DNA interaction, in vitro antimicrobial and SOD-like activity of copper(II) complexes with norfloxacin and terpyridines

Article abstract of DOI:10.1016/j.jorganchem.2011.11.022

The novel octahedral copper(II) complexes with the second generation fluoroquinolone, norfloxacin and terpyridine derivatives were prepared and characterized. The antimicrobial efficiency of the complexes were tested on five different microorganisms and s

Full text of DOI:10.1016/j.jorganchem.2011.11.022

Journal of Organometallic Chemistry 701 (2012) 8e16
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
DNA interaction, in vitro antimicrobial and SOD-like activity of copper(II)
complexes with norfloxacin and terpyridines
,
1
1
Mohan N. Patel 1 , 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
Article history:
The novel octahedral copper(II) complexes with the second generation fluoroquinolone, norfloxacin and
terpyridine derivatives were prepared and characterized. The antimicrobial efficiency of the complexes
were tested on five different microorganisms and showed good biological activity. Viscosity measure-
ment and absorption titration were employed to determine the mode of binding of complexes with DNA
whereas the cleavage efficacy of the complexes towards pUC19 DNA was determined by electrophoresis
in presence of ethidium bromide. SOD mimic behaviour was actively sought for clinical and mechanistic
purposes under a nonenzymatic system (NBT/NADH/PMS), and was found to have good antioxidant
activity.
Received 27 July 2011
Received in revised form
12 November 2011
Accepted 17 November 2011
Keywords:
Terpyridines
Norfloxacin
Antibacterial activity
DNA interaction
SOD mimic
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
[9e13]. It was discovered that a fluorine atom at position 6 and
a piperazine ring at position 7 greatly enhance the spectrum of
Synthesis of molecules which can serve as a probe for nucleic
acid structure or which can show nuclease property has gained
attention of many researchers [1]. Transition metal complexes have
been widely exploited for these purposes not only because of their
unique spectral and electrochemical signatures but also due to the
fact that by changing the ligand environment one can tune the DNA
binding and cleaving ability of a metal complex. Even though
interaction of DNA with complexes of many transition metal ions
have been investigated [2e4]. Copper(II) complexes are the
preferred molecules for bringing about DNA cleavage. This is due to
the fact that copper(II) complexes can not only bring about oxida-
tive cleavage of DNA but also hydrolytic, photolytic and electrolytic
cleavage of DNA [5,6]. Copper(II) is known to play a significant role
in naturally occurring biological systems as well as a pharmaco-
logical agent [7,8].
activity. Fluoroquinolones are extremely useful for the treatment
of variety of infections [14e16]. Norfloxacin (Fig. 1b),
a
a fluoroquinolone, is a 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-
piperazinyl)-3-quinolinecarboxylic acid. It functions by inhibiting
DNA gyrase, a type II topoisomerase, and topoisomerase IV,
enzymes necessary to separate bacterial DNA. Several human
diseases such as cardiovascular and neurodegenerative disorders,
cancer, inflammatory and ageing processes involve some form of
oxidation at the subcellular level. Cells and tissues injury (oxidative
stress) results from the imbalance between pro and antioxidant
species [17]. Superoxide dismutase activity (SOD) in conjunction
with catalase appears to be the most effective enzymatic defence
against the toxicity of oxygen metabolism [18e20]. Among the
known SOD enzymes, Cu2Zn2(SOD) is the most efficient catalytic
species. It catalysed the disproportionation of the cytotoxic super-
oxide radical Oꢀ2ꢁ, to oxygen and hydrogen peroxide, through one-
electron redox cycle involving its copper centre.
It is known since three decades ago that cancer cells have less
than normal SOD activity and the treatment with bovine native
CueSOD decreased the growth of several solid tumours [18].
Furthermore, low molecular weight compounds with superoxide
dismutase mimetic activity have potential use as antioxidant
pharmaceuticals in the treatment or prevention of several diseases
related with the overproduction of an undesired Oꢀ2ꢁ. In particular,
some copper complexes with SOD mimetic activity have
The term quinolones is commonly used for the quinolone-
carboxylic acids or 4-quinolones, which are a group of synthetic
antibacterial agents containing a 4-oxo-1,4-dihydroquinoline skel-
eton (Fig. 1a). Quinolone antibiotics are chelating agents for
a variety of metal ions well known for their biological activity
* Corresponding author.
