DNA interactions and promotion in antibacterial activities of the norfloxacin drug due to formation of mixed-ligand copper(II) complexes

Article abstract of DOI:10.1007/s00706-013-1086-4

Novel neutral mononuclear copper(II) complexes with the antibacterial drug norfloxacin in the presence of nitrogen donor heterocyclic (bipyridines) ligands were synthesized and characterized by elemental analysis, magnetic moment measurement, thermal anal

Full text of DOI:10.1007/s00706-013-1086-4

Monatsh Chem (2014) 145:369–381
DOI 10.1007/s00706-013-1086-4
ORIGINAL PAPER
DNA interactions and promotion in antibacterial activities
of the norfloxacin drug due to formation of mixed-ligand
copper(II) complexes
•
Mohan N. Patel Anshul P. Patidar
Received: 8 May 2013 / Accepted: 4 September 2013 / Published online: 8 October 2013
Ó Springer-Verlag Wien 2013
Abstract Novel neutral mononuclear copper(II) com-
plexes with the antibacterial drug norfloxacin in the
presence of nitrogen donor heterocyclic (bipyridines)
ligands were synthesized and characterized by elemental
analysis, magnetic moment measurement, thermal analysis
(TG), IR, LC–MS, and electronic spectra. The complexes
were tested against selective gram-positive and -negative
organisms for antibacterial activity. DNA binding studies
were carried out using viscosity measurement and
absorption titration. A DNA cleavage experiment was
performed using supercoiled double-stranded DNA plas-
mids in order to compare the cleavage efficiency of
synthesized copper(II) complexes in the different ligand
environments. Superoxide dismutase mimetic behavior was
actively sought for clinical and mechanistic purposes under
a nonenzymatic system and was found to have good anti-
oxidant activity. The in vitro cytotoxic properties of the
synthesized complexes were analyzed analogous to the
brine shrimp bioassay.
Introduction
During the past decades, metal complexes have been the
subjects of tremendous research interest owing to the
interactions of complexes with nucleic acids [1–3]. 2,20-
Bipyridines (bpy), which act as N,N donor ligands, are well
known because of their remarkable chemistry as hetero-
cyclic entities and because of their exceptional
coordination chemistry [4–7]. The study of ternary com-
plexes of metal ions and two different types of ligands is a
topic of interest due to their presence in biological systems.
Quinolone antibiotics are chelating agents for a variety of
metal ions well known for their biological activity [8–12].
In particular, copper(II) complexes are known to play
significant roles as both naturally occurring biological
systems and pharmacological agents [13]. Low molecular
weight copper(II) complexes have been proved to be ben-
eficial against several diseases such as tuberculosis,
rheumatoid arthritis, gastric ulcers, and cancers [14–17].
There have been several reports about the synthesis of
metal complexes with norfloxacin (NFL) [18–20]. In early
studies, it was postulated that DNA gyrase was the
molecular target [21]. However, in 1985, Shen and
coworkers found that norfloxacin binds the DNA double
helix instead of DNA gyrase [22]. Even in earlier reports,
Crumplin and coworkers reported that nalidixic acid binds
to single-stranded DNA through the presence of an excess
of copper ions [23]. These two hypotheses apparently
became antagonists. Therefore, Shen and coworkers
described their new proposal in 1989: that the mechanism
of action is due to a quinolone-DNA cooperative binding
for the inhibition of DNA gyrase [24].
Keywords Bipyridines ꢀ Norfloxacin ꢀ
Calf thymus DNA ꢀ Superoxide dismutase ꢀ Cytotoxicity
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-013-1086-4) contains supplementary
material, which is available to authorized users.
M. N. Patel (&) ꢀ A. P. Patidar
Superoxide dismutase (SOD) is an essential enzyme that
eliminates the superoxide radical anion Oꢀ2- and thus pro-
tects cells from damage induced by reactive oxygen species
Department of Chemistry, Sardar Patel University, Vallabh
Vidyanagar 388 120, Gujarat, India
e-mail: jeenen@gmail.com
123

