Antibacterial, DNA interaction and superoxide dismutase activity of drug based copper(II) coordination compounds

Article abstract of DOI:10.1016/j.poly.2010.08.037

The novel neutral mononuclear copper(II) complexes with the quinolone antibacterial drugs ciprofloxacin in the presence of the nitrogen donor heterocyclic ligand of terpyridine have been synthesized and characterized by elemental analysis, reflectance spe

Full text of DOI:10.1016/j.poly.2010.08.037

Polyhedron 29 (2010) 3238–3245
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Antibacterial, DNA interaction and superoxide dismutase activity of drug based
copper(II) coordination compounds
⇑
Mohan N. Patel , Promise A. Dosi, Bhupesh S. Bhatt
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120 Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel neutral mononuclear copper(II) complexes with the quinolone antibacterial drugs ciprofloxacin
in the presence of the nitrogen donor heterocyclic ligand of terpyridine have been synthesized and char-
acterized by elemental analysis, reflectance spectra, IR and mass spectroscopy. The antibacterial activities
of the newly synthesized compounds were evaluated and correlated with their physicochemical proper-
ties. Results revealed that the tested compounds exhibited better inhibitory activities than the reference
antibacterial quinolone drugs against Gram(+ve) and Gram(ꢀve) bacteria. The coordination compounds can
act as catalysts for the dismutation of superoxide anion radicals (O2ꢁꢀ). The detection of the rate constant
of the reaction of superoxide ion with nitro blue tetrazolium (NBT) is inhibited by superoxide dismutase
(SOD). The interaction of the complex with DNA was investigated using viscosity, absorption titration and
DNA melting temperature techniques. The results indicate that the complexes bind to DNA with interca-
lative mode and they have rather high DNA-binding constants. DNA-cleavage study showed better cleav-
ing ability of the complexes compare to metal salt and standard drugs.
Received 28 May 2010
Accepted 31 August 2010
Available online 7 September 2010
Keywords:
Copper(II)
Terpyridines
Ciprofloxacin
Antibacterial activity
DNA interaction
SOD
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
like hydroxyl radical and hydrogen peroxide. To control this, nat-
ure has created a family of enzymes which remove them from
The interaction and reaction of transition metal complexes with
DNA have been long intensively investigated in relation to applica-
tions in the fields of molecular biology, biotechnology and medi-
cine [1–3]. DNA provides a range of binding sites and binding
modes for covalent and non-covalent interactions, including inter-
calation, groove bindings, electrostatic forces and hydrogen bonds
with metal complexes. Interest in the fashion of metal complex
binding to DNA has been motivated not only by a desire to under-
stand the basics of these interaction modes but also by the devel-
opment of metal complexes into anti-inflammatory, antifungi,
antibacteria or anticancer reagents [4]. Hence, much of the atten-
tion has been targeted on the design of metal-based complexes,
predominantly copper(II) complexes, which can bind and cleave
DNA. Copper is a bioessential element in all living systems. Re-
cently, numerous research groups have reported novel copper(II)
complexes with organic ligands showing antifungal and antibacte-
rial properties against several pathogenic fungi and bacteria [5,6].
Superoxide dismutase (SOD) is an essential enzyme that elimi-
the cellular environment. One of these, superoxide dismutase
(SOD), catalyzes disproportionation of superoxide to dioxygen
and hydrogen peroxide, which is decomposed via catalyzes to
water and dioxygen [9].
Here, we report the synthesis and characterization of drug
based coordination compounds using ciprofloxacin and tridentates
terpyridines. The antimicrobial efficiency of complexes has also
been tested on five different microorganisms. Rate constant for
the reaction of superoxide radicals was detected using nitroblue
tetrazolium (NBT), which was inhibited by superoxide dismutase.
The DNA-binding and cleavage properties of the complexes have
been investigated using absorption titration, viscosity measure-
ments DNA melting temperature and gel electrophoresis method.
2. Experiments
2.1. Reagent
ꢁꢀ
nates superoxide radical anion O2 and thus protects cells from
All the chemicals and solvent used were of analytical grade. Cip-
rofloxacin hydrochloride was purchased from Bayer AG (Wupper-
tal, Germany). Pyridine, 2-acetyl pyridine, p-chloro benzaldehyde,
m-chloro benzaldehyde, p-bromo benzaldehyde, m-bromo benzal-
dehyde and p-fluoro benzaldehyde were purchased from Loba Che-
mie Pvt. Ltd. (India). Ethidium bromide and Luria broth were
purchased from Himedia, India. Acetic acid and EDTA were
damage induced by reactive oxygen species (ROS) [7,8]. The super-
oxide radical anion O2ꢁꢀ is formed as a byproduct of normal cellular
respiration. Its decomposition produces undesired harmful species
⇑
Corresponding author. Tel.: +91 2692 226856x220.
