Synthesis, crystal structure and bio-macromolecular interaction studies of pyridine-based thiosemicarbazone and its Ni(ii) and Cu(ii) complexes

Article abstract of DOI:10.1039/c3ra40904j

A new pyridine-based heterocyclic thiosemicarbazone ligand and its Ni(ii) and Cu(ii) complexes have been synthesized and characterized by structural, analytical and spectral methods. The mono-deprotonated anionic form of the ligand coordinates via NNS donor atoms to yield an octahedral Ni(ii) complex and distorted square planar Cu(ii) complex. UV-visible and fluorescence-based spectroscopic techniques revealed that both metal complexes interact with double stranded DNA via intercalation. A comparative assessment indicated that the Ni(ii) complex displayed superior DNA binding. The interaction of these compounds with bovine serum albumin (BSA) suggested that the ligand and its Cu(ii) complex quenched the intrinsic fluorescence of BSA in a static quenching process, whereas for the Ni(ii) complex, fluorescence quenching of BSA was a combination of both static and collision/dynamic quenching processes. The quenching of the fluorescence of BSA is owing to energy transfer from the tryptophan residues of BSA to the compounds bound to BSA. Cytotoxicity tests based on the standard MTT assay revealed that the Cu(ii) complex displayed prominent anti-proliferative activity against HeLa cells.

Full text of DOI:10.1039/c3ra40904j

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PAPER
Synthesis, crystal structure and bio-macromolecular
interaction studies of pyridine-based
Cite this: RSC Advances, 2013, 3,
1
4088
thiosemicarbazone and its Ni(II) and Cu(II) complexes3
a
b
a
b
Arghya Basu, Durairaj Thiyagarajan, Chirantan Kar, Aiyagari Ramesh*
a
and Gopal Das*
A new pyridine-based heterocyclic thiosemicarbazone ligand and its Ni(II) and Cu(II) complexes have been
synthesized and characterized by structural, analytical and spectral methods. The mono-deprotonated
anionic form of the ligand coordinates via NNS donor atoms to yield an octahedral Ni(II) complex and
distorted square planar Cu(II) complex. UV-visible and fluorescence-based spectroscopic techniques
revealed that both metal complexes interact with double stranded DNA via intercalation. A comparative
assessment indicated that the Ni(II) complex displayed superior DNA binding. The interaction of these
compounds with bovine serum albumin (BSA) suggested that the ligand and its Cu(II) complex quenched
the intrinsic fluorescence of BSA in a static quenching process, whereas for the Ni(II) complex, fluorescence
quenching of BSA was a combination of both static and collision/dynamic quenching processes. The
quenching of the fluorescence of BSA is owing to energy transfer from the tryptophan residues of BSA to
the compounds bound to BSA. Cytotoxicity tests based on the standard MTT assay revealed that the Cu(II)
complex displayed prominent anti-proliferative activity against HeLa cells.
Received 21st February 2013,
Accepted 20th May 2013
DOI: 10.1039/c3ra40904j
www.rsc.org/advances
practices. Recently, Natarajan and coworkers have shown that
metal complexes derived from thiosemicarbazones with a
Introduction
Thiosemicarbazones constitute a class of ligand which holds
considerable interest to medicinal chemists owing to their
2-oxoquinoline heterocycle unit exhibit outstanding DNA/
protein binding and antitumor activities, and this improved
biological activity could be attributed to the presence of the
1
,2
therapeutic potential. Over the last few decades, thiosemi-
carbazones and their transition metal complexes have received
substantial attention not only due to their coordination
16–18
heterocycles present in the thiosemicarbazones.
The development of transition metal complexes with DNA
binding abilities has been an active area of research due to
their potential applications as metallo-drugs, tools for mole-
cular biology, a regulator of gene expression and others.
Recently, substantial numbers of studies have been carried out
on transition metal complexes for their ability to mediate
oxidative damage to nucleobases and/or to the 2-deoxyribose
moiety in the presence of an oxidizing or reducing agent, light
2
behavior, but also their valuable pharmacological properties,
such as antitumor, antibacterial, antiviral and antimalarial
3
–14
activities.
Additionally, thiosemicarbazones can also
undergo ring closure processes through the action of bases,
acids or oxidants. Recently, we have shown that oxidative
cyclization of thiosemicarbazone can be used for selective
1
5
1
5h
chemodosimetric sensing of Cu(II) ions.
With regard to biological activity, metal complexes of
heterocyclic thiosemicarbazones exhibit more promising
results compared to other non heterocyclic thiosemicarba-
19
or metal centers with potential redox activity. It is well
documented in the literature that metal complexes interact
with DNA in a non-covalent fashion, such as electrostatic
binding, groove binding and intercalation. The development
of metal complexes with intercalation or a groove binding
1
6
zones.
Many heterocyclic thiosemicarbazones, such as
4
e
4b–d
triapine or marboran,
are already employed in medical
1
9e
mode of interaction with DNA are more promising
a
Department of Chemistry, Indian Institute of Technology, Guwahati.
E-mail: gdas@iitg.ernet.in; Fax: +91-361-2582349; Tel: +91-361-2582313
compared to those metal complexes which interact with DNA
through weak electrostatic interactions.
b
Department of Biotechnology, Indian Institute of Technology, Guwahati.
E-mail: aramesh@iitg.ernet.in; Fax: +91-361-258-2249; Tel: +91-361-258-2205
In the context of therapeutic applications of a potential
drug, it is pertinent to investigate the protein binding levels of
the drug, especially to serum albumins, as these proteins may
influence the metallo-pharmaceutical pharmacokinetics and
3
Electronic supplementary information (ESI) available: Compound
characterization spectra, crystallographic files in CIF format, additional
crystallographic data, characterization data. CCDC 905339 905341 905340. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3ra40904j
2
0
structure–activity relationships. Bovine serum albumin (BSA)
1
4088 | RSC Adv., 2013, 3, 14088–14098
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has been widely used as a model protein to study drug
interactions due to its structural resemblance to human serum
albumin (HSA). Additionally, serum albumins are also
identified as major transport proteins in blood plasma for
many compounds, such as fatty acids, which are otherwise
insoluble in plasma, hormones, bilirubin and many drugs.
