Studies on the effect of metal ions of hydrazone complexes on interaction with nucleic acids, bovine serum albumin and antioxidant properties

Article abstract of DOI:10.1016/j.ica.2011.11.033

Three new transition metal complexes of the type ML₂ (where M = Ni(II), Co(II) or Cu(II); HL = N′-[phenyl(pyridin-2-yl)methylidene]furan- 2-carbohydrazide]) have been prepared by treating [NiCl₂(PPh ₃)₂], [CoCl₂(PPh₃)₂] or [CuCl₂(PPh₃)₂] with N′-[phenyl(pyridin-2- yl)methylidene]furan-2-carbohydrazide derived from furoic acid hydrazide and 2-benzoyl pyridine wherein the hydrazone ligand (L) coordinated to the respective metal ions in 1:2 stoichiometry to mononuclear octahedral complex. The crystal structure of the complexes [NiL₂] (1), [CoL₂] (2) and [CuL₂] (3) solved using single crystals revealed a distorted octahedral geometry around the metal ion involving the coordination of an azomethine nitrogen, a pyridine nitrogen and an enolic oxygen derived from deprotonation of the ligand. From the bioinorganic application point of view, a detailed work on the binding of the complexes 1, 2 and 3 with CT DNA as well as BSA was undertaken along with DNA cleavage. In vitro assay on the antioxidant activity of the above complexes and hydrazone ligand revealed that they possess significant antioxidant activity. However, among the newly synthesized hydrazone complexes, complex 3 having coordinated Cu²⁺ ion in its molecular structure exhibited superior activity in all the biological studies in comparison with the other two complexes possessing nickel and cobalt ions with same ligand (L).

Full text of DOI:10.1016/j.ica.2011.11.033

Inorganica Chimica Acta 384 (2012) 83–96
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Studies on the effect of metal ions of hydrazone complexes on interaction
with nucleic acids, bovine serum albumin and antioxidant properties
a
a
a
b
Palanisamy Sathyadevi , Paramasivam Krishnamoorthy , Eswaran Jayanthi , Rachel R. Butorac ,
b
a,
⇑
Alan H. Cowley , Nallasamy Dharmaraj
a
Department of Chemistry, Bharathiar University, Coimbatore 641046, India
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
b
a r t i c l e i n f o
a b s t r a c t
0
Article history:
Received 15 August 2011
Received in revised form 11 November 2011
Accepted 12 November 2011
Available online 19 November 2011
Three new transition metal complexes of the type ML2 (where M = Ni(II), Co(II) or Cu(II); HL = N -
[
[
phenyl(pyridin-2-yl)methylidene]furan-2-carbohydrazide]) have been prepared by treating
0
2
NiCl (PPh
)
3 2
], [CoCl
2
(PPh
3
)
2
] or [CuCl
2 3
(PPh )
2
] with N -[phenyl(pyridin-2-yl)methylidene]furan-2-car-
bohydrazide derived from furoic acid hydrazide and 2-benzoyl pyridine wherein the hydrazone ligand
L) coordinated to the respective metal ions in 1:2 stoichiometry to mononuclear octahedral complex.
The crystal structure of the complexes [NiL ] (1), [CoL ] (2) and [CuL ] (3) solved using single crystals
revealed a distorted octahedral geometry around the metal ion involving the coordination of an azome-
thine nitrogen, a pyridine nitrogen and an enolic oxygen derived from deprotonation of the ligand. From
the bioinorganic application point of view, a detailed work on the binding of the complexes 1, 2 and 3
with CT DNA as well as BSA was undertaken along with DNA cleavage. In vitro assay on the antioxidant
activity of the above complexes and hydrazone ligand revealed that they possess significant antioxidant
activity. However, among the newly synthesized hydrazone complexes, complex 3 having coordinated
(
2
2
2
Keywords:
DNA binding
Protein binding
Hydrazones
Transition metal complexes
Crystal structure
Antioxidant properties
2
+
Cu ion in its molecular structure exhibited superior activity in all the biological studies in comparison
with the other two complexes possessing nickel and cobalt ions with same ligand (L).
Ó 2011 Elsevier B.V. All rights reserved.
1
. Introduction
Recent years have witnessed an unprecedented progress in bio-
and the elucidation of the mechanisms of interaction of transition
metal complexes with DNA and their subsequent applications in
molecular biology continue to attract significant attention. In par-
ticular, considerable interest has been generated in DNA binding
and DNA cleavage by redox and photoactive metal complexes in
order to explore the sequence specificities of DNA binding using
a variety of intercalating ligands [10–12]. Moreover, certain metal
complexes have been shown to be capable of cleaving DNA strands.
In the case of cancer genes, the cleavage of the DNA double strands
can destroy their replication ability. Free radicals can adversely
affect lipids, proteins and DNA and have been implicated in the
aging process and in a number of human diseases. Antioxidants
are capable of neutralizing these reactive species in terms of
prevention, interception and damage repair.
Serum albumin is the most abundant protein in animals includ-
ing human circulatory system that is in-charge for the transport,
distribution and deposition of a variety of endogenous and exoge-
nous substances in body [13]. Knowledge of interaction mecha-
nisms between drugs and plasma proteins is very important to
understand the pharmacodynamics and pharmacokinetics of a
drug. Drug binding influences their distribution, excretion, metab-
olism and interaction with the target tissues [14]. Bovine serum
albumin (BSA) has been proven to have a high homology and a
similarity to human serum albumin (HSA) in sequence and
logical applications of inorganic pharmaceuticals because of their
key role in clinical therapy. Important examples include cis-platin
and corresponding second generation alternatives such as oxalipl-
atin and carboplatin those serve as chemotherapeutic agents for
solid malignancies and as diagnostic contrast agents such as MRI
[
1–3]. Transition metals are particularly suitable for this purpose
because they can adopt a wide variety of coordination numbers,
geometries and oxidation states in comparison with carbon and
other main group elements [4]. We were particularly attracted to
the transition metal complexes of aryl hydrazones because such
compounds have been reported to function as antibacterial, antivi-
ral and antifungal agents [5–8]. A further reason for using metal-
containing compounds as structural scaffolds relates to the kinetic
stabilities of their coordination spheres in the biological environ-
ment [9].
Investigation of the interactions of DNA with small molecules
can serve as a foundation for the design of new types of
pharmaceutical molecules. The development of interaction models
⇑
Corresponding author. Tel.: +91 422 2428316; fax: +91 422 2422387.
E-mail address: dharmaraj@buc.edu.in (N. Dharmaraj).
0
020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.11.033

