Synthesis, characterization, antibacterial activity, SOD mimic and interaction with DNA of drug based copper(II) complexes

Article abstract of DOI:10.1016/j.saa.2010.11.056

Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with copper(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis reflectance, IR and mass spectroscopy. Complexes have been

Full text of DOI:10.1016/j.saa.2010.11.056

Spectrochimica Acta Part A 78 (2011) 763–770
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, characterization, antibacterial activity, SOD mimic and interaction
with DNA of drug based copper(II) complexes
Mohan N. Patela,∗, Promise A. Dosia, Bhupesh S. Bhatta, Vasudev R. Thakkarb
a Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
b BRD School of Bioscience, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2010
Received in revised form 23 October 2010
Accepted 15 November 2010
Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with cop-
per(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis
reflectance, IR and mass spectroscopy. Complexes have been screened for their in-vitro antibacterial activ-
ity against two Gram(+ve) Staphylococcus aureus, Bacillus subtilis, and three Gram(−ve) Serratia marcescens,
Escherichia coli and Pseudomonas aeruginosa organisms using the double dilution technique. The binding
of this complex with CT-DNA has been investigated by absorption titration, salt effect and viscosity mea-
surements. Binding constant is ranging from 1.3 × 104–3.7 × 104. The cleavage ability of complexes has
been assessed by gel electrophoresis using pUC19 DNA. The catalytic activity of the copper(II) complexes
towards the superoxide anion (O2•−) dismutation was assayed by their ability to inhibit the reduction of
nitroblue tetrazolium (NBT).
Keywords:
Enrofloxacin
Antibacterial activity
DNA interaction
Salt effect
IC50
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
break the DNA chain in the presence of H2O2 and reducing agents
[9].
Fluoroquinolones suppress cell growth by inhibiting activity of
DNA gyrase, an essential bacterial enzyme that maintains super-
helical twists in DNA [1]. The detailed mechanism of the biological
action of the compounds is not completely understood at present,
and many different explanations of the activity of fluoroquinolones
have been proposed. Some evidence suggests that the drugs inter-
act directly with DNA, blocking the activity of DNA-gyrase repair
enzymes [2,3].
Enrofloxacin is a typical second-generation antimicrobial drug
with a broad spectrum of activity against a wide range of Gram-
negative and Gram-positive bacteria, including those resistant to
ˇ-lactam antibiotics and sulfonamides. Enrofloxacin is the first
fluoroquinolone developed for veterinary application and is poten-
tially available for the treatment of urinary tract, respiratory tract
and skin infectious diseases in pets and livestock [4]. The knowl-
edge of structure of DNA and its interactions with other biological
compounds can lead to advance in pharmacology and diagnosis
basis of many diseases [5,6]. Sigman et al. [7] discovered in 1979
that complexes of copper(II) with 1,10-phenanthroline were capa-
ble of cleaving DNA. Copper complexes with a nitrogen donor
heterocyclic ligand have been widely used to improve nuclease
activity [8], mostly because of their high nucleolytic efficiency to
In recent years, the study of quinolones-copper-1,10-
phenanthroline complexes becomes an increasingly important
field due to antibacterial properties of the compounds [10]. In
recent years, particular interest has been paid to synthetic analogs
of CuZn-SOD [11]. In this perspective, the coordination and redox
potentials of each Cu(II) ion in the synthesized copper(II) com-
plexes which can be considered to possess SOD-mimic activity
were found to play a significant role [12]. The central biological
role of phosphate esters as constituents of nucleic acid chains has
prompted the development of drugs able to cleave this class of
compounds with a view to future application in biotechnology
and medicine, to permit gene manipulation, and eventually novel
therapeutic agents [13–15].
Herein, we represent the interaction of Cu(II) with the second-
generation quinolone enrofloxacin and bidentate ligands and an
attempt to examine the mode of coordination and biological prop-
erties of resultant complexes.
2. Experiments
2.1. Materials
All the chemicals for the synthesis of the compounds were used
as purchased. Enrofloxacin was purchased from Bayer AG (Wupper-
tal, Germany). Cupric chloride was purchased from E. Merck (India)
Ltd. Mumbai. 1,10-Phenanthroline and Luria Broth were purchased
∗
Corresponding author. Tel.: +91 2692 226856x202.
E-mail address: jeenen@gmail.com (M.N. Patel).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.11.056

764
M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
from Himedia, India. 2,9-Dimethyl-1,10-phenanthroline (A4) and
2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (A5) were pur-
chased from Loba chemie PVT. LTD. (India). Organic solvents were
purified by standard method.
2.4.4. [Cu(erx)(A4)Cl]
It was prepared using 5-bromo-1,10-phenantroline (1.5 mmol).
Yield: 69.8%, m.p.: 186 ◦C, ꢁeff:1.84 B.M. ꢂmax
, 281 nm. ε,
71,739 L mol−1 cm−1; ꢂmax, 314 nm. ε, 35,714 L mol−1 cm−1. Anal.
Calc. for: C31H28BrClFCuN5O3 (716.49): C, 51.97; H, 3.94; N, 9.77;
Cu, 8.87. Found: C, 52.06; H, 3.99; N, 9.75; Cu, 8.98%.