These facts reiterate the importance of studying the interac-
tion behavior of potential drugs with BSA protein whilst
assessing their biological properties. In this context, we have
previously reported protein sensing and detailed interaction
studies with structurally diverse amphiphilic organic
2
1
ligands.
Considering the above objectives, herein, we report the
synthesis and characterization of a newly synthesized pyridine-
based heterocyclic thiosemicarbazone ligand and its mono-
nuclear Cu(II) and Ni(II) complexes and demonstrate their
potential biological activity. The ligand and its complexes are
structurally characterized by single-crystal X-ray crystallogra-
phy. The study highlights the DNA and protein binding
characteristics of the test compounds as well as their anti-
proliferative effects on human cervical carcinoma HeLa cell
lines.
Scheme 1 Synthesis of the ligand LH and its Cu(II) and Ni(II) complexes.
Experimental
(
0.560 mL, 5 mmol) was added over a solution of 4-(1-
Materials
naphthyl)-3-thiosemicarbazide (1.085 g, 5 mmol) in ethanol
(15 mL). The light yellow solution was stirred at room
temperature for 3 h. The white solid formed was collected by
filtration, washed thoroughly with ethanol and diethyl ether
and dried in vacuo. Yield 87%. H NMR (400 MHz, DMSO-d
All reagents were obtained from commercial sources and used
as received. Solvents were freshly distilled following standard
procedures. Napthylamine, carbondisulphide, triethylamine,
1
iodine, dimethyl sulfoxide (DMSO), hydrated CuCl
2
and
6
) d-
hydrated NiCl were purchased from Merck, India and used
12.18(s, 1H) 10.59(s, 1H), 8.60(s, 1H), 8.5(d, 1H), 8.27(s, 1H),
2
1
3
as received. BSA was purchased from Fluka, Germany and was
used as supplied. Calf thymus DNA (CT-DNA), ethidium
bromide (EtBr), Dulbecco’s Modified Eagle Medium (DMEM),
trypsin-EDTA and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
7.99(m, 1H), 7.9–7.8(m, 3H), 7.6–7.5(m, 4H), 7.4(m, 1H),
NMR (100 MHz, DMSO-d ) d- 178.35, 153.38, 149.36, 143.05,
136.50, 135.65, 133.75, 130.61, 128.06, 127.14, 126.58, 126.18,
C
6
2
1
126.12, 125.51, 124.23, 123.45, 120.67 IR (KBr, cm ): 3348(N–
H), 1540(CLN, sym), 1504 (CLS) and 780(CLS, asym) ESI mass
2H-tetrazolium bromide (MTT) were procured from Sigma
3
+
+
Aldrich, USA and plasmid DNA pUC18 was purchased from
Bangalore Genei, India. Fetal calf serum (FCS) was procured
from PAA Laboratories, USA. The synthetic route for the ligand
and its complexes is shown in Scheme 1.
calcd for 322.4. [M + H ]; found 322.4 [M + H ]. MP 194 uC.
Synthesis of [Ni(L) ] (2). The complex Ni(L) was synthesized
2
2
by stirring an ethanolic solution of NiCl ?6H O (0.24 g, 1
2
2
mmol, 10 mL) and LH (0.64 g, 2.5 mmol, 25 mL) in the
presence of triethylamine (1 ml). The stirring was continued
for 5 h. The yellowish green precipitated complex was collected
Synthesis and characterization
Synthesis of 1-naphthylisothiocyanate. This compound was
synthesized by a previously reported literature procedure.
by filtration, washed with a cold 1 : 1 EtOH and H O mixture
to remove any impurities and finally dried in vacuum over
2
2
6
Synthesis of 4-(1-naphthyl)-3-thiosemicarbazide. In a 100 ml
round bottom flask, 1.85 g of naphthyl isothiocyanate (10
mmol) was dissolved in 50 mL of dry THF. Then, excess
hydrazine hydrate (30 mmol, 0.9 ml) was added to the solution
at once. After stirring under ice cold conditions for 2 h, the
precipitate was filtered and washed with 5 mL of diethyl ether
three times. A colorless solid was obtained after drying the
precipitate in a vacuum over silica gel. Yield 85%.
silica gel. It is important to mention here, we have also tried to
prepare a 1 : 1 Ni(II)-thiosemicarbazone complex by varying
the metal–ligand stoichiometry but in all the cases, a 1 : 2
(metal : ligand) complex was obtained, which was confirmed
by single crystal X-ray diffraction and elemental analysis. Yield
62%. Calc. for C H N NiOS : C, 60.25; H, 4.79; N, 16.64; S,
3
8
36
9
2
2
1
8.47 Found: C, 59.94; H, 4.49; N, 16.75; S, 8.36, IR (KBr, cm ):
3348(N–H), 1674(CLN), 1597(CLS) and 772(CLS, asym), MP 264
2
1
2
Synthesis of pyridine-2-carboxaldehyde-4-(1-naphthyl)-3-
thiosemicarbazone [LH] (1). The compound LH was prepared
by the condensation of pyridine-2-carboxyaldehyde and 4-(1-
B M
uC. meff (300 K): 2.98 m , L (S mol cm ): 0.66.
Synthesis of [Cu(L)Cl] (3). The complex Cu(L)Cl was
synthesized by refluxing an equimolar amount of ethanolic
naphthyl)-3-thiosemicarbazide.
Pyridine-2-carboxyaldehyde
solutions of CuCl
2
?2H
2
O (0.085 g, 0.5 mmol) and the ligand LH
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2
3
(
(
0.115 gm, 0.5 mmol) in the presence of Et N (0.5 ml) for 2 h
Scheme 1). The brownish red precipitated complex was
were applied to the data using the program SADABS. The
3
2
4a
structures were solved by direct methods using SHELXS-97
and refined with full-matrix least squares on F using the
2
collected by filtration, washed with cold ethanol to remove
any unreacted starting materials and impurities and finally
dried in a vacuum over silica gel. Yield 62%. Calc. for
C H13ClCuN S: C, 50.49; H, 3.24; N, 13.86; S, 7.93 Found: C,
17 4
5
2
4b
SHELXL-97 program package. All non-hydrogen atoms were
refined anisotropically. The hydrogen atoms attached to all
carbon and nitrogen atoms were geometrically fixed and the
positional and temperature factors were refined isotropically.