8
4
P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
conformation [15]. Therefore, it is always selected as a particularly
relevant protein and has become the best-studied model of general
drug–protein interactions.
(0.653 g; 1 mM) in 40 mL of methanol (Scheme 1). After 30 min, a
few drops of methanolic KOH were added to the reaction mixture,
following which it was refluxed for an additional 5 h. After cooling
the reaction mixture to room temperature, the resulting precipi-
tate was filtered, washed with methanol and dried in vacuo for
24 h. Following a purity check by TLC, crystals of 1 suitable for sin-
gle-crystal X-ray diffraction studies were obtained by recyrstalliza-
It has been reported that hydrazone complexes of transition
metals exhibit DNA interactions, DNA cleavage, as well as antitu-
mor and antimicrobial properties [16–19], but it appeared that
very less attention was only being paid so far to investigate sys-
tematically the protein binding ability of such similar systems.
However, to the best of our knowledge, there was no literature
available on the synthesis, structural characterization and interac-
tion studies of Ni(II), Co(II) and Cu(II) complexes containing
tion from a MeOH/CHCl
brown, mp: 270–272 °C. Anal. Calc. for NiC34
Wt. = 711.35): C, 57.40; H, 4.53; N, 11.81. Found: C, 57.24; H,
3
solvent mixture. Yield: 47%. Color: dark
32 6 8
H N O
(Mol.
ꢁ1
4.81; N, 11.52%. Selected IR bands (mmax/cm ): 1587 & 1501
0
N -[phenyl(pyridin-2-yl)methylidene]furan-2-carbohydrazide as
(C@N–N@C); 1309 (C–O); 1084 (N–N).
ligand. The foregoing facts stimulated our interest on the synthe-
ses, structure, DNA binding, cleavage, protein binding and antioxi-
dant properties of bivalent Ni, Co and Cu complexes containing the
above hydrazide ligand. The structures of the new complexes were
established on the basis of single-crystal X-ray diffraction studies
and spectroscopic data. In addition, detailed investigations on the
effect of metal ions on the biological potential of the hydrazone
complexes were also undertaken.
2
.2.2. Synthesis of [Co(L)
The complex [Co(L)
0.653 g; 1 mM) and the ligand (H-FBP) (0.291 g; 1 mM) using a
similar procedure to that described for 1 (Scheme 1). Slow evapo-
ration of a MeOH/CHCl solution of 2 afforded a crop of brown
crystals suitable for X-ray diffraction study. Yield: 48% mp: 280–
82 °C. Anal. Calc. for CoC34 (Mol. wt. = 657.540): C,
62.10; H, 3.98; N, 12.78. Found: C, 61.94; H, 3.91; N, 12.96%.
2
] (2)
2
] (2) was prepared from [CoCl
2 3 2
(PPh ) ]
(
3
2
26 6 5
H N O
2
. Experimental
ꢁ
1
Selected IR bands (
(C–O); 1078 (N–N).
mmax/cm ): 1586 & 1497 (C@N–N@C); 1311
2.1. Materials and physical measurements
The following reagent grade chemicals were procured commer-
cially and used without subsequent purification: NiCl
2
ꢀ6H
2
O,
2
.2.3. Syntheses of [Cu(L)
Complex [Cu(FBP)
0.658 g; 1 mM) and the (H-FBP) ligand (0.291 g; 1 mM) using a
2
] (3) and [Cu(Cl)(PPh
3 3
) ] (3a)
CoCl O, CuCl O and triphenylphosphine (Sigma–Aldrich);
2
ꢀ6H
2
2
ꢀ2H
2
3
2
]
was prepared from [CuCl
2 3 2
(PPh ) ]
0
furoic acid hydrazide and benzoyl pyridine (Alfa Aesar), 2,2 -
(
azino-3-ethylbenzthiazoline-6-sulfonic acid diammonium salt,
similar procedure to that described for 1 (Scheme 1). However,
analysis of the product by TLC revealed the presence of two differ-
ent complexes, namely 3 and 3a. Complex 3 was eluted from the
column using a 75:25 petroleum ether and ethyl acetate mixture
as the eluent. Slow evaporation of a MeOH/CHCl
afforded a crop of violet crystals suitable for X-ray diffraction
study. Yield: 38%, mp: 276–278 °C. Anal. Calc. for CuC35
0
1
-1 -diphenyl-2-picrylhydrazyl, 2-thiobarbituric acid, 2,4,6-tripyr-
idyl-S-triazine (Sigma–Aldrich). Calf-thymus (CT DNA) and bou-
vine serum albumin (BSA) were purchased from Himedia. The
plasmid supercoiled (SC) pUC19 DNA was purchased from Banga-
lore GeNei, Bangalore, India. All other chemicals and reagents used
for biological studies were obtained commercially.
3
solution of 3
28 6 5
H N O
Microanalyses (% C, H and N) were performed on a Vario EL III
CHNS analyzer and all IR spectra were recorded as KBr pellets on
(
Mol. wt. = 676.180): C, 62.17; H, 4.17; N, 12.42. Found: C, 61.95;
ꢁ1
H, 4.04; N, 12.53%. Selected IR bands (mmax/cm ): 1584 &
1
a
4
Nicolet Avatar instrument in the frequency range 400–
000 cm . The H NMR spectrum of the ligand was recorded on
505(C@N–N@C); 1309 (C–O); 1082 (N–N).
Complex 3a was obtained by using an 85:15 petroleum ether/
ꢁ1
1
a Bruker AMX 500 spectrometer operating at 500 MHz and using
tetramethylsilane as the internal standard. The chemical shift ra-
tios are reported in parts per million (ppm) relative to SiMe
ethyl acetate mixture as the eluent. A crop of crystals suitable for
single crystal X-ray diffraction study was obtained in a similar
fashion to that described above for 3. The structure of 3a was
4
(d
0
.00). The electronic absorption and emission spectra were
3 3
shown to identical to that of [Cu(Cl)(PPh ) ] [23].
recorded in DMSO:buffer (1:99) solution on Jasco V-630 spectro-
photometer and Jasco FP 6600 spectrofluorometer, respectively.
The cyclic voltammetric study was carried out with a CH Instru-
ments electrochemical analyzer in dichloromethane using tetrabu-
tylammonium perchlorate (TBAP) as the supporting electrolyte. A
three-electrode configuration cell comprising a glassy carbon
working electrode, a platinum wire auxiliary electrode and an
Ag/AgCl reference electrode was employed in this study. All solu-
tions were purged with nitrogen gas prior to making measure-
ments at room temperature.
2
.3. X-ray crystallography
For each compound, a crystal of suitable quality was removed
from a vial, covered with mineral oil and mounted on a nylon
thread loop. The X-ray diffraction data for compounds 1, 2 and
3
a were collected on a Rigaku AFC-12 Saturn 724+CCD diffractom-
eter equipped with a graphite-monochromated Mo K radiation
a
source (k = 0.71073 Å) and a Rigaku XStream low-temperature
device cooled to 100 K. The X-ray diffraction data for complex 3
were collected on a Rigaku SCX-Mini diffractometer equipped with
2.2. Syntheses of metal–hydrazone complexes
a Mercury CCD, a graphite-monochromated Mo K
a radiation
The requisite precursor metal complexes [NiCl
2
(PPh
3
)
2
],
source (k = 0.71073 Å) and a Rigaku Tech50 low-temperature
device cooled to 223 K. Corrections were applied for Lorentz and
polarization effects for each compound. Each structure was solved
by direct methods and refined by full-matrix least-squares cycles
0
[
2
CoCl (PPh
3
)
2
] and [CuCl
2 3
(PPh )
2
] and the ligand N -[phenyl(pyri-
din-2-yl)methylidene]furan-2-carbohydrazide (H-FBP) (HL) were
prepared according to the literature methods [20–22].
2
on F using the Siemens SHELXTL PLUS 5.0 (PC) software package
2
.2.1. Synthesis of [Ni(L)
Complex [Ni(FBP) ] (1) was prepared by refluxing equimolar
quantities of the ligand (H-FBP) (0.291 g; 1 mM) and [NiCl (PPh
2
] (1)
[24,25] and PLATON [26]. All non-hydrogen atoms were refined
anisotropically and hydrogen atoms were placed in fixed, calcu-
lated positions using a riding model.
2
2
3 2
) ]