E-mail address: jeenen@gmail.com (M.N. Patel).
Tel.: þ91 2692 226856x218.
1
0022-328X/$ e see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2011.11.022

M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
9
to an ethanolic solution of various aldehydes (10.0 mmol in 70 mL
EtOH). KOH pellets (26 mmol) and aqueous NH3 (25%, 0.425 mol)
were added to the solution and was stirred at room temperature for
8 h. An off-white solid formed which was collected by filtration,
followed by washings with H2O (3 ꢂ 10 mL) and EtOH (2 ꢂ 5 mL).
Crystallization from CHCl3eMeOH system gives a white crystalline
solid. The proposed reaction is shown in Scheme 1.
Fig 1. (a) Basic skeleton of quinolones; 4-oxo-1,4-dihydroquinoline. (b) 1-Ethyl-6-
fluro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline-3-carboxylic; norfloxacin (NFLH).
(c) 1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline-3-carboxylate;
deprotonated norfloxacin (NFL).
2.4. Synthesis of complexes
2.4.1. [Cu(NFL)(L1)Cl]
Methanolic solution of CuCl2$2H2O (1.5 mmol) was added to
a methanolic solution of 40-(4-chlorophenyl)-2,20:60,200-terpyridine
(L1) (1.5 mmol), followed by the addition of a previously prepared
methanolic solution of norfloxacin (1.5 mmol) in presence of
CH3ONa (1.5 mmol). The pH of the reaction mixture was adjusted to
w6.8. The resulting solution was refluxed for 2 h on a water bath,
followed by concentrating to half of its volume. A fine, green
amorphous product obtained was washed with ether/hexane and
dried in a vacuum desiccator. (Scheme 2) Yield: 65.2%, m.p.:
>300 ꢃC, meff: 1.90 B.M. Anal. Calcd. for: C38H31Cl2FCuN6O3 (761.13):
C, 58.39; H, 4.11; N, 11.04; Cu, 8.35%. Found: C, 58.29; H, 4.02; N,
10.95; Cu, 8.29%.
demonstrated to possess antiinflammatory activity, anticarcino-
genic and antimutagenic effects [19,20]. These facts encouraged the
investigation of model complexes, especially copper(II) with SOD
mimetic activity [21,22] and have motivated us to include the study
of low molecular weight copper complexes with antioxidant
compounds in our research on metal coordination compounds with
biological relevant ligands.
Herein, we have studied the biological properties of terpyr-
idinesenorfloxacinecopper(II) complexes including DNA interac-
tion and enzymatic activity.
2. Experiments
2.4.2. [Cu(NFL)(L2)Cl]
It was synthesized using 40-(4-bromophenyl)-2,20:60,200-terpyr-
idine (L2) (1.5 mmol). Yield: 64.5%, m.p.: 289 ꢃC, meff: 1.89 B.M. Anal.
Calcd. for: C37H31BrClFCuN6O3 (805.58): C, 55.16; H, 3.88; N, 10.43;
Cu, 7.89%. Found: C, 55.05; H, 3.78; N, 10.31; Cu, 7.80%.
2.1. Reagent
All reagents, chemicals and solvents used were of analytical
reagent grade; double-distilled water was used throughout. Nor-
floxacin was generously supplied by Bayer AG (Wuppertal, Ger-
many). Cupric chloride dihydrate was purchased from E. Merck Ltd,
Mumbai (India). 2-Acetyl pyridine, p-chloro benzaldehyde, p-
methyl benzaldehyde, p-bromo benzaldehyde, p-fluoro benzalde-
hyde, pyridine-2-carbaldehyde, thiophene-2-carbaldehyde, p-
benzyloxy benzaldehyde, nicotinamide adenine dinucleotide
reduced (NADH), nitro blue tetrazolium (NBT) and phenazine
methosulphate (PMS) were purchased from Loba Chemie PVT. LTD.
(India). Ethidium bromide, bromophenol blue, agarose and Luria
Broth (LB) were purchased from Himedia (India).
2.4.3. [Cu(NFL)(L3)Cl]
It was synthesized using 40-(4-fluorophenyl)-2,20:60,200-terpyr-
idine (L3) (1.5 mmol). Yield: 66.7%, m.p.: 279 ꢃC, meff: 1.92 B.M. Anal.
Calcd. for: C37H31ClF2CuN6O3 (744.68): C, 59.68; H, 4.20; N, 11.29;
Cu, 8.53%. Found: C, 59.58; H, 4.09; N, 11.18; Cu, 8.45%.
