Copper(II) complexes with norfloxacin and neutral terpyridines: Cytotoxic, antibacterial, superoxide dismutase and DNA-interaction approach

Article abstract of DOI:10.1016/j.poly.2012.03.050

Cu(II) complexes of the type [Cu(NFL)(Lⁿ)Cl] (Lⁿ = substituted terpyridines, NFL = norfloxacin) were synthesised and characterized. Antibacterial activity was assayed against selective Gram^(+ve) and Gram^(-ve) mi

Full text of DOI:10.1016/j.poly.2012.03.050

Polyhedron 40 (2012) 159–167
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Polyhedron
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Copper(II) complexes with norfloxacin and neutral terpyridines: Cytotoxic,
antibacterial, superoxide dismutase and DNA-interaction approach
⇑
Mohan N. Patel , Hardik N. Joshi, Chintan R. Patel
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, 388120 Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Cu(II) complexes of the type [Cu(NFL)(Ln)Cl] (Ln = substituted terpyridines, NFL = norfloxacin) were syn-
thesised and characterized. Antibacterial activity was assayed against selective Gram(+ve) and Gram(ꢀve)
microorganisms using the double dilution technique. The binding behavior of the complexes toward Her-
ring Sperm (HS) DNA was determined using absorption titration and hydrodynamic measurements,
whereas the cleavage efficacy of the complexes toward pUC19 DNA was determined by the gel electro-
phoresis technique. The brine shrimp bioassay was carried out to study the in vitro cytotoxic properties of
the synthesized metal complexes. The superoxide dismutase (SOD) like activity of the complexes was
measured using an NBT/NADH/PMS system and is expressed in term of the concentration of complex
which terminates the formation of formazan by 50% (IC50 value). The IC50 values were observed to be
Article history:
Received 2 December 2011
Accepted 27 March 2012
Available online 21 April 2012
Keywords:
Octahedral Cu(II) complex
LC50
IC50
HS DNA
Norfloxacin
in the range 0.653–1.344 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
metalloenzymes, known as superoxide dismutases (SODs). SODs
are dedicated to keeping the concentration of O2 in controlled
Åꢀ
The study of the interaction between drugs and transition metal
ions is an important and active research area in bioinorganic
chemistry [1–3]. It is well known that the action of many drugs de-
pends on the coordination with metal ions and/or the inhibition on
the formation of metalloenzymes. Therefore, metal ions might play
a vital role during the biological process of drug utilization in the
body.
low limits, thus protecting biological molecules from oxidative
damage. These SODs disproportionate the toxic O2 radical to
molecular oxygen and hydrogen peroxide [21,22]. All SODs employ
the two step ping-pong mechanism shown in Eqs. (1) and (2):
Åꢀ
O2Åꢀ þ CuII ! O2 þ CuI
ð1Þ
O2Åꢀ þ CuI þ 2Hþ ! H2O2 þ CuII
ð2Þ
Quinolones, a commonly used term for quinolonecarboxylic
acids or 4-quinolones, are a group of synthetic antibacterial agents
containing a 4-oxo-1,4-dihydroquinoline skeleton [4–6]. They can
act as antibacterial drugs that effectively inhibit DNA replication
and are commonly used in the treatment of many infections [7,8].
Ciprofloxacin and norfloxacin are amongst the most common and
widely used quinolones. The study of the biological properties of
quinolones has been focused on their interaction with DNA [9], anti-
bacterial activity tests on diverse microorganisms [10,11], cytotox-
icity [11,12] and potential antitumor activity [13]. The study of the
biological properties of quinolone metal complexes comprises
mainly their antibacterial activity against diverse microorganisms
[14–16] and, in some cases, their interaction with DNA [17,18].
The superoxide radical anion (O2Åꢀ) is an inevitable by-product of
aerobic metabolism which if not eliminated may cause significant
cellular damage; consequently, the superoxide radical has been
implicated in numerous medical disorders [19,20]. To avoid such
harmful consequences, all oxygen metabolizing organisms possess
The synthesis and characterization of new metal complexes
with norfloxacin and other quinolone antibacterial agents are of
great importance for understanding the drug–metal ion interaction
and pharmacological applications [23–30].
In continuation of our previous work [31,32], we report here the
synthesis and characterization of some new copper (II) complexes
with their in vitro antimicrobial activity against two Gram(+ve) and
three Gram(ꢀve) microorganisms. The DNA binding and cleavage
properties of the complexes have been investigated using absorp-
tion titration, viscosity measurements and the gel electrophoresis
method. The cytotoxic activities of all the complexes have been
measured. The antioxidant properties of the complexes have been
checked using the non-enzymatic NBT/NADH/PMS system.
2. Experiments
2.1. Reagents
⇑
All chemicals and solvents used were of analytical grade. The
drug norfloxacin was purchased from Bayer AG (Wuppertal,
Corresponding author. Tel.: +91 2692 226856x218.
E-mail address: jeenen@gmail.com (M.N. Patel).
