Effect of substitution and planarity of the ligand on DNA/BSA interaction, free radical scavenging and cytotoxicity of diamagnetic Ni(ii) complexes: A systematic investigation

Article abstract of DOI:10.1039/c1dt10767d

Four new bivalent nickel hydrazone complexes have been synthesised from the reactions of [NiCl₂(PPh₃)₂] with H ₂L {L = dianion of the hydrazones derived from the condensation of salicylaldehyde or o-hydroxy acet

Full text of DOI:10.1039/c1dt10767d

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PAPER
Effect of substitution and planarity of the ligand on DNA/BSA interaction,
free radical scavenging and cytotoxicity of diamagnetic Ni(II) complexes: A
systematic investigation†
Palanisamy Sathyadevi,a Paramasivam Krishnamoorthy,a Rachel R. Butorac,b Alan H. Cowley,b
Nattamai S. P. Bhuvaneshc and Nallasamy Dharmaraj*a
Received 27th April 2011, Accepted 24th June 2011
DOI: 10.1039/c1dt10767d
Four new bivalent nickel hydrazone complexes have been synthesised from the reactions of
[NiCl2(PPh3)2] with H2L {L = dianion of the hydrazones derived from the condensation of
salicylaldehyde or o-hydroxy acetophenone with p-toluic acid hydrazide (H2L1) (1), (H2L2) (2) and
o-hydroxy acetophenone or o-hydroxy naphthaldehyde with benzhydrazide (H2L3) (3) and (H2L4) (4)}
and formulated as [Ni(L1)(PPh3)] (5), [Ni(L2)(PPh3)] (6), [Ni(L3)(PPh3)] (7) and [Ni(L4)(PPh3)] (8).
Structural characterization of complexes 5–8 were accomplished by using various physico-chemical
techniques. In order to study the influence of substitution in the ligand and its planarity on the
biological activity of complexes 5–8 containing them, suitable hydrazone ligands 1–4 have been selected
in this study. Single crystal diffraction data of complexes 5, 7 and 8 proved the geometry of the
complexes to be distorted square planar with a 1 : 1 ratio between the metal ion and the coordinated
hydrazones. To provide more insight on the mode of action of complexes 5–8 under biological
conditions, additional experiments involving their interaction with calf thymus DNA (CT DNA) and
bovine serum albumin (BSA) were monitored by UV-visible and fluorescence titrations respectively.
Further, the ligands 1–4 and corresponding nickel(II) chelates 5–8 have been tested for their scavenging
-
effect towards OH and O2 radicals. The effect of complexes 5–8 to arrest the growth of HeLa and
Hep-2 tumour cell lines has been studied along with the cell viability against the non-cancerous NIH
3T3 cells under in vitro conditions.
cations are trafficked and stored prior to their incorporation
Introduction
into different metalloproteins.1 Metal chelation is an excellent
Transition metal chelates that play a key role in bio-inorganic
ways to increase the lipophilic character of the organic moiety.
chemistry and redox enzyme systems serve as the basis of models
In fact, on coordination, ligands might improve their bioactivity
for active sites in biologically important compounds. Despite
profiles, while some inactive ligands may acquire pharmacological
many efforts in trying to unravel the role of metal ions in
properties and consequently they have become an important class
biology, much remains to be learned about how these metal
of structure-selective binding agents for nucleic acids. Compounds
that can selectively recognize a non-duplex structure involved
in the control of gene expression have considerable potential
as chemotherapeutic agents for a variety of diseases. Metal
complexes can interact with non-duplex structures through the
same mechanisms as duplex DNA and RNA, i.e. covalent binding,
intercalation and groove binding.
aDepartment of Chemistry, Bharathiar University, Coimbatore, 641 046,
India. E-mail: dharmaraj@buc.edu.in; Fax: +91 422 2422387; Tel: +91 422
2428316
bDepartment of Chemistry and Biochemistry, University of Texas at Austin,
Austin, Texas, 78712, U.S.A
cDepartment of Chemistry, Texas A&M University, College Station, TX
77843, U.S.A
Among these DNA-targeted guest molecules, ruthenium, cobalt
and copper complexes are numerous in the literature.2–5 Though
nickel is an essential element related to life process and an active
component in various types of enzymes, studies on the interactions
of nickel(II) complexes with DNA are comparably less.6–8 In recent
years, reports on the role of nickel in bioinorganic chemistry have
been rapidly expanding. The interaction of nickel(II) complexes
with DNA has been mainly dependent on the structure of the
ligand exhibiting intercalative behavior and/or DNA cleavage
† Electronic supplementary information (ESI) available: Crystal packing
diagram of the unit cell of complexes 5, 7 and 8 (Figures S1, S2 and S3),
Electronic absorption spectra of binding of complexes 5, 6 and 7 with DNA
(Figures S4, S5 and S6), Emission spectra of DNA–EB in the presence of
complexes 5, 6 and 7 (Figures S7, S8 and S9), Emission spectra of binding
of complexes 5, 6 and 7 with BSA (Figures S10, S11 and S12), Absorption
spectra of binding of complexes 5, 6 and 7 with BSA (Figures S13, S14 and
S15), Synchronous spectra (Dl = 15 and 60 nm) of binding of complexes 5,
6 and 7 with BSA (Figures S16, S17 and S18). CCDC reference numbers
751231, 780528 and 822887. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c1dt10767d
9690 | Dalton Trans., 2011, 40, 9690–9702
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ability.9–13 Particulate nickel compounds (such as Ni2S3) enter a
cell by phagocytosis, find their way to the nucleus, and cause
oxidative DNA strand breaks, DNA–DNA cross-links and DNA–
protein cross-links.14–17 The mechanism by which this occurs is
of considerable interest not only to toxicologists investigating
carcinogenesis, but also to chemists designing DNA targeted
drugs.
Sigma–Aldrich and used as received. All the chemicals and
reagents used for DNA binding, protein binding, antioxidant and
cytotoxicity assays were of high quality and available from reputed
suppliers.
Synthesis of ligands (1–4) and corresponding nickel hydrazone
complexes (5–8)
Proteins play an important role in transportation and deposition
of endogenous and exogenous substances such as fatty acids,
harmones and medicinal drugs. Drug interactions at the protein
binding level will significantly affect the apparent distribution vol-
ume of the drugs and also the elimination rate of drugs. Therefore,
studies on this aspect are undertaken with a view to derive some
vital information regarding the structural features that determine
the therapeutic effectiveness of a drug, and have been an interesting
research field in life sciences, chemistry and clinical medicine.18
Studies on the binding of albumins from blood plasma with
pyridoxal phosphate, cystein, glutathione, Schiff bases, various
metal ions such as Cu(II), Ni(II), Mn(II), Co(II), Hg(II), Zn(II),
metal complexes and metal-binding metallothionein proteins are
reported in the literature. Although studies have been carried out
to understand the interaction of metal complexes, metal ions and
Schiff base ligands with albumins, corresponding investigations
on nickel hydrazone complexes seems to be scanty.19–27
Based on the above facts and considering the role and activity
of nickel and its complexes in biological systems, along with
the significance of hydrazones in medicine, we present in this
work a systematic study on the synthesis and molecular structure
of nickel(II) complexes containing hydrazone ligands and their
interaction with nucleic acids (DNA) and proteins, along with
cytotoxicity and antioxidant assays. Further, the effect of the
substituent group present on the ligand as well as planarity of
the molecule on the above said properties were also studied in
detail.
The reactions involved in the synthesis of hydrazone ligands and
corresponding nickel complexes are given in Scheme 1.
