Copper(i) and nickel(ii) complexes with 1:1 vs. 1:2 coordination of ferrocenyl hydrazone ligands: Do the geometry and composition of complexes affect DNA binding/cleavage, protein binding, antioxidant and cytotoxic activities?

Article abstract of DOI:10.1039/c2dt11938b

A new series of geometrically different complexes containing ferrocenyl hydrazone ligands were synthesised by reacting suitable precursor complex [MCl₂(PPh₃)₂] with the ligands HL¹ or HL² (where M = Cu(ii) or Ni(ii); HL¹ = [Cp ₂Fe(CHN-NH -CO- C₆H₅)] (1) and HL² = [Cp₂Fe(CHN-NH-CO-C₅H₄N)]) (2). The new complexes of the composition [Cu(L¹)(PPh₃)₂], (3) [Cu(L²)(PPh₃)₂] (4), [Ni(L ¹)₂] (5) and [Ni(L²)₂] (6) were characterised by various spectral studies. Among them, complexes 3 and 5 characterised by single crystal X-ray diffraction showed a distorted tetrahedral structure for the former with 1:1 metal-ligand stoichiometry, but a distorted square planar geometry with 1:2 metal-ligand stoichiometry in the case of the latter. Systematic biological investigations like DNA binding, DNA cleavage, protein binding, free radical scavenging and cytotoxicity activities were carried out using all the synthesised compounds and the results obtained were explained on the basis of structure-activity relationships. The binding constant (K_b) values of the synthesised compounds are found to be in the order of magnitude 10³-10⁵ M^-1 and also they exhibit significant cleavage of supercoiled (SC) pUC19 DNA in the presence of H₂O₂ as co-oxidant. The conformational changes of bovine serum albumin (BSA) upon binding with the above complexes were also studied. In addition, concentration dependent free radical scavenging potential of all the synthesised compounds (1-6) was also carried out under in vitro conditions. Assays on the cytotoxicity of the above complexes against HeLa and A431 tumor cells and NIH 3T3 normal cells were also carried out.

Full text of DOI:10.1039/c2dt11938b

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PAPER
Copper(I) and nickel(II) complexes with 1 : 1 vs. 1 : 2 coordination of
ferrocenyl hydrazone ligands: Do the geometry and composition of complexes
affect DNA binding/cleavage, protein binding, antioxidant and cytotoxic
activities?†
Paramasivam Krishnamoorthy,a Palanisamy Sathyadevi,a Rachel R. Butorac,b Alan H. Cowley,b Nattamai
S. P. Bhuvaneshc and Nallasamy Dharmaraj*a
Received 13th October 2011, Accepted 12th December 2011
DOI: 10.1039/c2dt11938b
A new series of geometrically different complexes containing ferrocenyl hydrazone ligands were
synthesised by reacting suitable precursor complex [MCl2(PPh3)2] with the ligands HL1 or HL2 (where M
= Cu(II) or Ni(II); HL1 = [Cp2Fe(CHvN–NH –CO– C6H5)] (1) and HL2 = [Cp2Fe(CHvN–NH–CO–
C5H4N)]) (2). The new complexes of the composition [Cu(L1)(PPh3)2], (3) [Cu(L2)(PPh3)2] (4), [Ni(L1)2]
(5) and [Ni(L2)2] (6) were characterised by various spectral studies. Among them, complexes 3 and 5
characterised by single crystal X-ray diffraction showed a distorted tetrahedral structure for the former
with 1 : 1 metal–ligand stoichiometry, but a distorted square planar geometry with 1 : 2 metal–ligand
stoichiometry in the case of the latter. Systematic biological investigations like DNA binding, DNA
cleavage, protein binding, free radical scavenging and cytotoxicity activities were carried out using all the
synthesised compounds and the results obtained were explained on the basis of structure–activity
relationships. The binding constant (Kb) values of the synthesised compounds are found to be in the order
of magnitude 103–105 M−1 and also they exhibit significant cleavage of supercoiled (SC) pUC19 DNA in
the presence of H2O2 as co-oxidant. The conformational changes of bovine serum albumin (BSA) upon
binding with the above complexes were also studied. In addition, concentration dependent free radical
scavenging potential of all the synthesised compounds (1–6) was also carried out under in vitro
conditions. Assays on the cytotoxicity of the above complexes against HeLa and A431 tumor cells and
NIH 3T3 normal cells were also carried out.
researchers due to their well known chelating capability and
Introduction
structural flexibility that can provide rigidity to the skeletal fra-
mework of the prepared metal complexes.1–5 Hydrazones and
Schiff bases together with various metals have been extensively
used as building blocks to produce a great variety of topologies.
Among them, hydrazones have attracted special attention from
analogous compounds play an important role in improving the
selectivity and toxicity profile of certain antitumor agents by
forming drug carrier systems employing suitable carrier pro-
teins.6 The real impetus towards developing the coordination
chemistry of these hydrazones has been provided by the remark-
able biological activities exhibited by them that are related to
their metal complexing ability.
Large numbers of ferrocene-containing metal chelates are in
fact multinuclear molecules possessing features of both organo-
metallic and coordination chemistry.7,8 Aroyl hydrazones
attached to a ferrocenyl molecule have been studied in detail
owing to their plentiful coordination chemistry and antibiotic
activity as well as the fact that azomethine ligands are models for
metal–ligand binding sites in several enzymes.9–15 Complexes of
aroyl hydrazones containing both heterocycles and the ferrocenyl
group within the same molecule have been scarcely investigated
so far and therefore we felt that it appropriate to undertake a sys-
tematic study involving such a novel system and to understand
their behaviour towards a series of biological experiments.
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, USA
cDepartment of Chemistry, Texas A&M University, College Station, TX
77843, USA
†Electronic supplementary information (ESI) available: Crystal packing
diagram of the unit cell of complexes 3 and 5 (Fig. S1 and S2), Elec-
tronic absorption spectra of binding of ligands 1 and 2 with DNA
(Fig. S3), Electronic absorption spectra of binding of complexes 3, 4
and 5 with DNA (Fig. S4 and S5), Circular dichroism spectra of DNA
and ligands 1 and 2 (Fig. S6), Circular dichroism spectra of DNA and
complexes 3, 4 (Fig. S7), Emission spectra of binding of complexes 3, 4
and 5 with BSA (Fig. S8 and S9), Synchronous spectra (Δλ = 15 and
60 nm) of binding of complexes 3, 4 and 5 with BSA (Fig. S10, S11
and S12). CCDC reference numbers 796259 and 822888 for complexes
3 and 5. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2dt11938b
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Further, most of the first row transition metals either alone or
in their complex form are biologically essential with a number of
known bioactivities. Among them, copper and nickel are
especially notable as they are involved in many biological
processes.16–25 The interactions between bio-macromolecules
and drugs have attracted special interest among both chemistry
and biology researchers during the past few decades.26–28 Since
DNA is one of the main molecular targets in the design of antic-
ancer compounds, interaction between nucleic acids and other
molecules is a fundamental issue in life science that relates the
replication, transcription, mutation of genes and related vari-
ations of species in character, origin of some diseases and mech-
anism of interaction of some DNA-targeted drugs.29–32 Another
biomolecule, serum albumin (BSA) is a major soluble protein
constituent of the circulatory system and has many physiological
functions, e.g. it serves as a depot protein for many exogenous
compounds.33,34 Significant interaction of any drug with a
protein may result in the formation of a stable protein–drug
complex, which has an important effect on the distribution,
metabolism and the efficacy of drugs, etc. Copper and nickel
being bio-essential elements, it is considered that their com-
plexes can find more applications in various biological
processes.
were recorded on a THERMO Finnigan LCQ Advantage max
ion trap mass spectrometer. The electronic absorption and emis-
sion spectra were recorded in DMSO–buffer (1 : 99) solution on
a Jasco V-630 spectrophotometer and Jasco FP 6600 spectrofluo-
rometer respectively at room temperature. The circular dichroism
experiments were carried out using a Jasco J-810 spectropolari-
meter at room temperature.