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.03.050

160
M.N. Patel et al. / Polyhedron 40 (2012) 159–167
Germany). 2-Acetylpyridine, m-chloro benzaldehyde, p-methoxy
benzaldehyde, m-bromo benzaldehyde, benzaldehyde, pyridine-
3-carbaldehyde, 9-anthraldehyde and m-benzyloxy benzaldehyde
were purchased from Loba Chemie Pvt. Ltd. (India). Ethidium bro-
mide and Luria Broth (LB) were purchased from Himedia, India.
Acetic acid and EDTA were purchased from Sd fine Chemicals
(India).
2.2. Physical measurements
The elemental analyses (C, H and N) of the synthesized com-
plexes were performed with a model 240 Perkin Elmer elemental
analyzer. The metallic content of the complexes was determined
after decomposing them in an acid mixture by heating and then
titrating against an EDTA solution volumetrically [33]. Room tem-
perature magnetic measurements of the complexes were carried
out using a Gouy magnetic balance. The Gouy tube was calibrated
using mercury(II)tetrathiocyanatocobaltate(II) as the calibrant
(v
g = 16.44 ꢁ 10ꢀ6 cgs units at 20 °C) The electronic spectra were
recorded on a UV-160A UV–Vis spectrophotometer, Shimadzu,
Kyoto (Japan). Infrared spectra were recorded on a FT-IR ABB Bo-
men MB 3000 spectrophotometer as KBr pellets in the range
4000–400 cmꢀ1. The LC–MS were recorded using a Thermo Scien-
tific mass spectrophotometer (USA). The minimum inhibitory con-
centration (MIC) study was performed by means of a laminar air
flow cabinet Toshiba, Delhi (India).
Scheme 1. Synthesis of 40-(3-chloro phenyl)-2,20:60,200-terpyridine.
2.3. Synthesis of the ligands
All the tridentate ligands were synthesized by following litera-
ture procedures [34]. 2-Acetylpyridine (20.0 mmol) was added to
an ethanolic solution of the various aldehydes (10.0 mmol). KOH
pellets (26.0 mmol) and 30 mL aqueous NH3 were added to the
solution, followed by stirring at room temperature for 8 h. An
off-white solid formed, which was collected by filtration, followed
by washings with H2O (3 ꢁ 10 mL) and EtOH (2 ꢁ 5 mL). Crystalli-
zation from the CHCl3/MeOH system gave a white crystalline solid.
The proposed reaction is shown in Scheme 1.
2.4.3. [Cu(NFL)(L3)Cl]
This was synthesized using 40-(3-chloro phenyl)-2,20:60,200-ter-
pyridine (1.5 mmol) as the ligand (L3). Yield: 64.7%, m.p.: >300 °C
decomposed,
leff: 1.89 B.M. Anal. Calc. for C38H31Cl2FCuN6O3
(761.13): C, 58.39; H, 4.11; N, 11.04; Cu, 8.35. Found: C, 58.28;
H, 4.01; N, 11.15; Cu, 8.24%.
2.4.4. [Cu(NFL)(L4)Cl]
This was synthesized using 40-(benzaldehyde)-2,20:60,200-terpyr-
idine (1.5 mmol) as the ligand (L4). Yield: 67.8%, m.p.: 279 °C
decomposed,
leff: 1.93 B.M. Anal. Calc. for C37H32ClFCuN6O3
2.4. Synthesis of the complexes
(726.69): C, 61.15; H, 4.44; N, 11.56; Cu, 8.74. Found: C, 61.05;
H, 4.34; N, 11.45; Cu, 8.63%.
2.4.1. [Cu(NFL)(L1)Cl]
A methanolic solution of CuCl2ꢂ2H2O (1.5 mmol) was added to a
methanolic solution of 40-(4-methoxy phenyl)-2,20:60,200-terpyri-
dine (L1) (1.5 mmol), followed by the addition of a previously pre-
pared methanolic solution of norfloxacin (1.5 mmol) in the
presence of CH3ONa (1.5 mmol). After mixing the above solutions,
the pH of solution was in the basic range (>6.8). Therefore, the pH
of the reaction mixture was adjusted to ꢃ6.8 by addition of dilute
hydrochloric acid. The resulting solution was refluxed for 2 h on a
water bath, followed by concentrating it to half of its volume. The
fine, green, amorphous product thus obtained was washed with
ether/hexane and dried in a vacuum desiccator (Scheme 2).
2.4.5. [Cu(NFL)(L5)Cl]
This was synthesized using 40-(3-pyridyl)-2,20:60,200-terpyridine
(1.5 mmol) as the ligand (L5). Yield: 65.0%, m.p.: >300 °C decom-
posed,
leff: 1.94 B.M. Anal. Calc. for C36H31ClFCuN7O3 (727.67): C,
59.42; H, 4.29; N, 13.47; Cu, 8.73. Found: C, 59.29; H, 4.17; N,
13.32; Cu, 8.60%.