Structural illustrations have been drawn with MERCURY for
Windows. A summary of the crystal data and relevant
refinement parameters are given in Table 1. CCDC 905339
2
1
0.54; H, 3.19; N, 13.75; S, 8.06, IR (KBr, cm ): 3348(N–H),
2
5
1
601(CLN), 1477(CLS) and 764(CLS, asym) MP 205 uC. meff (300
2
1
2
B M
K): 1.72 m , L (S mol cm ): 1.25.
Instruments
IR spectra were recorded on a Perkin-Elmer-Spectrum One FT-
(
1), 905340 (3) and 905341 (2) contain the supplementary
crystallographic data for this paper.
2
1
IR spectrometer with KBr disks in the range 4000–400 cm
.
UV-visible and fluorescence spectra were recorded with a
Perkin-Elmer Lambda-25 and FlouroMax-3, HORIBA spectro-
1
DNA binding studies
fluorometer, respectively. H NMR spectra were recorded at
1
3
4
00 MHz and C NMR spectra were recorded at 100 MHz
UV-visible spectroscopy
using a Varian AS400 spectrometer. Chemical shifts were
recorded in parts per million (ppm) on the scale solvent peak
as a reference. Elemental analyses were obtained from a
Perkin-Elmer Series II Analyzer. The magnetic moments of the
complexes were measured on a Cambridge Magnetic Balance.
The DNA interaction experiments were carried out in sterile
phosphate buffered saline (PBS) whose pH was adjusted to 7.4.
For the calf-thymus DNA, a molar extinction coefficient value
2
1
21
of 6600 M cm at 260 nm was used to determine the DNA
1
7
molarity.
Stock solutions of the test compounds were
Solution electrical conductivity was measured using
a
prepared in DMSO and diluted to the appropriate concentra-
tions in PBS prior to the experiments, while a stock solution of
CT-DNA was prepared in sterile PBS. For the UV-visible
absorption titration experiments, 30 mM of each test com-
pound was interacted with various concentrations of CT DNA
Systronic 305 conductivity bridge in a 5% aqueous DMSO
solution. ESI-MS spectra were recorded in a WATERS LC-MS/
MS system, Q-Tof Premier in the Central Instrument Facility
(CIF) of IIT Guwahati.
X-ray crystallography
(3 mM–30 mM). In these experiments, the DNA solution was
Intensity data were collected using a Bruker SMART APEX-II
CCD diffractometer equipped with fine focus 1.75 kW sealed
tube Mo–Ka radiation (l) 0.71073 (Å) at 298 K, with increasing
v (width of 0.3u per frame) at a scan speed of 5 s per frame.
The SMART software was used for the data acquisition. Data
integration and reduction were performed with the SAINT and
added drop-wise to the test compound, mixed gently and then
the absorbance of the solution was measured. Control samples
consisting of only the DNA solution or the test compounds
were also included in the experiments.
2
2
XPREP software. Multi-scan empirical absorption corrections
Table 1 Crystal parameters and refinement data
Compound
[LH] (1)
[Ni(L)
2
] (2)
NiOS
[Cu(L)Cl] (3)
4
13ClCuN S
Formula
17
C H
14
N
4
S
37
C H
33
N
9
2
C
17
H
Formula weight
Crystal system
Space group
306.39
Monoclinic
P 21
742.55
Monoclinic
P 21
404.38
Monoclinic
P 21/c
a/Å
b/Å
c/Å
b (u)
V (Å)
Z
9.5334(4)
6.4275(2)
13.0570(5)
108.293(2)
759.65(5)
2
9.0267(10)
33.436(3)
12.2036(11)
100.916(6)
3616.6(6)
4
7.5413(4)
13.9846(8)
15.6339(9)
101.290(3)
1616.88(16)
4
T/K
m/cm
dcal (g cm
Cryst dimens (mm )
no. of reflns collected
no. of unique reflns
no. of params
298(2)
0.214
1.339
0.28 6 0.26 6 0.22
3313
2999
207
298(2)
0.696
1.364
0.42 6 0.34 6 0.29
17 936
16 528
905
298(2)
1.651
1.661
0.26 6 0.19 6 0.17
3978
2648
221
2
1
23
)
3
R
1
; wR2 (I . 2s(I))
0.0444, 0.1197
0.0786
0.909
0.0879, 0.2039
0.102
0.857
0.0306, 0.0721
0.0719
0.912
R(int)
GOF (F )
2
Flack prameter
CCDC NO.
20.15(7)
905339
0.009(6)
905341
—
905340
1
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Fluorescence spectroscopy
protein. The background intensities of the buffer blanks were
subtracted from each sample spectrum to cancel out any
contribution due to the solvent.
For the fluorescence emission spectroscopic measurements,
30 mM of the test compounds were interacted with varying CT-
DNA concentrations (6 mM–30 mM). As mentioned before, the
emission titration experiments were performed by the drop-
wise addition of a CT-DNA solution to the test compounds
followed by gentle mixing and then the fluorescence emission
spectra of the samples were recorded in a scanning mode from
Cytotoxicity assay
The cytotoxicity assay for the test compounds was performed
on human cervical carcinoma (HeLa) cell lines by a standard
MTT assay following the manufacturer’s instructions (Sigma-
Aldrich, MO, USA). HeLa cells were initially propagated in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal calf serum (FCS). Subsequently, HeLa cells were
seeded in a 96 well tissue culture plate (10 cells well ) and
varying concentrations of the test compounds (15 mM, 30 mM
and 60 mM) constituted in DMEM devoid of FCS were added to
the cells. The cells were incubated for a period of 24 h in a CO2
335 nm to 600 nm by setting the excitation wavelength at 320
nm in a spectrofluorometer. Control samples consisting of
only the DNA solution or the test compounds were also
included in the experiments.
