Study of SOD mimic and nucleic acid interaction activity exerted by enrofloxacin-based copper(II) complexes

Article abstract of DOI:10.1002/cbdv.201100372

Five new copper(II) complexes of type [Cu(erx)(L)Cl] (erx, enrofloxacin; thiophene-2-carbaldehyde (L¹); pyridine-2-carbaldehyde (L ²); 2,2′-dipyridylamine (L³); 4,5-diazafluoren-9-one (L⁴); bis(3,5-dimethyl-1-py

Full text of DOI:10.1002/cbdv.201100372

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Study of SOD Mimic and Nucleic Acid Interaction Activity Exerted by
Enrofloxacin-Based Copper(II) Complexes
by Mohan N. Patel*, Bhupesh S. Bhatt, and Promise A. Dosi
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar–388120, Gujarat, India
(phone: (þ 912692)226856*218; e-mail: jeenen@gmail.com)
Five new copper(II) complexes of type [Cu(erx)(L)Cl] (erx, enrofloxacin; thiophene-2-carbalde-
hyde (L1); pyridine-2-carbaldehyde (L2); 2,2’-dipyridylamine (L3); 4,5-diazafluoren-9-one (L4); bis(3,5-
dimethyl-1-pyrazolyl)methane (L5)) have been synthesized and characterized by elemental analysis,
reflectance, IR, and FAB-MS. Complexes have been investigated for their interaction with calf thymus
(CT) DNAutilizing the absorption-titration method, viscometric and DNA thermal denaturation studies.
The cleavage reaction on pUC19 DNA has been monitored by agarose gel electrophoresis. The results
indicated that the CuII complexes can more effectively promote the cleavage of plasmid DNA at
physiological pH and superoxide dismutase. The (SOD) activity of the complexes has been evaluated by
the nitroblue tetrazolium assay, and the complexes catalyzed the dismutation of superoxide at pH 7.8 with
IC50 values of 0.35–1.25 mm. The complexes have also been screened for their antibacterial activity
against five pathogenic bacteria.
Introduction. – The study of DNAꢀmetal interactions constitutes a growing
research area. In addition to their potential use as therapeutic agents, they are being
considered as tools for biochemistry and molecular biology [1]. DNA plays an
important role in the life process, because it bears genetic information and instructs the
biological synthesis of proteins and enzymes through the replication and transcription
of genetic information in living cells. DNA is a particularly good target for metal
complexes, as it offers a wide variety of potential metal-binding sites [2–5]. Such sites
include the electron-rich DNA bases or phosphate groups that are available for direct
covalent coordination to the metal center. There are noncovalent binding modes as
well, such as H-bonding and electrostatic binding to grooved regions of the DNA and
intercalation of planar aromatic ligands into the stacked base pairs.
Copper is found in a variety of enzymes, including the Cu-centers of cytochrome-c
oxidase and the enzyme superoxide dismutase (SOD; containing Cu and Zn), and is
the central metal in the oxygen-carrying pigment hemocyanin. In addition to its
enzymatic roles, Cu is used for biological electron transport. Copper-superoxide
dismutase (Cu-SOD) is believed to protect the cell against oxidative damage and
inflammation due to toxic oxygen intermediates by the catalytic dismutation of
superoxide radicals to O2 molecules and H2O2 [6].
Fluoroquinolone antibacterial agents represent a fast-growing group of antibiotics;
various derivatives were synthesized and tested for their antimicrobial activities. The
new generations of fluoroquinolones achieved significant improvement in potency,
spectrum, and physicochemical properties [7]. ꢀQuinolonesꢁ is a term commonly used
ꢂ 2012 Verlag Helvetica Chimica Acta AG, Zꢃrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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for the quinolonecarboxylic acids or 4-quinolones, a group of synthetic antibacterial
agents containing a 1,4-dihydro-4-oxoquinoline skeleton [8–10]. Quinolones are
extremely useful for the treatment of diverse infections [11–13]. The activity of
quinolones as antibacterial drugs is due to the effective inhibition of DNA replication.
The proposed mechanism of the interaction between quinolone and metal ions was
chelation between the metal, and the 4-oxo and adjacent COOH groups [14].
Enrofloxacin is a typical second-generation quinolone antimicrobial drug with a broad
spectrum of activity against a wide range of Gram-negative and Gram-positive
bacteria, including those resistant to b-lactam antibiotics and sulfonamides [15][16]. It
is also used for the treatment of urinary-tract infections, pyelonephritis, sexually
transmitted diseases, prostatitis, skin and tissue infections, and urethral and cervical
gonococcal infections [17–19]. Due to such biological activities of enrofloxacin and
extensive bioavailability of CuII, we have synthesized five mononuclear CuII complexes
with enrofloxacin and different heterocyclic units expecting that coordination could
enhance the biological activity of enrofloxacin by increasing lipophilicity of the drug.
This article mainly focuses on exploring the trend in DNA-binding affinities of
complexes and the important differences in some related properties. Understanding the
features that contribute to recognition of DNA by small ligands or metal complexes is
crucial for the development of drugs targeted at DNA.
Results and Discussion. – Characterization of Complexes. All the complexes were
characterized using elemental analysis, conductance, magnetic measurements, reflec-
tance, and IR and FAB-MS. The elemental analysis is in accordance with the proposed
1:1:1 metal/erx/Ln ratio. The molar-conductance values were determined as described
by Kulkarni et al. using DMF as solvent [20]. The conductance values were too low to
account for presence of any hydrolyzable chloride ions in complexes, indicating the
nonelectrolytic nature of the complexes.
The diffuse reflectance spectra of CuIIꢀenrofloxacin complexes consist of one
asymmetric broad band center around 15,000 cmꢀ1 for all the compounds, character-
istic of distorted square-pyramidal geometry [21].
The observed magnetic moments of CuII complexes are given in Table 1. The best
compilation of the results on the magnetic behavior of Cu compounds was provided by
Figgis, Nyholm, and co-workers [22]. At room temperature, the magnetic moments of
the complexes lie in the range of 1.81–1.90 B.M., which is higher than the spin-only
value of 1.73 B.M.
A comparison of the IR spectra of the complexes with those of the free ligands
revealed interesting features related to the metalꢀligand interactions. In the IR
spectrum of enrofloxacin, the valence vibration of the carboxylic stretch n(C¼O) was
found at 1733 cmꢀ1 and the pyridone stretch n(C¼O) at 1622 cmꢀ1. The character-
ization of quinolone metal complexes could be achieved by studying the most typical
vibrations that are characteristic of the coordination type of quinolones. In Table 2, the
characteristic absorptions in the IR spectra of the complexes are collected. In the IR
spectra of the complexes 1–5, the absorption at n(C¼O) is replaced by two very strong
characteristic bands in the range of 1559–1578 and 1348–1374 cmꢀ1 assigned to
asymmetric (n(COO)as) and symmetric (n(COO)s) vibrations. The difference D¼
n(COO)as ꢀn(COO)s is a useful characteristic for determining the coordination mode