2.2. Instrumentation
2.4.5. [Cu(erx)(A5)Cl]
Infrared spectra were measured in the 4000–400 cm−1 range
on FT-IR Shimadzu spectrophotometer using KBr pellets. C, H
and N elemental analyses were performed on a model Perkin
Elmer 240 elemental analyzer. The metal contents of the com-
plexes were analyzed by EDTA titration [16] after decomposing
the organic matter with a mixture of HClO4, H2SO4, and HNO3
(1:1.5:2.5). The UV–Vis–near IR absorption spectra were recorded
with a Perkin-Elmer Lambda-9 spectrophotometer, using the
reflectance technique. Magnetic susceptibilities were measured
on a Faraday magnetometer balance using Hg[Co(SCN)4] as a
standard(ꢀg = 16.44 × 10−6 cgs unit at 20 ◦C), and diamagnetic cor-
rectionswere madebyPascal’s constants [17]. The FABmass spectra
were recorded on a Jeol SX 120/Da-600 mass spectrometer/Data
system using Argon/Xenon(6 kV, 10 mA) as FAB gas. The accel-
erating voltage was 10 kV and spectra were recorded at room
temperature.
It was prepared using 5-nitro-1,10-phenantroline (1.5 mmol).
Yield: 67.8%, m.p.: 185 ◦C, ꢁeff:1.87 B.M. ꢂmax
, 281 nm. ε,
69,255 L mol−1 cm−1; ꢂmax, 313 nm. ε, 34,161 L mol−1 cm−1. Anal.
Calc. for: C31H28ClCuFN6O5 (682.59): C, 54.55; H, 4.13; N, 11.72;
Cu, 9.31. Found: C, 54.49; H, 4.06; N, 11.79; Cu, 9.23%.
2.5. Minimum inhibitory concentration
The antibacterial activity of the compounds (ligands, metal salts
and complexes) was screened against two Gram(+ve) Staphylococ-
cus aureus, Bacillus subtilis, and three Gram(−ve) Serratia marcescens,
Escherichia coli and Pseudomonas aeruginosa organisms. Screening
was performed by determining the minimum inhibitory concen-
tration (MIC). Two different media [2% Luria Broth medium] were
prepared. All cultures were incubated at 37 ◦C. Control tests with no
active ingredients were also performed. The MIC was determined
using twofold serial dilutions in liquid media of the compound
being tested. The solvent used was DMSO.
2.3. Ligand preparation
A preculture of bacteria was grown in LB (Luria Broth) overnight
at optimal temperature of each species. This culture was used as a
control to examine if growth of the bacteria tested is normal. In a
similar second culture, 20 ␮L of the bacteria as well as the tested
compound at desired concentration was added. We monitored bac-
terial growth by measuring turbidity of the culture after 18 h. If a
certain concentration of a compound inhibited bacterial growth,
half the concentration of compound was tested. This procedure was
carried on to a concentration that bacteria grow normally. The low-
est concentration that inhibited bacterial growth was determined
as MIC value. All equipment and culture media were sterile.
1,10-Phenanthroline-5,6-dione (A2), 5-bromo-1,10-phenen-
troline(A4) and 5-nitro-1,10-phenanthroline (A5) were prepared as
per the reported method [18–20].
2.4. Preparation of complexes
2.4.1. [Cu(erx)(A1)Cl]
A methanolic solution of CuCl2·2H2O (1.5 mmol) was added
to methanolic solution of 2,9-dimethyl-1,10-phenantroline
(1.5 mmol), followed by the addition of a previously prepared
solution of enrofloxacin (1.5 mmol) in methanol in the presence
of CH3ONa (1.5 mmol). The pH was adjusted at ∼6.8 using dilute
solution of CH3ONa. The resulting solution was refluxed for 2 h on a
water bath, followed by concentrating it to half of its volume. A fine
green colored amorphous product obtained which was washed
with ether/hexane and dried in vacuum desiccators (Scheme 1).
Yield: 65.7%, m.p.: 220 ◦C, ꢁeff: 1.79 B.M. ꢂmax, 283 nm. ε,
87,888 L mol−1 cm−1; ꢂmax, 318 nm. ε, 42,857 mol−1 cm−1. Anal.
Calc. for: C33H33ClCuFN5O3 (665.64): C, 59.54; H, 5.00; N, 10.52;
Cu, 9.55%. Found: C, 59.46; H, 4.92; N, 10.48; Cu, 9.60.
2.6. DNA interaction activity
2.6.1. Viscosity measurements
Viscometric studies were done using the Ubbelohde viscome-
ter that was thermostated at 27 0.1 ◦C in a constant temperature
bath. DNA samples approximately 200 bp in length were prepared
by sonicating in order to minimize complexities arising from DNA
flexibility [21].
The concentration of DNA was 100 ␮M in NP and the flow
times were measured with an automated timer. Each sample was
measured 3 times. The rate of flow of sodium phosphate buffer
(pH∼7.2), DNA (100 ␮M) and DNA with the copper complexes at
various concentrations were measured. The data were presented
as (Á/Á0)1/3 versus [complex]/[DNA] [22], where Á is the viscos-
ity of DNA in the presence of complex and Á0 is viscosity of DNA
in the absence of complex. Viscosity values were calculated from
the flow time of DNA-containing solutions (t) corrected for that of
buffer alone (t0): Á = (t − t0).
In similar way, complexes 2–5 were prepared with the use of
corresponding ligands.
2.4.2. [Cu(erx)(A2)Cl]
It was prepared using 1,10-phenantroline-5,6-dione (1.5 mmol).
Yield: 66.8%, m.p.: 207 ◦C, ꢁeff:1.82 B.M. ꢂmax
, 284 nm. ε,
77,148 L mol−1 cm−1; ꢂmax, 318 nm. ε, 41,237 L mol−1 cm−1. Anal.
Calc. for: C31H27ClCuFN2O5 (667.57): C, 55.77; H, 4.08; N, 10.49;
Cu, 9.52. Found: C, 55.71; H, 4.17; N, 10.45; Cu, 9.49%.
