Variation in the biomolecular interactions of nickel(II) hydrazone complexes upon tuning the hydrazide fragment

Article abstract of DOI:10.1039/c2dt30121k

Three 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 o-hydroxynaphthaldehyde with fur

Full text of DOI:10.1039/c2dt30121k

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PAPER
Variation in the biomolecular interactions of nickel(II) hydrazone complexes
upon tuning the hydrazide fragment†
a
a
b
b
Paramasivam Krishnamoorthy, Palanisamy Sathyadevi, Rachel R. Butorac, Alan H. Cowley,
c
a
Nattamai S. P. Bhuvanesh and Nallasamy Dharmaraj*
Received 18th January 2012, Accepted 20th March 2012
DOI: 10.1039/c2dt30121k
Three new bivalent nickel hydrazone complexes have been synthesised from the reactions of
NiCl (PPh ) ] with H L {L = dianion of the hydrazones derived from the condensation of
[
2
3 2
2
1
2
o-hydroxynaphthaldehyde with furoic acid hydrazide (H L ) (1)/thiophene-2-acid hydrazide (H L )
2
2
3
1
2
(
[
2)/isonicotinic acid hydrazide (H L ) (3)} and formulated as [Ni(L )(PPh )] (4), [Ni(L )(PPh )] (5) and
2 3 3
Ni(L )(PPh )] (6). Structural characterization of these compounds 4–6 were accomplished by using
3
3
various physico-chemical techniques. Single crystal X-ray diffraction data of complexes 4 and 5 proved
their distorted square planar geometry. In order to ascertain the potential of the above synthesised
compounds towards biomolecular interactions, additional experiments involving interaction with calf
thymus DNA (CT DNA) and bovine serum albumin (BSA) were carried out. All the ligands and
−
corresponding nickel(II) chelates have been screened for their scavenging effect towards O , OH and NO
2
radicals. The efficiency of complexes 4–6 to arrest the growth of HeLa, HepG-2 and A431 tumour cell
lines has been studied along with the cell viability test against the non-cancerous NIH 3T3 cells under
in vitro conditions.
number of attractive features such as the degree of rigidity, a con-
jugated π-system and an NH unit that readily participates in
hydrogen bonding and may be a site of protonation–deprotona-
tion. It is well established that the formation of metal complexes
plays an important role to enhance the biological activity of free
Introduction
The bioinorganic chemistry of nickel has been rapidly expanded
due to the increasing interest in nickel complexes
been shown to act as antiepileptic, anticonvulsant agents or vita-
mins or have shown antibacterial, antifungal, antimicrobial and
anticancer/antiproliferative activity.
an environment similar to the one present in biological systems
usually by making coordination through oxygen and nitrogen
atoms. Various important properties of carbonic acid hydrazides,
along with their applications in medicine and analytical chem-
istry, have led to increased interest in their complexation charac-
1
–3
that have
18
hydrazones.
4
–16
Hydrazone ligands create
Due to the key role of DNA in cell life and pathological pro-
cesses, the design of specific chemical nucleases, DNA probes
and alkylating agents is an important research area for the devel-
opment of new therapeutic agents and tools in biochemistry.
Hence, the interaction of small molecules with DNA has
attracted in particular a great deal of attention. The two most
common non-covalent binding motifs for such systems are the
groove binding and intercalating modes. Many therapeutic
agents, particularly anticancer drugs, are known to bind DNA via
either of these motifs and the possibility of gene modulation by
specific sequence binding of small molecules to DNA has also
been explored. The interaction of Ni(II) complexes with DNA
has been mainly dependent on the structure of the ligand exhibit-
1
7
teristics with transition metal ions. The hydrazone unit offers a
a
Department of Chemistry, Bharathiar University, Coimbatore - 641
0
46, India. E-mail: dharmaraj@buc.edu.in; Fax: +91 422 2422387;
Tel: +91 422 2428316
b
Department of Chemistry and Biochemistry, University of Texas at
Austin, Austin, Texas 78712, U.S.A
c
Department of Chemistry, Texas A&M University, College Station, TX
19–23
24,25
ing intercalative behavior
and/or DNA cleavage ability.
7
7843, U.S.A
Serum albumin is the major soluble protein constituent in the
circulatory system of a wide variety of organisms and it has the
ability to reversibly bind to a large variety of endogenous and
exogenous ligands such as fatty acids, drugs, and metal ions in
†
Electronic supplementary information (ESI) available: crystal packing
diagram of the unit cell of complexes 4 and 5 (Fig. S1 and S2), elec-
tronic absorption spectra of compounds 1–5 with DNA (Fig. S3 and
S4), emission spectra of DNA-EB in the presence of compounds 1–5
(Fig. S5 and S6), emission spectra of binding of compounds 1–5 with
26,27
the bloodstream.
The drug–protein complex not only
BSA (Fig. S7 and S8), synchronous spectra (Δλ = 15 and 60 nm) of
binding of compounds 1–5 with BSA (Fig. S9 and S10), % cell inhi-
bition of NIH 3T3, HeLa, HepG-2 and A431 cell lines by nickel hydra-
zones 4, 5 and 6 (Fig. S11). CCDC reference numbers 796261 and
strongly affected the absorption, distribution, metabolism and
excretion properties of drugs, but also influenced the drug stabil-
ity and toxicity during the chemotherapeutic process. Therefore,
knowledge of the mechanism of interaction between the selected
8
51587 complexes 4 and 5. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2dt30121k
6842 | Dalton Trans., 2012, 41, 6842–6854
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drug and protein has become very vital to the design of several
Synthesis of furan-2-carboxylic acid (3-hydroxy-naphthalen-2-
2
8,29
1
new drugs with improved potential.
In the current work, the
ylmethylene)-hydrazide (H
2
L ) (1). The ligand furan-2-car-
authors have chosen bovine serum albumin (BSA) as a model
protein to investigate its interaction with the newly synthesised
nickel hydrazone chelates 4–6.
The present study was undertaken to establish the bio-
inorganic potential of the titled compounds for nucleic acid inter-
actions, protein interactions, free radical scavenging and cytotox-
icity. The effect of changing the hydrazide moieties of
hydrazones upon the above mentioned properties were also dis-
cussed in line with the electronegativity of the hetero atoms as
well as the size of heterocyclic ring of hydrazides.
boxylic acid (3-hydroxy-naphthalen-2-ylmethylene)-hydrazide
(1) was prepared by refluxing an equimolar mixture of o-hydrox-
ynaphthaldehyde (0.172 g; 1 mM) and furoic acid hydrazide
(0.126 g; 1 mM) in 50 mL of absolute ethanol for 8 h as given
in Scheme 1. The reaction mixture was cooled to room tempera-
ture and the solid obtained was filtered, washed several times
with distilled water and recrystallized from EtOH to afford the
ligand 1 in pure form with 83% yield. Mp 131–133 °C. Anal.
Found: C, 67.9; H, 4.0; N, 9.6%. Calc. for C16
H N O
12 2 3
(Mol. wt = 280.278): C, 68.6; H, 4.3; N, 10.0%. ESI-MS
(
2
MeOH–buffer): Found m/z = 280 (M + H) (calculated m/z =
79 for M ). Selected IR bands (νmax/cm ): 3440 (phenolic,
+
−1
OH); 3215 (NH); 1640 (CvO); 1604 (CvN); 1016 (N–N);
240 (phenolic, C–OH). δH (500 MHz; DMSO-d ; Me Si):
Experimental section
1
6
4
Materials and reagents
12.24 (1H, s, OH); 9.43 (1H, s, NH); 8.05 (1H, s, CHvN);
7
.86–6.64 (9H, m, Ar–H).