P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
85
Scheme 1. Synthesis of complexes 1, 2, 3 and 3a.
2
2
.4. DNA binding experiments
classical Stern–Volmer equation [28]: I
0
/I = K
q
[Q] + 1; I
0
is the
emission intensity in the absence of quencher, I is the emission
intensity in the presence of quencher, K is the quenching constant,
[Q] is the quencher concentration (10 M). K is the slope, obtained
from the plot of I /I versus [Q]. The apparent binding constant
.4.1. Electronic absorption titration
Electronic absorption titrations were performed with a fixed
q
l
q
concentration of metal complexes (25
lM) but by varying nucleo-
0
tide concentration from 0 to 25 M. The absorbance (A) band of the
l
(Kapp) has been calculated from the equation, KEB [EB] = Kapp[com-
7
ꢁ1
complexes that get shifted significantly due to the addition of CT
DNA was chosen to monitor as an indication of the binding be-
tween them. From the absorption titration data, the intrinsic bind-
plex] with [EB] = 10
lM and KEB = 1 ꢃ 10 M
.
2.5. DNA cleavage experiments
ing constant (K
b
) of the metal complexes with CT DNA was
determined using the equation [27],
The extent of DNA cleavage induced by the test compounds was
monitored by agarose gel electrophoresis. A solution containing
L of pUC19 DNA (1 g), HCl (50 mM, pH 7.2), NaCl (50 mM),
the metal complex (35 M), and H (60 M) was incubated at
37 °C for 1 h. Subsequently, 4
ing 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 lg/mL of ethidium bromide. Electrophoresis was
performed at 5 V/cm for 2 h in a TBE buffer and the bands were
visualized under UV light and photographed. The cleavage efficien-
cies were measured by determination of the ability of each com-
plex to convert the super coiled DNA (SC) to the open circular
form (OC). After electrophoresis, the proportion of both the cleaved
and uncleaved DNA in each fraction was quantitatively estimated
on the basis of the band intensities using the BIORAD Gel Docu-
mentation 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 [29].
½
DNAꢂ=½ ꢂ ¼ ½DNAꢂ=½ ꢂ þ 1=K
e
a
ꢁ
e
f
e
b
ꢁ
e
f
b
½
e
b
ꢁ
e
f
ꢂ
2
5
l
l
l
where, [DNA] is the concentration of DNA in base pairs,
e
a
is the
2
O
2
l
extinction coefficient of the complex at a given DNA concentration,
is the extinction coefficient of the complex in free solution and
is the extinction coefficient of the complex when fully bound to
DNA. A plot of [DNA]/[ ] versus [DNA] gave a slope and an
intercept equal to 1/[ ] and (1/K )[ ], respectively. The
is the ratio of the slope to the
l
L of 6ꢃ DNA loading buffer contain-
e
f
e
b
e
b
ꢁ
e
f
e
a
ꢁ
e
f
b
e
b
ꢁ
e
f
intrinsic binding constant K
b
intercept.
2.4.2. Competitive binding fluorescence measurements
The apparent binding constant (Kapp) of the complexes was
determined by fluorescence spectral technique using ethidiumbro-
mide (EB)-bound CT DNA solution in Tris–HCl buffer (pH, 7.2). The
changes in fluorescence intensities at 605 nm (545 nm excitation)
of EB bound to DNA were measured with respect to concentration
of the complex. EB was non-emissive in Tris–HCl buffer solution
(
pH 7.2) due to fluorescence quenching of the free EB by the sol-
2.6. Protein binding studies
vent molecules. In the presence of DNA, EB showed enhanced
emission intensity due to its intercalative binding to DNA. A com-
petitive binding of the metal complexes to CT DNA resulted in the
displacement of the bound EB, thereby decreasing its emission
Binding of metal hydrazone complexes with bovine serum albu-
min (BSA) was studied using fluorescence spectra recorded with
excitation at 280 nm and corresponding emission at 345 nm
assignable to that of bovine serum albumin (BSA). The excitation
q
intensity. The quenching constant (K ) was calculated using the

8
6
P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
and emission slit widths and scan rates were constantly main-
tained for all the experiments. Samples were carefully degassed
using pure nitrogen gas for 15 min by using quartz cells (4 ꢃ 1 ꢃ
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 me-
tal complexes were prepared by dissolving them in DMSO: phos-
phate buffer (1:99) and diluted suitably with phosphate buffer to
required concentrations. 2.5 ml of BSA solution (
by successive additions of a 25 l stock solution of complexes
10 M) using a micropipette. Synchronous fluorescence spectra
was also recorded using the same concentration of BSA and com-
plexes as mentioned above with two different k (difference be-
lM) was titrated
l
ꢁ4
(
D
tween the excitation and emission wavelengths of BSA) values
such as 15 and 60 nm.
2.7. Antioxidant assays
The free radical (DPPH, ABTS, FRAP, OH and NO) scavenging
activities of the samples were determined according to the re-
ported methods [30–34]. The lipid peroxidation was evaluated by
the modified method of Druper and Hadly in ex vivo and it was
determined on the basis of the formation of thiobarbituric acid-
reactive substances by an iron/ascorbate system with liver micro-
somes [35,36]. For each of the above assays, the tests were run in
triplicate and various concentrations of the complexes were used
to fix a concentration at which each complex showed approxi-
mately 50% activity. The percentage activities were calculated
Fig. 1. Molecular structure of complex 1 showing the atom-numbering scheme
with ellipsoid of 50% probability and solvent molecules were omitted for clarity.
Ni1–N2 [1.991(2)]. The [N2–Ni1–N5] trans angle is 176.9(1)°,
whereas the other trans angles [N1–Ni1–O1] 156.31(8)° and [N4–
Ni1–O3] 156.39(8)° are constrained within the meridional ligands.
The forgoing observations suggest that the coordination geometry
is considerably distorted from that of a perfect octahedron [38].
The Ni(II) center is shared by four fused five-membered chelate
rings and the bicyclic chelate system {Ni1O3C30N6N5C23C22}
with its counterpart {Ni1O1C13N3N2C6C5N1}. One of the furan
moieties attached to the Ni(II) center was found to be disordered
with respect to rotation about the C30 to C31 bond. The asymmetric
unit also contains four solvated water molecules. One of the hydro-
gens on O8 could not be located in the difference map hence it was
not included in the ORTEP diagram. The crystal–packing diagram evi-
dences substantial H-bonding. A perspective view of the unit cell
packing and the hydrogen bond network of 1 are shown in Fig. S1.
using the formula, % activity = [(A
0
ꢁ A
C
)/A
0
] ꢃ 100 where A
0
and
A
C
represent the absorbance in the absence and presence of the
tested complex, respectively. The 50% activity (IC50) can be calcu-
lated using the percentage of activity results.
3
. Results and discussion
The analytical data obtained for the metal hydrazone complexes
1–3 are in satisfactory agreement with the proposed structures and
it is clear that in each case the hydrazone ligand coordinates to the
metal ion in 1:2 instead of 1:1 M ratio.
3
3
.1. X-ray crystallography
2
.1.1. Crystal structure of [Ni(L) ] (1)
The molecular structure of complex 1 is depicted in Fig. 1 along
3.1.2. Crystal structure of [Co(L)
2
] (2)
with the atom labeling scheme. The crystallographic data and per-
tinent bond lengths and angles are listed in Tables 1 and 2. Com-
pound 1 crystallizes in the monoclinic space group P2 /n. The
1
Ni(II) center possesses a distorted octahedral geometry that com-
prises two equivalent monoanionic ligands that are coordinated
in a meridional fashion such that the cis pyridyl nitrogen, the trans
azomethine nitrogen and cis enolate oxygen atoms are essentially
perpendicular to each other.
The X-ray crystal structure of [Co(L) ] (2) is presented as an
2
ORTEP diagram in Fig. 2 along with an atom numbering scheme. List-
ings of pertinent bond distances and angles are presented in Tables
1 and 2. Analogously to the complex 1, the Co atom in complex 2 is
hexacoordinated by two units of the tridentate hydazone ligand.
Two monodeprotonated ligands coordinate to the Co(II) center in
a tridentate fashion and feature enolate O, azomethine N and pyr-
idine N-donor atoms.
Due to keto-enol tautomerism it is well known that the imino
tautomers can exist as two geometrical isomers, namely the syn
The Co(II) center is shared by four fused five-membered chelate
rings and the bicyclic chelate system {Co1O3C31N6N5C23C24}
with its counterpart {Co1O1C13N3N2C6C5N1}. Furthermore, one
of the furan moieties attached to the Co(II) center is disordered
by rotation about the C31–C32 bond. The observed torsion angles
of –176.9(2)° for C5–C6–N2–N3, and ꢁ175.0(2)° for C23–C24–
N5–N6 imply that the ligand adopts the E-conformation upon
coordination. The coordination environment around the cobalt
atom is distorted octahedral and the two oxygen atoms and four
nitrogen atoms occupy the coordination sites with varying li-
gand–metal–ligand bite angles; 78.50(7)° [N1–Co1–N2], 76.98(6)°
[N2–Co1–O1], 78.37(6)° [N5–Co1–O3] and 78.72(7)° [N4–Co1–
N5]. The two central bonds Co1–N1 [2.099(2)] and Co–O1
(
Z) and anti (E) forms. However, in this case, only the E isomer is ob-
served. The torsion angle of 178.6(2)° for C5–C6–N2–N3 and
78.0(2)° for C22–C23–N5–N6 support the view that the ligands
1
adopt the E conformation upon coordination [37]. The coordination
environment around the nickel center is distorted octahedral and
comprises two oxygen and four nitrogen atoms. The ligand–
metal–ligand bite angles are 79.00(9)° [N1–Ni1–N2], 77.32(8)°
[
N2–Ni1–O1], 77.42(9)° [N5–Ni1–O3] and 79.04(9)° [N4–Ni1–N5].
The two central coordinating bonds Ni1–N1 [2.077(2)] and Ni1–
O1 [2.083(2)] are comparable in length to those of the basal planar
bonds Ni1–O3 [2.083], Ni1–N5 [1.985(2)], Ni1–N4 [2.084(3)] and