E-mail address: jeenen@gmail.com (M.N. Patel).
0277-5387/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2010.08.037

M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
3239
purchased from Sd fine chemicals, India. CT-DNA was purchased
from Sigma Chemical Co., India.
and ammonia solution (10 mL). After stirring for 4 h at room tem-
perature, the resultant orange precipitate was isolated by filtration
and recrystallized from MeOH. Ligands were characterized using
1H NMR and 13C NMR spectra.
2.2. Physical measurement
Metal contents of the complexes were analyzed by EDTA titra-
tion [10] after decomposing the organic matter with a mixture of
HClO4, H2SO4, and HNO3 (1:1.5:2.5). C, H and N elemental analyses
were performed on a model Perkin–Elmer 240 elemental analyzer.
Magnetic moments were measured by Gouy’s method using
2.4. Synthesis of complexes
2.4.1. [Cu(cpf)(A1)Cl]
Methanolic solution of CuCl2ꢁ2H2O (1.5 mmol) was added to
methanolic solution of 40-(4-chloro phenyl)-2,20:60,200-terpyridine
(1.5 mmol), followed by addition of a previously prepared solution
of ciprofloxacin (1.5 mmol) in methanol in presence of CH3ONa
(1.5 mmol). The pH was adjusted at ꢃ6.8 using dilute solution of
CH3ONa. Resulting solution was refluxed for 2 h on water bath, fol-
lowed by concentrating it to half of its volume. A fine amorphous
product of green color was obtained which was washed with
ether/hexane and dried in vacuum desiccators. The proposed reac-
tion is shown in Scheme 1.
mercury tetrathiocyanatocobaltate(II) as the calibrant
(vg =
16.44 ꢂ 10ꢀ6 cgs units at 20 °C), on Citizen Balance. The diamag-
netic correction was made using Pascal’s constant. IR spectra were
recorded on a FT-IR Shimadzu spectrophotometer with sample
prepared as KBr pellets in the range of 4000–400 cmꢀ1. The reflec-
tance spectra were recorded on a LAMBDA 19 UV–Vis–NIR, Spec-
trophotometer, Perkin–Elmer (USA). The 1H NMR and 13C NMR
were recorded on a Bruker Avance (400 MHz). The FAB-mass spec-
tra were recorded on a Jeol SX 120/Da-600 mass spectrometer/
Data system using Argon/Xenon(6 kV, 10 mA) as the FAB gas. The
accelerating voltage was 10 kV and spectra were recorded at room
temperature.
Yield: 64.7%, m.p.: 230 °C, leff
: 1.89 B.M. Anal. Calc. for
C38H31Cl2CuFN6O3 (733.14): C, 59.03; H, 4.04; N, 10.87; Cu, 8.22.
Found: C, 58.94; H, 3.97; N, 10.96; Cu, 8.46%.
In similar way, complexes 2–5 were prepared with the use of
corresponding ligands.
2.3. Synthesis of ligands
2.4.2. [Cu(cpf)(A2)Cl]
All tridentate ligands were synthesized similarly by following
literature procedure [11]. Aqueous NaOH (10 mL 1.5 M solution)
It was prepared using 40-(3-chloro phenyl)-2,20:60,200-terpyri-
dine (1.5 mmol). Yield: 65.6%, m.p.: 228 °C,
leff: 1.92 B.M. Anal.
was added to
a
stirred solution of 2-acetylpyridine (1.20 g,
Calc. for C38H31Cl2CuFN6O3 (733.14): C, 59.03; H, 4.04; N, 10.87;
l0 mmol) and different aldehyde (10 mmol) in ethanol (20 mL)
Cu, 8.22. Found: C, 59.24; H, 3.78; N, 10.76; Cu, 8.15%.
Scheme 1. Structure of the title complex [Cu(cpf)(A1)Cl]ꢁ5H2O.

3240
M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
2.4.3. [Cu(cpf)(A3)Cl]
2.6. DNA interaction activity
It was prepared using 40-(4-bromo phenyl)-2,20:60,200-
terpyridine (1.5 mmol). Yield: 67.8%, m.p.: 238 °C,
l
eff: 1.89 B.M.