Synthesis of nickel precursor complex. The precursor metal
complex [NiCl2(PPh3)2] was prepared according to the literature
method.28
Synthesis of [4-methyl-benzoic acid (2-hydroxy-benzylidene)-
hydrazide] (H2L1) (1). The ligand 4-methyl-benzoic acid (2-
hydroxy-benzylidene)-hydrazide (1) was prepared by refluxing a
mixture of salicylaldehyde (0.122 g; 1 mM) and p-toluic hydrazide
(0.150 g; 1 mM) in 50 mL of absolute ethanol for 8 h as given in
Scheme 1. The reaction mixture was cooled to room temperature
and the solid obtained was filtered, washed several times with dis-
tilled water and recrystallized from EtOH to afford ligand 1 in pure
form with 85% yield. Anal. Found (%) for C15H14N2O2 (Mol. wt =
254.28): C, 70.93%; H, 5.32%; N, 11. 27%. Calculated (%) for C,
70.85%; H, 5.55%; N, 11.02%. Selected IR bands (n in cm-1): 3388
(phenolic, –OH); 3215 (–NH); 1622 (>C O); 1572(>C N–);
1035 ( N–N<); 1270 (phenolic, C–OH).1H NMR (DMSO-D6,
ppm): 12.10 (s, 1H, –OH); 11.20 (s, 1H, –NH); 8.24 (s, 1H, –
CH N–); 7.84–6.88 (m, 8H, Ar–H); 2.44 (s, 3H, –CH3).
Synthesis of [4-methyl-benzoic acid [1-(2-hydroxy-phenyl)-
ethylidene]-hydrazide] (H2L2) (2). Ligand 2 was prepared by
a procedure similar to the one described above utilizing o-
hydroxyacetophenone (0.136 g; 1 mM) and p-toluic hydrazide
(0.150 g; 1 mM) (Scheme 1). Anal. Found (%) for C16H16N2O2
(Mol. wt = 268.31): C, 71.93%; H, 6.12%; N, 10.77%. Calculated
(%) for C, 71.62%; H, 6.01%; N, 10.44%. Selected IR bands (n
in cm-1): 3342 (phenolic, –OH); 3206 (–NH); 1661 (>C O);
1608(>C N–); 1005 ( N–N<); 1269 (phenolic, C–OH). 1H
NMR (DMSO-D6, ppm): 12.72 (s, 1H, –OH); 8.94 (s, 1H, –NH);
7.78–6.90 (m, 8H, Ar–H); 2.46 (s, 3H, CH3C N–); 2.42 (s, 3H,
–CH3).
Experimental section
Materials and reagents
Reagent grade chemicals NiCl2·6H2O, triphenylphosphine, sali-
cylaldehyde, o-hydroxy acetophenone, o-hydroxy napthaldehyde,
benzhydrazide and p-toluic hydrazide were purchased from
Scheme 1
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Synthesis of [benzoic acid [1-(2-hydroxy-phenyl)-ethylidene]-
hydrazide] (H2L3) (3). Ligand 3 was prepared using a similar pro-
cedure as described above with o-hydroxyacetophenone (0.136 g;
1 mM) and benzhydrazide (0.136 g; 1 mM) as suitable reactants.
(Scheme 1). Anal. Found (%) for C15H14N2O2 (Mol. wt = 254.28):
C, 69.93%; H, 4.32%; N, 14. 27%. Calculated (%) for C, 70.85%; H,
5.55%; N, 11.02%. Selected IR bands (n in cm-1): 3328 (phenolic,
–OH); 3213 (–NH); 1649 (>C O); 1605 (>C N–); 1026 ( N–
N<); 1269 (phenolic, C–OH). 1H NMR (DMSO-D6, ppm): 13.32
(s, 1H, –OH); 11.30 (s, 1H, –NH); 7.94–6.87 (m, 9H, Ar–H); 2.49
(s, 3H, CH3C N–).
(>C N–N C<); 1346 (phenolic –C–O); 1329 (enolic –C–O);
1094 ( N–N ).1H NMR (DMSO-D6, ppm): 7.82–6.57 (m, 24H,
Ar–H); 2.54 (s, 3H, CH3–C N–).
Synthesis of [Ni(L4)(PPh3)] (8). Complex 8 was also prepared
in a manner similar to that mentioned in the previous cases by
treating [NiCl2(PPh3)2] (0.653 g; 1 mM) with ligand 4 (0.290 g;
1 mM). However, the reaction mixture after work-up afforded
needle like violet crystals of complex 8 which were analysed
through single crystal XRD to determine its geometry. Yield: 53%,
Color: violet, mp: 180 ◦C. Anal. Found (%) for NiC37H30N2O2
(Mol. wt. = 624.31) C, 61.94%; H, 3.91%; N, 12.96%. Calculated
(%) for C, 71.18%; H, 4.84%; N, 4.49%. Selected IR bands (n
in cm-1): 1597 & 1530 (>C N–N C<); 1345 (phenolic –C–
O); 1328 (enolic –C–O); 1095 ( N–N ).1H NMR (DMSO-D6,
ppm): 9.62 (s, 1H, –CH N–); 8.18–6.61 (m, 26H, Ar–H).
Synthesis of [benzoic acid(3-hydroxy-naphthalen-2-ylmethylene)-
hydrazide] (H2L4) (4). As described in the above cases, ligand 4
was prepared from o-hydroxynaphthaldehyde (0.172 g; 1 mM)
and benzhydrazide (0.136 g; 1 mM) (Scheme 1). Anal. Found (%)
for C18H14N2O2 (Mol. wt = 290.31): C, 69.93%; H, 4.32%; N, 14.
27%. Calculated (%) for C, 74.47%; H, 4.86%; N, 9.65%. Selected
IR bands (n in cm-1): 3486 (phenolic, –OH); 3223 (–NH); 1616
(>C O); 1570(>C N–); 1030 ( N–N<); 1290 (phenolic, C–
Physical measurements and instrumentation
Elemental analyses (% C, H & N) were performed on a Vario
EL III CHNS analyzer and all IR spectra were recorded as KBr
pellets on a Nicolet Avatar instrument in the frequency range
400–4000 cm-1. Both electronic and fluorescence spectra of the
complexes were recorded in DMSO : buffer (1 : 99) using Jasco
V-630 spectrophotometer and Jasco FP 6600 spectrofluorometer
1
OH). H NMR (DMSO-D6, ppm): 12.76 (s, 1H, –OH); 12.18 (s,
1H, –NH); 9.49 (s, 1H, CH N–); 8.22–7.22 (m, 11H, Ar–H).
Synthesis of the new nickel(II) hydrazone complex [Ni(L1)(PPh3)]
(5). Complex 5 was synthesised by refluxing a methanolic
solution of [NiCl2(PPh3)2] (0.653 g; 1 mM) and ligand 1 (0.254 g;
1 mM) (40 mL) in the presence of KOH (Scheme 1) for 5 h. After
cooling the reaction mixture to room temperature, the precipitate
formed was filtered, washed with methanol and dried in vacuo.
The purity of the complex was checked by TLC, following which
tetragonal shaped crystals of complex 5 suitable for single-crystal
X-ray diffraction studies were obtained by recrystallisation in
dichloromethane. Yield: 58%, Color: red, mp: 220 ◦C. Anal. Found
(%) for NiC34H30N2 O2P (Mol. Wt. = 588.28): C, 57.24%; H, 4.81%;
N, 11.52%. Calculated (%) for C, 69.42%; H, 5.14%; N, 4.76%.
Selected IR bands (n in cm-1): 1597 & 1501 (>C N–N C<);
1336 (phenolic –C–O); 1302 (enolic –C–O); 1099 ( N–N ). 1H
NMR (DMSO-D6, ppm): 8.31 (s, 1H, HC N–); 7.85–6.52 (m,
23H, Ar–H); 2.31 (s, 3H, –CH3).
1
respectively. H NMR spectra of the ligands and corresponding
nickel(II) complexes were recorded on a Bruker AMX 500 MHz
spectrometer using tetramethylsilane as an internal standard and
DMSO as a solvent. The X-ray diffraction data of complexes 5
and 7, were collected at 100 and 293 K respectively with Mo-Ka
˚
radiation (l = 0.71075 and 0.71073 A) using a Bruker Smart APEX
II CCD diffractometer equipped with a graphite monochromator.