Reagent grade chemicals NiCl ·6H O, triphenylphosphine,
2
2
o-hydroxynaphthaldehyde, furoic acid hydrazide, thiophene-2-
carboxylic acid hydrazide and isonicotinic acid hydrazide were
purchased from Sigma-Aldrich and used as received. Calf
thymus DNA (CT DNA) and bovine serum albumin (BSA) were
purchased from Himedia Company. The human cervical cancer
cell line (HeLa), human skin cancer cell line (A431), HepG2
Synthesis of thiophene-2-carboxylic acid (3-hydroxy-naphtha-
2
len-2ylmethylene)-hydrazide (H L ) (2). Ligand 2 was prepared
2
by a procedure similar to the one described above utilizing
o-hydroxynaphthaldehyde (0.172 g; 1 mM) and thiophene-2-
carboxylic acid hydrazide (0.142 g; 1 mM) (Scheme 1). Mp
138–140 °C. Anal. Found: C, 64.45; H, 3.62; N, 8.97; S,
10.05%. Calc. for C H N O S (Mol. wt = 296.343): C, 64.85;
(human liver hepatocellular carcinoma cells) and NIH
16 12 2 2
3
T3 mouse embryonic fibroblasts was obtained from National
H, 4.08; N, 9.45, S, 10.82%. ESI-MS (MeOH–buffer): Found
+
Centre for Cell Science (NCCS), Pune, India. All the other
chemicals and reagents used for DNA binding, protein binding,
antioxidant, cytotoxicity assays were of high quality and avail-
able from reputed suppliers.
m/z = 296 (M + H) (calculated m/z = 295 for M ). Selected IR
−1
bands (νmax/cm ): 3405 (phenolic, OH); 3225 (NH); 1634
(CvO); 1586 (CvN); 1037 (N–N); 1282 (phenolic, C–OH). δH
(500 MHz; DMSO-d ; Me Si): 13.18 (1H, s, OH); 9.28 (1H, s,
6
4
NH); 8.34 (1H, s, CHvN); 7.85–7.17 (9H, m, Ar–H).
Synthesis of ligands (1–3) and corresponding nickel hydrazone
complexes (4–6). Chemical reactions involved in the synthesis of
hydrazone ligands and corresponding nickel complexes were
given in Scheme 1.
Synthesis of isonicotinic acid (3-hydroxy-naphthalen-2-
3
ylmethylene)-hydrazide (H L ) (3). Ligand 3 was prepared using
2
a similar procedure as described above with o-hydroxynaphth-
aldehyde (0.172 g; 1 mM) and isonicotinic acid hydrazide
(0.137 g; 1 mM) as suitable reactants (Scheme 1). Mp
Synthesis of nickel precursor complex. The precursor metal
150–153 °C. Anal. Found: C, 69.93; H, 4.32; N, 14. 27%. Calc.
complex [NiCl (PPh ) ] was prepared according to the literature
method.
for C H N O (Mol. wt = 291.304): C, 70.09; H, 4.50; N,
14.42%. ESI-MS (MeOH–buffer): Found m/z = 291 (M + H)
2
3 2
17 13 3 2
30
Scheme 1
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+
(
3
1
calculated m/z = 290 for M ). Selected IR bands (νmax/cm−1):
were recorded in DMSO–buffer (5 : 95) solution on a Jasco
V-630 spectrophotometer and Jasco FP 6600 spectrofluorometer
respectively at room temperature. H NMR spectra of the ligands
442 (phenolic, OH); 3212 (NH); 1640 (CvO); 1606 (CvN);
017 (N–N); 1241 (phenolic, C–OH). δ (500 MHz; DMSO-d ;
1
H
6
Me Si): 12.64 (1H, s, OH); 9.51 (1H, s, NH); 8.23 (1H, s,
CHvN); 8.01–6.75 (10H, m, Ar–H).
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 electrospray
mass spectra of the complexes and ligands (ESI-MS) in
methanol–buffer (5 : 95) was recorded on a THERMO Finnigan
LCQ Advantage max ion trap mass spectrometer. The X-ray dif-
fraction data of complexes 4 and 5 were collected at 100 K
respectively with MoKα radiation (λ = 0.71073 and 0.71069 Å)
using a Bruker Smart APEX II CCD diffractometer equipped
with graphite monochromator. The structures were solved by
4
1
Synthesis of new nickel(II) hydrazone complex [Ni(L )(PPh
3
)]
(4). Complex 4 was synthesised by refluxing a methanolic sol-
ution of [NiCl (PPh ) ] (0.653 g; 1 mM) and the ligand 1
2
3 2
(0.280 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 4 suitable
for single-crystal X-ray diffraction studies were obtained by
recrystallisation in dichloromethane. Yield: 74%. Color: dark
red, mp 257–259 °C. Anal. Found: C, 67.46; H, 3.81; N, 4.02%.
Calc. for NiC H N O P (Mol. wt = 599.24): C, 68.15; H,
2
direct methods and refined full-matrix least squares (on F )
31
SHELXS97 and SHELXL97 and the graphics were produced
32
using PLATON97. All the non-hydrogen atoms were refined
anisotropically and the hydrogen atoms were positioned geome-
trically and refined as riding model.
34 25 2 3
4
.21; N, 4.67%. ESI-MS (MeOH–buffer): Found m/z = 599
+
(
(
M + H) (calculated m/z = 598 for M ). Selected IR bands
νmax/cm ): 1614 & 1519 (CvN–NvC); 1360 (phenolic,
−1
DNA binding studies
C–O); 1320 (enolic, C–O); 1034 (N–N). δ (500 MHz; DMSO-
H
Electronic absorption spectroscopy. All the experiments
involving the interaction of the hydrazone ligands 1–3 and their
nickel complexes 4–6 with DNA were carried out using 10 mM
Tris buffer at pH 7.2. Solutions of calf thymus DNA in the
buffer showed UV-visible absorption bands at 260 and 280 nm
with a ratio of 1.9 : 1, indicating the protein free nature of
d ; Me Si): 8.16 (1H, s, HCvN); 7.87–6.37 (24H, m, Ar–H).
6
4
2
Synthesis of [Ni(L )(PPh
procedure similar to that given in the case of 4, from
NiCl (PPh ) ] (0.653 g; 1 mM) and ligand 2 (0.296 g; 1 mM)
3
)] (5). Complex 5 was prepared by a
[
2
3 2
as reactants (Scheme 1). The purity of the complex was checked
by TLC following which tetragonal shaped crystals of complex
33
DNA. The DNA concentration per nucleotide was determined
5
suitable for single-crystal X-ray diffraction studies were
by absorption spectroscopy using the molar absorption coeffi-
cient (6600 M− cm ) at 260 nm. Electronic absorption titra-
tion experiments were performed by maintaining the
concentration of test compounds as constant (25 μM) but with
variable nucleotide concentration from 0 to 25 μM. While
measuring the absorption spectra, equal amounts of DNA were
added to both test compounds and reference solutions to elimin-
ate the absorbance of DNA itself. The data were then fit into the
1
−1
34
obtained by recrystallisation in dichloromethane. Yield: 75%.