2.4.4. [Cu(NFL)(L4)Cl]
It was synthesized using 40-(4-methylphenyl)-2,20:60,200-terpyr-
idine (L4) (1.5 mmol). Yield: 68.3%, m.p.: >300 ꢃC, meff: 1.95 B.M.
Anal. Calcd. for: C38H34ClFCuN6O3 (740.71): C, 61.62; H, 4.63; N,
11.35; Cu, 8.58%. Found: C, 61.50; H, 4.52; N, 11.46; Cu, 8.49%.
2.2. Physical measurement
Microanalyses (C, H, and N) were done using a 240 Perkin
Elmer elemental analyzer. Metal content of the complexes was
determined by EDTA titration [23] after decomposing the organic
matter with a mixture of HClO4, H2SO4, and HNO3 (1:1.5:2.5).
Room temperature magnetic measurement for the complexes was
made using Gouy magnetic balance. The Gouy tube was calibrated
using mercury(II)tetrathiocyanatocobaltate(II),
(
a
calibrant
cg ¼ 16.44 ꢂ 10ꢁ6 cgs units at 20 ꢃC). Electronic spectra were
recorded on a UV-160A UVeVis spectrophotometer, Shimadzu
(Japan). Infrared spectra were recorded on an FTeIR Shimadzu
spectrophotometer as KBr pellets in the range 4000e400 cmꢁ1
.
The minimum inhibitory concentration (MIC) study was carried
out by means of laminar air flow cabinet Toshiba, Delhi (India).
The LCeMS were recorded using Thermo mass spectrophotom-
eter (USA). Photoquantization of the gel after electrophoresis was
done using AlphaDigiDocÔ RT, version 4.0.0 PC-Image software
(California, USA).
2.3. Synthesis of ligands
All tridentate ligands were synthesized similarly by following
literature procedure [24]. 2-Acetyl pyridine (20.0 mmol) was added
Scheme 1. General synthesis of Ligand.

10
M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
continued to a concentration at which bacteria grow normally. The
lowest concentration that inhibits the bacterial growth was
considered as the MIC value. Surroundings were maintained
sterile throughout.
The bactericidal action of all compounds was evaluated against
same microorganisms. The inoculum was prepared by diluting an
overnight culture grown in Luria Broth to obtain 106 viable
bacteria/mL. Bacteria were exposed to various concentrations of
compounds. Compound-free control tubes were included in each
run. The final volume was 1 mL. Cultures were incubated at 37 ꢃC
for 2 h. The 100 mL bacterial culture from each dilution was taken
and spread over previously prepared agar plate. The plates were
incubated for 24 h and colony counts were performed on plates
yielding 30e300 colonies.
2.6. DNA interaction activity
2.6.1. Absorption titration
Binding of Herring Sperm DNA via intercalative mode usually
results in hypochromism and bathochromism [25,26] because the
intercalation mode involves a strong stacking interaction between
an aromatic chromophore and the DNA base pair [27]. After addi-
tion of an equivalent amount of DNA to the reference cell, it was
kept for 10 min incubation at room temperature followed by
absorption measurement around an appropriate absorbance peak.
This was specifically done to enable direct comparison between the
assays which was required to interpret the results obtained. The
intrinsic binding constant, Kb for each complex was determined by
making it subject to the following equation [28]:
Scheme 2. General synthesis of complex.
2.4.5. [Cu(NFL)(L5)Cl]
[DNA]/(εa ꢁ εf) ¼ [DNA]/(εb ꢁ εf) þ 1/Kb(εb ꢁ εf)
It was synthesized using 40-(2-pyridyl)-2,20:60,200-terpyridine
(L5) (1.5 mmol). Yield: 62.7%, m.p.: 285 ꢃC, meff: 1.97 B.M. Anal. Calcd.
for: C36H31ClFCuN7O3 (727.67): C, 59.42; H, 4.29; N, 13.47; Cu,
8.73%. Found: C, 59.31; H, 4.36; N, 13.56; Cu, 8.63%.
where, [DNA] is the concentration of Herring Sperm DNA in base
pairs, εa corresponds to the apparent extinction coefficient obtained
by calculating Aobsd/[complex], εf corresponds to the extinction
coefficient of the complex in its free form and εb refers to the
extinction coefficient of the complex in the fully bound form. The
data were fitted to the above equation to obtain straight line with
a slope value corresponding to the term 1/(εa ꢁ εf) and the y-
intercept to 1/Kb(εb ꢁ εf). The Kb was determined from the ratio of
slope to the y-intercept.
