Synthesis, structure, DNA and protein binding studies, and cytotoxic activity of nickel(II) complexes containing 3,3-dialkyl/aryl-1-(2,4- dichlorobenzoyl)thiourea ligands

Article abstract of DOI:10.1016/j.poly.2014.03.010

A new series of nickel(II) complexes of the type [Ni(L)₂] (1-6) has been synthesized by using the 3,3-dialkyl/aryl-1-(2,4-dichlorobenzoyl) thiourea ligands, and characterized by analytical and spectral (NMR, FT-IR and UV-Vis) techniques. The st

Full text of DOI:10.1016/j.poly.2014.03.010

Polyhedron 75 (2014) 95–109
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, structure, DNA and protein binding studies, and cytotoxic
activity of nickel(II) complexes containing 3,3-dialkyl/
aryl-1-(2,4-dichlorobenzoyl)thiourea ligands
N. Selvakumaran a, N.S.P. Bhuvanesh b, A. Endo c, R. Karvembu a,
⇑
a Department of Chemistry, National Institute of Technology, Tiruchirappalli 620015, India
b Department of Chemistry, Texas A & M University, College Station, TX 77842, USA
c Department of Materials and Life Sciences, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of nickel(II) complexes of the type [Ni(L)2] (1–6) has been synthesized by using the 3,3-
dialkyl/aryl-1-(2,4-dichlorobenzoyl)thiourea ligands, and characterized by analytical and spectral (NMR,
FT-IR and UV–Vis) techniques. The structures of the ligands (HL1, HL2 and HL3) and complexes (2 and
5) have been confirmed by single crystal X-ray diffraction studies. The interaction of the nickel(II) com-
plexes (2–5) with calf thymus (CT) DNA and bovine serum albumin (BSA) protein was investigated using
UV–Vis and fluorescence spectroscopic methods. Absorption and emission spectral studies indicate that
complex 2 interacts with CT DNA and BSA protein more strongly than the other complexes (3, 4 and 5).
Received 3 January 2014
Accepted 10 March 2014
Available online 20 March 2014
Keywords:
Acylthiourea
Ni(II) complex
DNA binding
BSA interaction
Cytotoxicity
Complexes 2 and 5 exhibited good cytotoxicity against A549 and HT29 cell lines at 50 lM concentration.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
against MFC cell lines and also bind DNA via a groove binding
mode; further, these complexes can cleave pBR322 DNA [24]. Nick-
el(II) complexes of benzoic acid(2-hydroxy-benzylidine)hydrazide
Recently, several highly potent platinum metal-based drugs are
used in the cancer treatment, with cisplatin, carboplatin, and oxa-
liplatin being widely used worldwide. However, several side effects
and frequent development of resistance phenomena complicate
and hamper the clinical applications [1,2]. A very promising strat-
egy to overcome these obstacles is the use of specific carriers and
the change from platinum to other transition metals [3–5]. The
transition metal complexes play an important role in nucleic acids
chemistry for their diverse applications such as foot printing
agents, sequence specific binding, structural probes, and therapeu-
tic agents [6–10]. Nickel is one of the most essential elements for
biological systems and is present in the trace quantities [11]. The
biological activity of the nickel has been rapidly expanded due to
the increasing interest in nickel complexes, which have been
shown to act as antiepileptic, anticonvulsant agents or vitamins
or have shown antibacterial, antifungal, antimicrobial and
anticancer/antiproliferative activities [12–19]. Several reports
described the reactivity of DNA with mononuclear nickel(II) com-
plexes [20–23]. Nickel(II) complexes with 2-phenylquinoline-4-
carboxylic acid hydrazide ligand showed significant cytotoxicity
ligands bind to DNA base pairs via intercalation and p–p stacking
interactions; in vitro free radical scavenging and cytotoxic potential
have also been investigated [25]. Nickel(II) complexes containing
N-substituted heterocyclic thiosemicarbazones were found to ex-
hibit excellent DNA/protein binding and antioxidant properties;
the binding ability of these complexes varies with the N-terminal
substituents [26].
DNA binding ability and cytotoxic activity of thiourea com-
plexes have also been investigated. A new class of hybrid platinum
compounds containing acridinylthiourea as intercalating group
and its analogs exhibit promising activity toward several tumor
cell lines, and show only partial cross resistance with cisplatin
[27–29]. Acylthiourea ligands are of considerable interest to inor-
ganic chemists because of their variable coordination behavior
and promising biological activities [30–38].
A survey of the literature revealed that there is no benzoyl thio-
urea ligand reported with two chloro substituents in the benzoyl
moiety. Binzet et al. reported the synthesis and characterization of
copper(II) and nickel(II) complexes of some 4-bromo-N-(di(alkyl/
aryl)carbamothioyl)benzamide derivatives [39]. Arslan et al. pre-
pared copper(II), nickel(II) and cobalt(II) complexes of 4-chloro-N-
(di(alkyl/aryl)carbamothioyl)benzamide, and copper(II) and zinc(II)
⇑
Corresponding author. Tel.: +91 431 2503631; fax: +91 431 2500133.
E-mail address: kar@nitt.edu (R. Karvembu).
http://dx.doi.org/10.1016/j.poly.2014.03.010
0277-5387/Ó 2014 Elsevier Ltd. All rights reserved.