Color: dark red, mp 248–251 °C. Anal. Found: C, 65.94; H,
3
.97; N, 4.06, S, 4.86%. Calc. for NiC H N O PS (mol. wt =
34 25 2 2
6
15.30): C, 66.37; H, 4.10; N, 4.55; S, 5.21%. ESI-MS (MeOH–
buffer): Found m/z = 615 (M + H) (calculated m/z = 614 for
+
−1
M ). Selected IR bands (νmax/cm ): 1612 & 1527 (CvN–
NvC); 1398 (phenolic, C–O); 1328 (enolic, C–O); 1094
(
N–N). δ (500 MHz; DMSO-d ; Me Si): 9.23 (1H, s, HCvN);
H
6
4
following equation and the intrinsic binding constant K was cal-
b
35
7
.88–6.60 (24H, m, Ar–H).
culated in each case.
3
Synthesis of [Ni(L )(PPh )] (6). Complex 6 was prepared by a
3
½DNAꢀ=½ε ꢁ ε ꢀ ¼ ½DNAꢀ=½ε ꢁ ε ꢀ þ 1=K ½ε ꢁ ε ꢀ
where, [DNA] is the concentration of DNA in base pairs, ε is
a
f
b
f
b
b
f
procedure similar to that given in the case of 5, from
NiCl (PPh ) ] (0.653 g; 1 mM) and ligand 3 (0.291 g; 1 mM)
as reactants (Scheme 1). Attempts made to grow suitable crystals
of complex 6 suitable for single crystal XRD studies were unsuc-
cessful. Yield: 73%. Color: dark red, mp 260–263 °C. Anal.
Found: C, 68.14; H, 3.97; N, 6.02%. Calc. for NiC H N O P
mol. wt = 610.267): C, 68.88; H, 4.29; N, 6.89%. ESI-MS
MeOH–buffer): Found m/z = 610 (M + H) (calculated m/z =
09 for M ). Selected IR bands (νmax/cm ): 1612 & 1526
[
a
2
3 2
the extinction coefficient of the ligand/complex at a given DNA
concentration, ε is the extinction coefficient of the complex in
f
free solution and ε is the extinction coefficient of the ligand/
b
complex when fully bound to DNA. A plot of [DNA] versus
3
5 26 3 2
[
1
DNA]/[εb − ε ] gave a slope and an intercept equal to
(
(
6
f
/[εa − ε ] and (1/K )[ε − ε ], respectively. The intrinsic
f
b
b
f
+
−1
binding constant K is the ratio of the slope to the intercept.
b
(CvN–NvC); 1398 (phenolic, C–O); 1327 (enolic, C–O);
1
096 (N–N). δH (500 MHz; DMSO-d ; Me Si): 9.32 (1H, s,
6 4
Competitive binding fluorescence measurements
HCvN); 7.86–6.37 (25H, m, Ar–H).
The apparent binding constant (Kapp) of the ligands 1–3 and
their complexes 4–6 with DNAwas determined by a fluorescence
spectral technique using ethidiumbromide (EB) bound CT DNA
solution in Tris–HCl buffer solution (pH, 7.2). The changes in
fluorescence intensities at 604 nm (515 nm excitation) of EB
bound to DNA were measured with respect to different concen-
tration of the compounds (0–150 μM). EB was non-emissive in
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
1
4
00–4000 cm− . The electronic absorption and emission spectra
6844 | Dalton Trans., 2012, 41, 6842–6854
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Tris–HCl buffer solution (pH, 7.2) due to fluorescence quench-
ing of free EB by the solvent molecules. In the presence of
DNA, EB showed enhanced emission intensity due to its interca-
lative binding to DNA. A competitive binding of the ligand/
metal complexes to CT DNA resulted in the displacement of the
bound EB thereby decreasing its emission intensity. The quench-
where, A and A represent the absorbance in the absence and
0
C
presence of the test compounds, respectively. The 50% activity
(IC ) can be calculated from the result of percentage activity.
50
Cytotoxicity
ing constant (K ) was calculated using the classical Stern–
q
36
Volmer equation.
In vitro cytotoxicity (IC ) of the synthesised compounds was
50
I0=I ¼ Kq½Qꢀ þ 1
performed on HeLa (human cervical cancer cells), A431 (human
skin cancer cells), HepG2 (human liver hepatocellular carcinoma
cells) and normal NIH 3T3 (mouse embryonic cells) cell lines
I0 is the emission intensity in the absence of quencher, I is the
emission intensity in the presence of quencher, K is the quench-
ing constant, [Q] is the quencher (complexes) concentration and
K is the slope, obtained from the plot of I /I vs. [Q]. The appar-
40
using MTT assay method. Tumour cell lines (HeLa, A431 and
q
HepG2) 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 μl of respective
medium containing 10% FBS, at a plating density of 10 000
q
0
ent binding constant (Kapp) has been calculated from the
equation,
KEB½EBꢀ ¼ Kapp½complexꢀ
cells per well and incubated at 37 °C, 5% CO , 95% air and
2
1
00% relative humidity for 24 h prior to addition of test
The ligand/complex concentration was obtained from the
compounds. The test compounds were dissolved in dimethyl-
sulfoxide and diluted in respective serum free medium. After
24 h, 100 μl of the medium containing the test compounds at
various concentrations (in the range of 15 to 500 μM for all the
ligands and precursor complex to HeLa, HepG-2 and NIH 3T3
normal cell lines whereas 6.25–200 μM of complex 4 and
value at a 50% reduction of the fluorescence intensity of EB,
7
−1
KEB = 1.0 × 10 M and [EB] = 5 μM.
Protein binding studies
3
.12–100 μM concentration of complexes 5 and 6 to the A431
Binding of ligands 1–3 and its corresponding nickel hydrazone
complexes 4–6 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 maintained for all the
experiments. Samples were carefully degassed using pure nitro-
gen gas for 15 minutes by using quartz cells (4 × 1 × 1 cm) with
high vacuum Teflon stopcocks. Stock solution of BSA was pre-
pared in 50 mM phosphate buffer (pH, 7.2) and stored in dark at
cell lines) was added and incubated at 37 °C, 5% CO , 95% air
2
and 100% relative humidity for 48 h. Triplicate was maintained
and the medium containing no test compounds served as control.
After 48 h, 15 μl of MTT (5 mg ml− ) in phosphate buffered
saline (PBS) was added to each well and incubated at 37 °C for
1
4
h. The medium with MTT was then flicked off and the formed
formazan crystals were solubilized in 100 μl of DMSO and the
absorbance at 570 nm was measured using micro plate reader.
The % cell inhibition was determined using the following
formula, % cell inhibition = 100 − Abs(drug)/Abs(control) × 100
and a graph was plotted between % cell inhibition and concen-
tration of the drug (complexes). From the graph, IC50 values
were also calculated.
4
°C for further use. Concentrated stock solutions of test com-
pounds were prepared by dissolving them in DMSO–phosphate
buffer (5 : 95) and diluted suitably with phosphate buffer to get
required concentrations. 2.5 ml of BSA solution (1 μM) was
−4
titrated by successive additions of a 10 M stock solution of
ligands/complexes using a micropipette. Synchronous fluor-
escence spectra were also recorded using the same concentration
of BSA and complexes as mentioned above with two different
Δλ (difference between the excitation and emission wavelengths
of BSA) values such as 15 and 60 nm.