2.4.6. [Cu(NFL)(L6)Cl]
This was synthesized using 40-(anthracene-9-yl)-2,20:60,200-ter-
pyridine (1.5 mmol) as the ligand (L6). Yield: 65.0%, m.p.: 291 °C
Yield: 67.8%, m.p.: >300 °C,
leff: 1.96 B.M. Anal. Calc. for
decomposed,
leff: 1.90 B.M. Anal. Calc. for C45H36ClFCuN6O3
C38H34ClFCuN6O4 (756.71): C, 60.31; H, 4.53; N, 11.11; Cu, 8.40.
(826.80): C, 65.37; H, 4.39; N, 10.16; Cu, 7.69. Found: C, 65.25;
H, 4.28; N, 10.02; Cu, 7.57%.
Found: C, 60.42; H, 4.40; N, 11.00; Cu, 8.49%.
2.4.2. [Cu(NFL)(L2)Cl]
2.4.7. [Cu(NFL)(L7)Cl]
This was synthesized using 40-(3-bromo phenyl)-2,20:60,200-ter-
This was synthesized using 40-(3-benzyloxyphenyl)-2,20:60,200-
pyridine as the ligand (L2) (1.5 mmol). Yield: 65.6%, m.p.: 286 °C
terpyridine (1.5 mmol) as the ligand (L7). Yield: 62.5%, m.p.:
decomposed,
leff: 1.91 B.M. Anal. Calc. for C37H31BrClFCuN6O3
>300 °C, leff: 1.95 B.M. Anal. Calc. for C44H38ClFCuN6O4 (832.81):
(805.58): C, 55.16; H, 3.88; N, 10.43; Cu, 7.89. Found: C, 55.02; H,
C, 63.46; H, 4.60; N, 10.09; Cu, 7.63%. Found: C, 63.34; H, 4.49;
3.75; N, 10.30; Cu, 7.75%.
N, 9.98; Cu, 7.51%.

M.N. Patel et al. / Polyhedron 40 (2012) 159–167
161
Scheme 2. Synthesis of [Cu(NFL)(L3)Cl].
2.5. In vitro antimicrobial screening
diluting an overnight culture of microorganisms grown in LB to
obtain 106 viable bacteria/mL. The bacteria were exposed to
various concentrations of the compounds. Control tubes without
compound were included in each run. The final volume was
1 mL. The cultures were incubated at 37 °C for 2 h. One hundred
microliters bacterial culture from each dilution was taken, spread
over a previously prepared agar plate and then the plates were
incubated for 24 h. The numbers of colonies present on the plates
were counted. The number of colonies should be in the range
30–300.
The antibacterial activities of the compounds were studied
against various microorganisms, i.e. Escherichia coli (MTCC 433),
Pseudomonas aeruginosa (MTCC P09), Serratia marcescens (MTCC
7103), Bacillus subtilis (MTCC 7193) and Staphylococcus aureus
(MTCC 3160). Screening was performed by determining the mini-
mum inhibitory concentration (MIC) using LB as a medium. Cul-
tures for Gram(+ve) and Gram(ꢀve) microorganisms were incubated
at 37 °C. Since the compounds are water insoluble, the samples
were dissolved in DMSO. A control test without any active ingredi-
ent was also performed [35]. The MIC was determined using two-
fold serial dilutions in liquid media containing the test compound.
A preculture of bacteria was grown in LB overnight at the optimal
temperature for each species. Bacterial growth was monitored by
measuring the turbidity of the culture after 18 h. If a certain
concentration of a compound inhibited the bacterial growth, half
the concentration of compound was then tested. This procedure
was carried out until a concentration was reached where the bac-
teria grew normally. The lowest concentration which inhibits bac-
terial growth was determined as the MIC value. All equipment and
culture media used were sterile.
2.6. DNA interaction study
2.6.1. Absorption titration
DNA-mediated hypochromic and bathochromic shifts under the
influence of the complexes were measured with the help of UV–Vis
absorbance spectra [36–39]. After addition of an equivalent
amount of DNA to a reference cell, it was incubated for 10 min at
room temperature, followed by absorbance measurement. The
DNA-mediated hypochromism (decrease in absorbance) for the
test compounds was calculated. Absorption titration experiments
were done using different concentrations of HS DNA, by keeping
the concentration of the complexes constant, with due correction
for the absorbance of the HS DNA itself. Samples were equilibrated
The bactericidal action of all the compounds was evaluated
against the same microorganisms. The inoculum was prepared by

162
M.N. Patel et al. / Polyhedron 40 (2012) 159–167
before recording each spectrum. The intrinsic binding constant Kb
was determined using the following equation [40]:
using the artificial seawater. The number of survivors was counted
after 24 h. Data were analyzed by a simple method to determine
the LC50 values, in which the log values of the concentration of
the samples were plotted against the percentage of mortality of
nauplii [44].