2.4.6. [Cu(NFL)(L6)Cl]
It was synthesized using 40-(thiophen-2-yl)-2,20:60,200-terpyr-
idine (L6) (1.5 mmol). Yield: 63.1%, m.p.: >300 ꢃC, meff: 1.87 B.M.
Anal. Calcd. for: C35H30ClFCuN6O3S (732.71): C, 57.37; H, 4.13; N,
11.47; Cu, 8.67%. Found: C, 57.44; H, 4.20; N, 11.38; Cu, 8.58%.
2.4.7. [Cu(NFL)(L7)Cl]
2.6.2. DNA binding study by hydrodynamic volume measurement
Ubbelohde viscometer immersed in a thermostatic bath main-
tained at 27 ꢄ 0.1 ꢃC was used to measure the change in hydro-
dynamic volume with change in complex concentration. Digital
It was synthesized using 40-(4-benzyloxyphenyl)-2,20:60,200-ter-
pyridine (L7) (1.5 mmol). Yield: 64.2%, m.p.: >300 ꢃC, meff: 1.93 B.M.
Anal. Calcd. for: C44H38ClFCuN6O4 (832.81), C, 63.46; H, 4.60; N,
10.09; Cu, 7.63%. Found: C, 63.38; H, 4.70; N, 9.98; Cu, 7.54%.
stopwatch with least count of 0.01 s was engaged for flow times
1/3
measurement with accuracy of ꢄ0.1 s. Plot of (
h
/h0
)
versus
[complex]/[DNA] is used to study the behavior of binding, where
h
2.5. Antibacterial activity
is the viscosity of DNA in presence of complex and h0 is the viscosity
of DNA alone. Viscosity values were calculated from the observed
flow time of DNA-containing solutions (t) corrected for that of the
The complexes were screened for their in vitro antibacterial
activity against two Gram(þve) i.e., Staphylococcus aureus, Bacillus
subtilis, and three Gram(ꢁve) i.e., Serratia marcescens, Pseudomonas
aeruginosa, and Escherichia coli species by minimum inhibitory
concentration (MIC) using Broth dilution method. Two percent of
sterile LB was used as a medium which consists of 1% w/v tryp-
tone, 0.5% w/v NaCl, and 0.5% w/v yeast extract. All the cultures
were incubated at 37 ꢃC. Control tests with no active ingredients
and the solvent alone were also performed. Preculture 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
concentration of compound inhibits the bacterial growth, half the
concentration of the compound was tried. The procedure was
buffer alone (t0),
h
¼ (t e t0) [29].
2.6.3. DNA cleavage study by gel electrophoresis
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). 15
(10 mM Tris, 1 mM EDTA, pH 8.0) and 200
were allowed to proceed for 24 h at 37 ꢃC. All reactions were
quenched by addition of 5 L loading buffer (0.25% bromophenol
m
L reaction mixture containing plasmid DNA in TE buffer
mM complex. Reactions
m
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 1X TAE buffer. Gel was stained with 0.5
mg/mL
EtBr and was photographed on a UV illuminator. The percentage of

M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
11
Table 1
complex was determined by plotting the graph of percentage
inhibition of NBT reduction against increase in the concentration of
complex. Concentration of the complex which causes 50% inhibi-
Change in IR bands for interaction of norfloxacin with Cu(II) in addition to terpyr-
idines (4000e400 cmꢁ1).
Compounds
n
(C]O) cmꢁ1
pyridine
n
(COO)asym
n
(COO)sym
Δn
n
(MeN)
n(MeO)
tion of NBT reduction is reported as IC50
.
cmꢁ1
cmꢁ1
cmꢁ1 cmꢁ1
cmꢁ1
Norfloxacin 1730
1642
1336
306
e
e
3. Result and discussion
Complex-1 1620
Complex-2 1615
Complex-3 1630
Complex-4 1622
Complex-5 1628
Complex-6 1625
Complex-7 1635
1568
1588
1570
1577
1591
1572
1594
1368
1385
1365
1369
1381
1360
1387
200
203
205
208
210
212
207
534
543
539
536
541
546
549
516
507
512
510
518
520
523
3.1. Magnetic and electronic behaviour
The magnetic moments of copper(II) in any of its geometry lies
around 1.8 B.M. which is very close to spin-only value i.e. 1.73 B.M.