The structures were solved by direct methods and refined full-
matrix least squares (on F2) SHELXS97 and SHELXL9729 and
the graphics were produced using PLATON97.30 All the non-
hydrogen atoms were refined anisotropically and the hydrogen
atoms were positioned geometrically and refined as riding model.
The XRD data of the complex 8 was collected at 110 K with
Cu-Ka radiation l = 1.5418 using a BRUKER GADDS X-ray
(three-circle) diffractometer. The structure was refined (weighted
least squares refinement on F2) to convergence.29 X-seed was
employed for the final data presentation and structure plots.31
Integrated intensity information for each reflection was obtained
by reduction of the data frames with APEX2.32 The integration
method employed a three dimensional profiling algorithm and all
data were corrected for Lorentz and polarization factors, as well
as for crystal decay effects. Finally the data was merged and scaled
to produce a suitable data set. SADABS33 was used to correct the
data for absorption effects. A solution was obtained readily using
XS29 as implemented in SHELXTL. Hydrogen atoms bound to
carbon were placed in idealized positions, and were set riding
on the parent atom. All non-hydrogen atoms were refined with
anisotropic thermal parameters.
Synthesis of [Ni(L2)(PPh3)] (6). Complex 6 was prepared by a
procedure similar to that given in the case of 5, from [NiCl2(PPh3)2]
(0.653 g; 1 mM) and ligand 2 (0.268 g; 1 mM) as reactants. The
work-up of the reaction mixture did not yield any crystals suitable
for single crystal XRD and hence this was not recorded. Yield:
55%, Color: red, mp: 212 ◦C. Anal. Found (%) for NiC35H32N2O2
(Mol. wt. = 602.30) C, 61.94%; H, 3.91%; N, 12.96%. Calculated
(%) for C, 69.79%; H, 5.36%; N,4.65%. Selected IR bands (n
in cm-1): 1579 & 1532 (>C N–N C<); 1356 (phenolic –C–
O); 1306 (enolic –C–O); 1098 ( N–N ). 1H NMR (DMSO-D6,
ppm): 7.75–6.40 (m, 23H, Ar–H); 2.52 (s, 3H, CH3C N–); 2.41
(s, 3H, –CH3).
Synthesis of [Ni(L3)(PPh3)] (7). Reaction of [NiCl2(PPh3)2]
(0.653 g; 1 mM) with ligand 3 (0.254 g; 1 mM) under identical
experimental conditions as reported for the synthesis of complex
5 yielded tetragonal shaped crystals of complex 7 which were
analysed by single crystal XRD. Yield: 59%, Color: red, mp:
DNA binding studies
Electronic absorption spectroscopy. Experiments involving
the interaction of nickel(II) hydrazone complexes with
DNA were carried out in twice distilled water containing
tris(hydroxymethyl)amino methane buffer (Tris, 5 mM) and NaCl
◦
208 C. Anal. Found (%) for NiC34H30N2O2 (Mol. wt. = 588.28)
C, 61.94%; H, 3.91%; N, 12.96%. Calculated (%) for C, 69.42%;
H, 5.14%; N, 4.76%. Selected IR bands (n in cm-1): 1574 & 1527
9692 | Dalton Trans., 2011, 40, 9690–9702
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(50 mM) and the pH was adjusted to 7.2 with hydrochloric acid. A
solution of calf thymus DNA (CT DNA) in the above buffer with
a ratio of about 1.8–1.9 : 1 corresponding to the absorptions at 260
and 280 nm in the UV-visible absorbance indicating the sufficient
protein free nature of DNA.34 The DNA concentration per
nucleotide was determined from the molar extinction coefficient
(6600 M-1 cm-1) of the absorption band observed at 260 nm.35
Electronic absorption titration experiments were performed by
maintaining the concentration of the metal complex as constant
(25 mM) but with variable nucleotide concentrations ranging from
0 to 25 mM. While measuring the absorption spectra, equal
amounts of DNA were added to both complex and reference
solutions to eliminate the absorbance of DNA itself. The data
were then fit into the following equation and the intrinsic binding
constant Kb was calculated in each case.36
maintained for all the experiments. Samples were carefully de-
gassed using pure nitrogen gas for 15 min by using quartz cells (4
¥ 1 ¥ 1 cm) with high vacuum Teflon stopcocks. Stock solution
of BSA was prepared in 50 mM phosphate buffer (pH = 7.2)
and stored in the dark at 4 ◦C for further use. Concentrated
stock solutions of metal complexes were prepared by dissolving
them in DMSO : phosphate buffer (1 : 100) and diluted suitably
with phosphate buffer to get required concentrations. 2.5 ml of
BSA solution (mM) was titrated by successive additions of a
5 ml stock solution of complexes (10-4 M) using a micropipette.
Synchronous fluorescence spectra was also recorded using the
same concentration of BSA and complexes as mentioned above
with two different Dl (difference between the excitation and
emission wavelengths of BSA) values such as 15 and 60 nm.
Antioxidant studies
[DNA]/[ea - ef] = [DNA]/[eb - ef] + 1/Kb[eb - ef]
where [DNA] is the concentration of DNA in base pairs, ea
is the extinction coefficient of the complex at a given DNA
concentration, ef is the extinction coefficient of the complex in free
solution and eb is the extinction coefficient of the complex when
fully bound to DNA. A plot of [DNA]/[eb - ef] versus [DNA] gave
a slope and an intercept equal to 1/[ea - ef] and (1/Kb)[eb - ef],
respectively. The intrinsic binding constant Kb is the ratio of the
slope to the intercept.
The free radical scavenging ability of the free hydrazone ligands
and nickel(II) complexes were determined against both hydroxyl
and superoxide. The hydroxyl radical scavenging activity of the
compounds was investigated using the method described by
-
Nash,38 whereas superoxide (O2 ) radical scavenging assay was
undertaken based on the capacity of the complexes to inhibit for-
mazan formation by scavenging the superoxide radicals generated
in the riboflavin–light–NBT system.39 For each of the above assays,
the tests were run in triplicate by varying the concentration of the
complexes, ranging from 10–50 mM. The percentage activity was
calculated by using the formula, % activity = [(A0 - AC)/A0] ¥
100, where A0 and AC represent the absorbance in the absence and
presence of the test complex, respectively. The 50% activity (IC50)
is calculated from the result of percentage activity.
Competitive binding fluorescence measurements. The apparent
binding constant (Kapp) of the complexes was determined by
a fluorescence spectral technique using ethidium bromide (EB)
bound CT DNA solution in Tris-HCl buffer solution (pH, 7.10).
The changes in fluorescence intensities at 605 nm (545 nm
excitation) of EB bound to DNA were measured with respect to
different concentrations of the complex. EB was non-emissive in
Tris-HCl buffer solution (pH 7.10) due to fluorescence quenching
of free EB by the solvent molecules. In the presence of DNA,
EB showed enhanced emission intensity due to its intercalative
binding to DNA. A competitive binding of the metal complexes
to CT DNA resulted in the displacement of the bound EB, thereby
decreasing its emission intensity. The quenching constant (Kq) was
calculated using the classical Stern–Volmer equation:37
Cytotoxicity
In vitro cytotoxicity (IC50) was performed on HeLa (human
cervical cancer cells), Hep-2 (human epidermoid cancer cells)
and normal NIH 3T3 (mouse embryonic cells) cell lines using
the MTT assay method.40 Tumour cell lines (HeLa and Hep-
2) used in this work were grown in Eagles Minimum Essential
Medium containing 10% fetal bovine serum (FBS) and NIH 3T3
fibroblasts were grown in Dulbeccos Modified Eagles Medium
(DMEM) containing 10% FBS. For screening studies, the cells
were seeded into 96-well plates in 100 ml of respective medium
containing 10% FBS, at plating density of 10 000 cells/well and
incubated at 37 ◦C, 5% CO2, 95% air and 100% relative humidity
for 24 h prior to addition of test compounds. The test compounds
were dissolved in dimethylsulfoxide and diluted in respective serum
free medium. After 24 h, 100 ml of the medium containing the test
compounds at various concentrations (0.031, 0.062, 0.125, 0.5,
1 mM) was added and incubated at 37 ◦C, 5% CO2, 95% air and
100% relative humidity for 48 h. Experiments were carried out in
triplicate and the medium containing no test compounds served
as a control.