2.6.2. Absorption titration
A solution of CT DNA in 0.5 mM NaCl/5 mM Tris–HCl (pH 7.0)
gave ratio of UV absorbance at 260 and 280 nm, A260/A280 of 1.8–1.9,
indicating DNA was sufficiently free of protein. So, no further effort
was made to purify commercially obtained DNA. The concentra-
tion of DNA was determined by absorption spectroscopy using the
ε value of 6600 mol−1 cm−1 L−1 at 260 nm [23]. The absorption titra-
tion of copper(II) complex in buffer (5 mM Tris–HCl, 50 mM NaCl,
pH 7.0) was performed by using a fixed complex concentration to
2.4.3. [Cu(erx)(A3)Cl]
It was prepared using 2,9-dimethyl-4,7-diphenyl-1,10-
phenantroline (1.5 mmol). Yield: 67.8%, m.p.: 182 ◦C, ꢁeff:1.88
B.M. ꢂmax
, ; ꢂmax, 319 nm. ε,
283 nm. ε, 76,211 L mol−1 cm−1
41,053 L mol−1 cm−1. Anal. Calc. for: C45H41ClCuFN5O3 (817.59): C,
66.09; H, 5.05; N, 8.56; Cu, 7.77. Found: C, 66.02; H, 5.08; N, 8.51;
Cu, 7.71%.

M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
765
Scheme 1. Structure of the title complex [Cu(erx)(A1)Cl]·5H2O.
(OH• radical) and NaN3 (1O2 radical) which were added to SC DNA
prior to the addition of the complex.
which increments of DNA stock solution were added. The concen-
tration of copper(II) solution was 15 ␮M and DNA was added to
a ratio of 6:1 [DNA]/[Cu]. Complex-DNA solutions were allowed
to incubate for 5 min before the absorption spectra were recorded.
The intrinsic binding constant Kb of copper(II) complex to DNA was
calculated from Eq. (1) [24]
2.7. Determination of SOD-like activity
SOD-like activity of all the complexes was determined by
NBT/NADH/PMS system [28]. The superoxide radial produce by
79 ␮M NADH, 30 ␮M PMS, system containing 75 ␮M NBT, phos-
phate buffer (pH = 7.8), and 0.25–3.0 ␮M tested compound. The
amount of reduced NBT was spectrophotometrically detected by
monitoring the concentration of blue formazan form which absorbs
at 560 nm. The reduction rate of NBT was measured in the presence
and absence of test compounds at various concentration of complex
in the system. All measurements were carried out at room temper-
ature. IC50 value of all the complexes was determined by plotting
graph of percentage inhibition of NBT reduction against increase in
concentration of the complex. Concentration of the complex which
[DNA]
εa − εf
[DNA]
εb − εf
1
=
+
(1)
kb(εb − εf)
where [DNA] is the concentration of CT-DNA in base pairs,
the apparent absorption coefficients εa, εf, and εb correspond
to Aobsd/[Cu], the absorbance for free copper complex, and the
absorbance for copper complex in the fully bounded form, respec-
tively. Kb is the equilibrium binding constant in M−1
.
2.6.3. Measurements of salt dependence in DNA binding
The equilibrium binding constant (Kb) of copper(II) complex to
CT-DNA determined by spectrophotometric titration over the con-
centration range 0.005–0.100 M NaCl. A fixed amount of copper(II)
complex in phosphate buffer at pH 7.2 and various concentra-
tions of NaCl was titrated with increasing amounts of CT-DNA
stock solutions (10−6–10−4 M) and the hypochromicity in the ␮L
at 281–284 nm. The Kb values at various NaCl concentrations were
calculated on the basis of Eq. (1) [25–27].
The salt concentration dependence of Kb for the copper(II)
complexes was evaluated by plotting log Kb versus log[Na+] to
obtain SK value, which is essential for polyelectrolyte analysis. Each
measured point was the average value of at least three separate
measurements with a relative standard deviation (RSD) normally
less than 15%.
causes 50% inhibition of NBT reduction is reported as IC50
.
3. Result and discussion
3.1. Characterization of complexes
All the complexes were analyzed using elemental analysis,
magnetic measurements, reflectance, IR, FAB-mass spectroscopy.
The elemental analysis is in concurrence with proposed 1:1:1,
metal:erx:An formulation and theoretical expectation.
3.2. IR spectra
In Table 1 the characteristic absorptions of the IR spectra of
the complexes are listed. In the IR spectra of complexes 1–5, the
absorption at 1733 cm−1 in the spectrum of enrofloxacin attributed
to the absorption of ꢃ(C O)carb has disappeared. Two very strong
characteristic bands are present in the range 1560–1675 cm−1
and 1345–1380 cm−1 that could be assigned as ꢃ(COO) asym-
metric and symmetric stretching vibrations, respectively, whereas
ꢃ(C O)p is shifted from 1615–1635 cm−1 upon bonding. The ꢄ val-
ues (ꢄ = ꢃ(COO)as − ꢃ(COO)s, a useful characteristic for determining
the coordination mode of ligands) fall in the range 197–214 cm−1
indicating a monodentate coordination mode of the carboxylato
group of enrofloxacinato ligand [29].