96
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
complexes with N-pyrrolidine-N0-(2-chlorobenzoyl)thiourea deriv-
atives[40]. Chelatingdifluoro-substitutedacylthiourealigandswere
also reported [41]. In our previous reports, we have disclosed the
synthesis and cytotoxicity of palladium(II) and nickel(II) complexes
containing 3,3-dialkyl/aryl-1-benzoylthiourea ligands [42,43]. We
report here the synthesis and characterization of six 3,3-diaryl/al-
kyl-1-(2,4-dichlorobenzoyl)thiourea ligands and their nickel(II)
complexes. We have also established the potential of these com-
plexes in DNA and protein interactions, and cytotoxicity.
2.2.3. 3,3-Dibenzyl-1-(2,4-dichlorobenzoyl)thiourea (HL3)
Yield: 76%. Greenish yellow. m.p. 168 °C. Anal. Calc. for
C
22H18Cl2N2OS (%): C, 53.18; H, 6.14; N, 7.75; S, 8.87. Found:
C, 53.22; H, 6.10; N, 7.69; S, 8.92. FTꢀIR (KBr):
, cmꢀ1 1702
(C O), 1243 (C
S), 3257 (N–H). 1H NMR (400 MHz, CDCl3):
m
d, ppm 8.70 (s, 1H), 7.57 (d, J = 6.8 Hz, 1H), 7.43 (t, J = 1.2 Hz, 1H),
7.36ꢀ7.38 (m, 10 H), 7.12 (s, 1H), 5.17 (s, 2H), 4.75 (s, 2H).
13C NMR (100 MHz, CDCl3): d, ppm 180.6 (C
S), 162.3
(C
O), 138.2, 132.2, 131.7, 131.6, 130.5, 129.1, 128.9, 128.4,
127.9, 127.8 (C6H5), 56.4, 55.9 (CH2).
2. Experimental
2.2.4. 3,3-Diphenyl-1-(2,4-dichlorobenzoyl)thiourea (HL4)
Yield: 72%. White. m.p. 161 °C. Anal. Calc. for C20H14Cl2N2OS (%):
2.1. Materials and methods
C, 59.86; H, 3.52; N, 6.98; S, 7.99. Found: C, 59.82; H, 3.55; N, 7.01;
S, 7.95%. FT-IR (KBr):
m
, cmꢀ1 1693 (C
O), 1371 (C
S), 3374
(N–H). 1H NMR (400 MHz, CDCl3): d, ppm 8.57 (s, 1H), 7.62 (t,
J = 1.2 Hz, 1H), 7.51 (t, J = 1.2 Hz, 1H), 7.42ꢀ7.48 (m, 10 H), 7.21
All the chemicals were purchased from Sigma Aldrich and used
as received. A549 (human lung cancer cell line) and HT29 (human
colon adenocarcinoma cell line) were purchased from NCCS, Pune
for cell culture experiments. Solvents were purified according to
the standard procedures. Elemental analysis was carried out on a
Vario EL-III elemental analyzer. Fourier transform infrared (FTꢀIR)
spectra were obtained on a NicoletꢀiS5 spectrophotometer as KBr
pellets. UVꢀVis spectra were recorded using a Shimadzu-2600
spectrophotometer operating in the range of 200ꢀ900 nm. Emis-
sion spectra were measured on a Jasco V-630 spectrophotometer
using 5% DMF in buffer as the solvent. NMR spectra were recorded
in CDCl3 by using TMS as an internal standard on a Bruker 400 MHz
spectrometer.
(s, 1H). 13C NMR (100 MHz, CDCl3): d, ppm 180.6 (C
S), 162.3
(C
O), 138.2, 132.2, 131.7, 131.6, 130.5, 129.1, 128.9, 128.4,
127.9, 127.8 (C6H5).
2.2.5. 3,3-Di-n-butyl-1-(2,4-dichlorobenzoyl)thiourea (HL5)
Yield: 64%. White. m.p. 158 °C. Anal. Calc. for C16H22Cl2N2OS (%):
C, 53.18; H, 6.14; N, 7.75; S, 8.87. Found: C, 53.15; H, 6.09; N, 7.78; S,
8.84%. FT-IR (KBr):
m
, cmꢀ1 1677 (C
O), 1202 (C
S), 3186 (N–
H). 1H NMR (400 MHz, CDCl3): d, ppm 8.56 (s, 1H), 7.60 (d, J = 6.4 Hz,
1H), 7.45 (s, 1H), 7.35ꢀ7.33 (dd, J = 7.6 & 1.2 Hz, 1H), 3.92 (s, 2H),
3.58 (t, J = 6.4 Hz, 2H), 1.76ꢀ1.66 (m, 4H), 1.44ꢀ1.28 (m, 4H),
0.99ꢀ0.91 (m, 6H). 13C NMR (100 MHz, CDCl3): d, ppm 178.3
(C
S), 161.9 (C
O), 137.9, 132.1, 132.0, 131.3, 130.3, 127.7
2.2. Synthesis of the ligands
(C6H5), 53.2 (CH2ꢀN), 30.1, 28.3, 20.1, 20.6 (CH2), 13.8, 13.7 (CH3).
A solution of 2,4-dichlorobenzoyl chloride (2.0946 g, 10 mmol)
in acetone (60 mL) was added drop wise to a suspension of potas-
sium thiocyanate (0.9718 g, 10 mmol) in anhydrous acetone
(60 mL). The reaction mixture was heated under reflux for 45 min-
utes and then cooled to room temperature. A solution of secondary
amine (0.7314–1.9728 g, 10 mmol) in acetone (60 mL) was added
and the resulting mixture was stirred for 2 h at 27 °C. Hydrochloric
acid (0.1 N, 500 mL) was added and the resulting white solid was
filtered off, washed with water and dried in vacuo. Single crystals
for X-ray diffraction studies were grown at room temperature from
acetonitrile solutions of the HL1, HL2 and HL3.
2.2.6. 3,3-Diisopropyl-1-(2,4-dichlorobenzoyl)thiourea (HL6)
Yield: 74%. Pale yellow. m.p. 152 °C. Anal. Calc. for C14H18Cl2N2OS
(%): C, 50.45; H, 5.44; N, 8.41; S, 9.62. Found: C, 50.37; H, 5.40; N,
8.35; S, 9.21. FTꢀIR (KBr):
m
, cmꢀ1 1664 (C
O), 1213 (C
S),
3171 (N–H). 1H NMR (100 MHz, CDCl3): d, ppm 8.35 (s, 1H), 7.62
(d, J = 8.4 Hz, 1H), 7.44 (d, J = 1.6 Hz, 1H), 7.33 (dd, J = 8.4 &
2.0 Hz, 1H), 3.42-3.31 (m, 2H), 1.71ꢀ1.29 (m, 12H). 13C NMR
(100 MHz, CDCl3): d, ppm 172.8 (C
S), 161.8 (C
O),
137.7, 132.4, 132.0, 131.3, 130.3, 127.6 (C6H5), 48.2 (CH), 20.0,
19.3 (CH3).
2.3. Synthesis of nickel(II) complexes
2.2.1. 3,3-Diethyl-1-(2,4-dichlorobenzoyl)thiourea (HL1)
3,3-Dialkyl/aryl-1-(2,4-dichlorobenzoyl)thiourea
(HL1ꢀHL6)
Yield: 82%. White. m.p. 142 °C. Anal. Calc. for C12H14Cl2N2OS (%):
C, 47.22; H, 4.62; N, 9.18; S, 10.51. Found: C, 47.19; H, 4.58; N,
(0.6080ꢀ0.85651 g, 2 mmol) dissolved in ethanol (30 mL) was
added to ethanol (30 mL) solution of NiCl2ꢁ6H2O (0.2377 g,
1 mmol). Then, NaOAc (0.1620 g, 4 mmol) dissolved in the mini-
mum amount of water was added. The reaction mixture was stir-
red at 27 °C for 2 h; a solid product was formed. The product was
isolated by filtration, washed with diethyl ether and dried in vacuo.
Suitable crystals for X-ray diffraction studies were grown at room
temperature from the dichloromethane solutions of 2 and 5.
9.15; S, 10.55%. FT-IR (KBr):
(C
S), 3237 (N–H). 1H NMR (400 MHz, CDCl3): d, ppm 8.45 (s,
1H), 7.61ꢀ7.32 (m, 3H), 3.98ꢀ3.64 (m, 4H), 1.32ꢀ1.31 (m, 6H).
13C NMR (100 MHz, CDCl3): d, ppm 177.9 (C
S), 162.2
(C O), 138.1, 132.2, 132.1, 131.5, 130.4, 127.8 (C6H5), 47.8,
m,
cmꢀ1 1654 (C
O), 1222
48.1 (CH2), 13.4, 11.4 (CH3).
2.2.2. 3,3-Diisobutyl-1-(2,4-dichlorobenzoyl)thiourea (HL2)
2.3.1. [Ni(L1)2] (1)
Yield: 68%. White. m.p. 153 °C. Anal. Calc. for C16H22Cl2N2OS (%):
Yield: 65%. Dark brown. m.p. 166 °C. Anal. Calc. for C24H26Cl4Ni
N4O2S2 (%): C, 43.21; H, 3.93; N, 8.40; S, 9.61. Found: C, 43.18;
H, 3.95; N, 8.38; S, 9.58%. UVꢀVis (5% DMF in buffer): kmax, nm
C, 61.54; H, 4.23; N, 6.52; S, 7.47. Found: C, 61.51; H, 4.19; N, 6.55;
S, 7.45. FT-IR (KBr):
m
, cmꢀ1 1701 (C
O), 1210 (C
S), 3181
(N–H). 1H NMR (400 MHz, CDCl3): d, ppm 8.65 (s, 1H), 7.62ꢀ7.60
(d, J = 8.4, 1H), 7.46ꢀ7.45 (d, J = 1.6 Hz, 1H), 7.36ꢀ7.33 (dd, J = 8.4
& 1.6 Hz, 1H), 2.36ꢀ2.29 (m, 4H), 2.16ꢀ2.09 (m, 2H), 1.02 (d,
J = 6.4 Hz, 6H), 0.91–0.93 (d, J = 6.4 Hz, 6H). 13C NMR (100 MHz,
(e
, dm3 molꢀ1 cmꢀ1) 247 (31250), 295 (43200), 371 (14924). FT-
IR (KBr):
m,
cmꢀ1 1375 (C
O), 1205 (C
S). 1H NMR
(400 MHz, CDCl3): d, ppm 7.65ꢀ7.16 (m, 6H), 3.73ꢀ3.72 (m, 8H),
1.27ꢀ1.24 (t, J = 5.6 Hz, 6H), 1.16-1.13 (t, J = 5.6 Hz, 6H). 13C NMR
CDCl3): d, ppm 179.1 (C
S), 161.3 (C
O), 137.9, 132.2,
(100 MHz, CDCl3): d, ppm 172.5 (C
S), 171.9 (C
O), 135.8,
132.0, 131.2, 130.3, 127.7 (C6H5), 61.7 (CH2ꢀN), 60.7 (CH2ꢀN),
135.2, 133.5, 132.3, 130.4, 126.5 (C6H5), 46.0, 45.5 (CH2), 13.3,
12.4 (CH3).
27.8, 26.0 (CH), 20.1, 20.0 (CH3).