2.6.1. Absorption titration
Anal. Calc. for C38H31BrClCuFN6O3 (817.59): C, 55.82; H, 3.82; N,
10.28; Cu, 7.77. Found: C, 55.62; H, 4.06; N, 10.11; Cu, 7.65%.
The DNA-binding experiments were performed in Tris–HCl/
NaCl buffer (50 mM Tris–HCl/1 mM NaCl buffer, pH 7.5). The
concentration of calf thymus (CT) DNA was determined measur-
ing absorption intensity at 260 nm with
a e value [12] of
2.4.4. [Cu(cpf)(A4)Cl]
6600 Mꢀ1 cmꢀ1
.
Absorption titration experiments were made
It was prepared using 40-(3-bromo phenyl)-2,20:60,200-terpyri-
using different concentrations of CT-DNA, keeping the concentra-
tion of the complexes constant, with due correction for the absor-
bance of the CT-DNA itself. Samples were equilibrated before
recording each spectrum.
dine (1.5 mmol). Yield: 67.8%, m.p.: 234 °C,
Calc. for C38H31BrClCuFN6O3 (817.59): C, 55.82; H, 3.82; N, 10.28;
Cu, 7.77. Found: C, 55.72; H, 3.71; N, 10.15; Cu, 7.94%.
leff: 1.97 B.M. Anal.
To compare quantitatively the affinity of the complexes bound
to DNA, the intrinsic-binding constants Kb of the complexes were
obtained by monitoring the changes in absorbance with increasing
concentration of DNA using the following Eq. (1) [13].
2.4.5. [Cu(cpf)(A5)Cl]
It was prepared using 40-(4-fluoro phenyl)-2,20:60,200-terpyridine
(1.5 mmol). Yield: 65.0%, m.p.: 218 °C,
leff: 1.87 B.M. Anal. Calc. for
½DNAꢄ=ðea
ꢀ
ef Þ ¼ ½DNAꢄ=ðeb
ꢀ
ef Þ þ 1=Kbðeb
ꢀ
ef
Þ
ð1Þ
C38H31ClCuF2N6O3 (756.69): C, 60.32; H, 4.13; N, 11.11; Cu, 8.40.
Found: C, 60.11; H, 4.34; N, 10.98; Cu, 8.31%.
where, [DNA] is the concentration of DNA in base pair, ea
,
ef and eb
correspond to the apparent absorption coefficient Aobs/[Cu], the
extinction coefficient for the free complex and the extinction coef-
ficient for the complex in the fully bound form, respectively.
2.5. Antibacterial activity
Antibacterial activity of the compounds (metal salts, standard
drugs and complexes) was screened against two Gram(+ve)
Staphylococcus aureus, Bacillus subtilis, and three Gram(ꢀve) Serratia
marcescens, Escherichia coli and Pseudomonas aeruginosa. Screening
was performed by determining the minimum inhibitory concen-
tration (MIC). All cultures were incubated at 37 °C. Control tests
with no active ingredients were also performed. The MIC was
determined using twofold serial dilutions in liquid media of the
compound being tested. The solvent used was DMSO.
2.6.2. Viscosity measurements
Viscosity measurements were carried out using an Ubbelodhe
viscometer maintained at a constant temperature of 27.0 0.1 °C
in a thermostatic bath. DNA samples approximately 200 base pairs
in average length were prepared by sonicating it to minimize com-
plexities arising from DNA flexibility [14]. Flow time was measured
with a digital stopwatch, and each sample was measured three
times, and an average flow time was calculated. The rate of flow of
sodium phosphate buffer (pH ꢃ7.2), DNA (100
lM) and DNA with
A preculture of bacteria was grown in LB (Luria Broth) overnight
at the most favorable temperature of each species. This culture was
used as a control to examine if the growth of bacteria tested is nor-
the copper complexes at various concentrations were measured.
1/3
Data are presented as (
g
/g0)
versus binding ratio [15], where g
is the viscosity of DNA in the presence of complex and
cosity of DNA alone.
g0 is the vis-
mal. In a similar second culture, 20 lL of the bacteria as well as the
tested compound at the desired concentration were added. We
monitored bacterial growth by measuring turbidity of the culture
after 18 h. If a certain concentration of a compound inhibited bac-
terial growth, half the concentration of the compound was tested.