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Table 2. IR-Spectral Data of Complexes and Enrofloxacin
Compounds
n(C¼O) [cmꢀ1
]
n(COO)asy
n(COO)sym
Dn
[cmꢀ1
n(MꢀN)
n(MꢀO)
of pyridone
[cmꢀ1
]
[cmꢀ1
]
]
[cmꢀ1
]
[cmꢀ1
]
Enrofloxacin
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
1733
1621
1619
1618
1621
1617
1622
1561
1578
1569
1569
1559
1340
1359
1374
1371
1355
1348
284
202
204
198
214
211
–
532
535
538
545
541
–
507
506
510
511
513
of the ligands. The D values fall in the range of 202–214 cmꢀ1 indicating a monodentate
coordination mode of the carboxylato group [23–27]. The pyridone stretch, n(C¼O), is
slightly shifted from 1617 to 1621 cmꢀ1 upon bonding. The overall changes of the IR
spectra indicate that enrofloxacin is coordinated to the metal via the pyridone and one
carboxylate O-atoms.
All the synthesized complexes show the molecular-ion peaks associated with
different number of H-atoms. Fig. 1 represents the FAB-MS of [Cu(erx)(L1)Cl] (1),
obtained using 3-nitro-benzyl alcohol as matrix. Peaks at m/z 136, 137, 154, 289, and 307
are due to the matrix. Doublets appeared at 568, 570; 456, 458; and 209, 211 for the
fragments with a single Cl-atom, respectively. Loss of the Cl-atom gave a fragment-ion
peak at m/z 533, which also confirms that the Cl-atom was attached to metal ion
with covalent bond. Several other peaks for fragments with m/z 421, 359, 174, and 112
were also detected. Similar patterns were also observed for the other synthesized
complexes.
Fig. 1. FAB Mass spectrum of [Cu(erx)(L1)Cl] (1) obtained using 3-nitrobenzyl alcohol
Antibacterial Activity. The efficiency of the ligands and the complexes have been
tested against three Gram-negative (Escherichia coli, Serratia marcescens, and
Pseudomonas aeruginosa) and two Gram-positive (Staphylococcus aureus and Bacillus
subtilis) microorganisms. The results of the minimum inhibitory concentration (MIC)