4
–
D1
Ethidium bromide displacement experiment
Initially, a 3 mM CT-DNA solution (taken in sterile PBS, pH 7.4)
was incubated with a 0.5 mM EtBr solution for 1 h at 37 uC in
an amber colored microcentrifuge tube. Subsequently, varying
concentrations of the test compounds (0.6 mM–3 mM) were
gradually dispensed into the tubes, incubated for 5 min at
room temperature and then the fluorescence emission spectra
of the samples were recorded in a scanning mode from 530 nm
to 720 nm by setting the excitation wavelength at 515 nm in a
spectrofluorometer. Emission spectra were also recorded for
DNA-bound EtBr alone in the absence of the test compounds.
2
incubator with 5% CO . Control samples consisting of the cells
alone and DMSO-treated cells (solvent control) were also
included in each assay. Following incubation, the growth
media was aspirated, a fresh DMEM-containing MTT solution
was added and the plate was incubated for 4 h at 37 uC.
Subsequently, the supernatant was gently removed and the
insoluble colored formazan product was solubilized in DMSO
and measured spectrophotometrically at 550 nm in
a
mutiplate reader (Perkin Elmer, USA). Every sample in the
MTT assay was taken in six sets. To determine the statistical
significance of the results, the obtained data were subjected to
analysis of the variance (ANOVA) using a Sigma plot.
Plasmid cleavage experiment
To determine the DNA cleavage activity of the compounds,
purified pUC18 plasmid DNA (60 mM) was taken in a 5 mM
Tris-HCl–50 mM NaCl buffer and incubated with varying
concentrations of the test compounds (60 mM and 180 mM) for
Results and discussion
1
h and 2 h. Control samples were also included, which
Crystal structure of [LH] (1)
consisted of pUC18 plasmid DNA treated with equivalent
concentrations of the Cu(II) and Ni(II) salts and DMSO
separately. Following incubation for the specified time, a
loading buffer (0.05% bromophenol blue, 50% glycerol and 2
mM EDTA) was added to each reaction tube. Subsequently, the
samples were subjected to agarose gel (0.8%) electrophoresis
followed by staining the gel with an EtBr solution and the DNA
bands were visualized in a gel documentation system.
The structure of LH was established accurately by X-ray
diffraction of single crystals. Light yellow block shaped crystals
of the ligand were grown by slow driven crystallization from a
saturated solution of DMSO. It crystallized in the space group
1
P2 with Z = 2. The molecular structure of LH along with the
atom numbering scheme is depicted in Fig. 1. The detail
structural analysis of LH shows the thiosemicarbazone ligand
exists in its thione form, confirmed by the presence of
hydrazinic hydrogen. The sulfur atom S1 and the hydrazone
nitrogen N3 are in the E configuration with respect to the C11–
N2 bond. This lower energy configuration helps the formation
BSA interaction studies
A 10 mM phosphate buffer of pH 7.4 was prepared in double-
distilled water for all the experiments. A stock solution of BSA
(10 mM) was prepared in an aqueous solution of a 10 mM
phosphate buffer of pH 7.4, while stock solutions of the test
compounds were prepared in dimethyl sulfoxide (DMSO). In a
1.0 mL aqueous buffer solution of BSA (1 mM), different
concentrations of compounds were added (0 to 40 mM), so that
the total volume of DMSO did not exceed 15%. The presence of
2
1a
1
5% DMSO induces no major BSA structural changes. Each
solution was mixed thoroughly before the spectral measure-
ments at room temperature (RT). For the fluorescence
resonance energy transfer (FRET) experiments, the concentra-
tion of BSA and the ligands were kept at 3 mM. The absorption
spectra were recorded at 298 K in the range of 200–450 nm.
Fluorescence measurements were taken at 298 K with a slit
width of 2 nm. An excitation wavelength of 295 nm was
applied to selectively excite the tryptophan residues in the
Fig. 1 Molecular structure of LH with an atom numbering scheme.
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…
of N2–H N4 hydrogen bond interactions, which subsequently
form a 1D hydrogen-bonding chain along the crystallographic
Table 2 Comparison of selected bond lengths (Å) and bond angles (u) of LH,
[Ni(L) ] and [Cu(L)Cl]
2
3
b axis (Fig. S8, ESI ). Probably, the presence of the strong
[
LH] (1)
[Ni(L)
2
] (2)
[Cu(L)Cl](3)
H-bond accepting pyridine N(4) nitrogen prevents the forma-
…
tion of centrosymmetric dimeric interactions through N–H S
N1–C11
C11–N2
N2–N3
N3–C12
1.350(3)
1.350(3)
1.377(3)
1.278(3)
1.670(2)
114.2(2)
114.3(2)
119.4(1)
1.387(1)
1.322(2)
1.365(1)
1.279(2)
1.738(1)
115(1)
1.362(3)
1.313(2)
1.374(2)
1.284(3)
1.745(2)
114.3(2)
119.0(2)
125.9(2)
hydrogen bonds, which is very common for a thiosemicarba-
zone type of ligand. The N2–N3 (1.377(3) Å) and N2–C11
(1.350(3) Å) bond distances in LH are in between the ideal
C11–S1
values corresponding single [N–N, 1.45 Å; C–N, 1.47 Å] and
double bonds [NLN, 1.25 Å, CLN, 1.28 Å], as evident for an
extended p-delocalization throughout the thiosemicarbazone
function. The elaborate 3D network of LH is mainly governed
N4–C13–C12
C12–N3–N2
N2–C11–S1
116.6(9)
128.1(9)
…
by the combination of H-bonding and C–H p interactions.
firmed by
a detailed bond length comparison of the
Crystal structure of [Ni(L) ?DMF] (2)
2
thiosemicarbazone functions of the coordinated ligands
Table 2). Interestingly, two symmetry independent complex
molecules in the asymmetric unit form dimeric interaction via
The isolated yellowish green Ni(II) complex was crystallized by
isothermal slow evaporation of a saturated DMF solution. It
(
crystallized in the space group P2 with Z = 4. The molecular
1
…
2
two N5–H N10 hydrogen bonds, forming an R (8) ring motif
2
2
structure of Ni(L) along with the atom numbering scheme is
(
3
Fig. S9, ESI ). The free DMF solvents are stabilized by the
depicted in Fig. 2 and selected bond lengths and bond angles
are given in Table 2 and Table 3. The asymmetric unit is
composed of two symmetry-independent octahedral complex
molecules along with two crystallized DMF molecules exhibit-
ing conformational isomorphism. Their occurrence is gener-
ally controlled by both kinetic and thermodynamic crystal
stability because these factors are mostly considered to be the
consequences of interrupted crystallization. Both complexes in
the asymmetric unit are almost identical, therefore, in this
crystallographic description section, we only consider the first
complex unit.