Results and discussion
The reactions of [NiCl (PPh ) ] with the respective hydrazone
ligands, furan-2-carboxylic acid (3-hydroxy-naphthalen-2-
ylmethylene)-hydrazide (H L ) (1) (or) thiophene-2-carboxylic
acid (3-hydroxy-naphthalen-2ylmethylene)-hydrazide (H L ) (2)
2
3 2
1
2
2
2
Antioxidant studies
(or) isonicotinic acid (3-hydroxy-naphthalen-2-ylmethylene)-
3
1–3
hydrazide (H L ) (3) yielded complexes of the type [Ni(L
)
The superoxide, hydroxyl and nitric radical scavenging activities
of all the three ligands and their corresponding complexes were
determined by the methods described by Beauchamp et al.,
2
(PPh )] (4–6) (Scheme 1). The analytical data of the above said
3
complexes were in good agreement with the proposed molecular
formulae of 1 : 1 metal to ligand stoichiometries (given under
the experimental part). All the synthesised compounds are quite
stable in air and light and soluble in most of the organic solvents
such as MeOH, EtOH, CH Cl , CHCl , DMF and DMSO and
were well characterised using several physico-chemical
techniques.
3
7–39
Nash and Green et al., respectively.
For each of the above
assays, tests were run in triplicate by varying the concentration
of compounds ranging from 10–50 μM.
The percentage activity was calculated by using the formula,
2
2
3
%
activity ¼ ½ðA0 ꢁ ACÞ=A0ꢀ ꢂ 100
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Infrared spectroscopy
Table 1 Crystal structure data of complexes 4 and 5
Complex 4 Complex 5
2
NiO NiO PS
The IR spectra of the metal hydrazone complexes were com-
pared with those of respective free hydrazone ligands in the
Formula
Formula weight
Temperature (K)
λ (Å)
C
34
H
25
N
2
3
P
C
34
H
25
N
2
−
1
region 4000–400 cm . The spectra of the ligands 1–3 displayed
characteristic absorption bands in the range of 3405–3442,
599.24
100(2)
0.71073
615.30
100(2)
0.71069
3
1
212–3225, 1639–1640, 1586–1606, 1240–1282 and
016–1037 cm−1 due to ν(O–H), ν(N–H), ν(CvO), ν(CvN) ν(C–OH)
Crystal system
Space group
Cell dimensions
a (Å)
b (Å)
c (Å)
Monoclinic
P21/c
Monoclinic
P21/c
and ν(N–N) vibrations, respectively. The bands due to ν(N–H) and
ν(CvO) vibrations of the free ligands were absent for complexes
10.379(2)
14.886(3)
19.785(6)
90
15.499(3)
10.844(5)
21.084(4)
90
4
–6, thus indicating that enolisation and deprotonation had taken
place prior to coordination. This fact was further confirmed by
the appearance of two new bands in the spectra of complexes
α (°)
β (°)
γ (°)
116.60(2)
90
125.601(15)
90
−
1
−1
around 1614–1519 cm and 1360–1398 cm that corresponds
to ν(CvN–NvC) and ν(C–O) stretching vibrations, respectively. The
bands attributed to ν(CvN) stretching were shifted to higher fre-
quencies while a positive shift of about ≈18–80 cm− was
observed for the ν(N–N) stretching vibration in comparison with
that of their respective free ligands, thus implying that the nitro-
gen atom of the azomethine group is coordinated to the metal in
these complexes. All these facts suggested that the hydrazone
ligands behave as a monobasic bidentate (NO) chelating ligands
in all the three complexes 4–6.
Z
4
4
hkl limits
−13 ≤ h ≥ 13
−19 ≤ k ≥ 15
−25 ≤ l ≥ 25
1.456
−20 ≤ h ≥ 20
−14 ≤ k ≥ 12
−27 ≤ l ≥ 27
1.418
1
3
D
calcd (Mg m−
)
F(000)
Crystal size (mm )
Independent reflections 6193 [R(int) = 0.0666] 6598 [R(int) = 0.0294]
1240
0.11 × 0.06 × 0.05
1272
0.28 × 0.15 × 0.14
3
Data/
6193/0/370
6598/0/370
restraints/parameters
Goodness-of-fit on F
Final R indices
2
1.503
1.081
R
1
= 0.0880,
wR = 0.1057
= 0.1854,
wR = 0.1279
R
1
= 0.0284,
wR = 0.0723
= 0.0322,
wR = 0.0738
[I > 2σ(I)]
2
2
R indices (all data)
R
1
R
1
1
H NMR spectroscopy
2
2
1
H NMR spectra of the free hydrazone ligands and their com-
plexes were assigned on the basis of observed chemical shifts.
The spectra of the ligands 1–3 displayed a weak singlet respect-
ively at 9.43, 9.28 and 9.51 ppm due to the NH proton. The
other characteristic resonance due to an OH proton (formed via
keto–enol tautomerization of the hydrazone ligand) appears in
the range of 13.18–12.24 ppm. But, the NMR spectra of com-
plexes 4–6 did not register any signal corresponding to NH,
revealing that the ligands adopted an enol form, followed by
deprotonation prior to coordination with the metal ion. In
addition, all the ligands showed a sharp singlet for azomethine
(HCvN) proton respectively at 8.05, 8.34 and 8.23 ppm.
However, in the case of spectra of nickel hydrazone complexes,
the signal corresponding to the azomethine protons gets shifted
downfield due to the participation of azomethine nitrogen in
coordination with metal ion and observed at 8.16, 9.23 and
9
.32 ppm, respectively. The signals corresponding to the protons
of aromatic moieties of the ligands and their complexes were
observed as multiplets in the range of 6.37–8.01 ppm.
Description of solid state structures
Fig. 1 The molecular structure of complex 4, with displacement ellip-
soids drawn at the 50% probability level.
The solid-state structure of complexes 4 and 5 were analysed
with single crystal X-ray diffraction study. Details of the data
collection conditions and the parameters of refinement process
are given in Table 1. The perspective ORTEP views of the com-
plexes 4 and 5 with atom numbering scheme are depicted in
Fig. 1 and 2 with corresponding selected geometric parameters
given in Table 2.
distorted square planar in which the dianionic ligand acted as a
planar tridentate forming a five-membered and a six-membered
metallocycle involving the metal ion. As expected, the
hydrazone ligand is bonded to the nickel ion via both phenolate
and enolate oxygen atoms and the imine nitrogen atom, the cor-
responding bite angles being 94.5(1)°, 84.3(1)° and 94.54(6)°,
84.16(6)° for [O1–Ni1–N1] and [N1–Ni1–O2] respectively for
complexes 4 and 5. The ONO donor set of atoms of the ligand
Dark-red, tetragonal shaped crystals of both complexes 4 and
5
belong to the monoclinic crystal system with P21/c space
1
2
group. In both the complexes [Ni(L )(PPh )] and [Ni(L )-
3
(
PPh )], the coordination geometry around the nickel ion is
3
6846 | Dalton Trans., 2012, 41, 6842–6854
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Fig. 3 Electronic absorption spectra of complex 6 (25 μM) in the
absence and presence of increasing amounts of CT DNA (2.5, 5.0, 7.5,
Fig. 2 The molecular structure of complex 5, with displacement ellip-
soids drawn at the 50% probability level.