P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
87
Table 1
Crystal data and structure refinement data.
Empirical formula
C
34
H
32
6
N NiO
8
(1)
C
34
6
H26CoN O
5
(2)
C
35
H
28CuN
6
O
5
(3)
Name
[Ni(FBP)
711.37
2
]ꢀ3H
2
Oꢀ(OH)
[Co(FBP)
657.54
2
]ꢀH
2
O
[Cu(FBP)
676.17
orthorhombic
Pbca
2
]ꢀMeOH
Formula weight
Crystal system
Space group
T (K)
monoclinic
P2 /n
monoclinic
P2 /c
1
1
100(2)
100(2)
223(2)
Wavelength (Å)
Unit cell dimensions
a (Å)
b (Å)
c (Å)
0.71075
0.71075
0.71069
8.7518(9)
27.268(3)
14.0131(15)
90.000
95.189(3)
90.000
dark brown
1.419
4
10.9462(5)
14.8724(7)
18.6278(9)
90.000
102.0440(10)
90.000
brown
1.473
15.3886(6)
19.6995(7)
20.7510(8)
90.000
90.000
90.000
violet
1.428
8
a
(°)
b (°)
(°)
Colour
c
D
Z
calc (Mg/m3)
4
F(0 0 0)
1480
1356
2792
Crystal size (mm3)
hkl limits
0.21 ꢃ 0.20 ꢃ 0.09
0.30 ꢃ 0.18 ꢃ 0.16
ꢁ14 6 h 6 14
ꢁ19 6 k 6 19
ꢁ24 6 l 6 24
3.22–27.46°
78524
0.18 ꢃ 0.13 ꢃ 0.10
ꢁ19 6 h 6 19
ꢁ25 6 k 6 25
ꢁ26 6 l 6 26
3.01–27.48°
64182
ꢁ11 6 h 6 11
ꢁ
ꢁ
35 6 k 6 35
18 6 l 6 8
h range for data collection
Reflections collected
R indices (all data)
3.01–27.49°
33288
R
1
= 0.0798
R
1
= 0.0457
R
1
= 0.0852
2
wR = 0.1268
2
wR = 0.1133
2
wR = 0.1146
Data/restraints /parameters
Independent reflections
Goodness-of-fit (GOF) on F
7633/142/489
7633 [R(int) = 0.0582]
1.041
6771/149/462
6771 [R(int) = 0.0361]
0.951
7205/0/429
7205 [R(int) = 0.0939]
1.055
2
Table 2
Selected bond lengths ( AÅ ) and bond angles (°) for 1 and 2.
0
C
34
H
32
N
6
NiO (1)
8
34 6 5
C H26CoN O (2)
Bond lengths
Bond angles
Bond lengths
Bond angles
Ni1–N1
Ni1–N2
Ni1–O3
Ni1–N5
Ni1–N4
Ni1–O1
2.077(2)
1.991(2)
2.074(2)
1.985(2)
2.084(3)
2.083(2)
N2–Ni1–O1
N2–Ni1–N1
N1–Ni1–N5
N5–Ni1–O1
N5–Ni1–O3
N5–Ni1–N4
O3–Ni–N4
N4–Ni1–O1
N1–Ni1–N4
N1–Ni1–O1
N5–Ni1–N2
77.32(8)
79.00(9)
99.36(9)
104.33(8)
77.42(9)
79.04(9)
156.39(8)
90.50(8)
93.67(9)
156.31(8)
176.93(1)
Co1–N1
Co1–N2
Co1–O3
Co1–N5
Co1–N4
Co1–O1
2.099(2)
1.973(2)
2.069(1)
1.956(2)
2.057(2)
2.105(1)
N2–Co1–O1
N2–Co1–N1
N1–Co1–N5
N5–Co1–O1
N5–Co1–O3
N5–Co1–N4
O3–Co1–N4
N4–Co1–O1
N1–Co1–N4
N1–Co1–O1
N5–Co1–N2
76.98(6)
78.50(7)
96.98(7)
107.32(6)
78.37(6)
78.72(7)
157.08(6)
90.55(6)
90.00(7)
155.32(6)
174.46(7)
[
2.105(1)] are comparable in length to those of the basal planar
35 1 6 5
C H28Cu N O . The Cu(II) coordination environment is distorted
bonds Co1–O3 [2.069(1)], Co1–N5 [1.956(2)], Co1–N4 [2.057(2)]
and Co1–N2 [1.973(2)]. The foregoing bond distances imply that
all the axial and basal planar bonds are essentially equal in length.
The [N2–Co1–N5] trans angle is 174.46(7)°; however, the trans an-
gles of 155.32(6)° [N1–Co1–O1] and 157.08(6)° [N4–Cl1–O3] are
constrained within the meridional ligands. These observations sug-
gest that the coordination geometry is significantly distorted from
that of a perfect octahedron. A perspective view of the unit cell
packing and the hydrogen bond network of 2 are shown in Fig. S2.
octahedral as evident from the bond angles subtended at the metal
center. The basal plane comprises an imine nitrogen (N2), an eno-
late oxygen (O2), an imine nitrogen (N6) and an pyridyl nitrogen
N(5). The pyridyl N1 and enolate O1 atoms occupy the apical posi-
tions thus completing the distorted octahedral coordination
environment.
The coordination environment around Cu(II) is distorted as evi-
denced by the observation that the bite angles for the two oxygen
and four nitrogen atoms vary between 75.97(9)° [N1–Cu1–N2],
7
4.59(8)° [N2–Cu1–O1], 79.54(9)° [N5–Cu1–N6] and 79.34(8)°
3
.1.3. Crystal structure of [Cu(L)
An ORTEP diagram of the structure of 3 is displayed in Fig. 3. The
2
] (3)
[N6–Cu1–O2]. The observed torsion angles of ꢁ177.5(2)° for C5–
C6–N2–N3 and ꢁ176.6(2)° for C23–C24–N6–N7 imply that the
ligand is coordinated to the metal in the E conformation [39].
The environment around the metal is distorted octahedral and
comprises two equivalent monoanionic ligands that are coordi-
nated in a tridentate fashion and orientated such that they are
essentially perpendicular to each other. The two central bonds
Cu1–N1 [2.267(2)] and Cu1–O1 [2.295(2)] are comparable in
length to those of the basal planar bonds. In turn, this implies that
crystallographic data, along with a selection of bond lengths and
bond angles, are presented in Tables 1 and 3, respectively. The X-
ray study of complex 3 revealed that the crystals are comprised
of orthorhombic unit cells in the space group Pbca with Z = 8.
The unit cell dimensions are a = 15.3886(6) Å, b = 19.6995(7) Å,
c = 20.7510(8) Å,
3
a
= b =
c
= 90°, V = 6290.6(4) Å ,
Dcalc = 1.428
3
l
g/m . The formula for the complex in crystalline form is