The found values in our case lie in the range, 1.87e1.96 B.M. These
values are typical for mononuclear copper(II) compounds having
d9-electronic configuration. The observed magnetic moments of all
the complexes correspond to typical high-spin octahedral
complexes. However, the values are slightly higher than the ex-
pected spin-only values due to spin orbit coupling contribution
[31]. Visible emission spectra of the copper(II) complexes, i.e. d9
system, were recorded in DMSO. Complexes exhibited one broad
band along with a shoulder at w14,000 cmꢁ1 and w11,000 cmꢁ1
respectively, attributed to the ded transition for Cu(II) atom in
a distorted octahedral geometry [32].
each form of DNA was quantities using AlphaDigiDocÔ RT. Version
V.4.0.0 PCeImage software.
2.7. Enzymatic behavior
NBT/NADH/PMS system was used to study SOD-like behavior of
the complexes. The superoxide radial produced by 79
30
M PMS in phosphate buffer (pH ¼ 7.8) was responsible for
reduction of 75 M NBT in system, and 0.5e3.0 M tested
mM NADH,
m
m
m
compound are responsible for retardation in the reduction rate of
NBT which was determined spectrophotometrically by monitoring
the concentration of blue formazan form which absorbs at 560 nm.
All measurements were carried out at room temperature. The %
inhibition (h) of NBT reduction was calculated using following
equation [30]:
3.2. IR spectra
Determination of the coordinating atoms was done on the basis
of the comparison of IR spectra of norfloxacin and of the complexes.
Significant wave numbers are given in Table 1. For norfloxacin, the
n
(C]O) stretching vibration band appears at 1730 cmꢁ1 whereas
for complexes it appears in the range 1615e1635 cmꢁ1. This shift in
band towards lower energy suggests that coordination occurs
through the pyridone oxygen atom. In case of norfloxacin, strong
absorption bands at 1642 cmꢁ1 and 1336 cmꢁ1 could be assigned
(% Inhibition of NBT reduction) ¼ (1 ꢁ k0/k) ꢂ 100
where, k0and k represent the slopes of the straight line of absor-
bance values as a function of time in presence and in absence of
SOD mimic or a model compound, respectively. IC50 value of the
for
n(COO)assy and n(COO)sym vibration respectively. While for metal
complexes these bands were observed in the range 1568e1594 and
Fig. 2. LC-mass spectrum of complex 1, that is [Cu(NFL)(L1)Cl].

12
M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
Scheme 3. Fragmentation pattern of [Cu(NFL)(L1)Cl].

M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
13
Table 2
Bacteriostatic concentration of CuCl2$2H2O, NFLH and complexes by Broth dilution
in terms of MIC (
m
mol Lꢁ1).
(þve)
(ꢁve)
Gram
Compounds Gram
S. aureus B. subtilis S. marcescens P. aeruginosa E. coli
CuCl2$2H2O
Norfloxacin
2698.0
2.4
2815.0
2.5
2756.0
3.9
2404.0
3.7
3402.0
2.7
1
2
3
4
5
6
7
0.65
1.0
0.5
1.45
2.5
0.7
1.05
1.25
1.1
1.6
0.9
2.4
3.9
3.15
4.1
0.9
1.2
0.7
1.9
2.9
2.3
3.2
1.7
1.0
2.6
4.2
3.3
4.5
0.6
1.5
2.6
2.0
2.8
1.9
2.75
Fig. 4. Electronic absorption spectra of 1 in phosphate buffer (Na2HPO4eNaH2PO4, pH
7.2) in the absence and presence of increasing amount of DNA. The [complex] ¼ 10
[DNA] ¼ 0e150
M. The incubation period is 10 min at 37 ꢃC. Inset: plot of [DNA]/
f3 ) versus [DNA]. Arrow shows the absorbance change upon increasing DNA
mM;
1360e1387 cmꢁ1. To determine coordination mode of ligands, the
m
difference Δ ¼
n(COO)assy e n(COO)sym is very useful. The Δ values
(
3
a
ꢁ
are greater than 200 cmꢁ1, which indicate the monodentate coor-
concentrations.
dination mode of carboxylato group of the ligands [33e37]. These
data are further supported by
n
(MeO), which appears in the range
(C]
3.4. Antibacterial activity
507e523 cmꢁ1 for complexes. In investigated complexes the
n
N) band of terpyridine appears at 1580 cmꢁ1. This band shift to
higher frequency at w1630 cmꢁ1 in complexes, which indicates the
tridentate NeN coordination of the ligand [38,39]. The N / M
Table 2 shows in vitro antibacterial activity data of the cupric
chloride dihydrate, norfloxacin and synthesized complexes (1e7).