1
0.0, 12.5, 15.0, 17.5 and 20.0, 22.5 and 25 μM). Arrows show the
changes in absorbance as a function of increasing DNA concentration
inset: plot of [DNA] vs. [DNA]/(ε − ε )).
(
a
f
occupies three of the four coordination sites of the square planar
arrangement with the remaining one occupied by the phosphorus
atom of the triphenylphosphine. The unequal bond lengths and
bond angles corresponding to complexes 4 and 5 (given in
Table 2) indicated a distortion from the regular square planar
geometry. The unit cell packing diagram of complexes 4 and 5 is
presented in Fig. S1 and S2.†
Further, the bands appeared around 370 and 450 nm for com-
plexes have been assigned to ligand-to-metal charge transfer
(
LMCT) as well as metal-to-ligand charge transfer (MLCT) tran-
42,43
sitions, respectively.
The electronic absorption spectra of
compounds (ligands/complexes) 1–6 without as well as with the
added CT DNA is shown in Fig. S3, S4† and 3.
From the electronic absorption spectral data, it is clear that
upon increasing concentration of DNA added to the ligands 1–3
showed only hypochromism without any shift in wavelength
whereas in metal complexes 4–6, all the above mentioned
absorption bands showed hypochromism accompanied with
small red or blue shifts in most of the absorption bands. The
nature of the shifts observed and the percentage of hypochro-
mism in the absorption bands in the case of all the compounds
after the addition of DNA given in Table 3 suggested that the
nickel hydrazone complexes bound more strongly to DNA than
their respective ligands via an intercalative mode. These obser-
vations were similar to those reported earlier for various metallo-
DNA binding studies
UV-visible absorption studies. The electronic absorption
spectra of hydrazone ligands 1–3 and respective nickel
hydrazone chelates 4–6 in DMSO–buffer displayed five or six
well-resolved bands in the range of 200 to 470 nm. The absorp-
tion bands in the range of 270–460 nm for ligands 1–3 have
been assigned to charge transfer transitions of the type π → π*
and n → π*. In the case of complexes 4–6, the high energy
absorption bands appeared between 250–300 nm and another
absorption around 320–360 nm were assigned to the intra-ligand
41
44
charge transfer transitions of the type π → π* and n → π*.
intercalators.
Table 2 Selected bond lengths (Å) and bond angles (°)
C
34
H
25
N
2
Ni O
3
P (4)
34 25 2 2
C H N Ni O P S (5)
Bond lengths
Bond angles
Bond lengths
Bond angles
Ni1–N1
Ni1–O1
Ni1–O2
Ni1–P1
C9–C10
C10–C11
N1–C11
N1–N2
1.847(4)
1.814(3)
1.842(3)
2.224(1)
1.454(7)
1.426(5)
1.296(6)
1.413(4)
1.307(6)
1.456(5)
O1–Ni1–N1
P1–Ni1–O1
P1–Ni1–O2
O2–Ni1–N1
P1–Ni1–N1
O1–Ni1–O2
C9–C10–C11–N1
N2–N1–C11–C10
C11–N1–N2–C12
N1–N2–C12–C13
94.5(1)
90.91(1)
90.2(1)
84.3(1)
173.1(1)
177.9(1)
178.2(4)
−179.7(4)
−175.8(4)
−176.1(4)
N1–Ni1
Ni1–O1
Ni1–O2
Ni1–P1
C9–C10
C10–C11
C11–N1
N1–N2
1.841(1)
1.824(2)
1.848(2)
2.216(7)
1.455(3)
1.428(3)
1.303(3)
1.401(2)
1.308(3)
1.463(3)
N1–Ni1–O1
O1–Ni1–P1
O2–Ni1–P1
N1–Ni1–O2
N1–Ni1–P1
O1–Ni1–O2
C9–C10–C11–N1
C10–C11–N1–N2
C11–N1–N2–C12
C13–C12–N2–N1
94.54(6)
89.39(4)
91.97(4)
84.16(6)
175.39(4)
178.13(5)
179.4(2)
178.4(1)
176.6(1)
−179.3(1)
N2–C12
C12–C13
C12–N2
C12–C13
This journal is © The Royal Society of Chemistry 2012
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Table 3 Nature of shift with % of hypochromism
Absorption
wavelength
nm) (εmax in
Shift of
wavelength
(nm)
(presence of
DNA)
(
−1
−1
cm mol
absence of
Complex DNA)
)
(
Nature
% of
of shift hypochromism
1
286 (9492)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3
—
1
—
—
—
2
1
—
1
2
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Blue
—
Red
—
—
—
Blue
Blue
—
Red
Red
Red
Blue
Blue
—
10.74
9.62
3
3
3
3
23 (9932)
34 (10 216)
78 (12 112)
98 (11 400)
11.92
11.82
11.65
10.74
13.89
9.07
2
270 (4820)
319 (3452)
333 (4096)
360 (4004)
376 (4104)
401 (3340)
12.86
4.24
14.97
11.65
12.29
11.20
10.09
11.09
11.77
16.81
18.15
16.99
16.64
13.99
18.13
19.02
17.60
20.08
17.72
6.96
3
313 (11 608)
3
3
3
4
4
27 (16 568)
46 (13 768)
67 (14 384)
39 (9008)
Fig. 4 Emission spectra of DNA–EB, in the presence of 0, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 μM of complex
6. Arrow indicates the change in the emission intensity as a function of
complex concentration (inset: Stern–Volmer plot of the fluorescence
titration data corresponding to the complex).
60 (7956)
4
5
282 (19 368)
312 (14 256)
379 (7980)
424 (10 652)
450 (9144)
283 (23 868)
their binding modes and only measures their ability to influence
45
3
3
3
4
4
19 (15 688)
33 (14 448)
80 (9800)
22 (12 528)
45 (9536)
the EB luminescence intensities in the EB–DNA complex. It
has been previously reported that the fluorescence intensity of
EB–DNA could be decreased by addition of the ligands/com-
plexes as quenchers, indicating the competition between the
ligands/complexes and EB in binding to DNA that proved the
intercalation of ligands/metal complexes to the base pairs of
6
281 (25 268)
20.34
17.19
14.14
19.32
18.48
4.93
3
3
3
4
4
19 (14 516)
33 (12 900)
79 (9212)
21 (121 200)
46 (86 680)
1
2
—
2
—
46,47
DNA.
Red
—
The fluorescence emission spectra of EB bound to DNA in
the absence and presence of the three complexes are shown in
Fig. S5, S6† and 4. From the figures, it is clear that an appreci-
able reduction in the fluorescence intensity at 604 nm of about
In order to determine quantitatively the binding strength of the
1
2.64, 16.40, 17.99, 41.55, 50.43 and 53.74% together with blue
ligands and its complexes with CT DNA, intrinsic binding con-
stants were obtained by monitoring the changes in both the
wavelength as well as their corresponding intensity of absorption
of the high energy bands upon increasing concentration of added
DNA. The following equation was applied to calculate
the binding constant: [DNA]/[ε − ε ] = [DNA]/[ε − ε ] +
shifts of 0, 0, 0, 2, 5 and 5 nm, respectively, was observed on
addition of free ligands 1–3 and their Ni(II) hydrazone com-
plexes 4–6, respectively to DNA pre-treated with EB, indicating
the replacement of EB molecules accompanied by intercalation
of the ligands/complexes with DNA. From the plot of intensity
against the quencher concentration furnished in Fig. 4, S5 and
S6† (as insets), the values of the apparent DNA binding constant
a
f
b
f
1
/K [ε − ε ] and the value of the intrinsic binding constant (K )
b
b
f
b
corresponding to the respective ligands 1, 2 and 3 and their
48
2
(Kapp) were calculated using the equation.
nickel complexes 4, 5 and 6 was found to be 4.60 ± 0.16 × 10
−1
2
−1
3
−1
M , 5.14 ± 0.20 × 10 M , 6.64 ± 0.15 × 10 M , 3.00 ±
4
−1
4
−1
4
0
.12 × 10 M , 4.60 ± 0.19 × 10 M and 7.52 ± 0.22 × 10
KEB½EBꢀ ¼ Kapp½complexꢀ
−1
M . The magnitude of the binding constant value clearly
showed that complex 6 bound more strongly with CT DNA than
the rest of the complexes through an intercalative mode whereas
the free hydrazone ligands displayed a electrostatic binding
mode of interactions.