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 3. MIC Data of the Compounds [mm]
Compounds
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
CuCl2 ·2 H2O
Enrofloxacin
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
2698.00
1.9
2815.00
3.9
2756.00
1.7
2404.00
1.4
3402.00
1.4
1.0
0.7
0.5
1.2
2.1
1.2
1.5
1.7
1.3
0.7
0.9
0.8
1.4
1.2
1.2
0.9
0.7
0.3
0.5
0.8
0.9
1.9
0.7
1.1
0.7
expressed in mm are presented in Table 3. All compounds exhibited good activities
against all microorganisms compared to reference drugs. The complexes showed better
antimicrobial activities than the corresponding free ligands and enrofloxacin. The
higher antimicrobial activities can be mainly attributed to the presence of the
quinolone chelated with the complexes. Comparing the results with those of the
previously reported CuII complexes of pefloxacin and sparfloxacin with similar ligands
L1, L2, and L3 [26], we can state that replacement of pefloxacin and sparfloxacin with
enrofloxacin enhances antibacterial activities of all compounds except for [Cu(P-
FL)(A2)Cl]·5 H2O against P. aeruginosa and E. coli.
Chelation reduces the polarity [28][29] of the metal ion mainly because of the
partial sharing of its positive charge with the donor groups and possibly the p- electron
delocalization within the whole chelate ring system thus formed during coordination.
This process of chelation increases the lipophilic nature of the central metal atom,
which in turn favors its permeation through the lipoid layer of the membrane,
increasing the hydrophobic character and liposolubility of the molecule in crossing cell
membrane of the microorganism, and hence enhances the biological utilization ratio
and activity of the tested drugs/compounds.
In addition, our study regarding bactericidal activity in terms of colony-forming
units (CFU)/ml of the metal complexes against same microorganisms revealed
decrease in number of colonies with increasing concentration of compounds. The
results are shown in Fig. 2 for all the complexes against S. aureus. The number of
colonies counted in this technique was 30–250 CFU/ml.
DNA-Binding Properties. Electronic absorption spectroscopy is employed to
determine the binding characteristics of metal complex with DNA. Fig. 3 shows the
absorption spectra of complex 1 in the presence of increasing amounts of calf thymus
(CT) DNA. Absorption titration experiments of CuII complexes in buffer were
performed using a fixed copper concentration to which increments of the DNA stock
solution were added. The binding of CuII complexes to duplex DNA led to decrease in
the absorption intensities with a small amount of red shifts in the UV/VIS absorption
spectra. In the UV region, the intense absorption bands observed in the CuII complexes
are attributed to the intraligand p–p* transition of the coordinated groups. Addition of
increasing amounts of CT-DNA resulted in hypochromism and bathochromic shift in
the UV spectra of all complexes due to intercalative mode involving a strong stacking
interaction between an aromatic chromophore and the nucleobases of DNA base stack
[30].