370
M. N. Patel, A. P. Patidar
(ROS) [25, 26]. The superoxide radical anion Oꢀ2- is
formed as a byproduct of normal cellular respiration. Its
decomposition produces undesired harmful species such as
the hydroxyl radical and hydrogen peroxide. To control
this, nature has created a family of enzymes that removes
them from the cellular environment. One of these, super-
oxide dismutase (SOD), catalyzes disproportionation of
superoxide to dioxygen and hydrogen peroxide, which is
decomposed via catalysis to water and dioxygen [27].
Herein, in continuation of earlier work [28, 29], we
represent the interaction of the copper(II) ion with the
second-generation quinolone, i.e., norfloxacin (NFL), and
N,N donor bidentate ligands. More specifically, the com-
plexes have been synthesized and characterized with
diverse analytical and spectroscopic techniques [elemental
analysis, thermal analysis (TG), magnetic moment mea-
surement, electronic, IR, and mass spectroscopy]. The
biological activities of the complexes have been evaluated
by determining the minimum inhibitory concentration
(MIC) against five microorganisms. The interactions of the
complexes with calf-thymus (CT) DNA have also been
investigated with UV spectroscopy and viscosity mea-
surement. The antioxidant properties of the complexes
have been checked using the nonenzymatic NBT/NADH/
PMS system. The gel electrophoresis technique has been
carried out to determine the influence of complexes on
pUC19 DNA. The cytotoxic activities of all complexes
have been carried out using the brine shrimp bioassay.
corresponding to a characteristic d–d transition in the
tetragonal field, suggesting distorted square pyramidal
geometry for copper(II) complexes. The magnetic moment
measurement for any geometry in copper(II) complexes
generally results in 1.8 BM, which is very close to the spin-
only value, i.e., 1.73 BM. The observed values in our case
are very close to the spin-only values expected for s = ‘
system (1.73 BM), leading to the conclusion that the metal
center in synthesized complexes possesses one unpaired
electron responsible for s = ‘ system [30, 31].
IR spectra
The valence vibration of the carboxylic stretch m(C=O)carb
of norfloxacin in the IR spectrum is found at 1,730 cm-1
and the pyridone stretch m(C=O)p at 1,642 cm-1 (Table 1).
The characterization of quinolone metal complexes can be
carried out by studying the most typical vibrations char-
acteristic of the coordination type of quinolone.
In the IR spectra of complexes 1–6, the absorption of the
m(C=O)carb has disappeared. Two very strong characteristic
bands are observed in the range 1,560–1,590 cm–1 and
1,350–1,388 cm–1, assigned as m(O–C–O) asymmetric and
symmetric stretching vibrations, respectively. The band at
1,642 cm–1 responsible for m(C=O)p in norfloxacin is
observed between 1,620 and 1,635 cm–1 in case of com-
plexes. The difference Dm = m(CO2)asym-m(CO2)sym is a
useful characteristic for determining the coordination
mode of the ligands. The D values fall in the range
201–220 cm–1, indicating a monodentate coordination
mode of the carboxylato group of the norfloxacin [32, 33].
In the investigated complexes, the m(C=N) band of bipyr-
idine derivatives is observed at about 1,584 cm–1. This
band shift to a higher frequency at *1,626 cm–1 in com-
plexes suggests the bidentate N–N coordination of the
ligand [34, 35]. The m(M–N) band at *530–545 cm–1 is
assigned to N?M bonding of complexes [36]. The overall
changes in the IR spectra suggest that norfloxacin is
coordinated to copper via pyridone (Op) and one carbox-
ylate (Oc) oxygen.
Results and discussion
Electronic spectra and magnetism
At low temperature, copper(II) complexes, that is, the d9
system with a simple ligand, exhibit an absorption band
with a large width, making them very difficult to interpret.
Complexes of copper(II) with different coordination num-
bers result in different geometries. The copper(II)
complexes exhibit a broad band at *15,300 cm-1 [30],
Table 1 Change in IR bands for interaction of norfloxacin with copper(II) in addition to bipyridines (4,000–400 cm-1
)
Compounds
m(C=O) pyridone/cm-1
m(COO)asym/cm-1
m(COO)sym/cm-1
Dm/cm-1
m(M–N)/cm-1
m(M–O)/cm-1
Norfloxacin
1,642
1,628
1,630
1,623
1,635
1,625
1,620
1,730a
1,560
1,590
1,580
1,575
1,568
1,585
–
–
–
–
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
1,350
1,388
1,365
1,358
1,360
1,381
210
202
215
217
208
204
540
535
530
540
533
542
511
508
517
519
515
510
a
As m(COOH)
123