The test compound concentrations were obtained from the
value at 50% reduction of the fluorescence intensity of EB and
7
−1
KEB = 1.0 × 10 M and [EB] = 5 μM. The calculated Kapp
values for ligands 1–3 and their complexes 4–6 are 4.92 ± 0.11
4
−1
4
−1
4
−1
×
±
1
10 M , 6.55 ± 0.25 × 10 M , 7.42 ± 0.17 × 10 M , 2.40
5
−1
5
−1
0.10 × 10 M , 3.48 ± 0.15 × 10 M and 4.00 ± 0.14 ×
0 M , respectively. The fluorescence quenching spectra of
Competitive binding between EB and ligands/complexes for CT
DNA
5
−1
DNA-bound EB by ligands and their complexes illustrated that
the quenching of EB bound to DNA by the test compounds are
in good agreement with the linear Stern–Volmer equation
Fluorescence quenching of the EB–DNA complex is used to
monitor the binding of test compounds to DNA regardless of
6848 | Dalton Trans., 2012, 41, 6842–6854
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(Fig. S5, S6† and 4 as insets). The ratio of the slope to the inter-
cept obtained by plotting I /I vs. [Q] yielded the value of
0
quenching constant (K ) corresponding to the three ligands 1, 2,
q
and 3 and their nickel chelates 4, 5 and 6 as 9.77 ± 0.13 ×
2
−1
2
−1
3
−1
1
0
0 M , 1.31 ± 0.24 × 10 M , 1.48 ± 0.19 × 10 M , 4.73 ±
3
−1
3
−1
3
−1
.12 × 10 M , 7.04 ± 0.18 × 10 M and 7.76 ± 0.15 × 10 M ,
respectively. However, the hydrazone ligands 1–3 did not exhibit
much potential to leach out the EB molecules that were orig-
inally bound to DNA.
Also, it was assessed that complex 6 showed a higher quench-
ing efficiency than the other complexes 4 and 5 that reflected the
strong binding of complex 6 with DNA to leach out more
number of EB molecules originally bound to DNA. The binding
affinity of the complexes towards DNA increased in the order 4
<
5 < 6. The results of this study strongly support the nature of
4
9
binding of DNAwith complex is intercalation mode.
Fig. 5 Emission spectra of BSA (1 × 10− M; λexi = 280 nm; λemi
6
=
3
1
45 nm) as a function of concentration of the complex 6 (0, 2, 4, 6, 8,
Protein binding studies
0, 12 and 14 × 10− M). Arrow indicates the effect of metal complex 6
7
0
on the fluorescence emission of BSA (inset: plot of [Q] versus I /I).
Fluorescence quenching of BSA by ligands (1–3) and their
metal complexes (4–6). Serum albumins are proteins that are
amongst others involved in the transport of metal ions and metal
complexes with drugs through the blood stream. Binding to
these proteins may lead to loss or enhancement of the biological
properties of the original drug, or provide paths for drug trans-
portation. Hence, the binding experiments using the newly syn-
thesised hydrazone ligands 1–3 and their nickel hydrazone
complexes 4–6 with BSA were carried out. A generally known
fact is that the fluorescence of a protein is caused by three intrin-
sic characteristics of the protein, namely tryptophan, tyrosine
and phenylalanine residues. Fluorescence quenching refers to
any process that decreases the fluorescence intensity of a fluoro-
phores due to a variety of molecular interactions including
excited state reactions, molecular rearrangements, energy trans-
fer, ground state complex formation and collision quenching.
Qualitative analysis of hydrazone ligands and its bivalent nickel
hydrazones bound to BSA has been undertaken by examining
the respective fluorescence spectra. The intensity of the fluor-
escence band of BSA observed at 345 nm was quenched to an
extent of about 36.51, 40.98, 42.76, 66.96, 69.01 and 70.62%
from its initial intensity upon the addition of test compounds
−5
Fig. 6 The absorption spectra of BSA (1 × 10 M) and BSA–
−5
−6
complex 6 (BSA = 1 × 10 M and complex 6 = 1 × 10 M).
(ligands/complexes) 1–6 together with a hypsochromic shift of
0
, 1, 1, 5, 5 and 6 nm due to the formation of a hydrazone–BSA
1
0 M , 1.76 ± 0.19 × 10 M and 1.88 ± 0.15 × 10 M−1,
6
−1
6
−1
6
complex/nickel hydrazone–BSA complex. Fig. S7, S8† and 5
showed the effect of the increase in the concentration of test
compounds 1–6 on the emission intensity of BSA.
The fluorescence quenching is described by Stern–Volmer
relation,
respectively.
UV-visible absorption measurement is a simple method to
explore the structural changes and is useful to distinguish the
type of quenching that exists i.e., static or dynamic quenching.
Dynamic quenching only affects the excited states of the fluoro-
phore and there are no changes in the absorption spectra.
However, ground-state complex formation will frequently result
in perturbation of the absorption spectrum of the fluorophore.
From the representative absorption spectra of pure BSA and
BSA–complex 6 shown in Fig. 6, it was clear that the addition
of complex 6 to a fixed concentration of BSA led to a gradual
increase in intensity of BSA absorption but at the same wave-
length due to interaction between test compounds and protein. In
I0=I ¼ 1 þ KSV½Qꢀ
where, I and I are the fluorescence intensities of the fluorophore
0
in the absence and presence of quencher, KSV is the Stern–
Volmer quenching constant and [Q] is the quencher concen-
tration. The KSV value obtained as a slope from the plot of [Q]
vs. I /I (as insets of Fig. S7, S8† and 5) in respect of ligands 1–3
and their complexes 4–6 are found to be 4.19 ± 0.18 × 10 M
0
5
−1
,
5
−1
5
−1
4
.29 ± 0.12 × 10 M , 4.82 ± 0.21 × 10 M , 1.62 ± 0.23 ×
This journal is © The Royal Society of Chemistry 2012
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other words, the fluorescence quenching between complex 6 and
with both DNA and BSA than that of the free hydrazone
ligands. Generally, these types of binding affinities of metal che-
lates towards DNA/BSA were due to the strong chelation
phenomenon experienced by the hydrazone systems with the
bivalent nickel centers. However, among the three types of
nickel chelate complexes, the complex 6 showed strong binding
with both DNA and BSA than the other two complexes. The
observed strong binding efficiency of complex 6 is attributed to
the presence of a six membered pyridine ring at the hydrazide
part that has delocalization of electrons in the aromatic ring than
the complexes containing five membered systems (complexes 4
and 5). However, among the five membered systems 4 and 5, the
one containing a sulphur atom in its molecular architecture
showed a better binding efficiency than the other with oxygen.