X-Ray crystallography
For each compound, a crystal of suitable quality was removed
from the vial, covered with mineral oil and mounted on a nylon
thread loop. X-Ray diffraction data of the complexes 3 and 3a
were obtained using a Rigaku AFC-12 Saturn 724+ CCD dif-
fractometer equipped with a graphite-monochromated Mo-Kα
radiation source (λ = 0.7107 Å) whereas complex 5 was col-
lected on a BRUKER GADDS X-ray (three-circle) diffract-
ometer equipped with a Cu-Kα radiation source (λ = 1.5418 Å).
Corrections were applied for Lorentz and polarization effects for
each of the compounds. Each structure was solved by direct
methods and refined by full-matrix least-squares cycles on F2
using the Siemens SHELXTL PLUS 5.0 (PC) software
package35,36 and PLATON.37 All non-hydrogen atoms were
refined anisotropically and hydrogen atoms were placed in fixed,
calculated positions using a riding model.
Based on the above facts, we undertook this study to prepare
some new hydrazone complexes of copper and nickel from the
reactions of [MCl2(PPh3)2] with HLn (where M = Cu(II) or Ni
(II); n = 1 or 2) in 1 : 1 metal-to-ligand stoichiometry. Further, a
systematic investigation on the effects of the free ligands and the
corresponding newly synthesised copper/nickel hydrazone com-
plexes with respect to DNA binding, DNA cleavage, protein
interaction, in vitro free radical scavenging and cytotoxic activi-
ties has also been carried out.
Synthesis of starting metal complexes
The requisite precursor metal complexes [CuCl2(PPh3)2] and
[NiCl2(PPh3)2] were prepared according to the literature
methods.38,39
Synthesis of hydrazone ligands
Experimental
Synthesis of formyl ferrocene-2-benzoyl hydrazone (HL1) (1)
and formyl ferrocene 2-isonicotinyl hydrazone (HL2)
(2). Ligands 1 and 2 were prepared by refluxing equimolar sol-
utions of ferrocene-2-carbaldehyde (0.214 g; 1 mM) and benzhy-
drazide (0.136 g; 1 mM) or isonicotinic acid hydrazide (0.137 g;
1 mM) respectively in 50 mL of ethanol for 8 h as summarised
in Scheme 1. The reaction mixture was cooled to room tempera-
ture and the product formed was filtered, washed several times
with water, and then recrystallized from ethanol to afford the
desired product in pure form.
Materials and physical measurements
Reagent grade chemicals were used without further purification
in all the synthetic work. Solvents were purified by standard
methods. The compounds CuCl2·2H2O, NiCl2·6H2O, triphenyl-
phosphine, benzhydrazide and isonicotinic acid hydrazide pur-
chased from Sigma-Aldrich Chemie or Alfa Aesar were used as
received. Calf-thymus DNA (CT-DNA) and bovine serum
albumin (BSA) were purchased from Himedia Company. The
plasmid supercoiled (SC) pUC19 DNA was purchased from
Bangalore GeNei, Bangalore, India. The human cervical cancer
cell line (HeLa), human skin cancer cell line (A431) and mouse
embryonic fibroblasts (NIH 3T3) was obtained from the National
Centre for Cell Science (NCCS), Pune, India. All other chemi-
cals and reagents used for the biological studies were of high
quality and procured commercially from reputable suppliers.
Microanalyses (% C, H & N) were performed on a Vario EL
III CHNS analyzer. IR spectra of the samples were recorded as
KBr pellets on a Nicolet Avatar instrument in the frequency
HL1 (1): Yield: 259 mg (78%), Anal. Found (%) for
C18H16N2OFe (Mol. wt = 332.18): C, 64.78%; H, 5.12%; N,
8.75%. Calculated (%) C, 65.09%; H, 4.86%; N, 8.43%.
ESI-MS (MeOH): Found m/z = 333 (M + H) (calculated m/z =
332 for M+). Selected IR bands (ν in cm−1): 3224 (–NH); 1648
(sCvO); 1605 (sCvN–); 1044 (vN–Nr). 1H NMR
(500 MHz, CDCl3): δ (ppm): 9.04 (s, 1H, –NH); 8.31 (s, 1H,
–OH enolic form); 7.87 (s, 1H, –HCvN–); 7.57–7.48 (1d &1t,
5H, Ar–H); 4.73–4.24 (3 s, 9H, cp–H).
range of 400–4000 cm−1. H NMR spectra of ligands and metal
HL2 (2): Yield: 239 mg (72%), Anal. Found (%) for
C17H15N3OFe (Mol. wt = 333.17): C, 60.99%; H, 4.32%; N,
12.89%. Calculated (%) C, 61.29%; H, 4.54%; N, 12.61%.
ESI-MS (MeOH): Found m/z = 334 (M + H) (calculated m/z =
333 for M+). Selected IR bands (ν in cm−1): 3235 (–NH); 1636
1
complexes were recorded on a Bruker AMX 500 spectrometer
operating at 500 MHz using CDCl3 as solvent and tetramethylsi-
lane as an internal standard. The electronspray ionization mass
(ESI-MS) spectra of the complexes and ligands in methanol
4424 | Dalton Trans., 2012, 41, 4423–4436
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Scheme 1
(sCvO); 1592 (sCvN–); 1035 (= N–Nr). 1H NMR
(500 MHz, CDCl3): δ (ppm): 9.12 (s, 1H, –NH); 8.54 (s, 1H,
–OH enolic form); 7.88 (s, 1H, –HCvN–); 7.31–7.16 (2d, 4H,
Ar–H); 4.56–3.81 (3 s, 9H, cp–H).
corresponding to complex 4 were as follows: Yield: (377 mg)
41%. mp: 285 °C Anal. Found (%) for C53H44N3OFeP2Cu (Mol.
wt = 920.28): C, 59.98%; H, 4.12%; N, 4.03%. Calculated (%)
for C, 69.17%; H, 4.82%; N, 4.57%. ESI-MS (MeOH): Found
m/z = 921 (M + H) (calculated m/z = 920 for M+). Selected IR
bands (ν in cm−1): 1583 & 1509 (sCvN–NvCr); 1362 (phe-
nolic –C–O); 1314 (enolic –C–O); 1092 (vN–Nv).1H NMR
(500 MHz, CDCl3): δ (ppm): 8.56 (s, 1H, –HCvN–);
7.40–7.22 (m, 34H, Ar–H); 4.70–3.82 (3 s, 9H, cp–H).
Complex 5 was prepared by a procedure similar to that
described for the above complexes 3 or 4 by utilising
[NiCl2(PPh3)2] (0.653 g; 1 mM) and the ligand 1 (0.332 g;
1 mM) as reactants (Scheme 1). Purity of the product 5 formed
in this reaction checked by TLC confirmed it to be a single spot.