It is clear from the data that complexes 1, 2, 3, 4, 6 are active
compare to NFLH for all the bacterial species employed.
Complexes 5 and 7 are less active than NFLH against all the
species. Out of all the active complexes, complex 3 is the most
active one. The observed higher antimicrobial activities of the
complexes can be explained on the basis of Tweedy’s chelation
theory [41]. The lipid membrane that surrounds the cell favours
the passage of only lipid soluble materials due to which lip-
ophilicity is an important factor which controls the antimicrobial
activity. On chelation, the polarity of the metal ion will be reduced
to a greater extent due to the overlap of the ligand orbitals and
partial sharing of the positive charge of the metal ion with the
bonding was supported by
complexes [40].
n
(MeN) band at w534e549 cmꢁ1 for
3.3. Mass spectra
The mass spectrum of complex 1 showed molecular ion peak at
m/z ¼ 761.14. The doublet observed at m/z ¼ 724.26 and 726.32 was
due to the presence of one chlorine atom. In case of cop-
pereterpyridine moiety, the doublet observed at m/z ¼ 443.05 and
445.05 confirms the presence of one chlorine atom. The doublet
was observed at m/z ¼ 406.02 and 408.02 corresponding to the
coppereterpyridine moiety. The doublet observed at m/z ¼ 416.03
and 418.03 also confirms the presence of one chlorine atom in case
of copperenorfloxacin moiety. The peak at m/z ¼ 381.15 was
observed due to loss of chlorine atom from the copperenorfloxacin
fragment. The doublet was observed at m/z ¼ 343.11 and 345.14
corresponding to presence of one chlorine atom in the terpyridine
moiety. Other peaks at m/z ¼ 266.08 and 156.28 were observed due
to fragmentation of terpyridine moiety. The peak was observed at
m/z ¼ 320.17 corresponding to norfloxacin. Fig. 2 represents the
mass spectrum of Complex 1. The proposed fragmentation pattern
of complex 1 is shown in Scheme 3.
donor groups. Further, it increases the delocalization of
p-elec-
trons over the whole chelate ring and hence enhances the lip-
osolubility of the complexes. This increased liposolubility
enhances the penetration of the complexes into the lipid
membrane; the lipophilic group to drive the compound through
the semipermeable membrane of the cell; and blocks the metal
binding sites in the enzymes of the microorganisms.
In addition, our study regarding bactericidal activity in terms of
CFU/mL of above metal complexes against same microorganisms
(three Gram(ꢁve) and two Gram(þve)) revealed decrease in number
of colonies with increase in the concentration of compounds. The
results are shown in Fig. 3 for all the complexes against S. aureus.
The number of colonies counted in this technique was 30e300.
From the minimum inhibitory concentration (MIC) values and
colony forming units (CFU), it is found that complex 3 is more
potent against all the five microorganisms.
Table 3
The binding constants (Kb) of complexes with DNA in
phosphate buffer pH 7.2.
Complexes
Kb (Mꢁ1
)
[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]
8.31 ꢂ 104
6.63 ꢂ 104
9.60 ꢂ 104
4.12 ꢂ 104
8.81 ꢂ 103
2.40 ꢂ 104
7.96 ꢂ 103
Fig. 3. Relationship between concentration and bactericidal activity of all complexes
against S. aureus.

14
M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
Table 4
Experimental values of IC50 obtain from nonenzymatic
SOD-like activities of synthesized complexes.
Complexes
IC50 (mM)
[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]
0.591
0.675
0.552
0.822
1.244
1.012
1.276
3.5.3. DNA cleavage study by gel electrophoresis
DNA cleavage accelerated by transition metal complexes is the
centre of interest [49]. Fig. 6 shows the electrophoretic separation
of pUC19 DNA reacted with complexes under aerobic conditions.