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fig. 2. Relationship between concentration and bactericidal activity of all complexes against S. aureus
(CFU, colony-forming unit)
Fig. 3. Electronic absorption titration curve of [Cu(erx)(L1)Cl] in absence and in presence of increasing
amount of CT-DNA in 5 mm Tris·HCl buffer (pH 7.2). [Complex]¼15 mm, [DNA]¼50–150 mm with an
incubation period of 30 min. at 278. Inset: Plot of [DNA]/(ea ꢀef) vs. [DNA].
The binding constants (Kb) of complexes are calculated in the range of 1.17ꢁ104 –
2.96ꢁ104 mꢀ1 (Table 4). The Kb values of metal complexes were greater than those
previously reported for CuII complexes of pefloxacin and sparfloxacin except for
[Cu(PFL)(A3)Cl]·5 H2O [26]. Comparing the intrinsic binding constant of complexes

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
with that of the DNA-intercalative [Ru(bpy)2ppd]þ2 (1.18ꢁ104 mꢀ1) complex [31], we
can deduce that all of the complexes bind moderately to DNA by intercalation. The Kb
values of complexes 1–5 are greater than those of CrIII and CrIV complexes reported by
Guo et al. [32], comparable to those of FeIII and CoII complexes reported by Psomas
[33], and ZnII complexes reported by Kessissoglou and co-workers [34] while lower
than those of ZnII complexes reported by Kessissoglou and co-workers [35]. The
spectral characteristics obviously indicate that the metal complexes interact with DNA
most likely through intercalation mode.
Table 4. Binding Constants, and IC50 and LC50 Values of CuII Complexes
Complexes
Kb [mꢀ1
]
IC50 [mm]
LC50 [mm]
[Cu(erx)(L1)Cl] (1)
[Cu(erx)(L2)Cl] (2)
[Cu(erx)(L3)Cl] (3)
[Cu(erx)(L4)Cl] (4)
[Cu(erx)(L5)Cl] (5)
1.94ꢁ104
2.96ꢁ104
1.52ꢁ104
1.17ꢁ104
1.68ꢁ104
0.50
0.35
1.00
1.25
0.75
21.06
21.70
14.51
12.71
9.56
To further clarify the nature of the interactions between the complexes and DNA,
viscosity measurements were carried out. Optical and photophysical probes provide
necessary, but not sufficient evidence to support the model of binding of the CuII
complexes with DNA. Hydrodynamic measurements that are sensitive to length
change (i.e., viscosity) are regarded as the least ambiguous and the most critical tests of
a binding model in solution in the absence of crystallographic structural data [36][37].
Intercalation is expected to lengthen the DNA helix, as the base pairs are pushed apart
to accommodate the bound ligand, leading to an increase in the DNA viscosity. In
contrast, a partial, non-classical intercalation of the ligand could bend (or kink) the
DNA helix, reduce its effective length and, concomitantly, its viscosity. The effects of
complexes, together with enrofloxacin and ethidium bromide (EB), on the viscosity of
rod-like DNA are shown in Fig. 4. The well-known DNA intercalator EB increases the
viscosity of DNA with increments of the concentration. Upon increasing the amount of
complexes, the relative viscosity of DNA increased steadily, but less than with EB. The
increase of the relative viscosity, expected to correlate with the DNA-intercalating
potential of a compound, follows the order EB >2>1>5>3>4 > enrofloxacin. The
results are consistent with previously reported metal complexes of type [Cu(PFL/
SPFL)(An)Cl]·5 H2O [26]. These results suggest that all the title complexes intercalate
between the base pairs of DNA, with the difference in binding strength probably being
caused by the different substituent on ancillary ligands.
The thermal behavior of DNA in the presence of complexes can provide insight into
their conformational changes when the temperature is raised, and offer information
about the interaction strength of the complexes with DNA. It is well-known that, when
the temperature of the solution is increased, the double-stranded DNA gradually
dissociates to single strands, generating a hyperchromic effect in the absorption spectra
of the DNA bases (lmax 260 nm). To identify this transition process, the melting
temperature, Tm, which is defined as the temperature where half of the total base pairs
are unbonded, is usually determined. According to the literature [38][39], the