The slow evaporation of complex 5 in MeOH/CHCl3 solution
afforded red crystals suitable for X-ray diffraction studies. Yield:
(346 mg) 48%. mp: >300 °C. Anal. Found (%) for C36H30N4O2
Fe2Ni (Mol. wt = 721.028): C, 59.02%; H, 3.97%; N, 7.12%.
Calculated (%) for C, 59.96%; H, 4.19%; N, 7.77%. ESI-MS
(MeOH): Found m/z = 722 (M + H) (calculated m/z = 721 for
M+). Selected IR bands (ν in cm−1): 1588 & 1518 (sCvN–
NvCr); 1357 (phenolic –C–O); 1305 (enolic –C–O); 1066
(vN–Nv).1H NMR (500 MHz, CDCl3): δ (ppm): 8.01 (s, 2H,
–HCvN–); 7.46–7.16 (1d &1t, 10H, Ar–H); 5.28–4.33 (3 s,
18H, cp–H).
Complex 6 was prepared by a procedure similar to that
described for complex 5 by utilizing [NiCl2(PPh3)2] (0.653 g;
1 mM) and ligand 2 (0.333 g; 1 mM) (Scheme 1). Attempts to
isolate crystals suitable for single crystal XRD were unsuccess-
ful. Yield: (318 mg) 44%. mp: >300 °C. Anal. Found (%) for
C34H28N6O2Fe2Ni (Mol. wt = 723.002): C, 55.98%; H, 3.11%;
N, 11.09%. Calculated (%) for C, 56.48%; H, 3.90%; N,
11.62%. ESI-MS (MeOH): Found m/z = 724 (M + H) (calculated
m/z = 723 for M+). Selected IR bands (ν in cm−1): 1599 & 1519
(sCvN–NvCr); 1372 (phenolic –C–O); 1305 (enolic –C–O);
1088 (vN–Nv).1H NMR (500 MHz, CDCl3): δ (ppm): 8.04
(s, 2H, –HCvN–); 7.49–7.18 (2d, 8H, Ar–H); 5.20– 4.35 (3 s,
18H, cp–H).
Synthesis of metal hydrazone complexes 3–6
Complexes [Cu(L1)(PPh3)2] (3) and [CuCl(PPh3)3] (3a) were
prepared by refluxing equimolar quantities of [CuCl2(PPh3)2]
(0.658 g; 1 mM) and the hydrazone ligand (HL1) (1) (0.332 g;
1 mM) in 40 mL of methanol (Scheme 1). After a few minutes
of mixing of the above reactants, 2 or 3 drops of methanolic
KOH was added to the reaction mixture and continuously
refluxed for 5 h. It is then cooled to room temperature, and the
resulting product was filtered off, washed with methanol and
dried under vacuum. Purity of the above product checked by
TLC revealed the presence of two distinct spots, i.e., complexes
3 and 3a that were subsequently separated by column chromato-
graphy using a mixture of petroleum ether and ethyl acetate as
an eluent. Slow evaporation of both 3 and 3a in MeOH/CHCl3
mixture afforded single crystals of respective complexes suitable
for X-ray diffraction studies. The data corresponding to complex
3 were as follows: Yield: (395 mg) 43%. mp: 270 °C. Anal.
Found (%) for C54H45N2OFeP2Cu (Mol. wt = 919.29): C,
70.08%; H, 4.12%; N, 2. 83%. Calculated (%) for C, 70.55%;
H, 4.93%; N, 3.05%. ESI-MS (MeOH): Found m/z = 920
(M + H) (calculated m/z = 919 for M+). Selected IR bands (ν in
cm−1): 1584 & 1505 (sCvN–NvCr); 1368 (phenolic –C–O);
1314 (enolic –C–O); 1093 (vN–Nv). 1H NMR (500 MHz,
CDCl3): δ (ppm): 8.50 (s, 1H, –HCvN–); 7.68–7.26 (1 m, 35H,
Ar–H); 4.72–3.88 (3 s, 9H, cp–H).
Complexes 4 and 4a were prepared by a procedure similar to
that described above using [CuCl2(PPh3)2] (0.658 g; 1 mM) and
ligand HL2 (2) (0.333 g; 1 mM) (Scheme 1). Isolation of the
crystals suitable for single crystal XRD studies were unsuccess-
ful in the case of complex 4 whereas complex 4a afforded white
crystals on crystallization and were identified to be [CuCl
(PPh3)3] as obtained in the previous reaction as 3a. The data
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DNA binding experiments
Protein binding studies
Electronic absorption titration. Electronic absorption titra-
tions were performed with a fixed concentration of ligands 1 and
2 and their metal complexes 3–6 (25 μM) but by varying nucleo-
tide concentration from 0 to 25 μM. The absorption band of the
compounds 1–6 that have undergone significant shift due to the
addition of CT DNA was chosen to monitor as an indication of
binding between them. From the absorption titration data, the
intrinsic binding constant (Kb) of the test compounds with CT
DNA was determined using the equation,40 [DNA]/[εa − εf] =
[DNA]/[εb − εf] + 1/Kb[εb − εf], where [DNA] is the concen-
tration of DNA, εa, εf and εb are the apparent extinction coeffi-
cient, the extinction coefficient for free test compounds and the
extinction coefficient for the test compound in the fully bound
form respectively. A plot of [DNA] vs. [DNA]/(εa − εf) gives
the intrinsic binding constant Kb as the ratio of slope to the
intercept.
Binding of metal hydrazone complexes with bovine serum
albumin (BSA) was studied from the fluorescence spectra
recorded with excitation at 280 nm and corresponding emission
at 345 nm assignable to that of bovine serum albumin (BSA).
Sample solutions were carefully degassed 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 the ligands
and its copper complexes were prepared by dissolving them in
DMSO: phosphate buffer (1 : 99) and diluted suitably with phos-
phate buffer to the required concentrations. 2.5 ml of BSA sol-
ution (μM) was titrated by successive additions of a 25 μl stock
solution of metal hydrazone complexes (10−4 M). Synchronous
fluorescence spectra were also recorded using the same concen-
tration of BSA and complexes as mentioned above.
Circular dichroism study. CD spectroscopic technique is
useful in monitoring the conformational changes induced by the
interaction of drugs as well as destabilization of the DNA helix.
The B-form conformation of DNA showed two consecutive CD
bands in the UV region, a positive band at 275 nm due to base
stacking and a negative band at 246 nm due to polynucleotide
helicity. The conformational changes upon the addition of test
compounds 1–6 was studied by keeping the concentration of
DNA constant at 200 μM and varying the test samples 1–6 with
a concentration of 20 μM in Tris-HCl buffer. The spectrum of
control DNA and with increasing amounts of the test compound
was monitored from 200 to 350 nm. It is known that the changes
in ellipticity are directly related to the conformational changes
observed in circular dichroism. The changes in CD signals of
DNA observed on intercalation with drugs may often be
assigned to the corresponding changes in the DNA structure.41
Thus, simple groove binding and electrostatic interaction of
small molecules show less or no perturbation on the base-stack-
ing and helicity bands, while intercalation enhances the intensi-
Antioxidant activity
The DPPH, OH and NO radical scavenging activities of the free
hydrazone ligands and corresponding metal complexes were
determined by the methods described by Blois, Nash and Green
et al., respectively.44–46 For each of the above assay, tests were
run in triplicate by varying the concentration of the ligands 1
and 2 and its corresponding complexes 3–6 ranging from
10–50 μM. The percentage activity was calculated by using the
formula, % activity = [(A0 – AC)/A0] × 100, where A0 and AC rep-
resent the absorbance in the absence and presence of the test
compounds, respectively. The 50% activities (IC50) were calcu-
lated from the results of percentage activity.