2.6.4. DNA cleavage study
Gel electrophoresis of plasmid DNA (pUC19 DNA) was carried
out in TAE buffer (0.04 M Tris–Acetate, pH 8, 0.001 M EDTA). 15 ␮L
reaction mixture containing plasmid DNA in TE buffer (10 mM Tris,
1 mM EDTA, pH 8.0) and 200 ␮M complex. Reactions were allowed
to proceed for 3 h at 37 oC. All reactions were quenched by the addi-
tion of 5 ␮L loading buffer (0.25% bromophenol blue, 40% sucrose,
0.25% xylene cyanole, and 200 mM EDTA). The aliquots were loaded
directly on to 1% agarose gel and electrophoresed at 50 V in 1X TAE
buffer. Gel was stained with 0.5 ␮g/mL ethidium bromide and was
photographed on a UV illuminator. The percentage of each form of
DNA was quantities using AlphaDigiDocTM RT. Version V.4.0.0 PC-
Image software. For mechanistic investigations, experiments were
carried out in the presence of radical scavenging agents, viz., DMSO
These changes in the IR spectra suggest that enrofloxacin is coor-
dinated to metal via pyridone and one carboxylate oxygen atoms
[30]. These data are further supported by ꢃ(M–O) [31] which appear

766
M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
Table 1
IR spectra data.
Compounds
ꢃ(C O)p (cm−1
)
ꢃ(COO)asy (cm−1
)
ꢃ(COO)sym (cm−1
)
ꢃ (cm−1
)
ꢃ(M–N) (cm−1
)
ꢃ(M–O) (cm−1
)
Enrofloxacin
1622
1634
1623
1622
1616
1620
1733
1570
1575
1575
1570
1562
–
1369
1378
1373
1370
1348
–
201
197
202
200
214
–
538
539
542
540
542
–
510
508
513
512
511
[Cu(erx)(A1)Cl]
[Cu(erx)(A2)Cl]
[Cu(erx)(A3)Cl]
[Cu(erx)(A4)Cl]
[Cu(erx)(A5)Cl]
at ∼512 cm−1. In investigated complexes, ꢃ(C N) band of ligands
appears at 1580 cm−1. N → M bonding was supported by ꢃ(M–N)
sphere and electronic properties of the metal ions and the factors
such as chelate formation, ring size and number of chelate rings.
Several workers have reported that heterocyclic rings containing
sulfur, nitrogen, and/or oxygen are responsible for the biological
activity of ligands and their metal complexes [39].
band [32] at ∼535 cm−1
.
3.3. Reflectance spectra and magnetic behaviour
The antibactericidal activity of the cupric chloride, fluoro-
quinolones and its complexes were tested against two Gram(+ve)
S. aureus, B. subtilis, and three Gram(−ve) S. marcescens, E. coli and P.
aeruginosa organisms using double dilution method. An acceptable
reason for this increase in bactericidal activity may be consid-
ered in the light of Overtone’s concept [40] and chelation theory
[41]. According to Overtone’s concept of cell permeability, the
lipid membrane that surrounds cell favors the passage of only
lipid soluble materials so that liposolubility is an important fac-
tor which controls bactericidal activity. On chelation, the polarity
of the cooper ion will be reduced to a greater extent due to the over-
lap of the ligand orbital and partial sharing of the positive charge of
the copper ion with donor groups. Further, it increases the delo-
calization of ␲-electrons over whole chelate ring and enhances
lipophilicity of the complexes. This increased lipophilicity enhances
the penetration of the complexes into lipid membranes and blocks
the metal binding sites in bacterial enzymes. These complexes
also disturb the respiratory processes of the cell and thus block
the synthesis of proteins which restricts further growth of the
organism.
The results concerning in vitro antimicrobial activity (MIC) of
ligands and their complexes are represented in Table 2. The antimi-
crobial activity of all complexes against five microorganisms is
much higher than metal salt. The complex shows better antimi-
crobial activity than the metal salt, free ligands, and enrofloxacin.
In case of S. aureus, all complexes have good activity compare to
enrofloxacin. In case of B. subtilis complexes II–IV have good activ-
ity compare to standard drug. In case of S. marcescens and E. coli
all complexes found more potent than enrofloxacin. In case of P.
aeruginosa complexes I–III have good activity compare to standard-
ized drug.
The reflectance spectra of Cu(II) complexes exhibit one asym-
metric broad band centred around 15,000 cm−1. These spectra
suggest that compounds have a distorted square-pyramidal geom-
etry arrangement [33].
The magnetic moments of the copper(II) complexes lie in the
1.79–1.88 BM range. These values are typical of mononuclear cop-
per(II) compounds with d9 electronic configuration. The observed
magnetic moments of all the complexes correspond to typical high-
spin distorted square-pyramidal complexes. However, the values
are slightly higher than the expected spin-only values due to
spin–orbit-coupling contribution [34].
3.4. FAB-mass
Fig. 1 represents the FAB-mass spectrum of complex 1, that is
[Cu(erx)(A1)Cl], obtained using m-nitro benzyl alcohol as matrix.
Peaks at 136, 137, 154, 289 and 306 m/z are due to usage of matrix.
The molecular ion peak is observed at m/z = 664 which is similar
to the molecular weight of complex. Loss of chlorine atom gave a
fragment ion peak at m/z = 630, which confirm that chlorine atom
attached to metal ion with covalent bond. Fig. 1 shows the frag-
ments corresponding to peaks at 456, 421, 359, 271 and 208 m/z
value.
3.5. Antibacterial activity
Coordination compounds have been studied for their antitu-
mour [35], antiviral [36] and antimalerial activity [37], which has
been related to the ability of metal ions to form stable complexes
[38]. The results have led to an understanding of coordination
Fig. 1. FAB-mass spectrum of complex 1, that is [Cu(erx)(A1)Cl], obtained using m-nitro benzyl alcohol.

M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
767
Table 2
MIC data of the compounds (␮M).