I0/I = Kq [Q] + 1
where I0 is the emission intensity in the absence of quencher, I
is the emission intensity in the presence of quencher, Kq is the
quenching constant and [Q] is the quencher concentration. Kq is
the slope, obtained from the plot of I0/I vs. [Q]. The apparent
binding constant (Kapp) has been calculated from the equation:
KEB [EB] = Kapp [complex]
The complex concentration was obtained from the value at a 50%
reduction of the fluorescence intensity of EB and KEB = 1.0 ¥ 107
M-1, ([EB] = 10 mM).
Protein binding studies
After 48 h, 15 ml of MTT (5 mg ml-1) in phosphate buffered saline
(PBS) was added to each well and incubated at 37 ◦C for 4 h. The
medium with MTT was then flicked off and the formed formazan
crystals were solubilized in 100 ml of DMSO and the absorbance
at 570 nm was measured using a micro plate reader. The % cell
inhibition was determined using the following formula and a graph
Binding of nickel hydrazone complexes with bovine serum albumin
(BSA) was studied using fluorescence spectra recorded with an
excitation at 280 nm and corresponding emission at 345 nm
assignable to that of free bovine serum albumin (BSA). The
excitation and emission slit widths and scan rates were constantly
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Table 1 Crystal structure data of complexes 5, 7 and 8
Complex 5
Complex 7
Complex 8
Formula
C33H27N2Ni O2P
573.25
100(2)
0.71075
Triclinic
¯
P1
C33H27N2Ni O2P
573.25
293(2)
0.71073
Triclinic
¯
P1
C36H27N2NiO2P
609.28
110(2)
1.54178
Monoclinic
Molecular weight
T (K)
˚
l (A)
Crystal system
Space group
P21/n
Cell dimensions
˚
a (A)
10.2703(10)
11.8729(11)
11.9851(11)
81.516(2)
68.128(2)
89.718(2)
11.5980(3)
15.2119(4)
17.2447(5)
106.718(2)
99.364(2)
104.284(2)
4
15.778(2)
8.8459(14)
20.488(3)
90
98.896(9)
90
˚
b (A)
˚
c (A)
a (◦)
b (◦)
g (◦)
Z
2
4
hkl limits
-12 ꢀ h ꢀ 12
-14 ꢀ k ꢀ 14
-13 ꢀ l ꢀ 14
1.738
-16 ꢀ h ꢀ 16
-20 ꢀ k ꢀ 21
-23 ꢀ l ꢀ 23
1.393
-17 ꢀ h ꢀ 17
-9 ꢀ k ꢀ 9
-23 ꢀ l ꢀ 22
1.432
Dcalcd (Mg m-3)
F(000)
596
1192
1264
Crystal size (mm3)
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
R indices (all data)
0.16 ¥ 0.10 ¥ 0.09
4696 [R(int) = 0.0311]
4696/0/200
1.072
R1 = 0.0584, wR2 = 0.1248
R1 = 0.0489, wR2 = 0.1179
0.220 ¥ 0.180 ¥ 0.120
15259 [R(int) = 0.042]
9357/0/705
1.016
R1 = 0.0453, wR2 = 0.1054
R1 = 0.0924, wR2 = 0.1302
0.08 ¥ 0.06 ¥ 0.03
4125 [R(int) = 0.0576]
4125/0/379
1.052
R1 = 0.0384, wR2 = 0.0906
R1 = 0.0548, wR2 = 0.0960
was plotted between % cell inhibition and concentration. From the
graph, IC50 values were calculated.
The crystal structures of complexes 5 and 8 consist of a single
molecular unit, while that of complex 7 consists of two molecular
units, namely Ni1 and Ni2, which are crystallographically indepen-
dent but chemically similar. All three complexes 5, 7 and 8 possess
distorted square planar geometry around the Ni(II) ion in which
the geometric plane is occupied by two oxygen atoms and one
nitrogen atom of the respective double negative ONO tridentate
hydrazone ligands 1, 3 and 4, while the fourth coordination site is
occupied by the phosphorous atom of triphenylphosphine.
% Cell inhibition = 100 - Abs(drug)/Abs(control) ¥ 100
Results and discussion
Description of solid state structures
In complex 5, the angles between the phenyl plane of hydrazide
(C9–C15) and the Ni(II) mean coordination plane (Ni, O1, O2,
N1, P1) are 4.96◦, while the corresponding angles between the
phenyl planes (C1A–C6A) and (C1B–C6B) and the Ni1 and Ni2
molecular units of 7 are 6.92◦ and 7.01◦, respectively. In complex
8, the corresponding angles between the phenyl plane of the
hydrazide (C13–C18) and the Ni(II) mean coordination plane (Ni,
O1, O2, N1, P1) are 6.95◦. The respective ligand–metal–ligand
bite angles, [N1–Ni1–O1] and [N1–Ni1–O2] in single monomeric
complexes 5 and 8 are found to be 95.7(1)◦, 83.5(1)◦ and 94.78(9)◦
and 83.72(9)◦. However, with complex 7, two monomeric units
showed their bite angles as 95.54(9)◦, 84.32(8)◦, 95.53(9)◦ and
84.76(9)◦ for [N1A–Ni1–O1A] and [N1B–Ni2–O2B] respectively,
which are close to that of the above monomeric unit complexes.
The bond distances of Ni–O1[1.820(2)] and Ni–O2 [1.844(2)]
in complex 5 are larger when compared to those of Ni–O1A
[1.802(2)], Ni–O2A [1.830(2)], Ni–O1B [1.796(2)] and Ni–O2B
[1.824(2)] in complex 7, and Ni–O1[1.816(2)] and Ni–O2[1.836(2)]
in complex 8, suggesting that the bond distances between the
central Ni(II) and the donor atoms are affected by the nature of
the substituents in the aroylhydrazone residue and the difference
in this case is due to the presence of electron donating group
(R = CH3) in complex 5 relative to that of the unsubstituted
complex (R = H) in 7 and 8.41 However, the bond distance of
The solid-state structure of complexes 5, 7 and 8 were analysed
by single crystal X-ray studies. Details of the data collection
conditions and the parameters of refinement process are given
in Table 1. The perspective ORTEP views of complexes 5, 7 and 8
with atom numbering schemes are depicted in Fig. 1, 2 and 3, with
corresponding selected geometric parameters given in Table 2.
Fig. 1 The molecular structure of complex 5, with displacement ellipsoids
drawn at the 50% probability level.
9694 | Dalton Trans., 2011, 40, 9690–9702
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Fig. 2 The molecular structure of 7, with displacement ellipsoids drawn at the 25% probability level.