The two deprotonated S,N,N–tridentate ligands chelate the
nickel atom, giving a distorted octahedral geometry around
the nickel centre. Moreover, the elongated C–S bonds in the
complex molecule compared to the free ligand also indicate
that the ligands are bound to nickel in the thiolate form
…
…
…
hydrogen bonding (N–H O and C–H O) and C–H p inter-
actions with the complex molecules.
Crystal structure of [Cu(L)Cl] (3)
The isolated brownish red Cu(II) complex was crystallized by
vapor diffusion of Et O into a 2 : 1 CHCl : DMF solution. The
2
3
1
complex crystallized into a monoclinic space group P2 /c with
Z = 4. The molecular structure of Cu(L)Cl along with the atom
numbering scheme is depicted in Fig. 3 and significant
structural parameters are given in Table 2 and Table 3. The
molecular structure of the complex shows that the mono-
negative thiosemicarbazone ligand (L) binds to the Cu(II)
center via N,N,S-donor atoms, resulting in two five membered
chelate rings. The fourth coordination site around Cu(II) is
occupied by a chloride anion, forming a distorted square
planer complex. The bonding parameters are in good agree-
ment with the coordinating pattern of a thiosemicarbazone via
the thiolate form. While coordinating in their iminothiolate
forms, the negative charges generated by deprotonation are
effectively delocalized in the C–N–N–C system, as shown by the
intermediate C12–N3 [1.284 Å], N3–N2 [1.374 Å] and N2–C11
(Table 2). The negative charge of the deprotonated ligand is
fully delocalized along the thiosemicarbazone moiety, con-
[1.313 Å] bond distances. The bond angles Cl1–Cu–S1 97.74(2),
Table 3 Metal–ligand bond lengths (Å) and angles (u) for the Ni(L)
2
and Cu(L)Cl
complexes
Ni(L)
S1–Ni1
N3–Ni1
N4–Ni1
S1–Ni1–S2
S1–Ni1–N3
S1–Ni1–N8
S1–Ni1–N7
N3–Ni1–N8
N3–Ni1–N4
Cu(L)Cl
2
2.419(4)
2.028(8)
2.11(1)
96.6(1)
80.1(3)
94.4(3)
103.4(3)
96.0(4)
79.2(4)
S2–Ni1
N7–Ni1
N8–Ni1
S2–Ni1–N3
S2–Ni1–N4
S2–Ni1–N7
N7–Ni1–N8
N7–Ni1–N4
N8–Ni1–N4
2.403(3)
2.04(1)
2.13(1)
104.1(3)
89.7(3)
80.3(3)
79.1(4)
97.1(4)
86.4(4)
S1–Cu1
Cl1–Cu1
Cl1–Cu1–S1
Cl1–Cu1–N4
2.2562(7)
2.2076(6)
97.74(2)
98.34(5)
N3–Cu1
N4–Cu1
S1–Cu1–N3
N3–Cu1–N4
1.970(2)
2.2027(2)
83.29(5)
80.69(6)
Fig. 2 Molecular structure of Ni(L)
2
with an atom numbering scheme. The other
unit and solvent (DMF) molecules are omitted for clarity.
1
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attributed to intercalation through stacking between the
planar aromatic chromophore of the test compound and the
2
8
bases present in DNA. In the present study, UV-visible
absorption spectroscopy of the complexes indicated that the
absorbance maxima of compounds 1, 2 and 3 were 317 nm,
432 nm and 459 nm, respectively. The UV-visible absorption
spectra of compounds 1–3 (30 mM) with increasing concentra-
tions of CT DNA (3 mM–30 mM) are indicated in Fig. 4. As
observed in Fig. 4A, the interaction of compound 1 with
increasing DNA concentrations resulted in marginal hypo-
chromism of 6.43% at the absorbance maxima (317 nm) of the
compound. In contrast to the ligand alone, the metal
complexes revealed a distinctly higher hypochromic shift of
the absorption bands upon interaction with incremental
concentrations of DNA, indicating that the binding strength
of the metal complexes is superior to that of the free ligand.
For instance, the extent of the hypochromism for compound 2
was around 25% at 432 nm, whereas compound 3 exhibited a
hypochromic shift of around 18% at 459 nm. The observed
hypochromic shifts for compound 2 and 3 perhaps indicate an
intercalative mode of binding with CT-DNA. The UV-visible
spectra of compound 2 and 3 (Fig. 4B and 4C) also reveal that
the binding of the complexes to CT-DNA led to an isosbestic
spectral change with an isosbestic point at 293 nm. It may also
be mentioned here that the binding of increasing concentra-
tions of CT-DNA lead to negligible shifts in the absorption
maxima for all the compounds. From the comparative
measurements of the hypochromic shifts following binding
with CT-DNA, it can be assumed that compound 2 has the
highest propensity to interact with DNA. The binding
Fig. 3 Molecular structure of Cu(L)Cl with an atom numbering scheme.
Cl1–Cu–N4 98.34(5), N3–Cu1–S1 83.295 and N3–Cu1–N4
80.69(6) illustrate that the complex is slightly distorted from
the square planar geometry (t = 0.12). In this complex, two
4
chelate rings are formed, which are almost planar with a
dihedral angle separation of 1.79u, the second chelate ring
being Cu1–N4–C13–C12–N3. A similar type of distorted square
2
8
4
planar Cu(II) thiosemicarbazone complex (t = 0.14) with a
coordinated linear isothiocyanate counter anion was pre-
viously reported by Kurup and coworkers, whereas in the case
of the chloride counter anion, they obtained a chloride
2
7
bridged
centrosymmetric
dimeric
Cu(II)
complex.