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
97
2.3.2. [Ni(L2)2] (2)
current of 40 mA) fitted with a graphite monochromator in the
parallel mode (175 mm collimator with 0.5 mm pinholes). 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. The unit cell was verified by exam-
ination of the h k l overlays on several frames of data by comparing
with both the orientation matrices. No super-cell or erroneous
reflections were observed. After careful examination of the unit
cell, a standard data collection procedure was initiated using ome-
ga scans. Integrated intensity information for each reflection was
obtained by reduction of the data frames with the program APEX2
[44]. The integration method employed a three dimensional profil-
ing algorithm and all data were corrected for Lorentz and polariza-
tion factors, as well as for crystal decay effects. Finally the data
were merged and scaled to produce a suitable data set. The absorp-
tion correction program SADABS [45] was employed to correct the
data for absorption effects. Systematic reflection conditions and
statistical tests of the data suggested the space group. Solution
was obtained readily using SHELXTL (XS) [46]. Hydrogen atoms were
placed in idealized positions and were set riding on the respective
parent atoms. All non-hydrogen atoms were refined with aniso-
tropic thermal parameters. The structure was refined (weighted
least squares refinement on F2) to convergence [46,47]. Olex2
was employed for the final data presentation and structure plots
[47]. The crystal data and refinement details of ligands (HL1, HL2
and HL3) and complexes (2 and 5) are listed in Table 1 and 2
respectively.
Yield: 68%. Brown. m.p. 176 °C. Anal. Calc. for C32H42Cl4Ni
N4O2S2 (%): C, 49.32; H, 5.43; N, 7.19; S, 8.23. Found: C, 49.35;
H, 5.41; N, 7.16; S, 8.25%. UVꢀVis (5% DMF in buffer): kmax, nm
(e
, dm3 molꢀ1 cmꢀ1) 243 (32000), 299 (48400), 382 (14796).
FTꢀIR (KBr):
m
, cmꢀ1 1415 (C
O), 1211 (C
S). 1H NMR
(400 MHz, CDCl3): d, ppm 7.66 (d, J = 8.4, 2H), 7.34 (d, J = 1.6 Hz,
2H), 7.19ꢀ7.17 (dd, J = 8.4 & 1.6 Hz, 2H), 3.56 (t, J = 7.2 Hz, 8H),
2.38ꢀ2.29 (m, 2H), 2.28ꢀ2.09 (m, 2H), 0.94ꢀ0.85 (m, 6H).
13C NMR (100 MHz, CDCl3): d, ppm 174.1 (C
S), 171.6
(C
O), 137.9, 132.2, 132.0, 131.2, 130.3, 127.7 (C6H5), 59.7,
59.2 (CH2ꢀN), 27.1, 26.0 (CH), 20.3, 20.18 (CH3).
2.3.3. [Ni(L3)2] (3)
Yield: 70%. Pink. m.p. 192 °C. Anal. Calc. for C44H34Cl4NiN4O2S2
(%): C, 57.73; H, 3.74; N, 6.12; S, 7.01. Found: C, 57.68; H, 3.71;
N, 6.15; S, 6.98%. UVꢀVis (5% DMF in buffer): kmax, nm (
molꢀ1 cmꢀ1
245 (27360), 314 (39840), 386 (15200). FT-IR
(KBr): O), 1242 (C
, cmꢀ1 1410 (C S). 1H NMR (400 MHz,
CDCl3): d, ppm 7.59–7.12 (m, 26H), 4.92 (s, 4H), 4.91 (s, 4H). 13C
NMR (100 MHz, CDCl3): d, ppm 175.4 (C S), 173.1 (C O),
e
, dm3 -
)
m
136.3, 136.1, 135.1, 133.8, 132.6, 131.7, 128.9, 128.8, 127.9, 127.6
(C6H5), 53.4, 52.0 (CH2).
2.3.4. [Ni(L4)2] (4)
Yield: 65%. Pink. m.p. 194 °C. Anal. Calc. for C40H26Cl4NiN4O2S2
(%): C, 55.91; H, 3.05; N, 6.52; S, 7.46. Found: C, 55.93; H, 3.07;
N, 6.48; S, 7.42%. UVꢀVis (5% DMF in buffer): kmax, nm (
molꢀ1 cmꢀ1
241 (35520), 282 (52800), 398 (16560). FT-IR
(KBr): O), 1301 (C
, cmꢀ1 1413 (C S). 1H NMR (400 MHz,
CDCl3): d, ppm 7.67–7.23 (m, 26H). 13C NMR (100 MHz, CDCl3): d,
ppm 174.1 (C S), 172.5 (C O), 138.2, 132.2, 131.7, 131.6,
e
, dm3 -
)
m
2.5. DNA binding studies
130.5, 129.1, 128.9, 128.4, 127.9, 127.8 (C6H5).
The experiments of DNA binding with the complexes were car-
ried out in TrisꢀHCl/NaCl buffer solution (pH 7.2). Concentrated
stock solutions of CT DNA were prepared in a 5 mM TrisꢀHCl/
50 mM NaCl buffer and sonicated for 25 cycles, where each cycle
consisted of 30 s with 1 min intervals. The absorption ratio of CT
DNA solutions at kmax 260 and 280 nm was found as 1.9:1. It
showed that DNA was sufficiently free from protein impurities
[48]. The concentration of DNA was determined using UVꢀVisible
2.3.5. [Ni(L5)2] (5)
Yield: 72%. Dark brown. m.p. 178 °C. Anal. Calcd. for C32H42Cl4Ni
N4O2S2 (%): C, 49.32; H, 5.43; N, 7.19; S, 8.23. Found: C, 49.28; H,
5.46; N, 7.16; S, 8.00%. UVꢀVis (5% DMF in buffer): kmax, nm (
e,
dm3 molꢀ1 cmꢀ1) 242 (29840), 284 (40120), 392 (16880). FTꢀIR
(KBr):
m
, cmꢀ1 1412 (C
O), 1148 (C
S). 1H NMR (400 MHz,
CDCl3): d, ppm 7.65 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 1.6 Hz, 2H),
absorbance and the molar absorption coefficient (6600 Mꢀ1 cmꢀ1
)
7.18ꢀ7.16 (dd, J = 8.4
&
1.6 Hz, 2H), 3.68ꢀ3.63 (m, 8H),
values at 260 nm [49]. The absorption titrations of the complexes
(25
M in TrisꢀHCl buffer containing 0.01% DMF) against CT
DNA were performed by monitoring their absorption spectra with
M concentration.
The spectra were recorded after equilibration for 3 minutes, allow-
ing the complexes to bind to the CT DNA. Concentrated stock solu-
tions of the nickel(II) complexes (2–5) were prepared by dissolving
calculated amounts of the complexes in a 5% DMF/5 mM TrisꢀHCl/
50 mM NaCl buffer to required concentrations for all the
experiments.
1.71ꢀ1.52 (m, 8H), 1.39ꢀ1.24 (m, 8H), 0.97ꢀ0.93 (t, J = 7.2 Hz,
l
6H), 0.90ꢀ0.86 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3): d,
ppm 172.8 (C
S), 171.7 (C
O), 135.7, 135.2, 133.4, 132.2,
incremental addition of CT DNA within 0ꢀ50
l
130.3, 126.4 (C6H5), 51.6, 50.9 (CH2ꢀN), 30.1, 29.2, 20.2, 20.1
(CH2), 13.8 (CH3).
2.3.6. [Ni(L6)2] (6)
Yield: 64%. Brown. m.p. 170 °C. Anal. Calc. for C28H34Cl4Ni
N4O2S2 (%): C, 46.50; H, 4.74; N, 7.75; S, 8.87. Found: C, 46.47; H,
4.56; N, 7.59; S, 8.81%. UVꢀVis (5% DMF in buffer): kmax, nm
(e
, dm3 molꢀ1 cmꢀ1) 246 (26240), 288 (38150), 376 (15478).
FTꢀIR (KBr):
m
, cmꢀ1 1412 (C
O), 1198 (C
S). 1H NMR
2.6. Competitive binding with ethidium bromide (EB)
(400 MHz, CDCl3): d, ppm 7.50 (d, J = 7.6 Hz, 2H), 7.32
(d, J = 1.2 Hz, 2H), 7.16ꢀ7.15 (dd, J = 7.6 & 1.2 Hz, 2H), 3.76ꢀ3.45
(m, 4H), 1.38ꢀ1.19 (m, 24H). 13C NMR (100 MHz, CDCl3): d, ppm
The apparent binding constant (Kapp) and quenching constant
(Kq) values of the complexes (2–5) were determined by a fluores-
cence spectral technique using EB-bound CT DNA solution in
TrisꢀHCl buffer (pH 7.2). The changes in fluorescence intensities
at 596 nm (510 nm excitation) of EB bound to DNA were recorded
with an increasing amount of the nickel(II) complex concentration
until maximum reduction in the intensity of fluorescence occurred.
In the presence of CT DNA, EB showed enhanced emission intensity
due to its intercalative binding to DNA. A competitive binding of
the nickel(II) complexes to CT DNA resulted in the displacement
of the bound EB and decrease of its emission intensity [50].
172.4 (C
S), 171.5 (C
O), 136.0, 135.1, 131.0, 130.9, 130.2,
126.3 (C6H5), 65.8, 52.7 (CH), 20.0, 19.8 (CH3).
2.4. Single crystal X-ray diffraction studies
A Bruker APEX2 X-ray (three-circle) diffractometer was em-
ployed for crystal screening, unit cell determination, and data col-
lection. The X-ray radiation employed was generated from a Mo
sealed X-ray tube (K = 0.70173 Å with a potential of 40 kV and a
a