Cytotoxicity studies
The in vitro cytotoxicity assay (IC50) was performed on the
human cervical cancer cell line (HeLa), human skin cancer cell
line (A431) and the mouse embryonic cell line (NIH 3T3). The
HeLa tumor cell lines used in this work were grown in Eagles
Minimum Essential Medium containing 10% fetal bovine serum
(FBS) and the NIH 3T3 fibroblasts were grown in Dulbeccos
Modified Eagles Medium (DMEM) containing 10% FBS. For
the screening experiments, the cells were seeded into 96-well
plates in 100 ml of the respective medium containing 10% FBS,
at a plating density of 10,000 cells well−1. The cells were incu-
bated at 37 °C in 5% CO2 and 95% air at a relative humidity of
100% for 24 h prior to the addition of the complexes. The com-
plexes were solubilized in dimethylsulfoxide and diluted in the
respective serum free medium. After 24 h, 100 ml of the
medium containing the test compounds with various concen-
trations (e.g. 12.5, 25, 50, 100, 200 and 400 μM) was added and
incubated at 37 °C in an atmosphere of 5% CO2 and 95% air
with 100% relative humidity for 48 h. All measurements were
made in triplicate and the medium containing no test complexes
ties of both the bands stabilizing the right-handed
B
conformation of DNA as observed for the classical intercalator
methylene blue.42
DNA cleavage experiments
The extent of DNA cleavage induced by the test compounds was
monitored by agarose gel electrophoresis. A solution containing
25 μL of pUC19 DNA (1 μg), HCl (50 mM, pH 7.5), NaCl
(50 mM), the metal complex (35 μM) and H2O2 (60 μM) was
incubated at 37 °C for 1 h. Subsequently, 4 μL of 6X DNA
loading buffer containing 0.25% bromophenol blue, 0.25%
xylene cyanol and 60% glycerol was added to the test solution
and then mixed with 1% agarose gel containing 1.0 μg mL−1 of
ethidium bromide. Electrophoresis was performed at 5 V cm−1
for 2 h in a TBE buffer and the bands were visualized under UV
light and photographed. The cleavage efficiencies were measured
using the BIORAD Gel Documentation System. The intensity of
each band relative to that of the plasmid supercoiled form was
multiplied by 1.43 to take account of the reduced affinity for
ethidium bromide.43
served as the control. 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 formazan crystals that had formed were solu-
bilized in 100 mL of DMSO and the absorbance at 570 nm was
4426 | Dalton Trans., 2012, 41, 4423–4436
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Table 1 Crystal and structure refinement data
Description
Complex 3
Complex 5
Formula
Formula weight
C54H45CuFeN2OP2
919.25
C37H31Cl3Fe2N4NiO2
840.42
T/K
λ/Å
100(2)
0.71075
110(2)
1.54178
Crystal system
Space group
Monoclinic
P21/c
Monoclinic
P21/n
Cell dimensions
a/Å
b/Å
c/Å
α (°)
β (°)
γ (°)
Z
13.7088(7)
17.8387(9)
20.7620(8)
90
119.129(2)
90
16.2656(8)
12.9068(6)
16.8967(8)
90
107.378(3)
90
4
4
hkl limits
−17 ≦ h ≦ 17
−23 ≦ k ≦ 23
−23 ≦ l ≦26
1.377
−18 ≦ h ≦ 18
−13 ≦ k ≦ 14
−18 ≦ l ≦ 18
1.649
D
calcd (Mg m−3
F(000)
)
1904
1712
Crystal size (mm3) 0.36 × 0.24 × 0.15
0.40 × 0.32 × 0.06
5006 [R(int) = 0.0805]
Independent
reflections
10114 [R(int) =
0.0792]
Data/restraints/
parameters
10114/0/550
5006/0/443
1.071
Fig. 1 The molecular structure of complex 3, with displacement ellip-
soids drawn at 50% probability level.
Goodness-of-fit on 1.056
F2
Final R indices [I > R1 = 0.0409, wR2 =
R1 = 0.0386, wR2 =
0.0875
2σ(I)]
0.1065
R indices (all data) R1 = 0.0447, wR2 =
R1 = 0.0448, wR2 =
0.0905
measured using a micro plate reader. The % cell inhibition was
determined using the following formula,
0.1091
% Cell inhibition ¼ 100 ꢀ Abs
=Abs
ꢁ 100:
ðsampleÞ
ðcontrolÞ
starting precursor. Furthermore, the bond angles of [O1–Cu–P1]
119.00(4)°, [N2–Cu–P1] 125.13(5)°, [O1–Cu–P2] 92.52(4)°,
[N2–Cu–P2] 113.59(5)°, [N2–Cu–O1] 79.08(6)° and [P1–Cu–
P2] 116.37(2)° are deviated from the theoretical value of
109.28°. The bond lengths of [Cu–O1] 2.111(1) Å, [Cu–N2]
2.052(1) Å, [Cu–P1] 2.2311(6) Å, and [Cu–P2] 2.3057(6) Å are
almost equal to the tetrahedral bond lengths and are comparable
with that of the other tetrahedral copper(I) complex.47 The unit
cell packing of complex 3 is shown in the Fig. S1.†
Structure of the minor product (3a) obtained along with 3 was
solved using XRD and found to be [CuCl(PPh3)3]. The crystal
structure, unit cell parameters, bond lengths of 3a was found to
be in good agreement with the Cu(I) monovalent species
reported earlier.48 Fig. 2 represents the crystal structure of
complex 3a.
The IC50 values were calculated from the graph plotted
between % cell inhibition and concentration.
Results and discussion
Characterisation of metal hydrazone complexes
The analytical data of the ligands and their corresponding com-
plexes summarized in the experimental section agreed well with
the theoretical values within the limit of experimental error and
confirmed the formulae [Cu(Ln)(PPh3)2] or [Ni(Ln)2] (n = 1 or
2) proposed for the new complexes. All these complexes are
insoluble in water but soluble in most of the common organic
solvents.