S. aureus B. subtilis S. marcescens P. aeruginosa E. coli
CuCl2·2H2O
2698.00 2815.00
2756.00
1.6
2404.00
1.4
3402.00
1.4
Ciprofloxacin
Gatifloxacin
Norfloxacin
1.6
1.1
5.1
4.0
2.9
4.1
1.0
3.8
2.9
2.8
2.5
2.5
Enrofloxacin
Pefloxacin
1.9
2.1
3.9
2.4
1.7
5.1
1.4
5.7
1.4
2.7
Levofloxacin
Sparfloxacin
Ofloxacin
1.7
1.3
1.9
2.2
2.0
1.4
1.7
1.5
1.7
1.7
1.5
2.2
1.0
1.3
1.4
[Cu(erx)(A1)Cl]
[Cu(erx)(A2)Cl]
[Cu(erx)(A3)Cl]
[Cu(erx)(A4)Cl]
[Cu(erx)(A5)Cl]
0.45
0.74
0.36
1.05
0.58
1.94
1.39
1.1
1.11
2.07
0.6
1.06
1.19
0.8
1.67
1.61
0.6
0.44
0.48
1.53
0.58
1.19
0.48
1.25
1.02
Fig. 2. Absorption titration curve of [Cu(erx)(A1)Cl] in absence and presence of
increasing amount of DNA; 50–150 ␮M in phosphate buffer(Na2HPO4/NaH2PO4, pH
7.2), [complex] = 15 ␮M at 281–284 nm, with incubation period of 30 min at 37 oC,
Inset: plot of [DNA]/(εa − εf) versus [DNA].
It was observed that all complexes were more bacteriostatic
than ligands. When antimicrobial activity of metal complexes is
investigated, the following principal factors [42] should be con-
sidered: (i) the chelate effect of the ligands; (ii) the nature of the
N-donor ligands; (iii) the total charge of the complex; (iv) the exis-
tence and the nature of the ion neutralizing the ionic complex; (v)
the nuclearity of the metal centre in the complex.
The first two of the five above-mentioned factors may be respon-
sible for higher antimicrobial activity; that is the chelate effect
provided by both the enrofloxacin ligand and the N-donor ligand
and the nature of the ligands.
Comparing the intrinsic binding constant of complexes with those
of DNA-intercalative [Ru(dmb)2(ipbp)]+2 (1.18 × 104 M−1) complex
[47], we can deduce that all the complex bind strongly to DNA
by intercalation. These spectral characteristics obviously suggest
that complexes interact with DNA most likely through a mode that
involves a stacking interaction between the aromatic chromophore
and the base pairs of DNA.
This is probably one of the main reasons for the diverse
antibacterial activities shown by the complexes. The significant
improvement of the activity of enrofloxacin when coordinated to
the copper complex is simply an evidence of the role of the coordi-
nated metal ion.
3.6.2. Viscosity measurement
Binding modes of the complexes were further investigated by
viscosity measurements. Though photophysical experiments, give
necessary information about binding modes of metal complexes
with DNA, they do not provide conclusive evidences for the exact
mode of binding. Hydrodynamic measurements such as viscosity
which is sensitive to length changes are regarded as the least uncer-
tain and the most critical tests of binding modes in solution [48].
Viscosity of DNA is increased in the case of classical intercalator due
to increase in the length of DNA helix, as base pairs are separated
to accommodate the intercalator.
There are many cases in which complexes having square pyra-
midal geometry and shows classical intercalation [49–51]. The
binding of DNA with complexes depends upon nature of ligands.
In present study, the bidentate ligands which have different sub-
stituent’s and intercalation in nature. Spectral characteristics are
consistent with a mode of interaction that involves a stacking inter-
action between the complex and the base pairs of DNA, which
means that the complexes can intercalate into the double helix
structure of DNA.
3.6. Complex-DNA interaction
3.6.1. Absorption titration
Complex binding with CT-DNA through intercalation usually
result in hypochromism and bathochromism, due to intercalative
mode involving a strong stacking interaction between an aromatic
chromophore and the base pairs of DNA [43,44].
The binding of Cu(II) complexes to duplex DNA led to decrease
in the absorption intensities with a small amount of red shifts in
the UV–Vis absorption spectra. After intercalating the base pairs of
DNA, the ␲* orbital of the intercalated ligand can couple with the
␲ orbital of base pairs, thus decreasing the ␲–␲* transition energy
and resulting in the bathochromism [45,46].
On the other hand, the coupling ␲ orbital is partially filled by
electrons, thus decreasing the transition probabilities and concomi-
tantly resulting in hypochromism. The binding constant (Kb) of the
complexes to DNA were determined by monitoring the changes of
absorbance at 281–284 nm with increasing concentration of DNA
(Fig. 2). The appreciable decrease in absorption intensity and sig-
nificant red shift of the ␲–␲* band of complexes is similar to that
observed for its interaction with DNA in DMSO solution, suggest-
ing that the complex bind to DNA strongly. The binding constant
(Kb) of complexes (Table 3) are in the range of 1.3 × 104–3.7 × 104.
From Fig. 3, as increasing the amounts of complexes, the viscos-
ity of DNA increases steadily, which is similar to that of the classical
intercalative complex [Ru(dmb)2(ipbp)]+2 [47]. The binding ability
of complexes is more compare to enrofloxacin and less compare
to classical intercalator ethidium bromide. Among all complexes,
complexes 2, 3 and 5 bind more strongly than 1 and 5.
3.6.3. Salt effect
The overturn salt titration is an efficient method to discrimi-
nate binding modes between the bound molecules and DNA. The
detailed results are collected in Table 4. The binding constants for
all the complexes reported were obtained over the concentration
range 0.005–0.100 M NaCl in order to apply polyelectrolyte the-
ory to the calculation of the non-electrostatic binding constants
and separate the binding free energy change into its electrostatic
and non-electrostatic contributions. The salt concentrations of
0.005–0.100 M were selected in this study because the polyelec-
trolyte theories which will be used for successive analysis are based
Table 3
Binding constants (Kb) and IC50 value of the complexes.