8
8
P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
Fig. 2. Molecular structure of complex 2 showing the atom-numbering scheme
Fig. 3. Molecular structure of complex 3 showing the atom-numbering scheme
with ellipsoid of 50% probability and solvent molecules were omitted for clarity.
with ellipsoid of 50% probability and solvent molecules were omitted for clarity.
Table 3
the Cu(II) complex adopts a tetragonally elongated structure. The
trans pair of bonds Cu1–N1 and Cu1–O1 are longer than the
remaining four bonds Cu1–N2, Cu1–O2, Cu1–N5 and Cu1–N6. This
distortion is a consequence of the Jahn–Teller effect that operates
0
Selected bond lengths ( ÅA ) and bond angles (°) for 3.
35 6 5
C H28CuN O (3)
Bond lengths
Bond angles
9
in the d electronic ground state of the six-coordinate complex.
Cu1–O1
Cu1–N2
Cu1–N1
Cu1–N6
Cu1–O2
Cu1–N5
2.295(2)
2.010(2)
2.267(2)
1.952(2)
2.028(2)
2.058(2)
O1–Cu1–N2
N2–Cu1–N1
N1–Cu1–N6
N6–Cu1–O1
N5–Cu1–N6
N6–Cu1–O2
N6–Cu1–N2
N1–Cu1–O1
O1–Cu1–O2
N1–Cu1–O2
O2–Cu1–N2
74.59(8)
75.97(9)
96.00(9)
113.49(8)
79.54(9)
79.34(8)
171.90(9)
150.35(8)
89.32(7)
93.33(8)
101.92(8)
In this case, one trans pair of coordinate bonds is elongated while
the remaining four bonds are shortened. It is clear from the bond
angles, N1–Cu1–N2 [75.97(9)°], N2–Cu1–O1 [74.59(8)°], O2–
Cu1–N6 [79.34(8)°] and N6–Cu1–N5 [79.54(9)°] that the
coordination geometry departs considerably from that of a perfect
octahedron. The Cu–N(py), Cu–N(imine) and Cu–O(enolate) bond
lengths fall within the ranges reported for similar complexes of
divalent metal ions. In general, the shortest coordinate bonds are
those to the central imine N donors. The bonds to the distal pyridyl
and carbonyl groups are significantly weaker. The latter
observation can be attributed to greater
p
-back bonding in the
3.2. Infrared spectra
Cu–N(imine) bond in comparison with that of the Cu–N(py) bond
in concert with the steric requirements of the meridionally
coordinated ligand [40]. A solvated methanol is involved in the
intermolecular hydrogen bonding between O1 of the enolate
oxygen of the hydazone ligand and the H50 hydrogen atom of
the solvated methanol molecule. A perspective view of the unit cell
packing and the hydrogen bond network of 3 is shown in Fig. S3.
The complex 3a was obtained as a minor product in the reaction
The IR spectrum of the free ligand was compared with those of
the metal complexes in order to study the binding mode(s) of the
hydrazone ligand to the metal. The ligand spectrum exhibits char-
ꢁ1
acteristic absorption bands at 3143, 1689, 1582 and 1072 cm due
to the (N–N) vibrations, respectively. The IR
m
(N–H) (C@O)
, m , m(C@N) and m
spectra of the complexes revealed significant differences from
those of the free ligand as described below. The bands due to the
of [CuCl
get suitable crystals for characterization using XRD. The single crys-
tal XRDdata proved thatthe molecular formulaof 3a is [CuCl(PPh
Fig. S4) without the coordination of the hydrazone moiety. It is
understood that the copper ion is reduced to 1+ oxidation state by
the hydrazone ligand which is normally a good reducing agent
2
3
(PPh )
2
] with the hydrazone ligand (HL) and crystallized to
m
(C@O) and
m
(N–H) stretching vibrations are absent in the IR spectra
of the complexes and two new bands appear between 1587–
1497 cm and 1309–1311 cm due to the m(C@N–N@C) and m
(C–O)
stretching vibrations, respectively, thus implying that deprotona-
tion of the N–H moiety has occurred, thereby confirming that the
hydrazone is coordinated in the enol form. Furthermore, the de-
ꢁ
1
ꢁ1
3
)
3
]
(
[
23]. The crystal structure, unit cell parameters, bond lengths of 3a
crease in the
m(C@N) stretching frequency involving the azomethine
was found to be in good agreement with the earlier report.
nitrogen indicates that the imine nitrogen is involved in coordina-

P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
89
tion to the metal ion [41]. The increase in the
m
(N–N) stretching fre-
Table 4
ꢁ1
Electronic and fluorescence spectral data for metal hydrazone complexes.
quency in comparison with that of the free ligand (ꢄ5–12 cm ) is
due to the increase in the double bond character offsetting the loss
of electron density via donation to the transition metal, thereby
providing further confirmation that the coordination of the hydra-
zone ligand involves the azomethine atom [42].
Complex
kmax(nm)
Fluorescencec data (kmax (nm))
a
b
b
b
1
2
3
373 , 290
458
498
476
a
364 , 266
a
373 , 282
a
b
c
LMCT.
⁄
p
?
p
.
3.3. Electronic spectra
Exicitation at LMCT.
The electronic spectra of all three complexes (1, 2 and 3), which
were recorded in DMSO:buffer solution, exhibit two bands in the
range 266–373 nm (Table 4). An intense band that is apparent in
the 364–373 nm region can be assigned to an ligand–metal charge
500 nm exhibited a positive shift of approximately 70–130 nm in
comparison with those of the excitation maxima. The CT lumines-
cence observed for these complexes may be due to the presence of
an imine functional group [44]. It is likely that this emission orig-
inates from the lowest energy ligand-to-metal charge transfer
(LMCT) state. The stronger blue luminescence observed may be
due to the coordination of the ligand to the metal center which in-
creases the rigidity of the ligand and reduces the loss of energy via
transfer (LMCT) transitions of the imine group and the higher
⁄
energy bands below 300 nm are attributable to
transitions [43].
p
?
p
intraligand
3.4. Photoluminescence studies
a non-radiative pathway, thus enhancing the probability of a li-
⁄
gand
p
–p
transition.
The luminescence behavior of complexes 1, 2, and 3 was also
investigated. When an excitation wavelength corresponding to
the highest energy absorption was used, none of the complexes
exhibited emission. However, the use of a lower energy excitation
wavelength resulted in an intense emission in each case (Table 4).
The emission maxima for the complexes fell in the range of 450–
3.5. Cyclic voltammetry
The electrochemical properties of complexes 1, 2, 3 and the
ligand HL were studied at a scan rate of 50 mVs . The HL ligand
ꢁ1
0.8
0.6
0.4
0.2
0.0
1
.5
1.4
.3
.2
.1
1.0
1
1.00
2
1
1
1
0
0
.75
.50
0
5
10
15
20
25
-6
[DNA] x 10 M
1
.25
.20
.15
.10
.05
1
1
1
0.25
0.00
1
0
5
10
15
20
25
-6
[DNA] x 10
M
250
300
350
400
450
500
250
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
1
1
1
0
0
0
0
.50
.25
.00
.75
.50
.25
.00
6
.0
5.5
.0
.5
.0
.5
3
5
4
4
3
0
5
10
15
20
M
25
-6
[DNA] x 10
2
50
300
350
400
450
500
Wavelength (nm)
Fig. 4. Electronic absorption spectra of complexes 1–3 (25
lM) 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 M). Arrows show the changes in absorbance with respect to an increase in the DNA concentration (inset: plot of [DNA] versus [DNA]/(e ꢁ e
l
a f
)).