This fact can be correlated to the electronegativities of oxygen
and sulphur atoms in the complexes. The overall order of inter-
action between the ligands/complexes and CT DNA as well as
BSA decreased in the order 6 > 5 > 4 > 3 > 2 > 1. The results of
5
0
BSA is mainly ascribed to be static quenching. Similar obser-
vation was also obtained for the rest of the complexes and the
free ligands under identical experimental conditions. The above
results can be rationalized in terms of the strong interaction
between nickel chelates and BSA that caused a change in the
conformation of BSA.
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
51
bound molecules is represented by the Scatchard equation.
log½F0 ꢁ F=Fꢀ ¼ log K þ n log½Qꢀ
where, K and n are the binding constant and the number of
binding sites, respectively.
Thus, a plot of log[Q] versus log(F − F)/F (Fig. 7) can be
0
the studies are compared with similar metal complexes reported
used to determine the values of both K and n and such values
calculated for test compounds 1–6 are listed in Table 4.
From the values of n, it is inferred that there is only one inde-
pendent class of binding sites for complexes on BSA and also a
direct relation between the binding constant and number of
binding sites.
52,53
earlier
and are listed in Table 4.
Characteristics of synchronous fluorescence spectra
Synchronous fluorescence spectroscopy provides information on
the molecular microenvironment, particularly in the vicinity of
The results of binding experiments clearly explored that all
the three nickel hydrazone chelates showed stronger interactions
54
the fluorophore functional groups. It is well known that the flu-
orescence of BSA may be due to presence of tyrosine, trypto-
phan and phenylalanine residues and hence spectroscopic
methods are usually applied to study the conformation of serum
55
protein. According to Miller, in synchronous fluorescence
spectroscopy, the difference between excitation and emission
wavelength (Δλ = λemi − λexc) reflects the spectra of a different
nature of chromophores, with large Δλ values such as 60 nm, the
synchronous fluorescence of BSA is characteristic of the trypto-
phan residue and the small Δλ value such as 15 nm is charac-
56
teristic of tyrosine. To explore the structural changes of BSA
due to the addition of free ligands and its nickel(II) hydrazone
complexes, we measured synchronous fluorescence spectra of
the former with respect to addition of the test compounds 1–6.
The synchronous fluorescence spectra of BSA with various con-
centrations of compounds were recorded at both Δλ = 15 nm and
6
0 nm. Upon increasing the concentration of test compounds,
the intensity of emission corresponding to tyrosine (at 302 nm)
was found to decrease slightly in the magnitude of 16.54, 20.38,
2
1.29, 22.05, 23.87 and 27.11% for respective ligands 1, 2, 3
Fig. 7 Plot of log[Q] vs. log[(F − F)/F].
0
and complexes 4, 5 and 6 without any shift in emission
Table 4 Comparison of interaction study results between compounds 1–6 on DNA and protein
DNA binding
Protein binding
(M−
1
)
K
(M−1
)
K
app (M−1
)
K
sv (M−1
)
K (M−1
)
n
Compounds
K
b
q
2
2
4
4
5
5
5
6
6
6
4
4
4
5
5
6
1
2
3
4
5
6
4.60 ± 0.16 × 102
5.14 ± 0.20 × 103
6.64 ± 0.15 × 104
3.00 ± 0.12 × 10
9.77 ± 0.13 × 102
1.31 ± 0.24 × 103
1.48 ± 0.19 × 103
4.73 ± 0.12 × 10
4.92 ± 0.11 × 10
4.19 ± 0.18 × 10
4.29 ± 0.12 × 10
4.82 ± 0.21 × 10
1.62 ± 0.23 × 10
1.76 ± 0.19 × 10
1.88 ± 0.15 × 10
1.50 ± 0.12 × 10
1.90 ± 0.08 × 10
2.02 ± 0.14 × 10
1.80 ± 0.10 × 10
5.67 ± 0.15 × 10
1.14 ± 0.12 × 10
0.6154
0.6274
0.7042
0.8688
0.9210
0.9648
0.9730
6.55 ± 0.25 × 10
7.42 ± 0.17 × 10
2.40 ± 0.10 × 10
3.48 ± 0.15 × 10
4.00 ± 0.14 × 10
4
5
5
5
4
3
4.60 ± 0.19 × 104
7.04 ± 0.18 × 103
7.52 ± 0.22 × 10
7.76 ± 0.15 × 10
3
3
3
5
4
Reported value
8.04 × 10
2.07 × 10
1.97 × 10
1.53 × 10
2.19 × 10
6850 | Dalton Trans., 2012, 41, 6842–6854
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View Article Online
wavelength was observed. The respective spectra of free hydra-
zone ligands 1, 2, 3 and complexes 4, 5 and 6 are given in
Fig. S9, S10† and 8. At the same time, the tryptophan fluor-
escence emission showed significant decrease in the intensity (at
The hydroxyl radical is one of the most aggressive oxidants
known which is capable of attacking any biological molecule. It
is a major product arising from the high-energy ionisation of
water and its most important biological source is the Haber–
Weiss and Fenton reaction, involving superoxide anion, hydro-
gen peroxide and transition metals. Hydroxyl radical is highly
reactive oxygen centered radical formed from the reactions of
various hydroperoxides with transition metal ions. Among all
the free radicals, hydroxyl radical is by far the most potent and
therefore the most dangerous oxygen metabolite and hence the
elimination of this radical is one of the major aims of antioxidant
3
42 nm) of about 35.27, 40.54, 46.08, 52.16, 66.71 and 70.45%,
without any change in the position of the emission band. These
experimental results indicate that the metal complexes did affect
microenvironments of both tyrosine and tryptophan residues
during the binding process. Hence, during the binding process
the polarity around the tyrosine and tryptophan residues were
decreased and the hydrophobicity around the same residues was
strengthened, however, the effect was more towards tryptophan
than tyrosine. The hydrophobicity observed in fluorescence and
synchronous measurements confirmed the effective binding of
all the complexes with the BSA and these results are comparable
58
administration. It attacks proteins, DNA, polyunsaturated fatty
59
acid in membranes and most biological molecules. Hydroxyl
radical is known to be capable of abstracting hydrogen atoms
from membrane lipids and brings about the peroxidic reaction of
lipids. The scavenging activity of the nickel hydrazones on the
hydroxyl radical has been investigated and their inhibitory effect
on the OH radical is shown in Fig. 9. The IC50 values of nickel
hydrazone complexes 4–6 are found to be 35.92 ± 0.33, 33.61 ±
57
with earlier reports.