98
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
Table 1
Crystal data and structure refinement for ligands.
Compound
HL1
HL2
HL3
Empirical formula
Formula weight
T (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C
12H14Cl2N2OS
C16H22Cl2N2OS
361.32
110(2)
C23H19.50Cl2N2.50OS
449.87
110(2)
305.21
150(2)
0.71073
monoclinic
P2(1)/c
0.71073
triclinic
0.71073
triclinic
ꢀ
ꢀ
P1
P1
14.225(5)
8.113(3)
10.136(7)
b (Å)
10.617(4)
10.561(4)
11.864(8)
c (Å)
9.986(3)
11.708(4)
18.820(13)
a
(°)
90
107.538(4)
102.749(7)
b (°)
108.166(4)
108.655(4)
96.811(8)
c
(°)
90
1432.9(8)
98.420(3)
872.9(5)
102.727(8)
2120(2)
V (Å3)
Z
4
2
4
Dcalc (Mg/m3)
1.415
0.588
632
1.375
0.494
380
1.410
0.424
932
Absorption coefficient (mmꢀ1
F(000)
)
Crystal size (mm3)
0.64 ꢂ 0.45 ꢂ 0.09
0.48 ꢂ 0.42 ꢂ 0.38
0.56 ꢂ 0.40 ꢂ 0.40
h Range for data collection (°)
2.44–27.50
2.10–27.49
1.13–27.50
Index ranges
ꢀ18 6 h 6 18, ꢀ13 6 k 6 13,
ꢀ12 6 l 6 12
ꢀ10 6 h 6 10, ꢀ13 6 k 6 13,
ꢀ15 6 l 6 15
10062
ꢀ13 6 h 6 13, -15 6 k 6 15,
ꢀ24 6 l 6 24
Reflections collected
23021
24513
Independent reflections (Rint
Completeness to theta = 27.50° (%)
Absorption correction
Maximum and minimum transmission
Refinement method
)
3264 (0.0298)
99.2
semi-empirical from equivalents
0.9490 and 0.7047
3953 (0.0157)
98.5
semi-empirical from equivalents
0.8344 and 0.7973
full-matrix least-squares on F2
3953/0/203
9594 (0.0178)
98.6
semi-empirical from equivalents
0.8487 and 0.7972
Data/restraints/parameters
3264/0/165
1.029
9594/0/533
1.026
Goodness-of-fit (GOF) on F2
1.059
Final R indices [I > 2
R indices (all data)
r
(I)]
R1 = 0.0257, wR2 = 0.0646
R1 = 0.0298, wR2 = 0.0676
0.274 and ꢀ0.243
R1 = 0.0262, wR2 = 0.0680
R1 = 0.0290, wR2 = 0.0693
0.393 and ꢀ0.205
R1 = 0.0314, wR2 = 0.0770
R1 = 0.0353, wR2 = 0.0791
0.738 and ꢀ0.627
Largest difference in peak and hole
(e Åꢀ3
)
Table 2
Crystal data and structure refinement for complexes.
Compound
2
5
Empirical formula
Formula weight
T (K)
C
32H42Cl4N4NiO2S2
C32H42Cl4N4NiO2S2
779.33
110(2)
779.33
110(2)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
0.71073
triclinic
P1
0.71073
monoclinic
P2(1)/c
ꢀ
8.919(4)
14.858(7)
15.272(7)
71.740(5)
75.954(5)
77.447(5)
1842.5(14)
2
8.6176(14)
16.711(3)
25.336(4)
90
95.928(2)
90
3629.2(10)
4
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dcalc (Mg/m3)
1.405
0.964
812
1.426
0.979
1624
Absorption coefficient (mmꢀ1
F(000)
)
Crystal size (mm3)
0.58 ꢂ 0.40 ꢂ 0.18
0.58 ꢂ 0.58 ꢂ 0.35
h Range for data collection (°)
Index ranges
Reflections collected
2.31–27.49
2.02–27.50
ꢀ11 6 h 6 11, ꢀ19 6 k 6 19, ꢀ19 6 l 6 19
21505
ꢀ11 6 h 6 11, ꢀ21 6 k 6 21, ꢀ32 6 l 6 31
35094
Independent reflections (Rint
)
8346 (0.0283)
8322 (0.0306)
Completeness to theta = 27.49° (%)
Absorption correction
Maximum and minimum transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
98.6
99.9
semi-empirical from equivalents
0.8456 and 0.6047
full-matrix least-squares on F2
8346/9/426
semi-empirical from equivalents
0.7256 and 0.6005
full-matrix least-squares on F2
8322/0/410
1.061
1.077
Final R indices [I > 2
r
(I)]
R1 = 0.0357, wR2 = 0.0837
R1 = 0.0442, wR2 = 0.0873
1.435 and ꢀ0.434
R1 = 0.0313, wR2 = 0.0670
R1 = 0.0367, wR2 = 0.0688
0.289 and ꢀ0.367
R indices (all data)
Largest difference in peak and hole (e Åꢀ3
)