DNA interactions and promotion in antibacterial activities
371
Thermal analysis
presence of one chlorine and one bromine atom. The peak
observed at m/z = 787.20 is due to loss of one chlorine
atom. Several other fragments at m/z = 469, 416, 404, 381,
319, and 156 are observed in the mass spectrum (Fig. 2).
The proposed fragmentation patterns of the complex are
shown in Scheme 1.
The objective of this section is to analyze the thermal
behavior of the complexes, having in view the composition
confirmation and the assessment of the role of water in the
crystallization molecule. For the complexes, the water
molecules are eliminated stepwise, namely the crystalli-
zation one up to 140 °C and the coordination one in the
140–200 °C temperature range, both steps being endo-
thermic [37–40]. TG was carried out for complex 1 at a
heating rate of 10 °C per minute in the range of 20–800 °C
under N2 atmosphere (Fig. 1).
Antibacterial activity
The antibacterial activity of CuCl2ꢀ2H2O, ligands, standard
drugs, and the complexes has been tested against two gram-
positive bacteria—Staphylococcus aureus and Bacillus
subtilis—and three gram-negative bacteria—Serratia
marcescens, Escherichia coli, and Pseudomonas aerugin-
osa—in terms of the minimum inhibitory concentration
(MIC). The MIC is the lowest concentration of an anti-
microbial that will inhibit the visible growth of a
microorganism after overnight incubation. The results
concerning in vitro antibacterial activity (MIC) are pre-
sented in Table 2.
On interpretation of the TG curve, three distinct mass
losses are observed. The first mass loss occurs between 40
and 130 °C, the second between 180 and 240 °C, and the
last between 260 and 450 °C. Mass loss occurring during
the first decomposition step corresponds to three molecules
of crystallization water, whereas mass loss during the
second step corresponds to decomposition of one chlorine
atom, and the third step corresponds to decomposition of
the NFL and neutral bidentate ligand, leaving behind CuO
as residue. A similar thermal degradation trend was
observed and reported in the literature for quinolone metal
complexes [41].
The antimicrobial activity of all the complexes against
the five microorganisms is much higher than that of
CuCl2ꢀ2H2O and free ligands. The inhibition activity seems
to be governed to a certain degree by the facility of coor-
dination at the metal center as well as the electronic nature
of the ligands. The biomolecular interaction with metal
complexes can be the cause of inhibition of biological
synthesis, and this prevents the organisms from reproduc-
ing [42]. It is clear from the data that complex 1 is more
active than NFL against all the bacterial species employed.
Mass spectra
The mass spectrum of complex 1 shows a molecular ion
peak at m/z = 822.12, 824.00, and 826.19, which are
assigned (M), (M ? 2), and (M ? 4) peaks, indicating the
Fig. 1 TGA of complex
[Cu(NFL)L1Cl]ꢀ3H2O (1)
123

372
M. N. Patel, A. P. Patidar
Fig. 2 LC-MS mass spectra of
complex 1
Complexes 2, 3, 4, 5, and 6 are moderately active com-
pared to NFL against all the bacterial species employed.
Out of all the active complexes, complex 1 is the most
active one. The MIC of norfloxacin ranges from 2.4 to
3.9 lM. Coordination of these drugs results in diverse
biological activity. This diverse biological activity may be
due to the chelation theory [43] or to the effect of the metal
ion on the normal cell process. All the complexes show
better antibacterial activity than [Cu(pr-norf)2(H2O)],
[Cu(pr-norf)(bipyam)Cl] against S. aureus and P. aeru-
ginosa [44]. The complexes show better antibacterial
activity than complexes of copper(II) with norfloxacin and
different phenanthroline derivatives against S. aureus, B.
subtilis, S. marcescens, and P. aeruginosa [28]. All com-
plexes also show better antibacterial activity than
[Cu(oxo)(bipy)Cl] against E. coli, P. aeruginosa, and S.
aureus [45].
bonds with metal ions [48]. Our results also supported the
fact that a metal ion is required for tight binding of DNA
with quinolone [49], since synthesized metal complexes or
chelates (metal-bonded quinolone) show better antimicro-
bial activity than free quinolone.
DNA-binding activity
Electron absorption study
The complexes directly binding to the CT DNA have been
studied by electronic absorption spectral studies. The
equilibrium DNA binding constants (Kb) of the complexes
to CT DNA were obtained by monitoring the change in the
absorption intensity of the spectral bands by subsequent
addition of CT DNA. A bathochromic shift along with
significant hypochromicity for all complexes has been
observed. The change in absorbance values of complexes
after mixing them with DNA indicates that the interaction
of complexes with DNA takes place by direct formation of
a new complex with double-helical DNA [50]. The binding
strength of complexes has been quantitatively determined
by calculating the intrinsic binding constants Kb of the
complexes by monitoring the change in absorbance at
various DNA concentrations. From the plot of [DNA]/(ea–
ef) versus [DNA], the binding constant values (Table 3)
were found in the range 1.55 9 104 to 1.89 9 104 M–1,
which is lower than those of the classical intercalator
(ethidium bromide), [Mn(sf)2(H2O)2] (1.17 9 105) [51],
[Cu(flmq)2(py)2] (8.12 9 105) [52], and [Cu(SPF)(-
pyc)Cl]ꢀ5H2O (7.93 9 104) [53]. Kb values of all the
complexes are higher than that of norfloxacin (1.13 9 103
M-1) and comparable to those of previously reported
copper(II) complexes (1.06–1.85 9 104) [54].
It has also been suggested [46, 47] that the ligands with
nitrogen donor systems might inhibit enzyme production,
since the enzymes that require these groups for their activity
appear to be especially more susceptible to deactivation by
the metal ions upon chelation. The chelation of copper with
ligand reduces the polarity of the metal ion by overlap of the
ligand’s orbital and partial sharing of the positive charge of
the metal ion with the donor groups. An increase in delo-
calization of p-electrons over the whole ligand enhances the
penetration of complexes into lipid membranes and block-
ing of the metal binding sites in the enzymes of
microorganisms. The complexes may also disturb the res-
piration process of the cell and thus block the synthesis of
proteins, which restricts further growth of the organism and
thus results in an overall gain in the biological potency. It
has been proposed that quinolone in solution forms stable
123