Complex 5. An ORTEP representation corresponding to the
structure of [Ni(L1)2] (5) inclusive of the atom numbering
scheme is shown in Fig. 3 and its selected bond lengths and
bond angles are presented in Tables 1 and 2. The title complex
crystallized in the monoclinic unit cell with space group of
P21/n. The dimensions of both of the coordinated ligands are
very similar and they adopted the enolate form. The Ni(II) center
of complex 5 contained two bidentate hydrazone ligands and its
crystallographic structural analysis revealed that the central metal
atom adopts a distorted square planar geometry shared by two
fused five-membered chelate rings {Ni1N1N2C1O1}with its
counterpart {Ni1N3N4C8O2}. Four atoms (O1, O2, N1, N3) of
the ligand (L1) define a basal plane of the square plane by the
replacement of both the chlorine and triphenylphosphines from
Single crystal X-ray studies
Structural description
Complex 3. An ORTEP diagram of complex 3 is displayed in
Fig. 1. The crystallographic data, along with a selection of bond
lengths and bond angles, are presented in Tables 1 and 2,
respectively. X-Ray results of complex 3 revealed that the crys-
tals are comprised of monoclinic unit cells in the space group
P21/c with Z = 4. The formula of the complex in the crystalline
form is C54H45CuFeN2OP2. The central copper(I) ion adopts a
distorted tetrahedral coordination sphere comprising an imine
nitrogen (N2), an enolate oxygen (O1) of the hydrazone ligand
and two phosphorus atoms of the triphenylphosphine of the
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Table 2 Selected bond lengths (Å) and bond angles (°)
C54H45CuFeN2OP2 (3)
C37H31Cl3Fe2N4NiO2 (5)
Bond lengths
Bond angles
Bond lengths
Bond angles
Cu1–P1
Cu1–O1
Cu1–P2
Cu1–N2
C7–O1
C7–N1
N1–N2
2.2311(6)
2.111(1)
2.3057(6)
2.052(1)
1.287(2)
1.327(2)
1.413(2)
O1–Cu1–P2
N2–Cu1–O1
N2–Cu1–P1
P1–Cu1–P2
N2–Cu1–P2
O1–Cu1–P1
C7–N1–N2–C8
C9–C8–N2–N1
92.52(4)
79.08(6)
125.13(5)
116.37(2)
113.59(5)
119.00(4)
179.4(2)
−178.8(2)
Ni1–N1
Ni1–O1
Ni1–O2
Ni1–N3
C8–O2
C8–N4
N3–N4
C1–O1
C1–N2
N1–N2
1.862(3)
1.850(2)
1.832(2)
1.850(2)
1.305(4)
1.302(4)
1.413(4)
1.300(4)
1.308(4)
1.407(4)
N1–Ni1–N3
N1–Ni1–O1
N1–Ni1–O2
N3–Ni1–O2
N3–Ni1–O1
O1–Ni1–O2
O1–Ni–N3–N4
C2–C1–O1–Ni
O2–Ni–N1–N2
C9–C8–O2–Ni
176.2(1)
83.5(1)
95.1(1)
83.9(1)
97.5(1)
177.7(1)
176.8(2)
−177.7(2)
−177.4(2)
−180.0(2)
square planar geometry. However, in the case of complex 5, the
reduction of diamagnetic Ni(II) from its d8 configuration with
6
2
t2g eg distribution to paramagnetic Ni(I) with d9 electronic
6
3
configuration corresponding to t2g eg did not occur. However,
similar 1 : 2 metal–ligand stoichiometry with square planar geo-
metry could have resulted in the case of copper also, if the
reduction of Cu(II) had not taken place.
Infrared spectroscopy
The IR spectra of the metal hydrazone complexes were com-
pared with that of the respective free hydrazone ligands in the
region 4000–200 cm−1. The spectra of the ligands 1 and 2 dis-
played characteristic absorption bands at 3224/3434, 1648/1636,
1555/1513 and 1044/1035 cm−1 due to ν(N–H), ν(CvO), ν(CvN)
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
3–6, 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 range 1599–1505 cm−1
and 1357–1372 cm−1 that corresponds to ν(CvN–NvC) and ν(C–O)
stretching vibrations, respectively. The bands attributed to ν(CvN)
stretching were shifted to lower frequencies while a positive shift
of about 40–50 cm−1 was observed for ν(N–N) stretching
vibration in comparison with that of their respective free ligands,
thus implying that the nitrogen atom of the azomethine group is
coordinated to the metal in these complexes. All these facts
suggested that the hydrazone ligands behaves as a monobasic
bidentate (NO) chelating ligand in each of the complexes 3, 4, 5
and 6.
Fig. 2 Molecular structure of the complex 3a showing the atom-num-
bering scheme with thermal ellipsoids at 25% probability level.
the starting precursor. Furthermore, the bond angles of [O1–Ni–
N1] 83.5(1)°, [N1–Ni–O2] 95.1(1)°, [O2–Ni–N3] 83.9(1)° and
[O1–Ni–N3] 87.5(1)° are found to be deviated from the theoreti-
cal value of 90°. The bond length of the basal planar bonds of
[Ni1–N1] 1.862(3) Å, [Ni1–N3] 1.863(3) Å, [Ni1–O1] 1.850(2)
Å, and [Ni1–O2] 1.832(2) Å are almost equal in length. These
observations indicate that the coordination geometry of Ni(II) ion
is a distorted square planar. In addition, a chloroform unit as a
solvent molecule is also found in the structure of the complex.
The unit cell packing of complex 5 is shown in Fig. S2.†
1H NMR spectroscopy
1H NMR spectra of the free hydrazone ligands and their com-
plexes were assigned on the basis of observed chemical shift.
The spectra of the ligands 1 and 2 displayed a weak singlet
respectively at 9.04 and 9.12 ppm due to –NH proton. Another
sharp singlet observed at 8.31 and 8.54 ppm are attributed to
enolic –OH protons of the ligands 1 and 2 respectively. But, the
NMR spectra of complexes 3–6 did not register any signals cor-
responding to either –NH or –OH and indicated that the ligands
adopted the enol form, followed by deprotonation prior to
coordination with the metal ion. In addition, both the ligands
A possible explanation for the observed variation in the metal–
ligand stoichiometry and the coordination geometry between
copper and nickel complexes could be offered as herein. The
observed reduction of Cu2+ to Cu1+ ion (complex 3) is accounted
partly due to the presence of hydrazone ligand which generally
acts as a good reducing agent 49 and also partly due to the attain-
6
4
ment of a more stable t2g eg (d10) configuration corresponding
to copper(I) rather than less favourable t2g6 eg3 (d9) configuration
of copper(II). Consequently, Cu(I) preferred tetrahedral over
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Fig. 3 The molecular structure of complex 5, with displacement ellipsoids drawn at the 50% probability level and the solvent molecule is omitted
for clarity.
showed a sharp singlet for azomethine (–HCvN–) proton at
7.87 and 7.88 ppm, three singlets in the range of 3.81–4.73 ppm
for cyclopentadienyl moieties, respectively. However, in the case
of spectra of complexes 3–6, the signal corresponds to the azo-
methine protons being shifted downfield due to the participation
of azomethine nitrogen in coordination with metal ions. Simi-
larly, the cyclopentadienyl signals also underwent a downfield
shift upon coordination of the ligands with that of the respective
metal ions. The signals corresponding to the protons of aromatic
moieties of the ligands and their complexes were observed as
multiplets in the range of 7–8 ppm.
DNA binding studies
Absorption spectral characteristics of DNA binding. Elec-
tronic absorption spectroscopy is usually employed to determine
the binding ability of metal complexes with the DNA helix.
Compounds that bound to DNA through intercalation are charac-
terized by a change in absorbance (hypochromism) and batho-
chromic shift in wavelength, due to a strong stacking interaction
Fig. 4 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,
10.0, 12.5, 15.0, 17.5 and 20.0, 22.5 and 25 μM). Arrows show the
between the aromatic chromophore of the test compounds and
DNA base pairs. The extent of hypochromism is commonly con-
sistent with the strength of intercalative interaction.50 The
absorption spectra of ligands 1 and 2 and metal hydrazone com-
plexes 3–6 in the absence and presence of CT DNA are shown
in Fig. S3, S4, S5† and 4, respectively. The electronic spectrum
of ligand 1 showed four well-resolved bands at 267, 301, 361
and 466 nm. Similarly, the spectrum of ligand 2 exhibited its
absorption bands at 262, 301, 372 and 470 nm. The bands
appearing in the range of 262–301 nm are assigned to intra-
ligand charge transfer (ILCT) transitions. A broad shoulder
found at 361 and 372 nm corresponds to ligand-to-metal (iron)
charge transfer (LMCT) transitions and the band at 466 and
470 nm was assigned to charge transfer transition from iron to
changes in absorbance with respect to an increase in the DNA concen-
tration (Inset: Plot between [DNA] and [DNA]/[εa − εf]).
either a non-bonding or an antibonding orbital of the cyclopenta-
dienyl ring.51 Corresponding electronic spectrum of complexes
3–6 exhibited two or three bands in the range of 267 to 476 nm.