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
99
2.7. Protein interaction studies
The excitation wavelength of BSA at 280 nm and the emission at
344 nm were monitored for the protein binding studies using fluo-
rescence spectra recorded with nickel(II) complexes (2–5). The
excitation and emission slit widths, and scan rates were constantly
maintained for all the experiments. Samples were carefully de-
gassed using pure nitrogen gas for 15 min by using quartz cells
(4 ꢂ 1 ꢂ 1 cm3) with high vacuum Teflon stopcocks. Stock solution
of BSA was prepared with phosphate buffer (50 mM, pH 7.2) and
stored in dark at 4 °C for further use. Concentrated stock solutions
of each test compound were prepared by dissolving them in DMF–
phosphate buffer (5:95) and diluted with phosphate buffer to get
required concentrations. 2.5 ml of BSA solution (1 lM) was titrated
by successive additions of a 10ꢀ6 M stock solution of the complexes
using a micropipette. Synchronous fluorescence spectra were also
recorded using the same concentration of BSA and the complexes
as mentioned above with two different
Dk (difference between
the excitation and emission wavelengths of BSA) values such as
15 and 60 nm.
Fig. 1. Molecular structure of HL1.
2.8. MTT assay
Nt is the absorbance of the cells treated with sample and Nc is the
absorbance of the untreated cells.
MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium]
assay was performed to evaluate the viability of the cells due to
the effect of the samples tested and it is a colorimetric test based
on the selective ability of viable cells to reduce the tetrazolium
component of MTT in to purple colored formazan crystals. Two dif-
ferent dilutions were prepared by dilution with DMSO and media.
To test the biocompatibility of the samples, cells were seeded at a
density of 10000 cells/well into a 96 well plate. After attaining 90%
confluency, the cells were incubated with different concentration
of the samples for a period of 24 h. Cells in media alone devoid
of test samples acted as a positive control and wells treated with
Triton X-100 (1%) as a negative control. The cells were incubated
with MTT solution for 4 h followed by 1 h incubation with solubili-
sation buffer. Then the absorbance of the solution was measured at
a wavelength of 570 nm using a Beckmann Coulter Elisa plate
reader (BioTek Power Wave XS). Triplicate samples were analyzed
for each experiment. Cell viability was expressed as the percentage
of the negative control calculated as, Viability (%) = (Nt/Nc) ꢂ 100;
3. Results and discussion
3.1. Synthesis
Dichloro substituted benzoyl thiourea ligands were synthesized
from 2,4-dichlorobenzoyl chloride, potassium thiocyanate and the
corresponding secondary amine in acetone (Scheme 1). The square
planar nickel(II) complexes were synthesized from the reactions
between NiCl2ꢁ6H2O and the ligands in ethanol (Scheme 2). All
the ligands and their corresponding complexes were obtained in
good yield and were characterized by elemental analyses, and
UVꢀVis, FT-IR, 1H and 13C NMR spectroscopic techniques. The
structures of the representative compounds were confirmed by
single crystal X-ray diffraction studies. Investigations showed that
3,3-dialkyl/aryl-1-(2,4-dichlorobenzoyl)thioureas act as mono an-
ionic bidentate ligands in the present case.
O
S
Cl
O
Cl
Cl
O
S
C
R
KSCN
HNR2
N
H
N
R
N
Cl
Acetone
Acetone
Reflux, 30 min
Cl
Cl
Cl
27 oC, 2 h
R = C2H5 (HL1), CH2CH(CH3)2 (HL2),
CH2C6H5 (HL3), C6H5 (HL4),
n-C4H9 (HL5) or CH(CH3)2 (HL6)
Scheme 1. Synthesis of ligands.
Cl
Cl
Cl
O
S
Cl
Cl
O
S
R
Ethanol / NaOAc
2 h, 27 oC, Stirring
O
N
N
R
NiCl2 6H2O
+
H
Ni
N
N
N
N
Cl
S
R
R
R
R
1
2
3
R = C2H5 ( ), CH2CH(CH3)2 ( ), CH2C6H5 ( ),
C6H5 (4), n-C4H9 (5) or CH(CH3)2 (6)
Scheme 2. Synthesis of Ni(II) complexes.