Complexes
Kb (M−1
)
IC50
[Cu(erx)(A1)Cl]
[Cu(erx)(A2)Cl]
[Cu(erx)(A3)Cl]
[Cu(erx)(A4)Cl]
[Cu(erx)(A5)Cl]
1.31 × 104
3.63 × 104
1.57 × 104
1.65 × 104
1.61 × 104
0.875
1.45
0.5
1.25
0.8

768
M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
where Z is estimated from the slope of the decay line in Fig. 4.
Z is partial charge on the binding ligand involved in the DNA
interaction, is the fraction of counterions coupled with each
DNA phosphate ( = 0.88 for double-stranded B-form DNA), ꢆ
is the mean activity coefficient at cation concentration M+, and
the remaining terms are constants for double stranded DNA in
B-form, i.e. ꢅ = 4.2 and ı = 0.56. Results of calculations are summa-
0
rized in Table 4 along with percentage of Kt contribution to the
total binding constants (Kb) at various concentrations of Na+. These
0
Kt can be taken as measure large non-electrostatic forces stabi-
lize the ligand–DNA interaction. In contrast to the Kb values which
0
are salt-dependent, the magnitude of Kt is constant throughout
the concentration of NaCl employed with the average value of
3.89 × 104 M−1 bp (RSD = 12.5%). Although the values of Kt0 are con-
0
stant throughout the concentrations of salt, the percentage of Kt
contributions to the Kb increases significantly and reach a maxi-
mum of 35.5% at [Na+] = 0.1 M. It can be expected that at higher
concentrations of salt.
Fig. 3. Effect on relative viscosity of DNA under the influence of increasing amount
of complexes at 27 0.1 oC in phosphate buffer.
Analysis is also possible to dissect the binding free energy
change ( G0) for the binding of copper(II) to CT-DNA into its elec-
trostatic ( Gpe0) and non-electrostatic ( Gt0) contributions at a
given concentration of NaCl. Table 5 summarizes the results of ener-
getics calculation for the binding of Cu(II) to CT-DNA in 0.050 M
NaCl along with those of other copper(II) complexes [53,54] for the
purpose of comparison. The total binding free energy changes listed
in Table 5 were calculated based on the standard Gibbs relation.
The observed binding constant Kb is a function of the charge
on the cation (Z), the fraction of counter ion associated with each
DNA phosphate ( ) which is generally taken to be 0.88 for double-
stranded B-form DNA. A slope in a plot of log Kb versus log[Na+] is
equal to SK in Eq. (3):
Table 4
Equilibrium binding constant (Kb) for the binding of complex-I to DNA.
0
[NaCl] (M)
Kb (M−1 bp) × 104
Kt (M−1 bp) × 104
Kt0/Kb (%)
0.005
0.025
0.050
0.075
0.100
3.367
2.020
1.662
1.273
0.9233
0.3626
0.4158
0.4438
0.3991
0.3278
10.96
20.58
26.67
31.35
35.5
Average: 0.389 × 104
on limiting laws that are exactingly applicable to salt concentra-
tions of lower than 0.100 M [52].
The plot of log[Na+] against log Kb for the binding of copper(II)
complexes to CT-DNA is given in Fig. 4. It is clear from the plots that
the binding constant decreases with increasing salt concentration.
Using the slope of Fig. 4, we calculated non-electrostatic binding
constant (Kt0) at various concentrations of NaCl ([M+]) using Eq.
(2):
ı log Kb
ı log [Na+]
SK =
= −Z
(3)
G0 = −RT ln Kb
Gp0e = SKRT ln[Na+]
(4)
(5)
Gt0
=
G0
−
Gp0e
(6)
0
ln Kb = ln Kt + Zꢅ−1 {ln(ꢆ ı)} + Z (ln[M+])
(2)
The binding free energy can be calculated from Eq. (4). Elec-
trostatic (ꢄGp0e) and nonelectrostatic (ꢄGt0) of the free energy can
be calculated from Eqs. (5) and (6), respectively. A nonelectrostatic
free energy ꢄGt0 was derived to be −20.4 kJ mol−1, and electrostatic
free energy ꢄGp0e of −6.3 kJ mol−1 in 50 mM NaCl. It is apparent
that electrostatic contribution to the free energy is much less than
nonelectrostatic portion, strongly supporting theory that the ter-
pyridine moiety is intercalated between base pairs of the DNA helix
[55].
The result of quantitative analysis suggests that the main
contribution to the stabilization of DNA binding comes from non-
electrostatic interaction as indicated by their Gt0 values is at 87%
relative to the total binding free energy change.
3.6.4. Gel electrophoresis
Cleavage of plasmid pUC19 DNA by synthesized complexes was
monitored by agarose gel electrophoresis technique. When plasmid
DNA was subjected to electrophoresis after interaction, upon illu-
mination of gel (Fig. 5) the fastest migration was observed for super
coiled (SC) Form I, where as the slowest moving was open circular
(OC) Form II and the intermediate moving is the linear (NC) Form III
generatedon cleavageofopen circular. Thedata ofplasmid cleavage
are presented in Table 6. All the complexes show higher DNA cleav-
age ability compare to the drug and metal salt. Complexes 1–5 do
not show inhibition of DNA cleavage in the presence of scavengers
of hydroxyl radicals (DMSO) (Supply 1) and singlet oxygen (sodium
azide) (Supply 2). This indicates that the cleavage of DNA probably
Fig. 4. The dependence of the DNA binding constant of the Cu(II) complex on the
salt concentrations.

M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
769
Table 5
Thermodynamic parameter for the binding of copper(II) complexes to CT-DNA.