Complex 3 exhibited three different absorptions positioned at
303, 343 and 384 nm whereas complex 4 showed only a couple
of bands at 292 and 365 nm. Similarly, complex 5 showed
absorption bands at 298, 343 and 476 nm, and complex 6 exhib-
ited absorption bands at 267, 298 and 378 nm. In general, the
bands appearing in the 267–343 nm range in the case of all the
complexes are due to intraligand π→π* and n→π* charge
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transfer (ILCT) transitions.52 The band at 384, 365 and 378 nm
corresponding to complexes 3, 4 and 6 are due to LMCT tran-
sitions. The band appearing at 476 nm in the electronic spectrum
of complex 5 is assigned to a metal-to-ligand charge transfer
(MLCT) transition. With increasing CT DNA concentration, the
absorption bands corresponding to hydrazone ligands 1 and 2
showed hypochromism of about 6.69, 9.05, 10.71, 13.5, 9.9, 9.9,
2.28 and 2.87% without any wavelength shift. However, in the
case of complexes 3 and 4, we observed the same phenomenon
of hypochromism (19.17, 30.04, 29.85, 43.51 and 42.68%) with
a bathochromic shift of about 0, 3, 3, 7 and 10 nm corresponding
to the absorptions at 303, 343, 384, 292 and 365 nm respect-
ively. Further, complex 5 exhibits hypochromism (68.43 and
65.53%) with a bathochromic shift of about 5 and 7 nm at 298
and 343 nm respectively, whereas 1 nm hypsochromic shift with
a hypochromism (61.53%) was observed at 476 nm. Complex 6
showed a significant hypochromism of about 109, 82.15 and
78.68% at 267, 299 and 387 nm together with a bathochromic
shift of 1, 11 and 9 nm, respectively. In order to compare the
DNA-binding affinity of these compounds quantitatively, their
intrinsic binding constants were calculated by the changes moni-
tored in absorption at the higher energy band with increasing
concentrations of DNA using the equation,
described herein. Addition of compounds 1–4 with DNA
decreased the intensity of both the positive and negative ellipti-
city bands revealing that there occurred an electrostatic mode of
binding between them without causing any conformational
changes to it even after interaction with a higher concentration of
the test solution. In addition, an increase in the molar ellipticity
values of the above mentioned bands of DNA was observed by
the addition of compounds 5 and 6 that reflected a single
binding mode. The enhancement of these bands is a character-
istic of groove binding that stabilizes the right handed B form of
DNA.58
DNA cleavage
The ability of metal hydrazone complexes to perform DNA clea-
vage is generally monitored by agarose gel electrophoresis and
in the present work pUC19 plasmid DNA was chosen to investi-
gate its cleavage. DNA cleavage was controlled by relaxation of
the supercoiled circular form of pUC19 into the open circular
form and linear form. When circular plasmid DNA is run on
horizontal gel using electrophoresis, the supercoiled form will
migrate first (Form I). If the strands of the selected DNA is
cleaved by interaction with the metal complexes, the supercoils
will relax to produce an open circular form (Form II) that moves
slower than form I, whereas with cleavage of both strands to a
linear form (Form III) the resulting system will migrate in
between the above two forms. The tests were performed under
aerobic conditions with H2O2 as a co-oxidant at a complex con-
centration of 35 μM. Neither H2O2 nor the metal complexes 3–6
yielded significant strand scission (not shown) when applied sep-
arately. These experimental facts demonstrated that a combi-
nation of both the metal hydrazone complexes and H2O2 are
required to show effective cleavage of plasmid DNA. Fig. 6
reveals that cleavage of pUC19 DNA induced by complexes 3–6
in the presence of H2O2 results in the conversion of Form I (lane
1) into Form II (lane 2). The extent of Form I diminished gradu-
ally upon partial conversion to Form II with simultaneous
increase in the intensity of the latter form. Based upon their
ability to convert the supercoiled form (Form I SC) to the open
circular form (Form II OC), it is obvious that complex 6 that pos-
sessed a hetero nitrogen containing pyridine moiety in its hydra-
zide part with square planar geometry which has more ability to
cleave the supercoiled plasmid DNA when compared to that of
the other complexes. These observations suggested that the clea-
vage of the supercoiled form to the open circular form increased
in the order 3 < 4 < 5 < 6. It is believed that the superior clea-
vage ability of complex 6 is due to the reaction of metal ions
with H2O2, which produces diffusible hydroxyl radicals or mol-
ecular oxygen at ease, which in turn damage DNA through
Fenton-type chemistry.59
½DNAꢂ=½εa ꢀ εf ꢂ ¼ ½DNAꢂ=½εb ꢀ εf ꢂ þ 1=Kb ½εb ꢀ εf ꢂ
Plotting [DNA] versus [DNA]/[εa − εf] gave a slope 1/[εa −
εf] and a y intercept equal to 1/Kb[εb − εf], respectively. The
intrinsic binding constants Kb were calculated using the above
plot and were found to be 3.874 0.050 × 103 M−1, 4.630
0.047 × 103 M−1, 2.449 0.042 × 104 M−1, 5.394 0.059 ×
104 M−1, 8.371 0.038 × 104 M−1 and 1.227 0.041 × 105
M−1 corresponding to the compounds 1–6, respectively. The
magnitude of the binding constant value clearly showed that
complex 6 bound more strongly with CT DNA than the rest of
the compounds. Once the test compound intercalates to the base
pairs of DNA, the π* orbital of the intercalators may couple with
the π orbital of the base pairs, thus decreasing the π→π* tran-
sition probabilities and hence a hypochromism is observed in the
above cases.53 These results are similar to those reported earlier
for various classical intercalators.54,55
Circular dichroism. The results of electronic spectral studies
of compounds 1–6 tentatively revealed an intercalative mode of
binding of them with DNA. However, circular dichroism is a
strong tool to identify the exact nature of binding between DNA
and small molecules in order to probe the asymmetry of the
system. The chirality of the DNA double helix originates from
DNA geometry, resulting from the coupling of bases, phosphate
backbone and chiral sugar units will be identified from CD spec-
tral measurement.56 Although CD spectral changes cannot be
interpreted on a quantitative basis, comparison of the experimen-
tal results with empirical spectra of representative DNA provides
useful structural information.57 Fig. S6, S7† and 5 show the
comparative CD spectra of pure CT-DNA and that of DNA
treated with different concentrations of test compounds 1–6. The
spectrum of pure DNA exhibited a positive band at 276 nm and
a negative band at 245 nm. Upon interaction of the test com-
pounds 1–6 with DNA, these bands underwent perturbations as
Protein binding studies
Fluorescence quenching of BSA by copper and nickel hydra-
zone complexes. 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 decreases the fluorescence
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Fig. 5 Circular dichroic spectra of CT DNA (200 μM) in the absence and presence of test complexes 5 and 6 (20 μM).
Fig. 6 Gel electrophoresis showing the chemical nuclease activity of
the pUC19 DNA incubated at 37 °C for a period of 1 h at a concen-
tration of 35 μM of complex 3–6 in the presence of H2O2 (60 μM) as a
co-oxidant: lane 1: C – pUC19 DNA + H2O2; lane 2: pUC19 DNA +
H2O2 + complex 3; lane 3: pUC19 DNA + H2O2 + complex 4; lane 4:
pUC19 DNA + H2O2 + complex 5; lane 5: pUC19 DNA + H2O2
complex 6.