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Fig. 4. Relative viscosity of DNA under the influence of increasing amounts of complexes at 27ꢂ0.18 in
5 mm Tris ·HCl buffer (pH 7.2) as a medium
intercalation of metallo-intercalators generally results in a considerable increase of Tm.
The melting curves of CT-DNA in the absence and presence of the complexes are
presented in Fig. 5. Here, the thermal denaturation experiment carried out for DNA in
the absence of the CuII complexes revealed a Tm value of 74.28 under our experimental
conditions. On addition of complexes 1–5, Tm of DNA increased to 79.2, 79.5, 78.4,
78.1, and 78.98, respectively, at a concentration ratio [DNA]/[Cu] of 5 :1. The large
increase, i.e. 3.9–5.3, in Tm values is comparable to that observed for classical
intercalators.
DNA-Cleavage Study. There has been considerable interest in nucleolytic cleavage
reactions that are activated by complexes [40–42]. The ability of the all metal
complexes to cleave DNA has been investigated by gel electrophoresis using pUC19
DNA. The cleavage of plasmid pUC19 DNA was monitored by gel electrophoresis to
investigate the ability of the present CuII complexes to serve as metallonucleases
(Fig. 6). The naturally occurring supercoiled form (Form I), when nicked, gives rise to
an open circular relaxed form (Form II) and further cleaves to a linear form
(Form III). When subjected to gel electrophoresis, Form I shows the fastest migration
compared to Forms II and III. Form II migrates very slowly prior to its relaxed
structure, whereas Form III migrates somewhere between the positions of Form I and
II [43]. The quantitative study of DNA cleavage in different forms is compiled in
Table 5.
Superoxide Dismutase (SOD). The mixed-ligand CuII complexes were synthesized
as a model for the active site of Cu-SOD. The SOD-like activities of the complexes
were investigated by nitroblue tetrazolium (NBT) assay and catalytic activities toward
the dismutation of superoxide anion were measured. The complexes provide a stable
environment similar to that in the active site in the native enzyme, ensuring the stable

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 5. Melting curves of CT-DNA in the absence and presence of complexes 1–5
Fig. 6. Interaction of pUC19 DNA (300 mg/ml) with a series of CuII complexes (200 mm). Lane 1, DNA
control; Lane 2, CuCl2 ·2 H2O; Lane 3, enrofloxacin; Lane 4, [Cu(erx)(L1)Cl]; Lane 5, [Cu(erx)(L2)Cl];
Lane 6, [Cu(L3)(erx)Cl]; Lane 7, [Cu(erx)(L4)Cl]; Lane 8, [Cu(erx)(L5)Cl].
Table 5. Gel-Electrophoresis Data
Compounds
% SC
% OC
% L
DNA control
DNAþMetal salt
DNAþEnrofloxacin
DNAþ1
77
75
45
29
28
30
26
27
23
25
34
43
48
49
48
49
–
–
11
28
24
21
26
25
DNAþ2
DNAþ3
DNAþ4
DNAþ5
existence of the complex at the investigated pH value in aqueous solution. In this work,
the SOD-like activities were measured at pH 7.8. The percentage inhibition of
formazan formation at various concentrations of complexes as a function of time was