DNA interactions and promotion in antibacterial activities
373
Scheme 1
F
F
F
N
N
N
Cl Cu
O
Br
N
N
Cu
O
Br
N
Cl Cu
O
N
O
F
Br
O
F
O
m/z=503.93
N
N
N
HN
m/z=822.05
HN
m/z=787.09
F
Cl Cu
O
F
O
N
N
N
N
Cu
Br
HN
m/z=468.96
m/z=416.02
F
Cu
O
O
F
O
N
N
N
N
Br
m/z=404.03
HN
m/z=381.05
H
O
O
N
N
F
O
m/z=156.07
N
N
HN
m/z=319.13
These spectral characteristics are consistent with a mode
of interaction that involves a stacking interaction between
the complex and the base pairs of DNA, which means that
the complexes can intercalate into the double-helix struc-
ture of DNA. Complex 1 interacts more strongly compared
to other complexes (Fig. 3).
123

374
M. N. Patel, A. P. Patidar
Table 2 MIC in lM
Compounds
Gram positive
Gram negative
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
CuCl2ꢀ2H2O
Norfloxacin
2,698.0
2.4
2,815.0
2.5
2,756.0
3.9
2,404.0
3.7
3,402.0
2.7
Ligand L1
Ligand L2
Ligand L3
Ligand L4
Ligand L5
Ligand L6
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
192.0
197.0
200.0
194.0
199.0
202.0
0.7
168.0
166.0
175.0
169.0
167.0
178.0
0.5
242.0
270.0
277.0
244.0
272.0
280.0
0.9
254.0
257.0
250.0
255.0
260.0
255.0
1.0
278.0
285.0
290.0
278.0
288.0
292.0
1.2
1.1
0.4
1.0
1.1
2.0
1.1
0.5
1.0
1.5
2.2
0.9
0.6
1.3
1.6
2.4
1.3
0.5
1.2
1.4
2.2
1.4
0.6
1.3
1.6
2.4
Table 3 The binding constant (Kb) values of complexes
DNA, which means that the complexes can intercalate into
the double helix structure of DNA.
Complexes
Kb/M-1
As shown in Fig. 4, with subsequently increasing
amounts of complexes, the viscosity of DNA increases
steadily, which is similar to what happens with the classical
intercalative complex [Ru(bpy)2(dppz)]?2 [59]. It is clear
from the figure that all these complexes show an increase in
the relative viscosity of CT DNA, but less so than with
ethidium bromide. This behavior is similar to that of cop-
per complexes reported by Ramana et al. [60] and Song
et al. [61]. The viscosity results may reflect the tendency of
each ligand to intercalate into DNA base pairs. The
increase in DNA viscosity observed in the complexes
suggests a classical intercalative mode. The increase in the
degree of viscosity of all complexes depends on their
affinity to DNA, with an order as follows: EtBr [ 1 [ 2 [
3 [ 4 [ 5 [ 6.
Norfloxacin
Ligand L1
Ligand L2
Ligand L3
Ligand L4
Ligand L5
Ligand L6
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
1.13 9 103
1.32 9 103
1.28 9 103
1.26 9 103
1.15 9 103
1.11 9 103
1.08 9 103
1.89 9 104
1.85 9 104
1.80 9 104
1.70 9 104
1.61 9 104
1.55 9 104
Chemical nuclease activity
Viscosity measurements
Chemical nuclease activity is an important technique for
monitoring the plasmid DNA cleavage reaction. When plas-
mid DNA was subjected to electrophoresis after interaction,
upon illumination of the gel (Fig. 5). The fastest migration
was observed for super coiled (SC) Form I. The slowest
moving was observed for open circular (OC) Form II. The
intermediate moving was observed for linear (L) Form III,
whichwasgeneratedoncleavageofopencircular. Thedataon
plasmid cleavage are presented in Table 4.
Viscosity measurements of the complexes were performed
with DNA by varying the concentrations of the com-
pounds. Hydrodynamic measurements that are sensitive to
length changes are regarded as the least ambiguous and the
most critical tests of a DNA-binding model in solution,
providing reliable evidence for the DNA-binding mode
[55]. Viscosity of DNA is increased in the case of a clas-
sical intercalator because of the increase in the length of
the DNA helix, as base pairs are separated to accommodate
the intercalator. There are many cases in which complexes
show classical intercalation [56–58]. In our case, the
increase in viscosity was observed because of the stacking
interaction between the complex and the base pairs of
Here all the complexes show an efficient cleavage
ability. The different DNA-cleavage efficiencies of the
complexes, metal salt, and drugs are due to the difference
in binding affinity of the complexes to DNA and the
structural dissimilarities of the ligands [62].
123