½DNAꢄ=ðea
ꢀ
ef Þ ¼ ½DNAꢄ=ðeb
ꢀ
ef Þ þ 1=Kbðeb
ꢀ
ef
Þ
ð3Þ
where [DNA] is the concentration of HS DNA in base pairs, ea is the
apparent extinction coefficient obtained by calculating Aobs./[com-
plex], eb refers to the extinction coefficient of the complex in the
fully bound form and ef corresponds to the extinction coefficient
of the complex in its free form. The data obtained were plotted in
accordance with the above equation to give a straight line with a
2.8. SOD-like activity
A non-enzymatic system containing 30
lM PMS, 79 lM NADH,
75
lM NBT and phosphate buffer (pH 7.8) was used to produce the
Åꢀ
superoxide anion (O2Åꢀ). The scavenging rate of O2 under the
slope value corresponding to the term 1/(ea
ꢀ
ef) and a y intercept
of 1/Kb(eb f). The Kb value was determined from the ratio of the
ꢀ
e
influence of 0.5–3.0 M tested compounds was determined by
l
slope to the intercept.
monitoring the reduction in the rate of transformation of NBT to
monoformazan dye [45]. The reactions were monitored at
560 nm with a UV–Vis spectrophotometer and the rate of absorp-
tion change was determined. The percentage inhibition of NBT
reduction was calculated using following equation [46]:
2.6.2. Viscosity titration
Viscosity measurement is regarded as a reliable tool to deter-
mine the binding mode in a solution state in the absence of crystal-
lographic structural data and NMR data [41]. Viscometric titrations
were performed using an Ubbelohde viscometer (Cannon, PA, USA)
immersed in a thermostatic bath maintained at 27 0.1 °C. The
flow time was measured with a digital stopwatch. Each sample
was measured in triplicate manner and an average flow time was
ð% InhibitionofNBTreductionÞ ¼ ð1 ꢀ k0=kÞ ꢁ 100
ð5Þ
where k0 and k represent slopes of the straight line of absorbance
values as a function of time in the presence and absence of SOD mi-
mic compounds, respectively. IC50 values of the complexes were
determined by plotting a graph of percentage inhibition of NBT
reduction against increasing concentrations of complex. The con-
centration of the complex which causes 50% inhibition of NBT
reduction is reported as IC50.
then calculated. The data are represented as (
plex]/[DNA], where is the viscosity of DNA in the presence of
g/g0)
1/3 versus [com-
g
complex and g0 is the viscosity of DNA alone. The viscosity values
were calculated from the observed flow time of the solutions con-
taining DNA (t) and corrected for that of the buffer alone (t0), using
the following equation [42]:
3. Result and discussion
g
¼ ðt ꢀ t0Þ
ð4Þ
3.1. Electronic spectra and magnetism
2.6.3. Gel electrophoresis: photo quantisation technique
The magnetic moments of the copper(II) complexes lie in the
range 1.89–1.96 B.M. These values are typical for mononuclear
copper(II) compounds having a d9 electronic configuration. The ob-
served magnetic moments of all the complexes correspond to typ-
ical high spin octahedral complexes. However, the values are
slightly higher than the expected spin-only values due to a spin–
orbit coupling contribution [47]. The UV–Vis spectra of the
complexes in the solid and solution states show a similar pattern
suggesting that the complexes retain their structures in solution.
The complexes exhibited one broad band along with a shoulder
at ꢃ14,000 and ꢃ11,000 cmꢀ1 respectively, which was attributed
to the d–d transition for the Cu(II) atom in a distorted octahedral
geometry [48–51].
For the gel electrophoresis experiment, a total volume of 15 lL
containing 300
1 mM EDTA, pH 8) was treated with different complexes
(200 M). The mixture was incubated for 24 h at 37 °C. Then the
samples were analyzed on the basis of their charge and size differ-
ence on a 1% agarose gel bed consisting of 0.5 g/mL of ethidium
bromide at 50 V, after quenching the reaction with 5 L loading
lg/mL of pUC19 DNA in TE buffer (10 mM Tris,
l
l
l
buffer (40% sucrose, 0.2% bromophenol blue). The whole bed was
immersed in 1ꢁ TAE buffer (0.04 M Tris–acetate, pH 8, 0.001 M
EDTA). The bands were visualized using UV light, then photo-
graphed. An estimation of the intensity of the DNA bands was done
using the AlphaDigiDoc™RT. Version V.4.0.0 PC-Image software gel
documentation system.
2.7. Brine shrimp assay
3.2. IR spectra
A brine shrimp assay was carried out according to the method
given by Meyer et al. [43]. Brine shrimp (Artemia cysts) eggs were
hatched in a shallow rectangular plastic dish (22 ꢁ 32 cm), filled
with artificial seawater. Artificial seawater was prepared using a
commercial salt mixture and double distilled water. 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 which was opened to ordinary light. After 48 h nau-
plii were collected by a pipette. A sample of the test compounds
was prepared by dissolving 10 mg of each compound in 10 mL of
DMSO. Solutions were transferred from the stock solutions to 18
vials in such an amount to make final concentrations of 2, 4, 8,
In the IR spectrum of norfloxacin the valence vibration of the
carboxylic stretch
m
(C@O)carb is found at 1730 cmꢀ1 and the pyri-
done stretch
m
(C@O)p at 1642 cmꢀ1 (Table 1). The characterization
of quinolone metal complexes can be achieved by studying the
most typical vibrations that are characteristic of the coordination
type of quinolones.