+
intensity of a fluorophore due to a variety of molecular inter-
actions including excited-state reactions, molecular rearrange-
ments, energy transfer, ground-state complex formation and
collision quenching. Qualitative analysis of chemical compounds
bound to BSA can be undertaken by examining the respective
fluorescence spectra. Addition of the hydrazone ligands 1 and 2
to the solution of BSA does not result in the quenching of its flu-
orescence emission and hence, further studies of protein binding
experiments were not carried out with respect to them. However,
addition of copper and nickel hydrazone complexes 3–6 to BSA
resulted in the quenching of fluorescence emission intensity
along with a hypsochromic shift of 4 to 6 nm due to the for-
mation of a complex between the metal hydrazone chelates and
BSA. Fig. S8, S9† and 7 show the effect of increasing the con-
centration of metal hydrazone complexes on the fluorescence
emission intensity of BSA.
Fig. 7 Emission spectra of BSA (1 × 10−6 M; λexci = 280 nm; λemi
345 nm) as a function of concentration of the complex 6 (0, 2, 4, 6, 8,
10, 12 and 14 × 10−7 M). Arrow indicates the effect of metal complex 6
on the fluorescence emission of BSA (Inset: Plot of [Q] vs. I0/I).
=
from the plot of [Q] vs. I0/I (as insets of Fig. S8, S9† and 7) was
found to be 6.985 0.140 × 104 M−1, 7.488 0.213 × 104 M−1
,
1.220 0.187 × 105 M−1 and 1.528 0.122 × 105 M−1 corre-
sponding to the respective metal hydrazone chelates 3–6.
Binding analysis. The binding constant of the metal hydra-
zone complexes on binding with BSA can be calculated using
the Scatchard equation,60
ðΔI0=IÞ=½Qꢂ ¼ nK ꢀ KðΔI0=IÞ
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 respectively, KSV is the Stern–Volmer quenching con-
stant and [Q] is the quencher concentration. KSV value obtained
where, n is the number of binding sites per albumin and K is the
association binding constant that is calculated from the slope in
plots (ΔI0/I)/[Q] versus (ΔI0/I) and n is given by the ratio of y
intercept to the slope. The K values of metal hydrazone
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Fig. 9 The absorption spectra of BSA (1 × 10−5 M) and BSA–
complex 6 (BSA = 1× 10−5 M and complex 6 = 1 × 10−6 M).
Fig. 8 Plot of (ΔI0/I)/[Q] versus (ΔI0/I).
Table 3 Comparison of interaction study results between compounds
1–6 on DNA and protein
copper complexes 3 and 4 and the overall order of binding
affinity of the complexes is found as 6 > 5 > 4 > 3. These results
did indicate that in addition to the planarity, the number of ferro-
cene units and the presence of hetero nitrogen atom in the struc-
ture of the complexes have also influenced their affinity towards
DNA/BSA binding.
Protein binding
DNA binding
Compounds Kb (M−1
)
Ksv (M−1
)
K (M−1
)
n
1
2
3
4
5
6
3.874 0.050
× 103
—
—
—
—
—
4.630 0.047
—
× 103
UV-visible absorption measurements. The quenching process
can occur by different mechanisms, 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 for-
mation in the ground state. A simple method to explore the type
of quenching is UV–visible absorption spectroscopy. The
absorption band obtained for BSA at 278 nm in the absence of
metal hydrazone complexes showed an increase in the intensity
of absorption after the addition of complexes 3, 4, 5 and 6
revealing that there exists a static interaction between BSA and
the respective complexes due to the formation of ground state
complexes of the type BSA–complex as reported earlier.61
Representative absorption spectra of pure BSA and BSA–
complex 6 are shown in Fig. 9.
2.449 0.042 6.985 0.140 1.260 0.010 0.6741
× 104 × 104 × 105
5.394 0.059 7.488 0.213 2.662 0.008 0.6930
× 104 × 104 × 105
8.371 0.038 1.220 0.187 3.725 0.021 0.7341
× 104 × 105 × 105
1.227 0.041 1.528 0.122 6.037 0.027 0.7613
× 105 × 105 × 105
complexes 3–6 obtained from the Scatchard plots (Fig. 8)
according to the above mentioned equation are found to be
1.260 0.010 × 105 M−1, 2.662 0.008 × 105 M−1, 3.725
0.021 × 105 M−1 and 6.037 0.027 × 105 M−1, respectively.
From the DNA binding and BSA binding results (Table 3), it
is clear that among the ligands HL1 and HL2, the latter with a
heterocyclic moiety containing a pyridine ring in its molecular
structure showed higher binding affinity with DNA when com-
pared to the former with the phenyl ring in its acid hydrazide
part. In general, the binding affinities of metal chelates towards
DNA are better than that of the ligands due to the chelate effect.
The overall order of binding affinity (DNA/BSA) increased in
the order 1< 2 < 3 < 4 < 5 < 6. This observed trend suggested
that the complexes 5 and 6 with more planar square planar geo-
metry exhibited strong binding affinity than the corresponding
copper complexes 3 and 4 possessing a less planar tetrahedral
arrangement. Also, it was found that complex 6 containing the
hetero nitrogen atom (pyridine ring) in its hydrazide moiety has
a strong interaction towards DNA/BSA than that of the other
(complex 5) that didn’t have a similar nitrogen atom in its hydra-
zide part. Similar observations have also been noted between the
Characteristics of synchronous fluorescence spectra. To inves-
tigate the structural changes which occurred to BSA upon the
addition of copper and nickel hydrazone complexes, we
measured synchronous fluorescence spectra of BSA respectively
before and after the addition of metal hydrazone complexes to
get valuable information on the molecular microenvironment,
particularly in the vicinity of the fluorophore functional
groups.62 It is a well known fact that the fluorescence of BSA is
normally due to the presence of tyrosine, tryptophan and phenyl-
alanine residues and hence spectroscopic methods are usually
applied to study the conformation changes of serum protein.
According to Miller,63 in synchronous fluorescence spec-
troscopy, the difference between excitation and emission wave-
length (Δλ = λemi − λexc) reflects the spectra of a different nature
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Fig. 10 Synchronous spectra of BSA (1 × 10−6 M) as a function of concentration of the complex 6 (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 × 10−7
M) with wavelength difference of Δλ = 15 nm (A) and Δλ = 60 nm (B). Arrow indicates changes in emission intensity w.r.t various concentrations of
complex 6.