This procedure was carried on to a concentration that bacteria
grow normally. The lowest concentration that inhibited bacterial
growth was determined as the MIC value. All equipment and cul-
ture media were sterilised.
The bactericidal action of all compounds was evaluated against
three Gram(ꢀve) and two Gram(+ve) bacteria. The inoculum was pre-
pared by diluting an overnight culture, grown in Luria broth, to ob-
tain 106 viable bacteria/mL, confirmed in each experiment by
colony counts. Bacteria were exposed to concentrations of 0.25–
2.6.3. DNA melting temperature
Thermal denaturation studies were performed with a Perkin–
Elmer Lambda 35 spectrophotometer equipped with a Peltier tem-
perature-controlling programmer ( 0.1 °C). The absorbance at
260 nm was continuously monitored for solutions of CT-DNA
(100 lM) in the absence and the presence of complex (20 lM).
The temperature of the solution was increased by 1 °C minꢀ1
.
2.6.4. DNA cleavage study
Gel electrophoresis of plasmid DNA (pUC19 DNA) was carried
out in TE buffer within 15 reaction mixture containing
g/mL plasmid DNA (10 mM Tris, 1 mM EDTA, pH 8.0) and
M complex. Reactions were allowed to proceed for 3 h at
L loading buf-
lL
1.75
lg/mL of compounds. The final volume was 1 mL. Cultures
300
200
l
l
were incubated at 37 °C for 2 h. The 100
lL bacterial culture from
above was taken and spread over previously prepared agar plate.
These were incubated for 24 h at 37 °C and the visual colonies were
calculated in order to check biocidal activity of metal complexes,
yielding 30–250 colonies.
37 °C. All reactions were quenched by addition of 5
l
fer (40% sucrose, 0.2% bromophenol blue). The aliquots were
loaded on to 1% agarose gel and electrophoresed at 50 V in 1X
TAE buffer. Gel was stained with 0.5 lg/mL ethidium bromide
Table 1
IR spectra data.
Compounds
m
(C@O) cmꢀ1 pyridone
m
(COO)as cmꢀ1
m
(COO)s cmꢀ1
D
m
(cmꢀ1
)
m
(M–N) cmꢀ1
m
(M–O) cmꢀ1
Ciprofloxacin
Complex-1
Complex-2
Complex-3
Complex-4
Complex-5
1708
1622
1617
1624
1619
1617
1624
1564
1578
1579
1564
1562
1340
1364
1377
1378
1374
1343
284
200
201
203
197
223
535
542
540
541
542
515
504
510
508
511

M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
3241
Table 2
MIC data of the compounds (
and was photographed on UV illuminator. The percentage of each
lM).
form of DNA was quantitised using AlphaDigiDoc™ RT. Version
V.4.0.0 PC-IMAGE software. The degree of DNA cleavage activity was
expressed in terms of the percentage of cleavage of the SC-DNA
according to the following equation [16]:
S. aureus B. subtilis S. marcescens P. aeruginosa E. coli
CuCl2ꢁ2H2O
Ciprofloxacin
Gatifloxacin
Norfloxacin
Enrofloxacin
Pefloxacin
Levofloxacin
Sparfloxacin
Ofloxacin
2698.00 2815.00
2756.00
1.6
2404.00
1.4
3402.00
1.6
5.1
2.5
1.9
2.1
1.7
1.3
1.9
1.1
4.0
2.5
3.9
2.4
2.2
2.0
1.4
1.4
2.9
2.8
1.4
2.7
1.0
1.3
1.4
2.9
4.1
1.7
5.1
1.7
1.5
1.7
1.0
3.8
1.4
5.7
1.7
1.5
2.2
%DNA cleavage activity
¼ f½ð% of SC-DNAÞcontrol
ꢀ ð% of SC-DNAÞsampleꢄ=ð%of SC-DNAÞcontrolg ꢂ 100
Complex-1
Complex-2
Complex-3
Complex-4
Complex-5
1.16
1.21
1.07
1.54
1.04
1.12
1.19
0.88
1.08
0.57
0.59
0.73
1.21
1.67
0.33
0.68
0.52
0.87
2.01
0.34
1.13
1.19
1.05
1.97
1.26
2.7. Determination of SOD-like activity
SOD–like activity of the complexes was determined using NBT/
NADH/PMS system [17]. The superoxide radical produce by 79
NADH, 30 M PMS, system containing 75 M NBT, phosphate buf-
fer (pH 7.8), and 0.25–3.0 M tested compound. The amount of re-
lM
l
l
l
duced NBT was spectrophotometrically detected by monitoring the
concentration of blue formazan form which absorbs at 560 nm. The
reduction rate of NBT was measured in presence and absence of
test compounds at various concentrations of complexes in the sys-
tem. All measurements were carried out at room temperature. IC50
value of the complexes was determined by plotting the graph of
percentage inhibition of NBT reduction against increase in concen-
tration of complexes. Concentration of complexes which causes
50% inhibition of NBT reduction is reported as IC50.
are obtained, that is, the only kmax at about 615 nm points toward
octahedral geometry.
