changes by adopting Hoechst 33258 staining. The results
revealed that the cell death mechanism was apoptosis
(
Fig. 8) [91].
Conclusions
Four indole-based thiosemicarbazone ligands and their
Ni(II) complexes were synthesized and characterized
with a view to evaluate their biological applications. The
complexes (1–4) bound to DNA via intercalation. Bind-
ing affinity of all the Ni(II) complexes with BSA was
1
3

J Biol Inorg Chem
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