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2819
determined by measuring the absorbances at 560 nm and plotted to give a straight line
(Fig. 7); with increase in concentration of tested complexes, a decrease in slope [m] was
observed. The relationship between the inhibition [%] and initial concentration of the
complex is depicted in Fig. 8.
Fig. 7. Absorbance values (Abs560) vs. time (t) at different concentrations [mm] of complex 1
Compounds exhibited SOD-like activities at biological pH with the IC50 values in
the range 0.35 to 1.25 mm. The superoxide scavenging data (Table 4) suggest that the
complexes 1–5 have higher activity than those of the reported complexes of type
[Cu(sftz)(py)3Cl] [44]. The chromophore concentration required to yield 50%
inhibition of the reduction of NBT (IC50) was determined as described in [45].
Complexes 1, 2, and 5 have better radical scavenging activities than previously reported
metal complexes of pefloxacin and sparfloxacin [26]. The result is reasonable, because
the complexes 1, 2, and 5 are square planar with vacant coordination site, and the Cu-
.
center is accessible to O2ꢀ
.
Cytotoxocity. Brine shrimp lethality bioassay is a recent development in the assay
procedure of bioactive compound, which indicates cytotoxicity as well as a wide range
of pharmacological activities (such as anticancer, antiviral, insecticidal, pesticidal,
AIDS, etc.) [46][47]. All the synthesized compounds were screened for their
cytotoxicity (brine shrimp bioassay) according to the protocol of Meyer et al. [46].
From the data presented in Table 4, it is evident that compounds 4 and 5 displayed most
potent cytotoxic activities against Artemia cysts, while the others were almost
moderately active.
Conclusions. – The molecular-ion peaks in FAB-mass spectra for metal complexes
account for attachment of neutral heterocyclic ligand, mono-anionic enrofloxacin, and

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 8. Percentage of inhibition of NBT (nitroblue tetrazolium) reduction vs. concentration of complex 1
Cl-atom in 1:1:1 ratio. IR-Spectral data suggest that monoanionic enrofloxacin is
coordinated to the metal via the pyridone and one carboxylate O-atoms. Furthermore,
conductance indicate the absence of any hydrolyzable Clꢀ ion and also nonelectrolytic
nature of metal complexes. The square pyramidal geometry suggested by reflectance
spectra can only be possible if Clꢀ ion is attached via covalent bond to the CuII in the
metal complex making it neutral. The DNA-binding experiments suggest classical
intercalation modes of binding. Nucleolytic experiments show sufficient interaction of
metal complexes with DNA and hence support the DNA-binding data. The brine
shrimp lethality assay shows the potent cytotoxic nature of the compounds. The SOD-
like activity indicates the ability of metal complexes to scavenge free radical anions at
their vacant coordination site. The SOD-mimic ability of title metal complexes is even
higher than some known systems.
Experimental Part
General. All the chemicals and solvents were reagent-grade and used as purchased. Enrofloxacin
hydrochloride was purchased from Bayer AG (D-Wuppertal). CuCl2 ·2 H2O, thiophene-2-carbaldehyde
(L1), pyridine-2-carbaldehyde (L2), 2,2’-dipyridylamine (L3), and CT-DNA were purchased from Sd fine
chemicals, India. Ethidium bromide (EB) and Luria Broth were purchased from Himedia, India. AcOH
and ethylenediaminetetraacetic acid (EDTA) were purchased from Sd fine chemicals, India.
Metal contents of the complexes were analyzed gravimetrically and volumetrically [48], after
decomposing the org. matter with acid mixture (HClO4, H2SO4, and HNO3). C, H, and N elemental
analyses: Perkin-Elmer 240 elemental analyzer. Magnetic moments were measured by Gouyꢁs method
using mercury tetrathiocyanatocobaltate(II) as the calibrant (cg ¼16.44ꢁ10ꢀ6 cgs units at 208), on
Citizen Balance. The diamagnetic correction was made using Pascalꢁs constant. The molar-conductance
measurements were carried out on Equip-Tronics EQ-660A, conductivity meter (India). IR Spectra: FT-