In the IR spectra of the complexes 1–7 the absorption of the
m
(C@O)carb has disappeared. Two very strong characteristic bands
are observed in the range 1565–1595 cmꢀ1 and 1364–1381 cmꢀ1
assigned as (O–C–O) asymmetric and symmetric stretching
vibrations, respectively, whereas (C@O)p is shifted from 1642
cmꢀ1 to 1618–1632 cmꢀ1 upon bonding. The difference
(CO2)asym (CO2)sym is a useful characteristic for determining the
coordination mode of the ligands. The
,
m
m
Dm = m
10, 12 and 16
lg/mL (three sets for each dilution were run for all
ꢀ
m
test samples and the mean of the three sets was taken for the cal-
culation of LC50). One vial was kept as a control, having the same
amount of DMSO only. When the shrimp nauplii were ready,
1 mL of artificial seawater and 10 shrimps were added to each vial
(30 shrimps/dilution). The volume was adjusted to 2.5 mL per vial
D values fall in the range
201–215 cmꢀ1, indicating a monodentate coordination mode of
the carboxylato group of norfloxacin [52,53]. The overall changes
of the IR spectra suggest that norfloxacin is coordinated to copper
via the pyridone (Op) and one carboxylate (Oc) oxygen.

M.N. Patel et al. / Polyhedron 40 (2012) 159–167
163
Table 1
Change in IR bands for the interaction of norfloxacin with Cu(II) in addition to terpyridines (4000–400 cmꢀ1).
Compounds
m
(C@O) (cmꢀ1) pyridone
m
(COO)asym (cmꢀ1
)
m
(COO)sym (cmꢀ1
)
D
m
(cmꢀ1
)
m
(M–N) (cmꢀ1
)
m
(M–O) (cmꢀ1
)
Norfloxacin
1642
1632
1618
1623
1626
1630
1627
1620
1730a
1565
1591
1575
1582
1595
1579
1568
–
–
–
–
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
[Cu(NFL)(L7)Cl]
1364
1381
1362
1373
1380
1367
1365
201
210
213
209
215
212
203
535
545
532
539
548
530
542
520
509
514
517
506
503
511
a
As m(COOH).
3.3. Mass spectra
and possible p-electron delocalization over the whole chelate ring.
Such a chelation could enhance the lipophilic character of the cen-
tral metal atom, which subsequently favors its permeation through
the lipid layers of the cell membrane [60] and blocking the metal
binding sites on the enzymes of microorganisms. The different
compounds exhibit microbial activity with small variations against
the bacterial species, and this difference in activity could be attrib-
uted to the impermeability of the cell of the microbes, which in the
case of Gram(+ve) is single layered and in the case of Gram(ꢀve) is a
multilayered structure [61], or differences in the ribosomes of the
microbial cells.
In addition to our study regarding MIC, we measured the bacte-
ricidal activity in terms of CFU/mL of the above metal complexes
against the same microorganisms (two Gram(+ve) and three
Gram(ꢀve)). The results reveal decrease in the number of colonies
with increasing concentration of the compounds. The results are
shown in Fig. 1 for all the complexes against S. aureus. The number
of colonies counted in this technique is in the range 30–300. From
the minimum inhibitory concentration (MIC) values and colony
forming units (CFU), it was found that complex 3 is more potent
against all the five microorganisms.
The mass spectrum of complex 1 shows molecular ion peak at
m/z = 755.21 and 757.21 due to the presence of one chlorine atom
and it also confirms that chlorine atom is attached to metal atom
through covalent bond. The peak observed at m/z = 720.10,
402.16, 340.18 and 320.23 are due to loss of one chlorine atom,
copper-terpyridine moiety, terpyridine moiety and norfloxacin,
respectively. The m/z values, i.e. 437.11, 439.12, 416.27, 418.27,
381.12, 262.14 and 156.31 correspond to peaks for particular frag-
ments are duly highlighted in fragmentation pattern of complex 1.
Figure of mass spectrum and the proposed fragmentation pattern
of complex 1 are kept in Supplementary material.
3.4. Antibacterial activity
The antibacterial activities of cupric chloride dihydrate, nor
floxacin and the complexes were tested against two Gram(+ve)
microorganisms, S. aureus and B. subtilis, and three Gram(ꢀve) micro-
organisms, S. marcescens, E. coli and P. aeruginosa, using the double
dilution method. The antimicrobial activities of all the complexes
against the five microorganisms are much higher than those of the
metal salt (Table 2). It is clear from the data that complex 3 is active
compared to NFLH against all the bacterial species employed. Com-
plexes 1, 2, 4, 5, 6 and 7 are moderate to less active compared to
NFLH against all the bacterial species employed. Out of all the active
complexes, complex 3 is the most active one. All the complexes
show better antibacterial activity than [Cu(pr-norf)2(H2O)] and
[Cu(pr-norf)(bipyam)Cl] against S. aureus and P. aeruginosa [54].