0
0
Binding ligand
Kb (104 M−1 bp)
G0
SK
G0
Kt0/103 (%Kt0/Kb) M−1 bp)
Gt (% Gt
/
Go)
pe
[Cu(erx)(A1)Cl]
[Cu(erx)(A2)Cl]
[Cu(erx)(A3)Cl]
[Cu(erx)(A4)Cl]
[Cu(erx)(A5)Cl]
1.662
1.522
1.680
1.544
1.589
−240.37
−239.19
−240.64
−238.55
−239.26
0.389
0.385
0.415
0.372
0.376
−28.82
−28.52
−30.75
−27.63
−27.86
3.626(26.67)
4.196(27.56)
4.119(24.51)
4.394(28.45)
4.460(28.06)
−211.55 (88.01)
−210.67 (88.07)
−209.89 (87.22)
−210.92 (88.41)
−211.40 (88.35)
Fig. 5. Photogenic view of interaction of pUC19 DNA (300 ␮g/mL) with of cop-
per(II) complexes (200 ␮M) using 1% agarose gel containing 0.5 ␮g/mL ethidium
bromide. All reactions were incubated in TE buffer (pH 8) in a final volume of 15 ␮L,
for 3h. at 37 ◦C. Lane 1, DNA control; Lane 2, CuCl2·2H2O; Lane 3, enrofloxacin;
Lane 4, [Cu(erx)(A1)Cl]; Lane 5, [Cu(erx)(A2)Cl]; Lane 6, [Cu(erx)(A3)Cl]; Lane 7,
[Cu(erx)(A4)Cl]; Lane 8, [Cu(erx)(A5)Cl].
Fig. 7. Plot of percentage of inhibiting NBT reduction with an increase in the con-
centration of complex 1.
Table 6
Gel electrophoresis data.
1. Compounds exhibit SOD-like activity at biological pH with their
IC50 values ranging from 0.5 to 1.45 ␮M. The superoxide scavenging
data are presented in Table 3. The higher IC50 can only be accredited
to the vacant coordination site facilitating the binding of super-
oxide anion, electrons of aromatic ligands that stabilize Cu–O2
interaction and not only to the partial dissociation of complex in
solution.
Compounds
% SC
% OC
% LC
% Cleavage
DNA control
DNA + Metal salt
DNA + Enrofloxacin
DNA + I
DNA + II
DNA + III
68
57
35
19
23
22
26
21
14
17
24
34
31
32
26
31
18
26
41
47
46
46
48
48
–
16.17
48.52
72.05
66.17
67.67
61.76
69.11
•−
DNA + IV
DNA + V
Acknowledgements
Authors thank Head, Department of Chemistry, Sardar Patel Uni-
versity, India, for making it convenient to work in laboratory and
U.G.C. for providing financial support under “UGC Research Fellow-
ship in Science for Meritorious Students” scheme.
follows a hydrolytic cleavage mechanism. Such DNA hydrolysis by
copper(II) complexes have been reported earlier [56].
3.7. SOD mimic activity
Appendix A. Supplementary data
The system used as a basis of superoxide radical generator in
order to check SOD like activity of the synthesized complexes was
NBT/NADH/PMS system. Absorbance at a function of time was plot-
ted to have a straight line obeying equation Y = mX + C (Fig. 6).
Fig. 7 shows percentage inhibition of reduction of nitro blue
tetrazolium (NBT) plotted against concentration of the complex-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2010.11.056.
References
[1] G. Klopman, S. Wang, M.R. Jacobs, S. Bajaksouzian, K. Edmonds, J.J. Ellner,
Antimicrob. Agents Chemother. 37 (1993) 1799–1806.
[2] L. Shen, A.G. Pernet, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 307–311.
[3] L.L. Shen, in: D.C. Hopper, J.S. Wolfson (Eds.), Quinolone Antimicrobial Agents,
2nd ed., American Society for Microbiology, Washington, DC, 1993, p. p77.
[4] A. Tarushi, C.P. Raptopoulou, V. Psycharis, A. Terzis, G. Psomas, D.P. Kessissoglo,
Bioorg. Med. Chem. 18 (2010) 2678–2685.
[5] K. Pinar, M. Burcu, Z. Aysin, O. Mehmet, Anal. Chim. Acta 518 (2004) 69–76.
[6] S. Pankaj, G.K. Werner, Anal. Chem. 69 (1997) 3552–3557.
[7] D.S. Sigman, D.R. Graham, V.D. Aurora, A.M. Stern, J. Biol. Chem. 254 (1979)
12269–12272.
[8] S.A. Ross, M. Pitie, B. Meunier, Eur. J. Inorg. Chem. 3 (1999) 557–563.
[9] L.Z. Li, C. Zhao, T. Xu, H. Ji, Y. Yu, G. Guo, H. Chao, J. Inorg. Biochem. 99 (2005)
1076–1082.
[10] J. Alos, Quinolones. Enferm. Infec. Microbiol. Clin. 21 (2003) 261–267.
[11] J. Casanova, G. Alzuet, J. Borrhs, J. Timoneda, S.G. Gradna, I.C. Gonzalex, J. Inorg.
Biochem. 56 (1994) 65–76.
[12] D.M. Miller, G.R. Buttner, S.D. Aust, Biol. Med. 8 (1990) 95–108.
[13] L.M. Rossi, A. Neves, A.J. Bortoluzzi, R. Horner, B. Szpoganicz, H. Terenzi, A.S.
Mangrich, E. Pereira-Maia, E.E. Castellano, W. Haase, Inorg. Chim. Acta 358
(2005) 1807–1822.