3.2. IR spectra
The determination of the coordinating atoms was made on the
basis of the comparison of the IR spectra of the ciprofloxacin and
of the complexes. Significant wave numbers are given in Table 1.
The
m
(C@O) stretching vibration band appears at 1708 cmꢀ1 for cip-
rofloxacin, where as for complexes it appear at 1617–1624 cmꢀ1
,
3. Result and discussion
this shift towards lower energy suggests that coordination occurs
through carbonyl oxygen of pyridine ring. Strong absorption band
at 1624 and 1340 cmꢀ1 in ciprofloxacin could be assigned for
3.1. Reflectance spectra and magnetism
The magnetic moments of the copper(II) complexes lie in the
1.87–1.97 BM range. These values are typical of mononuclear cop-
per(II) compounds with d9 electronic configuration. The observed
magnetic moments of all the complexes correspond to typical
high-spin octahedral complexes. However, the values are slightly
higher than the expected spin-only values due to spin–orbit-
coupling contribution [18].
m
(COO) asymmetric and symmetric vibration, respectively, while
in metal complexes these bands were observed at 1564 and
1364 cmꢀ1. The difference
as(COO) ꢀ s(COO) is useful for
values are
D
=
m
m
determining the coordination mode of ligands. The
D
greater than 200 cmꢀ1, indicating monodentate coordination mode
of carboxylato group [20–24] of the ligands. These data are further
supported by m
(M–O) which appear at 504–515 cmꢀ1 for complexes.
Copper(II) complexes with octahedral geometry are mock to
demonstrate the absorption spectra in visible region and only
one broad band about 660 nm [19]. Here in our case same results
In investigated complexes the m(C@N) band of terpyridine
appears at 1584 cmꢀ1. This band shift to higher frequency at
ꢃ1626 cmꢀ1 [25,26] in complexes indicates bidentate N–N
Fig. 1. FAB-mass spectrum of complex 1, that is [Cu(cpf)(A1)Cl], obtained using m-nitro benzyl alcohol.

3242
M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
ligands, there are two different type of ligands and both ligands
are responsible for higher antimicrobial activity; (iii) the total
charge of the complex; generally the antimicrobial efficiency de-
creases in the order cationic > neutral > anionic complex; (iv) the
nature of the ion neutralizing the ionic complex; (v) the nuclearity
of the metal center in the complex; dinuclear centers are usually
more active than mononuclear ones. The first two of the five
above-mentioned factors may be responsible for higher antimicro-
bial activity; that is the chelate effect provided by both the cipro-
floxacin ligand and the N-donor ligand and the nature of the
ligands.
This is probably one of the main reasons for the diverse antibac-
terial activities shown by the complexes. The significant improve-
ment of the activity of ciprofloxacin when coordinated to the
copper complex is simply an evidence of the role of the coordi-
nated metal ion [30].
It has also been suggested [31,32] that the ligands with nitrogen
donor systems might inhibit enzyme production, since the en-
zymes which require these groups for their activity appear to be
especially more susceptible to deactivation by the metal ions upon
chelation. Chelation reduces the polarity [31,32] of the metal ion
mainly because of the partial sharing of its positive charge with
Fig. 2. Relationship between concentration and bactericidal activity of all com-
plexes against S. aureus.
coordination of the ligand. N?M bonding was supported by
N) band [27] at ꢃ535–542 cmꢀ1 for complexes.
m(M–
3.3. Mass spectra
the donor groups and possibly the p-electron delocalization within
the whole chelate ring system thus formed during coordination.
This process of chelation thus increases the lipophilic nature of
the central metal atom, which in turn favors its permeation
through the lipoid layer of the membrane. This in turn is responsi-
ble for increasing the hydrophobic character and liposolubility of
the molecule in crossing cell membrane of the microorganism
and hence enhances the biological utilization ratio and activity of
the testing drug/compound.