◦
˚
Table 2 Selected bond lengths (A) and bond angles ( )
C33 H27 N2 Ni O2 P (5) C33 H27 N2 Ni O2 P (7)
C36 H27 N2 Ni O2 P (8)
Bond lengths
Bond angles
Bond lengths
Ni1–N1A 1.869(2) N1A–Ni1–O2A
86.93(7) Ni1–O1A 1.802(2) O2A–Ni1–P1
94.32(7) Ni1–O2A 1.830(2) P1–Ni1–O1A
Ni1–P1A 2.240(8) O1A–Ni1–N1A
174.75(8) N1A–N2A 1.405(3) P1–Ni1–N1A
174.5(1) O2A–C9A 1.303(3) O1A–Ni1–O2A
Bond angles
Bond lengths
Bond angles
Ni1–N1 1.854(2) N1–Ni1–O1
Ni1–O1 1.820(2) O1–Ni1–P1
Ni1–O2 1.844(1) P1–Ni1–O2
Ni1–P1 2.226(9) O2–Ni1–N1
C1–O1 1.333(5) N1–Ni1–P1
C1–C6 1.409(5) O2–Ni1–O1
95.7(1)
84.32(4) Ni1–N1 1.833(2) N1–Ni1–O1
87.88(6) Ni1–O1 1.816(2) O1–Ni1–P1
92.21(7) Ni1–O2 1.836(2) P1–Ni1–O2
95.54(9) Ni1–P1 2.235(9) O2–Ni1–N1
172.20(6) C1–O1 1.318(3) N1–Ni1–P1
175.56(9) C1–C6 1.404(4) O2–Ni1–O1
94.78(9)
86.36(6)
95.20(6)
83.72(9)
177.30(7)
177.86(9)
83.5(1)
C6–C7 1.442(2) C6–C7–N1–N2 178.9(3) O1A–C1A 1.318(3) N1B–Ni2–O2B
C7–N1 1.290(5) C9–C8–N2–N1 178.2(3) N1A–C7A 1.300(3) O2B–Ni2–P2
N1–N2 1.409(3) C2–C1–O1–Ni1 -175.3(2) N2A–C9A 1.302(3) P2–Ni2–O1B
N2–C8 1.314(5) C9–C8–O2–Ni1 -178.8(2) Ni1–N1B 1.855(2) O1B–Ni2–N1B
84.76(9) C6–C11 1.423(4) C6–C11–N1–N2 179.3(2)
92.07(7) C11–N1 1.304(4) C13–C12–N2–N1 -177.5(2)
87.64(7) N1–N2 1.407(3) C13–C12–O2–Ni1 176.9(2)
95.53(9) N2–C12 1.299(4) C2–C1–O1–Ni1 -179.1(2)
176.82(7) C12–O2 1.319(3)
C8–O2 1.309(4)
Ni1–O1B 1.796(2) N1B–Ni2–P2
Ni1–O2B 1.824(2) O2B–Ni2–O1B
174.96(9)
Ni1–P1B 2.222(8) C6A–C7A–N1A–N2A 176.5(2)
N1B–N2B 1.402(3) N1A–N2A–C9A–C10A 177.0(2)
N1B–C7B 1.303(3) C7A–N2A–N1A–C9A 176.5(2)
N2B–C9B 1.305(4) N2B–N1B–C7B–C6B -175.1(2)
O2B–C9B 1.315(3) N1B–N2B–C9B–C10B -177.4(2)
O1B–C1B 1.316(4) C9B–N2B–N1B–C7B -177.8(2)
Ni–N1[1.854(2)] in 5 and Ni–N1[1.833(2)] in 8 are lower when
compared to that of Ni1–N1A[1.869(2)] and Ni2–N1B[1.855(2)]
of complex 7 which may be due to the attachment of the electron
releasing –CH3 group on the azomethine carbon in the latter case
and the absence of the same in the former cases of 5 and 8. The
Ni(II) center is shared by one five-membered {Ni1N1N2C8O2}
and one six membered chelate ring {Ni1N1C7C6C1O1} in
complex 5, whereas in 7 it is shared by {Ni1N1AN2AC9AO2A}
{Ni2N1BN2BC9BO2B} five membered chelate rings and
{Ni1O1AC1AC6AC7AN1A} {Ni2O1BC1BC6BC7BN1B} six
membered chelate rings. Similarly, in the case of 8, the Ni(II)
center is shared by {Ni1N1N2C12O2} five membered ring and
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Fig. 3 The molecular structure of 8, with displacement ellipsoids drawn at the 50% probability level.
{Ni1O1C1C6C11N1} six membered rings. The unit cell packing
diagrams of the complexes are presented in Fig. S1, S2 and S3,
ESI.†
DNA binding studies
UV-visible absorption studies
The electronic absorption spectra of complexes 5–8 displayed three
to five well-resolved bands in the range of 200 to 470 nm out of
which the high energy absorption bands appeared between 250–
300 nm and another absorption around 320–360 were assigned
to the intra-ligand charge transfer transitions of the type p→p*
and n→p*.42 Further, the bands appeared around 370 and 420 nm
have been assigned to ligand-to-metal charge transfer (LMCT)
as well as metal-to-ligand charge transfer (MLCT) transitions,
respectively.43,44 A representative electronic absorption spectra of
complex 8 without as well as with the added CT DNA is shown
in Fig. 4 (similar spectra of complexes 5, 6 and 7 are presented as
S4, S5 and S6 in the ESI†).
From the electronic absorption spectral data, it was clear that
upon increasing the concentration of DNA added to the metal
complexes 6, 7 and 8, all of the above mentioned absorption
bands showed hypochromism accompanied with bathochromic
shifts in most of the absorption bands. However, addition of CT
DNA to complex 5 showed only hypochromism, without any shift
suggesting that the nickel hydrazone complexes bind strongly to
DNA in an intercalative mode. These observations are similar
to those reported earlier for various metallointercalators.45 The
nature of the shift observed and the percentage of hypochromism
Fig. 4 Electronic absorption spectra of complex 8 (25 mM) in the absence
and presence of increasing amounts of CT DNA (2.5, 5.0, 7.5, 10.0,
12.5, 15.0, 17.5 and 20.0, 22.5 and 25 mM). Arrows show the changes in
absorbance with respect to an increase in the DNA concentration (Inset:
Plot of [DNA] vs. [DNA]/(ea - ef)).
in the absorption bands in the case of all the complexes after the
addition of DNA are given in Table 3.
In order to determine quantitatively the binding strength of
the complexes with CT DNA, intrinsic binding constants were
obtained by monitoring the changes in both the wavelength, as well
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Table 3 Nature of shift with % of hypochromism
Absorption wavelength
(nm) (absence of DNA)
Shift of wavelength (nm)
(presence of DNA)
Complex
Nature of shift
% of hypochromism
5
305
372
415
264
303
368
409
268
308
371
411
272
330
380
420
441
—
—
—
6
6
3
5
8
9
3
5
1
3
2
3
5
—
—
—
31.44
38.85
36.05
41.95
40.43
36.33
30.30
85.60
51.65
42.90
32.62
43.90
57.46
59.86
58.78
46.15
6
7
8
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
bathochromic
as the corresponding intensity of the absorption of the high energy
bands of complexes 5, 6, 7 and 8 respectively, upon increasing the
concentration of added DNA. The following equation was applied
to calculate the binding constant: [DNA]/[ea - ef] = [DNA]/[eb -
ef] + 1/Kb[eb - ef] and the value of the intrinsic binding constant
(Kb) was found to be 4.694 ¥ 104 M-1, 7.258 ¥ 104 M-1, 7.420 ¥ 104
M-1 and 1.726 ¥ 105 M-1, corresponding to complexes 5, 6, 7 and 8,
respectively. The magnitude of the binding constant value clearly
showed that complex 8 bound more strongly with CT DNA than
complexes 5, 6 and 7 through an intercalative mode.
Competitive binding between EB and complexes for CT DNA
Generally, steady-state competitive binding experiments using
metal complexes as quenchers provide some information about
the binding of the complexes to DNA. Ethidium bromide (EB) is
a planar cationic dye that is widely used as a sensitive fluorescence
probe for native DNA. EB emits intense fluorescent light in
the presence of DNA due to its strong intercalation between
the adjacent DNA base pairs. This displacement technique is
based on the decrease of fluorescence intensity resulting from the
displacement of bound EB from a DNA sequence by a quencher
and the quenching is due to the reduction of the number of binding
sites on the DNA that is available to the EB. Hence, this method
serves as indirect evidence to identify intercalative binding modes.
In our study, changes in the intensity as well as the position of
the emission band of EB bound DNA were monitored after the
addition of metal complex solution. The above said emission
band in the spectra of combined nickel hydrazone complex–
DNA system showed a significant bathochromic shift with a
simultaneous reduction in the fluorescence intensity (34.76%,
36.51%, 36.85% and 39.26%, respectively) revealing that the EB
molecules are displaced from their DNA binding sites by the added
complexes.