9
0
P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
is neither reduced nor oxidized reversibly in the potential range
explored, hence the redox processes are assigned exclusively to
the transition metals. Peaks corresponding to the potentials
ior has been reported for related complexes, thus confirming the
occurrence of a slow chemical reaction pursuant to the electrode
process [46].
+1.211 V and 0.801 V were recorded for complex 1. The anodic re-
sponse detected at +1.211 V is believed to be due to Ni(II) ? Ni(III)
oxidation and the cathodic response at 0.801 V is attributable to
Ni(III) ? Ni(II) reduction. The large peak-to-peak separation
3.6. DNA binding
3.6.1. Absorption titration of the copper complex bound to DNA
Any change in the UV–Vis absorption spectra of metal com-
plexes upon the addition of DNA serve as an evidence for the exis-
P c a
(DE = 410 mV) and the observed i /i ratio due to cathodic and ani-
ꢁ1
odic sweeps at the scan rates employed (25–200 mVs ) implies
that this process can be best regarded as a quasi-reversible oxida-
tion. This behavior can be explained on the basis of slow electron
transfer and to adsorption of the complexes onto the electrode sur-
face [45]. The voltammogram for complex 2 exhibits an irreversible
oxidation peak at 0.641 V which is attributable to the oxidation of
Co(II) to Co(III). An additional peak detected at ꢁ0.303 V is as-
signed to the reduction of Co(II) to Co(I). Furthermore, complex 2
displays the corresponding oxidation peak at ꢁ0.181 V which is
assignable to Co(I) to Co(II) oxidation. This process is completely
reversible and is characterized by a peak-to-peak separation
tence of an interaction between them. In particular, hypochromism
⁄
due to
p
?
p
stacking interaction with a red-shift (bathochro-
mism) may appear in the case of an intercalative binding leading
to stabilization of DNA duplex [47]. The electronic absorption spec-
tra of complexes 1, 2 and 3 exhibited two well-resolved bands in
the range of 200–400 nm. Upon the addition of DNA, the band at
290 nm for the complex 1 exhibited hypochromism of about
11.40% together with 2 nm red shift and the band at 373 nm (com-
plex 1) showed only hypochromism (15.15%) without any shift. On
the other hand, the band centered at 266 nm corresponding to
complex 2 exhibited a slight hyperchromism during the initial
stages of DNA addition suggesting the tight binding and stabiliza-
tion whereas further addition of DNA results in a hypochromism
indicating the strong binding to CT DNA probably by intercalation.
Additionally, the band at 364 nm showed a hypochromism (37%)
accompanied with red-shift of 5 nm. A distinct isobestic point
p
(DE ) of 122 mV. The cyclic voltammogram for complex 3 exhibits
a reduction peak at ꢁ0.307 V which is assigned to the reduction of
Cu(II) to Cu(I). The corresponding oxidation peak is not observed,
thus implying that the hydrazone ligand stabilizes the +1 oxidation
state of Cu. The potential difference (
electrode couple increases with increasing scan rates. This behav-
D
E
p
= Ep
a
ꢁ Ep
c
) of the first
1
1
1
.3
.2
.1
1.3
1
2
8
6
4
2
00
00
00
00
0
800
1.2
1.1
1.0
600
400
200
0
1.0
0
25
50
75
100
125
150
0
25
50
75
100 125 150
-6
-6
[Complex] x 10
M
[Complex] x 10 M
550
600
650
Wavelength (nm)
700
750
550
600
650
Wavelength (nm)
700
750
1
1
1
1
1
.5
.4
.3
.2
.1
3
8
6
4
2
00
00
00
00
0
1.0
0
25
50
75
100
125
150
-
6
[Complex] x 10 M
550
600
650
700
750
Wavelength (nm)
Fig. 5. 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
lM of complexes 1–3. Arrow indicates the changes
in the emission intensity as a function of complex concentration. Inset: Stern–Volmer plot of the fluorescence titration data corresponding to the complexes 1–3.