DNA interactions and promotion in antibacterial activities
375
Fig. 3 Electronic absorption spectra of complex 1 in Tris-HCl buffer (pH 7.2) in the absence and presence of increasing amounts of CT DNA.
Inset: plot of [DNA]/(ea–ef) vs. [DNA]. Arrow shows the absorbance change upon increasing DNA concentrations
substituents. The synthesized copper(II) complexes show
good cytotoxic activity against brine shrimp, but this
investigation is a primary one, and further tests are required
to investigate its actual mechanism of cytotoxicity and its
probable effects on higher animal models and on cancer cell
lines. It suggests that the complexes can be used as potent
cytotoxic agents with the hope of adding to the arsenal of
weapons used against fatal cancer diseases.
SOD-like activity
SOD-like behavior was checked using the NBT/NADH/
PMS system. PMS is reduced by the hydrogen donor
NADH. The reduced PMS generates Oꢀ2- from dissolved
O2. NBT is reduced by O2ꢀ-, which results in a linear
Fig. 4 Effect on relative viscosity of CT DNA under the influence of
increasing amounts of complexes at 27 0.1 °C in Tris-HCl buffer
(pH 7.2)
accumulation of blue formazan with an increase in the
absorbance at 560 nm. The SOD activity data of the newly
synthesized complexes are collected in Table 6.
Cytotoxicity
On comparing the SOD activities of the present com-
plexes with the reported metal complexes of EDTA and
related chelators, our synthesized complexes exhibit sig-
nificant SOD-mimetic activities (using a modified NBT
assay) [65]. In a variety of complexes that act as SOD
enzymes [66], there is either one electron oxidation fol-
lowed by the reduction of the metal ion or the formation of
a superoxide complex that then gets reduced to peroxide by
another superoxide ion. Slopes of the straight line obtained
from absorption of monoformazan as a function of time for
different concentrations of 1 (Fig. 6 combined graphs) give
the plot for the percentage inhibition versus concentration
(Fig. 7 percentage inhibition).
The brine shrimp lethality bioassay is a recent development
in the assay procedures of bioactive compounds; it indicates
cytotoxicity as well as a wide range of pharmacological
activities (such as anticancer, antiviral, insecticidal, pesti-
cidal, AIDS, etc.) of the compounds [63, 64]. From the data
recorded in Table 5, it is evident that compound 1, having
less crowded structural units (–F), displayed potent cyto-
toxic activity against Artemia cysts. The order of potency of
compounds against brine shrimp is 1 [ 3 [ 2 [ 4 [ 6 [ 5.
The variation in lethality results may be due to the differ-
ence in the amount and kind of cytotoxic substances and
123

376
M. N. Patel, A. P. Patidar
Fig. 5 Photogenic view of cleavage of pUC19 DNA (300 lg/cm3)
with series of copper(II) complexes (200 lM) using 1 % agarose gel
containing 0.5 lg/cm3 ethidium bromide. All reactions were incu-
bated in TE buffer (pH 8) at a final volume of 15 mm3 for 24 h at
37 °C. Lane 1 DNA control, lane 2 CuCl2ꢀ2H2O, lane 3 norfloxacin,
lane 4 [Cu(NFL)(L1)Cl]ꢀ3H2O, lane 5 [Cu(NFL)(L2)Cl]ꢀ3H2O, lane 6
[Cu(NFL)(L5)Cl]ꢀ3H2O, lane 9 [Cu(NFL)(L6)Cl]ꢀ3H2O
[Cu(NFL)(L3)Cl]ꢀ3H2O, lane
7
[Cu(NFL)(L4)Cl]ꢀ3H2O, lane
8
Table 6 The IC50 values of complexes
Table 4 Complex-mediated DNA cleavage data by gel
electrophoresis
Complexes
IC50/lM
Lane Compound
Form I Form II Form III %
(SC)
Norfloxacin
161.07
0.494
0.550
0.673
0.510
0.594
0.740
1.310
5.170
5.860
(OC)
(LC)
Cleavage
[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(stz)(py)3Cl]
[Cu(Hstz)(MeOH)Cl2]
[CuL2(4-mHim)2]
1
2
3
4
5
6
7
8
9
Control
88
84
61
12
16
27
56
59
57
54
56
55
–
–
CuCl2ꢀ2H2O
Norfloxacin
–
4.55
12
20
14
13
20
16
14
30.68
73.93
69.66
66.29
70.78
68.53
65.17
[Cu(NFL)(L1)Cl] 24
[Cu(NFL)(L2)Cl] 27
[Cu(NFL)(L3)Cl] 30
[Cu(NFL)(L4)Cl] 26
[Cu(NFL)(L5)Cl] 28
[Cu(NFL)(L6)Cl] 31
Table 5 The LC50 values of complexes
Complexes
LC50/lM
Norfloxacin
194.98
7.964
[Cu(NFL)(L1)Cl]
[Cu(NFL)(L2)Cl]
[Cu(NFL)(L3)Cl]
[Cu(NFL)(L4)Cl]
[Cu(NFL)(L5)Cl]
[Cu(NFL)(L6)Cl]
10.139
9.843
12.034
12.994
12.438
SOD-like activity performed at biological pH results in
IC50 values ranging from 0.494 to 0.740 lM. All the
complexes have been compared with those reported by
Casanova et al. [67, 68]. The superoxide scavenging data
suggest that all complexes are found to be more active than
[Cu(glygly)(bipy)]ꢀ3H2O [69]. The IC50 values of the
copper(II) complexes are better than those recently repor-
ted for Ni(II) complexes [70]. The results show that
complexes exhibit greater scavenging activity toward
Fig. 6 Absorbance values (Abs560) as a function of time (t) plotted
for varying concentrations of complex 1 from 0.5 to 3 lM for which
good straight lines are observed
superoxide radicals, which may be accredited to the redox
potential of copper(II) complex, which depends on the
geometry at the metal center.
123