Antioxidant activities
0
.54 and 29.99 ± 0.73 μM respectively. Further, the free hydra-
In order to elucidate the ability of the nickel hydrazone com-
plexes to act toward different reactive radical species, we tested
zone ligands 1–3 failed to arrest the above said free radicals
under the studied concentration range.
some radical scavenging assay methods, in particular superoxide
Nitric oxide (NO) is a diffusible free radical which plays a
role as an effector in diverse biological systems including neur-
onal messenger, vasodilatation and antimicrobial and antitumor
−
(O2 ), hydroxyl (OH) and nitric oxide (NO) radical scavenging
assays. The hydrazone ligands and their metal complexes have
been tested in the range of 10–50 μM concentrations. The free
radical scavenger action is very important especially in the case
of superoxide anion, since it prevents or attenuates the formation
of peroxynitrite and hydroxyl radical. The over production of
peroxynitrite and hydroxyl radical is an important feature of
60
activities. Nitric oxide inhibitors were shown to have beneficial
effects on some aspect of inflammation and tissue damage seen
61
in inflammatory diseases. These compounds are responsible
for altering both the structural and functional behaviour of many
cellular components. The metal hydrazones have the property to
counteract the effect of NO formation and in turn possess con-
siderable interest in preventing the ill effects of excessive NO
generation in the human body. In order to understand the ability
of the nickel hydrazone complexes towards the reactive radical
species, a NO scavenging assay of them was carried out at differ-
ent concentration of complexes and the results are presented in
Fig. 9, and the corresponding IC50 values of the hydrazone
57
tissue damage mechanisms during pathological processes. In
this regard, the antioxidant potentials against superoxide (O2−
by the tested complexes were gave the IC50 values: 33.28 ±
.52, 30.84 ± 0.70 and 23.11 ± 0.42 μM respectively for com-
)
0
plexes 4–6 (Fig. 9), however the free hydrazone ligands do not
show significant activity against the same radical under identical
experimental conditions.
Fig. 8 Synchronous spectra of BSA (1 × 10− M) as a function of concentration of the complex 6 (0, 2, 4, 6, 8, 10, 12 and 14 × 10−7 M) with a
wavelength difference of Δλ = 15 nm (a) and Δλ = 60 nm (b). Arrow indicates the change in emission intensity w.r.t. various concentration of
complex.
6
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Fig. 9 Trends in the inhibition of super oxide, hydroxyl and nitric oxide radicals by synthesised compounds 1–6 at variable concentrations.
Table 5 Comparison of IC50 values of test compounds towards
selected free radicals
ligands 1–3 and its metal complexes 4–6 are 46.74 ± 0.87, 42.51
0.29, 39.97 ± 0.54, 28.43 ± 0.30, 27.05 ± 0.91 and 25.72 ±
.38. However, the nickel precursor complex did not register any
±
0
IC50 values (μM)
scavenging potentials under the same experimental conditions.
The antioxidant properties of the hydrazone ligands and its
nickel(II) complexes studied towards the above said radicals were
compared with standard antioxidant butylated hydroxy anisole
Test
compounds
Superoxide
radical
Hydroxyl
radical
Nitric oxide
radical
1
2
3
4
5
6
—
—
—
—
—
—
46.74 ± 0.87
42.51 ± 0.29
39.97 ± 0.54
28.43 ± 0.30
27.05 ± 0.91
25.72 ± 0.38
10.11
(
BHA).
Fig. 9 shows the plot of the hydroxyl radical scavenging effect
%) and it is found that the inhibitory effect of the compounds
33.28 ± 0.52
30.84 ± 0.70
23.11 ± 0.42
4
35.92 ± 0.33
33.61 ± 0.54
29.99 ± 0.73
38.46
(
−
tested with O2 , OH and NO radicals are concentration depen-
dent and their suppression ratio increases with increasing sample
concentrations in the tested range. From the above values
Reported value
(Table 5), it is found that all the metal complexes tested in this
study behaved as more effective inhibitors than the standard
−
scavenging agent, butylated hydroxy anisole (BHA) for O , OH
than the rest of the complexes with five membered furan and
thiophene rings in the complexes 4 and 5. Further, the nickel
hydrazone complexes exhibited better activity when compared to
that of free hydrazone ligands which might be due to the chelate
effect. Hence, we strongly believe that the present metal hydra-
zone complexes can be further evaluated as suitable candidates
leading to the development of new potential antioxidants and
therapeutic reagents for some diseases.
2
and NO radicals and are compared with that of the other nickel
53,62
complexes.
In general, the antioxidant activity of synthesised compounds
against the free radicals O2 , OH and NO decreased in the order
> 5 > 4 > 3 > 2 > 1. Among all three nickel hydrazones, the
one possessing six membered pyridine ring in the hydrazone
−
6
moiety (complex 6) showed more potential scavenging activity
6852 | Dalton Trans., 2012, 41, 6842–6854
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Table 6 Comparison of IC50 values between complexes 4–6
IC50 values in μM
and their interaction with biomolecules were presented. The
structure and geometry of the complexes analysed through single
crystal X-ray diffraction revealed a square planar geometry
around the Ni(II) ion in complexes 4 and 5. From the bio-
inorganic chemistry point of view, the binding interaction of the
synthesised compounds 1–6 with CT DNA and BSA protein has
been investigated and the order of binding affinity varies from a
magnitude of 10 to 10 . Also, their antioxidant and cytotoxicity
properties showed that they can be studied as potential drugs. To
summarise, complex 6 that possess a pyridine ring with deloca-
lized 6π electrons at the hydrazide part showed a better perform-
ance in biomolecular interactions than the other two complexes
Complex
HeLa
HepG-2
A431
NIH 3T3
4
5
6
185.6 ± 1.6 115.3 ± 1.3 45.7 ± 0.9 481.7 ± 2.4
125.9 ± 0.9 88.2 ± 1.2
116.6 ± 0.7 50.9 ± 0.8
29.3 ± 1.1 456.6 ± 1.8
19.1 ± 0.5 407.1 ± 1.5
2
5
Reported value 92.86
Reported value 49.48
—
—
80.72
35.13
312.44
188.53
Cytotoxicity
4
and 5 that have five membered heterocyclic rings. But, among
The cytotoxicity of the synthesised ligands 1–3, the starting pre-
cursor complex [NiCl (PPh ) ] and their hydrazone complexes
the complexes 4 and 5 with five membered heterocyclic moi-
eties, the former one with the more electronegative oxygen atom
displayed less potential with regard to biomolecular interactions
and cytotoxicity than the later one with a sulphur atom that
could have favoured the formation of an electron rich environ-
ment around the central nickel ion and thus led to a better inter-
action with the biomolecules.
2
3 2
4
–6 was tested with a MTT colorimetric assay that measures
mitochondrial dehydrogenase activity as an indication of cell
viability against HeLa (human cervical cancer cells), A431
(human skin cancer cells), HepG2 (human liver hepatocellular
carcinoma cells) and normal NIH 3T3 (mouse embryonic cells).
The test compounds were dissolved in DMSO and blank
samples containing the same amount of DMSO are taken as con-
trols. The effects of the compounds on the viability of these cells
were evaluated after an exposure period of 48 h. All the com-
plexes showed very good cytotoxic potencies under the studied
concentrations with the inhibition of cancer cell growth at the
Acknowledgements
We sincerely thank the University Grants Commission, New
Delhi for the award of a Fellowship in Science for Meritorious
Students (RFSMS) to one of the authors (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.
5
0% level.
Assays of nickel hydrazones against cancerous cell lines
HeLa, HepG-2 and NIH 3T3 normal cell lines were performed
in the concentration range of 15 to 500 μM whereas A431 cell
lines were performed in the concentration range of 6.25–200 for
complexes 4 and 5 and 3.12–100 μM for complex 6. Among the
cancerous cell lines, the IC50 values of the investigated com-
plexes 4–6 against A431 are found to be low which indicates its
higher activity when compared to that of the other cancerous cell
lines HeLa and HepG2 with high IC50 values (Table 6)
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