P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
91
observed at 397 nm which indicates the existence of single mode
of binding. However, the band at 282 nm for complex 3 exhibited
hypochromism (68.72%) together with 1 nm red shift and the band
at 373 nm also showed hypochromism of about 67.20% without
any shift in wavelength. The observed hypochromism could be
attributed to stacking interaction between the aromatic chro-
mophores of the complexes and DNA base pairs consistent with
the intercalative binding mode, while the red-shift is an evidence
of the stabilization of the CT DNA duplex. UV–Vis spectra of metal
complexes in the absence or presence of CT DNA derived for di-
verse R values are shown representatively for complexes 1, 2 and
tively. The Kapp values obtained for the three different complexes
using the equation KEB [EB] = Kapp[complex] was found as 2.193 ꢃ
5
ꢁ1
5
ꢁ1
5
ꢁ1
10 M , 2.222 ꢃ 10 M and 2.692 ꢃ 10 M . These values sug-
gested that the complex 3 containing bivalent copper ion showed
higher quenching efficiency than the respective nickel and cobalt
counterparts. However, neither the free hydrazone ligand nor the
starting precursor complexes displayed any affinity towards CT
DNA signifying the presence of metal ions in the hydrazone che-
lates is solely responsible for the kind of interaction exhibited by
them. The binding as well as quenching constants determined in
our experiments clearly demonstrates that the titled complexes
possess better DNA interaction than other similar complexes re-
ported in the literature [48].
3
in Fig. 4.
In order to compare quantitatively the binding ability of the
three different complexes containing same hydrazone ligand but
different metal ions with CT DNA, the intrinsic binding constants
were determined by monitoring the changes in lower energy bands
of complexes 1, 2 and 3, respectively, with increasing concentra-
3.7. DNA cleavage
tion of DNA using the equation: [DNA]/[
/K ]; [DNA] versus [DNA]/[
sets) gave a slope and the intercept which are equal to 1/[
and (1/K )[ ], respectively; K is the ratio of the slope to the
intercept. The magnitudes of intrinsic binding constants (K ) were
calculated to be 8.045 ꢃ 10 M , 2.528 ꢃ 10 M , and 3.135 ꢃ
e
a
ꢁ
e
f
] = [DNA]/[
e
b
ꢁ
e
f
] +
Gel electrophoresis is a technique that is based on the migration
of DNA under the influence of an electric potential. There are num-
ber of agents which exert their effect by inhibiting enzymes that
act upon DNA. These inhibitions result from the binding of such
agents to the enzyme site of interaction on the DNA rather than
to direct enzyme inactivation. Transition metals have been re-
ported to inhibit DNA repair enzymes. The DNA cleavage efficiency
of the complex is attributed to the different binding affinity of the
complex to DNA.
1
b
[e
b
ꢁ
e
f
e
b
ꢁ
f
e ] (shown in Fig. 4 as in-
e
a
ꢁ
f
e ]
b
e
b
ꢁ
e
f
b
b
3
ꢁ1
4
ꢁ1
4
ꢁ1
1
0 M corresponding to complexes 1, 2 and 3, respectively. The
observed binding constant (K ) revealed that the complex 3 con-
b
taining copper ion is strongly bound with CT DNA than the other
complexes containing nickel and cobalt ions as transition metal
counterpart, respectively.
For comparison purposes, the cleavage reactions for 1, 2 and 3
were carried out in the presence of H
M. Each complex was found to possess nuclease activity. Con-
trol experiments carried out using DNA or mixtures of H and
2 2
O at a concentration of
35 l
3.6.2. Competitive studies with ethidium bromide
2 2
O
Ethidium bromide (EB) serves as a typical indicator of intercala-
DNA do not show any apparent cleavage of DNA as is evident in
lanes 1 and 2, respectively (Fig. 6A). The cleavage efficiencies for
complexes 1, 2 and 3 (lanes 3, 4 and 5, respectively; Fig. 6A) in
tion since it can form soluble complexes with nucleic acids emit-
ting intense fluorescence in the presence of CT DNA due to the
intercalation of the planar phenenthridinium ring between adja-
cent base pairs on the double helix. EB does not show any apprecia-
ble emission in buffer solution due to fluorescence quenching of
the free EB by the solvent molecules and the fluorescence intensity
is highly enhanced upon addition of CT DNA, due to its strong
intercalation with DNA base pairs. Addition of a second molecule
2 2
the presence of H O are sufficient to exhibit nuclease activity.
Based upon their ability to convert the supercoiled form (Form I
SC) to the nicked circular (Form II NC), complex 3 was found to
be an efficient chemical nuclease for double strand cleavage of
(
complex in our case), that may bind to DNA more strongly than
(A)
EB results in lowering of DNA-induced EB emission intensity. The
changes observed in the fluorescence spectra of EB on its binding
to CT DNA are often used for the interaction study between DNA
and other compounds, such as metal complexes.
The emission spectra of EB bound to CT DNA in the absence and
presence of each compound have been recorded for [EB] = 10
and [DNA] = 10 M upon the addition of increasing amounts of
respective metal hydrazone complex [0–150 M]. In the case of
all the complexes 1, 2 and 3, the addition with respect to different
R values (Fig. 5) showed a significant decrease in the intensity of
the emission band of the DNA–EB system at 604 nm of about
lM
l
(
B) 18
Super coiled form Nicked form
l
16
14
1
2
0
8
6
4
2
0
2
8
4.65%, 26.69% and 29.17% with red shift of 5 nm, 7 nm and
nm, respectively, indicates that there existed a competition
1
between the metal hydrazone complexes and EB towards binding
to DNA. The observed significant quenching of DNA–EB fluores-
cence after the addition of the complexes 1, 2 and 3 proved that
they displace EB from the DNA–EB complex and they probably
interact with CT DNA by the intercalative mode [48]. The quench-
ing constant (K
shown as insets in Fig. 5) is used to evaluate the quenching effi-
ciency for each compound according to the equation: I /I = K
Q] + 1. The Stern–Volmer plots of DNA–EB illustrate that the
q 0
), obtained from the slope of the plot [Q] versus I /I
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
(
0
q
Fig. 6. (A) Gel-electrophoresis pictures for the metal hydrazone complexes
Photograph showing the effects of transition metal hydrazones on DNA of pUC19:
[
Lane 1: SC pUC19 DNA (0.5
(60 M); Lane 3: SC pUC19 DNA (0.5
: SC pUC19 DNA (0.5 g) + H (60 M) + (2)(30
0.5 g) + H (60 M) + (3)(30 M). (B) Relative amounts of different DNA forms
in the presence of complexes 1, 2 and 3.
l
g) alone; Lane 2: SC pUC19 DNA (0.5
g) + H (60 M) + (1)(30 M); Lane
M); Lane 5: SC pUC19 DNA
lg) +
quenching of EB bound to DNA by the compounds 1–3 is in good
agreement with the linear Stern–Volmer equation. Further, the val-
H
4
(
2
O
2
l
l
2
O
l
2
l
l
l
2
O
2
l
q
ues of K corresponding to the three complexes are found as
l
2
O
2
l
l
3
ꢁ1
3
ꢁ1
3
ꢁ1
2
.070 ꢃ 10 M , 2.083 ꢃ 10 M
and 2.610 ꢃ 10 M , respec-

9
2
P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
5
4
3
2
1
00
00
00
00
00
0
1
3.5
.0
2.5
2
2
2
1
1
.5
.0
.5
.0
6
00
3
2
1
1
.0
.5
.0
0
2
4
-66
8
10
400
0
2
4
[
6
-6
8
10
[
Q] x 10
M
Q] x 10
M
200
0
3
00
350
400
450
300
350
400
450
Wavelength (nm)
Wavelength (nm)
3.5
3
5
4
3
2
1
00
00
00
00
00
0
3.0
2.5
2.0
1.5
1.0
0
2
4
[
6
8
10
-
6
Q] x 10
M
3
00
350
400
450
Wavelength (nm)
Fig. 7. Emission spectrum of BSA (1 ꢃ 10 M; kexi = 280 nm; kemi = 345 nm) as a function of concentration of the complexes 1–3 (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ꢃ 10ꢁ6 M).
ꢁ6
Arrow indicates the effect of metal complexes on the fluorescence emission of BSA. (Inset: Stern–Volmer plot of the fluorescence titration data corresponding to the complex
1–3).
0.4
BSA + complex 3
BSA
0
.4
.3
.2
.1
.0
0.1
0.2
0.3
0.4
0.5
complex 1
complex 2
complex 3
0
0.3
0
0
0.2
0
-
-
0.1
-
-
-
0.0
250
275
300
325
-
5.0
-5.2
-5.4
-5.6
-5.8
-6.0
Wavelength (nm)
log [Q]
Fig. 9. The absorption spectra of BSA (1 ꢃ 10ꢁ M) and BSA–complex
5
3
ꢁ
5
ꢁ6
Fig. 8. Plot of log [(F
0
ꢁ F)/F] versus log [Q] for complexes 1, 2 and 3.
(BSA = 1 ꢃ 10 M and Complex 3= 1 ꢃ 10 M).

P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
93
400
1a
1b
400
300
300
200
200
100
100
0
0
2
80
300
300
300
320
340
340
340
360
300
300
300
320
320
320
340
360
380
Wavelength (nm)
Wavelength (nm)
5
00
600
2a
2b
5
00
4
00
4
00
3
00
3
00
2
00
2
00
1
00
1
00
0
0
2
80
320
360
340
360
380
Wavelength (nm)
Wavelength (nm)
5
00
4
00
3a
3b
4
00
3
00
3
00
2
00
2
00
1
00
1
00
0
0
2
80
320
360
340
360
380
Wavelength (nm)
Wavelength (nm)
ꢁ
6
ꢁ6
Fig. 10. Synchronous spectra of BSA (1 ꢃ 10 M) as a function of concentration of the complexes 1–3 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ꢃ 10 M) with wavelength difference of
D
k = 15 nm (a) and Dk = 60 nm (b). Arrow indicates the changes in emission intensity w.r.t various concentration of complexes 1–3.
DNA in comparison with complexes 1 and 2. The foregoing obser-
vations suggest that cleavage of the supercoiled form and forma-
tion of the nicked circular form increased in the order 1< 2< 3
3.8. Protein binding studies
3.8.1. Fluorescence quenching of BSA by metal complexes
(
Fig. 6B). It is believed that the superior cleavage ability of the cop-
BSA molecule contains three aromatic amino acids (phenylala-
nine, tyrosine and tryptophan) and the fluorescence of BSA can ap-
pear because of tryptophan and tyrosine residues [50]. If the
protein conformation changes, it will lead to the fluorescence
per complex is due to the reaction of copper ions with H , which
produces diffusible hydroxyl radicals or molecular oxygen, which
in turn damage DNA through Fenton-type chemistry [49].
2 2
O