DNA interactions and promotion in antibacterial activities
377
generously supplied on demand by Bayer AG (Wuppertal,
Germany). Cupric chloride dihydrate was purchased from
E. Merck, Ltd., Mumbai, India. 2-Acetylpyridine was
purchased from Spectrochem (India). Acetophenone,
4-fluorobenzaldehyde, 4-chlorobenzaldehyde, and 4-bro-
mobenzaldehyde were purchased from Sigma Aldrich.
Ethidium bromide (EB), bromophenol blue, agarose, and
Luria broth (LB) were purchased from Himedia (India).
Nicotinamide adenine dinucleotide reduced (NADH), nitro
blue tetrazolium (NBT), and phenazin methosulfate (PMS)
were purchased from Loba Chemie Pvt., Ltd. (India). CT
DNA was purchased from Banglore Genei (India).
Metal percentage of the complexes was determined by
EDTA titration [71] after decomposing the organic matter
with a mixture of HClO4, H2SO4, and HNO3 (1:1.5:2.5).
TG was obtained with a model Mettler Toledo TGA/DSC
1 thermogravimetric analyzer. The magnetic moments of
the complexes were measured by Gouy’s method using
mercury tetrathiocyanatocobaltate(II) as the calibrant
(vg = 16.44 9 10–6 cgs units at 20 °C) with a Citizen
Balance. Infrared spectra were recorded on a FT-IR ABB
Bomen MB 3000 spectrophotometer as KBr pellets in the
range 4,000–400 cm–1. The C, H, and N elemental ana-
lysis was carried out with a model 240 Perkin Elmer
elemental analyzer (MA, USA). The minimum inhibitory
concentration (MIC) study was carried out by means of a
laminar air flow cabinet (Toshiba, Delhi, India). The LC-
MS was recorded using a Thermo mass spectrophotometer
(USA). The electronic spectra were recorded on a UV-
160A UV-Vis spectrophotometer (Shimadzu, Japan).
Photo quantization of the gel after electrophoresis was
carried out on AlphaDigiDocTM RT, version V.4.0.0 PC-
Image software.
Fig. 7 Plot of percentage of inhibiting NBT reduction with an
increase in the concentration of complex 1
Conclusions
From the analytical and spectral investigations, we can
conclude the square pyramidal geometry of all copper(II)
complexes derived from bipyridine derivatives. The mea-
surement of the DNA-binding affinity of copper(II)
complexes toward DNA using absorption spectral analyses
suggests that the complexes bind with DNA via the clas-
sical intercalation mode. Complex 1 shows the highest Kb
(DNA binding constant) value while complex 6 shows the
lowest one because complex 1 is carrying a fluorine atom
as a substituent. The fluorine atoms act not only as
chemical isosteres for the oxygen atoms in the heterocyclic
base of thymidine, but also participate in ‘‘strong’’ F–H
bonding; as a result, complex 1 shows better antibacterial
and DNA interaction activity than complex 6. All the
complexes increase the relative viscosity of DNA. The
results from both absorption spectral and viscosity studies
reveal the complexes bind with DNA via intercalation
mode. Cytotoxic studies of metal complexes show potent
lethality (LC50) against brine shrimp. The DNA cleavage
study of pUC19 shows that all the complexes have high
cleavage ability compared to the metal salt and drug.
Ligands that can stabilize bonding between the metal
center and oxygen radical anion favor enzymatic behavior.
In our case, complex 1 showed the highest ability to
scavenge free radical anions of molecular oxygen.
Synthesis of ligands
All bidentate ligands L1 to L6 were prepared by reacting
the appropriate enone with pyridinium salt by following the
procedure described in the literature [72, 73].
Chloro[6-(4-bromophenyl)-4-(4-fluorophenyl)-2,20-
bipyridine][1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylato]copper(II) trihydrate
([Cu(NFL)L1Cl]ꢀ3H2O, 1, C38H37BrClCuF2N5O6)
A methanolic solution of CuCl2ꢀ2H2O (1.5 mmol) was
added to a methanolic solution of 6-(4-bromophenyl)-4-(4-
fluorophenyl)-2,20-bipyridine (1.5 mmol), followed by
addition of a previously prepared solution of NFL
(1.5 mmol) in methanol in the presence of CH3ONa
(1.5 mmol). The pH was adjusted at *6.8 using a dilute
CH3ONa solution. The resulting solution was refluxed for
3 h on a water bath, followed by concentrating it to half of
its volume. A fine amorphous product of green color was
Experimental
The chemicals employed were of analytical grade and were
used without further purification. Norfloxacin (NFL) was
123