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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IR Shimadzu spectrophotometer with sample prepared as KBr pellets in the range 4000–400 cmꢀ1
.
Reflectance spectra: LAMBDA 19 UV/VIS/NIR spectrophotometer, Perkin-Elmer (USA). FAB-MS:
Jeol SX 120/Da-600 mass spectrometer/data system using Ar/Xe (6 kV, 10 mA) as the FAB gas; the
accelerating voltage was 10 kV, and spectra were recorded at r.t.
Syntheses. The ligands 4,5-diazafluoren-9-one (¼ 5H-cyclopenta[1,2-b :5,4-b’]dipyridin-5-one; L4)
and bis(3,5-dimethyl-1-pyrazolyl)methane (¼1,1’-methanediylbis(3,5-dimethyl-1H-pyrazole); L5) were
synthesized according to literature methods [49][50].
The metal complex 1 was prepared by adding MeOH soln. of CuCl2 ·2 H2O (0.170 g, 0.001 mol) to
MeOH soln. of thiophene-2-carbaldehyde (L1) (0.112 g, 0.001 mol), followed by MeOH soln. of
enrofloxacin (0.374 g, 0.001 mol), while adjusting pH to 6.8. The resulting soln. was refluxed for 2 h on a
H2O bath, followed by concentrating it to half of its volume. A fine amorphous product of green color was
obtained which was washed with Et2O/hexane and dried in vacuum desiccators. The proposed reaction is
shown in the Scheme, and physical parameter of all the complexes are collected in Table 1. Remaining
complexes 2–5 were prepared by following the process described above.
Scheme. Formation of Complex [Cu(erx)(L1)Cl] (1)
In vitro Antibacterial Assay. Antibacterial screening on different bacterial strains was performed by
determining the minimum inhibitory concentration (MIC).The MIC value was defined as the lowest
concentration of antimicrobial agent showing complete inhibition of growth. The MIC values of the
reference drugs (ligands) were compared with those of the complexes. All cultures were incubated at 378.
Control tests with no active ingredients were also performed. The double dilution method was used for
MIC determination.
The bactericidal activities of all compounds were also evaluated against the same bacterial culture.
The inoculum was prepared by diluting a culture grown overnight in Luria Broth to obtain 106 viable
bacteria/ml, confirmed in each experiment by colony counts. Bacteria were exposed to concentrations of
0.25–1.75 mg mlꢀ1 of compounds. The final volume was 1 ml. Cultures were incubated at 378 for 2 h. The
100-ml bacterial cultures were taken and spread over previously prepared agar plates. These were
incubated for 24 h at 378, and the visual colonies were calculated in order to check biocidal activity of
metal complexes, yielding 30–250 colonies.
DNA-Binding Experiments. Viscosity Measurements. Viscosity experiments were carried on an
Ubbelohde viscometer, immersed in a thermostatic H2O-bath maintained at a constant temp. of 27ꢂ0.18.
DNA Samples of ca. 200 base pairs in average length were prepared by sonication in order to minimize
complexities arising from DNA flexibility [51]. Data were presented as (h/h0)1/3 vs. binding ratio [52],
where h is the viscosity of DNA in the presence of complex, and h0 is the viscosity of DNA alone.
Viscosity values were calculated from the observed flow time, t, of DNA-containing solns., t was
corrected for that of buffer alone, t0, h¼(t–t0).
Absorption Titration. Solns. of DNA in the buffer gave a ratio at UVabsorbances at 260 and 280 nm,
A260/A280, of 1.9, indicating that the DNA was sufficiently free of protein. The concentration of DNA was