Interestingly, in the present study, hydrogen bonding interac-
tions were not observed in the Cu(II) complex, whereas the 3D
…
network of Cu(L)Cl was mainly controlled by p p stacking
interactions (Fig. S11, ESI ).
3
constants (K ) for compounds 1, 2 and 3 were determined
b
from the UV spectroscopic studies by monitoring the change
in absorbance at 260 nm on increasing the concentration of
the DNA and calculating the ratio of the slope to the y intercept
DNA binding studies
6
h,17
in the plots of [DNA]/(e
a
2 e
f
) versus [DNA]
(insets in Fig. 4).
UV-visible spectroscopic studies
The values for the binding constants for compounds 1, 2 and 3
Absorption spectroscopy is a common tool to decipher the
mode of binding of test compounds to DNA. It is quite well
established that the binding of a compound to DNA may
typically lead to hypochromism, which can be largely
5
21
5
21
5
21
were 1.37 6 10 M , 1.15 6 10 M and 1.13 6 10 M
,
respectively. In the present study, the binding constant for the
Ni-complex (compound 2) was comparatively lower than the
binding constants reported earlier for analogous Ni(II)
Fig. 4 Absorbance titration spectra of (A) compound 1 (B) compound 2 (C) compound 3 with various concentrations (6 mM–30 mM) of CT-DNA in PBS pH 7.4. Inset
indicates binding isotherms with CT-DNA.
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h
thiosemicarbazone complexes, whereas in the case of the Cu-
complex (compound 3), the calculated binding constant was
observed to be higher than similar Cu(II) semicarbazone
provides a measure of the quenching constant K of the
q
1
7,29b,31
compounds.
–3 is shown in Fig. S13D (ESI
plot for each compound is consistent with the expected linear
pattern of a characteristic Stern–Volmer plot. The K values
obtained were 1.20 6 10 M , 2.26 6 10 M and 1.22 6
0
The plot of F /F versus [Q] for compounds
1
3
) and it is quite evident that the
1
7
complexes.
q
Fluorescence experiment
5
21
5
21
To acquire an insight into the mode of binding of the
compounds to CT-DNA, we performed fluorescence titration
experiments. As evident from Fig. S12A (ESI ), when com-
3
pound 1 was excited at 320 nm, a fluorescence emission peak
was observed at 439 nm. It was also observed that the
fluorescence of compound 1 was enhanced progressively upon
interaction with incremental concentrations of CT-DNA (6 mM–
5
21
10
M
for compound 1, 2 and 3, respectively. It may be
mentioned here that the K value obtained for the ligand
compound 1) was comparatively higher than a similar ligand
q
(
1
7
reported earlier. Likewise, the K value obtained for the Cu-
q
complex (compound 3) in the present study was relatively
1
7
higher than analogous Cu(II) semicarbazone complexes. The
values also seem to suggest that the quenching constant
K
q
3
0 mM). At the highest CT-DNA concentration of 30 mM, the
was highest for the the Ni-complex (compound 2) in
comparison to the other compounds.
fluorescence emission intensity of compound 1 was nearly
doubled. In the case of the Ni(II)-complex (compound 2), the
enhancement in the fluorescence emission intensity of the
compound with increasing concentrations of CT-DNA was
quite substantial, resulting in a nearly four-fold increase upon
interaction with 30 mM CT-DNA (Fig. 12B, ESI ). At this
3
concentration of CT-DNA, it was also noted that the
fluorescence emission maximum changed considerably from
57 nm for the compound alone to 436 nm following its
Plasmid cleavage experiments
Based on the strong propensity of compounds 1–3 to bind to
DNA, we were motivated to ascertain the DNA cleavage activity
of the compounds. To this end, varying concentrations of
compounds 1–3 (60 mM and 180 mM) were interacted with 60
mM purified pUC18 plasmid DNA for varying time periods (1 h
and 2 h). The result of the experiments is shown in Fig. S14,
4
interaction with CT-DNA. For compound 3, the increase in the
fluorescence intensity upon its interaction with CT-DNA was
minimal and was not accompanied by any peak shift in the
ESI
supercoiled (SC) and nicked circular (NC), were evident in the
untreated pUC18 plasmid (Fig. S14 A–D, Lane 1, ESI ). When
3
. Topologically different forms of plasmid DNA, namely
3
emission maximum (Fig. S12C, ESI ). The increase in the
3
fluorescence intensity of the ligand alone as well as the metal
complex upon its interaction with the DNA suggests that the
compounds perhaps gain access into the hydrophobic core of
the DNA bases upon intercalation, which may result in
the pUC18 plasmid DNA was treated with a concentration of
60 mM of compounds 1–3, DNA cleavage activity could not be
detected for any sample (Fig. S14 A–B, ESI ). At a higher
3
concentration of the compounds (180 mM), it was observed
that for compound 3, a stained band was detected in the well
of the agarose gel, indicating that the sample failed to migrate
2
8,29a,30
enhanced fluorescence.
On the basis of fluorescence
enhancement and the large shift in the fluorescence maxima,
it is quite evident that compound 2 interacts strongly with CT-
DNA, in comparison to compound 1 or compound 3.
3
during the electrophoretic run (Fig. S14 C–D, lane no. 4, ESI ).
The binding of high concentrations of the Cu(II)-complex to
DNA may lead to interference in the characteristic mobility of
the native DNA under the electrophoretic conditions and
consequently, the complex-bound DNA fails to migrate from
the well. The interaction of pUC18 plasmid DNA with 180 mM
of either the ligand or the Ni(II)-complex did not result in any
cleavage activity and the mobility of the plasmid DNA was also
Ethidium bromide (EB) displacement assay
The binding of compounds 1–3 to DNA via intercalation was
further verified by an EtBr displacement assay (Fig. S13, ESI ).
3
A systematic decrease in the fluorescence emission intensity of
DNA-bound EtBr on increasing the compound concentration
was observed for all the compounds. At the highest compound
concentration, the emission band of DNA-bound EtBr at 603
nm revealed a moderate hypochromic shift of the initial
3
unaffected (Fig. S14 C–D, lane nos. 2–3, ESI ).