of chromophores. If the Δλ value is small (15 nm) the synchro-
nous fluorescence of BSA is characteristic of a tyrosine residue
whereas a larger Δλ value of 60 nm is characteristic of trypto-
phan.64 The synchronous fluorescence spectra of BSA upon the
addition of copper and nickel hydrazone complexes recorded
with using two different Δλ values i.e., Δλ = 15 and 60 nm are
shown in Fig. S10, S11, S12† and 10. From these spectra, it
became clear that an increase in the concentration of the metal
hydrazone complexes added to BSA also increased (very
slightly) the intensity of the synchronous fluorescence spectral
band corresponding to the tyrosine residue in the case of com-
plexes 3 and 4 whereas complexes 5 and 6 showed an increase
in the intensity of the same band along with a bathochromic shift
of 1 nm. This shows that complexes 5 and 6 affected tyrosine
residues whereas complexes 3 and 4 did not affect the environ-
ment in the above said region. In addition, a significant decrease
of fluorescence intensity of tryptophan residues with a slight
blue–shift of emission wavelength was found after the addition
of complexes 3–6. These results indicated that though the com-
plexes 5 and 6 affected both the micro environments i.e., tyro-
sine and tryptophan residues, the effect was more towards
tryptophan residues than tyrosine residue whereas complexes 3
and 4 affected only the tryptophan residue but not the other. The
features of synchronous measurements confirmed that confor-
mational changes occurred in BSA upon interaction with the
complexes. Similar behaviour regarding the interaction between
the BSA and metal hydrazone complexes was reported earlier.65
4, 5 and 6 against DPPH, OH and NO radicals with respect to
different concentrations of the test compounds varying from 0 to
50 μM (Fig. 11) and the results were shown in Table 4. From
Table 4, it can be concluded that a much less or no scavenging
activity was exhibited by the free ligands when compared to that
of their corresponding metal hydrazone complexes which is due
to the chelation of them with the metal ions. Among the structu-
rally different copper and nickel complexes, the scavenging
potential of nickel complexes are better than the respective
copper hydrazone chelates that can be related to the ligand occu-
pancy with that of the metal ions. However, among the copper
and nickel hydrazone complexes, the one containing a pyridine
ring (complex 4 and 6) in the hydrazide moiety showed higher
antioxidant activity than the complexes 3 and 5 which did not
have such a molecular composition. The overall scavenging
activity of the tested compounds was found to increase in the
order of 1 < 2 < 3 < 4 < 5 < 6. Further, the results obtained
against the three different radicals confirmed that the complexes
are more effective to arrest the formation of DPPH than the
hydroxyl and nitric oxide radicals and are compared with that of
the other copper and nickel complexes.69 The lower IC50 values
observed in antioxidant assays did demonstrate that these com-
plexes have a strong potential to be applied as scavengers to
eliminate the radicals. Further, it is significant to mention that
the metal complexes synthesised herein possess superior antioxi-
dant activity against the above said radicals than that of the stan-
dard antioxidant butylated hydroxyl anisole (BHA).
Antioxidant activity
Cytotoxicity
It is a well documented fact that hydrazones and their corre-
sponding transition metal complexes displayed significant anti-
oxidant activity66–68 and therefore we undertook a systematic
investigation on the antioxidant potential of free hydrazone
ligands 1 and 2 and corresponding transition metal complexes 3,
Cytotoxicity is a common limitation in terms of the introduction
of new compounds into the pharmaceutical industry. In general,
heteronuclear organometallic complexes exhibit potential cyto-
toxicity with dose–response effects. In order to understand the
in vitro cytotoxicity of the metal hydrazone complexes,
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Fig. 11 Trends in the inhibition of DPPH, OH and NO radicals by ligands 1 and 2 and corresponding metal complexes 3, 4, 5 and 6 at various
concentrations.
Table 4 Comparison of IC50 values of test compounds towards
selected free radicals
Table 5 Comparison of IC50 values of complexes 5 and 6 on treatment
with NIH 3T3, HeLa and A431 cells
IC50 values (μM)
IC50 values (μM)
DPPH
radical
Hydroxyl
radical
Nitric oxide
radical
Complex
NIH 3T3
HeLa
A431
Test compounds
5
6
312.44
188.53
92.86
49.48
80.72
35.13
1
2
3
4
5
6
42.17
37.68
35.21
34.72
27.39
22.05
20.16
—
—
—
—
—
—
37.43
31.89
7.65
49.53
45.48
30.56
23.81
21.37
respectively. From the above IC50 values, it became clear that
complex 6 has more potential than complex 5 due to the pres-
ence of a pyridine ring in the hydrazide moiety of the coordi-
nated ligand in the former complex. Among the two different
cell lines used in this study, the proliferation of A431 was
arrested to a greater extent than HeLa cells by the complexes.
Though the above set of complexes are active against the cell
lines under in vitro cytotoxicity experiments, none of them could
reach the effectiveness shown by the standard drug cisplatin
(IC50 = 16.1 and 14.4 μM, respectively).70 Upon treatment of the
complexes 3–6 against normal NIH 3T3 cells, 3 and 4 did not
show any damage to healthy cells but very less damage was
induced by complexes 5 and 6 (IC50 = 312.44 and 188.53 μM,
respectively) to healthy cells. The results of the cell viability
tests imply that the complexes 5 and 6 have the ability to arrest
the proliferation of tumor cells without causing perceptible
damage to the normal cells.
Reported value
(Cu)
Reported value
(Ni)
35.66
38.46
10.11
experiments were carried out using a panel of cell lines namely
HeLa, A314 (tumor cells) and NIH 3T3 (normal cells) by colori-
metric (MTT) assay in which mitochondrial dehydrogenase
activity was measured as an indication of cell viability. Com-
plexes were dissolved in DMSO and the blank samples contain-
ing the same volume of DMSO were taken as controls to
identify the activity of solvent against the selected cell lines. Cis-
platin was used as a standard to assess the cytotoxicity of com-
plexes 3, 4, 5 and 6 (not shown).70,71 The capabilities of the
above said complexes to arrest the proliferation of tumor cells
without causing any damage to normal cells were evaluated after
48 h of incubation. The results were analyzed by means of cell
viability curves and expressed with IC50 values in the studied
concentration range from 12.5–400 μM. Upon increasing the
concentration of complexes, the results of MTT assays revealed
that complexes 3 and 4 completely failed to arrest the growth of
both HeLa and A431 tumor cell lines (up to a concentration of
400 μM) but complexes 5 and 6 (Table 5) showed notable
activity against both the cell lines mentioned above with respect-
ive IC50 values of 92.86, 49.48, 80.72 and 35.13 μM.72 A valid
reason for the inactiveness of copper complexes (3 and 4) but a
significant activity of nickel complexes (5 and 6) against the
selected tumour cell lines can be offered in relation to the vari-
able ligand occupancy with that of the respective metal ions i.e.,
copper and nickel such as 1 : 1 have a less planar tetrahedral geo-
metry and 2 : 1 have a more planar square planar geometry
Conclusion
In the present study, a set each of univalent copper and bivalent
nickel complexes containing ferrocene derived hydrazones have
been synthesized from the reactions of respective Cu(II) and Ni
(II) precursors and hydrazones in 1 : 1 stoichiometric ratio and
characterised using different physico-chemical techniques
including single crystal XRD. Interestingly, in the reactions
involving copper(II) starting precursor, reduced Cu(I) hydrazone
complexes of 1 : 1 metal–ligand occupancy with a tetrahedral
structure were formed, whereas, square planar complexes with
1 : 2 ligand occupancy was realized in the case of nickel(II) start-
ing precursor. The outcome of various biological experiments
did indicate that complexes 5 and 6 containing nickel(II) ion with
4434 | Dalton Trans., 2012, 41, 4423–4436
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Acknowledgements
We express our gratitude to Prof. A. Ramu and
Prof. K. Pitchumani, School of Chemistry, Madurai Kamaraj
University, India, for their help to record CD spectra. Also, we
thank University Grants Commission, New Delhi, India, for the
Fellowship in Science for Meritorious Students (RFSMS) to
Mr. P. Krishnamoorthy and Department of Science and Technol-
ogy, Government of India, New Delhi, for the award of Senior
Research Fellowship (SRF) to Ms.P. Sathyadevi. AHC is grateful
to the Robert A. Welch Foundation (Grant F–0003) for financial
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