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
determined from the band intensity at 260 nm with a known extinction coefficient value (e260 ¼6600mꢀ1
cmꢀ1). Absorption titration measurements were carried out by varying the concentration of CT-DNA
from 50 to 150 mm, while keeping the metal-complex concentration constant at 15 mm in the buffer soln.
Samples were incubated at 378 for 24 h before recording each spectrum. The intrinsic binding constant
(Kb) for the interaction of the complexes with CT-DNAwas determined from a plot of [DNA]/(ea ꢀef) vs.
[DNA] using absorption spectral titration data and the following equation [53]:
½DNAꢃ
½DNAꢃ
1
¼
þ
(1)
ðea ꢀ efÞ ðeb ꢀ efÞ Kbðeb ꢀ efÞ
where [DNA] is the concentration of DNA, the apparent absorption coefficients ea, ef, and eb correspond
to Aobs/[Cu], the extinction coefficient for the free CuII complex, and the extinction coefficient for the
CuII complex in the fully bound form, resp. The Kb value is given by the ratio of the slope to the intercept.
Thermal Denaturation. DNA Melting experiments were carried out by monitoring the absorption
intensity of CT-DNA (100 mm) at 260 nm in the temp. range of 35–908 with temp. increments of 18, both
in the absence and presence of the CuII complex (20 mm). Measurements were performed with an Agilent
8453 UV/VIS spectrophotometer. The melting temperature (Tm) of DNA was determined as the
midpoint of the optically detected transition curves. The DTm value was defined as the difference between
Tm of the free DNA and Tm of the bound DNA.
DNA Cleavage. For the gel-electrophoresis experiments, supercoiled pUC19 DNA (300 mg mlꢀ1
)
was treated with CuII complexes (200 mm) in 50 mm Tris, 18 mm NaCl buffer, pH 8.0, and the solns. were
incubated for 1 h in the dark, then irradiated at r.t. Reactions were allowed to proceed for 3 h at 378. All
reactions were quenched by addition of 5 ml of loading buffer (40% sucrose, 0.2% bromophenol blue).
The aliquots were loaded on to 1% agarose gel and submitted to electrophoresis at 50 V in 1X TAE
buffer. Gel was stained with 0.5 mg/ml ethidium bromide (EB) and photographed on UV illuminator. The
percentage of each form of DNA was quantified using AlphaDigiDocTM RT. Version V.4.1.0 PC-Image
software.
Superoxide Dismutase (SOD). The SOD activity was evaluated using the nitroblue tetrazolium
(NBT) assay according to the following scheme [53].
PMS=NADH
NBT
.
ꢀ
O2
O
blue formazan
(2)
ƒƒƒƒƒƒ!
ƒƒ!
2
Superoxide anions are produced from the NBT/NADH/PMS system. The indicator utilized in this
.
case is NBT, which reacts with O ꢀ2 to form blue formazan. The SOD model complex, added to the soln.,
inhibits the reaction by reacting with superoxide directly. The value of inhibition can thus be used to
determine activity of the SOD model complex.
Nonenzymatic system containing 30 mm PMS, 79 mm NADH, and 75 mm NBT, phosphate buffer
.
.
(pH 7.8) was used to produce superoxide anion (O ꢀ2 ) and the scavenging rate of O 2ꢀ was determined by
monitoring reduction rate of transformation of NBT to monoformazan dye under the influence of 0.25–
3.0 mm tested compound [45]. The concentration of the complex required to attain 50% inhibition of the
reduction (defined as IC50) was determined as an indication of SOD-like activity. The reactions were
monitored at 560 nm with a UV/VIS spectrophotometer, and the rate of absorption change was
determined. The % inhibition of NBT reduction was calculated using following equation.
% inhibition of NBT reduction¼(1ꢀk’/k)ꢁ100
(3)
where k’ and k are the slopes of the straight line of absorbance values as a function of time in the presence
and absence of SOD mimic compound, resp. The IC50 value of the complex was determined by plotting
the percentage of inhibition of NBT reduction vs. the increase in concentration of the complex. The
concentration of the complex which causes 50% inhibition of NBT reduction is defined as IC50
.
Cytotoxicity. Brine shrimp (Artemia cysts) eggs were hatched in a shallow rectangular plastic dish
(22ꢁ32 cm), filled with artificial seawater, which was prepared with commercial salt mixture and double

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2823
dist. H2O. An unequal partition was made in the plastic dish with the help of a perforated device.
Approximately 50 mg of eggs were sprinkled into the large compartment and was opened to ordinary
light. After 2 d, nauplii were collected by a pipette from the lighted side. A sample of the test compound
was prepared by dissolving 10 mg of each compound in 10 ml of DMSO. From these stock solns., solns.
were transfered to 18 vials to make final concentration 2, 4, 8, 12, 16, and 20 mg mlꢀ1 (three sets for each
dilutions were used for each test sample and mean of three sets was used for LC50 calculation), and three
vials were kept as control having same amount of DMSO only. When the nauplii were ready, 1 ml of
seawater and 10 shrimps were added to each vial, and the volume was adjusted with seawater to 2.5 ml
per vial. After 24 h, the number of survivors (Supplementary Table1)) was counted [46]. Data were
analyzed by simple logit method to determine the LC50 values (Table 4), in which log of concentration of
samples were plotted against percentage of mortality of nauplii [54].
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Received November 9, 2011