378
M. N. Patel, A. P. Patidar
obtained. It was washed with ether/hexane and dried in
vacuum desiccators (Scheme 2). The proposed structure of
complexes is shown in the Supplementary Material. Yield:
69.9 %; m.p.: 232 °C; leff: 1.85 BM.
It was synthesized using 4-(4-fluorophenyl)-6-phenyl-2,20-
bipyridine as ligand (L4) (0.5 mmol). Yield: 67.1 %; m.p.:
232 °C; leff: 1.84 BM.
Chloro[4-(4-chlorophenyl)-6-phenyl-2,20-bipyridine]-
[1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylato]copper(II) trihydrate
([Cu(NFL)(L5)Cl]ꢀ3H2O, 5, C38H38Cl2CuFN5O6)
It was synthesized using 4-(4-chlorophenyl)-6-phenyl-2,20-
bipyridine as ligand (L5). Yield: 68.3 %; m.p.: [239 °C;
leff: 1.87 BM.
Chloro[6-(4-bromophenyl)-4-(4-chlorophenyl)-2,20-
bipyridine][1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylato]copper(II)
trihydrate ([Cu(NFL)(L2)Cl]ꢀ3H2O, 2,
C38H37BrCl2CuFN5O6)
It was synthesized using 6-(4-bromophenyl)-4-(4-chloro-
phenyl)-2,20-bipyridine as ligand (L2). Yield: 69.03 %;
m.p.: 252 °C; leff: 1.89 BM.
Chloro[4,6-bis(4-bromophenyl)-2,20-bipyridine]-
[1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylato]copper(II) trihydrate
([Cu(NFL)(L3)Cl]ꢀ3H2O, 3, C38H37Br2ClCuFN5O6)
It was synthesized using 4,6-bis(4-bromophenyl)-2,20-
bipyridine as ligand (L3). Yield: 67.4 %; m.p.: 256 °C;
leff: 1.82 BM.
Chloro[4-(4-bromophenyl)-6-phenyl-2,20-bipyridine]-
[1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylato]copper(II) trihydrate
([Cu(NFL)(L6)Cl]ꢀ3H2O, 6, C38H38BrClCuFN5O6)
It was synthesized using 4-(4-bromophenyl)-6-phenyl-2,20-
bipyridine as ligand (L6) (0.5 mmol). Yield: 67.6 %; m.p.:
247 °C; leff: 1.90 BM.
In vitro antimicrobial screening
Chloro[4-(4-fluorophenyl)-6-phenyl-2,20-bipyridine]-
[1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylato]copper(II) trihydrate
([Cu(NFL)(L4)Cl]ꢀ3H2O, 4, C38H38ClCuF2N5O6)
The in vitro antibacterial activity of complexes was carried
out against two gram-positive—i.e., Staphylococcus aureus
and Bacillus subtilis—and three gram-negative—i.e.,
Scheme 2
R'
N
O
O
2- Acetylpyridine
R'
R
Pyridine
N
Iodine
NaOH
MeOH
N
R
I
N
N
Norfloxacin
CuCl2·2H2O
O
NaOMe
pH = 6.8
R
R'
O
Cl
N
N
O
O
Cu
Complex
R
R'
F
N
1
Br
2
3
4
5
6
Br
Br
H
H
H
Cl
Br
F
Cl
Br
F
N
NH
123