In addition, our study regarding bacteriocidal activity in terms
of CFU/mL of above metal complexes against same microorganisms
(three Gram(ꢀve) and two Gram(+ve)) revealed decrease in number
of colonies with increasing the concentration of compounds. The
results are shown in Fig. 2 for all the complexes against S. aureus.
The maximum concentration of all the complexes was 1.75 CFU/
mL which gave lesser number of colonies. The number of colonies
counted in this technique was 30–250. Complexes 1, 2 and 5 had
lesser number of colonies than complexes 3 and 4. Results for
microorganism B. subtilis, S. marcescens, P. aeruginosa and E. coli
are kept in Supplementary data.
Fig. 1 represents the FAB-mass spectrum of complex 1, that is
[Cu(cpf)(A1)Cl], obtained using m-nitro benzyl alcohol as matrix.
Peaks at 136, 137, 154, 289 and 307 m/z are due to usage of matrix.
The molecular ion peak is observed at m/z = 773 which is equiva-
lent of its molecular weight. Loss of chlorine atom gave a fragment
ion peak at m/z = 736, which confirm that chlorine atom attached
to metal ion with covalent bond. The highest peak was observed
at m/z = 406. Other fragment corresponds to peaks at 661, 442,
393, 343 and 331 m/z value.
3.4. Antibacterial activity
A comparative study of MIC values of the fluoroquinolones and
its mixed ligand complexes indicate that the metal complexes have
better antibacterial activity than fluoroquinolones (Table 2). The
inhibition activity seems to be governed in certain degree by the
facility of coordination at the metal centre as well as bulkiness of
the ligands. This may support the argument that some type of
molecular binding to the metal ions or intercalation or electrostatic
interactions causing the inhibition of biological synthesis and pre-
venting the organisms from reproducing. The increased activity of
copper complexes compared to that of the free ligands may be ex-
plained in terms of chelation theory [28]. According to this theory,
formation of the chelate ring enhanced the lipophilicity of com-
plexes, which breaks down the permeability barrier of the cell
retarding the normal cell processes.
3.5. Metal DNA interaction
3.5.1. Photophysical measurement
A classical intercalation model demands that the DNA helix
must lengthen as base pairs are separated to accommodate the
Efficiencies of ligands and complexes have been tested against
three Gram(ꢀve), E. coli, S. marcescens and P. aeruginosa and two
Gram(+ve)
, S. aureus and B. subtilis microorganisms. In case of
S. aureus, all compounds shows good activity compare to reference
drugs. In case of B. subtilis, all compounds exhibit good activity
compare to fluoroquinolones. In case of S. marcescens, compounds
I, II, III and V found more potent than reference drugs. In case of
P. aeruginosa, compounds I, II, III and V exhibit good activity
compare to fluoroquinolones. In case of E. coli, compounds I, II, III
and IV shows more potency than reference drugs.
In general, antimicrobial activity of the metal complexes de-
pends on following five factors [29]: (i) the chelate effect, that is,
ligands that are bound to metal ions in a bidentate and tridentate
fashion, such as the quinolones and the N-donor ligands (terpy),
show higher antimicrobial efficiency towards complexes than uni-
dentate N-donor ligands, e.g., pyridine; (ii) the nature of the
Fig. 3. Effect on relative viscosity of DNA under the influence of increasing amount
of complexes at 27 0.1 °C in phosphate buffer as a medium.

M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
3243
binding ligand, leading to an increase of the viscosity of the DNA
solution [33]. Binding modes of the complexes were further inves-
tigated by viscosity measurements. Though photophysical experi-
ments, give necessary information about binding modes of metal
complexes with DNA, they do not provide conclusive evidences
for the exact mode of binding. Hydrodynamic measurements such
as viscosity which is sensitive to length changes are regarded as
the least ambiguous and the most critical tests of binding modes
in solution [34]. Viscosity of DNA is increased in case of classical
intercalators due to increase in the length of DNA helix, as base
pairs are separated to accommodate the intercalator. In contrast,
decrease in DNA viscosity is noted in partial or non-classical inter-
calator, because it may bend (or kink) DNA helix, thereby decreas-
ing its effective length. From Fig. 3 as increasing the amounts of
complexes, viscosity of DNA increases steadily, which is similar
to that of the classical intercalative complex [Ru(phen)2(DPPZ)]2+
.