9
4
P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
160
140
120
100
L
1
2
3
134.12
122.25
80
6
0
52.41
4
8.31
49.57
3
8.46
35.66
40
31.49
26.45
2
17.11
16.56
20.16
1.37
20
9.85
10.11
8.80
1
5.47
7
.28
7.65
4
.59
0
DPPH
ABTS Cationc
Hydroxyl
Nitric oxide
Lipid peroxidation
Radical
ꢀ
ꢀ+
ꢀ
Fig. 11. Trends in the inhibition of DPPH , ABTS , OH , NO and lipid peroxidation by ligand L and complexes 1, 2 and 3.
483.7
of metal complex were recorded and a representative spectrum of
complex 3 is presented in Fig. 9 which revealed that the absor-
bance intensity of BSA appeared at 280 nm has been increased
after the addition of metal complex due to the formation of BSA–
complex 3 [54]. Similar behavior was observed for other complexes
and hence, these results confirm that static quenching exists in the
interaction between BSA–complex.
500.0
400.0
300.0
200.0
100.0
245.9
219.3
193.3
3.8.3. Characteristics of synchronous fluorescence spectra
Since the synchronous fluorescence spectra can give informa-
tion about the molecular environment in the vicinity of fluoro-
phore functional groups, it is a useful method to evaluate the
conformational changes of BSA. Moreover, it has several advanta-
ges, such as spectral simplification, spectral bandwidth reduction,
and avoiding different perturbing effects. The shift in maximum
emission position corresponds to the change in the polarity around
the chromophore molecule. Thus, the environment of amino acid
residues can be studied by measuring the shift in wavelength emis-
0
.0
L
1
2
3
Fig. 12. Ferric reducing antioxidant power (FRAP) of ligand L and complexes 1, 2
and 3.
emission change [51]. Fig. 7 shows the effect of increasing the con-
centration of metal complexes on the fluorescence emission of BSA.
Addition of metal complexes to the solution of BSA resulted in the
quenching of its fluorescence emission with blue shift suggesting
that the complex formed between the metal hydrazones and BSA
is responsible for the quenching of BSA. The fluorescence quench-
sion maximum. When the wavelength interval (
Dk) is 15 nm or
6
0 nm, the synchronous fluorescence offers the characteristic
information of tyrosine residues or tryptophan residues [55]. The
synchronous fluorescence spectroscopy of BSA upon addition of
metal-complexes gained at
in Fig. 10.
Dk = 15 nm and Dk = 60 nm are shown
ing is described by Stern–Volmer relation: I
and I are the fluorescence intensities of the fluorophore in the ab-
0
/I = 1 + KSV[Q]; where
I
0
To investigate the structural changes occurred to BSA upon the
addition of metal complexes 1–3, we measured synchronous fluo-
rescence spectra of BSA before and after the addition with respect
to all the three complexes. From the spectra, we understand that
an increase in the concentration of metal complexes resulted in a
decrease in the intensity of the synchronous fluorescence spectral
band corresponding to tyrosine residue with a slight red shift
sence and presence of quencher, respectively, KSV is the Stern–Vol-
mer quenching constant and [Q] is the quencher concentration. The
K
0
SV value obtained from the plot of [Q] versus I /I (shown in Fig. 7
5
ꢁ1
5
ꢁ1
as insets) was found to be 1.528 ꢃ 10 M , 1.979 ꢃ 10 M and
5
ꢁ1
2
.049 ꢃ 10 M
corresponding to the complexes 1, 2 and 3,
respectively.
When small molecules bind independently to a set of equiva-
lent sites on a macromolecule, the equilibrium between free and
bound molecules is represented by the Scatchard equation [52]:
(
1 nm), except in the case of complex 1 which displayed hypochro-
mism only. In addition, a gradual decrease of fluorescence intensity
of tryptophan residues together with a small blue shift (1 nm) in
the emission wavelength were also observed after the addition
all the three complexes to BSA. These experimental results indicate
that the metal complexes do affect the microenvironment of both
tyrosine and tryptophan residues during the binding process and
synchronous measurements confirmed the effective binding of all
the complexes with BSA. Similar behavior was observed for the
interaction between the BSA and other metal complexes [56].
log[(F
0
ꢁ F)/F] = log[K] + n log[Q]; where K and n are the binding
constant and the number of binding sites, respectively. Binding
constants obtained from the plot of log[Q] versus log[(F
0
ꢁ F)/F]
(
3
Fig. 8) corresponding to the complexes 1,
2
and
3
were
3
ꢁ1
3
ꢁ1
4
ꢁ1
.754 ꢃ 10 M , 8.775 ꢃ 10 M
and 1.604 ꢃ 10 M , respec-
tively .
3.8.2. UV–Vis absorption measurement of BSA by metal complexes
UV–Vis absorption measurement is a very simple and effective
method in exploring the structural change and detecting the com-
plex formation [53]. For confirming the type of quenching (i.e., sta-
tic or dynamic) effect may exist in the system of metal hydrazones
and BSA, the absorption spectra of BSA in the presence and absence
3.9. Antioxidant activity
The antioxidant activities of complexes 1, 2 and 3 were evalu-
ated in a series of in vitro assays involving DPPH radicals, ABTS

P. Sathyadevi et al. / Inorganica Chimica Acta 384 (2012) 83–96
95
cationic radicals, hydroxyl radicals, nitric oxide radicals and on the
Acknowledgments
basis of reducing power. The complexes were also studied for lipid
peroxidation by thiobarbituric acid reactive substances (TBARS)
using rat liver. The three complexes exhibited a scavenging effect
at a fixed concentration of 50 lM.
IC50 values of the ligand (L) on DPPH, ABTS cationic, OH, NO rad-
icals and lipid peroxidation assays are 134.12, 52.41, 122.25, 48.31
We sincerely thank Dr. Abraham John, Gandhigram University,
Gandhigram, India for carrying out the electrochemical measure-
ments. We are also grateful to Prof. S. Manian, Department of Bot-
any, Bharathiar University for his help with the biological activity
studies. We also offer our sincere thanks to the University Grants
Commission, New Delhi for the award of Research 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 No. F-0003)
for financial support of this work.
and 49.57
lM, respectively, whereas, the complexes 1, 2 and 3
showed their IC50 values at 35.66, 9.85, 38.46, 10.11, 17.11,
3
1
1.41, 7.28, 26.45, 8.80, 16.56, 20.16, 4.59, 21.37, 7.65 and
5.47 M, respectively. The results of these experiments were
l
shown in Fig. 11. In order to assess the ferric reducing abilities,
3
+
2+
the Fe ? Fe transformation was investigated in the presence
of L and 1, 2 and 3. The reducing capacity of a compound serves
as a significant indicator of its potential antioxidant activity. The
reductive effects of L and 1, 2 and 3 are summarized in Fig. 12.
In general, the antioxidant activity of the ligand and its M(II)
complexes [where M = Ni, Co and Cu] against the free radicals
i.e., DDPH, ABTS cationic, NO, OH, FRAP and lipid peroxidation
was found to decrease in the order of 3> 2> 1>L. The results indi-
cated that the metal complexes exhibited greater antioxidant
activity than the free ligand. Among the tested complexes, complex
Appendix A. Supplementary material
CCDC 787858, 796257 and 779830 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.ica.2011.11.033.
3
displayed very high scavenging activity due to the presence of
Cu(II) ion as described in the previous section. Further, the results
obtained against the different radicals confirmed that the com-
plexes are more effective to arrest the formation of the ABTS than
the other radicals studied. In addition, the results obtained in this
study imply that the metal complexes possess excellent antioxi-
dant activities that are superior to those of standard antioxidants
such as butylated hydroxyl anisole (BHA) and quercetin. The ob-
served lower IC50 values in antioxidant assays did demonstrate
that these complexes have the potential as drugs to eliminate the
radicals.
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