100
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
3.2. Spectroscopy
ligands showed N–H stretching vibrations at 3161ꢀ3257 cmꢀ1
,
which disappeared in the corresponding nickel(II) complexes
indicating enolization followed by deprotonation prior to coordi-
nation. This is consistent with the observation made in 1H NMR
spectral studies. The signals of the N–H protons were observed
as singlet at d 8.35ꢀ8.70 in the spectra of ligands, which disap-
peared in that of nickel(II) complexes. The resonances of aromatic
and aliphatic protons of the ligands were appeared at d 7.12–7.63
and d 0.91–5.17 respectively. The signals due to aromatic and ali-
phatic protons did not undergo a significant shift in the nickel(II)
complexes. The occurrence of sharp and non-shifted peaks in the
1H NMR spectra of all the complexes reveals the diamagnetic
The electronic spectra of the complexes measured in 5% DMF in
buffer showed three bands in the regions 241ꢀ247, 282ꢀ314 and
382ꢀ398 nm, which were assigned to intra ligand, charge-transfer
(L ? M), and forbidden d ? d transitions respectively. In the FTꢀIR
spectra of all the ligands, bands were observed at 1654ꢀ1702 and
1222ꢀ1265 cmꢀ1, which were assigned to C = O and C = S stretch-
ing vibrations respectively. These bands underwent a shift after
coordination of the ligands with nickel via O and S donor atoms
and appeared respectively at 1376ꢀ1440 and 1157ꢀ1243 cmꢀ1
in the spectra of the complexes. The FT-IR spectra of the acylthiourea
Fig. 2. Molecular structure of HL2. (The amide hydrogen is eclipsed behind the nitrogen atom)
Fig. 3. Molecular structure of HL3.

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
101
nature of the low-spin Ni(II) (d8) ion with a square planar
3.3. Crystal structures
geometry. The resonances due to carbons in the 13C NMR spectra
of the ligands and the complexes were observed in the expected
regions.
The ORTEP diagrams of ligands (HL1, HL2 and HL3) and com-
plexes (2 and 5) with the atomic labeling schemes are shown in
Fig. 4. Molecular structure of 2.
Fig. 5. Molecular structure of 5.

102
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
the Figs. 1–5. Selected interatomic bond lengths and bond angles
are in Table 3. Half a molecule of CH3CN was found solvated per
molecule of the HL3. C19–C21 atoms in 5 were found disordered
between two positions and were modeled successfully with a ratio
of 0.74:0.26. Structural analysis revealed that 2 and 5 have a square
planar geometry with a cis-NiO2S2 chromophore, in which two acy-
lthiourea (HL) ligands were coordinated to nickel centre as mono
for 2 is 0.028, and for 5 it is 0.0196, suggesting square planar geom-
etry around Ni [51]. Comparing the C–O and C–S bond lengths of 2
with HL2, there was a considerable elongation of these bonds in the
complex. On the other hand, N–C bonds of the chelate rings in 2
were short compared to the N–C bond distances of the correspond-
ing ligand. These results are consistent with the fact that the ligand
underwent deprotonation during the reaction and coordinated to
nickel ion via O and S donor atoms. Further, there was a significant
anionic bidentate ligands. Further, the structural index value (s4
)
1.0
1.0
(B)
(A)
0.8
0.8
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
250
300
350
400
450
500
550
250
300
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
(C)
(D)
250
300
350
400
450
500
550
250
300
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
Fig. 6. Absorption spectra of complexes 2 (A), 3 (B), 4 (C) and 5 (D) in Tris–HCl buffer upon addition of CT-DNA. [Complex] = 2.5 ꢂ 10ꢀ5 M, [DNA] = 0–50
lM. Arrow shows
that the absorption intensities decrease upon increasing DNA concentration.
Table 3
Selected bond lengths (Å) and angles (°).
Ligand
HL1
HL2
HL3
Complex
2
5
Cl(1)–C(4)
S(1)–C(8)
O(1)–C(7)
N(1)–C(7)
N(1)–C(8)
N(1)–H(1)
N(2)–C(8)
N(2)–C(9)
N(2)–C(11,13, 16)
C(7)–N(1)–C(8)
C(8)–N(1)–H(1)
C(8)–N(2)–C(9)
C(9)–N(2)–C(11)
C(1,5)–C(2,4)–Cl(1)
O(1)–C(7)–N(1)
O(1)–C(7)–C(1)
N(1)–C(7)–C(1)
N(2)–C(8)–N(1)
N(2)–C(8)–S(1)
N(1)–C(8)–S(1)
1.7367(14)
1.6670(13)
1.2283(14)
1.3486(15)
1.4343(15)
0.8470
1.3245(16)
1.4655(16)
1.4783(16)
121.70(9)
117.0
121.22(10)
115.30(10)
120.80(9)
122.64(11)
121.00(11)
116.32(10)
115.15(11)
126.45(9)
118.38(9)
1.7317(13)
1.6819(12)
1.2107(15)
1.3906(15)
1.4057(15)
0.8999
1.3263(15)
1.4750(15)
1.4776(15)
122.28(10)
118.5
1.7338(17)
1.6796(16)
1.2088(18)
1.3868(18)
1.4032(19)
0.8800
1.3271(19)
1.4740(18)
1.4646(18)
123.10(12)
118.5
124.87(12)
120.94(11)
118.56(12)
122.54(13)
124.04(13)
113.34(12)
116.92(12)
125.39(11)
117.67(10)
Ni(1)–O(1)
Ni(1)–O(2)
Ni(1)–S(2)
Ni(1)–S(1)
Cl(1)–C(3)
S(1)–C(8)
O(1)–C(1)
N(1)–C(1)
1.8500(16)
1.8535(15)
2.1391(11)
2.1472(9)
1.739(2)
1.737(2)
1.263(2)
1.319(2)
1.352(2)
83.30(6)
177.89(5)
94.89(5)
94.89(5)
178.06(5)
86.93(3)
108.78(7)
109.67(7)
133.38(13)
133.00(14)
1.8609(12)
1.8459(12)
2.1322(5)
2.1426(5)
1.7311(17)
1.7271(17)
1.2633(19)
1.318(2)
1.345(2)
83.73(5)
177.63(4)
94.25(4)
95.89(4)
179.61(4)
86.13(2)
108.10(6)
109.32(6)
131.85(11)
134.42(11)
N(1)–C(8)
O(1)–Ni(1)–O(2)
O(1)–Ni(1)–S(2)
O(2)–Ni(1)–S(2)
O(1)–Ni(1)–S(1)
O(2)–Ni(1)–S(1)
S(2)–Ni(1)–S(1)
C(8)–S(1)–Ni(1)
C(17)–S(2)–Ni(1)
C(1)–O(1)–Ni(1)
C(26)–O(2)–Ni(1)
120.10
115.31(9)
118.39(9)
122.54(11)
122.60(10)
114.73(10)
117.46(10)
124.14(9)
118.38(9)