Amongst all, complexes 1, 2 and 5 bind more strongly than 3 and
4. The increased degree of viscosity, which may depend on the
binding affinity to DNA, follows the order of increasing viscosity
is EB > [Cu(cpf)(An)Cl] > Ciprofloxacin. These results suggest that
the all title complexes intercalate between the base pairs of DNA
and the binding affinity of complexes is higher than ciprofloxacin
but less than ethidium bromide.
Fig. 4. Electronic absorption titration curve of [Cu(cpf)(A1)Cl]ꢁ5H2O in absence and
in presence of increasing amount of DNA; 50–150
l
M
in phosphate buffer
M with incuba-
ꢀ e
f) vs. [DNA].
(Na2HPO4/NaH2PO4, pH 7.2), [complex] = 15 lM, [DNA] = 50–150 l
tion period of 30 min at 37 °C, Inset: Plot of [DNA]/(ea
3.5.2. UV–Vis absorption spectra
DNA can provide three distinctive binding sites for the quino-
lone complexes; namely, groove binding, binding to phosphate
groups, and intercalation. This behavior is of great importance with
regard to the biological role of fluoroquinolone antibiotics in the
human body [35].
Table 3
Binding constant and IC50 values of copper(II) complexes.
Complexes
Kb (M–1
)
IC50
Interaction of metal complexes with DNA can be monitored by
absorption spectral titration [36]. In Fig. 4, with increasing concen-
tration of CT-DNA, absorption bands of the complexes are affected,
resulting in hypochromism with bathochromic shift. The change in
absorbance values were used to evaluate intrinsic-binding con-
stant Kb for the complexes (Table 3). Binding constants of the par-
ent complexes are sufficiently high due to their octahedral nature
[37]. To compare binding strength of complexes quantitatively, the
intrinsic binding constants Kb of complexes were determined by
monitoring the changes of absorbance at 274 nm with increasing
[Cu(cpf)(A1)Cl] (1)
[Cu(cpf)(A2)Cl] (2)
[Cu(cpf)(A3)Cl] (3)
[Cu(cpf)(A4)Cl] (4)
[Cu(cpf)(A5)Cl] (5)
2.66 ꢂ 104
2.62 ꢂ 104
1.67 ꢂ 104
1.27 ꢂ 104
4.73 ꢂ 104
0.750
1.25
1.35
1.5
0.5
concentration of DNA. From the plot of [DNA]/(ea
ꢀ
ef) versus
[DNA], Kb value of all complexes (Table 3) were found to be in
range of 1.2 ꢂ 104–4.73 ꢂ 104 Mꢀ1
.
3.5.3. Thermal denaturation study
Thermal behaviors of DNA in the presence of complexes can
give insight in to their conformational changes when temperature
is raised, and offer information about the interaction strength of
complexes with DNA. It is well known that when the temperature
in the solution increases, the double stranded DNA gradually disso-
ciates to single strands and generates a hyperchromic effect on the
absorption spectra of DNA bases (kmax = 260 nm). In order to iden-
tify this transition process, the melting temperature Tm, which is
defined as the temperature where half of the total base pairs are
unbounded, is usually introduced. According to the literature
Fig. 5. Melting curves of CT-DNA in the absence and presence of complexes 1–5.
the interaction of metallointercalators generally results in
a
Fig. 6. Photogenic view of interaction of pUC19 DNA (450
All reactions were incubated in TE buffer (pH 8) in a final volume of 15
[Cu(cpf)(A1)Cl]; Lane 5, [Cu(cpf)(A2)Cl]; Lane 6, [Cu(L)(cpf)Cl]; Lane 7, [Cu(cpf)(A4)Cl]; Lane 8[Cu(cpf)(A5)Cl].
l
g/mL) with series of copper(II) complexes (200
lM) using 1% agarose gel containing 0.5 lg/mL ethidium bromide.
l
L, for 3 h at 37 °C. Lane 1, DNA control; Lane 2, CuCl2ꢁ2H2O; Lane 3, ciprofloxacin; Lane 4,

3244
M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
considerable increase in melting temperature (Tm). DNA (100 lM)
melting experiments revealed that Tm of CT-DNA is 74.2 1 °C in
the absence of the complex (Fig. 5). However, with addition of
complexes 1, 2 and 5 the Tm increased to 78.9 1 °C, 78.8 1 °C
and 79.0 1 °C, respectively and for complexes 3 and 4 the Tm in-
creased to 78.3 1 °C and 78.1 1 °C, respectively. The increased
Tm (3.9–4.8 °C) value of the DNA after addition of the complexes
is comparable to that observed for classical intercalators [38].