All the complexes are less active than [Cu(pr-norf)2(H2O)], [Cu
(pr-norf)(bipy)Cl] and [Cu(erx)2(H2O)] against E. coli [55,56]. The
complexes show better activity than [Cu(oxo)2(H2O)], [Cu(oxo)(bip-
yam)Cl], [Cu(oxo)(bipy)Cl] and [Cu(oxo)(phen)Cl] against S. aureus,
P. aeruginosa and E. coli [57].
3.5. DNA interaction study
3.5.1. Absorption titration
Mixed-ligand complexes can bind to DNA via three distinct
binding sites namely, groove-binding, binding to the phosphate
group and intercalation [62]. These behaviors are of great
importance with respect to the relevant biological role of quino-
lone antibiotics in the body [63,64]. The changes observed in the
UV spectra of the complexes after mixing them with DNA (either
increase or decrease in the intensity or shift in the wavelength)
indicate the interaction of the complexes with DNA, due to the
formation of a new complex with the double-helical DNA [65].
With an increasing concentration of HS DNA, the absorption bands
Chelation could facilitate the ability of a complex to cross a cell
membrane [58] and can be explained by Tweedy’s chelation theory
[59]. Chelation considerably reduces the polarity of the metal ion
because of partial sharing of its positive charge with donor groups
Table 2
MIC in terms of
lg/mL.
Compounds
Gram positive
Gram negative
S. aureus B. subtilis S. marcescens P. aeruginosa E. coli
CuCl2ꢂ2H2O
459.95
0.77
0.98
0.87
0.61
1.24
1.60
2.81
2.50
479.90
0.80
1.02
0.97
0.65
1.31
1.67
2.98
2.66
469.84
1.25
1.66
1.57
1.14
2.14
2.62
4.22
4.00
409.83
1.18
1.59
1.45
0.99
2.03
2.51
3.97
3.66
579.97
0.86
1.21
1.13
0.76
1.53
1.86
3.22
2.83
Norfloxacin
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
[Cu(NFL)(L7)Cl]
Fig. 1. Relationship between the concentration and bactericidal activity of all the
complexes against S. aureus.

164
M.N. Patel et al. / Polyhedron 40 (2012) 159–167
Fig. 2. Electronic absorption spectra of [Cu(NFL)(L3)Cl] in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2) in the absence and presence of increasing amount of DNA. Inset: Plot
of [DNA]/(ea
ꢀ ef) vs. [DNA]. Arrow shows the absorbance change upon increasing the DNA concentration.
Table 3
The binding constants (Kb) of the complexes.
Complexes
Kb (Mꢀ1
)
% hypochromism
Norfloxacin
2.43 ꢁ 103
5.06 ꢁ 104
6.03 ꢁ 104
7.19 ꢁ 104
3.47 ꢁ 104
1.03 ꢁ 104
3.68 ꢁ 103
5.85 ꢁ 103
6.86
18.19
12.40
5.67
5.99
15.38
5.13
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
[Cu(NFL)(L7)Cl]
5.88
of the complexes are affected. This results in hypochromism with a
bathochromic shift. The extent of the binding strength of the com-
plexes is quantitatively determined by calculating the intrinsic
binding constants Kb of the complexes. This is done by monitoring
the change in absorbance at various concentrations of DNA (Fig. 2).
Fig. 3. Effect on the relative viscosity of DNA under the influence of increasing
amounts of the complexes at 27 0.1 °C in phosphate buffer (Na2HPO4/NaH2PO4,
pH 7.2).
From the plot of [DNA]/(ea
ꢀ
ef) versus [DNA] (inset in Fig. 2), the
Kb values of the complexes were determined and were found to
be in the range 3.68 ꢁ 103–7.19 ꢁ 104 (Table 3). The Kb values of
all the complexes are higher than that of norfloxacin (2.43 ꢁ
103 Mꢀ1). These values are much lower than those of classical
intercalators, e.g. ethidium bromide (7.16 ꢁ 105 Mꢀ1), but are com-
parable to those of [Ni(erx)2(H2O)2], [Ni(erx)2(phen)], [Ni(erx)2
(bipy)] (1.63–4.09 ꢁ 104 Mꢀ1), [66] [Zn(oxo)(bipy)Cl], [Zn(oxo)2
Fig. 4. Photogenic view of the cleavage of pUC19 DNA (300
copper(II) complexes (200 M) using 1% agarose gel containing 0.5
bromide. All the reactions were incubated in TE buffer (pH 8) in a final volume of
l
g/mL) with a series of
l
lg/mL ethidium
15
lL, for 24 h at 37 °C: lane 1, DNA control; lane 2, CuCl2ꢂ2H2O; lane 3, norfloxacin;
(phen)] (5.91 and 9.60 ꢁ 104 Mꢀ1
)
[67] and [SPFX–Mg]
lane 4, [Cu(NFL)(L1)Cl]; lane 5, [Cu(NFL)(L2)Cl]; lane 6, [Cu(NFL)(L3)Cl]; lane 7, [Cu
(NFL)(L4)Cl]; lane 8, [Cu(NFL)(L5)Cl]; lane 9, [Cu(NFL)(L6)Cl]; lane 10, [Cu(NFL)
(L7)Cl].