Fig. 5 Emission spectra of DNA-EB (10 mM), in the presence of 0, 10, 20,
30, 40, 50, 60, 70, 80, 90 and 100 mM of complex 8. The arrow indicates the
changes in the emission intensity as a function of complex concentration.
Inset: Stern–Volmer plot of the fluorescence titration data corresponding
to complex 8.
equation. The calculated apparent binding constant values, Kapp
,
corresponding to different nickel hydrazone complexes 5–8 was
5.423 ¥ 105 M-1, 5.622 ¥ 105 M-1, 5.762 ¥ 105 M-1 and 6.380
¥ 105 M-1 respectively, whereas the quenching constant Kq was
determined to be 5.730 ¥ 103 M-1, 5.746 ¥ 103 M-1, 6.215 ¥ 103
M-1 and 6.584 ¥ 103 M-1. This data showed the higher quenching
efficiency of complex 8 compared to that of the other complexes
under investigation, reflecting a strong binding of the former with
DNA than the rest of the complexes, similar to the results of the
absorption spectral study.
The fluorescence spectrum of EB bound-DNA quenched by
complex 8 is shown in Fig. 5 (respective spectra of complexes 5, 6
and 7 are presented as S7, S8 and S9 in the ESI†) with the Stern–
Volmer plot as an inset to the figures. The observed linearity in the
plot supported the fact that the quenching of EB bound to DNA by
the test complex is in good agreement with the linear Stern–Volmer
Protein binding studies
Fluorescence quenching of BSA by metal complexes (5–8)
Qualitative analysis of the binding of chemical compounds to
BSA is generally detected by examining its fluorescence spectra.
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Generally, the fluorescence of a protein is caused by three intrinsic
characteristics of the protein, namely tryptophan, tyrosine, and
phenylalanine residues. Fluorescence quenching refers to any
process that reduces the fluorescence intensity of the fluorophore
due to a variety of molecular interactions such as excited-state
reactions, molecular rearrangement, energy transfer ground-state
complex formation and collision quenching. Fig. 6 shows the
effect of increasing the concentration of added complex 8 on the
fluorescence emission of BSA (for complexes 5–7, see S10, S11 and
S12, ESI†). Addition of respective nickel hydrazone complexes to
BSA resulted in the quenching of fluorescence emission intensity
along with a hypsochromic shift of 6 to 8 nm, revealing quenching
due to the complex formed between the nickel(II) hydrazone
chelates and BSA.
similar behaviour of intensity increase with a slight hypsochromic
shift was observed, revealing that there exists a static interaction
between BSA and the added complexes due to the formation of a
ground state complex of the type BSA–metal complex as reported
earlier.46 A representative absorption spectrum of pure BSA and
BSA–complex 8 is shown in Fig. 7 (for complexes 5–7, see S13,
S14 and S15, ESI†).
Fig. 7 The absorption spectra of BSA (1 ¥ 10-5 M) and BSA–Complex 8
(BSA = 1 ¥ 10-5 M and Complex 8 = 1 ¥ 10-6 M).
Binding constant and number of binding sites
When small molecules bind independently to a set of equivalent
sites on a macromolecule, the equilibrium between free and bound
molecules is represented by the Scatchard equation:47,48
log [F0 - F/F] = log K + n log [Q]
Fig. 6 Emission spectrum of BSA (1 ¥ 10-6 M; lexi = 280 nm; lemi = 345
nm) as a function of concentration of complex 8 (0, 2, 4, 6, 8, 10, 12 and
14 ¥ 10-7 M). The arrow indicates the effect of metal complex 8 on the
fluorescence emission of BSA.
where K and n are the binding constant and the number of binding
sites, respectively.
Thus, a plot of log (F0 - F)/F versus log [Q] (Fig. 8) can be used
to determine the values of both K and n and such values calculated
for complexes 5–8 are listed in Table 4.
The fluorescence quenching is described by the Stern–Volmer
relation,
I0/I = 1+ KSV[Q]
where I0 and I are the fluorescence intensities of the fluorophore
in the absence and presence of quencher, KSV is the Stern–Volmer
quenching constant and [Q] is the quencher concentration. The
K
SV value obtained as a slope from the plot of I0/I vs. [Q] in respect
to complexes 5, 6, 7 and 8 were found to be 1.256 ¥ 106 M-1, 1.727
¥ 106 M-1, 1.788 ¥ 106 M-1 and 1.839 ¥ 106 M-1, respectively.
Quenching can occur by different mechanisms which are usually
classified as dynamic quenching and static quenching. Dynamic
quenching refers to a process in which the fluorophore and the
quencher come into contact during the transient existence of
the excited state, while static quenching refers to fluorophore–
quencher complex formation in the ground state. A simple method
to differentiate between the above types of quenching is UV-visible
absorption spectroscopy. The absorption band obtained in the
spectra of BSA at 278 nm in the absence of metal complexes
showed an increase in the intensity without any shift after the
addition of all the complexes except complex 8, whereupon a
Fig. 8 Plot of log [(F0 - F)/F] vs. log [Q].
From the values of n, it is inferred that there is only one indepen-
dent class of binding sites for complexes on BSA and also a direct
relation between the binding constant and number of binding sites.
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Table 4 Binding constant and number of binding sites for the interactions
of nickel hydrazone complexes 5–8 with BSA
cence of BSA may be due to the presence of tyrosine, tryptophan
and phenylalanine residues and hence spectroscopic methods are
usually applied to study the conformation of serum proteins. Ac-
cording to Miller,52 in synchronous fluorescence spectroscopy, the
difference between excitation and emission wavelength (Dl = lemi
- lexc) reflects the spectra of a different nature of chromophores,
with large Dl values such as 60 nm, the synchronous fluorescence
of BSA is characteristic of tryptophan residue and the small Dl
values such as 15 nm is characteristic of tyrosine.53 To explore the
structural changes of BSA due to the addition of nickel(II) hydra-
zone complexes, we measured synchronous fluorescence spectra
with respect to addition of complexes 5–8. The synchronous
fluorescence spectra of BSA with various concentrations of metal
complexes were recorded at both Dl = 15 nm and 60 nm. Upon
increasing the concentration of nickel hydrazone complexes, the
intensity of emission corresponding to tryptophan was found
to decrease with a hypsochromic shift of emission wavelength.
The representative spectrum of complex 8 is given in Fig. 9 (for
complexes 5, 6 and 7, see S16, S17 and S18, ESI†).
At the same time, the tyrosine fluorescence emission showed
very little decrease in the intensity without a significant change
in the position of the emission band. These experimental results
indicate that the metal complex does not affect the microenvi-
ronment of tyrosine residues during the binding process and the
polarity around the tryptophan residues is decreased and the
hydrophobicity around the tryptophan residues is strengthened.
The hydrophobicity observed in fluorescence and synchronous
measurements confirmed the effective binding of all the complexes
with the BSA.
System
K (M-1)
n
BSA + complex 5
BSA + complex 6
BSA + complex 7
BSA + complex 8
5.670 ¥ 105
8.300 ¥ 105
1.221 ¥ 106
1.086 ¥ 107
0.9403
0.9442
1.0232
1.1312
The results of binding experiments clearly proved that among
complexes 5 and 6, the later showed stronger binding with
both DNA and BSA compared to the former, which can be
attributed to the presence of an electron releasing methyl group
at the azomethine carbon of the coordinated hydrazone ligand
in complex 6. The electron releasing nature of the CH3 group
towards the Ni ion through the coordinated azomethine nitrogen
in complex 6 may assist the respective interactions between
the complex and either DNA or protein. However, complex 7,
containing a coordinated hydrazone possessing similar methyl
substitution at the azomethine carbon but with a lack of CH3
substitution at the phenyl ring of the acid hydrazide portion,
exhibited greater binding with DNA and proteins than complexes
5 and 6, which may be due to the greater planarity of complex 7.