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
103
the charge transfer band (282ꢀ314 nm) shows increasing hypo-
chromism (
, 19ꢀ54%) with a small red shift. As the extent of
6
5
4
3
2
1
0
D
e
(2)
(3)
(4)
hypochromism is commonly associated with the strength of DNA
interaction, the observed decrease in order of hypochromism,
2 > 5 > 3 > 4, reflects the decrease in DNA binding affinities of the
complexes in this order. These results proposed association of
the compounds with CT DNA, and it also probably that these com-
pounds interact with the helix via intercalation. The DNA binding
affinities of the complexes are compared quantitatively by obtain-
ing the intrinsic binding constant Kb using the following equation:
(
5)
½DNAꢃ=ðea
ꢀ
ef Þ ¼ ½DNAꢃ=ðeb
ꢀ
ef Þ þ 1=Kbðea
ꢀ
ef
Þ
where [DNA] is the concentration of DNA in base-pairs, ea is the
apparent extinction coefficient obtained by calculating absorbance/
[complex], ef is the extinction coefficient of the complex in its free
5
10
15
20
25
30
form, and
form. Each set of data, when fitted into the above equation, gave a
straight line with a slope of 1/(eb f) and an intercept of 1/Kb(eb ef)
eb is the extinction coefficient of the complex in the bound
[DNA] (μM)
ꢀ
e
ꢀ
Fig. 7. Plots of [DNA]/(ea
ꢀ ef) vs. [DNA] for the titration of the complexes with CT
and the value of Kb was determined from the ratio of slope to inter-
cept (Fig. 7). The observed Kb values (2.51–7.57 ꢂ 104 Mꢀ1, Table 4)
of the nickel(II) complexes are higher than those of previously re-
ported [53–56]. The complexes 2 (Kb = 7.57 ꢂ 104 Mꢀ1) and 5
(Kb = 5.62 ꢂ 104 Mꢀ1) showed higher DNA binding affinity than the
complexes 3 (Kb = 2.51 ꢂ 104 Mꢀ1) and 4 (Kb = 2.91 ꢂ 104 Mꢀ1).
Interestingly, the complex 2 showed better DNA binding property
than the complex 5; this may be due to the rigid nature of the isobutyl
group, which favors stronger interaction with DNA [57].
DNA.
delocalization of p-electron density over the six atoms comprising
the respective chelate rings in the complex. The coordination geom-
etries and bonding characteristics of 2 and 5 are similar to that of
previously reported nickel(II) acylthiourea complexes [43].
3.4. DNA binding studies
3.4.1. Electronic absorption titration
3.4.2. Fluorescence spectroscopic studies
Absorption titration technique has been used to monitor the
mode of interaction of complexes (2–5) with CT DNA (Fig. 6)
[52]. Upon the incremental addition of CT DNA to the complexes
In an effort to understand the interaction pattern of the com-
plexes with CT DNA more clearly, fluorometric competitive binding
experiment was carried out using EB as a probe. The intrinsic
600
600
EB + DNA
EB + DNA
(B)
(A)
500
500
400
300
400
300
200
200
100
0
EB
EB
100
0
550
600
650
700
550
600
650
700
Wavelength (nm)
Wavelength (nm)
600
500
400
300
200
100
0
EB + DNA
EB + DNA
600
500
400
300
200
100
0
(D)
(C)
EB
EB
550
600
650
700
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Fig. 8. Fluorescence quenching curves of EB bound to DNA in the presence of complexes 2 (A), 3 (B), 4 (C) and 5 (D). [DNA] = 5 lM, [EB] = 5 lM and [complex] = 0–30 lM.

104
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
Table 4
fluorescence intensities of DNA and that of EB in TrisꢀHCl buffer
are low. However, EB emits intense fluorescent light in the pres-
ence of DNA due to its strong intercalation between the adjacent
DNA base pairs [58,59]. If the complexes can intercalate into
DNA, the binding sites of DNA available for EB will be decreased,
hence the fluorescence intensity of EB will be quenched. This is a
proof that the complexes intercalate between the base pairs of
DNA [60,61]. The quenching extent of fluorescence of EB bound
DNA was used to determine the extent of binding between the
complex and DNA. The emission spectra of EB binding to DNA in
the absence and presence of the complexes are given in Fig. 8.
The addition of the complexes to DNA pretreated with EB causes
an appreciable reduction in the emission intensity, indicating the
replacement of EB by the complexes. The SternꢀVolmer quenching
constant [62] for each complex was calculated using the equation
given as Fo/F = 1 + Kq [Q]. Here, Fo and F are the fluorescence inten-
sity in the absence and presence of complexes, respectively, Kq is a
linear SternꢀVolmer quenching constant, and [Q] is the total con-
centration of a complex to that of DNA. The value of Kq is given by
the ratio of slope to intercept in a plot of Fo/F versus [Q] (Fig. 9). The
quenching constant values are listed in Table 4. The apparent bind-
ing constant (Kapp) values of DNA were calculated by using the
equation KEB [EB] = Kapp [complex], where the complex concentra-
tion is the values at 50% reduction in the fluorescence intensity of
EB, KEB (1.0 ꢂ 107 Mꢀ1) is the DNA binding constant of EB (Table 4).
Complexes 2 and 5 represented a slightly more pronounced
quenching effect on fluorescence intensity of EB and DNA, under-
scoring its tighter binding to DNA as compared to the rest of com-
plexes (3 and 4). The observed quenching constant and binding
constant values of the nickel(II) complexes proposed that the com-
plexes interact with CT DNA through intercalation.
DNA binding constant (Kb), quenching constant (Kq) and apparent binding constant
(Kapp) values.
Complex
Kb (Mꢀ1
)
Kq (Mꢀ1
)
Kapp (Mꢀ1
)
2
3
4
5
7.57 ꢂ 104
2.51 ꢂ 104
2.91 ꢂ 104
5.62 ꢂ 104
2.86 ꢂ 104
2.16 ꢂ 104
2.19 ꢂ 104
2.69 ꢂ 104
1.43 ꢂ 106
1.08 ꢂ 106
1.09 ꢂ 106
1.35 ꢂ 106
2.0
1.8
1.6
1.4
1.2
1.0
(2)
(3)
(4)
(5)
5
10
15
20
25
30
[Q] (μM)
Fig. 9. Stern-Volmer plots of fluorescence titrations of the complexes with CT DNA.
700
(A)
(B)
700
600
600
500
400
300
200
100
0
500
400
300
200
100
0
300
325
350
375
400
425
450
300
350
400
450
Wavelength (nm)
Wavelength (nm)
700
600
500
400
300
200
100
0
800
700
600
500
400
300
200
100
0
(C)
(D)
300
350
400
450
300
350
400
450
Wavelength (nm)
Wavelength (nm)
Fig. 10. Fluorescence quenching curves of BSA in the absence and presence of complexes 2 (A), 3 (B), 4 (C) and 5 (D). [BSA] = 1 lM and [complex] = 0–30 lM.