[14] M. Roy, S. Saha, A.K. Patra, M. Nethaji, A.R. Chakravarty, Inorg. Chem. 46 (2007)
4368–4370.
[15] M. Roy, T. Bhowmick, R. Santhanagopal, S. Ramakumar, A.R. Chakravarty, Dalton
Trans. (2009) 4671–4682.
Fig. 6. Plot of absorbance values(Abs560) against time (t).

770
M.N. Patel et al. / Spectrochimica Acta Part A 78 (2011) 763–770
[16] A.I. Vogel, Textbook of Quantitative Inorganic Analysis, 4th ed., ELBS and Long-
man, London, 1978.
[17] P. Pascal, Compt. Rend. 57 (1944) 218.
[18] L.J. Henderson Jr., F.R. Fronczek, W.R. Cherry, J. Am. Chem. Soc. 106 (1984)
5876–5879.
[19] C. Hiort, P. Lincoln, B. Norden, J. Am. Chem. Soc. 115 (1993) 3448–3454.
[20] G.F. Smith, Jr. Cagle, J. Org. Chem. 12 (1947) 781–784.
[21] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933–3940.
[22] G. Cohen, H. Eisenberg, Biopolymer 8 (1969) 45–55.
[23] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047–3053.
[24] M.T. Carter, M. Rodriguez, A. Bard, J. Am. Chem. Soc. 111 (1989) 8901–8911.
[25] R.E. Holmlin, E.D.A. Stemp, J.K. Barton, Inorg. Chem. 37 (1998) 29–34.
[26] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3051–3058.
[37] D.L. Klayman, J.P. Sconill, J.F. Bafosevich, J. Bruce, J. Med. Chem. 26 (1983) 35–39.
[38] K.C. Agrawal, A.C. Santorelli, J. Med. Chem. 21 (1978) 218–221.
[39] R.C. Sharma, S.P. Trpathi, R.S. Sharma, Curr. Sci. 50 (1981) 748–750.
[40] Y. Anjaneyula, R.P. Rao, Synth. React. Inorg. Met. Org. Chem. 16 (1986) 257.
[41] P.K. Panchal, P.B. Pansuriya, M.N. Patel, J. Enzyme Inhib. Med. Chem. 21 (2006)
453–458.
[42] H.W. Rossmore, Disinfection, Sterilization and Preservation, 4th ed., Lea &
Febiger, Philadelphia, 1991, pp. 290–321.
[43] B. Peng, H. Chao, B. Sun, H. Li, F. Gao, L.-N. Ji, J. Inorg. Biochem. 100 (2006)
1487–1494.
[44] P.U. Maheswari, M. Palaniandavar, J. Inorg. Biochem. 98 (2004) 219–230.
[45] V. Uma, M. Kanthimathi, T. Weyhermuller, B. Unni Nair, J. Inorg. Biochem. 99
(2005) 2299–2307.
[46] S. Ramakrishnan, M. Palaniandavar, J. Chem. Sci. 117 (2005) 179–186.
[47] Y.J. Liu, X.Y. Guan, X.Y. Wei, L.X. He, W.J. Mei, J.H. Yao, Transit. Met. Chem. 33
(2008) 289–294.
[27] S. Mahadevan, M. Palaniandavar, Inorg. Chem. 37 (1998) 693–700.
[28] V. Ponti, M.V. Dianzaini, K.J. Cheesoman, T.F. Stater, Chem. Biol. Interact. 23
(1978) 281–291.
[48] G. Yang, J.Z. Wu, L. Wang, L.N. Ji, X. Tian, J. Inorg. Biochem. 66 (1997) 141–144.
[49] R. Rao, A.K. Patra, P.R. Chetana, Polyhedron 27 (2008) 1343–1352.
[29] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Com-
pounds, 4th ed., Wiley, New York, 1986.
[50] P.R. Reddy, N. Raju, P. Manjula, K.V.G. Reddy, Chem. Biodivers.
1565–1577.
4 (2007)
[30] I. Turel, Coord. Chem. Rev. 232 (2002) 27–47.
[51] Y.-L. Song, Y.-T. Li, Z.-Y. Wu, J. Inorg. Biochem. 102 (2008) 1691–1699.
[31] I. Turel, I. Leban, N. Bukovec, J. Inorg. Biochem. 66 (1997) 241–247.
[32] H.H. Freedman, J. Am. Chem. Soc. 83 (1961) 2900–2905.
[33] A.B.P. Lever, Inorganic Electronic Spectroscopy, 2nd ed., Elsevier, Amsterdam,
1984.
[52] I. Haq, P. Lincoln, D. Suh, B. Norden, B.Z. Chowdhry, J.B. Chaires, J. Am. Chem.
Soc. 117 (1995) 4788–4796.
[53] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992)
9319–9324.
[34] R. Carballo, A. Castineiras, B. Covelo, E. Garcia-Martinez, J. Niclos, E.M. Vazquez-
Lopez, Polyhedron 23 (2004) 1505–1518.
[35] W.E. Anthroline, J.M. Knight, D.H. Petering, Inorg. Chem. 16 (1977) 569–574.
[36] D.H. Jones, R. Slack, S. Squires, K.R.H. Wooldrige, J. Med. Chem. 8 (1965)
676–680.
[54] K. Mudasir, N. Wijaya, H. Yoshioka, J. Inorg. Biochem. 94 (2003) 263–271.
[55] Mudasir, K. Wijaya, E.T. Wahyuni, N. Yoshioka, H. Inoue, Biophys. Chem. 121
(2006) 44–50.
[56] A. Barve, A. Kumbhar, M. Bhat, B. Joshi, R. Butcher, U. Sonawane, R. Joshi, Inorg.
Chem. 48 (2009) 9120–9132.