In the case of complex 8, containing the coordinated hydrazone
with substitution of a phenyl ring in the carbonyl carbon with
a napthyl group, showed the highest level of binding among all
complexes in this study. The higher binding affinity of complex 8
towards both CT DNA and BSA could be explained in terms of
the planarity of the ligand coordinated to nickel.49,50 The overall
order of interaction between the complexes and CT DNA as well
as BSA decreased in the order 8 > 7 > 6 > 5.
Antioxidant
Characteristics of synchronous fluorescence spectra
The radical scavenging property of hydrazone derivatives have
attracted the interest of researchers and are thus investigated
in in vitro systems.54,55 Both the hydroxyl (OH) and superoxide
Synchronous fluorescence spectrum provides information on the
molecular microenvironment, particularly in the vicinity of the
fluorophore functional groups.51 It is well known that the fluores-
-
(O2 ) radicals are clinically important reactive species produced in
Fig. 9 Synchronous spectra of BSA (1 ¥ 10-6 M) as a function of concentration of complex 8 (0, 2, 4, 6, 8, 10, 12 and 14 ¥ 10-7 M) with a wavelength
difference of Dl = 15 nm (A) and Dl = 60 nm (B). The arrow indicates the decrease in emission intensity accompanied by hypochromic shift (B) w.r.t
various concentration of complex 8.
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most organ systems and participate in various physiological and
pathophysiological processes, such as carcinogenesis, aging, viral
infection, inflammation, among others.56,57 Hence, the antioxidant
properties of the ligands (1–4) and corresponding nickel(II) com-
plexes (5–8) were studied towards the above mentioned radicals
and compared with the standard antioxidant butylated hydroxy
anisole (BHA).
Cytotoxicity
Preliminary up-to-date results are extremely positive, thus sup-
porting our facts and confirming the enormous potential of this
class of Ni(II) complexes as anticancer agents. Nickel hydrazone
complexes 5–8 were evaluated for their cytotoxicity against NIH
3T3 normal cell lines and HeLa and Hep-2 tumour cell lines
by colorimetric assays (MTT assay) in which mitochondrial
dehydrogenase activity was measured as an indication of cell
viability. Complexes 5–8 were dissolved in DMSO and blank
samples containing the same amount of DMSO were taken as
controls. The effects of the complexes on the viability of these cells
evaluated after an exposure period of 48 h showed antitumour
activity and their corresponding IC50 values, related to inhibition
of tumour cell growth at the 50% level, are noted.
Surprisingly, complexes 5, 6 and 7 completely failed to arrest the
growth of both HeLa and Hep-2 cells, even up to a concentration of
1000 mM. However, complex 8 showed a significant, concentration
dependent, activity in arresting the growth of the selected cells.
Upon increasing the concentration of complex 8 (Fig. 11) from
63 mM to 1000 mM, an increase in the percentage of cell inhibition
Fig. 10 shows the plot of the hydroxyl radical scavenging effect
(%) and it is found that the inhibitory effect of the compounds
-
tested with OH and O2 radicals are concentration dependent
and their suppression ratio increases with increasing sample
concentrations in the tested range. The IC50 values exhibited by
ligands 1–4 to scavenge hydroxyl radicals are 57.81, 53.65, 50.1
and 38.55 mM, respectively, whereas for superoxide radicals the
IC50 values are found to be 41.95, 41.92, 39.51 and 36.84 mM in
the order of 1 > 2 > 3 > 4. The IC50 values of newly synthesised
nickel(II) complexes containing the above ligands against OH and
O2- radicals are found to be 27.74, 24.42, 21.37, 20.09, 26.82, 23.75,
21.06 and 18.19 mM with the order of 5 > 6 > 7 > 8. From the
above values, it is found that all of the metal complexes and ligands
tested in this study behaved as more effective inhibitors than the
standard scavenging agent, butylated hydroxy anisole (BHA) for
-
OH and O2 radicals.
In general, the antioxidant activity of Ni(II) complexes as well
-
as free ligands against the free radicals OH and O2 , decreased
in the order 8 > 7 > 6 > 5 > 4 > 3 > 2 > 1. However,
the radical scavenging effects of nickel chelates are higher than
that of the corresponding free ligands due to chelation with
nickel ions in complexation that exert different and selective
effects on scavenging radicals of the biological system. Among
all the ligands and their corresponding nickel complexes, the
one possessing a more planar hydrazone moiety ie., complex 8
containing ligand 4, showed greater potential scavenging activity
than the rest of the complexes with their respective ligands.
Further, the results obtained for the different radicals confirmed
that the complexes are more effective at arresting the formation
-
of O2 than OH radicals. Hence, we strongly believe that the
present metal hydrazone complexes can be further evaluated as
suitable candidates leading to the development of new potential
antioxidants and therapeutic reagents for some diseases.
Fig. 11 % inhibition of NIH 3T3 and HeLa cell lines as a function of
concentration of complex 8.
-
Fig. 10 Trends in the inhibition of OH (A) and O2 (B) radicals by ligands 1, 2, 3 and 4 and the corresponding metal complexes 5, 6, 7 and 8 at variable
concentrations.
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was observed, with respective IC50 values of 104 mM and > 500 mM
for HeLa and Hep-2 cells (data not presented in Fig. 11) and they
are comparable with that of earlier report.58 Hence, it is clear
that complex 8 selectively arrested the growth of HeLa cells only.
A simple structure activity relationship suggest that among the
investigated complexes, complex 8, which contains a coordinated
hydrazone with a more planar napthyl group, is responsible for the
selectivity and the potential inhibition against the tumour cells. All
complexes did not cause any damage to the healthy cells (NIH 3T3)
(complex 8: IC50 = 937 mM), indicating that they are non-toxic to
normal cells, as expected for a better drug.
5 A. A. Tak, F. Arjmand and S. Tabassum, Transition Met. Chem., 2002,
27, 741–747.
6 N. Valls, I. Uso´n, C. Gouyette and J. A. Subirana, J. Am. Chem. Soc.,
2004, 126, 7812–7816.
7 S. Arounaguiri and B. G. Maiya, Inorg. Chem., 1996, 35, 4267–4270.
8 Q. L. Zhang, J. G. Liu, J. Liu, G. Q. Xue, H. Li and J. Z. Liu et al., J.
Inorg. Biochem., 2001, 85, 291–296.
9 K. C. Skyrianou, C. P. Raptopoulou, V. Psycharis, D. P. Kessissoglou
and G. Psomas, Polyhedron, 2009, 28, 3265–3271.
10 Y. Jin, M. A. Lewis, N. H. Gokhale, E. C. Long and J. A. Cowan, J.
Am. Chem. Soc., 2007, 129, 8353–8361.
11 F. Bisceglie, M. Baldini, M. Belicchi-Ferrari, E. Buluggiu, M. Careri,
G. Pelosi, S. Pinelli and P. Tarasconi, Eur. J. Med. Chem., 2007, 42,
627–634.
12 G. Barone, N. Cambino, A. Ruggirello, A. Silvestri, A. Terenzi and V.
T. Liveri, J. Inorg. Biochem., 2009, 103, 731–737.
13 S. S. Matkar, L. A. Wrischnik, P. R. Jones and U. Hellmann-Blumberg,
Biochem. Biophys. Res. Commun., 2006, 343, 754–761.
14 K. S. Kasprzak, Chem. Res. Toxicol., 1991, 4, 604–615.
15 R. B. Ciccarelli and K. E. Wetterhahn, Cancer Res., 1984, 44, 3892–
3897.
Conclusion
In this work, a systematic approach to the synthesis of four new
bivalent nickel complexes containing double negative tridentate
ONO chelating hydrazone ligands, differing in the nature of sub-
stitution and planarity, is presented. The structure and geometry
of the complexes analysed through single crystal X-ray diffraction
revealed square planar geometry around the Ni(II) ion, with a
slight distortion in complexes 5, 7 and 8. From the bio-inorganic
chemistry point of view, binding interaction of all the newly
synthesised complexes with CT DNA and BSA protein has been
carried out. Binding experiments revealed that the nature of the
substitution group and planarity of the hydrazone coordinated to
the nickel ion in the complexes showed a pronounced effect on their
binding ability with both DNA and proteins. Also, the antioxidant
and cytotoxicity properties of the newly synthesised complexes
showed that they can be investigated in detail as potential drugs.