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
105
3.5. Protein binding studies
0.4
(
2
)
3)
)
3.5.1. Fluorescence quenching of BSA by nickel(II) complexes
(
(
(
0.2
0.0
Bovine serum albumin (BSA) is the most extensively studied
serum albumin, owing to its structural homology with human ser-
um albumin (HSA). It is well known that the transport of drugs
through the bloodstream is affected via the interaction of drugs
with blood plasma proteins. Binding to these proteins may lead
to loss or enhancement of the biological properties of the original
compounds, or may provide paths for their transportation. Fluores-
cence quenching analyses are usually employed to analyze the
interaction of chemical compounds with BSA. A solution of BSA
4
5
)
-0.2
-0.4
-0.6
-0.8
(1
(0–30
l
M) was titrated with various concentrations of the complexes
M). Fluorescence spectra were recorded in the range from
l
290 to 450 nm upon excitation at 280 nm. The effect of complexes
(2–5) on the fluorescence spectrum of BSA is shown in Fig. 10.
Upon the addition of the nickel(II) complexes to the solution of
BSA, fluorescence intensity at 344 nm decreases up to 74.5%,
58.4%, 46.6% and 64.8%, from the initial intensity of BSA, accompa-
nied by a hypsochromic shift of 6, 4 and 5 nm for complexes 2, 3
and 5 respectively, and bathochromic shift of 4 nm for complex
4. The observed hypochromicity with blue or red shift has revealed
that the complexes interact hydrophobically with the BSA protein
[63]. The fluorescence quenching data were analyzed by Stern–
Volmer and Scatchard equations. The quenching constant (Kq)
can be calculated using the plot of log (Fo/F) versus log [Q]
(Fig. 11). If it is assumed that the binding of complexes with BSA
occurs at equilibrium, the equilibrium binding constant can be
analyzed according to the Scatchard equation log [(Fo–F)/F] = log
-5.7 -5.6 -5.5 -5.4 -5.3 -5.2 -5.1 -5.0 -4.9
log [Q] (μM)
Fig. 12. Scatchard plots of the fluorescence titrations of the complexes with BSA.
0.7
BSA
0.6
0.5
0.4
0.3
0.2
0.1
0.0
BSA + 2
BSA + 3
BSA + 4
BSA + 5
K
b+n log [Q], where Kb is the binding constant of the compound
with BSA and n is the number of binding sites. From the plot of
log [(Fo–F)/F] versus log [Q] (Fig. 12), the number of binding sites
(n) and the binding constant (Kb) values have been obtained. The
calculated Kq, Kb and n values are given in Table 5. The calculated
value of n is around 1 for all of the complexes. The values of Kq
and Kb suggested the binding of complexes with BSA. Among the
four complexes, complex 2 exhibited better interaction with BSA
compared to other complexes (3, 4 and 5).
Generally, quenching occurs through either dynamic or static
mode. The dynamic quenching is a process in which the fluoro-
phore and the quencher come into contact during the transient
existence of the excited state, whereas static quenching refers to
the formation of flourophore–quencher complex in the ground
state. The easiest method to determine the type of quenching is
240
260
280
300
320
Wavelength (nm)
Fig. 13. The absorption spectra of BSA (10 lM) and BSA with complexes 2-5 (4 lM).
Table 5
Protein binding constant (Kb), quenching constant (Kq) and number of binding sites
(n) values.
Complex
Kb (Mꢀ1
)
Kq (Mꢀ1
)
n
2
3
4
5
4.07 ꢂ 105
4.61 ꢂ 104
4.41 ꢂ 104
1.48 ꢂ 105
1.85 ꢂ 105
7.47 ꢂ 104
5.71 ꢂ 104
1.14 ꢂ 105
1.11
0.94
0.91
0.97
3.5
(2)
3.0
(3)
(4)
(5)
2.5
UV–Vis absorption spectroscopy. The UV–Vis spectra of BSA in
the absence and the presence of complexes are shown in Fig. 13,
which indicates that the addition of nickel(II) complexes to a fixed
concentration of BSA led to a gradual increase in the intensity of
BSA absorption but at the same wavelength due to the interaction
between test compounds and protein. In other words, the fluores-
cence quenching between complexes and BSA is mainly ascribed to
be static quenching [64]. The above results revealed the strong
interaction between nickel(II) complexes and BSA that caused a
change in the conformation of BSA.
2.0
1.5
1.0
2
4
6
8
10
12
[Q] (μM)
3.5.2. Characteristics of synchronous fluorescence spectra
Synchronous fluorescence spectroscopy provides information
on the molecular microenvironment, particularly in the vicinity
Fig. 11. Stern-Volmer plots of the fluorescence titrations of the complexes with
BSA.

106
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
400
(A)
(B)
400
300
200
100
0
300
200
100
0
280
290
300
310
320
280
290
300
310
320
Wavelength (nm)
Wavelength (nm)
400
300
200
100
0
400
(D)
(C)
300
200
100
0
280
290
300
310
320
280
290
300
310
320
Wavelength (nm)
Wavelength (nm)
Fig. 14. Synchronous spectra of BSA (1
lM) as a function of concentration of the complexes 2 (A), 3 (B), 4 (C) and 5 (D) (0–30
lM) with Dk = 15 nm.
700
700
(A)
(B)
600
600
500
400
300
200
100
0
500
400
300
200
100
0
325
350
375
400
325
350
375
400
Wavelength (nm)
Wavelength (nm)
700
600
500
400
300
200
100
0
700
600
500
400
300
200
100
0
(C)
(D)
325
350
375
400
325
350
375
400
Wavelength (nm)
Wavelength (nm)
Fig. 15. Synchronous spectra of BSA (1
l
M) as a function of concentration of the complexes 2 (A), 3 (B), 4 (C) and 5 (D) (0–30
l
M) with Dk = 60 nm.

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
107
of the fluorophore functional groups [65]. The fluorescence of BSA
both 15 and 60 nm
D
k with the addition of the nickel(II) complexes
might be due to the presence of tyrosine, tryptophan and phenyl-
alanine residues. According to Miller [66], the difference between
(2–5) in various concentrations to understand the structural
changes occurred in BSA (Figs. 14 and 15). While increasing the
concentration of the complexes, the intensity of emission corre-
sponding to tyrosine (at 302 nm) was found to decrease in the
magnitude of 43.7%, 31.6%, 24.8% and 37.4% for complexes 2, 3, 4
and 5 respectively without any shift in emission wavelength. The
excitation and emission wavelengths (
chromophores. Large (ꢄ60 nm) and small (ꢄ15 nm)
D
k) reflects the nature of
k values
D
are characteristic of tryptophan and tyrosine residues respectively
[67]. Synchronous fluorescence spectra of BSA were recorded at
Fig. 16. Cytotoxicity of HL1–HL6 (A and B) and 1–6 (C and D) after 24 h incubation on A549 cell lines.

108
N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
tryptophan fluorescence emission showed significant decrease in
the intensity (at 342 nm) of about 77.5%, 62.7%, 50.4% and 66.8%
for complexes 2, 3, 4 and 5 respectively, without any change in
the position of the emission band. These experimental results indi-
cate that although the complexes affected the microenvironments
of both tyrosine and tryptophan during the binding process, the
effect was more towards tryptophan than tyrosine.
3.6. Cytotoxic activity
The cytotoxicity of the ligands (HL1-HL6) and their nickel(II)
complexes (1–6) toward A549 (human lung cancer) and HT29 cells
(human colon adenocarcinoma) has been examined in comparison
with cisplatin (IC50 = 25
MTT assay [26]. Fig. 16 depicts the cytotoxic effect of the ligands
lM) under identical conditions by using
Fig. 17. Cytotoxicity of HL1–HL6 (A and B) and 1–6 (C and D) after 24 h incubation on HT29 cell lines.

N. Selvakumaran et al. / Polyhedron 75 (2014) 95–109
109
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4. Conclusion
In conclusion, we have synthesized and characterized new nick-
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urea. UV–Vis and fluorescence spectral studies indicated the
binding of the complexes with CT DNA. Fluorescence quenching
experiments revealed the interaction of the complexes with BSA
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Appendix A. Supplementary material
Crystallographic data for the structures reported in this paper
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