3.5.4. Gel electrophoresis
Fig.
6 illustrates gel electrophoretic separations showing
cleavage of pUC19 DNA induced by the complexes under aerobic
conditions [39]. When circular plasmid DNA is conducted by elec-
trophoresis, the fastest migration will be observed for supercoiled
form-I (SC). If one strand is cleaved, the supercoiled will relax to
produce a slower-moving open circular form-II (OC). If both
strands are cleaved, linear form-III (LC) will be generated that mi-
grates in between. This clearly shows that the relative binding effi-
cacy of complexes to DNA is much higher than the binding efficacy
of metal salt or ciprofloxacin (Table 4). Different DNA-cleavage effi-
ciency of the complexes was due to different binding affinity of
complexes to DNA, which has been observed in other cases.
Fig. 8. Plot of percentage of inhibiting NBT reduction with an increase in the
concentration of complex 1.
Copper gives good SOD activity, although it structure is totally
unrelated with native enzyme. The ping-pong mechanism of SOD
activity is given in Eqs. (2) and (3).
O2ꢀ þ CuII ! O2 þ CuI
ð2Þ
ð3Þ
O2ꢀ þ CuI þ 2Hþ ! H2O2 þ CuII
Fig. 7 represents plot of absorbance values (Aobs560) against time (t)
with varying concentration of complex 1 from 0.25 to 5 M. Fig. 8
3.6. Free radical scavenger activity
l
The superoxide radicals (O2ꢁꢀ) were generated in vitro using a
non-enzymatic (NBT/NADH/PMS) system and determined spectro-
photometrically. The system used as a source of superoxide radical
generator was NBT/NADH/PMS system in order to check SOD-like
activity of the synthesized complexes. The SOD activities of com-
plexes were investigated by NBT assay. SOD data for the complexes
have been compiled in Table 3 along with SOD activity value of
other similar complexes and containing copper metal ion [40].
illustrate that chromophore requires to yield 50% inhibition of the
reduction of NBT (IC50) value of complexes are higher than exhib-
ited by the copper salt. The copper(II) complexes showed SOD-like
activity which was evaluated by the scavenger concentration causes
50% inhibition in the detector formation, IC50. Compounds exhibit
SOD-like activity at biological pH with IC50 values ranging from
0.5 to 1.5 lM. Complexes 1 and 5 were found even more active
compare to 2–4.
3.7. Structure–activity relationship
Table 4
Gel electrophoresis data.
The quinolone/naphthyridone ring system can be modified at
various sites and consequently, over the years, hundreds of analogs
have been investigated in an attempt to increase efficacy and re-
duce adverse effects. Features that enhance the effectiveness of
the quinolones most are a halogen (F or Cl) at C8, which improves
oral absorption and activity against anaerobes; alkylated pyrroli-
dine or piperazine at C7, which increases serum half-life and activ-
ity against gram-positive microorganisms; and a cyclopropyl group
at N1 and an amino substituent at C5, which improve overall po-
tency [41].
Compounds
%SC
%OC
%LC
% Cleavage
DNA control
DNA + metal salt
DNA + CPFH
DNA + I
DNA + II
DNA + III
75
72
67
16
15
20
29
14
25
28
33
52
52
50
56
56
4.00
10.66
78.66
80.00
73.33
61.33
81.33
32
33
30
15
30
DNA + IV
DNA + V
The electron-withdrawing substituent on the intercalative li-
gand can improve the DNA interaction affinity, free radical scav-
enging activity and antibacterial activity of the original complex,
whereas the electron-pushing substituent is reverse. Such a trend
suggests that the DNA interaction affinity, free radical scavenging
activity and antibacterial activity of the complex can be effectively
controlled by the substituents. In our case there are three different
substituents and all are electron withdrawing in nature. Increasing
ability of electron-withdrawing power of substituents in order of
F > Cl > Br. In case of antibacterial activity, DNA interaction and
superoxide dismutase, compound containing electron-withdraw-
ing substituents (F) has better activity then compound containing
substituents Cl and Br. The ascending order of complexes towards
the biological activities is V > I > II > III > IV.
Acknowledgements
Authors thank Head, Department of Chemistry, Sardar Patel
University, India for making it convenient to work in laboratory
Fig. 7. Plot of absorbance values (Aobs560) against time (t).

M.N. Patel et al. / Polyhedron 29 (2010) 3238–3245
3245
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.poly.2010.08.037.
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