Among the complexes synthesised in this work, complex 8, which
possesses a more planar napthyl unit instead of the phenyl ring
available in the other complexes, showed greater efficiency at
arresting the growth of cancer cells, as well as in inhibiting the
production of free radicals.
16 R. B. Ciccarelli and K. E. Wetterhahn, Cancer Res., 1982, 42, 3544–
3549.
17 R. B. Ciccarelli, R. H. Hampton and K. W. Jennette, Cancer Lett.,
1981, 12, 349–354.
18 L. Jiaquin, T. Jianniao, J. Zhang, Z. Hu and C. Xingguo, Anal. Bioanal.
Chem., 2003, 376, 864–867.
19 J. S. Stamler, D. J. Singel and J. Loscalzo, Science, 1992, 258, 1898–1902.
20 H. Y. Shrivastava, M. Kanthimathi and B. U. Niar, Biochem. Biophys.
Res. Commun., 1999, 265, 311–314.
21 S. J. Lan, T. P. A. Kruck and B. Sarkar, J. Biol. Chem., 1974, 249,
5878–5884.
22 C. Tanford, J. Am. Chem. Soc., 1952, 74, 211–215.
23 H. A. Saroof and H. J. Mark, J. Am. Chem. Soc., 1953, 75, 1420–1426.
24 F. Friedberg, FEBS Lett., 1975, 59, 140–141.
25 M. C. Beinfield, D. A. Bryce, D. Kahavy and A. Mortonosi, J. Biol.
Chem., 1975, 250, 6282–6287.
26 B. K. Jin and L. P. Lu, Chin. Chem. Lett., 2001, 12, 989–992.
27 N. S. Quiming, R. B. Vergel, M. G. Nicolas and J. A. Villanueva, J.
Health Sci., 2005, 51, 8–15.
28 L. M. Venanzi, J. Chem. Soc., 1958, 719–724.
29 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008,
64, 112–122.
30 A. L. Spek, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2009, 65,
From the results obtained in various biological studies, it
became clear that among complexes 5, 6, 7 and 8, the one
(complex 8) that possesses a more planar napthyl unit instead
of the phenyl ring available in other complexes (5, 6 and 7)
showed higher efficiency. Hence, we believe that detailed in vivo
experiments utilizing these nickel hydrazone complexes could lead
to a significant outcome in further expanding their scope.
148–155.
31 L. J. Barbour, J. Supramol. Chem., 2001, 1, 189–191.
32 APEX2, Program for Data Collection and Integration on Area Detectors,
BRUKER AXS Inc, 5465, East Cheryl Parkway, Madison, WI 53711–
5373, USA.
33 G. M. Sheldrick, SADABS (version 2008/1), Program for Absorption
Correction for Data from Area Detector Frames, University of Gottin-
gen, 2008.
34 J. Marmur, J. Mol. Biol., 1961, 3, 208–218.
35 M. E. Reichmann, S. A. Rice, C. A. Thomas and P. Doty, J. Am. Chem.
Soc., 1954, 76, 3047–3053.
36 A. Wolfe, G. H. Shimer and T. Meehan, Biochemistry, 1987, 26, 6392–
6396.
37 M. Lee, A. L. Rhodes, M. D. Wyatt, S. Forrow and J. A. Hartley,
Biochemistry, 1993, 32, 4237–4245.
38 T. Nash, Biochem. J., 1953, 55, 416–421.
39 C. Beauchamp and I. Fridovich, Anal. Biochem., 1971, 44, 276–287.
40 M. Blagosklonny and W. S. EI-diery, Int. J. Cancer, 1996, 67, 386–
392.
Acknowledgements
We sincerely thank the University Grants Commission, New
Delhi, India for the award of a Fellowship in Science for
Meritorious Students (RFSMS) to P. Krishnamoorthy under the
UGC-SAP-DRS programme. AHC is grateful to the Robert A.
Welch Foundation (Grant F-0003) for financial support of this
work.
41 N. M. H. Salem, L. El-Sayed, S. Foro, W. Haase and M. F. Iskander,
Polyhedron, 2007, 26, 4161–4172.
42 K. Chichak, U. Jacquenard and N. R. Branda, Eur. J. Inorg. Chem.,
References
2002, 357–368.
43 T. Ghosh, B. Mondal, Tarun Ghosh, M. Sutradhar, G. Mukherjee and
M. G. B. Drew, Inorg. Chim. Acta, 2007, 360, 1753–1761.
44 M. Kandaz, I. Yilmaz, S. Keskin and A. Koca, Polyhedron, 2002, 21,
825–834.
45 W. J. Mei, J. Liu, K. C. Zheng, L. J. Lin, H. Chao, A. X. Li, F. C. Yun
and L. N. Ji, Dalton Trans., 2003, 1352–1359.
1 R. McRae, P. Bagchi, S. Sumalekshmy and C. J. Fahrni, Chem. Rev.,
2009, 109, 4780–4827.
2 E. V. Bichenkova, X. Yu, P. Bhadra, H. Heissigerova, S. J. A. Pope and
B. J. Coe, Inorg. Chem., 2005, 44, 4112–4114.
3 F. Westerlund, L. M. Wilhelmsson, B. Norden and P. Lincoln, J. Phys.
Chem. B, 2005, 109, 21140–21144.
4 G. O. Ahn, K. J. Botting, A. V. Patterson, D. C. Ware, M. Tercel and
W. R. Wilson, Biochem. Pharmacol., 2006, 71, 1683–1694.
46 Y. Hu, Y. Yang, C. Dai, Y. Liu and X. Xiao, Biomacromolecules, 2010,
11, 106–112.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 9690–9702 | 9701
©

View Online
47 J. R. Lakowicz, Fluorescence Quenching: Theory and Applications.
Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum
Publishers, New York, 1999, pp. 53–127.
52 J. N. Miller, Proc. Anal. Div. Chem. Soc., 1979, 16, 203–208.
53 J. H. Tang, F. Luan and X. G. Chen, Bioorg. Med. Chem., 2006, 14,
3210–3217.
48 X. Z. Feng, Z. Yang, L. J. Wang and C. Bai, Talanta, 1998, 47, 1223–
54 Yong Li, Zheng-Yin Yang and Ming-Fang Wang, Eur. J. Med. Chem.,
1229.
2009, 44, 4585–4595.
49 S. Ramakrishnan, V. Rajendiran, M. Palaniandavar, V. S. Periasamy, B.
S. Srinag, H. Krishnamurthy and M. A. Akbarsha, Inorg. Chem., 2009,
48, 1309–1322.
50 V. Rajendiran, M. Murali, E. Suresh, M. Palaniandavar, V. S. Periasamy
and M. A. Akbarsha, Dalton Trans., 2008, 2157–2170.
51 G. Z. Chen, X. Z. Huang, J. G. Xu, Z. Z. Zheng and Z. B. Wang,
Methods of Fluorescence Analysis, second ed., Science Press, Beijing,
1990.
55 Bao-dui Wang, Zheng-Yin Yang, Patrick Crewdson and Da-qi wang,
J. Inorg. Biochem., 2007, 101, 1492–1504.
56 S. Satyanarayana, J. C Dabrowiak and J. B. Chaires, Biochemistry, 1993,
32, 2573–2584.
57 A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, N. J. Turro and
J. K. Barton, J. Am. Chem. Soc., 1989, 111, 3051–3058.
58 Zeng-Chen Liu, Bao-Dui Wang, Bo Li, Qin Wang, Zheng-Yin Yang,
Tian-Rong Li and Yong Li, Eur. J. Med. Chem., 2010, 45, 5353–5361.
9702 | Dalton Trans., 2011, 40, 9690–9702
This journal is
The Royal Society of Chemistry 2011
©