DNA interactions and promotion in antibacterial activities
379
Serratia marcescens, Pseudomonas aeruginosa, and
Escherichia coli—species by minimum inhibitory con-
centration (MIC) using the LB dilution method. Two
percent of sterile LB was used as a medium for MIC. All
the cultures were incubated at 37 °C. Control tests with no
active ingredients and the solvent alone were also per-
formed. Precultures of the bacteria used were grown in LB
overnight at an optimum temperature for each species.
Bacterial growths were measured by the turbidity of the
culture after incubation for 18 h. If a particular concen-
tration of compound inhibited the bacterial growth, half the
concentration of the compound was tried. The procedure
was continued to a concentration at which bacteria grow
normally. The lowest concentration that inhibited the
bacterial growth was considered the MIC value. Sur-
roundings were maintained sterile throughout.
bath. The concentration of DNA was 200 lM in NP, and
the flow times were measured with a digital timer. Each
sample was measured three times, and an average flow
time was calculated. Data were presented as (g/g0)1/3 ver-
sus [complex]/[DNA], where g is the viscosity of DNA in
the presence of complex and g0 that of DNA alone. Vis-
cosity values were calculated from the observed flowing
time of DNA-containing solutions (t) corrected for that of
buffer alone (t0), gµ(t–t0) [75].
Chemical nuclease activity
Gel electrophoresis of plasmid DNA (pUC19 DNA) was
carried out in Tris-acetate-EDTA (TAE) buffer (0.04 M
TAE, pH 8, 0.001 M EDTA). The reaction mixture
(15 mm3) contained plasmid DNA in TE buffer (10 mM
Tris, 1 mM EDTA, pH 8.0) and 200 lM complex. The
reaction mixture was allowed to incubate for 24 h at 37 °C.
All reactions were quenched by the addition of 5 mm3
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 electro-
phoresed at 50 V in 19 TAE buffer. Gel was stained with
0.5 lg/cm3 EtBr and was photographed on a UV illumi-
nator. The percentage of each form of DNA was quantified
using AlphaDigiDocTM RT, version V.4.0.0, PC-Image
software.
The bactericidal action of all compounds was also
evaluated against the same bacterial culture. The inocu-
lums were prepared by diluting an overnight culture
grown in Luria broth to obtain 106 viable bacteria/cm3
confirmed in each experiment by colony counts. Bacteria
were exposed to concentrations of 0.25–1.75 lg/cm3 of
compounds. The final volume was 1 cm3. Cultures were
incubated at 37 °C for 2 h. The 100 mm3 bacterial culture
from above was taken and spread over a previously pre-
pared agar plate. The incubation conditions comprised
using Luria broth as the medium for culturing bacteria
at 37 °C. The visual colonies were calculated in order
to check biocidal activity of metal complexes, yield-
ing 30–300 colonies (Fig. shown in Supplementary
Material).
Brine shrimp bioassay
Brine shrimp assay was carried out according to the
method given by Mayer et al. [76]. Brine shrimp (Artemia
cysts) eggs were hatched in a shallow rectangular plastic
dish (22 9 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 was sprinkled into the large
compartment and opened to ordinary light. A sample of the
test compounds was prepared by dissolving 10 mg of each
compound in 10 cm3 of DMSO. Solutions were transferred
from the stock solutions to 15 vials in amounts to yield
final concentrations of 4, 8, 12, 16, and 20 lg/cm3 (three
sets for each dilution were run for all test samples, and a
mean of three sets was taken for calculation of LC50). One
vial was kept as control having the same amount of DMSO
only. After 48 h, nauplii were ready to use; 1 cm3 of
artificial seawater and ten nauplii were added to each vial.
The total volume was adjusted to 2.5 cm3 per vial using
artificial seawater. The number of live and dead nauplii
was counted after 24 h. Data were analyzed by a simple
method to determine the LC50 values, in which the log of
sample concentration was plotted against percentage of
mortality of nauplii [77].
DNA interaction activity
Absorption titration
The UV absorbance at 260 and 280 nm of the CT DNA
solution in 5 mM Tris-HCl buffer (pH 7.2) gave a ratio of
1.9, indicating the DNA was free of protein. The con-
centration of CT DNA was measured from the band
intensity at 260 nm with a known value (6,600 M–1 cm–1)
[74]. Absorption titration measurements were carried out
by varying the concentration of CT DNA, keeping the
metal complex concentration constant in 5 mM Tris-HCl/
5 mM NaCl buffer (pH 7.2). Samples were kept for
equilibrium before recording the spectrum. The intrinsic
binding constants (Kb) were determined from a plot of
[DNA]/(ea–ef) versus [DNA] using absorption spectral
titration data.
Viscosity measurement
The viscosity measurementwas were performed with an
Ubbelodhe automated viscometer. The viscometer was
thermostated at 27 0.1 °C in a constant temperature
123

380
M. N. Patel, A. P. Patidar
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ð1Þ
where k’ and k represent slopes of the straight line of
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