Protein binding studies
fluorescence emission intensity (Fig. S13 A–C, ESI
3
). This
Studies related to the binding of small ligands with BSA are
usually examined by fluorescence spectroscopic techniques.
The fluorescence of BSA is commonly attributed to two
fluorophoric amino acid residues of the protein, namely
tryptophan and tyrosine. Changes in the protein conforma-
tion, subunit associations, substrate binding or denaturation
usually affect the emission pattern of tryptophan. Therefore,
the tryptophan fluorescence of BSA can provide structural
information of the protein and it can be employed in
understanding the protein folding mechanism and association
reactions. As the excitation wavelength of tyrosine is less
(y280 nm) than the excitation wavelength of tryptophan
(y295 nm), thus, the excitation wavelength of 295 nm was
chosen to avoid the contribution from any of the tyrosine
residues in BSA. We have studied the interaction of BSA with
reduction in the fluorescence of DNA-bound EtBr suggests that
compounds 1–3 compete for the binding site on DNA, which
results in the displacement of EtBr, as reported in earlier
1
7,31
studies.
The quenching of the EtBr fluorescence by the compounds
was analyzed by the standard Stern–Volmer equation which
may be represented as:
F /F = K [Q] + 1
(1)
0
q
where F is the initial DNA-bound EtBr emission intensity, F is
0
fluorescence intensity in the presence of a quencher, Kq
represents the quenching constant, and [Q] is the concentra-
tion of the quencher. The slope of the plot of F /F versus [Q]
0
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compounds 1, 2 and 3 by fluorescence measurements at room
temperature. A decrease in the fluorescence intensity of the
tryptophan moiety at 346 nm in the order of 75%, 87.55% and
The number of binding sites (n) and the binding constant
(Kbin) have been calculated from the plot of log [(F – F)/F] vs.
0
3
q
app
log[Q] (Fig. S16, ESI ). The calculated K , K and Kbin values
97.03% was observed upon the interaction of BSA with
of compounds 1, 2 and 3 are given in Table 4. These values are
in general agreement with the K values reported in the
literature for analogous protein–ligand or protein–metal
compounds 1, 2 and 3, respectively. The diminution of the
emission intensity is accompanied by the significant develop-
ment of a new hump at a red shifted wavelength (Fig. S15,
6h,18,33
complex interactions.
3
ESI ). The shifting of the emission maxima towards a higher
Generally, quenching of the emission intensity of
a
wavelength along with the formation of an iso-emissive point
is a clear indication of the probable energy transfer from the
indole unit of the tryptophan to the protein bound com-
pounds.
Furthermore, the fluorescence quenching data were studied
using the Stern–Volmer equation, a modified Stern–Volmer
equation and Scatchard equation. On the basis of the
relationship between the quenching of the excited states and
the quencher concentration, the Stern–Volmer equation is
fluorophore occurs by either dynamic or static quenching
mechanisms. In a typical dynamic quenching, the fluorophore
and the quencher come into contact during the transient
presence of the excited state, while in case of static quenching,
fluorophore–quencher complex formation occurs in the
ground state. One of the simple and decisive methods to
differentiate between static and dynamic quenching is UV-
visible absorption spectroscopy. The UV-visible spectra of BSA
with the incremental addition of compounds 1, 2 and 3 show
an increase in the absorption intensity of BSA as compounds
given by eqn (1). Here, F
intensities in the absence and presence of quencher, respec-
tively, and [Q] is the concentration of the quencher. K is the
0
and F are the relative fluorescence
3
1, 2 and 3 are added (Fig. S17, ESI ). The significant changes
q
observed in the UV-visible spectra of BSA clearly suggest that
fluorophore–quencher complex formation is occurring in the
ground state for compound 1 and 3, wherein static quenching
is exclusively occurring. In the case of compound 2, the
combined results from the UV-visible and fluorescence
experiments indicate that static quenching of BSA occurs in
the presence of compound 2 along with an adequate
contribution from collision/dynamic quenching.
Stern–Volmer quenching constant, which measures the
efficiency of the quenching. In the case of compound 2, a
plot with an upward curvature, concave towards the y-axis, was
obtained. This type of positive deviation from the Stern–
Volmer equation is frequently observed when the extent of the
quenching is large and is due to the quenching of the
fluorophore by collision and by complex formation with the
3
2
same quencher. For the above mentioned phenomenon, the
Stern–Volmer plot can be analyzed using a modified Stern–
Volmer equation, as stated below in eqn (2). The modified
Stern–Volmer equation is second order in [Q], which accounts
for the upward curvature observed when static and dynamic
quenching occur for the same fluorophore.
Distance measurement using resonance energy transfer
between quenchers and BSA
The FRET (Fluorescence Resonance Energy Transfer) techni-
que has emerged as a successful tool to determine the spatial
distance between a donor and an acceptor in proteins and
FRET distances have been extensively used to study protein
folding pathways, conformational changes upon ligand bind-
ing, protein–protein interactions and monomer–dimer equili-
F
0
/F = Kapp [Q] + 1
(2)
where, Kapp = (KD + K ) + K K [Q]
34
S
D
S
bria. According to F ¨o rster’s theory, the efficiency of FRET
Here, KD is the dynamic quenching constant and K is the
S
depends mainly on the following factors: (i) the extent of the
overlap between the donor emission and the acceptor
absorption, (ii) the orientation of the transition dipole of the
donor and acceptor, and (iii) the distance between the donor
static quenching constant.
The equilibrium binding constant was analyzed using the
Scatchard equation (eqn (3)):
3
5
and the acceptor. In a proteinaceous environment, the
proximity of a guest molecule to the tryptophan moiety is often
determined through a FRET study. For this purpose, the donor
log [(F
0
2 F)/F] = log Kbin + n log [Q]
(3)
where Kbin is the binding constant of the compound with BSA
and n is the number of binding sites. The modified Scatchard
equation used in the present study is similar to that used in
earlier investigations to determine the binding interaction
(
BSA) was excited at 295 nm, where the absorbance of the
acceptors (ligands) was negligible. On the gradual addition of
guest molecules, the fluorescence intensity of the tryptophan
residue present in BSA decreased along with the development
of a new red shifted hump, which was accompanied by the
6
h,18,33
between a protein (BSA) and ligand (or metal complex).