When the plasmid DNA was subjected to electrophoresis upon
reacting with complexes, the fastest migration was observed for the
supercoiled (SC) form (form I), the slowest moving open circular
(OC) form (form II) produced upon relaxing SC and the interme-
diate moving linear (LC) form (form III), generated upon the
cleavage of circular form. Data for the cleavage study are presented
in Table 5. The difference in the DNA cleavage efficiency of
complexes was due to the difference in the binding affinity of the
complexes to DNA.
Fig. 5. Effect on relative viscosity of DNA under the influence of increasing amount of
complexes at 27 ꢄ 0.1 ꢃC in phosphate buffer (Na2HPO4eNaH2PO4, pH 7.2).
3.5. DNA interaction study
3.5.1. Absorption titration experiment
DNA can provide three distinct binding sites for quinolone metal
complexes, namely, groove, phosphate group and intercalation
[42]. This behaviour is of great importance with respect to the
relevant biological role of quinolone antibiotics in the body [43]. An
absorption spectrum of complexes with Herring Sperm DNA was
recorded for a constant concentration of complexes with varying
concentration of DNA to obtain different DNA:complex mixing
ratio. A representative titration curve is shown in Fig. 4. The
changes observed in the UV spectra of the complexes after mixing it
with DNA (either the increase in intensity or the shift in the
wavelength) indicate that the interaction of complexes with DNA
takes place by a direct formation of a new complex with double-
helical DNA [44]. The extent of the binding strength of complexes
was quantitatively determined by calculating intrinsic binding
constant (Kb) of the complexes by monitoring the change in
absorbance at various concentrations of DNA. From the plot of
[DNA]/(εa ꢁ εf) versus [DNA] (inset, Fig. 4), the Kb values of
complexes were determined and were found in the range
7.96 ꢂ 103e9.60 ꢂ 104 Mꢁ1 (Table 3). The Kb value and red shift
clearly indicates that the synthesized complexes bind to DNA via
intercalation mode but the values are much lower compared with
ethidium bromide, a classical intercalator [45].
Fig. 7. Absorbance (Abs560) as a function of time (t) plotted for varying concentration
of complex 1 from 0.5 mMe3 mM.
3.5.2. DNA binding study by hydrodynamic volume measurement
Viscosity measurement can be regarded as a reliable tool in the
absence of crystallographic and NMR data [46]. The intercalation of
a molecule into DNA could result in lengthening, unwinding and
stiffening of the helix and is usually accompany by increase in
solution viscosity [47,48]. In this case, an increase in viscosity was
observed hence the complexes were bound to DNA via an inter-
calation mode and out of all the complexes, complex 3 interacted
more strongly compare to the others (Fig. 5).
Fig. 6. Separation of cleaved pUC19 DNA (150
complexes (200
mol Lꢁ1) using 1% agarose gel stained with 0.5
bromide. Interaction between DNA and complexes was allowed for 24 h at 37 ꢃC in TE
mg/mL) with series of copper(II)
m
mg/mL ethidium
buffer (pH 8) with a final volume of 15 mL: Lane 1, DNA control; Lane 2, CuCl2$2H2O;
Lane 3, norfloxacin; 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].
Fig. 8. Plot of percentage of inhibiting NBT reduction with an increase in the
concentration of complex 1.

M.N. Patel et al. / Journal of Organometallic Chemistry 701 (2012) 8e16
15
Table 5
strongly than rest of the complexes. The electronic absorption
data are in good accordance with viscosity study. The DNA
cleavage study of pUC19 shows that all complexes have high
cleavage ability than metal salt and drug. Upon determination of
antioxidant activity in NBT/NADH/PMS system, complex 3 shows
the highest scavenging ability for oxygen radical. The preliminary
studies encourage for carrying out further in vivo experiments
and seeming helpful in generating database for preparation of
effective DNA probe.
Complex mediated DNA cleavage data by gel electrophoresis.
Lane No.
Compound
Form I (SC)
Form II (OC)
Form III (LC)
1
2
3
4
5
6
7
8
9
Control
CuCl2$2H2O
Norfloxacin
91
87
63
27
25
20
28
33
30
35
9
13
22
56
59
62
53
52
51
49
e
e
15
17
16
18
19
15
19
16
[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]
Conflict of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
10
Acknowledgements
The authors thank the Head, Department of Chemistry, Sardar
Patel University, India, for making it convenient to work in
laboratory.
Appendix. Supplementary Data
Supplementary data related to this article can be found online at
doi:10.1016/j.jorganchem.2011.11.022.
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