(7.558 ꢁ 103 Mꢀ1) [68]. Therefore, the above results indicate that
the complexes may bind with DNA by the intercalative mode.
The binding mode is further confirmed by viscosity measurements.
of DNA increases steadily, which is similar to that of the classical
intercalative complex [Ru(bpy)2 ppd]+2 {ppd = pteridino[6,7-f]-
[1,10]phenanthroline-11,13(10H,12H)-dione} [70]. This is similar
to the behavior of EtBr, which indicates that the complexes bind
with DNA through the classical intercalation mode. The increase in
the degree of viscosity may depend on the affinity to DNA, and
follows the order: EtBr > 3 > 2 > 1 > 4 > 5 > 7 > 6.
3.5.2. Viscosity titrations
The binding modes of the Cu(II) complexes were further investi-
gated by viscosity measurements. Viscosity measurements are sen-
sitive to DNA length change, and are regarded as the least ambiguous
and most critical test for measuring the binding mode of DNA in
solution. A classical intercalative mode demands that the DNA helix
must lengthen as base pairs are separated to accommodate the bind-
ing ligand, which leads to an increase in DNA viscosity [69]. The ef-
fects of the complexes, EtBr and NFLH on the viscosity of DNA are
shown in Fig. 3. EtBr increases the relative specific viscosity by
lengthening the DNA double helix through the intercalation mode.
With increasing the amounts of the complexes, the relative viscosity
3.5.3. Gel electrophoresis; photo quantisation technique
Agarose gel electrophoresis was used as a base for monitoring
the plasmid DNA cleavage reaction. Circular plasmid DNA, when
subjected to electrophoresis, undergoes a relatively fast migration

M.N. Patel et al. / Polyhedron 40 (2012) 159–167
165
Table 4
presented in Table 4. Complex 3 shows the maximum cleavage abil-
ity compared to all the synthesized complexes. All the complexes
show a higher DNA cleavage ability compare to NFLH and the metal
salt.
Complex mediated DNA cleavage data by gel electrophoresis.
Lane Compound
Form I
(SC)
Form II
(OC)
Form III
(LC)
%
cleavage
1
2
3
4
5
6
7
8
9
Control
88
84
61
12
16
27
52
56
57
53
51
55
50
–
–
–
4.55
CuCl2ꢂ2H2O
3.6. Brine shrimp assay
Norfloxacin
12
18
17
19
16
14
12
11
30.68
65.91
69.32
72.73
64.77
60.23
62.50
55.68
[Cu(NFL)(L1)Cl] 30
[Cu(NFL)(L2)Cl] 27
[Cu(NFL)(L3)Cl] 24
[Cu(NFL)(L4)Cl] 31
[Cu(NFL)(L5)Cl] 35
[Cu(NFL)(L6)Cl] 33
[Cu(NFL)(L7)Cl] 39
A general bioassay that appears capable of detecting a broad
spectrum of the bioactivity present in a complex is the brine shrimp
lethality bioassay. The technique is easily mastered, is of little cost
and utilizes a small amount of test material. The aim of this method
is to provide a front-line screen that can be backed up by more
specific and more expensive bioassays after isolating the active
compounds. The brine shrimp lethality bioassay (BSLA) is a develop-
ment in the assay procedure of a bioactive compound, which indi-
cates cytotoxicity as well as a wide range of pharmacological
activities (such as anticancer, antiviral, insecticidal, pesticidal, AIDS,
etc.) of the compound. All the synthesized compounds were
screened for their cytotoxicity (brine shrimp bioassay) using the
protocol of Meyer et al. [43]. From the data recorded in Table 5,
10
for the intact supercoil form (SC; Form I). Scission on one strand
(nicking) will relax the supercoil to generate a slower moving open
circular form (OC; Form II). If both strands are cleaved, a linear form
(LC; Form III) that migrates between Form I and Form II will be gen-
erated [71]. Data of the cleavage study obtained from Fig. 4 are
Table 5
Effect of compounds on brine shrimp lethality bioassay.
Compound
Concentration
g mL-1
Log(Conc.)