Table 4 Quenching and energy transfer parameters of different BSA–compound composites
Stern–Volmer constant (K or Kapp Binding constant (Kbin
q
)
)
R
0
(nm)
E
4
21
21
4
21
21
L
8.3 6 10 M
1.4 6 10 M
2.49
3.09
0.75
0.87
5
5
L–Ni Complex
1.8 6 10 M
4.2 6 10 M
app
(K )
1.2 6 10 M
5
21
5
21
L–Cu Complex
2.5 6 10 M
2.43
0.80
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formation of a distinct iso-emissive point (Fig. S15, ESI ).
Taken together, this result indicates that the quenching of the
tryptophan fluorescence may be due to a non-radiative energy
transfer between the tryptophan and the guest molecules. As
per F ¨o rster’s theory, the efficiency of the energy transfer (E) is
related to the distance r between the donor and acceptor by the
following equation:
6
6
0
6
E = R
0
/(R
+ r )
(4)
where r is the distance between the acceptor and donor and R
0
is the critical distance when the transfer efficiency is 50% and
is given by the following equation:
2
24
1/6
R = 0.211[k n WD J(l)]
(5)
0
2
where n is the refractive index of the medium, k is the
orientation factor and WD is the quantum yield of the donor.
The spectral overlap integral (J) between the donor emission
spectrum and the acceptor absorbance spectrum was approxi-
mated by the following summation:
Fig. 6 MTT-based cell viability assay of the compounds on HeLa cell lines after
24 h incubation. * indicates a p value , 0.001 in ANOVA.
The calculated values of R and E using F ¨o rster’s theory are
0
presented in Table 4.
4
J(l) = #F
d
(l)e
a
(l)l dl/#F
d
(l)dl
(6)
where F (l) and e (l) represent the fluorescence intensity of
d
a
Cytotoxicity assay
the donor and the molar extinction coefficient of the acceptor,
respectively, at the wavelength l. The value of J can be
evaluated by integrating the overlapped portion of the spectra
Based on the premise that the test compounds displayed an
appreciable interaction with biological macromolecules, such
as DNA and proteins, it was pertinent to investigate the
cytotoxic effect of these compounds on model human cell
lines. As evident from Fig. 6, the ligand as well as the Ni(II)-
complex failed to exert any cytotoxic effect on human cervical
carcinoma (HeLa) cell lines. In contrast, the Cu(II)-complex
revealed a dose-dependent cytotoxic effect, with an IC50 value
in Fig. 5 and Fig. S18 (ESI
3
). If both the donor and acceptor are
tumbling rapidly and free to assume any orientations, then the
2
36a
dipole orientation factor, k , equals 2/3. In the present case,
3
6
n = 1.36 and W
obtained by measuring the change in donor fluorescence. The
donor fluorescence intensity (F ) was measured in the absence
and presence (F ) of acceptor. The efficiency of transfer (E) was
calculated using
D
= 0.118. The energy transfer data were
d
a
3
of 31.27 mM (Fig. S19, ESI ). It can be mentioned here that the
viability of cells treated with the Cu(II)-complex was signifi-
cantly low (p value , 0.001 based on ANOVA) compared to the
viability observed in the case of cells treated with the ligand or
Ni(II)-complex. The anti-proliferative effect of the Cu-complex
against HeLa cells indicates that the complex has the potential
to act as an anti-cancer chemotherapeutic agent. There are
reports which suggest that Cu(II) complexes are cytotoxic and
this phenomenon has been attributed to their DNA cleavage
E = 1 2 F /Fd
(7)
a
3
7
activity. However, in the present study, it was observed that
the Cu(II)-complex failed to display any DNA cleavage activity
3
even at a concentration of 60 mM (Fig. S14A–B, ESI ). These
results suggest that in the case of the Cu(II)-complex, the
observed cytotoxicity is probably attributed to a mechanism
other than DNA cleavage and warrants further investigation.
Conclusions
In summary, a new pyridine-based thiosemicarbazone ligand
LH and its Cu(II) and Ni(II) complexes have been synthesized
and characterized by structural, analytical and detailed
spectroscopic methods. The results of DNA binding experi-
Fig. 5 The overlap of the fluorescence spectra of BSA (red line) and the
absorption spectra of compound 1 (blue line).
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ments revealed that the free ligand and its Ni(II) and Cu(II)
complexes significantly bind to double stranded DNA, possibly
by intercalation. Their interactions with plasmid DNA and
agarose gel electrophoresis suggested that none of the
complexes could induce DNA cleavage, although the Cu(II)
complex was able to strongly bind to DNA and interfere with
its electrophoretic mobility. The protein binding studies of the
compounds suggested a static quenching interaction with BSA
for compound 1 and 3 whereas the fluorescence quenching of
BSA in the presence of compound 2 was a combined effect of
both static and collision/dynamic quenching processes.
Interestingly, the fluorescence quenching of BSA in the
presence of compounds 1, 2 and 3 was due to the spectral
overlap between the donor’s fluorescence and acceptor’s
absorption, suggesting the quenching of the fluorescence of
BSA is attributed to an energy transfer process from the
tryptophan residues to the BSA bound compounds. Amongst
the test compounds, only the Cu-complex displayed prominent
cytotoxic activity against HeLa cells. This compound holds
promise as an anti-cancer chemotherapeutic agent and in
future studies, our endeavour would be to unravel the
mechanism of the cytotoxic activity of this compound.
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F. Zani, J. Inorg. Biochem., 2005, 99, 2231–2239.
6
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Acknowledgements
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G. D. acknowledges the Council of Scientific and Industrial
Research (EMR/01/3664/12) and the Department of Science
and Technology (DST), New Delhi, India for financial support,
CIF and IIT Guwahati for providing instrument facilities and
DST FIST for the single crystal X-ray diffraction facility. A. R
thanks the Department of Biotechnology (DBT), Government
of India, for a research grant (BT/01/NE/PS/08). A. B, D. T and
C. K are thankful to IIT Guwahati for a research fellowship. We
are also thankful to Debraj Biswas for his kind cooperation.
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