No. of nauplii
taken
No. of nauplii
dead
% of mortality
LC50(l )
g mL-1
(
l
)
Norfloxacin
10
50
1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
1
2
3
4
5
6
1
3
5
6
7
8
1
3
6
7
8
9
2
4
6
7
8
9
1
2
4
6
7
8
1
2
4
5
7
8
0
1
2
4
5
7
0
2
4
5
6
7
10
20
30
40
50
60
10
30
50
60
70
80
10
30
60
70
80
90
20
40
60
70
80
90
10
20
40
60
70
80
10
20
40
50
70
80
0
1.698
2
100
150
200
240
2
4
8
10
12
16
2
4
8
10
12
16
2
4
8
10
12
16
2
4
8
10
12
16
2
4
8
10
12
16
2
4
8
10
12
16
2
4
8
10
12
16
194.98
6.93
6.36
5.24
7.82
8.1
2.176
2.301
2.380
0.301
0.602
0.903
1
1.079
1.204
0.301
0.602
0.903
1
1.079
1.204
0.301
0.602
0.903
1
1.079
1.204
0.30
0.602
0.903
1
1.079
1.204
0.301
0.602
0.903
1
1.079
1.204
0.301
0.602
0.903
1
1.079
1.204
0.301
0.602
0.903
1
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
[Cu(NFL)(L7)Cl]
10
20
40
50
70
0
20
40
50
60
70
11.96
11.3
1.079
1.204

166
M.N. Patel et al. / Polyhedron 40 (2012) 159–167
Table 6
The IC50 values of the complexes.
Complexes
IC50
161.1
0.784
(
l
M)
Norfloxacin
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
[Cu(NFL)(L7)Cl]
[Ni(sf)2(bipy)]
[Ni(erx)2(bipy)]
[Ni(oxo)2(bipy)]
[Ni(flmq)2(bipy)]
0.713
0.653
0.876
1.145
1.344
1.319
28.30a
26.54a
27.45a
12.69a
Scheme 3. Mechanism for SOD-like activity performed using the NBT/NADH/PMS
system.
a
In mM.
complexes can be used as potent cytotoxic agents with the hope
of adding to the arsenal of weapons used against the fatal disease
cancer.
3.7. SOD-like activity
The SOD-like behavior was checked using the NBT/NADH/PMS
system. PMS gets reduced by the hydrogen donor NADH. The re-
Åꢀ
duced PMS generates O2 from dissolved O2. NBT is reduced by
O2Åꢀ, which results in a linear accumulation of blue formazan with
an increase in the absorbance at 560 nm. SOD or a model com-
Åꢀ
pound when present in the reaction medium scavenges O2
,
resulting in a decrease in the formation of formazan. The mecha-
nism is shown in Scheme 3. The percentage inhibition of formazan
formation at various concentrations of the complexes as a function
of time is calculated by measuring the absorbance at 560 nm, and
the results are plotted to give a straight line (Fig. 5). With an in-
crease in the concentration of the tested compounds, a decrease
in slope (m) is observed. The percentage inhibition of the reduction
of NBT is plotted against the concentration of the complex (Fig. 6).
The compounds exhibit SOD-like activity at biological pH, with IC50
Fig. 5. Absorbance values (Abs560) as a function of time (t) plotted for varying
concentrations of complex 3 from 0.5 to 3 lM, for which good straight lines are
observed.
values in the range 0.653–1.344 lM (Table 6). The IC50 values of
the copper(II) complexes are better than those of the Ni(II) com-
plexes recently reported [72].
4. Conclusions
The data of magnetic behavior and electronic spectral measure-
ment point towards a d9 system with a distorted octahedral geom-
etry. The antibacterial activity of norfloxacin is changed upon
coordination with the copper(II) ion. From the viscosity data of
the complexes, it is clear that all the complexes show a classical
intercalative mode of binding. Complex 3 is bound more strongly
than the other complexes. The absorption titration data are in good
accordance with the viscosity titration curves. All the complexes
exhibit good cytotoxic activity. The DNA cleavage study of pUC19
shows that all the complexes have a high cleavage ability com-
pared to the metal salt and the drug. Upon determination of the
antioxidant activity in the NBT/NADH/PMS system, complex 3
shows the highest scavenging ability for the oxygen radical.
Fig. 6. Plot of percentage of inhibiting NBT reduction with an increase in the
concentration of complex 3.
Conflict of interest
complex 3 is the most potent amongst all the compounds. The order
of potency of compounds is: EtBr > 3 > 2 > 1 > 4 > 5 > 7 > 6. All the
complexes show better cytotoxic activity than norfloxacin. The
variation in the BSLA results may be due to the difference in the
amount and kind of cytotoxic substances. The tested copper(II)
complexes have strong cytotoxic activities, but this investigation
is a primary one and further tests are required to investigate the ac-
tual mechanism of the cytotoxicity and the probable effects on high-
er animal models and on cancer cell lines. It suggests that the
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
Acknowledgments
The authors thank the Head, Department of Chemistry, Sardar
Patel University, India, for making it convenient to work in the

M.N. Patel et al. / Polyhedron 40 (2012) 159–167
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.poly.2012.03.050.
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